Patent application title: VACCINE COMPRISING AN ATTENUATED PESTIVIRUS
Inventors:
Gregor Meyers (Waldorfhaeslach, DE)
Boehringer Ingelheim Vetmedica Gmbh (Ingelheim, DE)
Andreas Ege (Teubingen, DE)
Christiane Meyer (Meunster, DE)
Martina Von Freyburg (Heibronn, DE)
Assignees:
BOEHRINGER INGELHEIM VETMEDICA GMBH
IPC8 Class: AC12N704FI
USPC Class:
4242201
Class name: Virus or component thereof togaviridae or flaviviridae, except hepatitis c virus (e.g., yellow fever virus, bovine viral diarrhea virus, dengue virus, equine viral arteritis virus, equine encephalitis virus, japanese b encephalitis virus, sindbis virus, flavivirus, etc.) hog cholera virus
Publication date: 2013-02-14
Patent application number: 20130039946
Abstract:
Attenuated pestiviruses, in particular attenuated BVDV, wherein at least
one mutation is in the coding sequence for glycoprotein Ems and at
least another mutation in the coding sequence for Npro which
preferably leads to combined inactivation of the RNase activity residing
in glycoprotein Ems in addition to the inactivation of the
(hypothesized) immunomodulating activity residing in Npro. Methods
for attenuating pestiviruses such as BVDV, nucleic acids encoding the
pestiviruses, in particular BVDV, compositions and vaccines comprising
the attenuated pestiviruses, in particular BVDV, of the invention.Claims:
1. An attenuated classical swine fever virus (CSFV) having at least one
mutation in the coding sequence for glycoprotein Ems and at least
another mutation in the coding sequence for Npro.
2. The classical swine fever virus according to claim 1, wherein the mutation in the coding sequence for glycoprotein Ems leads to inactivation of RNase activity residing in Ems and/or the mutation in the coding sequence for Npro leads to inactivation of the Npro.
3. The classical swine fever virus according to claim 1, wherein the mutations are selected from the group of deletions, insertion mutations, and substitution mutations.
4. The classical swine fever virus according to claim 2, wherein the mutations are selected from the group of deletions, insertion mutations, and substitution mutations.
5. The classical swine fever virus according to claim 1, wherein the mutation(s) are deletions.
6. The classical swine fever virus according to claim 2, wherein the mutation(s) are deletions.
7. The classical swine fever virus according to claim 1, wherein the mutation(s) in the coding sequence for glycoprotein Ems are located in the encoding nucleotide sequence corresponding to amino acids at position 295 to 307 and/or position 338 to 357.
8. The classical swine fever virus according to claim 1, wherein the mutation in the coding sequence for glycoprotein Ems is a deletion or substitution of the histidine at position 346.
9. The classical swine fever virus according to claim 1, wherein the mutation(s) in the coding sequence for glycoprotein Ems are located in the nucleotide sequence coding for the conserved Ems sequence SLHGIWPEKIC (SEQ ID NO: 15) and/or RHEWNKHGWCNW (SEQ ID NO: 16).
10. The classical swine fever virus according to claim 1, wherein the mutation(s) in the coding sequence for glycoprotein Erns are two mutations located in the nucleotide sequence coding for the conserved Ems sequence SLHGIWPEKIC (SEQ ID NO: 15) and/or RHEWNKHGWCNW (SEQ ID NO: 16).
11. The classical swine fever virus according to claim 1, wherein the mutation in the coding sequence for glycoprotein Ems is a single mutation located in the conserved Ems sequence SLHGIWPEKIC (SEQ ID NO: 15) or RHEWNKHGWCNW (SEQ ID NO: 16).
12. The classical swine fever virus according to claim 1, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]x-[PS]y-[C-term] wherein: [Npro] is the Npro portion of the polyprotein, wherein x is the number of amino acids of the Npro present in the polyprotein; [PS] is a processing signal selected from the group consisting of: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16 or GABA(A)RAP), Intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus; [C-term] is the complete virus polyprotein except for Npro, but including the capsid (C)-protein and any other protein present in the virus polyprotein including the carboxyterminal NSSB; y is 0 or 1, where 0 means [PS] is absent and 1 means [PS] is present; and x is 0 to 12 amino acids if y is 0, or 0 to 168 amino acids if y is 1.
13. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]1-[PS]0-[C-term].
14. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]3-[PS]0-[C-term].
15. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]3-[PS]0[-C-term] and the mutation in the coding sequence for glycoprotein Ems is a single mutation located in the conserved Ems sequence SLHGIWPEKIC (SEQ ID NO: 15) or RHEWNKHGWCNW (SEQ ID NO: 16).
16. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]4-[PS]0-[C-term].
17. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]6-[PS]0-[C-term].
18. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula: [Npro]4-[PS]0-[C-term], wherein [C-term]* is [C-term] wherein in the C-protein the amino acid at position 2 is changed from D to N.
19. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro leads to an encoded polyprotein is characterized by the following formula: [Npro]x-[PS]1-[C-term], wherein PS is ubiquitin or LC3.
20. The classical swine fever virus according to claim 12, wherein mutation(s) in the coding sequence for Npro leads to an encoded polyprotein as characterized by a formula selected from the group consisting of: M-[PS]0-[C-term]; MEL-[PS]0-[C-term]; MELN-[PS]0-[C-term]; MELNH-[PS]0-[C-term]; MELHF-[PS]0-[C-term]; MELNHFE-[PS]0-[C-term]; MELNHFEL-[PS]0-[C-term]; MELNHFELL-[PS]0-[C-term]; MELNHFELLY-[PS]0-[C-term]; MELNHFELLYK-[PS]0-[C-term]; and MELNHFELLYKT-[PS]0-[C-term].
21. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro leads to an encoded polyprotein as characterized by the following formula: [Npro]x-[PS]0-ME-[PS]0-[C-term*], wherein [C-term]* is [C-term] wherein in the C-protein the amino acid at position 2 is changed from D to N.
22. The classical swine fever virus according to claim 12, wherein the mutation(s) in the coding sequence for Npro leads to an encoded polyprotein as characterized by the following formula: [Npro]22-[PS]1-[C-term], wherein PS is ubiquitin or LC3.
23. The classical swine fever virus according to one of claims 12, wherein the [PS]0 is replaced by [PS]1, and wherein the PS is selected from the group of consisting of: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16, GABA(A)RAP, intein, picornavirus 3C, caridovirus 2A, and p15 of rabbit hemorrhagic disease virus.
24. A composition comprising the virus according to claim 1 and a solution.
25. The composition according to claim 24, which induces an immunological response in an animal.
26. The composition according to claim 24, which is a vaccine.
27. The composition according to claim 26, further comprising a pharmaceutically acceptable carrier or excipient.
28. A nucleic acid molecule comprising the nucleic acid encoding a classical swine fever virus according to claim 1, or a fragment, functional variant, variant based on the degenerative nucleic acid code, fusion molecule, or a chemical derivative thereof.
29. The nucleic acid molecule according to claim 28, wherein the nucleotide molecule is DNA.
30. The nucleic acid molecule according to claim 29, wherein the nucleotide molecule is RNA.
31. A method for attenuating a classical swine fever virus, wherein at least one mutation in the coding sequence for glycoprotein Ems and at least another mutation in the coding sequence for Npro is generated in a pestivirus.
32. The method according to claim 31, the method comprising: (a) reversely transcribing a wild type classical swine fever to obtain a cDNA; (b) cloning the cDNA; (c) introducing mutations selected from deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein Ems and the protease Nxo; and (d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of the classical swine fever virus cDNA into RNA in vitro or upon infection of suitable cells.
33. A method of treatment of disease caused by classical swine fever virus, the method comprising administering to an animal in need thereof an effective amount of the attenuated classical swine fever virus according to claim 1.
34. A method for attenuating a classical swine fever virus, wherein at least one mutation in the coding sequence for glycoprotein Ems and at least another mutation in the coding sequence for Npro is generated in the classical swine fever virus and wherein the attenuated virus does not cross the placenta in animals infected with the attenuated virus.
35. The method according to claim 34, the method comprising: (a) reversely transcribing a wild type classical swine fever virus to obtain a cDNA; (b) cloning the cDNA; (c) introducing mutations selected from deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein Ems and the protease NPpro; and (d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of classical swine fever virus cDNA into RNA in vitro or upon infection of suitable cells.
Description:
RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Ser. No. 60/589,361, filed Jul. 20, 2004, and claims priority to German Application No. 10 2004 025 452.4, filed May 19, 2004, each of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of animal health and in particular to attenuated pestiviruses such as bovine viral diarrhea virus (BVDV).
BACKGROUND OF THE INVENTION
[0003] Pestiviruses are causative agents of economically important diseases of animals in many countries worldwide. Presently known virus isolates have been grouped into four different species which together form one genus within the family Flaviviridae. [0004] I/II. Bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and type 2 (BVDV-2) cause bovine viral diarrhea (BVD) and mucosal disease (MD) in cattle (Baker, 1987; Moennig and Plagemann, 1992; Thiel et al., 1996). The division of BVDV into 2 species is based on significant differences at the level of genomic sequences (summarized in Heinz et al., 2000) which are also obvious from limited cross neutralizing antibody reactions (Ridpath et al. 1994). [0005] III. Classical swine fever virus (CSFV), formerly named hog cholera virus, is responsible for classical swine fever (CSF) or hog cholera (HC) (Moennig and Plagemann, 1992; Thiel et al., 1996). [0006] IV. Border disease virus (BDV) is typically found in sheep and causes border disease (BD). After intrauterine infection of lambs with BDV persistently infected lambs can be born that are weak and show different abnormalities among which the "hairy shaker" syndrome is best known (Moennig and Plagemann, 1992; Thiel et al., 1996).
[0007] Pestiviruses are small enveloped viruses with a single stranded RNA genome of positive polarity lacking both 5' cap and 3' poly(A) sequences. The viral genome codes for a polyprotein of about 4000 amino acids giving rise to final cleavage products by co- and post-translational processing involving cellular and viral proteases. The viral proteins are arranged in the polyprotein in the order NH2--Npro--C-Ems-E1-E2-p7-NS2-NS3-NS4A-NS4B--NS5A-NS5B--CO- OH (Lindenbach and Rice, 2001). Protein C (=core- or capsidprotein) and the glycoproteins Ems, E1, and E2 represent structural components of the pestivirus virion as demonstrated for CSFV (Thiel et al., 1991). This also holds true for BVDV. E2 and, to a lesser extent, Ems were found to be targets for antibody neutralization (Donis et al., 1988; Paton et al., 1992; van Rijn et al., 1993; Weiland et al., 1990, 1992). Ems lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). The function of this enzymatic activity for the viral life cycle is presently unknown. The enzymatic activity depends on the presence of two stretches of amino acids conserved between the pestivirus Ems and different known RNases of plant and fungal origin. Both of these conserved sequences contain a histidine residue (Schneider et al., 1993). Exchange of each of these residues against lysine in the Ems protein of a CSFV vaccine strain resulted in the destruction of RNase activity (Hulst et al., 1998). Introduction of these mutations into the genome of the CSFV vaccine strain did not influence viral viability or growth properties but led to a virus exhibiting a cytopathogenic phenotype (Hulst et al., 1998). Similarly, Meyers et al. showed that an RNase negative variant of the virulent CSFV strain Alfort/Tubingen was fully viable. However, the respective virus mutant showed no cytopathogenic phenotype (Meyers et al., 1999).
[0008] Npro represents the first protein encoded by the long open reading frame in the pestivirus RNA. Npro represents a nonstructural protein that has protease activity and cleaves itself of the nascent polyprotein (Stark et al., 1993; Wiskerchen et al., 1991) presumably already during translation. Npro is a cysteine protease (Rumenapf et al., 1998) that is not essential for virus replication (Tratschin et al., 1998). Recently, it was shown that Npro somehow interferes with the cellular antiviral defense so that it can be hypothesized to modulate the immune system within an infected host (Ruggli et al., 2003). Mayer and coworkers presented indications for an attenuation of CSFV in consequence of a deletion of the Npro gene (Mayer et al., 2004).
[0009] Present BVDV vaccines for the prevention and treatment of BVDV infections still have drawbacks (Oirschot et al., 1999). Vaccines against the classical BVDV-1 provide only partial protection from BVDV-2 infection, and vaccinated dams may produce calves that are persistently infected with virulent BVDV-2 (Bolin et al., 1991; Ridpath et al., 1994). This problem is probably due to the great antigenic diversity between type 1 and type 2 strains which is most pronounced in the glycoprotein E2, the major antigen for virus neutralization (Tijssen et al., 1996). Most monoclonal antibodies against type 1 strains fail to bind to type 2 viruses (Ridpath et al., 1994).
[0010] Vaccines comprising attenuated or killed viruses or viral proteins expressed in heterologous expression systems have been generated for CSFV and BVDV and are presently used. Killed vaccines (inactivated whole virus) or subunit vaccines (conventionally purified or heterologously expressed viral proteins) are most often inferior to live vaccines in their efficacy to produce a full protective immune response even in the presence of adjuvants.
[0011] The structural basis of the attenuation of BVDV used as live vaccines is not known. These vaccines, although attenuated, are most often associated with safety problems. The vaccine viruses may cross the placenta of pregnant animals, e.g., cows, and lead to clinical manifestations in the fetus and/or the induction of persistently infected calves. Therefore, they cannot be applied to breeding herds that contain pregnant cows. Pregnant cows have to be kept separate from vaccinated cattle to protect fetuses and must not be vaccinated themselves. Furthermore, revertants of attenuated live BVDV pose a serious threat to animals. For conventionally derived attenuated viruses wherein the attenuation is achieved by conventional multiple passaging, the molecular origin as well as the genetic stability of the attenuation remains unknown and reversion to the virulent wild-type is unpredictable.
[0012] Because of the importance of an effective and safe as well as detectable prophylaxis and treatment of pestiviral infections, there is a strong need for improved attenuated pestiviruses, such as BVDV, with a high potential for induction of immunity as well as a defined basis of attenuation which can also be distinguished from pathogenic pestiviruses, such as BVDV, as well as compositions and vaccines comprising the attenuated pestiviruses, such as BVDV.
[0013] Therefore, the technical problem underlying the present invention is to provide improved attenuated pestivirus, preferably an attenuated BVDV for use as live attenuated vaccines. Such improved attenuated pestivirus, preferably BVDV, should especially (i) not cross the placenta themselves, and (ii) induce an immunity that prevents viral transmission across the placenta and thereby prevents pregnancy problems like abortion of the fetus or birth of persistently infected host such calves in the case of BVDV infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the serum neutralization against NY93/C (BVDV type II);
[0015] FIG. 2 shows the serum neutralization assay against KE9 (BVDV type I); and
[0016] FIG. 3 shows the serum neutralization assay against NY93/C (BVDV type II).
[0017] All subsequent sequences show the deleted regions indicated with dashes (-), which are also numbered, whereas the sequences in the sequence listing attached hereto are continuously numbered without the deleted regions or amino acid codons.
[0018] SEQ ID NO:1 XIKE-A-cDNA sequence
[0019] SEQ ID NO:2 XIKE-A-NdN-cDNA sequence
[0020] SEQ ID NO:3 XIKE-B-cDNA sequence
[0021] SEQ ID NO:4 XIKE-B-NdN-cDNA
[0022] SEQ ID NO:5 XIKE-A amino acid sequence
[0023] SEQ ID NO:6 XIKE-A-NdN amino acid sequence
[0024] SEQ ID NO:7 XIKE-B amino acid sequence
[0025] SEQ ID NO:8 XIKE-B-NdN amino acid sequence
[0026] SEQ. ID NO:9 XIKE-C-NdN amino acid sequence
[0027] SEQ ID NO:10 XIKE-C-NdN-cDNA sequence
[0028] SEQ ID NO:11 XIKE-C-cDNA sequence
[0029] SEQ ID NO:12 XIKE-C amino acid sequence
SUMMARY OF THE INVENTION
[0030] The present invention relates to attenuated pestivirus, preferably to attenuated BVDV, wherein at least one mutation is in the coding sequence for glycoprotein Ems and at least another mutation in the coding sequence for Npro which preferably leads to combined inactivation of the RNase activity residing in glycoprotein Ems in addition to the inactivation of the (hypothesized) immunomodulating activity residing in Npro. The invention also relates to methods for attenuating pestivirus in such that the attenuation results in an attenuated pestivirus, preferably in an attenuated BVDV, as described above. The present invention furthermore relates to nucleic acid molecules encoding the attenuated pestiviruses, preferably encoding attenuated BVDV, compositions and vaccines comprising the attenuated pestivirus, preferably BVDV as disclosed herein.
DETAILED DESCRIPTION OF THE INVENTION
Definitions of Terms Used in the Description
[0031] Before the embodiments of the present invention it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a BVDV" includes a plurality of such BVDV, reference to the "cell" is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
[0032] The term "pestivirus" as used herein refers to all members of the genus Pestivirus, including BVDV, CSFV, and BDV, within the family Flaviviridae.
[0033] The term "CSFV" as used herein refers to all viruses belonging to species of classical swine fever virus (CSFV) in the genus Pestivirus within the family Flaviviridae.
[0034] The term "BVDV" as used herein refers to all viruses belonging to species bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and BVDV type 2 (BVDV-2) in the genus Pestivirus within the family Flaviviridae (Heinz et al., 2000). The more classical BVDV type 1 strains and the more recently recognized BVDV type 2 strains display some limited but distinctive differences in nucleotide and amino acid sequences.
[0035] The term "NP'" as understood herein relates to the first protein encoded by the viral open reading frame and cleaves itself from the rest of the synthesized polyprotein (Stark et al., J. Virol. 67:7088-7093 (1993); Wiskerchen et al., Virol. 65:4508-4514 (1991)). The term, depending on the context, may also relate to the remaining "Npro" amino acids after mutation of the encoding nucleotide sequence or to the coding nucleotide sequence for the protein itself. "Protease activity residing in Npro" relates to the polypeptide cleavage activity of the "Npro".
[0036] "Ems" as used herein relates to the glycoprotein Ems which represents a structural component of the pestivirus virion (Thiel et al., 1991). Ems lacks a typical membrane anchor and is secreted in considerable amounts from the infected cells; this protein has been reported to exhibit RNase activity (Hulst et al., 1994; Schneider et al., 1993; Windisch et al., 1996). It should be noted that the term glycoprotein E0 is often used synonymously to glycoprotein Ems in publications. The term, depending on the context, may also relate to the mutated "Ems" protein after mutation of the encoding nucleotide sequence or to the coding nucleotide sequence for the protein itself. "RNase activity residing in glycoprotein Ems" relates to the RNA cleavage activity of the glycoprotein, i.e., the ability of the glycoprotein Ems to hydrolyze RNA. The term "inactivation of the RNase activity residing in the glycoprotein" refers to the inability or reduced capability of a modified glycoprotein Ems to hydrolyze RNA as compared to the unmodified wild-type of the glycoprotein Ems.
[0037] "An attenuated pestivirus or BVDV particle" as used herein means that there is a statistically significant difference between the virulence of attenuated pestivirus or BVDV particles of the present invention, wherein the attenuated viral particles being attenuated by a method described herein, and wild-type pestivirus or BVDV isolates from which the attenuated pestivirus or BVDV particles have been derived, for the predominant clinical parameters, in case of BVDV for diarrhea, pyrexia, and lethality in animals infected with the same dose, preferably 6×106 TCID50. Thus, the attenuated BVDV particles do not cause diarrhea, pyrexia, and lethality and thus may be used in a vaccine.
[0038] "Inactivation of Ems" as used herein means RNase activity not significantly above the level measured for noninfected control cells in an RNase assay as described in Meyers et al., 1999. "Not significantly above the level measured for noninfected control cells in an RNase assay as described in Meyers et al., 1999," means for example, that the RNase activity is less than 150% compared to the noninfected control cells.
[0039] "Inactivation of Npro" as used herein means the prevention or considerable reduction of the probable immunomodulating activity of Npro by mutation. In a preferred embodiment, this mutation prevents or considerably reduces the interference of Npro with the induction of an interferon response by the infected cells as described by Ruggli et al., 2003. In this case, the inactivation of Npro would allow the cell to mount a normal interferon response.
[0040] "Processing signal" as used herein relates to a substance that ensures the generation of a functional N-terminal of the C protein of the pestivirus, preferably of BVDV, in particular a substance selected from the group of ubiquitin, LC3, SUMO-1, NEDD8, GATE-16, and GABA(A)RAP. Also proteases selected from the group of intein, picornavirus 3C, caridovirus 2A, and p15 of rabbit hemorrhagic disease virus are understood as "processing signals" as used herein. Any other similar processing signal known to the skilled person that ensures the generation of a functional N-terminal of the C protein shall also be comprised in the term "processing signal".
[0041] "Protein C" or "C protein" or "C-protein" as used herein relates to a structural component of the pestivirus virion (Thiel et al., 1991). "Protein C" is the capsid or core protein of pestiviruses. The term, depending on the context, may also relate to the "Protein C" with one or several amino acids exchanges resulting from mutation of the encoding nucleotide sequence.
[0042] A "fragment" according to the invention is any subunit of a polynucleotide molecule according to the invention, i.e., any subset. For DNA, the fragment is characterized in that it is shorter than the DNA covering the full length viral genome.
[0043] A "functional variant" of the nucleotide molecule according to the invention is a nucleotide molecule which possesses a biological activity (either functional or structural) that is substantially similar to the nucleotide molecule according to the invention. The term "functional variant" also includes "a fragment", "a functional variant", "variant based on the degenerative nucleic acid code", or "chemical derivative". Such a "functional variant", e.g., may carry one or several nucleotide exchanges, deletions, or insertions. The functional variant at least partially retains its biological activity, e.g., function as an infectious clone or a vaccine strain, or even exhibits improved biological activity. "Possess a biological activity that is substantially similar" means with respect to the pestiviruses provided herewith, for example, that the pestivirus is attenuated in a manner described herein and result in an non-pathogenic virus suitable for the production of live attenuated virus, which loss ability to pass the placenta but mediates an immune response after vaccination.
[0044] A "variant based on the degenerative nature of the genetic code" is a variant resulting from the fact that a certain amino acid may be encoded by several different nucleotide triplets. The variant at least partially retains its biological activity, or even exhibits improved biological activity.
[0045] A molecule is "substantially similar" to another molecule if both molecules have substantially similar nucleotide sequences or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein if the nucleotide sequence is not identical, and two molecules which have a similar nucleotide sequence are considered variants as that term is used herein even if their biological activity is not identical.
[0046] A mutation as used herein relates to modifications in the nucleic acid molecules encoding the proteins/amino acids according to the invention. The mutations relate to, but are not limited to, substitutions (replacement of one or several nucleotides/base pairs), deletions (removal of one or several nucleotides/base pairs), and/or insertions (addition of one or several nucleotides/base pairs). As used herein, mutation may be a single mutation or several mutations, therefore, often the term "mutation(s)" is used and relates to both a single mutation and several mutations. The mutations include, but are not limited to point mutations (single nucleotide mutations) or larger mutations wherein, e.g., parts of the encoding nucleic acid molecules are deleted, substituted, and/or additional coding nucleic acid is inserted. The mutations may result in a modified expressed polypeptide due to the change in the coding sequence. Such modified polypeptides are desired, as set out in the disclosure of the invention as set out below.
[0047] The term "vaccine" as used herein refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly but not necessarily one or more additional components that enhance the immunological activity of the active component. A vaccine may additionally comprise further components typical to pharmaceutical compositions. The immunologically active component of a vaccine may comprise complete virus particles in either their original form or as attenuated particles in a so called modified live vaccine (MLV) or particles inactivated by appropriate methods in a so called killed vaccine (KV). In another form the immunologically active component of a vaccine may comprise appropriate elements of the organisms (subunit vaccines) whereby these elements are generated either by destroying the whole particle or the growth cultures containing such particles and optionally subsequent purification steps yielding the desired structure(s), or by synthetic processes including an appropriate manipulation by use of a suitable system based on, for example, bacteria, insects, mammalian, or other species plus optionally subsequent isolation and purification procedures, or by induction of the synthetic processes in the animal needing a vaccine by direct incorporation of genetic material using suitable pharmaceutical compositions (polynucleotide vaccination). A vaccine may comprise one or simultaneously more than one of the elements described above. The term "vaccine" as understood herein is a vaccine for veterinary use comprising antigenic substances and is administered for the purpose of inducing a specific and active immunity against a disease provoked by a pestivirus infection, preferably by a BVDV infection. The attenuated pestivirus, in particular the attenuated BVDV as described herein, confer active immunity that may be transferred passively via maternal antibodies against the immunogens it contains and sometimes also against antigenically related organisms. A vaccine of the invention refers to a vaccine as defined above, wherein one immunologically active component is a BVDV or of pestiviral origin or derived from a nucleotide sequence that is more than 70% homologous to any known pestivirus sequence (sense or antisense).
[0048] The term "live vaccine" refers to a vaccine comprising a living, in particular, a living viral active component.
[0049] Additional components to enhance the immune response are constituents commonly referred to as "adjuvants", e.g., aluminum hydroxide, mineral or other oils, or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins, or growth factors.
[0050] A "pharmaceutical composition" essentially consists of one or more ingredients capable of modifying physiological, e.g., immunological functions, of the organism it is administered to, or of organisms living in or on the organism. The term includes, but is not restricted to, antibiotics or antiparasitics, as well as other constituents commonly used to achieve certain other objectives such as, but not limited to, processing traits, sterility, stability, feasibility to administer the composition via enteral or parenteral routes such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal, or other suitable route, tolerance after administration, or controlled release properties. One non-limiting example of such a pharmaceutical composition, solely given for demonstration purposes, could be prepared as follows: cell culture supernatant of an infected cell culture is mixed with a stabilizer (e.g., spermidine and/or bovine serum albumin (BSA)) and the mixture is subsequently lyophilized or dehydrated by other methods. Prior to vaccination, the mixture is then rehydrated in aqueous (e.g., saline, phosphate buffered saline (PBS)) or non-aqueous solutions (e.g., oil emulsion, aluminum-based adjuvant).
[0051] The solution to the above technical problem is achieved by the description and the embodiments characterized in the claims.
[0052] It has surprisingly been found that pestiviruses, in particular BVDV, can be more effectively attenuated by introducing at least one mutation in the coding sequence for glycoprotein Ems and at least another mutation in the coding sequence for Npro which preferably leads to combined inactivation of the RNase activity residing in glycoprotein Ems in addition to the inactivation of the immunomodulating activity residing in Npro. An immunomodulating effect in one aspect is indicated but not limited to the indicated function for one pestivirus in an exemplary manner by Ruggli et al., 2003.
[0053] A pestivirus, in particular BVDV, attenuated in accordance with the present invention may be advantageously used in vaccines. The attenuated pestivirus, in particular the attenuated BVDV, now provide live vaccines of high immunogenicity. Surprisingly, the pestivirus, in particular the BVDV, according to the invention furthermore are safe for use in pregnant animals as they do not cross the placenta. This is exemplified in a non-limiting manner for BVDV in Example 3.
[0054] Furthermore, live vaccines with defined mutations as a basis for attenuation will allow to avoid the disadvantages of the present generation of vaccines, e.g., the risk of reversion to an more pathogenic strain. A further advantage of the attenuating mutations lies in their molecular uniqueness which allows to use them as distinctive labels for an attenuated pestivirus, in particular BVDV, and to distinguish them from pestivirus, in particular BVDV, from the field. Therefore, in one aspect the present invention provides an attenuated pestivirus, in particular an attenuated BVDV, having at least one mutation in the coding sequence for glycoprotein Ems and at least another mutation in the coding sequence for Npro. Preferably, in such attenuated pestivirus, preferably in such attenuated BVDV, the mutation in the coding sequence for glycoprotein Ems leads to inactivation of the RNase activity residing in Ems and/or the mutation in the coding sequence for Npro leads to inactivation of the Npro. The inactivation may take place by any mutation known to the person skilled in the art of the Ems- and the Npro-coding sequence, wherein the mutations are any mutation as defined in the Definitions of Terms Used in the Description section above, such as deletions, insertion mutations, and/or substitution mutations. Most preferably, the mutation(s) are deletions, as the likelihood for revertation to the wild-type is the lowest for deletions.
[0055] It has been shown that the glycoprotein Ems forms a disulfide-bonded homodimer of about 97 kD, wherein each monomer consists of 227 amino acids corresponding to the amino acids 268 to 494 of the CSFV polyprotein as described by Rumenapf et al., 1993. The genome sequence of the Alfort/Tubingen strain of CSFV is available in the GenBank/EMBL data library under accession number J04358; alternatively, the amino acid sequence for the BVDV strain CP7 can be accessed in the GenBank/EMBL data library (accession number U63479); in the BVDV CP7 polyprotein, the Ems protein corresponds to residues 271 to 497. Two regions of amino acids are highly conserved in glycoprotein Ems as well as in some plant and fungal RNase-active proteins (Schneider et al., 1993). These two regions are of particular importance to the RNase enzymatic activity. The first region consists of the region at the amino acids at position 295 to 307 (298 to 310 for BVDV strain CP7) and the second region consists of the amino acids at position 338 to 357 (341 to 360 for BVDV strain CP7) of the viral polyprotein as exemplified for the Alfort strain of CSFV in Meyers et al., 1999 (numbering according to the published deduced amino acid sequence of CSFV strain Alfort/Tubingen (Meyers et al., 1989). The amino acids of particular importance to the RNase activity as mentioned above are by no means limited to the exact position as defined for the Alfort/Tubingen strain of CSFV but are simply used in an exemplary manner to point out the preferred amino acids being at that position or corresponding to that position in other strains such as found in BVDV, BDV, and pestiviruses in general since they are highly conserved. For pestiviruses other than the CSFV Alfort/Tubingen strain, the numbering of the positions of the preferred amino acids can be different but an expert in the field of the molecular biology of pestiviruses will easily identify these preferred amino acids by the high degree of conservation of this amino acid sequence and the position of these motifs in the sequence context. In one particular non-limiting example, the position of CSFV Alfort/Tubingen 346 is identical to position 349 of BVDV strain CP7.
[0056] As a consequence, the present invention preferably relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for glycoprotein Ems are located in the encoding nucleotide sequence corresponding to amino acids at position 298 to 310 and/or position 341 to 360. Preferably, such mutations are (where amino acids are given in the one letter symbols; the amino acid before the position number indicates the amino acid to be substituted, the amino acid after the position number the substituting amino acid (del indicates deletion): for example, H300L means histidine 300 was substituted by leucine):
[0057] Suitable modification of the glycoprotein Ems are for example, the single substitutions/deletions: S298G, H300K, H300L, H300R, H300del, W303G, P304de1, E305A, C308G, R343G, E345del, W346G, K348A, H349K, H349L, H349de1, H349Q, H349SV (mutation H349S and insertion of V), K348R, W351P, W351G, W351L, W351K, W351H; the double substitutions/deletions: H300L/H349L, K348del/H349del, H349del/G350de1, E345del/H349de1, W303G/E305A, H300K/H349K, H300K/H349L and the triple deletions: L299de1/H300del/G300del, K348del/H349del/G350de1. Numbering is according to the published amino acid sequence of BVDV CP7 for all the mutants listed above (the given numbers minus 3 would correspond to the equivalent residues of the CSFV Alfort/Tubingen amino acid sequence). All the above-listed mutants were at least tested as respective CSFV or BVDV mutants without mutations in the Npro region. Suitable mutants of the pestiviral glycoprotein Ems are provided, for example, by WO 99/64604, which is incorporated herein in its entirety. It should be noted, however, that according to the present invention, at least one additional mutation in the Npro region, as disclosed in further detail below, must be present.
[0058] It was particularly found that deletion or substitution of the histidine residue at position 346 (CSFV) or 349 (BVDV) leads to effective inactivation of Ems and therefore leads to particularly useful pestiviral live vaccines. The present invention demonstrates that pestiviruses are viable and code for an Ems protein without RNase activity when the histidine residue at position 346 of the viral polyprotein (numbering according to the published sequence of CSFV Alfort/Tubingen (Meyers et al., 1989)), or at position 349 (numbering according to the published sequence of BVDV CP7 (Meyers et al., 1996b)) if the pestivirus is BVDV, which represents one of the conserved putative active site residues of the Ems RNase, is deleted. Thus, preferably, the invention also relates to a BVDV according to the invention, wherein the mutation in the coding sequence for glycoprotein Ems is a deletion or substitution of the histidine residue at position 349. Even more specifically, the putative active site of the RNase is represented by the conserved Ems sequences SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW (sequence of the BVDV-2 NewYork93 protein given here in an exemplary manner; minor changes can possibly be found in other pestivirus sequences but the identity of the motif will always be obvious for an expert in the field. As an example, the corresponding amino acid sequences of BVDV-1 CP7 would be SLHGIWPEKICTG and/or LQRHEWNKHGWCNWYNIEPW and that of CSFV Alfort/Tubingen SLHGIWPEKICKG and/or LQRHEWNKHGWCNWYNIDPW). Thus, preferably, the invention further relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for glycoprotein Ems are located in the nucleotide sequence coding for the conserved Ems sequence SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW. These sequences are representing the putative active site of the RNase. The sequences SLHGIWPEKIC and RHEWNKHGWCNW of the putative Ems active site are even more conserved across pestiviruses. Thus, preferably, the invention also relates to a pestivirus, in particular to BVDV, having at least one mutation in the coding sequence of the Npro protein and the glycoprotein Ems, wherein the mutation(s) in the coding sequence for glycoprotein Ems are located in the nucleotide sequence coding for the conserved Ems sequence SLHGIWPEKIC and/or RHEWNKHGWCNR. Preferably, the mutation is located in only one of the sequences. Thus the invention also relates to a pestivirus, in particular to BVDV, having at least one mutation in the coding sequence of the Npro protein and the glycoprotein Ems, wherein the mutation(s) in the coding sequence for glycoprotein Ems are located in the nucleotide sequence coding for the conserved Ems sequence SLHGIWPEKIC or RHEWNKHGWCNR. Preferably, such mutations concern two different amino acids, i.e., are double mutations. Thus, the mutations may be 1 to 3 nucleotide mutations in two different triplets encoding two amino acids. Thus, the invention also relates to a pestivirus, in particular to BVDV having at least one mutation in the coding sequence of the Npro protein and the glycoprotein Ems, wherein the mutation(s) in the coding sequence for glycoprotein Ems are two mutations located in the nucleotide sequence coding for the conserved Ems sequence SLHGIWPEKIC and/or RHEWNKHGWCNR. Preferably, such mutations concern a single amino acid. Thus, the mutation may be 1 to 3 nucleotide mutations in one triplet encoding one amino acid. Thus, the invention also relates to a pestivirus, in particular to BVDV, having at least one mutation in the coding sequence of the Npro protein and the glycoprotein Ems, wherein a single mutation is located in the conserved Ems sequence SLHGIWPEKIC or RHEWNKHGWCNR.
[0059] As mentioned above, the attenuated pestiviruses provided by the present invention, having at least on mutation in the coding sequence of the glycoprotein Ems and in the coding sequence of the Npro protein, wherein the mutation preferably result in inactivation of the RNase activity residing in the glycoprotein Ems and of the immunomodulating activity residing in Npro. Inactivation of the Npro is achieved in pestiviruses, in particular BVDV, of the specified formula described more in detail below, wherein between 0 and all amino acids of Npro are present; ubiquitin or LC3 or another sequence serving as processing signal (e.g., SUMO-1, NEDD8, GATE-16, GABA(A)RAP, or proteases, e.g., intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus) is present or absent. In case a processing signal is present, the coding sequence of the processing signal is inserted at or close to the C-terminal end of the (remaining part of the) Npro-protein. Only in the case that a processing signal is present, any number of amino acids coding for Npro (═Npro amino acids) may be present. In case no processing signal sequence is inserted, a maximum of 12 amino acids, preferably aminoterminal amino acids, of Npro may be present, the remaining amino acids have to be deleted. Furthermore, other than the Ems mutations as disclosed above (at least one of which has to be present in the pestivirus, in particular in BVDV according to the invention), the remaining sequences of the pestivirus, in particular BVDV may remain unchanged, i.e., are not mutated, or may also have mutations close to the N-terminal end of the C-protein. A number of more specific embodiments as disclosed below exemplify this.
[0060] Thus, the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]x-[PS]y-[C-term],
wherein: [0061] [Npro] relates to the Npro portion of the polyprotein, wherein x represents the number of amino acids of the Npro present in the polyprotein; [0062] [PS] relates to a processing signal selected from: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16 or GABA(A)RAP) or proteases, e.g., intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus, or the like, or any processing signal known to the skilled person that ensures the generation of a functional N-terminal of the C-protein and y may be 0, which means that no processing signal is present (i.e., that PS is absent), or y may be 1, which means that a processing signal is present (i.e., that PS is present); [0063] [C-term] relates to the complete pestivirus, in particular the complete BVDV polyprotein except for Npro, but including the capsid (C)-protein and any other protein present in the pestivirus polyprotein, in particular in the BVDV polyprotein including the carboxyterminal NS5B. Preferably, the glycoprotein Ems in the [C-term] is mutated, in such that the RNase activity residing in the glycoprotein Ems is inactivated. The term "any other protein present in the pestivirus polyprotein/BVDV polyprotein" relates to Ems, E1, E2, p7, NS2, NS3, NS4A, NS4B, and NS5A, wherein glycoprotein Ems is mutated, preferably as disclosed herein (see above), in such that the RNase activity residing in the glycoprotein Ems is inactivated. Preferably, the pestivirus, in particular the BVDV, according to the invention has a C-protein which is not mutated except for the amino acid at position 2 which is changed from D to N. Therefore, [C-term*] is the same as [C-term] but with a mutation at position 2 of the C-protein (N instead of D); [0064] if y is 0 (which means that no [PS] is present) then x is 0 to 12, (which means no Npro specific amino acid or 1 to 12 amino acids of Npro, preferably of the N-terminus of Npro, are present); and [0065] if y is 1 (which means that [PS] is present) then x is 0 to 168; (which means no Npro specific amino acid or 1 to all 168 amino acids of Npro, preferably of the N-terminus of Npro, are present).
[0066] Also more preferably, the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]1-[PS]0-[C-term],
wherein the definitions are as defined above.
[0067] A specific example thereof is disclosed below, wherein the N-terminal methionine is followed by the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a pestivirus, in particular BVDV, according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
M[C-term],
wherein the definitions are as defined above.
[0068] Also more preferably, the invention relates to a pestivirus, in particular to BVDV, according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]3-[PS]0-[C-term],
wherein the definitions are as defined above.
[0069] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the Npro sequence EL and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
MEL-[C-term],
wherein the definitions are as defined above.
[0070] Also more preferably, the invention relates to a pestivirus, in particular to BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]4-[PS]0-[C-term],
wherein the definitions are as defined above.
[0071] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the Npro sequence ELF and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
MELF-[C-term],
wherein the definitions are as defined above.
[0072] Also more preferably, the invention relates to a pestivirus, in particular to BVDV, according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]6-[PS]0-[C-term],
wherein the definitions are as defined above.
[0073] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the Npro sequence ELFSN and the C-protein and any other protein present in the polyprotein including the carboxyterminal NS5B. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
MELFSN-[C-term],
wherein the definitions are as defined above.
[0074] Also more preferably, the invention relates to a pestivirus, in particular to BVDV, according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]4-[PS]0-[C-term*],
wherein the definitions are as defined above except for the fact that the aminoterminal part of the C-protein is changed.
[0075] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by the Npro sequence ELF and in the C-protein sequence, the amino acid at position 2 is changed from D to N. Therefore, the aminoterminal C-protein sequence is SNEGSK . . . instead of SDEGSK. Hence, most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
MELF-[C-term*],
wherein in the C-protein the amino acid at position 2 is changed from D to N, and the definitions are as defined above.
[0076] Also more preferably, the invention relates to a pestivirus, in particular BVDV, according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]x-[PS]1-[C-term],
wherein the definitions are as defined as above, and PS is any of the PS disclosed above, preferably selected from the group of ubiquitin or LC3.
[0077] A specific example of BVDV is disclosed below, wherein the N-terminal methionine is followed by any 21 or 28 Npro amino acids, ubiquitin, or LC3 and the C-protein. Hence most preferably, the invention relates to a BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula:
[Npro]22-[PS]1-[C-term], wherein preferably the PS is ubiquitin or LC3, or
[Npro]29-[PS]1-[C-term], wherein preferably the PS is ubiquitin or LC3.
[0078] Ubiquitin is a well known highly conserved cellular protein of 76 amino acids. Among other functions, ubiquitin is a key player in protein catabolism since conjugation with ubiquitin can mark a protein for degradation via the proteasome. Ubiquitin conjugated with or fused to other proteins via the carboxyterminal glycine can be cleaved off by cellular ubiquitin-specific proteases. Thus, fusion of a protein to the carboxyterminus of ubiquitin will usually result in defined proteolytic cleavage of the fusion protein into its components when expressed within a cell.
[0079] LC3 (light chain 3 of microtubule associated proteins) represents a cellular protein of 125 amino acids that serves a variety of functions (length given for bovine LC3). Recently, a fundamental role of the protein in autophagy has been defined. During this process, LC3 is activated by carboxyterminal cleavage. Thereby, a new carboxyterminus is generated that consists of glycine. LC3 is then conjugated via the carboxyterminal glycine to phosphatidylethanolamine present in the membranes of autophagic vesicles. Because of this process, a protein fused to the carboxyterminus of LC3 will be cleaved off by a cellular protease at a defined position.
[0080] Also more preferably, the invention relates to a pestivirus, preferably to BVDV according to the invention, wherein the mutation(s) in the coding sequence for Npro lead to an encoded polyprotein as characterized by the following formula selected from the group of:
[pro]2-[PS]y-[C-term] and preferably ME-[PS]y-[C-term];
[Npro]5-[PS]y-[C-term] and preferably MELFS-[PS]y-[C-term];
[Npro]7-[PS]y-[C-term] and preferably MELFSNE-[PS]y-[C-term];
[Npro]8-[PS]y[-C-term] and preferably MELFSNEL-[PS]r[C-term];
[Npro]9-[PS]y-[C-term] and preferably MELFSNELL-[PS]y-[C-term];
[Npro]10-[PS]y[C-term] and preferably MELFSNELLY-[PS]y-[C-term];
[Npro]11-[PS]y-[C-term] and preferably MELFSNELLYK-[PS]y-[C-term]; and
[Npro]12-[PS]y-[C-term] and preferably MELFSNELLYKT-[PS]y-[C-term],
wherein the definitions are as defined as above. The preferably disclosed embodiments refers to BVDV. Most preferably, y is 0 (i.e., no PS is present).
[0081] Also more preferably, the BVDV according to the invention as described supra is a BVDV type 1 BVDV. Most preferably, the BVDV according to the invention as described supra is a BVDV type 2 BVDV. BVDV-1 and BVDV-2 are differentiated according to features of their genomic sequences (Heinz et al., 2000 and references therein). BVDV-1 as disclosed herein may be used in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 infections in breeding stocks of cattle, in pregnant cows and in the induction of fetal protection against BVDV type 1 infection is pregnant cows. Surprisingly, a BVDV-2 as disclosed herein may be used in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 infections in breeding stocks of cattle. In particular, the invention relates to the use of a BVDV type 2 according to the invention in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 infections in pregnant cows. Preferably, the BVDV type 2 according to the invention may be used in the manufacture of a composition for use in the induction of fetal protection against BVDV type 1 infections in pregnant cows. Surprisingly also, a BVDV-1 as disclosed herein may be used in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 2 infections in breeding stocks of cattle. In particular, the invention relates to the use of a BVDV type 1 according to the invention in the manufacture of a composition for use in the prevention and/or treatment of BVDV type 2 infections in pregnant cows. Preferably, the BVDV type 1 according to the invention may be used in the manufacture of a composition for use in the induction of fetal protection against BVDV type 2 infections in pregnant cows. Most preferred is the use of BVDV type 1 and type 2 in combination for the manufacture of a composition for use in the prevention and/or treatment of BVDV type 1 and or type 2 infections in breeding stocks of cattle, in pregnant cows and in the induction of fetal protection against BVDV type 1 and/or type 2 infections is pregnant cows.
[0082] Most preferably, the wild-type BVDV according to the invention which is to be mutated as disclosed herein corresponds to amino acid sequence SEQ ID NO:5 (termed XIKE-A) or is a functional variant thereof. Most preferably also, the BVDV according to the invention has a Npro mutation according to the invention and corresponds to amino acid sequence SEQ ID NO:6 (termed XIKE-A-NdN) or is a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the amino acid sequence disclosed herein. On the amino acid level, homologies are very roughly: BVDV-1/-BVDV-1: 93%; BVDV-1/-BVDV-2: 84%; BVDV-2/-BVDV-2: 98%. Therefore, more preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the amino acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the amino acid sequence disclosed herein.
[0083] Most preferably also, the BVDV according to the invention has a Ems mutation according to the invention which has a deletion of the codon coding for histidine 349, and corresponds to amino acid sequence SEQ ID NO:7 (termed XIKE-B) or is a functional variant thereof. Most preferably also, the BVDV according to the invention has both a Ems mutation and a Npro mutation according to the invention, wherein the codon coding for histidine 349 of Ems is deleted and also the complete Npro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of Npro remain. The mutant corresponds to amino acid sequence SEQ ID NO:8 (termed XIKE-B-NdN) or is a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the amino acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the amino acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the amino acid sequence disclosed herein.
[0084] Most preferably also, the BVDV according to the invention has a Ems mutation according to the invention which has a substitution of the codon coding for histidine 300 by the codon coding for leucine and corresponds to amino acid sequence SEQ ID NO:9 (termed XIKE-C) or is a functional variant thereof. Most preferably also, the BVDV according to the invention has both a Ems mutation and a Npro mutation according to the invention, wherein the codon coding for histidine 300 is substituted by the codon coding for leucine and also the complete Npro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of Npro remain. The mutant corresponds to amino acid sequence SEQ ID NO:10 (termed XIKE-C NdN) or is a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the amino acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the amino acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the amino acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the amino acid sequence disclosed herein.
[0085] Another important embodiment of the invention described herein is a composition comprising a pestivirus, in particular a BVDV according to the invention and a solution. The skilled person knows additional components which may be comprised in the composition (see also Remington's Pharmaceutical Sciences, 18th ed. Mack Publ., Easton (1990)). The expert may use known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as, e.g., saline or corresponding plasma protein solutions, are readily available. The pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g., as a kit of parts.
[0086] The final preparation of the compositions of the present invention are prepared for, e.g., injection by mixing the pestivirus, preferably BVDV according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or/and additives (e.g., serum albumin, dextrose, sodium bisulfite, EDTA). The solution may be based on a physiologically acceptable solvent, e.g., an aqueous solution between pH 7 and 8. The pH may be stabilized by a pharmaceutically acceptable buffer. The solution may also contain further stabilizing agents like a detergent like Tween 20, serum albumin such as bovine serum albumin (BSA), ascorbic acid, and/or spermidine. The composition may also comprise adjuvants, e.g., aluminum hydroxide, mineral or other oils or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins, or growth factors.
[0087] For example, in a composition according to the invention, the pestivirus, in particular BVDV may be solved in:
TABLE-US-00001 Pestivirus (preferably BVDV) 102-108 TCID50 SGS* 25% v/v Cell culture medium qsp 1 dose *SGS: Composition per 2 mL Sucrose 75 mg Gelatin 20 mg Potassium hydroxide 0.274 mg L-glutamic acid 0.72 mg Potassium dihydrogen phosphate 0.516 mg Dipotassium phosphate 1.254 mg Water for injection qsp 2 mL
[0088] If the composition is first lyophilized or dehydrated by other methods, then, prior to vaccination, the composition is rehydrated in aqueous (e.g., saline, phosphate buffered saline (PBS)) or non-aqueous solutions (e.g., oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based), aluminum-based, carbomer based adjuvant).
[0089] Preferably, the composition according to the invention induces an immunological response in an animal. More preferred, the composition according to the invention is a vaccine. A vaccine as understood herein comprises a pestivirus, in particular BVDV according to the invention and is defined above in the Definitions of Terms Used in the Description section.
[0090] Most preferred, the composition according to the invention further comprises a pharmaceutically acceptable carrier or excipient. Several carriers or excipients are disclosed above. The composition may comprise, if aimed at injections or infusion, substances for preparing isotonic solutions, preservatives such as p-hydroxybenzoates, stabilizers such as alkali salts of ethylendiamintetracetic acid, possibly also containing emulsifying and/or dispersing agents.
[0091] The composition according to the invention may be applied intradermally, intratracheally, or intravaginally. The composition preferably may be applied intramuscularly or intranasally. In an animal body, it can prove advantageous to apply the pharmaceutical compositions as described above via an intravenous or by direct injection into target tissues. For systemic application, the intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred. A more local application can be effected subcutaneously, intradermally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily, or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, or epithelial tissue). Depending on the desired duration and effectiveness of the treatment, the compositions according to the invention may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks, or months and in different dosages.
[0092] The invention also relates to the use of a pestivirus, in particular BVDV, according to the invention in the manufacture of a vaccine for the prophylaxis and treatment of pestiviral infections, in particular of BVDV infections.
[0093] Another important part of the invention is a polynucleotide molecule comprising the nucleic acid coding for a pestivirus, in particular for a BVDV, according to the invention, or a fragment, functional variant, variant based on the degenerative nucleic acid code, fusion molecule, or a chemical derivative thereof. Preferably, the polynucleotide molecule is DNA. Also preferably, the polynucleotide molecule is RNA. In a more preferred embodiment, the polynucleotide molecule also comprises the nucleotide sequence of a functional 5'- and/or 3'-non-translated region of a pestivirus, in particular of BVDV.
[0094] There are several nucleotide sequences known in the art, which represents the basis for the production of a polynucleotide molecule coding for a pestivirus attenuated according to the present invention, having at least one mutation in the coding sequence of Npro and at least one in the coding sequence of glycoprotein Ems, wherein the mutations result in an combined inactivation of the RNase activity residing in glycoprotein Ems and in the inactivation of the immunomodulating activity residing in Npro. Examples of nucleic acid sequences of wild-type sequences of several members of pestiviruses are listed below:
Border Disease Virus
TABLE-US-00002 [0095] Strain BD31 NCBI GenBank Accession No. [U70263] Strain X818 NCBI GenBank Accession No. [AF037405]
Bovine Viral Diarrhea Virus 1
TABLE-US-00003 [0096] Strain NADL NCBI GenBank Accession No. [M31182] Strain Osloss NCBI GenBank Accession No. [M96687] Strain SD-1 NCBI GenBank Accession No. [M96751] Strain CP7 NCBI GenBank Accession No. [U63479]
Bovine Viral Diarrhea Virus 2
TABLE-US-00004 [0097] Strain 890 NCBI GenBank Accession No. [U18059] Strain C413 NCBI GenBank Accession No. [AF002227]
Classical Swine Fever Virus
TABLE-US-00005 [0098] Strain Alfort/187 NCBI GenBank Accession No. [X87939] Strain Alfort-Tubingen NCBI GenBank Accession No. [J04358] Strain Brescia NCBI GenBank Accession No. [M31768] Strain C strain NCBI GenBank Accession No. [Z46258]
[0099] The mutations/modifications according to the invention relating to the coding sequence of Npro and Ems are described above more in detail. Having this information, a person skilled in the art is able to realize the manufacture of any polynucleotide/polynucleic acid coding for a pestivirus according to the present invention. Furthermore, this person is able to manufacture an attenuated pestivirus according to the invention. Molecular method for introducing a mutation into a polynucleotide sequence, cloning, and amplification of the mutated polynucleotide are for example provided by Sambrook et al., 1989 or Ausubel et al., 1994.
[0100] Most preferably, the wild-type BVDV according to the invention which is to be mutated as disclosed herein is encoded by the nucleic acid sequence SEQ ID NO: 1 (termed XIKE-A) or a functional variant thereof. Most preferably also, the BVDV according to the invention has a Npro mutation according to the invention and is encoded by nucleic acid sequence SEQ ID NO:2 (termed XIKE-A-NdN) or a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the nucleic acid sequence disclosed herein. On the nucleic acid level, homologies are very roughly: BVDV-1/-BVDV-1: 80%; BVDV-1/-BVDV-2: 70%; BVDV-2/-BVDV-2: 96%. Therefore, more preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the nucleic acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the nucleic acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the nucleic acid sequence disclosed herein.
[0101] Most preferably also, the BVDV according to the invention has a Ems mutation according to the invention which has a deletion of codon H349 and is encoded by nucleic acid sequence SEQ ID NO:7 (termed XIKE-B) or by a functional variant thereof. Most preferably also, the BVDV according to the invention has both a Ems mutation and a Npro mutation according to the invention, wherein the codon coding for histidine 349 of Ems is deleted and also the complete Npro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of Npro remain. The mutant is encoded by nucleic acid sequence SEQ ID NO:8 (termed XIKE-B-NdN) or by a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the nucleic acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the nucleic acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the nucleic acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the nucleic acid sequence disclosed herein.
[0102] Most preferably also, the BVDV according to the invention has a Ems mutation according to the invention which is a substitution of codon "H300" by a leucine codon, and is encoded by nucleic acid sequence SEQ ID NO:11 (termed XIKE-C) or a functional variant thereof. Most preferably also, the BVDV according to the invention has both a Ems mutation and a Npro mutation according to the invention, wherein the codon coding for histidine 300 is substituted by the codon coding for leucine and also the complete Npro coding region is deleted, except for codons 1 to 4, thus amino acids MELF of Npro remain. The mutant is encoded by nucleic acid sequence SEQ ID NO:12 (termed XIKE-C-NdN) or by a functional variant thereof. Preferably, such a functional variant is at least 65% homologous to the nucleic acid sequence disclosed herein. More preferable, such a functional variant is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% homologous to the nucleic acid sequence disclosed herein. More preferably also, such functional variant is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% homologous to the nucleic acid sequence disclosed herein. Most preferably, such functional variant is at least 99% or 99.9% homologous to the nucleic acid sequence disclosed herein.
[0103] Another important aspect of the invention is a method for attenuating a pestivirus, characterized in that at least one mutation in the coding sequence for glycoprotein Ems and at least another mutation in the coding sequence for Npro is generated in a pestivirus genome. According to a preferred embodiment, the pestivirus is BVDV.
[0104] According to a more preferred embodiment, the method comprises the steps: [0105] a) reverse transcription of a wild-type pestivirus nucleotide sequence into a cDNA; [0106] b) cloning the cDNA; [0107] c) introducing mutations selected from the group of deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein Ems and the protease Npro; and [0108] d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of pestivirus cDNA into RNA in vitro or upon infection of suitable cells.
[0109] Regarding the method for attenuating a BVDV according to the invention, the preferred methods comprises the steps: [0110] a) reverse transcription of a wild-type BVDV nucleotide sequence into a cDNA; [0111] b) cloning the cDNA; [0112] c) introducing mutations selected from the group of deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein Ems and the protease Npro ; and [0113] d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of pestivirus cDNA into RNA in vitro or upon infection of suitable cells.
[0114] Yet another important embodiment of the invention is a method of treatment of disease caused by a pestivirus, wherein a pestivirus according to the invention or a composition according to the invention, wherein the pestivirus or the composition is administered to an animal in need thereof at a suitable doses as known to the skilled person and the reduction of symptoms of the pestivirus infection.
[0115] Yet another important embodiment of the invention is a method of treatment of disease caused by BVDV, wherein a BVDV according to the invention or a composition according to the invention, wherein the BVDV or the composition is administered to an animal in need thereof at a suitable doses as known to the skilled person and the reduction of symptoms of BVDV infection such as viremia and leukopenia and/or pyrexia and/or diarrhea is monitored.
EXAMPLES
[0116] The following examples serve to further illustrate the present invention; but the same should not be construed as limiting the scope of the invention disclosed herein.
Example 1
BVDV XIKE-B
Fetopathogenicity Assessment in Pregnant Heifers
[0117] BVDV XIKE-B, an RNase negative mutant of the highly pathogenic BVDV type 2 isolate NewYork93/C was recovered from the infectious cDNA clone pKANE40B and showed wild-type-like (wt-like) growth characteristics in tissue culture. In animal experiments the mutant virus was found to be considerably attenuated so that it represented a promising candidate for development of a live attenuated vaccine virus (Meyer et al., 2002). To test whether this attenuated virus is still able to cross the placenta and infect the fetus, pregnant heifers were infected with XIKE-B. As a control, wild-type BVDV recovered from cDNA clone pKANE40A was used. The respective virus named XIKE-A expresses an active Emn RNase in the infected cell. The study aimed to assess the safety of XIKE-A and XIKE-B in pregnant animals.
Experimental Design
[0118] Ten pregnant heifers were selected from a BVDV negative herd. The following groups of 5 heifers were included in the trial:
TABLE-US-00006 No. Inoculation Virus Group 1: 5 One i.n. administration, XIKE-A 3 mL in each nostril Group 2: 5 One i.n. administration, XIKE-B 3 mL in each nostril
[0119] Heifers were moved to the experimental facilities 8 days before inoculations. Pregnancy status was confirmed after transport into the experimental facility. Heifers were between days 60 and 90 of gestation on the day of inoculation. Inoculation took place for all animals at one point of time.
[0120] Heifers were monitored for the presence of clinical signs of BVDV infection including abortions during the observation period. Blood samples were collected from the animals for serology, antigen detection, and white blood cells were counted. The experiment was terminated 9 weeks after infection. Non-aborted cows were slaughtered, the uterus examined, and collected. Fetal organ samples were collected during routine necropsy and examined for BVDV infection.
[0121] The presence of fetal infection was the main evaluation parameter, composed from the number of BVDV-related cow mortality, the number of BVDV-related abortions, and the number of BVDV positive fetuses at termination. In addition to the main parameter, clinical signs characteristic for BVDV infection, viremia, and white blood cell counts in cows and rectal temperature after challenge were evaluated.
Animals
[0122] Heifers were purchased from a farm free of BVDV. Only animals which met the following inclusion criteria were used.
Inclusion Criteria
[0123] Free of BVD antibodies; each individual was tested in the serum antibody test prior to transport and at the initiation of the study (at the animal test facility). [0124] Free of BVDV; plasma and/or buffy-coat preparation from each individual was tested by a suitable test. [0125] Clinically healthy at the initiation of the study judged upon physical examination. The health examination of the animals was accomplished in accordance with the current, generally accepted veterinary practice. [0126] Pregnancy confirmed by physical examination before inoculation. Pregnancy was between 60-90 days at the time of inoculation, proven by insemination records.
TABLE-US-00007 [0126] Test Strain A Description: XIKE-A, live virus BVDV strain Composition: Experimental material comprising of cell culture supernatant of low passaged XIKE-A BVD components: BVDV type II strain: XIKE-A Supplied by: Dr. Gregor Meyers, "Bundesforschungsanstalt fur Viruskrankheiten der Tiere" (BFAV), Paul- Ehrlich-Stra e 28, 72076 Tubingen, Germany Applied BVD Type 1 strain: 105 TCID50/6 mL (TCID = Tissue virus dose: Culture Infective Dose) Applied 3 mL per nostril vaccine volume: Application Intranasal route: Preparation The inoculum was sent in a pre-diluted frozen form of dosage in a 50 mL vial on dry ice and was to be stored form: at -70° C. before inoculation. Immediately before inoculation of Group 1 heifers, the material was thawed avoiding local temperatures above 37° C. After no ice was visible in the fluid, material was gently stirred and immediately used for inoculation of the animals. Unused inoculum: The volume of the unused material was be measured and split on two aliquots before immediate freezing in dry ice or liquid nitrogen and stored for re-titration purposes. Virus and contaminated plastic or glassware were incubated with an appropriate volume of an 8-10% formaldehyde solution for at least 24 hours at room temperature before discarding in order to inactivate viruses.
TABLE-US-00008 Test Strain B Description: XIKE-B, live virus BVDV strain Composition: Experimental material comprising of cell culture supernatant of low passaged XIKE-B BVD components: BVDV type II strain: XIKE-B Supplied by: Dr. Gregor Meyers, "Bundesforschungsanstalt fur Viruskrankheiten der Tiere" (BFAV), Paul- Ehrlich-Stra e 28, 72076 Tubingen, Germany Applied BVD Type 1 strain: 105 TCID50/6 mL (TCID = Tissue virus dose: Culture Infective Dose) Applied 3 mL per nostril vaccine volume: Application Intranasal route: Preparation The inoculum was sent in a pre-diluted frozen form of dosage in a 50 mL vial on dry ice and was stored at -70° C. form: before inoculation. Immediately before inoculation of Group 2 heifers, the material was thawed avoiding local temperatures above 37° C. After no ice was visible in the fluid, material was gently stirred and immediately used for inoculation of the animals. Unused vaccine: The volume of the unused material was measured and split on two aliquots before immediate freezing in dry ice or liquid nitrogen and stored for re- titration purposes. Virus and contaminated plastic or glassware was incubated with an appropriate volume of an 8-10% formaldehyde solution for at least 24 hours at room temperature before discarding in order to inactivate viruses.
Pregnancy Control
[0127] Pregnancy was confirmed immediately before inoculation.
Inoculation of Heifers
[0128] The inoculation is Day 0 of the experiment.
[0129] In each nostril, 3 mL of the test material was administered intranasally by syringe without needle. Each time a new sterile syringe was taken. Administration was performed during the aspiration phase in order to minimize loss of fluid via expiration of material.
Post-Inoculation Observations
Collection and Examination of Blood Samples
[0130] Blood was collected following standard, aseptic procedures (disinfecting the bleeding site). A new sterile syringe and needle was used for each animal.
Blood Collection to Prepare Serum
[0131] At least 10 mL blood was collected from the heifers immediately before inoculation, then weekly after infection and at the termination of the study. Serum was stored at -20° C. until required.
Blood Collection for Leukocyte Counts and Buffy Coat Preparations
[0132] For leukocyte counting, 3 mL blood was transferred immediately after collection to suitable sterile vessels (Venoject, Terumo Europe N.V., Leuven, Belgium), pre-filled with 0.06 mL EDTA (0.235 MOL/L).
[0133] For buffy coat preparations, at least 15 mL blood was transferred immediately after collection to suitable sterile vessels, pre-filled with 0.1 mL Heparin solution (Na-heparin for inj., 5000 IU/mL lot A7B163A, exp. date: 11/2000: Gedeon Richter R T, Budapest, Hungary) yielding at least 20 IU Heparin per mL blood in the blood sample. The content was carefully mixed thereafter.
[0134] For preparation of buffy coats and leukocyte counting, blood was collected from the heifers on every day, between Day 0 and Day 14 after infection; and on every second day, between Day 15 and Day 40, or until all animals were negative for virus isolation for three consecutive sampling time points.
Preparation of Serum
[0135] Blood was allowed to clot at room temperature, and separated by centrifugation. Each serum sample was divided into two aliquots of at least 2 mL each. One set of aliquots was assayed for BVDV specific antibodies by ELISA. The rest of the sera was frozen and stored at -20° C. until required.
Leukocyte Counts
[0136] Leukocyte counts was determined with a coulter-counter semi-automated electronic device (Diatron Minicell-16, Messtechnik GmbH, Wien, Austria) with a claimed accuracy of 0.1×109/1,100/μL. The instrument was used (calibration and leukocyte-counts) according to the manufacturer's recommendations.
Preparation of Buffy Coats
[0137] Heparin blood samples was transported to the laboratory as soon as possible. Buffy coat preparation procedure, following a standard laboratory procedure, was performed under aseptic conditions (sterile pipettes, handling, clean bench, etc.).
[0138] The obtained buffy coats were re-suspended in a small volume (2 mL) of RPMI 1640 and frozen at -70° C. in two aliquots of 0.5 mL. The residual 1 mL buffy coats was immediately used for determination of blood cell associated BVDV by co-cultivation in a permissive cell culture.
BVD Serum Antibody ELISA-Test
[0139] Each serum sample was tested for the presence of BVDV-antibodies using a suitable and validated ELISA test (Svanovir® BVDV antibody test Cat#10-2200-10). Test was validated and performed according to the manufacturer's recommendations. Positive samples were diluted according to the log2 scale to determine BVDV antibody titers.
BVD Antigen Assay(s)
[0140] Each buffy coat sample was assayed for the presence of BVDV by co-cultivation of the freshly prepared buffy-coats with susceptible cells or a cell-line. No freezing was allowed before co-cultivation. Plasma was collected and provided to Man-Gene from each sample.
Clinical Observations
Observation of Heifers
[0141] Animals were examined daily from Day 0-42 post-inoculation for the presence of clinical symptoms by a sufficiently trained veterinarian.
[0142] All clinical signs were recorded and described by its nature, consistence/touch, severity (mild, medium or severe) location, size of the area affected, and they will be scored according to agreed and standard definitions. Special attention was paid to respiratory signs (respiration, its rate; nasal or ocular discharge; conjunctivitis, sneezing, coughing, etc.) and diarrhea.
Rectal Temperatures
[0143] Rectal temperatures were measured daily in each heifer, at the same hour of the day (preferably in the morning) from 5 days prior to the inoculation till 21 days post-infection. Daily measurement of rectal temperature was continued until each animal had rectal temperatures below or equal to 39° C. for at least 3 consecutive days.
Detection of Interrupted Pregnancy
[0144] Pregnancy was confirmed and suspicion for abortion or resorption of the fetus was established by rectal examination. A trained veterinarian examined all animals at inoculation, 1 and 2 months post-inoculation. The examination was carried out according to the generally accepted veterinary practice. Heifers were examined daily for any sign of abortion until termination of the study (8-12 weeks post-challenge).
Termination of the Study
[0145] The study was terminated by slaughtering the heifers and extracting the fetuses. Fetuses and fetal material were transferred into closed transport containers marked with the number of the cow and the date/time. Containers were transported to a selected necropsy room. Necropsy of the heifers was not required. Necropsy was performed on fetuses, findings recorded, and a panel of samples collected as described below.
Post-Mortem Examination
[0146] A detailed necropsy of the experimental animals was done in each case of death. Post-mortem examinations were carried out by an experienced veterinary surgeon and the data were recorded on appropriate data sheets. Further laboratory tests were performed according to the clinical signs and lesions observed. If the diagnosis of the necropsy referred to a disease caused by microbial agent the diagnosis was verified by an appropriate test, specific for the agent. Each tissue sample was collected in at least 2 separate, labeled containers and snap-frozen in liquid nitrogen. Samples were stored at -70° C. until required.
Aborted Fetuses and Study Termination
[0147] At least the following tissue samples were collected from the fetuses: exudate from the peritoneal cavity or thorax, if present; mesenteric lymph nodes; spleen; thymus; cerebellum; kidney; bone marrow from the sternum; and sample from the placenta, if available.
Dead or Sacrificed Heifers
[0148] At least the following tissue samples were collected: blood for buffy coat, if available; blood for serum, if available; Peyer's patches; mesenteric lymph nodes; spleen; kidney; uterus, including a sample from the placenta, if available.
Storage and Transport of Samples
TABLE-US-00009 [0149] Samples: Storage: Serum -20° C. Buffy coat -70° C. Virus -70° C. Tissue from heifers -70° C. Tissue from fetuses -70° C.
[0150] Samples were sent for laboratory analysis as required by the sponsor. The choice of samples and the timing of transport were agreed with the study monitor or the project manager. As a matter of general principle, samples coming from aborted material or from new-born calves were investigated as soon as possible.
Results
Mortality
[0151] Heifer No. 626 (Group 1) died on Day 13 PI (post-inoculation). The following table summarizes the observed clinical signs and lesions revealed during necropsy:
TABLE-US-00010 Heifer In-life observations Post-mortem findings No. 626 signs of disease from 7 DPI dehydration lachrymation, nasal discharge on hemorrhages on the 7-12 DPI serous membranes loss of appetite from 8-12 DPI hyperemia of the diarrhea on 11-12 DPI Peyer's patches elevated respiratory rate on 9-10 edema of the lung and 12 DPI coughing on 9 DPI abnormal breathing on 12 DPI
[0152] These clinical and gross-pathological findings are consistent with BVDV induced lesions, therefore it may be concluded that the reason of death was the BVDV infection.
Abortions After Infection
[0153] One heifer had clinical abortion in each group. Heifer No. 615 (Group 1) aborted on Day 38 PI, Heifer No. 469 (Group 2) aborted on Day 39 PI. Both fetuses showed the signs of autolysis, and they were estimated to die at least 3-7 days before the abortion (around 32-35 DPI). In Group 1, no fetus was found in Heifer No. 526 during the slaughter examination at termination. Gross-pathology of the uterus revealed the following: the right uterine horn was slightly enlarged, and the remains of placenta with progressed autolysis was retained in the lumen. The findings on the uterus of Heifer No. 526 is consistent with a "silent" abortion, most likely due to the BVD infection.
Clinical Observation of Heifers
[0154] A summary of the clinical observation data and duration of clinical signs in the groups are presented below.
Clinical Signs and the Days Post-Inoculation (DPI) When They Were Observed
[0155] Group 1 (XIKE-A)
TABLE-US-00011 Animal ID 526 598 615 618 626* Clinical sign DPI Loss of appetite 8-13 8-18 8-18 8-16 8-12 Lachrymation 7-10 7-8, 10-12 8-10 8-10 7-8 Conjunctivitis 9-10 9-12 9-11 9-11 -- Nasal discharge 7-13 7-9, 11-12 8-13 8-12 7-12 Oral erosion -- -- -- -- -- Oral hemorrhage -- -- -- -- -- Diarrhea -- 11-16 10-15 11-15 11-12 Coughing 9 10, 15 9 8-9, 13 9 Abnormal breathing -- 11-14 12-14 12-14 12 Elevated respiratory -- 10-13 9-13 8-13 9-10, 12 rate Hoof erosion -- -- -- -- -- *Heifer No. 626 died on Day 13 PI
[0156] Group 2 (XIKE-B)
TABLE-US-00012 Animal ID Clinical sign 469 588 565 608 619 Loss of appetite -- -- -- -- -- Lachrymation -- -- -- -- -- Conjunctivitis -- -- -- -- -- Nasal discharge -- -- -- -- -- Oral erosion -- -- -- -- -- Oral hemorrhage -- -- -- -- -- Diarrhea -- -- -- -- -- Coughing -- -- -- -- -- Abnormal breathing -- -- -- -- -- Elevated respiratory -- -- -- -- -- rate Hoof erosion -- -- -- -- --
[0157] All Group 1 animals infected with XIKE-A exhibited a broad spectrum of clinical signs. Respiratory signs appeared first accompanied by loss of appetite, and a few days later heifers developed diarrhea with the exception of Heifer No. 526. One heifer died and another one aborted (see before) after infection. All these signs are consistent with the symptoms expected after infection with a virulent BVDV strain.
[0158] All Group 2 animals infected with XIKE-B were free of clinical signs. At the same time, one heifer had abortion during the observation period.
Rectal Temperatures
[0159] No abnormal temperature changes were detected before the infection of the animals. In Group 2, all temperature values remained within the physiological range from Day 0 to Day 21 after infection. All Group 1 animals showed elevated rectal temperature after infection that were detected between Days 7-11 PI.
Findings at Study Termination
[0160] At study termination, fetuses were examined at slaughter. No fetus was recovered from Heifer No. 526 (see section 10.2 "Abortions after Infection"). The following findings were observed at the necropsy of the fetuses:
TABLE-US-00013 Animal No. Findings Conclusion Group 1 598 Ascites, general edema, autolysis Died at least 2 weeks earlier 618 Ascites, general edema, autolysis Died at least 3 weeks earlier Group 2 565 Ascites, general edema, liver degeneration Fetus considered non-viable 588 Normal -- 608 Normal, perirenal edema -- 619 General autolysis Died 3-6 weeks earlier
[0161] The findings suggest that 2 Group 1 animals (Heifers No. 598 and No. 618) and one Group 2 animal (Heifer No. 619) died several weeks before extraction, and so they can be considered abortions.
Abortions Modified by Post-Mortem Findings
[0162] After the post-mortem examination it was not clear why some of the heifers had not had abortions. Dead fetuses should be considered as abortions, therefore the clinical picture was modified after the termination of the study as follows:
[0163] Group 1
TABLE-US-00014 Animal No. Conclusion 526 BVD abortion (uterus with placenta post-mortem) 598 BVD abortion (fetus post-mortem) 615 Clinical BVD abortion 618 BVD abortion (fetus post-mortem) 626 Died due to BVD
[0164] Group 2
TABLE-US-00015 Animal No. Conclusion 469 Clinical BVD abortion 565 Expected BVD abortion; non-viable fetus 588 Normal 608 Normal 619 BVD abortion (fetus post-mortem)
Examination of Blood Samples
Leukocyte Count
[0165] WBC counting was interrupted on Day 26 PI, as all animals became negative for virus isolation for this time point. 0 DPI values were considered as individual baseline for comparison. In Group 2, the leukocyte counts never went to 40% or more below the baseline value until the end of the observation period (26 DPI). In Group 1, one animal (Heifer No. 598) had WBC count below the 40% baseline for one day.
Serology
[0166] None of the selected animals had BVDV specific antibody in their sera before the infection. After infection, all surviving Group 1 heifers developed BVDV specific antibodies detected from 3 weeks PI and lasted until the end of the observation period in all study animals. In Group 2, 4 out of the 5 heifers had BVDV specific antibodies detected from 4 weeks PI. Measurable antibody response lasted only in 3 animals until the end of the observation period. Titers were lower in Group 2 than in Group 1.
Virus Detection by Co-Cultivation
Buffy Coats
[0167] BVDV was detected in both groups. The duration of virus detection is summarized below. All samples were co-cultivated immediately after collection, i.e., without freezing.
TABLE-US-00016 Animal No. DPI when BVDV was detected Group 1 615 5-12 526 5-9 626 5-12 618 5-11, 14 598 5-11, 13 Group 2 565 7-9 588 8 608 6-9 469 8 619 5-11
Tissue Samples
[0168] The presence of BVD virus in the dead heifer and the fetuses is summarized below:
[0169] Heifer:
TABLE-US-00017 Animal No. BVDV in tissue samples Group 1 626 Present#
[0170] Fetuses:
TABLE-US-00018 Animal No. BVDV in tissue samples Group 1 615 Not present# 526 NT 626 Present# 618 Not present 598 Present Group 2 565 Present 588 Not present 608 Present 469 Not present# 619 Not present NT = Not tested
[0171] Samples were co-cultivated immediately after collection (i.e., without freezing), except "#" marked ones, from which only frozen samples were available.
Summary of BVD Related Clinical and Laboratory Data
[0172] Group 1
TABLE-US-00019 Animal No. Conclusion BVD 526 BVD abortion (uterus with NT (no sample found) placenta post-mortem) 598 BVD abortion (fetus post-mortem) +(fetus)* 615 Clinical BVD abortion -(fetus)* 618 BVD abortion (fetus post-mortem) -(fetus)* 626 Died due to BVD +(fetus)/+(heifer) NT = not tested
[0173] Group 2
TABLE-US-00020 Animal No. Conclusion BVD 469 Clinical BVD abortion -(fetus)* 565 Expected BVD abortion; non-viable fetus +(fetus) 588 Normal -(fetus) 608 Normal +(fetus) 619 BVD abortion (fetus post-mortem) -(fetus)* *Fetuses were autolyzed at the time of sampling
Conclusion
[0174] The study aimed to assess the safety of XIKE-A and XIKE-B in pregnant animals. Ten pregnant heifers were selected from a BVDV negative herd. Two groups of 5 heifers were included in the trial: one was inoculated with XIKE-A the other with XIKE-B virus strain. Heifers were between days 60 and 90 of gestation on the day of inoculation. Heifers were monitored for the presence of clinical signs of BVDV infection including abortions during the observation period. Blood samples were collected from the animals for serology, antigen detection and white blood cells were counted. The experiment was terminated 9 weeks after infection. Non-aborted cows were slaughtered and the uterus examined and collected. Fetal organ samples were collected during routine necropsy and examined for BVDV infection.
[0175] The presence of fetal infection was the main evaluation parameter, composed from the number of BVDV-related cow mortality, the number of BVDV-related abortions and the number of BVD positive fetuses at termination. In addition to the main parameter, clinical signs characteristic for BVDV infection, viremia, and white blood cell count in cows and rectal temperature after challenge were evaluated. The XIKE-B virus proved to be less pathogenic than XIKE-A, nevertheless BVD-related abortion and infection of the fetus was observed in the XIKE-B group, too. Therefore it can be concluded that the inactivation of the Ems RNase does not prevent fetal infection.
Example 2
BVDV XIKE-A-NdN
Fetopathogenicity Assessment in Pregnant Heifers
[0176] The Npro gene has been shown to be nonessential for growth of CSFV in tissue culture (Tratschin et al., 1998). Even though a proof for BVDV attenuation in consequence of Npro deletion is still missing, a role of this protein in the interaction between virus and host seemed to be possible and was actually indicated by recent experiments for CSFV (Mayer et al., 2004; Ruggli et al., 2003). We therefore investigated whether the deletion of the major part of the Npro coding sequence leads to a virus that no longer infects the fetus in pregnant heifers. The Npro gene except for the 5' terminal 4 codons was deleted from the full length cDNA clone pKANE40A according to standard procedures. The resulting mutant full length clone was used as template for in vitro transcription and the resulting cRNA was transfected into MDBK cells as described (Meyer et al., 2002). The recovered virus was amplified in tissue culture and then used in the animal experiment described below. BVDV XIKE-B served as a control since it was shown before that it is able to cross the placenta (Example 1).
Objective(s)/Purpose of the Study
[0177] The study aims to assess the safety of a live attenuated BVDV with a genomic deletion of most of the Npro coding region in pregnant animals.
[0178] Materials and Methods applied are as described in Example 1
Study Design
[0179] Eight pregnant heifers were assigned at random to two groups. They were treated and observed according to the following schedule:
TABLE-US-00021 Group 1 Group 2 N 5 3 Treatments XIKE-A-NdN XIKE-B/control Route Intramuscular Vaccination time between days 60 and 90 of pregnancy (day 0 of the study) Observations Clinical signs Post-vaccination Serum at days 0, 14, 28, 42 and at termination (in life) WBC at day 0 and then daily for 14 days Buffy coat at day 0 and then daily for 14 days Post-mortem Gross-pathology (day 60) Organ panel for virus isolation Type of study: open controlled clinical study Experimental unit: Individual animal Method of blinding: Partial blinding. No detailed procedures for blinding and access to treatment schedule were applied. The observing veterinarian at the study location and the pathologist were not be aware of the treatment; they only received a protocol extract relevant to their tasks. Vaccination was performed by the investigator or his assignee. Samples for virus isolation were coded by the investigator until all results are available.
Results
[0180] All heifers were healthy and pregnant at study start. All animals proved to be free of BVDV and BVDV antibodies before the initiation of the study.
Preparation and Control of the Virus used for the Infection
[0181] Samples were collected throughout the dilution steps and assayed on the day of preparation, i.e., without freezing by co-cultivation on suitable tissue culture. The results of virus titration are shown in the following table.
TABLE-US-00022 Log10 Sample ID Virus strain Dilution/description titer/mL VT1a XIKE-A/NdN (S) 1:2 (at 4° C.) 4.4 VT1b #2a on ice without 4.0 opening VT1c Return of #2b 2.8 VT2a XIKE-B 1:2.2 (at 4° C.) 2.3 VT2b #3a on ice without 2.8 opening VT2c Return of #3b Negative
Clinical Symptoms of BVDV Infection
[0182] The table below gives a summary about the animals that had clinical signs during the observation period.
Clinical Signs and the Days Post-Inoculation (DPI) When They Were Observed
TABLE-US-00023 [0183] Group 1 (XIKE-A NdN) Group 2 (XIKE-B) Animal ID Animal ID Clinical sign 1583 1438 1585 Loss of appetite 8 -- 10 Lachrymation -- -- -- Conjunctivitis -- -- -- Nasal discharge -- -- -- Oral erosion -- -- -- Oral hemorrhage -- -- -- Diarrhea -- -- -- Coughing -- 12 10-13 Abnormal breathing -- -- -- Elevated respiratory rate -- -- -- Hoof erosion -- -- --
[0184] Only mild and transient clinical signs were observed in some of the animals in each group. In Group 1, one out of the 5 heifers had loss of appetite on day 8 PI. In Group 2, two out of the 3 animals had clinical signs. Both heifers experienced coughing around day 21 PI that was accompanied with loss of appetite in one of the animals.
Rectal Temperatures
[0185] No abnormal temperature changes were detected before the inoculation of the animals. The few cases of elevated temperatures measured after the inoculation are summarized in the table below.
TABLE-US-00024 Group Animal ID Temperature (° C.) PI day 1 1583 39.9 8 1621 39.0 5 2 1438 39.0 2 1585 40.8 9
[0186] One animal had slightly elevated temperature in each group, and also one animal had fever in each group. Fever was detected on day 8 or 9 PI. Temperature values always returned to normal value on the following day.
Leukocyte Counts
[0187] Some leukopenia was observed in all groups between PI days 3-8. The number of animals with at least 40% reduction in white blood cell count was the following:
TABLE-US-00025 Group Number of animals having leukopenia/total 1 3/5 (60%) 2 1/3 (33%)
Serology (BVDV Antibodies)
[0188] In compliance with the study protocol, all heifers were free of BVDV antibodies before vaccination. In Group 1 (inoculated with XIKE-A NdN) and Group 2 (inoculated with XIKE-B), complete seroconversion was detected only at study termination (2 months after inoculation).
BVD Virus Isolation from Buffy Coats
[0189] No viremia was detected
BVD Virus Isolation from Fetal Tissue Samples
TABLE-US-00026 Group 1 Group 2 N 5 3 Treatments XIKE-A-NdN XIKE-B/control Route Intramuscular Intramuscular Number of fetuses in which fetal 4 out of 5 2 out of 3 transmission was detected: fetuses infected fetuses infected Conclusion of the virus used for Fetal transmission Fetal transmission treatment has the potential to be for XIKE-A-NdN for XIKE-B transmitted over the placenta: observed observed
Conclusion
[0190] The Npro deletion resulted in a considerable attenuation of the BVDV in comparison to the parental virus XIKE-A that was shown to be highly pathogenic (Meyer et al., 2002). However, the Npro deletion alone is not preventing transmission of a NY93-based virus recombinant to the fetus after inoculation of pregnant cows.
Example 3
BVDV XIKE-B-NdN
Fetopathogenicity Assessment in Pregnant Heifers
[0191] To be able to test the potential of a combination of RNase inactivation and Npro deletion with regard to BVDV attenuation and fetal transmission, different BVDV-2 mutants with deletions within the Npro coding region were established based on the infectious cDNA clone pKANE40B, the RNase negative mutant of pKANE40A with a deletion of codon 349. The recovered viruses were analyzed with regard to presence of the desired mutations, the absence of second site mutations in the regions flanking the introduced changes, and their growth characteristics in tissue culture. XIKE-B-NdN (V-pK88C), a variant containing a deletion of the complete Npro coding region except for codons 1 to 4 in addition to the RNase inactivating deletion of codon 349 was chosen for an animal experiment since it combined the desired mutations with acceptable growth characteristics. The aim of the study was to assess the safety of a live attenuated BVDV isolate in pregnant animals.
[0192] Five BVDV-negative, pregnant heifers were inoculated intranasally with an infective dose of 105 TCID50/animal XIKE-B-NdN (back titration data are depicted in Table 3.1). Clinical data were recorded daily. Blood samples were collected for white blood cell counting, for buffy-coat preparation and serology. After termination of the study, fetal tissues were collected for virus isolation.
Materials and Methods
[0193] As detailed for Example 1.
Results
[0194] No clinical data were observed (data not shown). Leukocyte counts remained virtually unchanged except for a significant decrease by approximately 40% below the baseline value (day 0) in heifer No. 1015 on a single day (day 6 PI) (data not shown).
Analysis of Buffy Coat Preparations
[0195] Approximately 106 leukocytes were cultured in duplicates with MDBK-cells in 24-well tissue culture plates for 5 days. Samples were freeze-thawed twice. 100 μL aliquots of thawed samples were inoculated onto freshly seeded 24-well tissue culture plates and tested for virus by indirect immunofluorescence staining (mAb Code 4, directed against a conserved epitope in nonstructural protein NS3). No BVDV could be isolated from the buffy coat preparations of animals #921, 1013, 1015, 1055 and 1075 (Table 3.2) whereas positive controls clearly showed the correct conduction of the test.
Post-Mortem Examination of Fetal Tissues
[0196] After termination of the study the following fetal tissues were collected for virus isolation: spleen, kidney, thymus, sternum, cerebellum, placenta, intestine, and abdominal fluid. Briefly, tissue suspensions were made in a mortar using sterile sea sand and ice-cold PBS without Ca2+ and Mg2+. Mortars were rinsed with 1 mL ice-cold PBS without Ca2+ and Mg2+ and suspensions were centrifuged for 10 minutes at 2000×g (4° C.). The supernatant was first passed through a disposable 0.45 μm filter holder, followed by a second filter passage (0.2 μm pore size). Virus isolation was carried out in duplicates (400 μl, of fetal tissue suspension or 100 μL of fetal abdominal fluid) on a monolayer of MDBK-cells in a 24 wells tissue culture plate (37° C., 7% CO2). Tissue samples were controlled daily for cytopathic effects or bacterial contamination, and after an incubation time of 5 days plates were frozen and thawed twice. 100 μL of samples were passaged to freshly seeded MDBK-cells. Virus was detected by indirect immunofluorescence staining (mAb Code 4). No BVDV could be detected in the tissue samples or fetal abdominal fluid (Table 3.3).
Serological Findings
[0197] Serum neutralization titers were determined before inoculation, 1 month post-inoculation and at termination of the study. Sera from all animals were tested in triplicates for neutralizing antibodies against NY93/C, and the endpoint dilution was read by indirect immunofluorescence staining. Results were expressed as the endpoint dilution, which neutralized approximately 100 TCID50 and calculated by the method of Kaerber. No definite data could be obtained for day 0, and 1 and 2 weeks post-infection as the sera were toxic for MBDK-cells in dilutions up to 1:16 and no neutralization could be detected at higher dilutions. Starting with the third week post-vaccination all animals developed neutralizing antibodies against the homologous BVDV-2 virus NY93/C lasting till the end of the experiment (Table 3.4 and FIG. 1).
Conclusions
[0198] The data obtained during the animal study clearly show that BVDV XIKE-B-NdN represents a highly attenuated virus. In contrast to wild-type virus or the single mutants XIKE-B or XIKE-A-NdN that show fetal transmission in pregnant heifers at high rates, the double mutant did not cross the placenta. BVDV XIKE-B-NdN as well as similar double mutants are extremely suitable for the use in a live attenuated vaccine.
TABLE-US-00027 TABLE 3.1 Study No.: B01 BIVI020 and B01 BIVI022 Back Titration of Viruses Sample ID Virus Strain Dilution Titer 1a XIKE-B-NdN concentrated virus 105.44 TCID50/mL 1 1:4 104.86 TCID50/mL 6 residues of infection (#1) 104.27 TCID50/mL
TABLE-US-00028 TABLE 3.2 Detection of Viremia Animal Days after vaccination ID No. 0 1 2 3 4 5 6 7 8 0921 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1013 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1015 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1055 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1075 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Date 13 Dec. 14 Dec. 15 Dec. 16 Dec. 17 Dec. 18 Dec. 19 Dec. 20 Dec. 21 Dec. 2001 2001 2001 2001 2001 2001 2001 2001 2001 Animal Days after vaccination ID No. 9 10 11 12 13 14 0921 -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- 2. isolation 1013 -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- 2. isolation 1015 -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- 2. isolation 1055 -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- 2. isolation 1075 -- -- -- -- -- -- 1. isolation -- -- -- -- -- -- 2. isolation Date 22 Dec. 23 Dec. 24 Dec. 25 Dec. 26 Dec. 27 Dec. 2001 2001 2001 2001 2001 2001 -- = sample negative
TABLE-US-00029 TABLE 3.3 Analysis of fetus tissue samples for the presence of BVDV Mesen- Abdom- Thor- teric Bone Date of Animal inal acic lymph marrow Cere- tissue No. fluid Fluid nodes Spleen Kidney Thymus (sternum) bellum Placenta Intestine collection Isolation 0921 -- NC NC -- -- -- -- -- -- -- 12 Feb. 2002 1. isolation -- -- -- -- -- -- -- -- 2. isolation 1013 -- NC NC -- -- -- -- -- -- -- 12 Feb. 2002 1. isolation -- -- -- -- -- -- -- -- 2. isolation 1015 -- NC NC -- -- -- -- -- -- -- 12 Feb. 2002 1. isolation -- -- -- -- -- -- -- -- 2. isolation 1055 -- NC NC -- -- -- -- -- -- -- 12 Feb. 2002 1. isolation -- -- -- -- -- -- -- -- 2. isolation 1075 -- NC NC -- -- -- -- -- -- -- 12 Feb. 2002 1. isolation -- -- -- -- -- -- -- -- 2. isolation -- = sample negative NC = not collected
TABLE-US-00030 TABLE 3.4 B01 BIVI022/BVDV XIKE-B-NdN; fetal protection study Serum Neutralization Assay From the heifers at Animal selec- during ID No. tion ACC 1 wPV 2 wPV 3 wPV 4 wPV 5 wPV 6 wPV 7 wPV 8 wPV 0921 * * * 1:40.sup.(2) 1:161.sup.(1) 1:256.sup.(1) 1:323.sup.(1) 1:128.sup.(1) 1:256.sup.(1) 1013 * * * 1:3.sup.(2) NA 1:161.sup.(1) 1:323.sup.(1) 1:406.sup.(1) 1:256.sup.(1) 1015 * * * 1:64.sup.(2) 1:161.sup.(1) 1:256.sup.(1) 1:323.sup.(1) 1:406.sup.(1) 1:323.sup.(1) 1055 * * * 1:32.sup.(2) 1:40.sup.(2) 1:256.sup.(1) 1:323.sup.(1) 1:406.sup.(1) 1:406.sup.(1) 1075 * * * NA 1:128.sup.(2) 1:102.sup.(1) 1:203.sup.(1) 1:161.sup.(1) 1:406.sup.(1) Date 06 Dec. 2001 20 Dec. 2001 27 Dec. 2001 03 Jan. 2002 10 Jan. 2002 17 Jan. 2002 24 Jan. 2002 31 Jan. 2002 07 Feb. 2002 .sup.(1)SNT against 1456 Nase (=NY93/C) 102.03 TCID50/50 μL .sup.(2)SNT against 1456 Nase (=NY93/C) 101.57 TCID50/50 μL * Serum toxic for MBDK-cells in dilutions up to 1:16 no data available NA data not available The Serum Neutralization Assay against NY93/C is illustrated in FIG. 1
Efficacy and Crossprotection Study
[0199] Two possible problems have to be faced with regard to vaccination with attenuated virus mutants BVDV XIKE-B or BVDV XIKE-B-NdN. First, there is a general problem concerning crossprotection between BVDV-1 and BVDV-2. At least vaccination with inactivated BVDV-1 vaccines did not prevent the transmission of BVDV-2 to the fetus in pregnant animals. Since protection against fetal infection represents the major aim of anti-BVDV vaccination, such vaccines cannot be regarded to induce a protective immunity on a broad range. The question therefore was, whether vaccination with live attenuated BVDV-2 can prevent virus transmission to the fetus. Second, the reduced growth rates of BVDV XIKE-B-NdN might result in only a low level of protection not able to prevent transplacental infection of the fetus in pregnant heifers. To address these problems, an animal study was started. The animals (2 groups of 10 animals each) were vaccinated either with BVDV XIKE-B or XIKE-B-NdN (intended dosage: 1 mL of supernatant with 105 TCID50 of virus; backtitration is shown in Table 3.5). None of the animals showed significant clinical signs after the vaccination except for one animal of the nonvaccinated control group with mild coughing for one day. Rectal temperature values were below 39° C. except for one animal of the nonvaccinated control group that had 39.1° C. for one day. Buffy coat samples prepared after vaccination were analyzed for the presence of virus as described above. The experiments showed that only 5 of the 20 animals contained virus in the blood for 1 or 2 days at 4 to 8 days post-infection (Table 3.6).
TABLE-US-00031 TABLE 3.5 Back Titration of Viruses used for vaccination Sample ID Virus Strain Dilution Titer 1a XIKE-B-NdN concentrated virus 105.44 TCID50/mL 1 1:4 104.86 TCID50/mL 6 residues of infection (#1) 104.27 TCID50/mL 3 XIKE-B 1:11 105.76 TCID50/mL 4 1:110 104.92 TCID50/mL 5 residues of infection (#4) 104.27 TCID50/mL
TABLE-US-00032 TABLE 3.6 Study No./Id.: B01 BIVI020/BVDV T{umlaut over (u )} XIKE-B/XIKE-B-NdN; fetal protection study Inoculation with white blood cell(buffy coat) preparations collected after vaccination Animal Days After Vaccination ID No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1134.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1141.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1142.sup.(1) OO OO OO OO OO OO OO +O OO OO OO OO OO OO OO 1. isolation OO OO OO OO O+ OO OO OO OO OO OO OO OO OO OO 2. isolation 1145.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1149.sup.(1) OO OO OO OO +O OO OO OO O+ OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1151.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1152.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1156.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1158.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1160.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1197.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1200.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1206.sup.(2) OO OO OO OO OO OO OO O+ OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO O+ OO OO OO OO OO OO OO OO 2. isolation 1210.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1212.sup.(1) OO OO OO OO OO OO O+ OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1214.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1216.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1217.sup.(1) OO OO OO OO OO OO O+ OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1218.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1225.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation Date 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 2001 Immunofluorescence staining: Code 4 O sample negative + sample positive B bacterial contamination in well Code of animal numbers: .sup.(1)vaccination with BVDV XIKE-B (RNase mutant) .sup.(2)vaccination with BVDV XIKE-B-NdN (RNase and Npro double mutant)
[0200] Four weeks after vaccination, insemination of the animals was carried out. Challenge infections were performed 60 to 90 days later using either a BVDV-1 strain (BVDV KE-9, heterologous challenge, animals vaccinated with XIKE-B) or a heterologous BVDV-2 strain (BVDV KE-13, homologous challenge, animals vaccinated with XIKE-B-NdN) (intended dosage: 105 TCID50 in 6 mL; backtitration is shown in Table 3.7). From each group of vaccinated animals 5 pregnant heifers were randomly selected for the challenge infection. Animals vaccinated with BVDV XIKE-B were challenged with the BVDV-1 strain KE-9, whereas heifers vaccinated with BVDV XIKE-B/NdN were challenged with BVDV-2 KE-13. In addition, two nonvaccinated control animals were infected with each of the challenge viruses.
TABLE-US-00033 TABLE 3.7 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B-NdN; fetal protection study Back titration of challenge viruses Titer Mean Titer Virus Strain Sample ID (TCID50/mL) (TCID50/mL) KE 9 1 104.44 104.94 105.10 2 104.69 * 104.44 3 ** KE13 1 104.69 104.76 104.82 2 104.57 104.63 104.69 3 *** 103.5 103.5 Sample 1: stock of inoculate Sample 2: stock of inoculate returned from the stable Sample 3: excess inoculate * Second inoculation of KE9, sample 2 wasn't interpretable because of cell death. ** KE9, sample 3 wasn't interpretable because of cell death or bacterial contamination. *** First inoculation of KE13, sample 3 wasn't interpretable because of bacterial contamination.
[0201] The vaccinated animals did not show viremia or clinical symptoms upon challenge infection. The challenge was successful as all non-vaccinated controls were BVDV positive (Table 3.8). Only mild signs of disease were observed in the control groups. The white blood cell counts were nearly normal (not shown).
TABLE-US-00034 TABLE 3.8 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B/XIKE-B-NdN; fetal protection study Inoculation with white blood cell (buffy coat) preparations collected after challenge Animal Days After Challenge ID No. 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Isolation 1104.sup.(3) OO OO OO O+ ++ ++ O+ OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO +O ++ OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1108.sup.(3) OO OO OO OO ++ +O OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1126.sup.(3) OO OO ++ +O ++ O+ OO O+ O+ OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation 1145.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation 1151.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation OO OO OO OO OO OO OO OO OO OO OO OO 3. isolation 1152.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation OO OO OO 3. isolation 1156.sup.(2) OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO 2. isolation OO OO OO OO OO OO OO OO OO OO OO OO 3. isolation 1197.sup.(1) 1. isolation OO OO OO O+ ++ ++ O+ OO OO OO OO OO OO OO OO OO 2. isolation OO OO OO +O ++ OO OO OO OO OO OO OO OO OO OO OO 3. isolation 1200.sup.(1) OO OO OO OO ++ +O OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation 1214.sup.(1) 1. isolation OO OO ++ +O ++ O+ OO O+ O+ OO OO OO OO OO OO OO 2. isolation OO OO 3. isolation 1216.sup.(2) 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation OO 3. isolation 1217.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO 2. isolation OO OO OO OO OO OO OO OO OO OO OO OO 3. isolation 1218.sup.(1) OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO 2. isolation 1249.sup.(3) OO OO OO OO OO OO OO OO OO OO OO OO OO OO 1. isolation OO OO OO OO OO OO OO OO OO OO OO OO 2. isolation Date Apr. Apr. Apr. Apr. May May May May May May May May May May May May 22 25 27 29 01 03 05 07 09 11 13 15 17 19 21 23 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 2002 Immunofluorescence staining: Code 4 O sample negative + sample positive B bacterial contamination in well Code of animal numbers: .sup.(1)vaccination with BVDV XIKE-B (RNase mutant) .sup.(2)vaccination with BVDV XIKE-B-NdN (RNase and Npro double mutant) .sup.(3)nonvaccinated controls
[0202] Serum neutralization titers were determined before inoculation, 1 month post-inoculation, before challenge, 1 month after challenge and at termination of the study. Sera from all animals were tested in triplicates for neutralizing antibodies against KE9 and NY93/C (1456 Nase), and the endpoint dilution was read by indirect immunofluorescence staining. Results were expressed as the endpoint dilution, which neutralized approximately 100 TCID50 and calculated by the method of Kaerber. At some of the higher antibody titers, the used endpoint dilution was not high enough. Against KE9, only animals vaccinated with XIKE-B developed low antibody titers starting about week 4. At challenge, all animals had antibody titers, which increased considerably starting around week 4 post-challenge. XIKE-B vaccinated animals had higher antibody titers then those vaccinated with XIKE-B-NdN vaccinated. All animals developed about the same neutralization titer against NY93/C four weeks post-vaccination, with marginally lower titers in XIKE-B-NdN vaccinated animals. After challenge all animals had high antibody titers. FIG. 2 shows the serum neutralization assay against KE9 (BVDV-1) and FIG. 3 shows the serum neutralization assay against NY93/C (BVDV-2).
[0203] Analysis of tissue samples obtained after termination of the study from the fetuses revealed that the material obtained from the vaccinated animals gave negative results whereas transmission had occurred in all 4 control animals (Table 3.9). Thus, it is clear that the established BVDV-2 mutants are well suited as efficient cross protective vaccine viruses.
TABLE-US-00035 TABLE 3.9 Study No./Id.: B01 BIVI020/BVDV Tu XIKE-B/XIKE-B-NdN; fetal protection study Analysis of fetus tissue samples for the presence of BVDV Mesenteric Bone Animal Abdominal Thoracic lymph Small marrow No. Fluid fluid nodes intestine Spleen Thymus Kidney (sternum) Cerebellum Placenta Date 1214.sup.(1) NA NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1126.sup.(3) ++ *** ++ ++ ++ ++ ++ ++ ++ ++ 21 Jun. 2002 1249.sup.(3) ++ NA ++ ++ ++ ++ ++ ++ ++ O+ 21 Jun. 2002 1218*.sup.(1) NA NA NA NA NA NA NA NA NA OO** 21 Jun. 2002 1197.sup.(1) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1217.sup.(1) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1200.sup.(1) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1145.sup.(2) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1108.sup.(3) +O NA ++ ++ ++ ++ ++ ++ ++ OO 21 Jun. 2002 1156.sup.(2) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1104.sup.(3) ++ NA ++ ++ ++ ++ ++ ++ ++ ++ 21 Jun. 2002 1216.sup.(2) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1151.sup.(2) NA NA OO OO OO OO OO OO OO OO 21 Jun. 2002 1152.sup.(2) OO NA OO OO OO OO OO OO OO OO 21 Jun. 2002 NA = not available *No fetus was found in the uterus of heifer #1218 **Endometrium (also collected for histology) *** Sample was not sent to BFA Tubingen Code of animal numbers: .sup.(1)vaccination with BVDV XIKE-B (RNase mutant) .sup.(2)vaccination with BVDV XIKE-B-NdN (RNase and Npro double mutant) .sup.(3)nonvaccinated controls
Conclusion
[0204] The challenge was successful as all non-vaccinated controls were BVDV viremic and fetuses of all non-vaccinated controls were BVDV positive.
[0205] Both isolates gave full protection under the present test and assay conditions. Isolate XIKE-B, with the single genetic marker was shown to cross-protect against type 1 BVDV challenge in terms of BVD viremia and transmission to the fetus after challenge. Isolate XIKE-B-NdN with the double genetic marker was able to fully protect against a heterologue type 2 BVDV challenge strain in terms of BVD viremia and transmission to the fetus after challenge.
[0206] Isolate XIKE-B (type 2 isolate) was shown to cross-protect against type 1 BVDV challenge in terms of BVD viremia and transmission to the fetus after challenge under the present test and assay conditions (n=4).
[0207] Isolate XIKE-B-NdN (type 2 isolate) fully protected against a heterologues type 2 BVDV challenge strain in terms of BVD viremia and transmission to the fetus after challenge under the present test and assay conditions (n=5).
Example 4
Establishment of Npro Mutants
[0208] Further analyses of BVDV-2 mutants with Npro deletions. Different mutants with deletions in the Npro-coding region of the genome were established. Initially, only true deletions or a deletion accompanied by a point mutation were introduced.
[Npro]1-[C-term]; A
[Npro]3-[C-term]; B
[Npro]4-[C-term]; C
[Npro]6-[C-term]; D
[Npro]4-[C-term*] E
[0209] In the formulas, [Npro]x represents the number of residues of the aminoterminus of Npro that are left in the mutated polyprotein amino acids, [C-term] is the complete polyprotein except for Npro (starting with the C protein and ending with NS5B), and [C-term*] is the same as [C-term] but with a mutation at position 2 of the C protein (N instead of D).
[0210] The growth rates of the recovered viruses were considerably lower than those of wild-type XIKE-A or the RNase negative mutant XIKE-B. There are two possible explanations for this finding: (i) dependent on the virus strain, sequences of variable length of the Npro-coding region are necessary for efficient translation initiation (Myers et al., 2001; Tautz et al., 1999), and (ii) the fusion of additional sequences to the aminoterminus of the capsid protein interferes with capsid protein function.
[0211] To obtain better growing Npro deletion mutants, a second set of mutants was generated with either a bovine ubiquitin gene or a fragment of the bovine LC3-coding sequence replacing the major part of the Npro gene. These constructs allow efficient translation and generate a capsid protein with the correct amino terminus.
[Npro]22-[PS]-[C-term]
wherein PS is ubiquitin or LC3 and C-term is the complete polyprotein except for Npro (starting with the C protein and ending with NS5B).
[0212] The growth rates of these mutants were more similar to what was determined for XIKE-A. It even seemed that the two RNase positive viruses according to the formula [Npro]22-[PS]-[C-term] named V-pK87F and V-pK87G showed no significant growth retardation at all, whereas the RNase negative counterpart V-pK88G once again was somewhat hampered in propagation but to a lesser extend than the formerly described mutants.
[0213] Further examples of Npro deletion mutants may be:
MESDEGSK . . . MELFSSDEGSK . . . MELFSNESDEGSK . . . MELFSNELSDEGSK . . . MELFSNELLSDEGSK . . . MELFSNELLYSDEGSK . . . MELFSNELLYKSDEGSK . . . MELFSNELLYKTSDEGSK . . . MELFSNELLYKT represents the aminoterminal sequence of Npro of the BVDV isolate NewYork93/C.
[0214] It may also be possible to use variants of this sequence with one or several mutations. Especially the naturally occurring variations as found in other pestiviruses can be expected to be functional. Therefore, the complete list of the tested or proposed variants with the different parts of the aminoterminal end of Npro can be enlarged by equivalent sets with amino acid exchanges. Below, typical examples of the respective sequences are given for several pestiviruses but the possible variations are not limited to these examples.
BVDV NewYork93/C: MELFSNELLYKT
BVDV CP13: MELISNELLYKT
BVDV SD1: MELITNELLYKT--
CSFV Brescia: MELNHFELLYKT
BDV X818: MELNKFELLYKT
[0215] Thus, these variants for example may include: MELI-[PS]0-[C-term];
MELIS-[PS]0-[C-term];
MELISN-[PS]0-[C-term];
MELISNE-[PS]0-[C-term];
MELISNEL-[PS]0-[C-term];
MELISNELL-[PS]0-[C-term];
MELISNELLY-[PS]0-[C-term];
MELISNELLYK-[PS]0-[C-term];
MELISNELLYKT-[PS]0-[C-term];
MELIT-[PS]0-[C-term];
MELITN-[PS]0-[C-term];
MELITNE-[PS]0-[C-term];
MELITNEL-[PS]0-[C-term];
MELITNELL-[PS]0-[C-term];
MELITNELLY-[PS]0-[C-term];
MELITNELLYK-[PS]0-[C-term];
MELITNELLYKT-[PS]0-[C-term];
[0216] These formulas may also have [PS]1, i.e., PS may also be one of the PS as described herein. Sequences belonging to the Npro protein are in italics. Amino acid exchanges with regard to the sequence of BVDV NewYork93/C are in bold.
[0217] Further examples can be found, e.g., by using the GenBank accession numbers given in Becher et al., 2003, Virology 311, 96-104) or by standard sequence data searches.
[0218] A further possibility could be the use of a processing signal (PS) inserted between the (residual) sequence and the aminoterminus of the capsid protein. The PS leads to a cleavage that generates a functional capsid protein. The configuration of such constructs could be as follows:
[Npro]22-PS-[C-term]
where PS is a processing signal and can either be a target for a protease (e.g., ubiquitin, LC3 as defined herein or a protease or an unstable peptide leading to processing at its own carboxyterminus like e.g., intein (Chong et al. 1998 and references therein) or 3C of picornaviruses, 2A of cardioviruses or aphtoviruses, p15 of rabbit hemorrhagic disease virus, or the corresponding protease of other caliciviruses (Proter, 1993, and references therein; Meyers et al., 2000 and references therein).
[0219] When using a PS, a large number of different variants are possible since the PS ensures the generation of the correct amino terminus of the capsid protein C. Thus, when using a PS construct, all kinds of deletions or mutations of the Npro sequence are expected to result in viable mutants as long as the reading frame is not shifted or translation stopped by an in frame stop codon. As an example we established a viable CSFV N' deletion mutant according to the formula
[Npro]29--PS--[C-term]
[0220] Especially interesting could be Npro mutations blocking the proteolytic activity of the protein. Rumenapf et al., 1998, have published the identification of the active site residues of the protease for CSFV Alfort Tubingen. The respective amino acids (glutamic acid at position 22, histidine at position 49 and cysteine at position 69) are conserved for other pestiviruses. Thus, exchanges of any amino acid expect for serine or threonine for the cysteine at position 69 will result in destruction of the protease activity. Similarly, changing the glutamic acid at position 22 will most likely result in inactivation of the protease unless the new amino acid is aspartic acid. Similarly most if not all exchanges at position 49 will lead to an inactive protease).
REFERENCES
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Theil, 1993, Processing of Pestivirus Polyprotein: Cleavage Site between Autoprotease and Nucleocapsid Protein of Classical Swine Fever Virus, J. Virol., 67: 7088-7095. [0250] H.-J. Theil, G. W. Plagemann, and V. Moennig, 1996, The Pestiviruses, in: Fields Virology, B. N. Fields, D. M. Knipe, and P. M. Howley (eds.), Lippincott-Raven, Philadelphia, pp. 1059-1073.
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Sequence CWU
1
16112332DNAWildtyp BVDV XIKE-A 1gtatacgaga ttagctaaag aactcgtata
tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg
ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc
agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact
ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta
acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat
agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga
gttgttttca aatgaacttt tatacaaaac 420atataaacaa aaaccagcag gcgtcgtgga
acctgtttac gacgtcaacg ggcgcccact 480gtttggagag agcagtgact tgcacccgca
gtcaacacta aaactaccac accaacgagg 540cagcgccaac atcctgacca atgctaggtc
cctaccgcgg aaaggtgact gccggagagg 600taatgtgtat ggaccggtga gtggcatcta
tatcaaacca ggaccgatct actaccagga 660ttatgtgggc cccgtctatc atagagcccc
actggaacta tgtagggagg caagtatgtg 720cgaaacaact aggagagttg gcagagtgac
cggtagtgat gggaaattat atcatatcta 780catctgcata gatgggtgta tcctcctgaa
gagggcgact aggaaccaac cagaagtcct 840gaaatgggta tacaacagat taaattgtcc
tttatgggtc accagctgct ccgatgaagg 900gagcaagggt gctacaagta agaagcagcc
taagccagat aggatagaaa aaggtaagat 960gaaaatagcc ccaaaagaga cagaaaaaga
ttgcaaaacc agaccccccg acgcgactat 1020agtagtagaa ggggttaagt accaggtgaa
gaaaaaagga aaggtaaggg gaaaaaatac 1080tcaagatggg ttatatcaca acaagaataa
gccccctgaa tcaagaaaaa aattggaaaa 1140ggcactgctg gcttgggcca tcttagcagc
ggtcctgctt cagctggtaa caggagagaa 1200tatcacccag tggaacttga tggacaacgg
caccgaggga atacagcaag cgatgttcct 1260aagaggggtg aacaggagtc tacatggaat
ttggccagag aaaatttgca ccggagtacc 1320aactcactta gcaacagact atgagcttaa
agagatagtg gggatgatgg acgcgagtga 1380gaagaccaac tacacgtgtt gcaggttgca
aagacatgag tggaataaac atggttggtg 1440taactggttt catatagaac cgtggatatg
gttgatgaac aaaacccaaa acaacctgac 1500agaagggcaa ccgcttaggg agtgtgctgt
gacttgtagg tatgacaagg aaacagaatt 1560gaacatcgtg acacaggcta gggacagacc
tacaactctg acaggttgca agaaaggcaa 1620gaatttctct ttcgcaggtg ttatactgga
tgggccctgt aactttaaag tatcggttga 1680agatgtgctg ttcaaggagc acgattgcgg
caacatgctg caagagaccg cgatacagct 1740actcgatggg gcaaccaaca ccattgaggg
agcaagggta gggacggcca agttgacaac 1800ctggttaggg aagcaattag ggatccttgg
taagaagttg gagaacaaaa gcaaagcatg 1860gtttggtgca catgcagcaa gtccatactg
cggagtggag aggaagatcg gttacgtatg 1920gtatacaaaa aactgcactc cagcttgcct
tccaagaaac actagaataa taggccccgg 1980gaaatttgat accaacgccg aagatggaaa
aatactccat gagatggggg ggcacctctc 2040agaatttgtc ctattgtcct tggtggttct
gtctgacttt gccccggaaa ccgcgagcgt 2100catctacttg gttctacatt ttgcgatccc
gcaaagccac gttgatgtag acacatgcga 2160caagaaccag ctgaatttaa cggtagcaac
cacagtagca gaggtcatac cagggacagt 2220gtggaaccta gggaagtatg tctgcataag
accagactgg tggccatatg agacgacgac 2280agtcttcgtc atagaggaag cagggcaagt
aatcaaattg atgctaaggg ccatcagaga 2340cttaactagg atatggaatg ctgccactac
cacagctttc ttaatctttt tagtaaaagc 2400actgagggga caactaatcc aagggctatt
gtggctgatg ctaataacag gagcacaggg 2460cttccctgaa tgcaaagagg gcttccaata
tgccatatct aaagacagga aaatggggtt 2520attggggcca gagagcttaa ctacaacatg
gcacctcccc accaaaaaaa tagtggattc 2580catggtgcat gtatggtgtg aaggaaaaga
cttgaaaata ttaaaaatgt gcacaaagga 2640agagaggtat ctagtggctg tgcacgagag
agccttatca accagtgccg agtttatgca 2700gatcagtgat gggacaatag gcccagacgt
gatagatatg cctgatgact ttgagtttgg 2760actctgccct tgtgactcaa aaccagtgat
aaagggcaaa tttaatgcca gcttactgaa 2820tggaccagct ttccagatgg tatgcccaca
ggggtggact ggtacaatag aatgcaccct 2880agcgaaccaa gacaccttgg acacaactgt
cattaggaca tatagaagaa ctaccccatt 2940tcagcggaga aaatggtgta cctatgaaaa
aataataggg gaagatatct atgaatgcat 3000tctaggtgga aactggacat gcataaccgg
tgaccatagc aggttgaaag acggacctat 3060caagaagtgt aagtggtgtg gccatgactt
cgtcaactca gaggggctac cacactaccc 3120aataggcaag tgcatgctca tcaacgagag
tgggtacagg tatgtagatg acacctcttg 3180cgataggggt ggtgtagcca tagttccatc
tggcaccgta aagtgtagaa taggtaacgt 3240cacggtgcaa gttatcgcta ctaacaatga
tctgggaccc atgccttgca gcccagctga 3300agtgatagca agtgaaggac cagtggaaaa
gactgcatgc acattcaact attcaaggac 3360tctacctaat aagtattatg agccaaggga
ccggtacttc caacaataca tgttaaaagg 3420ggagtggcaa tattggttcg acctggattc
tgtagaccac cacaaagact acttctcaga 3480gttcataatc atagcagtgg tcgccttgtt
gggtggtaag tacgtactgt ggctcttgat 3540aacatacaca atactgtctg agcagatggc
tatgggtgct ggagtgaata ctgaagagat 3600agtcatgata ggcaatttgc tgacagacag
tgatattgag gttgtggttt atttccttct 3660tctgtactta atagttaaag aggaactggc
gaggaaatgg attatactgg tataccacat 3720ccttgtagcc aaccctatga aaacaattgg
ggtcgtctta ctaatgctag ggggagtggt 3780gaaggccagc agaatcaatg ctgatgacca
aagtgctatg gacccatgct ttcttctcgt 3840gacaggcgta gtggctgttt tgatgatcgc
tagaagagaa cctgccacat taccactgat 3900tgtagcattg ctagcaataa gaacatcagg
attcctactg cccgctagca ttgatgtaac 3960tgtagcagta gtattaattg tacttttgtt
ggctagctac ataacagact actttagata 4020taaaaagtgg cttcaactct tatttagtct
gatagctggt atctttatta taaggagctt 4080aaaacatatc aaccagatgg aggtaccaga
aatatctatg ccaagttgga gacctctagc 4140tctggtcctt ttctatataa catctacagc
aataaccact aattgggaca ttgacttagc 4200aggcttcctg ctgcaatggg cgccagcagt
gatcatgatg gctaccatgt gggcagactt 4260tttgactctg atcatagtcc tgcccagtta
cgagttatct aagctttact tcctaaagaa 4320cgtcaggaca gacgtggaaa agaactggct
cggcaaagtg aaatacagac agatcagttc 4380agtttatgac atctgtgaca gtgaggaagc
agtgtaccta tttccatcaa ggcataagag 4440tggaagcagg ccagatttca tattaccttt
tttgaaagcc gtgttaataa gctgcatcag 4500cagccaatgg caagtggttt acatttctta
cctaatactg gaaattacat actatatgca 4560caggaaaatc atagatgagg tgtcaggagg
agcaaatttt ctatcaagac tcatagcagc 4620catcatagaa ttaaattggg ccatagatga
tgaggaatgt aaaggactga agaaactgta 4680tctcttgtca gggagagcga agaatttgat
agttaaacat aaggtaagaa atgaagccgt 4740ccacagatgg tttggtgagg aggaaatata
cggggcaccc aaggtgatca ctatcataaa 4800agctagtacc ctaagtaaaa acaggcactg
cataatctgc acgatctgtg aagggaaaga 4860atggaatgga gccaactgcc caaagtgtgg
aagacaagga aagcccataa catgtggaat 4920gacactcgca gactttgagg agaaacatta
caaaaagata tttataagag aagaatcttc 4980ttgtcctgtg ccttttgatc cttcttgcca
ttgtaattat tttcgccacg atgggccttt 5040caggaaagag tataagggtt acgtccaata
cacagccaga ggacaactct ttctgaggaa 5100cctaccaatt ctagcgacga agatgaagct
attaatggtg ggaaacctcg gcgcagaaat 5160tggcgacctg gaacatctag gatgggtact
gagagggcca gccgtgtgca aaaaaattac 5220caaccatgag aagtgccacg taaacatcat
ggataagcta actgcatttt ttggaatcat 5280gcctagaggc acgaccccta gggcacctgt
gaggttcccc acagcactac taaaagtgag 5340aagggggcta gagacgggat gggcttacac
gcaccaagga gggatcagct cggtagacca 5400tgtcacagcc ggaaaggatt tactagtgtg
tgacagtatg ggcaggacca gggttgtctg 5460tcatagtaac aataagatga ctgatgagac
tgagtatggc atcaagaccg actcagggtg 5520tcccgaaggt gcgaggtgtt acgtgctaaa
cccagaagct gttaacattt ctggcacaaa 5580aggagctatg gtacacctcc agaaaacggg
gggggagttc acatgtgtca ctgcctcagg 5640gaccccggct ttcttcgatc tgaaaaatct
aaaaggctgg tccgggctac caatttttga 5700agcatccagt ggcagggtgg ttggtagggt
gaaagtcggc aagaatgagg attccaagcc 5760caccaaacta atgagcggaa tccagacagt
gtctaagaac cagacagacc tagcggacat 5820cgtaaaaaaa ttgactagta tgaacagagg
agagttcaaa cagataacat tagccactgg 5880ggcaggaaaa actacggaac tgccaaggtc
cgtcatagag gagataggga ggcacaaaag 5940ggtcttagtc ctgataccat tgagagcagc
agcagagtca gtgtatcagt atatgagagt 6000gaagtaccca agtatatctt tcaatttgag
aataggagat atgaaggaag gtgacatggc 6060cactggtatc acctacgcct catatgggta
cttttgtcag cttcctcagc ccaaactgag 6120agctgccatg gtagagtact catatatatt
cttagatgag taccactgtg ctacacccga 6180gcaattagca ataattggaa agatacacag
gtttgctgaa aatcttagag tggtagcaat 6240gacagcaacc ccagctggaa cggtcacaac
gactggtcag aaacacccta tagaggagtt 6300catagcccca gaggtgatga aaggtgaaga
tctaggtagt gaatacttgg atattgcagg 6360gttgaagata ccgactgaag agatgaaagg
caacatgctc gtgttcgcgc caactaggaa 6420catggcagta gaaacagcta agaaattgaa
ggctaaggga tacaactctg gatactatta 6480cagtggggaa aacccagaga acttgagggt
ggtaacctcg caatccccgt atgtggtagt 6540agccaccaat gccatagagt caggtgtgac
attaccagac ttagacacag ttgtagacac 6600tggactaaag tgtgagaaga gggtgaggat
ttcttcaaaa atgcccttca ttgtaacagg 6660acttaagaga atggcagtca caatcggaga
gcaagcccag cgcaggggta gagtaggaag 6720agtcaagcca ggtaggtact ataggagtca
agaaacagct tcagggtcaa aagattacca 6780ttacgaccta ctgcaagccc agaggtacgg
aatagaagat ggaattaatg taacaaagtc 6840attcagggag atgaactatg attggagcct
ttacgaagag gacagcttga tgataactca 6900actcgaggtc cttaacaacc tccttatatc
agaagacctg cctgccgcag tgaagaacat 6960catggcccgg accgatcacc cagaacccat
acaactggcc tataacagtt atgaaaacca 7020aattccagtg ctgttcccaa agatcaaaaa
tggtgaggtg acagacagtt atgagaatta 7080cacatatctc aatgcaagaa aattaggaga
ggacgtgccg gcatatgtgt acgccacaga 7140ggatgaggat ctagcagtgg atcttctggg
tatggattgg ccggacccag gcaaccaaca 7200ggtggtagag acagggaggg cattaaaaca
agtaactggc ttatccacag cagaaaacgc 7260cctcttgata gccctattcg gctacgtcgg
gtaccagaca ctttcaaaaa ggcacatacc 7320catgattact gacatctata cacttgaaga
ccacaggctt gaggacacaa cccacctcca 7380gtttgcccca aacgctataa ggaccgacgg
caaggactca gagttgaagg aattagctgt 7440gggagacctt gataaatatg tggacgcact
ggtagactac tccaaacaag ggatgaaatt 7500catcaaagtc caagctgaaa aggtcagaga
ctcccagtct acgaaggaag gcttgcaaac 7560cattaaggag tatgtggata agtttataca
atcactaaca gagaataagg aggagatcat 7620caggtatgga ctatggggag ttcacacggc
actctacaaa agcttggcag cgagactggg 7680gcatgaaaca gcttttgcaa ctttagtggt
aaaatggttg gcttttgggg gcgaaacggt 7740atctgctcac atcaagcaag tagcagttga
tctagtagta tattatatca tcaacaaacc 7800atcttttcct ggagatacag agacccaaca
agaggggagg aagtttgtgg ctagtctttt 7860tatatctgca ctagcaacat acacatataa
aacctggaat tacaacaatc tgcaacgggt 7920tgtcgaacct gccttagctt acctcccata
tgctacaagt gccttgaagt tgttcacacc 7980cacaagatta gagagtgtgg tcatactcag
ttctacaatt tacaagacat acctctctat 8040aaggaagggt aagagtgacg gcttgttagg
tacaggcata agtgcagcca tggagatctt 8100aaaccaaaac ccaatctcag taggtatatc
tgtgatgctg ggggtaggtg ccatcgccgc 8160ccataatgca atagaatcta gtgaacagaa
aagaactttg ctgatgaagg tctttgtaaa 8220aaacttctta gaccaagcag caacagatga
gctagtcaaa gagaaccctg aaaaaataat 8280catggctcta tttgaagcag tccagaccat
aggaaacccc ctaagactca tctaccatct 8340gtacggggtg tactataagg ggtgggaagc
aaaagaactc gcagagaaaa ctgctggccg 8400caacttattc acattgatca tgtttgaggc
ctttgagctt ttaggtatgg actcagaagg 8460aaagataaga aacttgtcag gcaactacat
actggactta atcttcaact tgcataataa 8520attaaacaag gggctcaaaa aactagtcct
tgggtgggct cctgcacctt tgagctgtga 8580ttggacacca agtgatgaga gaataagcct
acctcataac aactacttaa gggtagaaac 8640caggtgtcct tgtggctatg agatgaaggc
aataaaaaat gttgctggta aattgacaaa 8700agttgaagaa aaggggtcct tcctatgcag
gaatagatta gggagaggac ctccaaactt 8760caaagtaaca aagttctatg atgataactt
gatagaagtc aagccagtag ctaggctaga 8820aggccaggtg gacctctatt acaagggagt
aacagctaag ttagactaca acaatgggaa 8880agtactgtta gctaccaaca agtgggaggt
ggaccacgct ttcctgacca gactagtaaa 8940gaagcacaca gggataggtt ttaaaggtgc
atatttgggt gaccgaccag accatcaaga 9000tcttgtcgat agagattgtg caactataac
gaagaactca gtacagttcc taaaaatgaa 9060gaagggttgc gctttcacat atgacctaac
aatctctaac cttgtcaggc ttattgaact 9120agtccataag aataatttac aagaaagaga
gatccctacc gtgacagtaa ctacttggct 9180tgcatattct tttgtcaatg aagacctggg
gactatcaag cctgtattgg gggagaaagt 9240catcccagaa ccccccgagg agttgagtct
ccaacccacc gtgagactag tcaccactga 9300aacagcaata accataacag gggaggctga
agtgatgacg acagggatca caccagtggt 9360agagatgaaa gaagaacctc agctggacca
ccagtcaact accctaaagg tagggttgaa 9420ggaaggggaa tatccagggc caggagttaa
ccctaaccat ttagcagagg tgatagatga 9480gaaagatgac aggccttttg tcctaatcat
cggtaacaaa ggttctacct cgaacagagc 9540aagaacggcc aagaatatac ggctgtacaa
aggaaacaac ccaagagaga tcagggatct 9600gatgagccaa ggaagaatat tgacggttgc
tctaaaagag ttggacccgg aattaaaaga 9660attagtagat tacaagggga cctttctcaa
tagggaagct ttagaagccc taagcttagg 9720taagccaatc aagaggaaaa ccacaacagc
aatgatcagg aggttaatag agccagaggt 9780tgaggaggaa ctaccagatt ggttccaagc
ggaagaaccc ctatttttgg aagcaaaaat 9840acagaatgac ttataccacc taattggcag
tgtagatagt ataaaaagca aagcaaagga 9900attaggggcc acagataaca caaagatagt
gaaggaagtt ggggctagga cctatacgat 9960gaaattgagc agctggagca cacaagttac
aaaaaaacag atgagtctag cccctctctt 10020tgaagagctg ttattaaagt gccctccatg
tagtaaaatt tcaaagggac atatggtgtc 10080agcataccaa ctggctcaag gaaactggga
acccctcggg tgtggggtct atatgggaac 10140cataccagct aggcgtctca agatccaccc
ttatgaggct taccttaaac tcaaagagct 10200ggtggaagtt gaatcttcga gggccactgc
aaaagaatcc atcataagag aacataacac 10260ctggatcctg cggaaggtga gacatgaagg
gaacctaaga accaaatcaa tgatcaaccc 10320tgggaaaata tcagatcagc tatgcagaga
tggacacaaa agaaacatat ataataagat 10380cataggctca acaatggcct ctgctggtat
taggctggag aaactgccag tagtccgagc 10440ccaaactgac acaaccagtt tccaccaagc
cataagagaa aaaattgata aaacagaaaa 10500caagcagacc cctgaattgc atgaagaact
aatgaaggtc ttcgactgct taaagatccc 10560agagctgaag gaatcgtatg atgaagtttc
atgggaacaa ttagaagccg ggataaaccg 10620taagggtgca gcaggctatc tagagagcaa
gaacataggg gaagtcctag acacagagaa 10680acacatagta gagcagctga tcaaggatct
gaggaagggg aagaagatta ggtactatga 10740aacagccatc cccaagaatg agaagagaga
cgtcagcgac gactgggaag ccggagagtt 10800cgttgatgaa aagaaaccaa gagtaatcca
gtacccggac gccaaggtga gactggccat 10860tacaaaagtg atgtacaaat gggtaaagca
aaaaccagtg gtgatacccg gctatgaagg 10920taaaacacct ctatttgaca tattcaacaa
agtgaagaag gaatgggatt cattccagga 10980ccccgtagca gtgagctttg acaccaaagc
gtgggataca caagtcacca gtagagacct 11040aatgttgata aaggatatcc agaaatatta
tttcaagaga agtatacaca aatttttaga 11100tacaataaca gaacacatgg tggaggtacc
tgtcattaca gcagacggtg aagtttacat 11160aaggaatggt cagaggggta gtggccaacc
cgacacaagt gctggtaata gtatgttgaa 11220tgtcctaacc atgatatatg ctttctgtaa
aagtacaggc ataccttaca ggggattcag 11280cagagtggca agaatccatg tgtgtggtga
tgatggcttt ttgataacag agagaggact 11340gggactgaaa ttctctgaga agggtatgca
gatattacat gaggccggga agccccagaa 11400aataactgaa ggggacaaaa tgaaagtggc
atacagattc gaggacatag agttttgttc 11460ccatactccc gtgccagtca gatgggcaga
taacaccagt agttacatgg cagggaggag 11520cacagccact atactagcta agatggcaac
caggctggat tccagcggag agaggggtag 11580cacagcttat gagaaggccg tagccttcag
cttccttttg atgtactcat ggaatcccgt 11640agttagaagg atctgcttac tggtgttgtc
acagtttcca gaaatatccc catccaaaaa 11700cacaatatac tactaccaag gggatcccat
agctgcgtac agagaagtga tagggaaaca 11760gctgtgtgaa ctgaaaagaa caggatttga
gaagctggct ggtctgaatt tgagtatgac 11820cactctaggc atctggacaa aacatactag
taaaagacta atccaagcct gtgtagaaat 11880aggtaagaga gaaggtacct ggttagttaa
tgctgacaga ctgattgcag gaaagactgg 11940gaagttttac atcccaagca ctggtgtcac
tctgttggga aaacactatg aggaaattaa 12000cttaaagcaa aaggcggcac aaccgccgat
agagggggtt gacagatata agttgggccc 12060catagttaat gttatcttga gaaggctgag
ggtgatgctg atgacagttg ccagcggaag 12120ctggtgaatc cgtccggagc gtcgtgccct
cactcaaggt ttttaattgt aaatattgta 12180aatagacagc taagatattt attgtagttg
gatagtaatg cagtgatagt aaatacccca 12240atttaacact acctccaatg cactaagcac
tttagctgtg tgaggttaac tcgacgtcca 12300cggttggact agggaagacc tctaacagcc
cc 12332211840DNAArtificial
SequenceMutated BVDV XIKE- A-NdN 2gtatacgaga ttagctaaag aactcgtata
tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg
ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc
agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact
ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta
acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat
agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga
gttgttttcc gatgaaggga gcaagggtgc 420tacaagtaag aagcagccta agccagatag
gatagaaaaa ggtaagatga aaatagcccc 480aaaagagaca gaaaaagatt gcaaaaccag
accccccgac gcgactatag tagtagaagg 540ggttaagtac caggtgaaga aaaaaggaaa
ggtaagggga aaaaatactc aagatgggtt 600atatcacaac aagaataagc cccctgaatc
aagaaaaaaa ttggaaaagg cactgctggc 660ttgggccatc ttagcagcgg tcctgcttca
gctggtaaca ggagagaata tcacccagtg 720gaacttgatg gacaacggca ccgagggaat
acagcaagcg atgttcctaa gaggggtgaa 780caggagtcta catggaattt ggccagagaa
aatttgcacc ggagtaccaa ctcacttagc 840aacagactat gagcttaaag agatagtggg
gatgatggac gcgagtgaga agaccaacta 900cacgtgttgc aggttgcaaa gacatgagtg
gaataaacat ggttggtgta actggtttca 960tatagaaccg tggatatggt tgatgaacaa
aacccaaaac aacctgacag aagggcaacc 1020gcttagggag tgtgctgtga cttgtaggta
tgacaaggaa acagaattga acatcgtgac 1080acaggctagg gacagaccta caactctgac
aggttgcaag aaaggcaaga atttctcttt 1140cgcaggtgtt atactggatg ggccctgtaa
ctttaaagta tcggttgaag atgtgctgtt 1200caaggagcac gattgcggca acatgctgca
agagaccgcg atacagctac tcgatggggc 1260aaccaacacc attgagggag caagggtagg
gacggccaag ttgacaacct ggttagggaa 1320gcaattaggg atccttggta agaagttgga
gaacaaaagc aaagcatggt ttggtgcaca 1380tgcagcaagt ccatactgcg gagtggagag
gaagatcggt tacgtatggt atacaaaaaa 1440ctgcactcca gcttgccttc caagaaacac
tagaataata ggccccggga aatttgatac 1500caacgccgaa gatggaaaaa tactccatga
gatggggggg cacctctcag aatttgtcct 1560attgtccttg gtggttctgt ctgactttgc
cccggaaacc gcgagcgtca tctacttggt 1620tctacatttt gcgatcccgc aaagccacgt
tgatgtagac acatgcgaca agaaccagct 1680gaatttaacg gtagcaacca cagtagcaga
ggtcatacca gggacagtgt ggaacctagg 1740gaagtatgtc tgcataagac cagactggtg
gccatatgag acgacgacag tcttcgtcat 1800agaggaagca gggcaagtaa tcaaattgat
gctaagggcc atcagagact taactaggat 1860atggaatgct gccactacca cagctttctt
aatcttttta gtaaaagcac tgaggggaca 1920actaatccaa gggctattgt ggctgatgct
aataacagga gcacagggct tccctgaatg 1980caaagagggc ttccaatatg ccatatctaa
agacaggaaa atggggttat tggggccaga 2040gagcttaact acaacatggc acctccccac
caaaaaaata gtggattcca tggtgcatgt 2100atggtgtgaa ggaaaagact tgaaaatatt
aaaaatgtgc acaaaggaag agaggtatct 2160agtggctgtg cacgagagag ccttatcaac
cagtgccgag tttatgcaga tcagtgatgg 2220gacaataggc ccagacgtga tagatatgcc
tgatgacttt gagtttggac tctgcccttg 2280tgactcaaaa ccagtgataa agggcaaatt
taatgccagc ttactgaatg gaccagcttt 2340ccagatggta tgcccacagg ggtggactgg
tacaatagaa tgcaccctag cgaaccaaga 2400caccttggac acaactgtca ttaggacata
tagaagaact accccatttc agcggagaaa 2460atggtgtacc tatgaaaaaa taatagggga
agatatctat gaatgcattc taggtggaaa 2520ctggacatgc ataaccggtg accatagcag
gttgaaagac ggacctatca agaagtgtaa 2580gtggtgtggc catgacttcg tcaactcaga
ggggctacca cactacccaa taggcaagtg 2640catgctcatc aacgagagtg ggtacaggta
tgtagatgac acctcttgcg ataggggtgg 2700tgtagccata gttccatctg gcaccgtaaa
gtgtagaata ggtaacgtca cggtgcaagt 2760tatcgctact aacaatgatc tgggacccat
gccttgcagc ccagctgaag tgatagcaag 2820tgaaggacca gtggaaaaga ctgcatgcac
attcaactat tcaaggactc tacctaataa 2880gtattatgag ccaagggacc ggtacttcca
acaatacatg ttaaaagggg agtggcaata 2940ttggttcgac ctggattctg tagaccacca
caaagactac ttctcagagt tcataatcat 3000agcagtggtc gccttgttgg gtggtaagta
cgtactgtgg ctcttgataa catacacaat 3060actgtctgag cagatggcta tgggtgctgg
agtgaatact gaagagatag tcatgatagg 3120caatttgctg acagacagtg atattgaggt
tgtggtttat ttccttcttc tgtacttaat 3180agttaaagag gaactggcga ggaaatggat
tatactggta taccacatcc ttgtagccaa 3240ccctatgaaa acaattgggg tcgtcttact
aatgctaggg ggagtggtga aggccagcag 3300aatcaatgct gatgaccaaa gtgctatgga
cccatgcttt cttctcgtga caggcgtagt 3360ggctgttttg atgatcgcta gaagagaacc
tgccacatta ccactgattg tagcattgct 3420agcaataaga acatcaggat tcctactgcc
cgctagcatt gatgtaactg tagcagtagt 3480attaattgta cttttgttgg ctagctacat
aacagactac tttagatata aaaagtggct 3540tcaactctta tttagtctga tagctggtat
ctttattata aggagcttaa aacatatcaa 3600ccagatggag gtaccagaaa tatctatgcc
aagttggaga cctctagctc tggtcctttt 3660ctatataaca tctacagcaa taaccactaa
ttgggacatt gacttagcag gcttcctgct 3720gcaatgggcg ccagcagtga tcatgatggc
taccatgtgg gcagactttt tgactctgat 3780catagtcctg cccagttacg agttatctaa
gctttacttc ctaaagaacg tcaggacaga 3840cgtggaaaag aactggctcg gcaaagtgaa
atacagacag atcagttcag tttatgacat 3900ctgtgacagt gaggaagcag tgtacctatt
tccatcaagg cataagagtg gaagcaggcc 3960agatttcata ttaccttttt tgaaagccgt
gttaataagc tgcatcagca gccaatggca 4020agtggtttac atttcttacc taatactgga
aattacatac tatatgcaca ggaaaatcat 4080agatgaggtg tcaggaggag caaattttct
atcaagactc atagcagcca tcatagaatt 4140aaattgggcc atagatgatg aggaatgtaa
aggactgaag aaactgtatc tcttgtcagg 4200gagagcgaag aatttgatag ttaaacataa
ggtaagaaat gaagccgtcc acagatggtt 4260tggtgaggag gaaatatacg gggcacccaa
ggtgatcact atcataaaag ctagtaccct 4320aagtaaaaac aggcactgca taatctgcac
gatctgtgaa gggaaagaat ggaatggagc 4380caactgccca aagtgtggaa gacaaggaaa
gcccataaca tgtggaatga cactcgcaga 4440ctttgaggag aaacattaca aaaagatatt
tataagagaa gaatcttctt gtcctgtgcc 4500ttttgatcct tcttgccatt gtaattattt
tcgccacgat gggcctttca ggaaagagta 4560taagggttac gtccaataca cagccagagg
acaactcttt ctgaggaacc taccaattct 4620agcgacgaag atgaagctat taatggtggg
aaacctcggc gcagaaattg gcgacctgga 4680acatctagga tgggtactga gagggccagc
cgtgtgcaaa aaaattacca accatgagaa 4740gtgccacgta aacatcatgg ataagctaac
tgcatttttt ggaatcatgc ctagaggcac 4800gacccctagg gcacctgtga ggttccccac
agcactacta aaagtgagaa gggggctaga 4860gacgggatgg gcttacacgc accaaggagg
gatcagctcg gtagaccatg tcacagccgg 4920aaaggattta ctagtgtgtg acagtatggg
caggaccagg gttgtctgtc atagtaacaa 4980taagatgact gatgagactg agtatggcat
caagaccgac tcagggtgtc ccgaaggtgc 5040gaggtgttac gtgctaaacc cagaagctgt
taacatttct ggcacaaaag gagctatggt 5100acacctccag aaaacggggg gggagttcac
atgtgtcact gcctcaggga ccccggcttt 5160cttcgatctg aaaaatctaa aaggctggtc
cgggctacca atttttgaag catccagtgg 5220cagggtggtt ggtagggtga aagtcggcaa
gaatgaggat tccaagccca ccaaactaat 5280gagcggaatc cagacagtgt ctaagaacca
gacagaccta gcggacatcg taaaaaaatt 5340gactagtatg aacagaggag agttcaaaca
gataacatta gccactgggg caggaaaaac 5400tacggaactg ccaaggtccg tcatagagga
gatagggagg cacaaaaggg tcttagtcct 5460gataccattg agagcagcag cagagtcagt
gtatcagtat atgagagtga agtacccaag 5520tatatctttc aatttgagaa taggagatat
gaaggaaggt gacatggcca ctggtatcac 5580ctacgcctca tatgggtact tttgtcagct
tcctcagccc aaactgagag ctgccatggt 5640agagtactca tatatattct tagatgagta
ccactgtgct acacccgagc aattagcaat 5700aattggaaag atacacaggt ttgctgaaaa
tcttagagtg gtagcaatga cagcaacccc 5760agctggaacg gtcacaacga ctggtcagaa
acaccctata gaggagttca tagccccaga 5820ggtgatgaaa ggtgaagatc taggtagtga
atacttggat attgcagggt tgaagatacc 5880gactgaagag atgaaaggca acatgctcgt
gttcgcgcca actaggaaca tggcagtaga 5940aacagctaag aaattgaagg ctaagggata
caactctgga tactattaca gtggggaaaa 6000cccagagaac ttgagggtgg taacctcgca
atccccgtat gtggtagtag ccaccaatgc 6060catagagtca ggtgtgacat taccagactt
agacacagtt gtagacactg gactaaagtg 6120tgagaagagg gtgaggattt cttcaaaaat
gcccttcatt gtaacaggac ttaagagaat 6180ggcagtcaca atcggagagc aagcccagcg
caggggtaga gtaggaagag tcaagccagg 6240taggtactat aggagtcaag aaacagcttc
agggtcaaaa gattaccatt acgacctact 6300gcaagcccag aggtacggaa tagaagatgg
aattaatgta acaaagtcat tcagggagat 6360gaactatgat tggagccttt acgaagagga
cagcttgatg ataactcaac tcgaggtcct 6420taacaacctc cttatatcag aagacctgcc
tgccgcagtg aagaacatca tggcccggac 6480cgatcaccca gaacccatac aactggccta
taacagttat gaaaaccaaa ttccagtgct 6540gttcccaaag atcaaaaatg gtgaggtgac
agacagttat gagaattaca catatctcaa 6600tgcaagaaaa ttaggagagg acgtgccggc
atatgtgtac gccacagagg atgaggatct 6660agcagtggat cttctgggta tggattggcc
ggacccaggc aaccaacagg tggtagagac 6720agggagggca ttaaaacaag taactggctt
atccacagca gaaaacgccc tcttgatagc 6780cctattcggc tacgtcgggt accagacact
ttcaaaaagg cacataccca tgattactga 6840catctataca cttgaagacc acaggcttga
ggacacaacc cacctccagt ttgccccaaa 6900cgctataagg accgacggca aggactcaga
gttgaaggaa ttagctgtgg gagaccttga 6960taaatatgtg gacgcactgg tagactactc
caaacaaggg atgaaattca tcaaagtcca 7020agctgaaaag gtcagagact cccagtctac
gaaggaaggc ttgcaaacca ttaaggagta 7080tgtggataag tttatacaat cactaacaga
gaataaggag gagatcatca ggtatggact 7140atggggagtt cacacggcac tctacaaaag
cttggcagcg agactggggc atgaaacagc 7200ttttgcaact ttagtggtaa aatggttggc
ttttgggggc gaaacggtat ctgctcacat 7260caagcaagta gcagttgatc tagtagtata
ttatatcatc aacaaaccat cttttcctgg 7320agatacagag acccaacaag aggggaggaa
gtttgtggct agtcttttta tatctgcact 7380agcaacatac acatataaaa cctggaatta
caacaatctg caacgggttg tcgaacctgc 7440cttagcttac ctcccatatg ctacaagtgc
cttgaagttg ttcacaccca caagattaga 7500gagtgtggtc atactcagtt ctacaattta
caagacatac ctctctataa ggaagggtaa 7560gagtgacggc ttgttaggta caggcataag
tgcagccatg gagatcttaa accaaaaccc 7620aatctcagta ggtatatctg tgatgctggg
ggtaggtgcc atcgccgccc ataatgcaat 7680agaatctagt gaacagaaaa gaactttgct
gatgaaggtc tttgtaaaaa acttcttaga 7740ccaagcagca acagatgagc tagtcaaaga
gaaccctgaa aaaataatca tggctctatt 7800tgaagcagtc cagaccatag gaaaccccct
aagactcatc taccatctgt acggggtgta 7860ctataagggg tgggaagcaa aagaactcgc
agagaaaact gctggccgca acttattcac 7920attgatcatg tttgaggcct ttgagctttt
aggtatggac tcagaaggaa agataagaaa 7980cttgtcaggc aactacatac tggacttaat
cttcaacttg cataataaat taaacaaggg 8040gctcaaaaaa ctagtccttg ggtgggctcc
tgcacctttg agctgtgatt ggacaccaag 8100tgatgagaga ataagcctac ctcataacaa
ctacttaagg gtagaaacca ggtgtccttg 8160tggctatgag atgaaggcaa taaaaaatgt
tgctggtaaa ttgacaaaag ttgaagaaaa 8220ggggtccttc ctatgcagga atagattagg
gagaggacct ccaaacttca aagtaacaaa 8280gttctatgat gataacttga tagaagtcaa
gccagtagct aggctagaag gccaggtgga 8340cctctattac aagggagtaa cagctaagtt
agactacaac aatgggaaag tactgttagc 8400taccaacaag tgggaggtgg accacgcttt
cctgaccaga ctagtaaaga agcacacagg 8460gataggtttt aaaggtgcat atttgggtga
ccgaccagac catcaagatc ttgtcgatag 8520agattgtgca actataacga agaactcagt
acagttccta aaaatgaaga agggttgcgc 8580tttcacatat gacctaacaa tctctaacct
tgtcaggctt attgaactag tccataagaa 8640taatttacaa gaaagagaga tccctaccgt
gacagtaact acttggcttg catattcttt 8700tgtcaatgaa gacctgggga ctatcaagcc
tgtattgggg gagaaagtca tcccagaacc 8760ccccgaggag ttgagtctcc aacccaccgt
gagactagtc accactgaaa cagcaataac 8820cataacaggg gaggctgaag tgatgacgac
agggatcaca ccagtggtag agatgaaaga 8880agaacctcag ctggaccacc agtcaactac
cctaaaggta gggttgaagg aaggggaata 8940tccagggcca ggagttaacc ctaaccattt
agcagaggtg atagatgaga aagatgacag 9000gccttttgtc ctaatcatcg gtaacaaagg
ttctacctcg aacagagcaa gaacggccaa 9060gaatatacgg ctgtacaaag gaaacaaccc
aagagagatc agggatctga tgagccaagg 9120aagaatattg acggttgctc taaaagagtt
ggacccggaa ttaaaagaat tagtagatta 9180caaggggacc tttctcaata gggaagcttt
agaagcccta agcttaggta agccaatcaa 9240gaggaaaacc acaacagcaa tgatcaggag
gttaatagag ccagaggttg aggaggaact 9300accagattgg ttccaagcgg aagaacccct
atttttggaa gcaaaaatac agaatgactt 9360ataccaccta attggcagtg tagatagtat
aaaaagcaaa gcaaaggaat taggggccac 9420agataacaca aagatagtga aggaagttgg
ggctaggacc tatacgatga aattgagcag 9480ctggagcaca caagttacaa aaaaacagat
gagtctagcc cctctctttg aagagctgtt 9540attaaagtgc cctccatgta gtaaaatttc
aaagggacat atggtgtcag cataccaact 9600ggctcaagga aactgggaac ccctcgggtg
tggggtctat atgggaacca taccagctag 9660gcgtctcaag atccaccctt atgaggctta
ccttaaactc aaagagctgg tggaagttga 9720atcttcgagg gccactgcaa aagaatccat
cataagagaa cataacacct ggatcctgcg 9780gaaggtgaga catgaaggga acctaagaac
caaatcaatg atcaaccctg ggaaaatatc 9840agatcagcta tgcagagatg gacacaaaag
aaacatatat aataagatca taggctcaac 9900aatggcctct gctggtatta ggctggagaa
actgccagta gtccgagccc aaactgacac 9960aaccagtttc caccaagcca taagagaaaa
aattgataaa acagaaaaca agcagacccc 10020tgaattgcat gaagaactaa tgaaggtctt
cgactgctta aagatcccag agctgaagga 10080atcgtatgat gaagtttcat gggaacaatt
agaagccggg ataaaccgta agggtgcagc 10140aggctatcta gagagcaaga acatagggga
agtcctagac acagagaaac acatagtaga 10200gcagctgatc aaggatctga ggaaggggaa
gaagattagg tactatgaaa cagccatccc 10260caagaatgag aagagagacg tcagcgacga
ctgggaagcc ggagagttcg ttgatgaaaa 10320gaaaccaaga gtaatccagt acccggacgc
caaggtgaga ctggccatta caaaagtgat 10380gtacaaatgg gtaaagcaaa aaccagtggt
gatacccggc tatgaaggta aaacacctct 10440atttgacata ttcaacaaag tgaagaagga
atgggattca ttccaggacc ccgtagcagt 10500gagctttgac accaaagcgt gggatacaca
agtcaccagt agagacctaa tgttgataaa 10560ggatatccag aaatattatt tcaagagaag
tatacacaaa tttttagata caataacaga 10620acacatggtg gaggtacctg tcattacagc
agacggtgaa gtttacataa ggaatggtca 10680gaggggtagt ggccaacccg acacaagtgc
tggtaatagt atgttgaatg tcctaaccat 10740gatatatgct ttctgtaaaa gtacaggcat
accttacagg ggattcagca gagtggcaag 10800aatccatgtg tgtggtgatg atggcttttt
gataacagag agaggactgg gactgaaatt 10860ctctgagaag ggtatgcaga tattacatga
ggccgggaag ccccagaaaa taactgaagg 10920ggacaaaatg aaagtggcat acagattcga
ggacatagag ttttgttccc atactcccgt 10980gccagtcaga tgggcagata acaccagtag
ttacatggca gggaggagca cagccactat 11040actagctaag atggcaacca ggctggattc
cagcggagag aggggtagca cagcttatga 11100gaaggccgta gccttcagct tccttttgat
gtactcatgg aatcccgtag ttagaaggat 11160ctgcttactg gtgttgtcac agtttccaga
aatatcccca tccaaaaaca caatatacta 11220ctaccaaggg gatcccatag ctgcgtacag
agaagtgata gggaaacagc tgtgtgaact 11280gaaaagaaca ggatttgaga agctggctgg
tctgaatttg agtatgacca ctctaggcat 11340ctggacaaaa catactagta aaagactaat
ccaagcctgt gtagaaatag gtaagagaga 11400aggtacctgg ttagttaatg ctgacagact
gattgcagga aagactggga agttttacat 11460cccaagcact ggtgtcactc tgttgggaaa
acactatgag gaaattaact taaagcaaaa 11520ggcggcacaa ccgccgatag agggggttga
cagatataag ttgggcccca tagttaatgt 11580tatcttgaga aggctgaggg tgatgctgat
gacagttgcc agcggaagct ggtgaatccg 11640tccggagcgt cgtgccctca ctcaaggttt
ttaattgtaa atattgtaaa tagacagcta 11700agatatttat tgtagttgga tagtaatgca
gtgatagtaa ataccccaat ttaacactac 11760ctccaatgca ctaagcactt tagctgtgtg
aggttaactc gacgtccacg gttggactag 11820ggaagacctc taacagcccc
11840312329DNAArtificial SequenceMutated
BVDV XIKE-B 3gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat
ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg
cccttagtag 120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc
gaacccctga 180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg
agatgccatg 240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg
ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc
tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga gttgttttca aatgaacttt
tatacaaaac 420atataaacaa aaaccagcag gcgtcgtgga acctgtttac gacgtcaacg
ggcgcccact 480gtttggagag agcagtgact tgcacccgca gtcaacacta aaactaccac
accaacgagg 540cagcgccaac atcctgacca atgctaggtc cctaccgcgg aaaggtgact
gccggagagg 600taatgtgtat ggaccggtga gtggcatcta tatcaaacca ggaccgatct
actaccagga 660ttatgtgggc cccgtctatc atagagcccc actggaacta tgtagggagg
caagtatgtg 720cgaaacaact aggagagttg gcagagtgac cggtagtgat gggaaattat
atcatatcta 780catctgcata gatgggtgta tcctcctgaa gagggcgact aggaaccaac
cagaagtcct 840gaaatgggta tacaacagat taaattgtcc tttatgggtc accagctgct
ccgatgaagg 900gagcaagggt gctacaagta agaagcagcc taagccagat aggatagaaa
aaggtaagat 960gaaaatagcc ccaaaagaga cagaaaaaga ttgcaaaacc agaccccccg
acgcgactat 1020agtagtagaa ggggttaagt accaggtgaa gaaaaaagga aaggtaaggg
gaaaaaatac 1080tcaagatggg ttatatcaca acaagaataa gccccctgaa tcaagaaaaa
aattggaaaa 1140ggcactgctg gcttgggcca tcttagcagc ggtcctgctt cagctggtaa
caggagagaa 1200tatcacccag tggaacttga tggacaacgg caccgaggga atacagcaag
cgatgttcct 1260aagaggggtg aacaggagtc tacatggaat ttggccagag aaaatttgca
ccggagtacc 1320aactcactta gcaacagact atgagcttaa agagatagtg gggatgatgg
acgcgagtga 1380gaagaccaac tacacgtgtt gcaggttgca aagacatgag tggaataaag
gttggtgtaa 1440ctggtttcat atagaaccgt ggatatggtt gatgaacaaa acccaaaaca
acctgacaga 1500agggcaaccg cttagggagt gtgctgtgac ttgtaggtat gacaaggaaa
cagaattgaa 1560catcgtgaca caggctaggg acagacctac aactctgaca ggttgcaaga
aaggcaagaa 1620tttctctttc gcaggtgtta tactggatgg gccctgtaac tttaaagtat
cggttgaaga 1680tgtgctgttc aaggagcacg attgcggcaa catgctgcaa gagaccgcga
tacagctact 1740cgatggggca accaacacca ttgagggagc aagggtaggg acggccaagt
tgacaacctg 1800gttagggaag caattaggga tccttggtaa gaagttggag aacaaaagca
aagcatggtt 1860tggtgcacat gcagcaagtc catactgcgg agtggagagg aagatcggtt
acgtatggta 1920tacaaaaaac tgcactccag cttgccttcc aagaaacact agaataatag
gccccgggaa 1980atttgatacc aacgccgaag atggaaaaat actccatgag atgggggggc
acctctcaga 2040atttgtccta ttgtccttgg tggttctgtc tgactttgcc ccggaaaccg
cgagcgtcat 2100ctacttggtt ctacattttg cgatcccgca aagccacgtt gatgtagaca
catgcgacaa 2160gaaccagctg aatttaacgg tagcaaccac agtagcagag gtcataccag
ggacagtgtg 2220gaacctaggg aagtatgtct gcataagacc agactggtgg ccatatgaga
cgacgacagt 2280cttcgtcata gaggaagcag ggcaagtaat caaattgatg ctaagggcca
tcagagactt 2340aactaggata tggaatgctg ccactaccac agctttctta atctttttag
taaaagcact 2400gaggggacaa ctaatccaag ggctattgtg gctgatgcta ataacaggag
cacagggctt 2460ccctgaatgc aaagagggct tccaatatgc catatctaaa gacaggaaaa
tggggttatt 2520ggggccagag agcttaacta caacatggca cctccccacc aaaaaaatag
tggattccat 2580ggtgcatgta tggtgtgaag gaaaagactt gaaaatatta aaaatgtgca
caaaggaaga 2640gaggtatcta gtggctgtgc acgagagagc cttatcaacc agtgccgagt
ttatgcagat 2700cagtgatggg acaataggcc cagacgtgat agatatgcct gatgactttg
agtttggact 2760ctgcccttgt gactcaaaac cagtgataaa gggcaaattt aatgccagct
tactgaatgg 2820accagctttc cagatggtat gcccacaggg gtggactggt acaatagaat
gcaccctagc 2880gaaccaagac accttggaca caactgtcat taggacatat agaagaacta
ccccatttca 2940gcggagaaaa tggtgtacct atgaaaaaat aataggggaa gatatctatg
aatgcattct 3000aggtggaaac tggacatgca taaccggtga ccatagcagg ttgaaagacg
gacctatcaa 3060gaagtgtaag tggtgtggcc atgacttcgt caactcagag gggctaccac
actacccaat 3120aggcaagtgc atgctcatca acgagagtgg gtacaggtat gtagatgaca
cctcttgcga 3180taggggtggt gtagccatag ttccatctgg caccgtaaag tgtagaatag
gtaacgtcac 3240ggtgcaagtt atcgctacta acaatgatct gggacccatg ccttgcagcc
cagctgaagt 3300gatagcaagt gaaggaccag tggaaaagac tgcatgcaca ttcaactatt
caaggactct 3360acctaataag tattatgagc caagggaccg gtacttccaa caatacatgt
taaaagggga 3420gtggcaatat tggttcgacc tggattctgt agaccaccac aaagactact
tctcagagtt 3480cataatcata gcagtggtcg ccttgttggg tggtaagtac gtactgtggc
tcttgataac 3540atacacaata ctgtctgagc agatggctat gggtgctgga gtgaatactg
aagagatagt 3600catgataggc aatttgctga cagacagtga tattgaggtt gtggtttatt
tccttcttct 3660gtacttaata gttaaagagg aactggcgag gaaatggatt atactggtat
accacatcct 3720tgtagccaac cctatgaaaa caattggggt cgtcttacta atgctagggg
gagtggtgaa 3780ggccagcaga atcaatgctg atgaccaaag tgctatggac ccatgctttc
ttctcgtgac 3840aggcgtagtg gctgttttga tgatcgctag aagagaacct gccacattac
cactgattgt 3900agcattgcta gcaataagaa catcaggatt cctactgccc gctagcattg
atgtaactgt 3960agcagtagta ttaattgtac ttttgttggc tagctacata acagactact
ttagatataa 4020aaagtggctt caactcttat ttagtctgat agctggtatc tttattataa
ggagcttaaa 4080acatatcaac cagatggagg taccagaaat atctatgcca agttggagac
ctctagctct 4140ggtccttttc tatataacat ctacagcaat aaccactaat tgggacattg
acttagcagg 4200cttcctgctg caatgggcgc cagcagtgat catgatggct accatgtggg
cagacttttt 4260gactctgatc atagtcctgc ccagttacga gttatctaag ctttacttcc
taaagaacgt 4320caggacagac gtggaaaaga actggctcgg caaagtgaaa tacagacaga
tcagttcagt 4380ttatgacatc tgtgacagtg aggaagcagt gtacctattt ccatcaaggc
ataagagtgg 4440aagcaggcca gatttcatat tacctttttt gaaagccgtg ttaataagct
gcatcagcag 4500ccaatggcaa gtggtttaca tttcttacct aatactggaa attacatact
atatgcacag 4560gaaaatcata gatgaggtgt caggaggagc aaattttcta tcaagactca
tagcagccat 4620catagaatta aattgggcca tagatgatga ggaatgtaaa ggactgaaga
aactgtatct 4680cttgtcaggg agagcgaaga atttgatagt taaacataag gtaagaaatg
aagccgtcca 4740cagatggttt ggtgaggagg aaatatacgg ggcacccaag gtgatcacta
tcataaaagc 4800tagtacccta agtaaaaaca ggcactgcat aatctgcacg atctgtgaag
ggaaagaatg 4860gaatggagcc aactgcccaa agtgtggaag acaaggaaag cccataacat
gtggaatgac 4920actcgcagac tttgaggaga aacattacaa aaagatattt ataagagaag
aatcttcttg 4980tcctgtgcct tttgatcctt cttgccattg taattatttt cgccacgatg
ggcctttcag 5040gaaagagtat aagggttacg tccaatacac agccagagga caactctttc
tgaggaacct 5100accaattcta gcgacgaaga tgaagctatt aatggtggga aacctcggcg
cagaaattgg 5160cgacctggaa catctaggat gggtactgag agggccagcc gtgtgcaaaa
aaattaccaa 5220ccatgagaag tgccacgtaa acatcatgga taagctaact gcattttttg
gaatcatgcc 5280tagaggcacg acccctaggg cacctgtgag gttccccaca gcactactaa
aagtgagaag 5340ggggctagag acgggatggg cttacacgca ccaaggaggg atcagctcgg
tagaccatgt 5400cacagccgga aaggatttac tagtgtgtga cagtatgggc aggaccaggg
ttgtctgtca 5460tagtaacaat aagatgactg atgagactga gtatggcatc aagaccgact
cagggtgtcc 5520cgaaggtgcg aggtgttacg tgctaaaccc agaagctgtt aacatttctg
gcacaaaagg 5580agctatggta cacctccaga aaacgggggg ggagttcaca tgtgtcactg
cctcagggac 5640cccggctttc ttcgatctga aaaatctaaa aggctggtcc gggctaccaa
tttttgaagc 5700atccagtggc agggtggttg gtagggtgaa agtcggcaag aatgaggatt
ccaagcccac 5760caaactaatg agcggaatcc agacagtgtc taagaaccag acagacctag
cggacatcgt 5820aaaaaaattg actagtatga acagaggaga gttcaaacag ataacattag
ccactggggc 5880aggaaaaact acggaactgc caaggtccgt catagaggag atagggaggc
acaaaagggt 5940cttagtcctg ataccattga gagcagcagc agagtcagtg tatcagtata
tgagagtgaa 6000gtacccaagt atatctttca atttgagaat aggagatatg aaggaaggtg
acatggccac 6060tggtatcacc tacgcctcat atgggtactt ttgtcagctt cctcagccca
aactgagagc 6120tgccatggta gagtactcat atatattctt agatgagtac cactgtgcta
cacccgagca 6180attagcaata attggaaaga tacacaggtt tgctgaaaat cttagagtgg
tagcaatgac 6240agcaacccca gctggaacgg tcacaacgac tggtcagaaa caccctatag
aggagttcat 6300agccccagag gtgatgaaag gtgaagatct aggtagtgaa tacttggata
ttgcagggtt 6360gaagataccg actgaagaga tgaaaggcaa catgctcgtg ttcgcgccaa
ctaggaacat 6420ggcagtagaa acagctaaga aattgaaggc taagggatac aactctggat
actattacag 6480tggggaaaac ccagagaact tgagggtggt aacctcgcaa tccccgtatg
tggtagtagc 6540caccaatgcc atagagtcag gtgtgacatt accagactta gacacagttg
tagacactgg 6600actaaagtgt gagaagaggg tgaggatttc ttcaaaaatg cccttcattg
taacaggact 6660taagagaatg gcagtcacaa tcggagagca agcccagcgc aggggtagag
taggaagagt 6720caagccaggt aggtactata ggagtcaaga aacagcttca gggtcaaaag
attaccatta 6780cgacctactg caagcccaga ggtacggaat agaagatgga attaatgtaa
caaagtcatt 6840cagggagatg aactatgatt ggagccttta cgaagaggac agcttgatga
taactcaact 6900cgaggtcctt aacaacctcc ttatatcaga agacctgcct gccgcagtga
agaacatcat 6960ggcccggacc gatcacccag aacccataca actggcctat aacagttatg
aaaaccaaat 7020tccagtgctg ttcccaaaga tcaaaaatgg tgaggtgaca gacagttatg
agaattacac 7080atatctcaat gcaagaaaat taggagagga cgtgccggca tatgtgtacg
ccacagagga 7140tgaggatcta gcagtggatc ttctgggtat ggattggccg gacccaggca
accaacaggt 7200ggtagagaca gggagggcat taaaacaagt aactggctta tccacagcag
aaaacgccct 7260cttgatagcc ctattcggct acgtcgggta ccagacactt tcaaaaaggc
acatacccat 7320gattactgac atctatacac ttgaagacca caggcttgag gacacaaccc
acctccagtt 7380tgccccaaac gctataagga ccgacggcaa ggactcagag ttgaaggaat
tagctgtggg 7440agaccttgat aaatatgtgg acgcactggt agactactcc aaacaaggga
tgaaattcat 7500caaagtccaa gctgaaaagg tcagagactc ccagtctacg aaggaaggct
tgcaaaccat 7560taaggagtat gtggataagt ttatacaatc actaacagag aataaggagg
agatcatcag 7620gtatggacta tggggagttc acacggcact ctacaaaagc ttggcagcga
gactggggca 7680tgaaacagct tttgcaactt tagtggtaaa atggttggct tttgggggcg
aaacggtatc 7740tgctcacatc aagcaagtag cagttgatct agtagtatat tatatcatca
acaaaccatc 7800ttttcctgga gatacagaga cccaacaaga ggggaggaag tttgtggcta
gtctttttat 7860atctgcacta gcaacataca catataaaac ctggaattac aacaatctgc
aacgggttgt 7920cgaacctgcc ttagcttacc tcccatatgc tacaagtgcc ttgaagttgt
tcacacccac 7980aagattagag agtgtggtca tactcagttc tacaatttac aagacatacc
tctctataag 8040gaagggtaag agtgacggct tgttaggtac aggcataagt gcagccatgg
agatcttaaa 8100ccaaaaccca atctcagtag gtatatctgt gatgctgggg gtaggtgcca
tcgccgccca 8160taatgcaata gaatctagtg aacagaaaag aactttgctg atgaaggtct
ttgtaaaaaa 8220cttcttagac caagcagcaa cagatgagct agtcaaagag aaccctgaaa
aaataatcat 8280ggctctattt gaagcagtcc agaccatagg aaacccccta agactcatct
accatctgta 8340cggggtgtac tataaggggt gggaagcaaa agaactcgca gagaaaactg
ctggccgcaa 8400cttattcaca ttgatcatgt ttgaggcctt tgagctttta ggtatggact
cagaaggaaa 8460gataagaaac ttgtcaggca actacatact ggacttaatc ttcaacttgc
ataataaatt 8520aaacaagggg ctcaaaaaac tagtccttgg gtgggctcct gcacctttga
gctgtgattg 8580gacaccaagt gatgagagaa taagcctacc tcataacaac tacttaaggg
tagaaaccag 8640gtgtccttgt ggctatgaga tgaaggcaat aaaaaatgtt gctggtaaat
tgacaaaagt 8700tgaagaaaag gggtccttcc tatgcaggaa tagattaggg agaggacctc
caaacttcaa 8760agtaacaaag ttctatgatg ataacttgat agaagtcaag ccagtagcta
ggctagaagg 8820ccaggtggac ctctattaca agggagtaac agctaagtta gactacaaca
atgggaaagt 8880actgttagct accaacaagt gggaggtgga ccacgctttc ctgaccagac
tagtaaagaa 8940gcacacaggg ataggtttta aaggtgcata tttgggtgac cgaccagacc
atcaagatct 9000tgtcgataga gattgtgcaa ctataacgaa gaactcagta cagttcctaa
aaatgaagaa 9060gggttgcgct ttcacatatg acctaacaat ctctaacctt gtcaggctta
ttgaactagt 9120ccataagaat aatttacaag aaagagagat ccctaccgtg acagtaacta
cttggcttgc 9180atattctttt gtcaatgaag acctggggac tatcaagcct gtattggggg
agaaagtcat 9240cccagaaccc cccgaggagt tgagtctcca acccaccgtg agactagtca
ccactgaaac 9300agcaataacc ataacagggg aggctgaagt gatgacgaca gggatcacac
cagtggtaga 9360gatgaaagaa gaacctcagc tggaccacca gtcaactacc ctaaaggtag
ggttgaagga 9420aggggaatat ccagggccag gagttaaccc taaccattta gcagaggtga
tagatgagaa 9480agatgacagg ccttttgtcc taatcatcgg taacaaaggt tctacctcga
acagagcaag 9540aacggccaag aatatacggc tgtacaaagg aaacaaccca agagagatca
gggatctgat 9600gagccaagga agaatattga cggttgctct aaaagagttg gacccggaat
taaaagaatt 9660agtagattac aaggggacct ttctcaatag ggaagcttta gaagccctaa
gcttaggtaa 9720gccaatcaag aggaaaacca caacagcaat gatcaggagg ttaatagagc
cagaggttga 9780ggaggaacta ccagattggt tccaagcgga agaaccccta tttttggaag
caaaaataca 9840gaatgactta taccacctaa ttggcagtgt agatagtata aaaagcaaag
caaaggaatt 9900aggggccaca gataacacaa agatagtgaa ggaagttggg gctaggacct
atacgatgaa 9960attgagcagc tggagcacac aagttacaaa aaaacagatg agtctagccc
ctctctttga 10020agagctgtta ttaaagtgcc ctccatgtag taaaatttca aagggacata
tggtgtcagc 10080ataccaactg gctcaaggaa actgggaacc cctcgggtgt ggggtctata
tgggaaccat 10140accagctagg cgtctcaaga tccaccctta tgaggcttac cttaaactca
aagagctggt 10200ggaagttgaa tcttcgaggg ccactgcaaa agaatccatc ataagagaac
ataacacctg 10260gatcctgcgg aaggtgagac atgaagggaa cctaagaacc aaatcaatga
tcaaccctgg 10320gaaaatatca gatcagctat gcagagatgg acacaaaaga aacatatata
ataagatcat 10380aggctcaaca atggcctctg ctggtattag gctggagaaa ctgccagtag
tccgagccca 10440aactgacaca accagtttcc accaagccat aagagaaaaa attgataaaa
cagaaaacaa 10500gcagacccct gaattgcatg aagaactaat gaaggtcttc gactgcttaa
agatcccaga 10560gctgaaggaa tcgtatgatg aagtttcatg ggaacaatta gaagccggga
taaaccgtaa 10620gggtgcagca ggctatctag agagcaagaa cataggggaa gtcctagaca
cagagaaaca 10680catagtagag cagctgatca aggatctgag gaaggggaag aagattaggt
actatgaaac 10740agccatcccc aagaatgaga agagagacgt cagcgacgac tgggaagccg
gagagttcgt 10800tgatgaaaag aaaccaagag taatccagta cccggacgcc aaggtgagac
tggccattac 10860aaaagtgatg tacaaatggg taaagcaaaa accagtggtg atacccggct
atgaaggtaa 10920aacacctcta tttgacatat tcaacaaagt gaagaaggaa tgggattcat
tccaggaccc 10980cgtagcagtg agctttgaca ccaaagcgtg ggatacacaa gtcaccagta
gagacctaat 11040gttgataaag gatatccaga aatattattt caagagaagt atacacaaat
ttttagatac 11100aataacagaa cacatggtgg aggtacctgt cattacagca gacggtgaag
tttacataag 11160gaatggtcag aggggtagtg gccaacccga cacaagtgct ggtaatagta
tgttgaatgt 11220cctaaccatg atatatgctt tctgtaaaag tacaggcata ccttacaggg
gattcagcag 11280agtggcaaga atccatgtgt gtggtgatga tggctttttg ataacagaga
gaggactggg 11340actgaaattc tctgagaagg gtatgcagat attacatgag gccgggaagc
cccagaaaat 11400aactgaaggg gacaaaatga aagtggcata cagattcgag gacatagagt
tttgttccca 11460tactcccgtg ccagtcagat gggcagataa caccagtagt tacatggcag
ggaggagcac 11520agccactata ctagctaaga tggcaaccag gctggattcc agcggagaga
ggggtagcac 11580agcttatgag aaggccgtag ccttcagctt ccttttgatg tactcatgga
atcccgtagt 11640tagaaggatc tgcttactgg tgttgtcaca gtttccagaa atatccccat
ccaaaaacac 11700aatatactac taccaagggg atcccatagc tgcgtacaga gaagtgatag
ggaaacagct 11760gtgtgaactg aaaagaacag gatttgagaa gctggctggt ctgaatttga
gtatgaccac 11820tctaggcatc tggacaaaac atactagtaa aagactaatc caagcctgtg
tagaaatagg 11880taagagagaa ggtacctggt tagttaatgc tgacagactg attgcaggaa
agactgggaa 11940gttttacatc ccaagcactg gtgtcactct gttgggaaaa cactatgagg
aaattaactt 12000aaagcaaaag gcggcacaac cgccgataga gggggttgac agatataagt
tgggccccat 12060agttaatgtt atcttgagaa ggctgagggt gatgctgatg acagttgcca
gcggaagctg 12120gtgaatccgt ccggagcgtc gtgccctcac tcaaggtttt taattgtaaa
tattgtaaat 12180agacagctaa gatatttatt gtagttggat agtaatgcag tgatagtaaa
taccccaatt 12240taacactacc tccaatgcac taagcacttt agctgtgtga ggttaactcg
acgtccacgg 12300ttggactagg gaagacctct aacagcccc
12329411837DNAArtificial SequenceMutated BVDV XIKE-B-NdN
4gtatacgaga ttagctaaag aactcgtata tggattggac gtcaacaaat ttttaattgg
60caacgtaggg aaccttcccc tcagcgaagg ccgaaaagag gctagccatg cccttagtag
120gactagcaaa agtaggggac tagcggtagc agtgagttcg ttggatggcc gaacccctga
180gtacagggga gtcgtcaatg gttcgacact ccattagtcg aggagtctcg agatgccatg
240tggacgaggg catgcccacg gcacatctta acccatgcgg gggttgcatg ggtgaaagcg
300ctattcgtgg cgttatggac acagcctgat agggtgtagc agagacctgc tattccgcta
360gtaaaaactc tgctgtacat ggcacatgga gttgttttcc gatgaaggga gcaagggtgc
420tacaagtaag aagcagccta agccagatag gatagaaaaa ggtaagatga aaatagcccc
480aaaagagaca gaaaaagatt gcaaaaccag accccccgac gcgactatag tagtagaagg
540ggttaagtac caggtgaaga aaaaaggaaa ggtaagggga aaaaatactc aagatgggtt
600atatcacaac aagaataagc cccctgaatc aagaaaaaaa ttggaaaagg cactgctggc
660ttgggccatc ttagcagcgg tcctgcttca gctggtaaca ggagagaata tcacccagtg
720gaacttgatg gacaacggca ccgagggaat acagcaagcg atgttcctaa gaggggtgaa
780caggagtcta catggaattt ggccagagaa aatttgcacc ggagtaccaa ctcacttagc
840aacagactat gagcttaaag agatagtggg gatgatggac gcgagtgaga agaccaacta
900cacgtgttgc aggttgcaaa gacatgagtg gaataaaggt tggtgtaact ggtttcatat
960agaaccgtgg atatggttga tgaacaaaac ccaaaacaac ctgacagaag ggcaaccgct
1020tagggagtgt gctgtgactt gtaggtatga caaggaaaca gaattgaaca tcgtgacaca
1080ggctagggac agacctacaa ctctgacagg ttgcaagaaa ggcaagaatt tctctttcgc
1140aggtgttata ctggatgggc cctgtaactt taaagtatcg gttgaagatg tgctgttcaa
1200ggagcacgat tgcggcaaca tgctgcaaga gaccgcgata cagctactcg atggggcaac
1260caacaccatt gagggagcaa gggtagggac ggccaagttg acaacctggt tagggaagca
1320attagggatc cttggtaaga agttggagaa caaaagcaaa gcatggtttg gtgcacatgc
1380agcaagtcca tactgcggag tggagaggaa gatcggttac gtatggtata caaaaaactg
1440cactccagct tgccttccaa gaaacactag aataataggc cccgggaaat ttgataccaa
1500cgccgaagat ggaaaaatac tccatgagat gggggggcac ctctcagaat ttgtcctatt
1560gtccttggtg gttctgtctg actttgcccc ggaaaccgcg agcgtcatct acttggttct
1620acattttgcg atcccgcaaa gccacgttga tgtagacaca tgcgacaaga accagctgaa
1680tttaacggta gcaaccacag tagcagaggt cataccaggg acagtgtgga acctagggaa
1740gtatgtctgc ataagaccag actggtggcc atatgagacg acgacagtct tcgtcataga
1800ggaagcaggg caagtaatca aattgatgct aagggccatc agagacttaa ctaggatatg
1860gaatgctgcc actaccacag ctttcttaat ctttttagta aaagcactga ggggacaact
1920aatccaaggg ctattgtggc tgatgctaat aacaggagca cagggcttcc ctgaatgcaa
1980agagggcttc caatatgcca tatctaaaga caggaaaatg gggttattgg ggccagagag
2040cttaactaca acatggcacc tccccaccaa aaaaatagtg gattccatgg tgcatgtatg
2100gtgtgaagga aaagacttga aaatattaaa aatgtgcaca aaggaagaga ggtatctagt
2160ggctgtgcac gagagagcct tatcaaccag tgccgagttt atgcagatca gtgatgggac
2220aataggccca gacgtgatag atatgcctga tgactttgag tttggactct gcccttgtga
2280ctcaaaacca gtgataaagg gcaaatttaa tgccagctta ctgaatggac cagctttcca
2340gatggtatgc ccacaggggt ggactggtac aatagaatgc accctagcga accaagacac
2400cttggacaca actgtcatta ggacatatag aagaactacc ccatttcagc ggagaaaatg
2460gtgtacctat gaaaaaataa taggggaaga tatctatgaa tgcattctag gtggaaactg
2520gacatgcata accggtgacc atagcaggtt gaaagacgga cctatcaaga agtgtaagtg
2580gtgtggccat gacttcgtca actcagaggg gctaccacac tacccaatag gcaagtgcat
2640gctcatcaac gagagtgggt acaggtatgt agatgacacc tcttgcgata ggggtggtgt
2700agccatagtt ccatctggca ccgtaaagtg tagaataggt aacgtcacgg tgcaagttat
2760cgctactaac aatgatctgg gacccatgcc ttgcagccca gctgaagtga tagcaagtga
2820aggaccagtg gaaaagactg catgcacatt caactattca aggactctac ctaataagta
2880ttatgagcca agggaccggt acttccaaca atacatgtta aaaggggagt ggcaatattg
2940gttcgacctg gattctgtag accaccacaa agactacttc tcagagttca taatcatagc
3000agtggtcgcc ttgttgggtg gtaagtacgt actgtggctc ttgataacat acacaatact
3060gtctgagcag atggctatgg gtgctggagt gaatactgaa gagatagtca tgataggcaa
3120tttgctgaca gacagtgata ttgaggttgt ggtttatttc cttcttctgt acttaatagt
3180taaagaggaa ctggcgagga aatggattat actggtatac cacatccttg tagccaaccc
3240tatgaaaaca attggggtcg tcttactaat gctaggggga gtggtgaagg ccagcagaat
3300caatgctgat gaccaaagtg ctatggaccc atgctttctt ctcgtgacag gcgtagtggc
3360tgttttgatg atcgctagaa gagaacctgc cacattacca ctgattgtag cattgctagc
3420aataagaaca tcaggattcc tactgcccgc tagcattgat gtaactgtag cagtagtatt
3480aattgtactt ttgttggcta gctacataac agactacttt agatataaaa agtggcttca
3540actcttattt agtctgatag ctggtatctt tattataagg agcttaaaac atatcaacca
3600gatggaggta ccagaaatat ctatgccaag ttggagacct ctagctctgg tccttttcta
3660tataacatct acagcaataa ccactaattg ggacattgac ttagcaggct tcctgctgca
3720atgggcgcca gcagtgatca tgatggctac catgtgggca gactttttga ctctgatcat
3780agtcctgccc agttacgagt tatctaagct ttacttccta aagaacgtca ggacagacgt
3840ggaaaagaac tggctcggca aagtgaaata cagacagatc agttcagttt atgacatctg
3900tgacagtgag gaagcagtgt acctatttcc atcaaggcat aagagtggaa gcaggccaga
3960tttcatatta ccttttttga aagccgtgtt aataagctgc atcagcagcc aatggcaagt
4020ggtttacatt tcttacctaa tactggaaat tacatactat atgcacagga aaatcataga
4080tgaggtgtca ggaggagcaa attttctatc aagactcata gcagccatca tagaattaaa
4140ttgggccata gatgatgagg aatgtaaagg actgaagaaa ctgtatctct tgtcagggag
4200agcgaagaat ttgatagtta aacataaggt aagaaatgaa gccgtccaca gatggtttgg
4260tgaggaggaa atatacgggg cacccaaggt gatcactatc ataaaagcta gtaccctaag
4320taaaaacagg cactgcataa tctgcacgat ctgtgaaggg aaagaatgga atggagccaa
4380ctgcccaaag tgtggaagac aaggaaagcc cataacatgt ggaatgacac tcgcagactt
4440tgaggagaaa cattacaaaa agatatttat aagagaagaa tcttcttgtc ctgtgccttt
4500tgatccttct tgccattgta attattttcg ccacgatggg cctttcagga aagagtataa
4560gggttacgtc caatacacag ccagaggaca actctttctg aggaacctac caattctagc
4620gacgaagatg aagctattaa tggtgggaaa cctcggcgca gaaattggcg acctggaaca
4680tctaggatgg gtactgagag ggccagccgt gtgcaaaaaa attaccaacc atgagaagtg
4740ccacgtaaac atcatggata agctaactgc attttttgga atcatgccta gaggcacgac
4800ccctagggca cctgtgaggt tccccacagc actactaaaa gtgagaaggg ggctagagac
4860gggatgggct tacacgcacc aaggagggat cagctcggta gaccatgtca cagccggaaa
4920ggatttacta gtgtgtgaca gtatgggcag gaccagggtt gtctgtcata gtaacaataa
4980gatgactgat gagactgagt atggcatcaa gaccgactca gggtgtcccg aaggtgcgag
5040gtgttacgtg ctaaacccag aagctgttaa catttctggc acaaaaggag ctatggtaca
5100cctccagaaa acgggggggg agttcacatg tgtcactgcc tcagggaccc cggctttctt
5160cgatctgaaa aatctaaaag gctggtccgg gctaccaatt tttgaagcat ccagtggcag
5220ggtggttggt agggtgaaag tcggcaagaa tgaggattcc aagcccacca aactaatgag
5280cggaatccag acagtgtcta agaaccagac agacctagcg gacatcgtaa aaaaattgac
5340tagtatgaac agaggagagt tcaaacagat aacattagcc actggggcag gaaaaactac
5400ggaactgcca aggtccgtca tagaggagat agggaggcac aaaagggtct tagtcctgat
5460accattgaga gcagcagcag agtcagtgta tcagtatatg agagtgaagt acccaagtat
5520atctttcaat ttgagaatag gagatatgaa ggaaggtgac atggccactg gtatcaccta
5580cgcctcatat gggtactttt gtcagcttcc tcagcccaaa ctgagagctg ccatggtaga
5640gtactcatat atattcttag atgagtacca ctgtgctaca cccgagcaat tagcaataat
5700tggaaagata cacaggtttg ctgaaaatct tagagtggta gcaatgacag caaccccagc
5760tggaacggtc acaacgactg gtcagaaaca ccctatagag gagttcatag ccccagaggt
5820gatgaaaggt gaagatctag gtagtgaata cttggatatt gcagggttga agataccgac
5880tgaagagatg aaaggcaaca tgctcgtgtt cgcgccaact aggaacatgg cagtagaaac
5940agctaagaaa ttgaaggcta agggatacaa ctctggatac tattacagtg gggaaaaccc
6000agagaacttg agggtggtaa cctcgcaatc cccgtatgtg gtagtagcca ccaatgccat
6060agagtcaggt gtgacattac cagacttaga cacagttgta gacactggac taaagtgtga
6120gaagagggtg aggatttctt caaaaatgcc cttcattgta acaggactta agagaatggc
6180agtcacaatc ggagagcaag cccagcgcag gggtagagta ggaagagtca agccaggtag
6240gtactatagg agtcaagaaa cagcttcagg gtcaaaagat taccattacg acctactgca
6300agcccagagg tacggaatag aagatggaat taatgtaaca aagtcattca gggagatgaa
6360ctatgattgg agcctttacg aagaggacag cttgatgata actcaactcg aggtccttaa
6420caacctcctt atatcagaag acctgcctgc cgcagtgaag aacatcatgg cccggaccga
6480tcacccagaa cccatacaac tggcctataa cagttatgaa aaccaaattc cagtgctgtt
6540cccaaagatc aaaaatggtg aggtgacaga cagttatgag aattacacat atctcaatgc
6600aagaaaatta ggagaggacg tgccggcata tgtgtacgcc acagaggatg aggatctagc
6660agtggatctt ctgggtatgg attggccgga cccaggcaac caacaggtgg tagagacagg
6720gagggcatta aaacaagtaa ctggcttatc cacagcagaa aacgccctct tgatagccct
6780attcggctac gtcgggtacc agacactttc aaaaaggcac atacccatga ttactgacat
6840ctatacactt gaagaccaca ggcttgagga cacaacccac ctccagtttg ccccaaacgc
6900tataaggacc gacggcaagg actcagagtt gaaggaatta gctgtgggag accttgataa
6960atatgtggac gcactggtag actactccaa acaagggatg aaattcatca aagtccaagc
7020tgaaaaggtc agagactccc agtctacgaa ggaaggcttg caaaccatta aggagtatgt
7080ggataagttt atacaatcac taacagagaa taaggaggag atcatcaggt atggactatg
7140gggagttcac acggcactct acaaaagctt ggcagcgaga ctggggcatg aaacagcttt
7200tgcaacttta gtggtaaaat ggttggcttt tgggggcgaa acggtatctg ctcacatcaa
7260gcaagtagca gttgatctag tagtatatta tatcatcaac aaaccatctt ttcctggaga
7320tacagagacc caacaagagg ggaggaagtt tgtggctagt ctttttatat ctgcactagc
7380aacatacaca tataaaacct ggaattacaa caatctgcaa cgggttgtcg aacctgcctt
7440agcttacctc ccatatgcta caagtgcctt gaagttgttc acacccacaa gattagagag
7500tgtggtcata ctcagttcta caatttacaa gacatacctc tctataagga agggtaagag
7560tgacggcttg ttaggtacag gcataagtgc agccatggag atcttaaacc aaaacccaat
7620ctcagtaggt atatctgtga tgctgggggt aggtgccatc gccgcccata atgcaataga
7680atctagtgaa cagaaaagaa ctttgctgat gaaggtcttt gtaaaaaact tcttagacca
7740agcagcaaca gatgagctag tcaaagagaa ccctgaaaaa ataatcatgg ctctatttga
7800agcagtccag accataggaa accccctaag actcatctac catctgtacg gggtgtacta
7860taaggggtgg gaagcaaaag aactcgcaga gaaaactgct ggccgcaact tattcacatt
7920gatcatgttt gaggcctttg agcttttagg tatggactca gaaggaaaga taagaaactt
7980gtcaggcaac tacatactgg acttaatctt caacttgcat aataaattaa acaaggggct
8040caaaaaacta gtccttgggt gggctcctgc acctttgagc tgtgattgga caccaagtga
8100tgagagaata agcctacctc ataacaacta cttaagggta gaaaccaggt gtccttgtgg
8160ctatgagatg aaggcaataa aaaatgttgc tggtaaattg acaaaagttg aagaaaaggg
8220gtccttccta tgcaggaata gattagggag aggacctcca aacttcaaag taacaaagtt
8280ctatgatgat aacttgatag aagtcaagcc agtagctagg ctagaaggcc aggtggacct
8340ctattacaag ggagtaacag ctaagttaga ctacaacaat gggaaagtac tgttagctac
8400caacaagtgg gaggtggacc acgctttcct gaccagacta gtaaagaagc acacagggat
8460aggttttaaa ggtgcatatt tgggtgaccg accagaccat caagatcttg tcgatagaga
8520ttgtgcaact ataacgaaga actcagtaca gttcctaaaa atgaagaagg gttgcgcttt
8580cacatatgac ctaacaatct ctaaccttgt caggcttatt gaactagtcc ataagaataa
8640tttacaagaa agagagatcc ctaccgtgac agtaactact tggcttgcat attcttttgt
8700caatgaagac ctggggacta tcaagcctgt attgggggag aaagtcatcc cagaaccccc
8760cgaggagttg agtctccaac ccaccgtgag actagtcacc actgaaacag caataaccat
8820aacaggggag gctgaagtga tgacgacagg gatcacacca gtggtagaga tgaaagaaga
8880acctcagctg gaccaccagt caactaccct aaaggtaggg ttgaaggaag gggaatatcc
8940agggccagga gttaacccta accatttagc agaggtgata gatgagaaag atgacaggcc
9000ttttgtccta atcatcggta acaaaggttc tacctcgaac agagcaagaa cggccaagaa
9060tatacggctg tacaaaggaa acaacccaag agagatcagg gatctgatga gccaaggaag
9120aatattgacg gttgctctaa aagagttgga cccggaatta aaagaattag tagattacaa
9180ggggaccttt ctcaataggg aagctttaga agccctaagc ttaggtaagc caatcaagag
9240gaaaaccaca acagcaatga tcaggaggtt aatagagcca gaggttgagg aggaactacc
9300agattggttc caagcggaag aacccctatt tttggaagca aaaatacaga atgacttata
9360ccacctaatt ggcagtgtag atagtataaa aagcaaagca aaggaattag gggccacaga
9420taacacaaag atagtgaagg aagttggggc taggacctat acgatgaaat tgagcagctg
9480gagcacacaa gttacaaaaa aacagatgag tctagcccct ctctttgaag agctgttatt
9540aaagtgccct ccatgtagta aaatttcaaa gggacatatg gtgtcagcat accaactggc
9600tcaaggaaac tgggaacccc tcgggtgtgg ggtctatatg ggaaccatac cagctaggcg
9660tctcaagatc cacccttatg aggcttacct taaactcaaa gagctggtgg aagttgaatc
9720ttcgagggcc actgcaaaag aatccatcat aagagaacat aacacctgga tcctgcggaa
9780ggtgagacat gaagggaacc taagaaccaa atcaatgatc aaccctggga aaatatcaga
9840tcagctatgc agagatggac acaaaagaaa catatataat aagatcatag gctcaacaat
9900ggcctctgct ggtattaggc tggagaaact gccagtagtc cgagcccaaa ctgacacaac
9960cagtttccac caagccataa gagaaaaaat tgataaaaca gaaaacaagc agacccctga
10020attgcatgaa gaactaatga aggtcttcga ctgcttaaag atcccagagc tgaaggaatc
10080gtatgatgaa gtttcatggg aacaattaga agccgggata aaccgtaagg gtgcagcagg
10140ctatctagag agcaagaaca taggggaagt cctagacaca gagaaacaca tagtagagca
10200gctgatcaag gatctgagga aggggaagaa gattaggtac tatgaaacag ccatccccaa
10260gaatgagaag agagacgtca gcgacgactg ggaagccgga gagttcgttg atgaaaagaa
10320accaagagta atccagtacc cggacgccaa ggtgagactg gccattacaa aagtgatgta
10380caaatgggta aagcaaaaac cagtggtgat acccggctat gaaggtaaaa cacctctatt
10440tgacatattc aacaaagtga agaaggaatg ggattcattc caggaccccg tagcagtgag
10500ctttgacacc aaagcgtggg atacacaagt caccagtaga gacctaatgt tgataaagga
10560tatccagaaa tattatttca agagaagtat acacaaattt ttagatacaa taacagaaca
10620catggtggag gtacctgtca ttacagcaga cggtgaagtt tacataagga atggtcagag
10680gggtagtggc caacccgaca caagtgctgg taatagtatg ttgaatgtcc taaccatgat
10740atatgctttc tgtaaaagta caggcatacc ttacagggga ttcagcagag tggcaagaat
10800ccatgtgtgt ggtgatgatg gctttttgat aacagagaga ggactgggac tgaaattctc
10860tgagaagggt atgcagatat tacatgaggc cgggaagccc cagaaaataa ctgaagggga
10920caaaatgaaa gtggcataca gattcgagga catagagttt tgttcccata ctcccgtgcc
10980agtcagatgg gcagataaca ccagtagtta catggcaggg aggagcacag ccactatact
11040agctaagatg gcaaccaggc tggattccag cggagagagg ggtagcacag cttatgagaa
11100ggccgtagcc ttcagcttcc ttttgatgta ctcatggaat cccgtagtta gaaggatctg
11160cttactggtg ttgtcacagt ttccagaaat atccccatcc aaaaacacaa tatactacta
11220ccaaggggat cccatagctg cgtacagaga agtgataggg aaacagctgt gtgaactgaa
11280aagaacagga tttgagaagc tggctggtct gaatttgagt atgaccactc taggcatctg
11340gacaaaacat actagtaaaa gactaatcca agcctgtgta gaaataggta agagagaagg
11400tacctggtta gttaatgctg acagactgat tgcaggaaag actgggaagt tttacatccc
11460aagcactggt gtcactctgt tgggaaaaca ctatgaggaa attaacttaa agcaaaaggc
11520ggcacaaccg ccgatagagg gggttgacag atataagttg ggccccatag ttaatgttat
11580cttgagaagg ctgagggtga tgctgatgac agttgccagc ggaagctggt gaatccgtcc
11640ggagcgtcgt gccctcactc aaggttttta attgtaaata ttgtaaatag acagctaaga
11700tatttattgt agttggatag taatgcagtg atagtaaata ccccaattta acactacctc
11760caatgcacta agcactttag ctgtgtgagg ttaactcgac gtccacggtt ggactaggga
11820agacctctaa cagcccc
1183753913PRTWildtyp BVDV XIKE-A 5Met Glu Leu Phe Ser Asn Glu Leu Leu Tyr
Lys Thr Tyr Lys Gln Lys 1 5 10
15 Pro Ala Gly Val Val Glu Pro Val Tyr Asp Val Asn Gly Arg Pro
Leu 20 25 30 Phe
Gly Glu Ser Ser Asp Leu His Pro Gln Ser Thr Leu Lys Leu Pro 35
40 45 His Gln Arg Gly Ser Ala
Asn Ile Leu Thr Asn Ala Arg Ser Leu Pro 50 55
60 Arg Lys Gly Asp Cys Arg Arg Gly Asn Val Tyr
Gly Pro Val Ser Gly 65 70 75
80 Ile Tyr Ile Lys Pro Gly Pro Ile Tyr Tyr Gln Asp Tyr Val Gly Pro
85 90 95 Val Tyr
His Arg Ala Pro Leu Glu Leu Cys Arg Glu Ala Ser Met Cys 100
105 110 Glu Thr Thr Arg Arg Val Gly
Arg Val Thr Gly Ser Asp Gly Lys Leu 115 120
125 Tyr His Ile Tyr Ile Cys Ile Asp Gly Cys Ile Leu
Leu Lys Arg Ala 130 135 140
Thr Arg Asn Gln Pro Glu Val Leu Lys Trp Val Tyr Asn Arg Leu Asn 145
150 155 160 Cys Pro Leu
Trp Val Thr Ser Cys Ser Asp Glu Gly Ser Lys Gly Ala 165
170 175 Thr Ser Lys Lys Gln Pro Lys Pro
Asp Arg Ile Glu Lys Gly Lys Met 180 185
190 Lys Ile Ala Pro Lys Glu Thr Glu Lys Asp Cys Lys Thr
Arg Pro Pro 195 200 205
Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys 210
215 220 Gly Lys Val Arg
Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys 225 230
235 240 Asn Lys Pro Pro Glu Ser Arg Lys Lys
Leu Glu Lys Ala Leu Leu Ala 245 250
255 Trp Ala Ile Leu Ala Ala Val Leu Leu Gln Leu Val Thr Gly
Glu Asn 260 265 270
Ile Thr Gln Trp Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln
275 280 285 Ala Met Phe Leu
Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro 290
295 300 Glu Lys Ile Cys Thr Gly Val Pro
Thr His Leu Ala Thr Asp Tyr Glu 305 310
315 320 Leu Lys Glu Ile Val Gly Met Met Asp Ala Ser Glu
Lys Thr Asn Tyr 325 330
335 Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys
340 345 350 Asn Trp Phe
His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln 355
360 365 Asn Asn Leu Thr Glu Gly Gln Pro
Leu Arg Glu Cys Ala Val Thr Cys 370 375
380 Arg Tyr Asp Lys Glu Thr Glu Leu Asn Ile Val Thr Gln
Ala Arg Asp 385 390 395
400 Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe
405 410 415 Ala Gly Val Ile
Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu 420
425 430 Asp Val Leu Phe Lys Glu His Asp Cys
Gly Asn Met Leu Gln Glu Thr 435 440
445 Ala Ile Gln Leu Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly
Ala Arg 450 455 460
Val Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile 465
470 475 480 Leu Gly Lys Lys Leu
Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His 485
490 495 Ala Ala Ser Pro Tyr Cys Gly Val Glu Arg
Lys Ile Gly Tyr Val Trp 500 505
510 Tyr Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg
Ile 515 520 525 Ile
Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu 530
535 540 His Glu Met Gly Gly His
Leu Ser Glu Phe Val Leu Leu Ser Leu Val 545 550
555 560 Val Leu Ser Asp Phe Ala Pro Glu Thr Ala Ser
Val Ile Tyr Leu Val 565 570
575 Leu His Phe Ala Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp
580 585 590 Lys Asn
Gln Leu Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile 595
600 605 Pro Gly Thr Val Trp Asn Leu
Gly Lys Tyr Val Cys Ile Arg Pro Asp 610 615
620 Trp Trp Pro Tyr Glu Thr Thr Thr Val Phe Val Ile
Glu Glu Ala Gly 625 630 635
640 Gln Val Ile Lys Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile
645 650 655 Trp Asn Ala
Ala Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala 660
665 670 Leu Arg Gly Gln Leu Ile Gln Gly
Leu Leu Trp Leu Met Leu Ile Thr 675 680
685 Gly Ala Gln Gly Phe Pro Glu Cys Lys Glu Gly Phe Gln
Tyr Ala Ile 690 695 700
Ser Lys Asp Arg Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr 705
710 715 720 Thr Trp His Leu
Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val 725
730 735 Trp Cys Glu Gly Lys Asp Leu Lys Ile
Leu Lys Met Cys Thr Lys Glu 740 745
750 Glu Arg Tyr Leu Val Ala Val His Glu Arg Ala Leu Ser Thr
Ser Ala 755 760 765
Glu Phe Met Gln Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp 770
775 780 Met Pro Asp Asp Phe
Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro 785 790
795 800 Val Ile Lys Gly Lys Phe Asn Ala Ser Leu
Leu Asn Gly Pro Ala Phe 805 810
815 Gln Met Val Cys Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr
Leu 820 825 830 Ala
Asn Gln Asp Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg 835
840 845 Thr Thr Pro Phe Gln Arg
Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile 850 855
860 Gly Glu Asp Ile Tyr Glu Cys Ile Leu Gly Gly
Asn Trp Thr Cys Ile 865 870 875
880 Thr Gly Asp His Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys
885 890 895 Trp Cys
Gly His Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro 900
905 910 Ile Gly Lys Cys Met Leu Ile
Asn Glu Ser Gly Tyr Arg Tyr Val Asp 915 920
925 Asp Thr Ser Cys Asp Arg Gly Gly Val Ala Ile Val
Pro Ser Gly Thr 930 935 940
Val Lys Cys Arg Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn 945
950 955 960 Asn Asp Leu
Gly Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser 965
970 975 Glu Gly Pro Val Glu Lys Thr Ala
Cys Thr Phe Asn Tyr Ser Arg Thr 980 985
990 Leu Pro Asn Lys Tyr Tyr Glu Pro Arg Asp Arg Tyr
Phe Gln Gln Tyr 995 1000 1005
Met Leu Lys Gly Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val
1010 1015 1020 Asp His His
Lys Asp Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val 1025
1030 1035 Val Ala Leu Leu Gly Gly Lys Tyr
Val Leu Trp Leu Leu Ile Thr 1040 1045
1050 Tyr Thr Ile Leu Ser Glu Gln Met Ala Met Gly Ala Gly
Val Asn 1055 1060 1065
Thr Glu Glu Ile Val Met Ile Gly Asn Leu Leu Thr Asp Ser Asp 1070
1075 1080 Ile Glu Val Val Val
Tyr Phe Leu Leu Leu Tyr Leu Ile Val Lys 1085 1090
1095 Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu
Val Tyr His Ile Leu 1100 1105 1110
Val Ala Asn Pro Met Lys Thr Ile Gly Val Val Leu Leu Met Leu
1115 1120 1125 Gly Gly
Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp Gln Ser 1130
1135 1140 Ala Met Asp Pro Cys Phe Leu
Leu Val Thr Gly Val Val Ala Val 1145 1150
1155 Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro
Leu Ile Val 1160 1165 1170
Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala Ser 1175
1180 1185 Ile Asp Val Thr Val
Ala Val Val Leu Ile Val Leu Leu Leu Ala 1190 1195
1200 Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys
Lys Trp Leu Gln Leu 1205 1210 1215
Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys
1220 1225 1230 His Ile
Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp 1235
1240 1245 Arg Pro Leu Ala Leu Val Leu
Phe Tyr Ile Thr Ser Thr Ala Ile 1250 1255
1260 Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu
Leu Gln Trp 1265 1270 1275
Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe Leu 1280
1285 1290 Thr Leu Ile Ile Val
Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr 1295 1300
1305 Phe Leu Lys Asn Val Arg Thr Asp Val Glu
Lys Asn Trp Leu Gly 1310 1315 1320
Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp
1325 1330 1335 Ser Glu
Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly 1340
1345 1350 Ser Arg Pro Asp Phe Ile Leu
Pro Phe Leu Lys Ala Val Leu Ile 1355 1360
1365 Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile
Ser Tyr Leu 1370 1375 1380
Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp Glu 1385
1390 1395 Val Ser Gly Gly Ala
Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile 1400 1405
1410 Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu
Glu Cys Lys Gly Leu 1415 1420 1425
Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile Val
1430 1435 1440 Lys His
Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu 1445
1450 1455 Glu Glu Ile Tyr Gly Ala Pro
Lys Val Ile Thr Ile Ile Lys Ala 1460 1465
1470 Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys
Thr Ile Cys 1475 1480 1485
Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly Arg 1490
1495 1500 Gln Gly Lys Pro Ile
Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1505 1510
1515 Glu Lys His Tyr Lys Lys Ile Phe Ile Arg
Glu Glu Ser Ser Cys 1520 1525 1530
Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg His
1535 1540 1545 Asp Gly
Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr 1550
1555 1560 Ala Arg Gly Gln Leu Phe Leu
Arg Asn Leu Pro Ile Leu Ala Thr 1565 1570
1575 Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala
Glu Ile Gly 1580 1585 1590
Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 1595
1600 1605 Lys Lys Ile Thr Asn
His Glu Lys Cys His Val Asn Ile Met Asp 1610 1615
1620 Lys Leu Thr Ala Phe Phe Gly Ile Met Pro
Arg Gly Thr Thr Pro 1625 1630 1635
Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg Arg
1640 1645 1650 Gly Leu
Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser 1655
1660 1665 Ser Val Asp His Val Thr Ala
Gly Lys Asp Leu Leu Val Cys Asp 1670 1675
1680 Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn
Asn Lys Met 1685 1690 1695
Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys Pro 1700
1705 1710 Glu Gly Ala Arg Cys
Tyr Val Leu Asn Pro Glu Ala Val Asn Ile 1715 1720
1725 Ser Gly Thr Lys Gly Ala Met Val His Leu
Gln Lys Thr Gly Gly 1730 1735 1740
Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp
1745 1750 1755 Leu Lys
Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala 1760
1765 1770 Ser Ser Gly Arg Val Val Gly
Arg Val Lys Val Gly Lys Asn Glu 1775 1780
1785 Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln
Thr Val Ser 1790 1795 1800
Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr Ser 1805
1810 1815 Met Asn Arg Gly Glu
Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala 1820 1825
1830 Gly Lys Thr Thr Glu Leu Pro Arg Ser Val
Ile Glu Glu Ile Gly 1835 1840 1845
Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala
1850 1855 1860 Glu Ser
Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser 1865
1870 1875 Phe Asn Leu Arg Ile Gly Asp
Met Lys Glu Gly Asp Met Ala Thr 1880 1885
1890 Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln
Leu Pro Gln 1895 1900 1905
Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe Leu 1910
1915 1920 Asp Glu Tyr His Cys
Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly 1925 1930
1935 Lys Ile His Arg Phe Ala Glu Asn Leu Arg
Val Val Ala Met Thr 1940 1945 1950
Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His Pro
1955 1960 1965 Ile Glu
Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu 1970
1975 1980 Gly Ser Glu Tyr Leu Asp Ile
Ala Gly Leu Lys Ile Pro Thr Glu 1985 1990
1995 Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr
Arg Asn Met 2000 2005 2010
Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser 2015
2020 2025 Gly Tyr Tyr Tyr Ser
Gly Glu Asn Pro Glu Asn Leu Arg Val Val 2030 2035
2040 Thr Ser Gln Ser Pro Tyr Val Val Val Ala
Thr Asn Ala Ile Glu 2045 2050 2055
Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly
2060 2065 2070 Leu Lys
Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe 2075
2080 2085 Ile Val Thr Gly Leu Lys Arg
Met Ala Val Thr Ile Gly Glu Gln 2090 2095
2100 Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro
Gly Arg Tyr 2105 2110 2115
Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His Tyr 2120
2125 2130 Asp Leu Leu Gln Ala
Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn 2135 2140
2145 Val Thr Lys Ser Phe Arg Glu Met Asn Tyr
Asp Trp Ser Leu Tyr 2150 2155 2160
Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn Asn
2165 2170 2175 Leu Leu
Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met 2180
2185 2190 Ala Arg Thr Asp His Pro Glu
Pro Ile Gln Leu Ala Tyr Asn Ser 2195 2200
2205 Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile
Lys Asn Gly 2210 2215 2220
Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala Arg 2225
2230 2235 Lys Leu Gly Glu Asp
Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp 2240 2245
2250 Glu Asp Leu Ala Val Asp Leu Leu Gly Met
Asp Trp Pro Asp Pro 2255 2260 2265
Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln Val
2270 2275 2280 Thr Gly
Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe 2285
2290 2295 Gly Tyr Val Gly Tyr Gln Thr
Leu Ser Lys Arg His Ile Pro Met 2300 2305
2310 Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu
Glu Asp Thr 2315 2320 2325
Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly Lys 2330
2335 2340 Asp Ser Glu Leu Lys
Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr 2345 2350
2355 Val Asp Ala Leu Val Asp Tyr Ser Lys Gln
Gly Met Lys Phe Ile 2360 2365 2370
Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys Glu
2375 2380 2385 Gly Leu
Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser 2390
2395 2400 Leu Thr Glu Asn Lys Glu Glu
Ile Ile Arg Tyr Gly Leu Trp Gly 2405 2410
2415 Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg
Leu Gly His 2420 2425 2430
Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe Gly 2435
2440 2445 Gly Glu Thr Val Ser
Ala His Ile Lys Gln Val Ala Val Asp Leu 2450 2455
2460 Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser
Phe Pro Gly Asp Thr 2465 2470 2475
Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe Ile
2480 2485 2490 Ser Ala
Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn 2495
2500 2505 Leu Gln Arg Val Val Glu Pro
Ala Leu Ala Tyr Leu Pro Tyr Ala 2510 2515
2520 Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu
Glu Ser Val 2525 2530 2535
Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg 2540
2545 2550 Lys Gly Lys Ser Asp
Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala 2555 2560
2565 Met Glu Ile Leu Asn Gln Asn Pro Ile Ser
Val Gly Ile Ser Val 2570 2575 2580
Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu Ser
2585 2590 2595 Ser Glu
Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn 2600
2605 2610 Phe Leu Asp Gln Ala Ala Thr
Asp Glu Leu Val Lys Glu Asn Pro 2615 2620
2625 Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln
Thr Ile Gly 2630 2635 2640
Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr Lys 2645
2650 2655 Gly Trp Glu Ala Lys
Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn 2660 2665
2670 Leu Phe Thr Leu Ile Met Phe Glu Ala Phe
Glu Leu Leu Gly Met 2675 2680 2685
Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu
2690 2695 2700 Asp Leu
Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys 2705
2710 2715 Lys Leu Val Leu Gly Trp Ala
Pro Ala Pro Leu Ser Cys Asp Trp 2720 2725
2730 Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn
Asn Tyr Leu 2735 2740 2745
Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala Ile 2750
2755 2760 Lys Asn Val Ala Gly
Lys Leu Thr Lys Val Glu Glu Lys Gly Ser 2765 2770
2775 Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly
Pro Pro Asn Phe Lys 2780 2785 2790
Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro Val
2795 2800 2805 Ala Arg
Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr 2810
2815 2820 Ala Lys Leu Asp Tyr Asn Asn
Gly Lys Val Leu Leu Ala Thr Asn 2825 2830
2835 Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu
Val Lys Lys 2840 2845 2850
His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg Pro 2855
2860 2865 Asp His Gln Asp Leu
Val Asp Arg Asp Cys Ala Thr Ile Thr Lys 2870 2875
2880 Asn Ser Val Gln Phe Leu Lys Met Lys Lys
Gly Cys Ala Phe Thr 2885 2890 2895
Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu Val
2900 2905 2910 His Lys
Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val 2915
2920 2925 Thr Thr Trp Leu Ala Tyr Ser
Phe Val Asn Glu Asp Leu Gly Thr 2930 2935
2940 Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu
Pro Pro Glu 2945 2950 2955
Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu Thr 2960
2965 2970 Ala Ile Thr Ile Thr
Gly Glu Ala Glu Val Met Thr Thr Gly Ile 2975 2980
2985 Thr Pro Val Val Glu Met Lys Glu Glu Pro
Gln Leu Asp His Gln 2990 2995 3000
Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly
3005 3010 3015 Pro Gly
Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys 3020
3025 3030 Asp Asp Arg Pro Phe Val Leu
Ile Ile Gly Asn Lys Gly Ser Thr 3035 3040
3045 Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu
Tyr Lys Gly 3050 3055 3060
Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg Ile 3065
3070 3075 Leu Thr Val Ala Leu
Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu 3080 3085
3090 Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg
Glu Ala Leu Glu Ala 3095 3100 3105
Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala Met
3110 3115 3120 Ile Arg
Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp 3125
3130 3135 Trp Phe Gln Ala Glu Glu Pro
Leu Phe Leu Glu Ala Lys Ile Gln 3140 3145
3150 Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser
Ile Lys Ser 3155 3160 3165
Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val Lys 3170
3175 3180 Glu Val Gly Ala Arg
Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser 3185 3190
3195 Thr Gln Val Thr Lys Lys Gln Met Ser Leu
Ala Pro Leu Phe Glu 3200 3205 3210
Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly
3215 3220 3225 His Met
Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro 3230
3235 3240 Leu Gly Cys Gly Val Tyr Met
Gly Thr Ile Pro Ala Arg Arg Leu 3245 3250
3255 Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys
Glu Leu Val 3260 3265 3270
Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile Arg 3275
3280 3285 Glu His Asn Thr Trp
Ile Leu Arg Lys Val Arg His Glu Gly Asn 3290 3295
3300 Leu Arg Thr Lys Ser Met Ile Asn Pro Gly
Lys Ile Ser Asp Gln 3305 3310 3315
Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile Ile
3320 3325 3330 Gly Ser
Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro 3335
3340 3345 Val Val Arg Ala Gln Thr Asp
Thr Thr Ser Phe His Gln Ala Ile 3350 3355
3360 Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr
Pro Glu Leu 3365 3370 3375
His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro Glu 3380
3385 3390 Leu Lys Glu Ser Tyr
Asp Glu Val Ser Trp Glu Gln Leu Glu Ala 3395 3400
3405 Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr
Leu Glu Ser Lys Asn 3410 3415 3420
Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln Leu
3425 3430 3435 Ile Lys
Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr 3440
3445 3450 Ala Ile Pro Lys Asn Glu Lys
Arg Asp Val Ser Asp Asp Trp Glu 3455 3460
3465 Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val
Ile Gln Tyr 3470 3475 3480
Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr Lys 3485
3490 3495 Trp Val Lys Gln Lys
Pro Val Val Ile Pro Gly Tyr Glu Gly Lys 3500 3505
3510 Thr Pro Leu Phe Asp Ile Phe Asn Lys Val
Lys Lys Glu Trp Asp 3515 3520 3525
Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp
3530 3535 3540 Asp Thr
Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile 3545
3550 3555 Gln Lys Tyr Tyr Phe Lys Arg
Ser Ile His Lys Phe Leu Asp Thr 3560 3565
3570 Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr
Ala Asp Gly 3575 3580 3585
Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp 3590
3595 3600 Thr Ser Ala Gly Asn
Ser Met Leu Asn Val Leu Thr Met Ile Tyr 3605 3610
3615 Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr
Arg Gly Phe Ser Arg 3620 3625 3630
Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr
3635 3640 3645 Glu Arg
Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln Ile 3650
3655 3660 Leu His Glu Ala Gly Lys Pro
Gln Lys Ile Thr Glu Gly Asp Lys 3665 3670
3675 Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe
Cys Ser His 3680 3685 3690
Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr Met 3695
3700 3705 Ala Gly Arg Ser Thr
Ala Thr Ile Leu Ala Lys Met Ala Thr Arg 3710 3715
3720 Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr
Ala Tyr Glu Lys Ala 3725 3730 3735
Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val Val
3740 3745 3750 Arg Arg
Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser 3755
3760 3765 Pro Ser Lys Asn Thr Ile Tyr
Tyr Tyr Gln Gly Asp Pro Ile Ala 3770 3775
3780 Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu
Leu Lys Arg 3785 3790 3795
Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr Thr 3800
3805 3810 Leu Gly Ile Trp Thr
Lys His Thr Ser Lys Arg Leu Ile Gln Ala 3815 3820
3825 Cys Val Glu Ile Gly Lys Arg Glu Gly Thr
Trp Leu Val Asn Ala 3830 3835 3840
Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser
3845 3850 3855 Thr Gly
Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu 3860
3865 3870 Lys Gln Lys Ala Ala Gln Pro
Pro Ile Glu Gly Val Asp Arg Tyr 3875 3880
3885 Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg
Leu Arg Val 3890 3895 3900
Met Leu Met Thr Val Ala Ser Gly Ser Trp 3905 3910
63749PRTArtificial SequenceMutated BVDV XIKE-A-NdN 6Met Glu
Leu Phe Ser Asp Glu Gly Ser Lys Gly Ala Thr Ser Lys Lys 1 5
10 15 Gln Pro Lys Pro Asp Arg Ile
Glu Lys Gly Lys Met Lys Ile Ala Pro 20 25
30 Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro
Asp Ala Thr Ile 35 40 45
Val Val Glu Gly Val Lys Tyr Gln Val Lys Lys Lys Gly Lys Val Arg
50 55 60 Gly Lys Asn
Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro Pro 65
70 75 80 Glu Ser Arg Lys Lys Leu Glu
Lys Ala Leu Leu Ala Trp Ala Ile Leu 85
90 95 Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu
Asn Ile Thr Gln Trp 100 105
110 Asn Leu Met Asp Asn Gly Thr Glu Gly Ile Gln Gln Ala Met Phe
Leu 115 120 125 Arg
Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys 130
135 140 Thr Gly Val Pro Thr His
Leu Ala Thr Asp Tyr Glu Leu Lys Glu Ile 145 150
155 160 Val Gly Met Met Asp Ala Ser Glu Lys Thr Asn
Tyr Thr Cys Cys Arg 165 170
175 Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Phe His
180 185 190 Ile Glu
Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn Asn Leu Thr 195
200 205 Glu Gly Gln Pro Leu Arg Glu
Cys Ala Val Thr Cys Arg Tyr Asp Lys 210 215
220 Glu Thr Glu Leu Asn Ile Val Thr Gln Ala Arg Asp
Arg Pro Thr Thr 225 230 235
240 Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Val Ile
245 250 255 Leu Asp Gly
Pro Cys Asn Phe Lys Val Ser Val Glu Asp Val Leu Phe 260
265 270 Lys Glu His Asp Cys Gly Asn Met
Leu Gln Glu Thr Ala Ile Gln Leu 275 280
285 Leu Asp Gly Ala Thr Asn Thr Ile Glu Gly Ala Arg Val
Gly Thr Ala 290 295 300
Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu Gly Lys Lys 305
310 315 320 Leu Glu Asn Lys
Ser Lys Ala Trp Phe Gly Ala His Ala Ala Ser Pro 325
330 335 Tyr Cys Gly Val Glu Arg Lys Ile Gly
Tyr Val Trp Tyr Thr Lys Asn 340 345
350 Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr Arg Ile Ile Gly
Pro Gly 355 360 365
Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met Gly 370
375 380 Gly His Leu Ser Glu
Phe Val Leu Leu Ser Leu Val Val Leu Ser Asp 385 390
395 400 Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr
Leu Val Leu His Phe Ala 405 410
415 Ile Pro Gln Ser His Val Asp Val Asp Thr Cys Asp Lys Asn Gln
Leu 420 425 430 Asn
Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro Gly Thr Val 435
440 445 Trp Asn Leu Gly Lys Tyr
Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr 450 455
460 Glu Thr Thr Thr Val Phe Val Ile Glu Glu Ala
Gly Gln Val Ile Lys 465 470 475
480 Leu Met Leu Arg Ala Ile Arg Asp Leu Thr Arg Ile Trp Asn Ala Ala
485 490 495 Thr Thr
Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu Arg Gly Gln 500
505 510 Leu Ile Gln Gly Leu Leu Trp
Leu Met Leu Ile Thr Gly Ala Gln Gly 515 520
525 Phe Pro Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile
Ser Lys Asp Arg 530 535 540
Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr Thr Trp His Leu 545
550 555 560 Pro Thr Lys
Lys Ile Val Asp Ser Met Val His Val Trp Cys Glu Gly 565
570 575 Lys Asp Leu Lys Ile Leu Lys Met
Cys Thr Lys Glu Glu Arg Tyr Leu 580 585
590 Val Ala Val His Glu Arg Ala Leu Ser Thr Ser Ala Glu
Phe Met Gln 595 600 605
Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp Met Pro Asp Asp 610
615 620 Phe Glu Phe Gly
Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly 625 630
635 640 Lys Phe Asn Ala Ser Leu Leu Asn Gly
Pro Ala Phe Gln Met Val Cys 645 650
655 Pro Gln Gly Trp Thr Gly Thr Ile Glu Cys Thr Leu Ala Asn
Gln Asp 660 665 670
Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg Thr Thr Pro Phe
675 680 685 Gln Arg Arg Lys
Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile 690
695 700 Tyr Glu Cys Ile Leu Gly Gly Asn
Trp Thr Cys Ile Thr Gly Asp His 705 710
715 720 Ser Arg Leu Lys Asp Gly Pro Ile Lys Lys Cys Lys
Trp Cys Gly His 725 730
735 Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys Cys
740 745 750 Met Leu Ile
Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys 755
760 765 Asp Arg Gly Gly Val Ala Ile Val
Pro Ser Gly Thr Val Lys Cys Arg 770 775
780 Ile Gly Asn Val Thr Val Gln Val Ile Ala Thr Asn Asn
Asp Leu Gly 785 790 795
800 Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu Gly Pro Val
805 810 815 Glu Lys Thr Ala
Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys 820
825 830 Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe
Gln Gln Tyr Met Leu Lys Gly 835 840
845 Glu Trp Gln Tyr Trp Phe Asp Leu Asp Ser Val Asp His His
Lys Asp 850 855 860
Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val Val Ala Leu Leu Gly Gly 865
870 875 880 Lys Tyr Val Leu Trp
Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln 885
890 895 Met Ala Met Gly Ala Gly Val Asn Thr Glu
Glu Ile Val Met Ile Gly 900 905
910 Asn Leu Leu Thr Asp Ser Asp Ile Glu Val Val Val Tyr Phe Leu
Leu 915 920 925 Leu
Tyr Leu Ile Val Lys Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu 930
935 940 Val Tyr His Ile Leu Val
Ala Asn Pro Met Lys Thr Ile Gly Val Val 945 950
955 960 Leu Leu Met Leu Gly Gly Val Val Lys Ala Ser
Arg Ile Asn Ala Asp 965 970
975 Asp Gln Ser Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val
980 985 990 Ala Val
Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile 995
1000 1005 Val Ala Leu Leu Ala
Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala 1010 1015
1020 Ser Ile Asp Val Thr Val Ala Val Val Leu
Ile Val Leu Leu Leu 1025 1030 1035
Ala Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln
1040 1045 1050 Leu Leu
Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu 1055
1060 1065 Lys His Ile Asn Gln Met Glu
Val Pro Glu Ile Ser Met Pro Ser 1070 1075
1080 Trp Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr
Ser Thr Ala 1085 1090 1095
Ile Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln 1100
1105 1110 Trp Ala Pro Ala Val
Ile Met Met Ala Thr Met Trp Ala Asp Phe 1115 1120
1125 Leu Thr Leu Ile Ile Val Leu Pro Ser Tyr
Glu Leu Ser Lys Leu 1130 1135 1140
Tyr Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu
1145 1150 1155 Gly Lys
Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys 1160
1165 1170 Asp Ser Glu Glu Ala Val Tyr
Leu Phe Pro Ser Arg His Lys Ser 1175 1180
1185 Gly Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys
Ala Val Leu 1190 1195 1200
Ile Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr 1205
1210 1215 Leu Ile Leu Glu Ile
Thr Tyr Tyr Met His Arg Lys Ile Ile Asp 1220 1225
1230 Glu Val Ser Gly Gly Ala Asn Phe Leu Ser
Arg Leu Ile Ala Ala 1235 1240 1245
Ile Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly
1250 1255 1260 Leu Lys
Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile 1265
1270 1275 Val Lys His Lys Val Arg Asn
Glu Ala Val His Arg Trp Phe Gly 1280 1285
1290 Glu Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr
Ile Ile Lys 1295 1300 1305
Ala Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile 1310
1315 1320 Cys Glu Gly Lys Glu
Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly 1325 1330
1335 Arg Gln Gly Lys Pro Ile Thr Cys Gly Met
Thr Leu Ala Asp Phe 1340 1345 1350
Glu Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser
1355 1360 1365 Cys Pro
Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg 1370
1375 1380 His Asp Gly Pro Phe Arg Lys
Glu Tyr Lys Gly Tyr Val Gln Tyr 1385 1390
1395 Thr Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro
Ile Leu Ala 1400 1405 1410
Thr Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile 1415
1420 1425 Gly Asp Leu Glu His
Leu Gly Trp Val Leu Arg Gly Pro Ala Val 1430 1435
1440 Cys Lys Lys Ile Thr Asn His Glu Lys Cys
His Val Asn Ile Met 1445 1450 1455
Asp Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr
1460 1465 1470 Pro Arg
Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg 1475
1480 1485 Arg Gly Leu Glu Thr Gly Trp
Ala Tyr Thr His Gln Gly Gly Ile 1490 1495
1500 Ser Ser Val Asp His Val Thr Ala Gly Lys Asp Leu
Leu Val Cys 1505 1510 1515
Asp Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys 1520
1525 1530 Met Thr Asp Glu Thr
Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys 1535 1540
1545 Pro Glu Gly Ala Arg Cys Tyr Val Leu Asn
Pro Glu Ala Val Asn 1550 1555 1560
Ile Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly
1565 1570 1575 Gly Glu
Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe 1580
1585 1590 Asp Leu Lys Asn Leu Lys Gly
Trp Ser Gly Leu Pro Ile Phe Glu 1595 1600
1605 Ala Ser Ser Gly Arg Val Val Gly Arg Val Lys Val
Gly Lys Asn 1610 1615 1620
Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val 1625
1630 1635 Ser Lys Asn Gln Thr
Asp Leu Ala Asp Ile Val Lys Lys Leu Thr 1640 1645
1650 Ser Met Asn Arg Gly Glu Phe Lys Gln Ile
Thr Leu Ala Thr Gly 1655 1660 1665
Ala Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile
1670 1675 1680 Gly Arg
His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala 1685
1690 1695 Ala Glu Ser Val Tyr Gln Tyr
Met Arg Val Lys Tyr Pro Ser Ile 1700 1705
1710 Ser Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly
Asp Met Ala 1715 1720 1725
Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro 1730
1735 1740 Gln Pro Lys Leu Arg
Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe 1745 1750
1755 Leu Asp Glu Tyr His Cys Ala Thr Pro Glu
Gln Leu Ala Ile Ile 1760 1765 1770
Gly Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met
1775 1780 1785 Thr Ala
Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His 1790
1795 1800 Pro Ile Glu Glu Phe Ile Ala
Pro Glu Val Met Lys Gly Glu Asp 1805 1810
1815 Leu Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys
Ile Pro Thr 1820 1825 1830
Glu Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn 1835
1840 1845 Met Ala Val Glu Thr
Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn 1850 1855
1860 Ser Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro
Glu Asn Leu Arg Val 1865 1870 1875
Val Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile
1880 1885 1890 Glu Ser
Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr 1895
1900 1905 Gly Leu Lys Cys Glu Lys Arg
Val Arg Ile Ser Ser Lys Met Pro 1910 1915
1920 Phe Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr
Ile Gly Glu 1925 1930 1935
Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg 1940
1945 1950 Tyr Tyr Arg Ser Gln
Glu Thr Ala Ser Gly Ser Lys Asp Tyr His 1955 1960
1965 Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly
Ile Glu Asp Gly Ile 1970 1975 1980
Asn Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu
1985 1990 1995 Tyr Glu
Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn 2000
2005 2010 Asn Leu Leu Ile Ser Glu Asp
Leu Pro Ala Ala Val Lys Asn Ile 2015 2020
2025 Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu
Ala Tyr Asn 2030 2035 2040
Ser Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn 2045
2050 2055 Gly Glu Val Thr Asp
Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala 2060 2065
2070 Arg Lys Leu Gly Glu Asp Val Pro Ala Tyr
Val Tyr Ala Thr Glu 2075 2080 2085
Asp Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp
2090 2095 2100 Pro Gly
Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln 2105
2110 2115 Val Thr Gly Leu Ser Thr Ala
Glu Asn Ala Leu Leu Ile Ala Leu 2120 2125
2130 Phe Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg
His Ile Pro 2135 2140 2145
Met Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp 2150
2155 2160 Thr Thr His Leu Gln
Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly 2165 2170
2175 Lys Asp Ser Glu Leu Lys Glu Leu Ala Val
Gly Asp Leu Asp Lys 2180 2185 2190
Tyr Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe
2195 2200 2205 Ile Lys
Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys 2210
2215 2220 Glu Gly Leu Gln Thr Ile Lys
Glu Tyr Val Asp Lys Phe Ile Gln 2225 2230
2235 Ser Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr
Gly Leu Trp 2240 2245 2250
Gly Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly 2255
2260 2265 His Glu Thr Ala Phe
Ala Thr Leu Val Val Lys Trp Leu Ala Phe 2270 2275
2280 Gly Gly Glu Thr Val Ser Ala His Ile Lys
Gln Val Ala Val Asp 2285 2290 2295
Leu Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp
2300 2305 2310 Thr Glu
Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe 2315
2320 2325 Ile Ser Ala Leu Ala Thr Tyr
Thr Tyr Lys Thr Trp Asn Tyr Asn 2330 2335
2340 Asn Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr
Leu Pro Tyr 2345 2350 2355
Ala Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser 2360
2365 2370 Val Val Ile Leu Ser
Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile 2375 2380
2385 Arg Lys Gly Lys Ser Asp Gly Leu Leu Gly
Thr Gly Ile Ser Ala 2390 2395 2400
Ala Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser
2405 2410 2415 Val Met
Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu 2420
2425 2430 Ser Ser Glu Gln Lys Arg Thr
Leu Leu Met Lys Val Phe Val Lys 2435 2440
2445 Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val
Lys Glu Asn 2450 2455 2460
Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile 2465
2470 2475 Gly Asn Pro Leu Arg
Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr 2480 2485
2490 Lys Gly Trp Glu Ala Lys Glu Leu Ala Glu
Lys Thr Ala Gly Arg 2495 2500 2505
Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly
2510 2515 2520 Met Asp
Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile 2525
2530 2535 Leu Asp Leu Ile Phe Asn Leu
His Asn Lys Leu Asn Lys Gly Leu 2540 2545
2550 Lys Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu
Ser Cys Asp 2555 2560 2565
Trp Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr 2570
2575 2580 Leu Arg Val Glu Thr
Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala 2585 2590
2595 Ile Lys Asn Val Ala Gly Lys Leu Thr Lys
Val Glu Glu Lys Gly 2600 2605 2610
Ser Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe
2615 2620 2625 Lys Val
Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro 2630
2635 2640 Val Ala Arg Leu Glu Gly Gln
Val Asp Leu Tyr Tyr Lys Gly Val 2645 2650
2655 Thr Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu
Leu Ala Thr 2660 2665 2670
Asn Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys 2675
2680 2685 Lys His Thr Gly Ile
Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg 2690 2695
2700 Pro Asp His Gln Asp Leu Val Asp Arg Asp
Cys Ala Thr Ile Thr 2705 2710 2715
Lys Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe
2720 2725 2730 Thr Tyr
Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu 2735
2740 2745 Val His Lys Asn Asn Leu Gln
Glu Arg Glu Ile Pro Thr Val Thr 2750 2755
2760 Val Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu
Asp Leu Gly 2765 2770 2775
Thr Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro 2780
2785 2790 Glu Glu Leu Ser Leu
Gln Pro Thr Val Arg Leu Val Thr Thr Glu 2795 2800
2805 Thr Ala Ile Thr Ile Thr Gly Glu Ala Glu
Val Met Thr Thr Gly 2810 2815 2820
Ile Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His
2825 2830 2835 Gln Ser
Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro 2840
2845 2850 Gly Pro Gly Val Asn Pro Asn
His Leu Ala Glu Val Ile Asp Glu 2855 2860
2865 Lys Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn
Lys Gly Ser 2870 2875 2880
Thr Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys 2885
2890 2895 Gly Asn Asn Pro Arg
Glu Ile Arg Asp Leu Met Ser Gln Gly Arg 2900 2905
2910 Ile Leu Thr Val Ala Leu Lys Glu Leu Asp
Pro Glu Leu Lys Glu 2915 2920 2925
Leu Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu
2930 2935 2940 Ala Leu
Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala 2945
2950 2955 Met Ile Arg Arg Leu Ile Glu
Pro Glu Val Glu Glu Glu Leu Pro 2960 2965
2970 Asp Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu
Ala Lys Ile 2975 2980 2985
Gln Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys 2990
2995 3000 Ser Lys Ala Lys Glu
Leu Gly Ala Thr Asp Asn Thr Lys Ile Val 3005 3010
3015 Lys Glu Val Gly Ala Arg Thr Tyr Thr Met
Lys Leu Ser Ser Trp 3020 3025 3030
Ser Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe
3035 3040 3045 Glu Glu
Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys 3050
3055 3060 Gly His Met Val Ser Ala Tyr
Gln Leu Ala Gln Gly Asn Trp Glu 3065 3070
3075 Pro Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro
Ala Arg Arg 3080 3085 3090
Leu Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu 3095
3100 3105 Val Glu Val Glu Ser
Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile 3110 3115
3120 Arg Glu His Asn Thr Trp Ile Leu Arg Lys
Val Arg His Glu Gly 3125 3130 3135
Asn Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp
3140 3145 3150 Gln Leu
Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile 3155
3160 3165 Ile Gly Ser Thr Met Ala Ser
Ala Gly Ile Arg Leu Glu Lys Leu 3170 3175
3180 Pro Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe
His Gln Ala 3185 3190 3195
Ile Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu 3200
3205 3210 Leu His Glu Glu Leu
Met Lys Val Phe Asp Cys Leu Lys Ile Pro 3215 3220
3225 Glu Leu Lys Glu Ser Tyr Asp Glu Val Ser
Trp Glu Gln Leu Glu 3230 3235 3240
Ala Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys
3245 3250 3255 Asn Ile
Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln 3260
3265 3270 Leu Ile Lys Asp Leu Arg Lys
Gly Lys Lys Ile Arg Tyr Tyr Glu 3275 3280
3285 Thr Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser
Asp Asp Trp 3290 3295 3300
Glu Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln 3305
3310 3315 Tyr Pro Asp Ala Lys
Val Arg Leu Ala Ile Thr Lys Val Met Tyr 3320 3325
3330 Lys Trp Val Lys Gln Lys Pro Val Val Ile
Pro Gly Tyr Glu Gly 3335 3340 3345
Lys Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp
3350 3355 3360 Asp Ser
Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala 3365
3370 3375 Trp Asp Thr Gln Val Thr Ser
Arg Asp Leu Met Leu Ile Lys Asp 3380 3385
3390 Ile Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys
Phe Leu Asp 3395 3400 3405
Thr Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp 3410
3415 3420 Gly Glu Val Tyr Ile
Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro 3425 3430
3435 Asp Thr Ser Ala Gly Asn Ser Met Leu Asn
Val Leu Thr Met Ile 3440 3445 3450
Tyr Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser
3455 3460 3465 Arg Val
Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile 3470
3475 3480 Thr Glu Arg Gly Leu Gly Leu
Lys Phe Ser Glu Lys Gly Met Gln 3485 3490
3495 Ile Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr
Glu Gly Asp 3500 3505 3510
Lys Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser 3515
3520 3525 His Thr Pro Val Pro
Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr 3530 3535
3540 Met Ala Gly Arg Ser Thr Ala Thr Ile Leu
Ala Lys Met Ala Thr 3545 3550 3555
Arg Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys
3560 3565 3570 Ala Val
Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val 3575
3580 3585 Val Arg Arg Ile Cys Leu Leu
Val Leu Ser Gln Phe Pro Glu Ile 3590 3595
3600 Ser Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly
Asp Pro Ile 3605 3610 3615
Ala Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys 3620
3625 3630 Arg Thr Gly Phe Glu
Lys Leu Ala Gly Leu Asn Leu Ser Met Thr 3635 3640
3645 Thr Leu Gly Ile Trp Thr Lys His Thr Ser
Lys Arg Leu Ile Gln 3650 3655 3660
Ala Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn
3665 3670 3675 Ala Asp
Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro 3680
3685 3690 Ser Thr Gly Val Thr Leu Leu
Gly Lys His Tyr Glu Glu Ile Asn 3695 3700
3705 Leu Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly
Val Asp Arg 3710 3715 3720
Tyr Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg 3725
3730 3735 Val Met Leu Met Thr
Val Ala Ser Gly Ser Trp 3740 3745
73912PRTArtificial SequenceMutated BVDV XIKE-B 7Met Glu Leu Phe Ser Asn
Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys 1 5
10 15 Pro Ala Gly Val Val Glu Pro Val Tyr Asp Val
Asn Gly Arg Pro Leu 20 25
30 Phe Gly Glu Ser Ser Asp Leu His Pro Gln Ser Thr Leu Lys Leu
Pro 35 40 45 His
Gln Arg Gly Ser Ala Asn Ile Leu Thr Asn Ala Arg Ser Leu Pro 50
55 60 Arg Lys Gly Asp Cys Arg
Arg Gly Asn Val Tyr Gly Pro Val Ser Gly 65 70
75 80 Ile Tyr Ile Lys Pro Gly Pro Ile Tyr Tyr Gln
Asp Tyr Val Gly Pro 85 90
95 Val Tyr His Arg Ala Pro Leu Glu Leu Cys Arg Glu Ala Ser Met Cys
100 105 110 Glu Thr
Thr Arg Arg Val Gly Arg Val Thr Gly Ser Asp Gly Lys Leu 115
120 125 Tyr His Ile Tyr Ile Cys Ile
Asp Gly Cys Ile Leu Leu Lys Arg Ala 130 135
140 Thr Arg Asn Gln Pro Glu Val Leu Lys Trp Val Tyr
Asn Arg Leu Asn 145 150 155
160 Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Glu Gly Ser Lys Gly Ala
165 170 175 Thr Ser Lys
Lys Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met 180
185 190 Lys Ile Ala Pro Lys Glu Thr Glu
Lys Asp Cys Lys Thr Arg Pro Pro 195 200
205 Asp Ala Thr Ile Val Val Glu Gly Val Lys Tyr Gln Val
Lys Lys Lys 210 215 220
Gly Lys Val Arg Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys 225
230 235 240 Asn Lys Pro Pro
Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala 245
250 255 Trp Ala Ile Leu Ala Ala Val Leu Leu
Gln Leu Val Thr Gly Glu Asn 260 265
270 Ile Thr Gln Trp Asn Leu Met Asp Asn Gly Thr Glu Gly Ile
Gln Gln 275 280 285
Ala Met Phe Leu Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro 290
295 300 Glu Lys Ile Cys Thr
Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu 305 310
315 320 Leu Lys Glu Ile Val Gly Met Met Asp Ala
Ser Glu Lys Thr Asn Tyr 325 330
335 Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys Gly Trp Cys
Asn 340 345 350 Trp
Phe His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn 355
360 365 Asn Leu Thr Glu Gly Gln
Pro Leu Arg Glu Cys Ala Val Thr Cys Arg 370 375
380 Tyr Asp Lys Glu Thr Glu Leu Asn Ile Val Thr
Gln Ala Arg Asp Arg 385 390 395
400 Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala
405 410 415 Gly Val
Ile Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp 420
425 430 Val Leu Phe Lys Glu His Asp
Cys Gly Asn Met Leu Gln Glu Thr Ala 435 440
445 Ile Gln Leu Leu Asp Gly Ala Thr Asn Thr Ile Glu
Gly Ala Arg Val 450 455 460
Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu 465
470 475 480 Gly Lys Lys
Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala 485
490 495 Ala Ser Pro Tyr Cys Gly Val Glu
Arg Lys Ile Gly Tyr Val Trp Tyr 500 505
510 Thr Lys Asn Cys Thr Pro Ala Cys Leu Pro Arg Asn Thr
Arg Ile Ile 515 520 525
Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His 530
535 540 Glu Met Gly Gly
His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val 545 550
555 560 Leu Ser Asp Phe Ala Pro Glu Thr Ala
Ser Val Ile Tyr Leu Val Leu 565 570
575 His Phe Ala Ile Pro Gln Ser His Val Asp Val Asp Thr Cys
Asp Lys 580 585 590
Asn Gln Leu Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro
595 600 605 Gly Thr Val Trp
Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp 610
615 620 Trp Pro Tyr Glu Thr Thr Thr Val
Phe Val Ile Glu Glu Ala Gly Gln 625 630
635 640 Val Ile Lys Leu Met Leu Arg Ala Ile Arg Asp Leu
Thr Arg Ile Trp 645 650
655 Asn Ala Ala Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu
660 665 670 Arg Gly Gln
Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly 675
680 685 Ala Gln Gly Phe Pro Glu Cys Lys
Glu Gly Phe Gln Tyr Ala Ile Ser 690 695
700 Lys Asp Arg Lys Met Gly Leu Leu Gly Pro Glu Ser Leu
Thr Thr Thr 705 710 715
720 Trp His Leu Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp
725 730 735 Cys Glu Gly Lys
Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu Glu 740
745 750 Arg Tyr Leu Val Ala Val His Glu Arg
Ala Leu Ser Thr Ser Ala Glu 755 760
765 Phe Met Gln Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile
Asp Met 770 775 780
Pro Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val 785
790 795 800 Ile Lys Gly Lys Phe
Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln 805
810 815 Met Val Cys Pro Gln Gly Trp Thr Gly Thr
Ile Glu Cys Thr Leu Ala 820 825
830 Asn Gln Asp Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg
Thr 835 840 845 Thr
Pro Phe Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly 850
855 860 Glu Asp Ile Tyr Glu Cys
Ile Leu Gly Gly Asn Trp Thr Cys Ile Thr 865 870
875 880 Gly Asp His Ser Arg Leu Lys Asp Gly Pro Ile
Lys Lys Cys Lys Trp 885 890
895 Cys Gly His Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile
900 905 910 Gly Lys
Cys Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp 915
920 925 Thr Ser Cys Asp Arg Gly Gly
Val Ala Ile Val Pro Ser Gly Thr Val 930 935
940 Lys Cys Arg Ile Gly Asn Val Thr Val Gln Val Ile
Ala Thr Asn Asn 945 950 955
960 Asp Leu Gly Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu
965 970 975 Gly Pro Val
Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu 980
985 990 Pro Asn Lys Tyr Tyr Glu Pro Arg
Asp Arg Tyr Phe Gln Gln Tyr Met 995 1000
1005 Leu Lys Gly Glu Trp Gln Tyr Trp Phe Asp Leu
Asp Ser Val Asp 1010 1015 1020
His His Lys Asp Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val Val
1025 1030 1035 Ala Leu Leu
Gly Gly Lys Tyr Val Leu Trp Leu Leu Ile Thr Tyr 1040
1045 1050 Thr Ile Leu Ser Glu Gln Met Ala
Met Gly Ala Gly Val Asn Thr 1055 1060
1065 Glu Glu Ile Val Met Ile Gly Asn Leu Leu Thr Asp Ser
Asp Ile 1070 1075 1080
Glu Val Val Val Tyr Phe Leu Leu Leu Tyr Leu Ile Val Lys Glu 1085
1090 1095 Glu Leu Ala Arg Lys
Trp Ile Ile Leu Val Tyr His Ile Leu Val 1100 1105
1110 Ala Asn Pro Met Lys Thr Ile Gly Val Val
Leu Leu Met Leu Gly 1115 1120 1125
Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp Gln Ser Ala
1130 1135 1140 Met Asp
Pro Cys Phe Leu Leu Val Thr Gly Val Val Ala Val Leu 1145
1150 1155 Met Ile Ala Arg Arg Glu Pro
Ala Thr Leu Pro Leu Ile Val Ala 1160 1165
1170 Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro
Ala Ser Ile 1175 1180 1185
Asp Val Thr Val Ala Val Val Leu Ile Val Leu Leu Leu Ala Ser 1190
1195 1200 Tyr Ile Thr Asp Tyr
Phe Arg Tyr Lys Lys Trp Leu Gln Leu Leu 1205 1210
1215 Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile
Arg Ser Leu Lys His 1220 1225 1230
Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp Arg
1235 1240 1245 Pro Leu
Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala Ile Thr 1250
1255 1260 Thr Asn Trp Asp Ile Asp Leu
Ala Gly Phe Leu Leu Gln Trp Ala 1265 1270
1275 Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp
Phe Leu Thr 1280 1285 1290
Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr Phe 1295
1300 1305 Leu Lys Asn Val Arg
Thr Asp Val Glu Lys Asn Trp Leu Gly Lys 1310 1315
1320 Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr
Asp Ile Cys Asp Ser 1325 1330 1335
Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly Ser
1340 1345 1350 Arg Pro
Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu Ile Ser 1355
1360 1365 Cys Ile Ser Ser Gln Trp Gln
Val Val Tyr Ile Ser Tyr Leu Ile 1370 1375
1380 Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile
Asp Glu Val 1385 1390 1395
Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile Ile 1400
1405 1410 Glu Leu Asn Trp Ala
Ile Asp Asp Glu Glu Cys Lys Gly Leu Lys 1415 1420
1425 Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys
Asn Leu Ile Val Lys 1430 1435 1440
His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu Glu
1445 1450 1455 Glu Ile
Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys Ala Ser 1460
1465 1470 Thr Leu Ser Lys Asn Arg His
Cys Ile Ile Cys Thr Ile Cys Glu 1475 1480
1485 Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys
Gly Arg Gln 1490 1495 1500
Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala Asp Phe Glu Glu 1505
1510 1515 Lys His Tyr Lys Lys
Ile Phe Ile Arg Glu Glu Ser Ser Cys Pro 1520 1525
1530 Val Pro Phe Asp Pro Ser Cys His Cys Asn
Tyr Phe Arg His Asp 1535 1540 1545
Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr Ala
1550 1555 1560 Arg Gly
Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala Thr Lys 1565
1570 1575 Met Lys Leu Leu Met Val Gly
Asn Leu Gly Ala Glu Ile Gly Asp 1580 1585
1590 Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala
Val Cys Lys 1595 1600 1605
Lys Ile Thr Asn His Glu Lys Cys His Val Asn Ile Met Asp Lys 1610
1615 1620 Leu Thr Ala Phe Phe
Gly Ile Met Pro Arg Gly Thr Thr Pro Arg 1625 1630
1635 Ala Pro Val Arg Phe Pro Thr Ala Leu Leu
Lys Val Arg Arg Gly 1640 1645 1650
Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser Ser
1655 1660 1665 Val Asp
His Val Thr Ala Gly Lys Asp Leu Leu Val Cys Asp Ser 1670
1675 1680 Met Gly Arg Thr Arg Val Val
Cys His Ser Asn Asn Lys Met Thr 1685 1690
1695 Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly
Cys Pro Glu 1700 1705 1710
Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala Val Asn Ile Ser 1715
1720 1725 Gly Thr Lys Gly Ala
Met Val His Leu Gln Lys Thr Gly Gly Glu 1730 1735
1740 Phe Thr Cys Val Thr Ala Ser Gly Thr Pro
Ala Phe Phe Asp Leu 1745 1750 1755
Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala Ser
1760 1765 1770 Ser Gly
Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu Asp 1775
1780 1785 Ser Lys Pro Thr Lys Leu Met
Ser Gly Ile Gln Thr Val Ser Lys 1790 1795
1800 Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu
Thr Ser Met 1805 1810 1815
Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala Gly 1820
1825 1830 Lys Thr Thr Glu Leu
Pro Arg Ser Val Ile Glu Glu Ile Gly Arg 1835 1840
1845 His Lys Arg Val Leu Val Leu Ile Pro Leu
Arg Ala Ala Ala Glu 1850 1855 1860
Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser Phe
1865 1870 1875 Asn Leu
Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala Thr Gly 1880
1885 1890 Ile Thr Tyr Ala Ser Tyr Gly
Tyr Phe Cys Gln Leu Pro Gln Pro 1895 1900
1905 Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile
Phe Leu Asp 1910 1915 1920
Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly Lys 1925
1930 1935 Ile His Arg Phe Ala
Glu Asn Leu Arg Val Val Ala Met Thr Ala 1940 1945
1950 Thr Pro Ala Gly Thr Val Thr Thr Thr Gly
Gln Lys His Pro Ile 1955 1960 1965
Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu Gly
1970 1975 1980 Ser Glu
Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr Glu Glu 1985
1990 1995 Met Lys Gly Asn Met Leu Val
Phe Ala Pro Thr Arg Asn Met Ala 2000 2005
2010 Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr
Asn Ser Gly 2015 2020 2025
Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu Arg Val Val Thr 2030
2035 2040 Ser Gln Ser Pro Tyr
Val Val Val Ala Thr Asn Ala Ile Glu Ser 2045 2050
2055 Gly Val Thr Leu Pro Asp Leu Asp Thr Val
Val Asp Thr Gly Leu 2060 2065 2070
Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe Ile
2075 2080 2085 Val Thr
Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln Ala 2090
2095 2100 Gln Arg Arg Gly Arg Val Gly
Arg Val Lys Pro Gly Arg Tyr Tyr 2105 2110
2115 Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr
His Tyr Asp 2120 2125 2130
Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn Val 2135
2140 2145 Thr Lys Ser Phe Arg
Glu Met Asn Tyr Asp Trp Ser Leu Tyr Glu 2150 2155
2160 Glu Asp Ser Leu Met Ile Thr Gln Leu Glu
Val Leu Asn Asn Leu 2165 2170 2175
Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met Ala
2180 2185 2190 Arg Thr
Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn Ser Tyr 2195
2200 2205 Glu Asn Gln Ile Pro Val Leu
Phe Pro Lys Ile Lys Asn Gly Glu 2210 2215
2220 Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn
Ala Arg Lys 2225 2230 2235
Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp Glu 2240
2245 2250 Asp Leu Ala Val Asp
Leu Leu Gly Met Asp Trp Pro Asp Pro Gly 2255 2260
2265 Asn Gln Gln Val Val Glu Thr Gly Arg Ala
Leu Lys Gln Val Thr 2270 2275 2280
Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe Gly
2285 2290 2295 Tyr Val
Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro Met Ile 2300
2305 2310 Thr Asp Ile Tyr Thr Leu Glu
Asp His Arg Leu Glu Asp Thr Thr 2315 2320
2325 His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp
Gly Lys Asp 2330 2335 2340
Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr Val 2345
2350 2355 Asp Ala Leu Val Asp
Tyr Ser Lys Gln Gly Met Lys Phe Ile Lys 2360 2365
2370 Val Gln Ala Glu Lys Val Arg Asp Ser Gln
Ser Thr Lys Glu Gly 2375 2380 2385
Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser Leu
2390 2395 2400 Thr Glu
Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp Gly Val 2405
2410 2415 His Thr Ala Leu Tyr Lys Ser
Leu Ala Ala Arg Leu Gly His Glu 2420 2425
2430 Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala
Phe Gly Gly 2435 2440 2445
Glu Thr Val Ser Ala His Ile Lys Gln Val Ala Val Asp Leu Val 2450
2455 2460 Val Tyr Tyr Ile Ile
Asn Lys Pro Ser Phe Pro Gly Asp Thr Glu 2465 2470
2475 Thr Gln Gln Glu Gly Arg Arg Phe Val Ala
Ser Leu Phe Ile Ser 2480 2485 2490
Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn Leu
2495 2500 2505 Gln Arg
Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala Thr 2510
2515 2520 Ser Ala Leu Lys Leu Phe Thr
Pro Thr Arg Leu Glu Ser Val Val 2525 2530
2535 Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser
Ile Arg Lys 2540 2545 2550
Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala Met 2555
2560 2565 Glu Ile Leu Asn Gln
Asn Pro Ile Ser Val Gly Ile Ser Val Met 2570 2575
2580 Leu Gly Val Gly Ala Ile Ala Ala His Asn
Ala Ile Glu Ser Ser 2585 2590 2595
Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn Phe
2600 2605 2610 Leu Asp
Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn Pro Glu 2615
2620 2625 Lys Ile Ile Met Ala Leu Phe
Glu Ala Val Gln Thr Ile Gly Asn 2630 2635
2640 Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr
Tyr Lys Gly 2645 2650 2655
Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn Leu 2660
2665 2670 Phe Thr Leu Ile Met
Phe Glu Ala Phe Glu Leu Leu Gly Met Asp 2675 2680
2685 Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly
Asn Tyr Ile Leu Asp 2690 2695 2700
Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys Lys
2705 2710 2715 Leu Val
Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp Trp Thr 2720
2725 2730 Pro Ser Asp Glu Arg Ile Ser
Leu Pro His Asn Asn Tyr Leu Arg 2735 2740
2745 Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys
Ala Ile Lys 2750 2755 2760
Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu Lys Gly Ser Phe 2765
2770 2775 Leu Cys Arg Asn Arg
Leu Gly Arg Gly Pro Pro Asn Phe Lys Val 2780 2785
2790 Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu
Val Lys Pro Val Ala 2795 2800 2805
Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr Ala
2810 2815 2820 Lys Leu
Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr Asn Lys 2825
2830 2835 Trp Glu Val Asp His Ala Phe
Leu Thr Arg Leu Val Lys Lys His 2840 2845
2850 Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp
Arg Pro Asp 2855 2860 2865
His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr Ile Thr Lys Asn 2870
2875 2880 Ser Val Gln Phe Leu
Lys Met Lys Lys Gly Cys Ala Phe Thr Tyr 2885 2890
2895 Asp Leu Thr Ile Ser Asn Leu Val Arg Leu
Ile Glu Leu Val His 2900 2905 2910
Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val Thr
2915 2920 2925 Thr Trp
Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly Thr Ile 2930
2935 2940 Lys Pro Val Leu Gly Glu Lys
Val Ile Pro Glu Pro Pro Glu Glu 2945 2950
2955 Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr
Glu Thr Ala 2960 2965 2970
Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr Thr Gly Ile Thr 2975
2980 2985 Pro Val Val Glu Met
Lys Glu Glu Pro Gln Leu Asp His Gln Ser 2990 2995
3000 Thr Thr Leu Lys Val Gly Leu Lys Glu Gly
Glu Tyr Pro Gly Pro 3005 3010 3015
Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys Asp
3020 3025 3030 Asp Arg
Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser Thr Ser 3035
3040 3045 Asn Arg Ala Arg Thr Ala Lys
Asn Ile Arg Leu Tyr Lys Gly Asn 3050 3055
3060 Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly
Arg Ile Leu 3065 3070 3075
Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu Val 3080
3085 3090 Asp Tyr Lys Gly Thr
Phe Leu Asn Arg Glu Ala Leu Glu Ala Leu 3095 3100
3105 Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr
Thr Thr Ala Met Ile 3110 3115 3120
Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp Trp
3125 3130 3135 Phe Gln
Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile Gln Asn 3140
3145 3150 Asp Leu Tyr His Leu Ile Gly
Ser Val Asp Ser Ile Lys Ser Lys 3155 3160
3165 Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile
Val Lys Glu 3170 3175 3180
Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser Thr 3185
3190 3195 Gln Val Thr Lys Lys
Gln Met Ser Leu Ala Pro Leu Phe Glu Glu 3200 3205
3210 Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys
Ile Ser Lys Gly His 3215 3220 3225
Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro Leu
3230 3235 3240 Gly Cys
Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg Leu Lys 3245
3250 3255 Ile His Pro Tyr Glu Ala Tyr
Leu Lys Leu Lys Glu Leu Val Glu 3260 3265
3270 Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile
Ile Arg Glu 3275 3280 3285
His Asn Thr Trp Ile Leu Arg Lys Val Arg His Glu Gly Asn Leu 3290
3295 3300 Arg Thr Lys Ser Met
Ile Asn Pro Gly Lys Ile Ser Asp Gln Leu 3305 3310
3315 Cys Arg Asp Gly His Lys Arg Asn Ile Tyr
Asn Lys Ile Ile Gly 3320 3325 3330
Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro Val
3335 3340 3345 Val Arg
Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala Ile Arg 3350
3355 3360 Glu Lys Ile Asp Lys Thr Glu
Asn Lys Gln Thr Pro Glu Leu His 3365 3370
3375 Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile
Pro Glu Leu 3380 3385 3390
Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu Ala Gly 3395
3400 3405 Ile Asn Arg Lys Gly
Ala Ala Gly Tyr Leu Glu Ser Lys Asn Ile 3410 3415
3420 Gly Glu Val Leu Asp Thr Glu Lys His Ile
Val Glu Gln Leu Ile 3425 3430 3435
Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr Ala
3440 3445 3450 Ile Pro
Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Glu Ala 3455
3460 3465 Gly Glu Phe Val Asp Glu Lys
Lys Pro Arg Val Ile Gln Tyr Pro 3470 3475
3480 Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met
Tyr Lys Trp 3485 3490 3495
Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys Thr 3500
3505 3510 Pro Leu Phe Asp Ile
Phe Asn Lys Val Lys Lys Glu Trp Asp Ser 3515 3520
3525 Phe Gln Asp Pro Val Ala Val Ser Phe Asp
Thr Lys Ala Trp Asp 3530 3535 3540
Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile Gln
3545 3550 3555 Lys Tyr
Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp Thr Ile 3560
3565 3570 Thr Glu His Met Val Glu Val
Pro Val Ile Thr Ala Asp Gly Glu 3575 3580
3585 Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln
Pro Asp Thr 3590 3595 3600
Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr Met Ile Tyr Ala 3605
3610 3615 Phe Cys Lys Ser Thr
Gly Ile Pro Tyr Arg Gly Phe Ser Arg Val 3620 3625
3630 Ala Arg Ile His Val Cys Gly Asp Asp Gly
Phe Leu Ile Thr Glu 3635 3640 3645
Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln Ile Leu
3650 3655 3660 His Glu
Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys Met 3665
3670 3675 Lys Val Ala Tyr Arg Phe Glu
Asp Ile Glu Phe Cys Ser His Thr 3680 3685
3690 Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser
Tyr Met Ala 3695 3700 3705
Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met Ala Thr Arg Leu 3710
3715 3720 Asp Ser Ser Gly Glu
Arg Gly Ser Thr Ala Tyr Glu Lys Ala Val 3725 3730
3735 Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp
Asn Pro Val Val Arg 3740 3745 3750
Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser Pro
3755 3760 3765 Ser Lys
Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile Ala Ala 3770
3775 3780 Tyr Arg Glu Val Ile Gly Lys
Gln Leu Cys Glu Leu Lys Arg Thr 3785 3790
3795 Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met
Thr Thr Leu 3800 3805 3810
Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu Ile Gln Ala Cys 3815
3820 3825 Val Glu Ile Gly Lys
Arg Glu Gly Thr Trp Leu Val Asn Ala Asp 3830 3835
3840 Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe
Tyr Ile Pro Ser Thr 3845 3850 3855
Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu Lys
3860 3865 3870 Gln Lys
Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg Tyr Lys 3875
3880 3885 Leu Gly Pro Ile Val Asn Val
Ile Leu Arg Arg Leu Arg Val Met 3890 3895
3900 Leu Met Thr Val Ala Ser Gly Ser Trp 3905
3910 83748PRTArtificial SequenceMutated BVDV
XIKE-B-NdN 8Met Glu Leu Phe Ser Asp Glu Gly Ser Lys Gly Ala Thr Ser Lys
Lys 1 5 10 15 Gln
Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met Lys Ile Ala Pro
20 25 30 Lys Glu Thr Glu Lys
Asp Cys Lys Thr Arg Pro Pro Asp Ala Thr Ile 35
40 45 Val Val Glu Gly Val Lys Tyr Gln Val
Lys Lys Lys Gly Lys Val Arg 50 55
60 Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn
Lys Pro Pro 65 70 75
80 Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Leu
85 90 95 Ala Ala Val Leu
Leu Gln Leu Val Thr Gly Glu Asn Ile Thr Gln Trp 100
105 110 Asn Leu Met Asp Asn Gly Thr Glu Gly
Ile Gln Gln Ala Met Phe Leu 115 120
125 Arg Gly Val Asn Arg Ser Leu His Gly Ile Trp Pro Glu Lys
Ile Cys 130 135 140
Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu Leu Lys Glu Ile 145
150 155 160 Val Gly Met Met Asp
Ala Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg 165
170 175 Leu Gln Arg His Glu Trp Asn Lys Gly Trp
Cys Asn Trp Phe His Ile 180 185
190 Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn Asn Leu Thr
Glu 195 200 205 Gly
Gln Pro Leu Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys Glu 210
215 220 Thr Glu Leu Asn Ile Val
Thr Gln Ala Arg Asp Arg Pro Thr Thr Leu 225 230
235 240 Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe
Ala Gly Val Ile Leu 245 250
255 Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp Val Leu Phe Lys
260 265 270 Glu His
Asp Cys Gly Asn Met Leu Gln Glu Thr Ala Ile Gln Leu Leu 275
280 285 Asp Gly Ala Thr Asn Thr Ile
Glu Gly Ala Arg Val Gly Thr Ala Lys 290 295
300 Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile Leu
Gly Lys Lys Leu 305 310 315
320 Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala Ala Ser Pro Tyr
325 330 335 Cys Gly Val
Glu Arg Lys Ile Gly Tyr Val Trp Tyr Thr Lys Asn Cys 340
345 350 Thr Pro Ala Cys Leu Pro Arg Asn
Thr Arg Ile Ile Gly Pro Gly Lys 355 360
365 Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu
Met Gly Gly 370 375 380
His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val Leu Ser Asp Phe 385
390 395 400 Ala Pro Glu Thr
Ala Ser Val Ile Tyr Leu Val Leu His Phe Ala Ile 405
410 415 Pro Gln Ser His Val Asp Val Asp Thr
Cys Asp Lys Asn Gln Leu Asn 420 425
430 Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro Gly Thr
Val Trp 435 440 445
Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr Glu 450
455 460 Thr Thr Thr Val Phe
Val Ile Glu Glu Ala Gly Gln Val Ile Lys Leu 465 470
475 480 Met Leu Arg Ala Ile Arg Asp Leu Thr Arg
Ile Trp Asn Ala Ala Thr 485 490
495 Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu Arg Gly Gln
Leu 500 505 510 Ile
Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly Ala Gln Gly Phe 515
520 525 Pro Glu Cys Lys Glu Gly
Phe Gln Tyr Ala Ile Ser Lys Asp Arg Lys 530 535
540 Met Gly Leu Leu Gly Pro Glu Ser Leu Thr Thr
Thr Trp His Leu Pro 545 550 555
560 Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp Cys Glu Gly Lys
565 570 575 Asp Leu
Lys Ile Leu Lys Met Cys Thr Lys Glu Glu Arg Tyr Leu Val 580
585 590 Ala Val His Glu Arg Ala Leu
Ser Thr Ser Ala Glu Phe Met Gln Ile 595 600
605 Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp Met
Pro Asp Asp Phe 610 615 620
Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly Lys 625
630 635 640 Phe Asn Ala
Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys Pro 645
650 655 Gln Gly Trp Thr Gly Thr Ile Glu
Cys Thr Leu Ala Asn Gln Asp Thr 660 665
670 Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg Thr Thr
Pro Phe Gln 675 680 685
Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile Tyr 690
695 700 Glu Cys Ile Leu
Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His Ser 705 710
715 720 Arg Leu Lys Asp Gly Pro Ile Lys Lys
Cys Lys Trp Cys Gly His Asp 725 730
735 Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly Lys
Cys Met 740 745 750
Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys Asp
755 760 765 Arg Gly Gly Val
Ala Ile Val Pro Ser Gly Thr Val Lys Cys Arg Ile 770
775 780 Gly Asn Val Thr Val Gln Val Ile
Ala Thr Asn Asn Asp Leu Gly Pro 785 790
795 800 Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser Glu
Gly Pro Val Glu 805 810
815 Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys Tyr
820 825 830 Tyr Glu Pro
Arg Asp Arg Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu 835
840 845 Trp Gln Tyr Trp Phe Asp Leu Asp
Ser Val Asp His His Lys Asp Tyr 850 855
860 Phe Ser Glu Phe Ile Ile Ile Ala Val Val Ala Leu Leu
Gly Gly Lys 865 870 875
880 Tyr Val Leu Trp Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln Met
885 890 895 Ala Met Gly Ala
Gly Val Asn Thr Glu Glu Ile Val Met Ile Gly Asn 900
905 910 Leu Leu Thr Asp Ser Asp Ile Glu Val
Val Val Tyr Phe Leu Leu Leu 915 920
925 Tyr Leu Ile Val Lys Glu Glu Leu Ala Arg Lys Trp Ile Ile
Leu Val 930 935 940
Tyr His Ile Leu Val Ala Asn Pro Met Lys Thr Ile Gly Val Val Leu 945
950 955 960 Leu Met Leu Gly Gly
Val Val Lys Ala Ser Arg Ile Asn Ala Asp Asp 965
970 975 Gln Ser Ala Met Asp Pro Cys Phe Leu Leu
Val Thr Gly Val Val Ala 980 985
990 Val Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu
Ile Val 995 1000 1005
Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala Ser 1010
1015 1020 Ile Asp Val Thr Val
Ala Val Val Leu Ile Val Leu Leu Leu Ala 1025 1030
1035 Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys
Lys Trp Leu Gln Leu 1040 1045 1050
Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys
1055 1060 1065 His Ile
Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser Trp 1070
1075 1080 Arg Pro Leu Ala Leu Val Leu
Phe Tyr Ile Thr Ser Thr Ala Ile 1085 1090
1095 Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu
Leu Gln Trp 1100 1105 1110
Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe Leu 1115
1120 1125 Thr Leu Ile Ile Val
Leu Pro Ser Tyr Glu Leu Ser Lys Leu Tyr 1130 1135
1140 Phe Leu Lys Asn Val Arg Thr Asp Val Glu
Lys Asn Trp Leu Gly 1145 1150 1155
Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp
1160 1165 1170 Ser Glu
Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser Gly 1175
1180 1185 Ser Arg Pro Asp Phe Ile Leu
Pro Phe Leu Lys Ala Val Leu Ile 1190 1195
1200 Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr Ile
Ser Tyr Leu 1205 1210 1215
Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp Glu 1220
1225 1230 Val Ser Gly Gly Ala
Asn Phe Leu Ser Arg Leu Ile Ala Ala Ile 1235 1240
1245 Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu
Glu Cys Lys Gly Leu 1250 1255 1260
Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile Val
1265 1270 1275 Lys His
Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly Glu 1280
1285 1290 Glu Glu Ile Tyr Gly Ala Pro
Lys Val Ile Thr Ile Ile Lys Ala 1295 1300
1305 Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile Cys
Thr Ile Cys 1310 1315 1320
Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly Arg 1325
1330 1335 Gln Gly Lys Pro Ile
Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1340 1345
1350 Glu Lys His Tyr Lys Lys Ile Phe Ile Arg
Glu Glu Ser Ser Cys 1355 1360 1365
Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg His
1370 1375 1380 Asp Gly
Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr Thr 1385
1390 1395 Ala Arg Gly Gln Leu Phe Leu
Arg Asn Leu Pro Ile Leu Ala Thr 1400 1405
1410 Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly Ala
Glu Ile Gly 1415 1420 1425
Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val Cys 1430
1435 1440 Lys Lys Ile Thr Asn
His Glu Lys Cys His Val Asn Ile Met Asp 1445 1450
1455 Lys Leu Thr Ala Phe Phe Gly Ile Met Pro
Arg Gly Thr Thr Pro 1460 1465 1470
Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg Arg
1475 1480 1485 Gly Leu
Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile Ser 1490
1495 1500 Ser Val Asp His Val Thr Ala
Gly Lys Asp Leu Leu Val Cys Asp 1505 1510
1515 Ser Met Gly Arg Thr Arg Val Val Cys His Ser Asn
Asn Lys Met 1520 1525 1530
Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys Pro 1535
1540 1545 Glu Gly Ala Arg Cys
Tyr Val Leu Asn Pro Glu Ala Val Asn Ile 1550 1555
1560 Ser Gly Thr Lys Gly Ala Met Val His Leu
Gln Lys Thr Gly Gly 1565 1570 1575
Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp
1580 1585 1590 Leu Lys
Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala 1595
1600 1605 Ser Ser Gly Arg Val Val Gly
Arg Val Lys Val Gly Lys Asn Glu 1610 1615
1620 Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile Gln
Thr Val Ser 1625 1630 1635
Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr Ser 1640
1645 1650 Met Asn Arg Gly Glu
Phe Lys Gln Ile Thr Leu Ala Thr Gly Ala 1655 1660
1665 Gly Lys Thr Thr Glu Leu Pro Arg Ser Val
Ile Glu Glu Ile Gly 1670 1675 1680
Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala Ala
1685 1690 1695 Glu Ser
Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile Ser 1700
1705 1710 Phe Asn Leu Arg Ile Gly Asp
Met Lys Glu Gly Asp Met Ala Thr 1715 1720
1725 Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln
Leu Pro Gln 1730 1735 1740
Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe Leu 1745
1750 1755 Asp Glu Tyr His Cys
Ala Thr Pro Glu Gln Leu Ala Ile Ile Gly 1760 1765
1770 Lys Ile His Arg Phe Ala Glu Asn Leu Arg
Val Val Ala Met Thr 1775 1780 1785
Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His Pro
1790 1795 1800 Ile Glu
Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp Leu 1805
1810 1815 Gly Ser Glu Tyr Leu Asp Ile
Ala Gly Leu Lys Ile Pro Thr Glu 1820 1825
1830 Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro Thr
Arg Asn Met 1835 1840 1845
Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser 1850
1855 1860 Gly Tyr Tyr Tyr Ser
Gly Glu Asn Pro Glu Asn Leu Arg Val Val 1865 1870
1875 Thr Ser Gln Ser Pro Tyr Val Val Val Ala
Thr Asn Ala Ile Glu 1880 1885 1890
Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr Gly
1895 1900 1905 Leu Lys
Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro Phe 1910
1915 1920 Ile Val Thr Gly Leu Lys Arg
Met Ala Val Thr Ile Gly Glu Gln 1925 1930
1935 Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys Pro
Gly Arg Tyr 1940 1945 1950
Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His Tyr 1955
1960 1965 Asp Leu Leu Gln Ala
Gln Arg Tyr Gly Ile Glu Asp Gly Ile Asn 1970 1975
1980 Val Thr Lys Ser Phe Arg Glu Met Asn Tyr
Asp Trp Ser Leu Tyr 1985 1990 1995
Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn Asn
2000 2005 2010 Leu Leu
Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile Met 2015
2020 2025 Ala Arg Thr Asp His Pro Glu
Pro Ile Gln Leu Ala Tyr Asn Ser 2030 2035
2040 Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys Ile
Lys Asn Gly 2045 2050 2055
Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala Arg 2060
2065 2070 Lys Leu Gly Glu Asp
Val Pro Ala Tyr Val Tyr Ala Thr Glu Asp 2075 2080
2085 Glu Asp Leu Ala Val Asp Leu Leu Gly Met
Asp Trp Pro Asp Pro 2090 2095 2100
Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln Val
2105 2110 2115 Thr Gly
Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu Phe 2120
2125 2130 Gly Tyr Val Gly Tyr Gln Thr
Leu Ser Lys Arg His Ile Pro Met 2135 2140
2145 Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg Leu
Glu Asp Thr 2150 2155 2160
Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly Lys 2165
2170 2175 Asp Ser Glu Leu Lys
Glu Leu Ala Val Gly Asp Leu Asp Lys Tyr 2180 2185
2190 Val Asp Ala Leu Val Asp Tyr Ser Lys Gln
Gly Met Lys Phe Ile 2195 2200 2205
Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys Glu
2210 2215 2220 Gly Leu
Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln Ser 2225
2230 2235 Leu Thr Glu Asn Lys Glu Glu
Ile Ile Arg Tyr Gly Leu Trp Gly 2240 2245
2250 Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg
Leu Gly His 2255 2260 2265
Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe Gly 2270
2275 2280 Gly Glu Thr Val Ser
Ala His Ile Lys Gln Val Ala Val Asp Leu 2285 2290
2295 Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser
Phe Pro Gly Asp Thr 2300 2305 2310
Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe Ile
2315 2320 2325 Ser Ala
Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn Asn 2330
2335 2340 Leu Gln Arg Val Val Glu Pro
Ala Leu Ala Tyr Leu Pro Tyr Ala 2345 2350
2355 Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu
Glu Ser Val 2360 2365 2370
Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile Arg 2375
2380 2385 Lys Gly Lys Ser Asp
Gly Leu Leu Gly Thr Gly Ile Ser Ala Ala 2390 2395
2400 Met Glu Ile Leu Asn Gln Asn Pro Ile Ser
Val Gly Ile Ser Val 2405 2410 2415
Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu Ser
2420 2425 2430 Ser Glu
Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys Asn 2435
2440 2445 Phe Leu Asp Gln Ala Ala Thr
Asp Glu Leu Val Lys Glu Asn Pro 2450 2455
2460 Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val Gln
Thr Ile Gly 2465 2470 2475
Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr Lys 2480
2485 2490 Gly Trp Glu Ala Lys
Glu Leu Ala Glu Lys Thr Ala Gly Arg Asn 2495 2500
2505 Leu Phe Thr Leu Ile Met Phe Glu Ala Phe
Glu Leu Leu Gly Met 2510 2515 2520
Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu
2525 2530 2535 Asp Leu
Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu Lys 2540
2545 2550 Lys Leu Val Leu Gly Trp Ala
Pro Ala Pro Leu Ser Cys Asp Trp 2555 2560
2565 Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His Asn
Asn Tyr Leu 2570 2575 2580
Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala Ile 2585
2590 2595 Lys Asn Val Ala Gly
Lys Leu Thr Lys Val Glu Glu Lys Gly Ser 2600 2605
2610 Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly
Pro Pro Asn Phe Lys 2615 2620 2625
Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro Val
2630 2635 2640 Ala Arg
Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val Thr 2645
2650 2655 Ala Lys Leu Asp Tyr Asn Asn
Gly Lys Val Leu Leu Ala Thr Asn 2660 2665
2670 Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg Leu
Val Lys Lys 2675 2680 2685
His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg Pro 2690
2695 2700 Asp His Gln Asp Leu
Val Asp Arg Asp Cys Ala Thr Ile Thr Lys 2705 2710
2715 Asn Ser Val Gln Phe Leu Lys Met Lys Lys
Gly Cys Ala Phe Thr 2720 2725 2730
Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu Val
2735 2740 2745 His Lys
Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr Val 2750
2755 2760 Thr Thr Trp Leu Ala Tyr Ser
Phe Val Asn Glu Asp Leu Gly Thr 2765 2770
2775 Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro Glu
Pro Pro Glu 2780 2785 2790
Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu Thr 2795
2800 2805 Ala Ile Thr Ile Thr
Gly Glu Ala Glu Val Met Thr Thr Gly Ile 2810 2815
2820 Thr Pro Val Val Glu Met Lys Glu Glu Pro
Gln Leu Asp His Gln 2825 2830 2835
Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly
2840 2845 2850 Pro Gly
Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu Lys 2855
2860 2865 Asp Asp Arg Pro Phe Val Leu
Ile Ile Gly Asn Lys Gly Ser Thr 2870 2875
2880 Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu
Tyr Lys Gly 2885 2890 2895
Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg Ile 2900
2905 2910 Leu Thr Val Ala Leu
Lys Glu Leu Asp Pro Glu Leu Lys Glu Leu 2915 2920
2925 Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg
Glu Ala Leu Glu Ala 2930 2935 2940
Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala Met
2945 2950 2955 Ile Arg
Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro Asp 2960
2965 2970 Trp Phe Gln Ala Glu Glu Pro
Leu Phe Leu Glu Ala Lys Ile Gln 2975 2980
2985 Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp Ser
Ile Lys Ser 2990 2995 3000
Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val Lys 3005
3010 3015 Glu Val Gly Ala Arg
Thr Tyr Thr Met Lys Leu Ser Ser Trp Ser 3020 3025
3030 Thr Gln Val Thr Lys Lys Gln Met Ser Leu
Ala Pro Leu Phe Glu 3035 3040 3045
Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly
3050 3055 3060 His Met
Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu Pro 3065
3070 3075 Leu Gly Cys Gly Val Tyr Met
Gly Thr Ile Pro Ala Arg Arg Leu 3080 3085
3090 Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys
Glu Leu Val 3095 3100 3105
Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile Arg 3110
3115 3120 Glu His Asn Thr Trp
Ile Leu Arg Lys Val Arg His Glu Gly Asn 3125 3130
3135 Leu Arg Thr Lys Ser Met Ile Asn Pro Gly
Lys Ile Ser Asp Gln 3140 3145 3150
Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile Ile
3155 3160 3165 Gly Ser
Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu Pro 3170
3175 3180 Val Val Arg Ala Gln Thr Asp
Thr Thr Ser Phe His Gln Ala Ile 3185 3190
3195 Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr
Pro Glu Leu 3200 3205 3210
His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro Glu 3215
3220 3225 Leu Lys Glu Ser Tyr
Asp Glu Val Ser Trp Glu Gln Leu Glu Ala 3230 3235
3240 Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr
Leu Glu Ser Lys Asn 3245 3250 3255
Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln Leu
3260 3265 3270 Ile Lys
Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu Thr 3275
3280 3285 Ala Ile Pro Lys Asn Glu Lys
Arg Asp Val Ser Asp Asp Trp Glu 3290 3295
3300 Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg Val
Ile Gln Tyr 3305 3310 3315
Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr Lys 3320
3325 3330 Trp Val Lys Gln Lys
Pro Val Val Ile Pro Gly Tyr Glu Gly Lys 3335 3340
3345 Thr Pro Leu Phe Asp Ile Phe Asn Lys Val
Lys Lys Glu Trp Asp 3350 3355 3360
Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala Trp
3365 3370 3375 Asp Thr
Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp Ile 3380
3385 3390 Gln Lys Tyr Tyr Phe Lys Arg
Ser Ile His Lys Phe Leu Asp Thr 3395 3400
3405 Ile Thr Glu His Met Val Glu Val Pro Val Ile Thr
Ala Asp Gly 3410 3415 3420
Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro Asp 3425
3430 3435 Thr Ser Ala Gly Asn
Ser Met Leu Asn Val Leu Thr Met Ile Tyr 3440 3445
3450 Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr
Arg Gly Phe Ser Arg 3455 3460 3465
Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile Thr
3470 3475 3480 Glu Arg
Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln Ile 3485
3490 3495 Leu His Glu Ala Gly Lys Pro
Gln Lys Ile Thr Glu Gly Asp Lys 3500 3505
3510 Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe
Cys Ser His 3515 3520 3525
Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr Met 3530
3535 3540 Ala Gly Arg Ser Thr
Ala Thr Ile Leu Ala Lys Met Ala Thr Arg 3545 3550
3555 Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr
Ala Tyr Glu Lys Ala 3560 3565 3570
Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val Val
3575 3580 3585 Arg Arg
Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile Ser 3590
3595 3600 Pro Ser Lys Asn Thr Ile Tyr
Tyr Tyr Gln Gly Asp Pro Ile Ala 3605 3610
3615 Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys Glu
Leu Lys Arg 3620 3625 3630
Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr Thr 3635
3640 3645 Leu Gly Ile Trp Thr
Lys His Thr Ser Lys Arg Leu Ile Gln Ala 3650 3655
3660 Cys Val Glu Ile Gly Lys Arg Glu Gly Thr
Trp Leu Val Asn Ala 3665 3670 3675
Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser
3680 3685 3690 Thr Gly
Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn Leu 3695
3700 3705 Lys Gln Lys Ala Ala Gln Pro
Pro Ile Glu Gly Val Asp Arg Tyr 3710 3715
3720 Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg Arg
Leu Arg Val 3725 3730 3735
Met Leu Met Thr Val Ala Ser Gly Ser Trp 3740 3745
93913PRTArtificial SequenceXIKE-C BVDV-Sequence 9Met Glu Leu
Phe Ser Asn Glu Leu Leu Tyr Lys Thr Tyr Lys Gln Lys 1 5
10 15 Pro Ala Gly Val Val Glu Pro Val
Tyr Asp Val Asn Gly Arg Pro Leu 20 25
30 Phe Gly Glu Ser Ser Asp Leu His Pro Gln Ser Thr Leu
Lys Leu Pro 35 40 45
His Gln Arg Gly Ser Ala Asn Ile Leu Thr Asn Ala Arg Ser Leu Pro 50
55 60 Arg Lys Gly Asp
Cys Arg Arg Gly Asn Val Tyr Gly Pro Val Ser Gly 65 70
75 80 Ile Tyr Ile Lys Pro Gly Pro Ile Tyr
Tyr Gln Asp Tyr Val Gly Pro 85 90
95 Val Tyr His Arg Ala Pro Leu Glu Leu Cys Arg Glu Ala Ser
Met Cys 100 105 110
Glu Thr Thr Arg Arg Val Gly Arg Val Thr Gly Ser Asp Gly Lys Leu
115 120 125 Tyr His Ile Tyr
Ile Cys Ile Asp Gly Cys Ile Leu Leu Lys Arg Ala 130
135 140 Thr Arg Asn Gln Pro Glu Val Leu
Lys Trp Val Tyr Asn Arg Leu Asn 145 150
155 160 Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Glu Gly
Ser Lys Gly Ala 165 170
175 Thr Ser Lys Lys Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met
180 185 190 Lys Ile Ala
Pro Lys Glu Thr Glu Lys Asp Cys Lys Thr Arg Pro Pro 195
200 205 Asp Ala Thr Ile Val Val Glu Gly
Val Lys Tyr Gln Val Lys Lys Lys 210 215
220 Gly Lys Val Arg Gly Lys Asn Thr Gln Asp Gly Leu Tyr
His Asn Lys 225 230 235
240 Asn Lys Pro Pro Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala
245 250 255 Trp Ala Ile Leu
Ala Ala Val Leu Leu Gln Leu Val Thr Gly Glu Asn 260
265 270 Ile Thr Gln Trp Asn Leu Met Asp Asn
Gly Thr Glu Gly Ile Gln Gln 275 280
285 Ala Met Phe Leu Arg Gly Val Asn Arg Ser Leu Leu Gly Ile
Trp Pro 290 295 300
Glu Lys Ile Cys Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu 305
310 315 320 Leu Lys Glu Ile Val
Gly Met Met Asp Ala Ser Glu Lys Thr Asn Tyr 325
330 335 Thr Cys Cys Arg Leu Gln Arg His Glu Trp
Asn Lys His Gly Trp Cys 340 345
350 Asn Trp Phe His Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr
Gln 355 360 365 Asn
Asn Leu Thr Glu Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys 370
375 380 Arg Tyr Asp Lys Glu Thr
Glu Leu Asn Ile Val Thr Gln Ala Arg Asp 385 390
395 400 Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly
Lys Asn Phe Ser Phe 405 410
415 Ala Gly Val Ile Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu
420 425 430 Asp Val
Leu Phe Lys Glu His Asp Cys Gly Asn Met Leu Gln Glu Thr 435
440 445 Ala Ile Gln Leu Leu Asp Gly
Ala Thr Asn Thr Ile Glu Gly Ala Arg 450 455
460 Val Gly Thr Ala Lys Leu Thr Thr Trp Leu Gly Lys
Gln Leu Gly Ile 465 470 475
480 Leu Gly Lys Lys Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His
485 490 495 Ala Ala Ser
Pro Tyr Cys Gly Val Glu Arg Lys Ile Gly Tyr Val Trp 500
505 510 Tyr Thr Lys Asn Cys Thr Pro Ala
Cys Leu Pro Arg Asn Thr Arg Ile 515 520
525 Ile Gly Pro Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly
Lys Ile Leu 530 535 540
His Glu Met Gly Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val 545
550 555 560 Val Leu Ser Asp
Phe Ala Pro Glu Thr Ala Ser Val Ile Tyr Leu Val 565
570 575 Leu His Phe Ala Ile Pro Gln Ser His
Val Asp Val Asp Thr Cys Asp 580 585
590 Lys Asn Gln Leu Asn Leu Thr Val Ala Thr Thr Val Ala Glu
Val Ile 595 600 605
Pro Gly Thr Val Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp 610
615 620 Trp Trp Pro Tyr Glu
Thr Thr Thr Val Phe Val Ile Glu Glu Ala Gly 625 630
635 640 Gln Val Ile Lys Leu Met Leu Arg Ala Ile
Arg Asp Leu Thr Arg Ile 645 650
655 Trp Asn Ala Ala Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys
Ala 660 665 670 Leu
Arg Gly Gln Leu Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr 675
680 685 Gly Ala Gln Gly Phe Pro
Glu Cys Lys Glu Gly Phe Gln Tyr Ala Ile 690 695
700 Ser Lys Asp Arg Lys Met Gly Leu Leu Gly Pro
Glu Ser Leu Thr Thr 705 710 715
720 Thr Trp His Leu Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val
725 730 735 Trp Cys
Glu Gly Lys Asp Leu Lys Ile Leu Lys Met Cys Thr Lys Glu 740
745 750 Glu Arg Tyr Leu Val Ala Val
His Glu Arg Ala Leu Ser Thr Ser Ala 755 760
765 Glu Phe Met Gln Ile Ser Asp Gly Thr Ile Gly Pro
Asp Val Ile Asp 770 775 780
Met Pro Asp Asp Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro 785
790 795 800 Val Ile Lys
Gly Lys Phe Asn Ala Ser Leu Leu Asn Gly Pro Ala Phe 805
810 815 Gln Met Val Cys Pro Gln Gly Trp
Thr Gly Thr Ile Glu Cys Thr Leu 820 825
830 Ala Asn Gln Asp Thr Leu Asp Thr Thr Val Ile Arg Thr
Tyr Arg Arg 835 840 845
Thr Thr Pro Phe Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile 850
855 860 Gly Glu Asp Ile
Tyr Glu Cys Ile Leu Gly Gly Asn Trp Thr Cys Ile 865 870
875 880 Thr Gly Asp His Ser Arg Leu Lys Asp
Gly Pro Ile Lys Lys Cys Lys 885 890
895 Trp Cys Gly His Asp Phe Val Asn Ser Glu Gly Leu Pro His
Tyr Pro 900 905 910
Ile Gly Lys Cys Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp
915 920 925 Asp Thr Ser Cys
Asp Arg Gly Gly Val Ala Ile Val Pro Ser Gly Thr 930
935 940 Val Lys Cys Arg Ile Gly Asn Val
Thr Val Gln Val Ile Ala Thr Asn 945 950
955 960 Asn Asp Leu Gly Pro Met Pro Cys Ser Pro Ala Glu
Val Ile Ala Ser 965 970
975 Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr
980 985 990 Leu Pro Asn
Lys Tyr Tyr Glu Pro Arg Asp Arg Tyr Phe Gln Gln Tyr 995
1000 1005 Met Leu Lys Gly Glu Trp
Gln Tyr Trp Phe Asp Leu Asp Ser Val 1010 1015
1020 Asp His His Lys Asp Tyr Phe Ser Glu Phe Ile
Ile Ile Ala Val 1025 1030 1035
Val Ala Leu Leu Gly Gly Lys Tyr Val Leu Trp Leu Leu Ile Thr
1040 1045 1050 Tyr Thr Ile
Leu Ser Glu Gln Met Ala Met Gly Ala Gly Val Asn 1055
1060 1065 Thr Glu Glu Ile Val Met Ile Gly
Asn Leu Leu Thr Asp Ser Asp 1070 1075
1080 Ile Glu Val Val Val Tyr Phe Leu Leu Leu Tyr Leu Ile
Val Lys 1085 1090 1095
Glu Glu Leu Ala Arg Lys Trp Ile Ile Leu Val Tyr His Ile Leu 1100
1105 1110 Val Ala Asn Pro Met
Lys Thr Ile Gly Val Val Leu Leu Met Leu 1115 1120
1125 Gly Gly Val Val Lys Ala Ser Arg Ile Asn
Ala Asp Asp Gln Ser 1130 1135 1140
Ala Met Asp Pro Cys Phe Leu Leu Val Thr Gly Val Val Ala Val
1145 1150 1155 Leu Met
Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro Leu Ile Val 1160
1165 1170 Ala Leu Leu Ala Ile Arg Thr
Ser Gly Phe Leu Leu Pro Ala Ser 1175 1180
1185 Ile Asp Val Thr Val Ala Val Val Leu Ile Val Leu
Leu Leu Ala 1190 1195 1200
Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr Lys Lys Trp Leu Gln Leu 1205
1210 1215 Leu Phe Ser Leu Ile
Ala Gly Ile Phe Ile Ile Arg Ser Leu Lys 1220 1225
1230 His Ile Asn Gln Met Glu Val Pro Glu Ile
Ser Met Pro Ser Trp 1235 1240 1245
Arg Pro Leu Ala Leu Val Leu Phe Tyr Ile Thr Ser Thr Ala Ile
1250 1255 1260 Thr Thr
Asn Trp Asp Ile Asp Leu Ala Gly Phe Leu Leu Gln Trp 1265
1270 1275 Ala Pro Ala Val Ile Met Met
Ala Thr Met Trp Ala Asp Phe Leu 1280 1285
1290 Thr Leu Ile Ile Val Leu Pro Ser Tyr Glu Leu Ser
Lys Leu Tyr 1295 1300 1305
Phe Leu Lys Asn Val Arg Thr Asp Val Glu Lys Asn Trp Leu Gly 1310
1315 1320 Lys Val Lys Tyr Arg
Gln Ile Ser Ser Val Tyr Asp Ile Cys Asp 1325 1330
1335 Ser Glu Glu Ala Val Tyr Leu Phe Pro Ser
Arg His Lys Ser Gly 1340 1345 1350
Ser Arg Pro Asp Phe Ile Leu Pro Phe Leu Lys Ala Val Leu Ile
1355 1360 1365 Ser Cys
Ile Ser Ser Gln Trp Gln Val Val Tyr Ile Ser Tyr Leu 1370
1375 1380 Ile Leu Glu Ile Thr Tyr Tyr
Met His Arg Lys Ile Ile Asp Glu 1385 1390
1395 Val Ser Gly Gly Ala Asn Phe Leu Ser Arg Leu Ile
Ala Ala Ile 1400 1405 1410
Ile Glu Leu Asn Trp Ala Ile Asp Asp Glu Glu Cys Lys Gly Leu 1415
1420 1425 Lys Lys Leu Tyr Leu
Leu Ser Gly Arg Ala Lys Asn Leu Ile Val 1430 1435
1440 Lys His Lys Val Arg Asn Glu Ala Val His
Arg Trp Phe Gly Glu 1445 1450 1455
Glu Glu Ile Tyr Gly Ala Pro Lys Val Ile Thr Ile Ile Lys Ala
1460 1465 1470 Ser Thr
Leu Ser Lys Asn Arg His Cys Ile Ile Cys Thr Ile Cys 1475
1480 1485 Glu Gly Lys Glu Trp Asn Gly
Ala Asn Cys Pro Lys Cys Gly Arg 1490 1495
1500 Gln Gly Lys Pro Ile Thr Cys Gly Met Thr Leu Ala
Asp Phe Glu 1505 1510 1515
Glu Lys His Tyr Lys Lys Ile Phe Ile Arg Glu Glu Ser Ser Cys 1520
1525 1530 Pro Val Pro Phe Asp
Pro Ser Cys His Cys Asn Tyr Phe Arg His 1535 1540
1545 Asp Gly Pro Phe Arg Lys Glu Tyr Lys Gly
Tyr Val Gln Tyr Thr 1550 1555 1560
Ala Arg Gly Gln Leu Phe Leu Arg Asn Leu Pro Ile Leu Ala Thr
1565 1570 1575 Lys Met
Lys Leu Leu Met Val Gly Asn Leu Gly Ala Glu Ile Gly 1580
1585 1590 Asp Leu Glu His Leu Gly Trp
Val Leu Arg Gly Pro Ala Val Cys 1595 1600
1605 Lys Lys Ile Thr Asn His Glu Lys Cys His Val Asn
Ile Met Asp 1610 1615 1620
Lys Leu Thr Ala Phe Phe Gly Ile Met Pro Arg Gly Thr Thr Pro 1625
1630 1635 Arg Ala Pro Val Arg
Phe Pro Thr Ala Leu Leu Lys Val Arg Arg 1640 1645
1650 Gly Leu Glu Thr Gly Trp Ala Tyr Thr His
Gln Gly Gly Ile Ser 1655 1660 1665
Ser Val Asp His Val Thr Ala Gly Lys Asp Leu Leu Val Cys Asp
1670 1675 1680 Ser Met
Gly Arg Thr Arg Val Val Cys His Ser Asn Asn Lys Met 1685
1690 1695 Thr Asp Glu Thr Glu Tyr Gly
Ile Lys Thr Asp Ser Gly Cys Pro 1700 1705
1710 Glu Gly Ala Arg Cys Tyr Val Leu Asn Pro Glu Ala
Val Asn Ile 1715 1720 1725
Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly Gly 1730
1735 1740 Glu Phe Thr Cys Val
Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp 1745 1750
1755 Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu
Pro Ile Phe Glu Ala 1760 1765 1770
Ser Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu
1775 1780 1785 Asp Ser
Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser 1790
1795 1800 Lys Asn Gln Thr Asp Leu Ala
Asp Ile Val Lys Lys Leu Thr Ser 1805 1810
1815 Met Asn Arg Gly Glu Phe Lys Gln Ile Thr Leu Ala
Thr Gly Ala 1820 1825 1830
Gly Lys Thr Thr Glu Leu Pro Arg Ser Val Ile Glu Glu Ile Gly 1835
1840 1845 Arg His Lys Arg Val
Leu Val Leu Ile Pro Leu Arg Ala Ala Ala 1850 1855
1860 Glu Ser Val Tyr Gln Tyr Met Arg Val Lys
Tyr Pro Ser Ile Ser 1865 1870 1875
Phe Asn Leu Arg Ile Gly Asp Met Lys Glu Gly Asp Met Ala Thr
1880 1885 1890 Gly Ile
Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys Gln Leu Pro Gln 1895
1900 1905 Pro Lys Leu Arg Ala Ala Met
Val Glu Tyr Ser Tyr Ile Phe Leu 1910 1915
1920 Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu Ala
Ile Ile Gly 1925 1930 1935
Lys Ile His Arg Phe Ala Glu Asn Leu Arg Val Val Ala Met Thr 1940
1945 1950 Ala Thr Pro Ala Gly
Thr Val Thr Thr Thr Gly Gln Lys His Pro 1955 1960
1965 Ile Glu Glu Phe Ile Ala Pro Glu Val Met
Lys Gly Glu Asp Leu 1970 1975 1980
Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Thr Glu
1985 1990 1995 Glu Met
Lys Gly Asn Met Leu Val Phe Ala Pro Thr Arg Asn Met 2000
2005 2010 Ala Val Glu Thr Ala Lys Lys
Leu Lys Ala Lys Gly Tyr Asn Ser 2015 2020
2025 Gly Tyr Tyr Tyr Ser Gly Glu Asn Pro Glu Asn Leu
Arg Val Val 2030 2035 2040
Thr Ser Gln Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu 2045
2050 2055 Ser Gly Val Thr Leu
Pro Asp Leu Asp Thr Val Val Asp Thr Gly 2060 2065
2070 Leu Lys Cys Glu Lys Arg Val Arg Ile Ser
Ser Lys Met Pro Phe 2075 2080 2085
Ile Val Thr Gly Leu Lys Arg Met Ala Val Thr Ile Gly Glu Gln
2090 2095 2100 Ala Gln
Arg Arg Gly Arg Val Gly Arg Val Lys Pro Gly Arg Tyr 2105
2110 2115 Tyr Arg Ser Gln Glu Thr Ala
Ser Gly Ser Lys Asp Tyr His Tyr 2120 2125
2130 Asp Leu Leu Gln Ala Gln Arg Tyr Gly Ile Glu Asp
Gly Ile Asn 2135 2140 2145
Val Thr Lys Ser Phe Arg Glu Met Asn Tyr Asp Trp Ser Leu Tyr 2150
2155 2160 Glu Glu Asp Ser Leu
Met Ile Thr Gln Leu Glu Val Leu Asn Asn 2165 2170
2175 Leu Leu Ile Ser Glu Asp Leu Pro Ala Ala
Val Lys Asn Ile Met 2180 2185 2190
Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr Asn Ser
2195 2200 2205 Tyr Glu
Asn Gln Ile Pro Val Leu Phe Pro Lys Ile Lys Asn Gly 2210
2215 2220 Glu Val Thr Asp Ser Tyr Glu
Asn Tyr Thr Tyr Leu Asn Ala Arg 2225 2230
2235 Lys Leu Gly Glu Asp Val Pro Ala Tyr Val Tyr Ala
Thr Glu Asp 2240 2245 2250
Glu Asp Leu Ala Val Asp Leu Leu Gly Met Asp Trp Pro Asp Pro 2255
2260 2265 Gly Asn Gln Gln Val
Val Glu Thr Gly Arg Ala Leu Lys Gln Val 2270 2275
2280 Thr Gly Leu Ser Thr Ala Glu Asn Ala Leu
Leu Ile Ala Leu Phe 2285 2290 2295
Gly Tyr Val Gly Tyr Gln Thr Leu Ser Lys Arg His Ile Pro Met
2300 2305 2310 Ile Thr
Asp Ile Tyr Thr Leu Glu Asp His Arg Leu Glu Asp Thr 2315
2320 2325 Thr His Leu Gln Phe Ala Pro
Asn Ala Ile Arg Thr Asp Gly Lys 2330 2335
2340 Asp Ser Glu Leu Lys Glu Leu Ala Val Gly Asp Leu
Asp Lys Tyr 2345 2350 2355
Val Asp Ala Leu Val Asp Tyr Ser Lys Gln Gly Met Lys Phe Ile 2360
2365 2370 Lys Val Gln Ala Glu
Lys Val Arg Asp Ser Gln Ser Thr Lys Glu 2375 2380
2385 Gly Leu Gln Thr Ile Lys Glu Tyr Val Asp
Lys Phe Ile Gln Ser 2390 2395 2400
Leu Thr Glu Asn Lys Glu Glu Ile Ile Arg Tyr Gly Leu Trp Gly
2405 2410 2415 Val His
Thr Ala Leu Tyr Lys Ser Leu Ala Ala Arg Leu Gly His 2420
2425 2430 Glu Thr Ala Phe Ala Thr Leu
Val Val Lys Trp Leu Ala Phe Gly 2435 2440
2445 Gly Glu Thr Val Ser Ala His Ile Lys Gln Val Ala
Val Asp Leu 2450 2455 2460
Val Val Tyr Tyr Ile Ile Asn Lys Pro Ser Phe Pro Gly Asp Thr 2465
2470 2475 Glu Thr Gln Gln Glu
Gly Arg Arg Phe Val Ala Ser Leu Phe Ile 2480 2485
2490 Ser Ala Leu Ala Thr Tyr Thr Tyr Lys Thr
Trp Asn Tyr Asn Asn 2495 2500 2505
Leu Gln Arg Val Val Glu Pro Ala Leu Ala Tyr Leu Pro Tyr Ala
2510 2515 2520 Thr Ser
Ala Leu Lys Leu Phe Thr Pro Thr Arg Leu Glu Ser Val 2525
2530 2535 Val Ile Leu Ser Ser Thr Ile
Tyr Lys Thr Tyr Leu Ser Ile Arg 2540 2545
2550 Lys Gly Lys Ser Asp Gly Leu Leu Gly Thr Gly Ile
Ser Ala Ala 2555 2560 2565
Met Glu Ile Leu Asn Gln Asn Pro Ile Ser Val Gly Ile Ser Val 2570
2575 2580 Met Leu Gly Val Gly
Ala Ile Ala Ala His Asn Ala Ile Glu Ser 2585 2590
2595 Ser Glu Gln Lys Arg Thr Leu Leu Met Lys
Val Phe Val Lys Asn 2600 2605 2610
Phe Leu Asp Gln Ala Ala Thr Asp Glu Leu Val Lys Glu Asn Pro
2615 2620 2625 Glu Lys
Ile Ile Met Ala Leu Phe Glu Ala Val Gln Thr Ile Gly 2630
2635 2640 Asn Pro Leu Arg Leu Ile Tyr
His Leu Tyr Gly Val Tyr Tyr Lys 2645 2650
2655 Gly Trp Glu Ala Lys Glu Leu Ala Glu Lys Thr Ala
Gly Arg Asn 2660 2665 2670
Leu Phe Thr Leu Ile Met Phe Glu Ala Phe Glu Leu Leu Gly Met 2675
2680 2685 Asp Ser Glu Gly Lys
Ile Arg Asn Leu Ser Gly Asn Tyr Ile Leu 2690 2695
2700 Asp Leu Ile Phe Asn Leu His Asn Lys Leu
Asn Lys Gly Leu Lys 2705 2710 2715
Lys Leu Val Leu Gly Trp Ala Pro Ala Pro Leu Ser Cys Asp Trp
2720 2725 2730 Thr Pro
Ser Asp Glu Arg Ile Ser Leu Pro His Asn Asn Tyr Leu 2735
2740 2745 Arg Val Glu Thr Arg Cys Pro
Cys Gly Tyr Glu Met Lys Ala Ile 2750 2755
2760 Lys Asn Val Ala Gly Lys Leu Thr Lys Val Glu Glu
Lys Gly Ser 2765 2770 2775
Phe Leu Cys Arg Asn Arg Leu Gly Arg Gly Pro Pro Asn Phe Lys 2780
2785 2790 Val Thr Lys Phe Tyr
Asp Asp Asn Leu Ile Glu Val Lys Pro Val 2795 2800
2805 Ala Arg Leu Glu Gly Gln Val Asp Leu Tyr
Tyr Lys Gly Val Thr 2810 2815 2820
Ala Lys Leu Asp Tyr Asn Asn Gly Lys Val Leu Leu Ala Thr Asn
2825 2830 2835 Lys Trp
Glu Val Asp His Ala Phe Leu Thr Arg Leu Val Lys Lys 2840
2845 2850 His Thr Gly Ile Gly Phe Lys
Gly Ala Tyr Leu Gly Asp Arg Pro 2855 2860
2865 Asp His Gln Asp Leu Val Asp Arg Asp Cys Ala Thr
Ile Thr Lys 2870 2875 2880
Asn Ser Val Gln Phe Leu Lys Met Lys Lys Gly Cys Ala Phe Thr 2885
2890 2895 Tyr Asp Leu Thr Ile
Ser Asn Leu Val Arg Leu Ile Glu Leu Val 2900 2905
2910 His Lys Asn Asn Leu Gln Glu Arg Glu Ile
Pro Thr Val Thr Val 2915 2920 2925
Thr Thr Trp Leu Ala Tyr Ser Phe Val Asn Glu Asp Leu Gly Thr
2930 2935 2940 Ile Lys
Pro Val Leu Gly Glu Lys Val Ile Pro Glu Pro Pro Glu 2945
2950 2955 Glu Leu Ser Leu Gln Pro Thr
Val Arg Leu Val Thr Thr Glu Thr 2960 2965
2970 Ala Ile Thr Ile Thr Gly Glu Ala Glu Val Met Thr
Thr Gly Ile 2975 2980 2985
Thr Pro Val Val Glu Met Lys Glu Glu Pro Gln Leu Asp His Gln 2990
2995 3000 Ser Thr Thr Leu Lys
Val Gly Leu Lys Glu Gly Glu Tyr Pro Gly 3005 3010
3015 Pro Gly Val Asn Pro Asn His Leu Ala Glu
Val Ile Asp Glu Lys 3020 3025 3030
Asp Asp Arg Pro Phe Val Leu Ile Ile Gly Asn Lys Gly Ser Thr
3035 3040 3045 Ser Asn
Arg Ala Arg Thr Ala Lys Asn Ile Arg Leu Tyr Lys Gly 3050
3055 3060 Asn Asn Pro Arg Glu Ile Arg
Asp Leu Met Ser Gln Gly Arg Ile 3065 3070
3075 Leu Thr Val Ala Leu Lys Glu Leu Asp Pro Glu Leu
Lys Glu Leu 3080 3085 3090
Val Asp Tyr Lys Gly Thr Phe Leu Asn Arg Glu Ala Leu Glu Ala 3095
3100 3105 Leu Ser Leu Gly Lys
Pro Ile Lys Arg Lys Thr Thr Thr Ala Met 3110 3115
3120 Ile Arg Arg Leu Ile Glu Pro Glu Val Glu
Glu Glu Leu Pro Asp 3125 3130 3135
Trp Phe Gln Ala Glu Glu Pro Leu Phe Leu Glu Ala Lys Ile Gln
3140 3145 3150 Asn Asp
Leu Tyr His Leu Ile Gly Ser Val Asp Ser Ile Lys Ser 3155
3160 3165 Lys Ala Lys Glu Leu Gly Ala
Thr Asp Asn Thr Lys Ile Val Lys 3170 3175
3180 Glu Val Gly Ala Arg Thr Tyr Thr Met Lys Leu Ser
Ser Trp Ser 3185 3190 3195
Thr Gln Val Thr Lys Lys Gln Met Ser Leu Ala Pro Leu Phe Glu 3200
3205 3210 Glu Leu Leu Leu Lys
Cys Pro Pro Cys Ser Lys Ile Ser Lys Gly 3215 3220
3225 His Met Val Ser Ala Tyr Gln Leu Ala Gln
Gly Asn Trp Glu Pro 3230 3235 3240
Leu Gly Cys Gly Val Tyr Met Gly Thr Ile Pro Ala Arg Arg Leu
3245 3250 3255 Lys Ile
His Pro Tyr Glu Ala Tyr Leu Lys Leu Lys Glu Leu Val 3260
3265 3270 Glu Val Glu Ser Ser Arg Ala
Thr Ala Lys Glu Ser Ile Ile Arg 3275 3280
3285 Glu His Asn Thr Trp Ile Leu Arg Lys Val Arg His
Glu Gly Asn 3290 3295 3300
Leu Arg Thr Lys Ser Met Ile Asn Pro Gly Lys Ile Ser Asp Gln 3305
3310 3315 Leu Cys Arg Asp Gly
His Lys Arg Asn Ile Tyr Asn Lys Ile Ile 3320 3325
3330 Gly Ser Thr Met Ala Ser Ala Gly Ile Arg
Leu Glu Lys Leu Pro 3335 3340 3345
Val Val Arg Ala Gln Thr Asp Thr Thr Ser Phe His Gln Ala Ile
3350 3355 3360 Arg Glu
Lys Ile Asp Lys Thr Glu Asn Lys Gln Thr Pro Glu Leu 3365
3370 3375 His Glu Glu Leu Met Lys Val
Phe Asp Cys Leu Lys Ile Pro Glu 3380 3385
3390 Leu Lys Glu Ser Tyr Asp Glu Val Ser Trp Glu Gln
Leu Glu Ala 3395 3400 3405
Gly Ile Asn Arg Lys Gly Ala Ala Gly Tyr Leu Glu Ser Lys Asn 3410
3415 3420 Ile Gly Glu Val Leu
Asp Thr Glu Lys His Ile Val Glu Gln Leu 3425 3430
3435 Ile Lys Asp Leu Arg Lys Gly Lys Lys Ile
Arg Tyr Tyr Glu Thr 3440 3445 3450
Ala Ile Pro Lys Asn Glu Lys Arg Asp Val Ser Asp Asp Trp Glu
3455 3460 3465 Ala Gly
Glu Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr 3470
3475 3480 Pro Asp Ala Lys Val Arg Leu
Ala Ile Thr Lys Val Met Tyr Lys 3485 3490
3495 Trp Val Lys Gln Lys Pro Val Val Ile Pro Gly Tyr
Glu Gly Lys 3500 3505 3510
Thr Pro Leu Phe Asp Ile Phe Asn Lys Val Lys Lys Glu Trp Asp 3515
3520 3525 Ser Phe Gln Asp Pro
Val Ala Val Ser Phe Asp Thr Lys Ala Trp 3530 3535
3540 Asp Thr Gln Val Thr Ser Arg Asp Leu Met
Leu Ile Lys Asp Ile 3545 3550 3555
Gln Lys Tyr Tyr Phe Lys Arg Ser Ile His Lys Phe Leu Asp Thr
3560 3565 3570 Ile Thr
Glu His Met Val Glu Val Pro Val Ile Thr Ala Asp Gly 3575
3580 3585 Glu Val Tyr Ile Arg Asn Gly
Gln Arg Gly Ser Gly Gln Pro Asp 3590 3595
3600 Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu Thr
Met Ile Tyr 3605 3610 3615
Ala Phe Cys Lys Ser Thr Gly Ile Pro Tyr Arg Gly Phe Ser Arg 3620
3625 3630 Val Ala Arg Ile His
Val Cys Gly Asp Asp Gly Phe Leu Ile Thr 3635 3640
3645 Glu Arg Gly Leu Gly Leu Lys Phe Ser Glu
Lys Gly Met Gln Ile 3650 3655 3660
Leu His Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys
3665 3670 3675 Met Lys
Val Ala Tyr Arg Phe Glu Asp Ile Glu Phe Cys Ser His 3680
3685 3690 Thr Pro Val Pro Val Arg Trp
Ala Asp Asn Thr Ser Ser Tyr Met 3695 3700
3705 Ala Gly Arg Ser Thr Ala Thr Ile Leu Ala Lys Met
Ala Thr Arg 3710 3715 3720
Leu Asp Ser Ser Gly Glu Arg Gly Ser Thr Ala Tyr Glu Lys Ala 3725
3730 3735 Val Ala Phe Ser Phe
Leu Leu Met Tyr Ser Trp Asn Pro Val Val 3740 3745
3750 Arg Arg Ile Cys Leu Leu Val Leu Ser Gln
Phe Pro Glu Ile Ser 3755 3760 3765
Pro Ser Lys Asn Thr Ile Tyr Tyr Tyr Gln Gly Asp Pro Ile Ala
3770 3775 3780 Ala Tyr
Arg Glu Val Ile Gly Lys Gln Leu Cys Glu Leu Lys Arg 3785
3790 3795 Thr Gly Phe Glu Lys Leu Ala
Gly Leu Asn Leu Ser Met Thr Thr 3800 3805
3810 Leu Gly Ile Trp Thr Lys His Thr Ser Lys Arg Leu
Ile Gln Ala 3815 3820 3825
Cys Val Glu Ile Gly Lys Arg Glu Gly Thr Trp Leu Val Asn Ala 3830
3835 3840 Asp Arg Leu Ile Ala
Gly Lys Thr Gly Lys Phe Tyr Ile Pro Ser 3845 3850
3855 Thr Gly Val Thr Leu Leu Gly Lys His Tyr
Glu Glu Ile Asn Leu 3860 3865 3870
Lys Gln Lys Ala Ala Gln Pro Pro Ile Glu Gly Val Asp Arg Tyr
3875 3880 3885 Lys Leu
Gly Pro Ile Val Asn Val Ile Leu Arg Arg Leu Arg Val 3890
3895 3900 Met Leu Met Thr Val Ala Ser
Gly Ser Trp 3905 3910
1012332DNAArtificial SequenceXIKE-C BVDV-Sequence 10gtatacgaga ttagctaaag
aactcgtata tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc
tcagcgaagg ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac
tagcggtagc agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg
gttcgacact ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg
gcacatctta acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac
acagcctgat agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat
ggcacatgga gttgttttca aatgaacttt tatacaaaac 420atataaacaa aaaccagcag
gcgtcgtgga acctgtttac gacgtcaacg ggcgcccact 480gtttggagag agcagtgact
tgcacccgca gtcaacacta aaactaccac accaacgagg 540cagcgccaac atcctgacca
atgctaggtc cctaccgcgg aaaggtgact gccggagagg 600taatgtgtat ggaccggtga
gtggcatcta tatcaaacca ggaccgatct actaccagga 660ttatgtgggc cccgtctatc
atagagcccc actggaacta tgtagggagg caagtatgtg 720cgaaacaact aggagagttg
gcagagtgac cggtagtgat gggaaattat atcatatcta 780catctgcata gatgggtgta
tcctcctgaa gagggcgact aggaaccaac cagaagtcct 840gaaatgggta tacaacagat
taaattgtcc tttatgggtc accagctgct ccgatgaagg 900gagcaagggt gctacaagta
agaagcagcc taagccagat aggatagaaa aaggtaagat 960gaaaatagcc ccaaaagaga
cagaaaaaga ttgcaaaacc agaccccccg acgcgactat 1020agtagtagaa ggggttaagt
accaggtgaa gaaaaaagga aaggtaaggg gaaaaaatac 1080tcaagatggg ttatatcaca
acaagaataa gccccctgaa tcaagaaaaa aattggaaaa 1140ggcactgctg gcttgggcca
tcttagcagc ggtcctgctt cagctggtaa caggagagaa 1200tatcacccag tggaacttga
tggacaacgg caccgaggga atacagcaag cgatgttcct 1260aagaggggtg aacaggagtc
tattaggaat ttggccagag aaaatttgca ccggagtacc 1320aactcactta gcaacagact
atgagcttaa agagatagtg gggatgatgg acgcgagtga 1380gaagaccaac tacacgtgtt
gcaggttgca aagacatgag tggaataaac atggttggtg 1440taactggttt catatagaac
cgtggatatg gttgatgaac aaaacccaaa acaacctgac 1500agaagggcaa ccgcttaggg
agtgtgctgt gacttgtagg tatgacaagg aaacagaatt 1560gaacatcgtg acacaggcta
gggacagacc tacaactctg acaggttgca agaaaggcaa 1620gaatttctct ttcgcaggtg
ttatactgga tgggccctgt aactttaaag tatcggttga 1680agatgtgctg ttcaaggagc
acgattgcgg caacatgctg caagagaccg cgatacagct 1740actcgatggg gcaaccaaca
ccattgaggg agcaagggta gggacggcca agttgacaac 1800ctggttaggg aagcaattag
ggatccttgg taagaagttg gagaacaaaa gcaaagcatg 1860gtttggtgca catgcagcaa
gtccatactg cggagtggag aggaagatcg gttacgtatg 1920gtatacaaaa aactgcactc
cagcttgcct tccaagaaac actagaataa taggccccgg 1980gaaatttgat accaacgccg
aagatggaaa aatactccat gagatggggg ggcacctctc 2040agaatttgtc ctattgtcct
tggtggttct gtctgacttt gccccggaaa ccgcgagcgt 2100catctacttg gttctacatt
ttgcgatccc gcaaagccac gttgatgtag acacatgcga 2160caagaaccag ctgaatttaa
cggtagcaac cacagtagca gaggtcatac cagggacagt 2220gtggaaccta gggaagtatg
tctgcataag accagactgg tggccatatg agacgacgac 2280agtcttcgtc atagaggaag
cagggcaagt aatcaaattg atgctaaggg ccatcagaga 2340cttaactagg atatggaatg
ctgccactac cacagctttc ttaatctttt tagtaaaagc 2400actgagggga caactaatcc
aagggctatt gtggctgatg ctaataacag gagcacaggg 2460cttccctgaa tgcaaagagg
gcttccaata tgccatatct aaagacagga aaatggggtt 2520attggggcca gagagcttaa
ctacaacatg gcacctcccc accaaaaaaa tagtggattc 2580catggtgcat gtatggtgtg
aaggaaaaga cttgaaaata ttaaaaatgt gcacaaagga 2640agagaggtat ctagtggctg
tgcacgagag agccttatca accagtgccg agtttatgca 2700gatcagtgat gggacaatag
gcccagacgt gatagatatg cctgatgact ttgagtttgg 2760actctgccct tgtgactcaa
aaccagtgat aaagggcaaa tttaatgcca gcttactgaa 2820tggaccagct ttccagatgg
tatgcccaca ggggtggact ggtacaatag aatgcaccct 2880agcgaaccaa gacaccttgg
acacaactgt cattaggaca tatagaagaa ctaccccatt 2940tcagcggaga aaatggtgta
cctatgaaaa aataataggg gaagatatct atgaatgcat 3000tctaggtgga aactggacat
gcataaccgg tgaccatagc aggttgaaag acggacctat 3060caagaagtgt aagtggtgtg
gccatgactt cgtcaactca gaggggctac cacactaccc 3120aataggcaag tgcatgctca
tcaacgagag tgggtacagg tatgtagatg acacctcttg 3180cgataggggt ggtgtagcca
tagttccatc tggcaccgta aagtgtagaa taggtaacgt 3240cacggtgcaa gttatcgcta
ctaacaatga tctgggaccc atgccttgca gcccagctga 3300agtgatagca agtgaaggac
cagtggaaaa gactgcatgc acattcaact attcaaggac 3360tctacctaat aagtattatg
agccaaggga ccggtacttc caacaataca tgttaaaagg 3420ggagtggcaa tattggttcg
acctggattc tgtagaccac cacaaagact acttctcaga 3480gttcataatc atagcagtgg
tcgccttgtt gggtggtaag tacgtactgt ggctcttgat 3540aacatacaca atactgtctg
agcagatggc tatgggtgct ggagtgaata ctgaagagat 3600agtcatgata ggcaatttgc
tgacagacag tgatattgag gttgtggttt atttccttct 3660tctgtactta atagttaaag
aggaactggc gaggaaatgg attatactgg tataccacat 3720ccttgtagcc aaccctatga
aaacaattgg ggtcgtctta ctaatgctag ggggagtggt 3780gaaggccagc agaatcaatg
ctgatgacca aagtgctatg gacccatgct ttcttctcgt 3840gacaggcgta gtggctgttt
tgatgatcgc tagaagagaa cctgccacat taccactgat 3900tgtagcattg ctagcaataa
gaacatcagg attcctactg cccgctagca ttgatgtaac 3960tgtagcagta gtattaattg
tacttttgtt ggctagctac ataacagact actttagata 4020taaaaagtgg cttcaactct
tatttagtct gatagctggt atctttatta taaggagctt 4080aaaacatatc aaccagatgg
aggtaccaga aatatctatg ccaagttgga gacctctagc 4140tctggtcctt ttctatataa
catctacagc aataaccact aattgggaca ttgacttagc 4200aggcttcctg ctgcaatggg
cgccagcagt gatcatgatg gctaccatgt gggcagactt 4260tttgactctg atcatagtcc
tgcccagtta cgagttatct aagctttact tcctaaagaa 4320cgtcaggaca gacgtggaaa
agaactggct cggcaaagtg aaatacagac agatcagttc 4380agtttatgac atctgtgaca
gtgaggaagc agtgtaccta tttccatcaa ggcataagag 4440tggaagcagg ccagatttca
tattaccttt tttgaaagcc gtgttaataa gctgcatcag 4500cagccaatgg caagtggttt
acatttctta cctaatactg gaaattacat actatatgca 4560caggaaaatc atagatgagg
tgtcaggagg agcaaatttt ctatcaagac tcatagcagc 4620catcatagaa ttaaattggg
ccatagatga tgaggaatgt aaaggactga agaaactgta 4680tctcttgtca gggagagcga
agaatttgat agttaaacat aaggtaagaa atgaagccgt 4740ccacagatgg tttggtgagg
aggaaatata cggggcaccc aaggtgatca ctatcataaa 4800agctagtacc ctaagtaaaa
acaggcactg cataatctgc acgatctgtg aagggaaaga 4860atggaatgga gccaactgcc
caaagtgtgg aagacaagga aagcccataa catgtggaat 4920gacactcgca gactttgagg
agaaacatta caaaaagata tttataagag aagaatcttc 4980ttgtcctgtg ccttttgatc
cttcttgcca ttgtaattat tttcgccacg atgggccttt 5040caggaaagag tataagggtt
acgtccaata cacagccaga ggacaactct ttctgaggaa 5100cctaccaatt ctagcgacga
agatgaagct attaatggtg ggaaacctcg gcgcagaaat 5160tggcgacctg gaacatctag
gatgggtact gagagggcca gccgtgtgca aaaaaattac 5220caaccatgag aagtgccacg
taaacatcat ggataagcta actgcatttt ttggaatcat 5280gcctagaggc acgaccccta
gggcacctgt gaggttcccc acagcactac taaaagtgag 5340aagggggcta gagacgggat
gggcttacac gcaccaagga gggatcagct cggtagacca 5400tgtcacagcc ggaaaggatt
tactagtgtg tgacagtatg ggcaggacca gggttgtctg 5460tcatagtaac aataagatga
ctgatgagac tgagtatggc atcaagaccg actcagggtg 5520tcccgaaggt gcgaggtgtt
acgtgctaaa cccagaagct gttaacattt ctggcacaaa 5580aggagctatg gtacacctcc
agaaaacggg gggggagttc acatgtgtca ctgcctcagg 5640gaccccggct ttcttcgatc
tgaaaaatct aaaaggctgg tccgggctac caatttttga 5700agcatccagt ggcagggtgg
ttggtagggt gaaagtcggc aagaatgagg attccaagcc 5760caccaaacta atgagcggaa
tccagacagt gtctaagaac cagacagacc tagcggacat 5820cgtaaaaaaa ttgactagta
tgaacagagg agagttcaaa cagataacat tagccactgg 5880ggcaggaaaa actacggaac
tgccaaggtc cgtcatagag gagataggga ggcacaaaag 5940ggtcttagtc ctgataccat
tgagagcagc agcagagtca gtgtatcagt atatgagagt 6000gaagtaccca agtatatctt
tcaatttgag aataggagat atgaaggaag gtgacatggc 6060cactggtatc acctacgcct
catatgggta cttttgtcag cttcctcagc ccaaactgag 6120agctgccatg gtagagtact
catatatatt cttagatgag taccactgtg ctacacccga 6180gcaattagca ataattggaa
agatacacag gtttgctgaa aatcttagag tggtagcaat 6240gacagcaacc ccagctggaa
cggtcacaac gactggtcag aaacacccta tagaggagtt 6300catagcccca gaggtgatga
aaggtgaaga tctaggtagt gaatacttgg atattgcagg 6360gttgaagata ccgactgaag
agatgaaagg caacatgctc gtgttcgcgc caactaggaa 6420catggcagta gaaacagcta
agaaattgaa ggctaaggga tacaactctg gatactatta 6480cagtggggaa aacccagaga
acttgagggt ggtaacctcg caatccccgt atgtggtagt 6540agccaccaat gccatagagt
caggtgtgac attaccagac ttagacacag ttgtagacac 6600tggactaaag tgtgagaaga
gggtgaggat ttcttcaaaa atgcccttca ttgtaacagg 6660acttaagaga atggcagtca
caatcggaga gcaagcccag cgcaggggta gagtaggaag 6720agtcaagcca ggtaggtact
ataggagtca agaaacagct tcagggtcaa aagattacca 6780ttacgaccta ctgcaagccc
agaggtacgg aatagaagat ggaattaatg taacaaagtc 6840attcagggag atgaactatg
attggagcct ttacgaagag gacagcttga tgataactca 6900actcgaggtc cttaacaacc
tccttatatc agaagacctg cctgccgcag tgaagaacat 6960catggcccgg accgatcacc
cagaacccat acaactggcc tataacagtt atgaaaacca 7020aattccagtg ctgttcccaa
agatcaaaaa tggtgaggtg acagacagtt atgagaatta 7080cacatatctc aatgcaagaa
aattaggaga ggacgtgccg gcatatgtgt acgccacaga 7140ggatgaggat ctagcagtgg
atcttctggg tatggattgg ccggacccag gcaaccaaca 7200ggtggtagag acagggaggg
cattaaaaca agtaactggc ttatccacag cagaaaacgc 7260cctcttgata gccctattcg
gctacgtcgg gtaccagaca ctttcaaaaa ggcacatacc 7320catgattact gacatctata
cacttgaaga ccacaggctt gaggacacaa cccacctcca 7380gtttgcccca aacgctataa
ggaccgacgg caaggactca gagttgaagg aattagctgt 7440gggagacctt gataaatatg
tggacgcact ggtagactac tccaaacaag ggatgaaatt 7500catcaaagtc caagctgaaa
aggtcagaga ctcccagtct acgaaggaag gcttgcaaac 7560cattaaggag tatgtggata
agtttataca atcactaaca gagaataagg aggagatcat 7620caggtatgga ctatggggag
ttcacacggc actctacaaa agcttggcag cgagactggg 7680gcatgaaaca gcttttgcaa
ctttagtggt aaaatggttg gcttttgggg gcgaaacggt 7740atctgctcac atcaagcaag
tagcagttga tctagtagta tattatatca tcaacaaacc 7800atcttttcct ggagatacag
agacccaaca agaggggagg aagtttgtgg ctagtctttt 7860tatatctgca ctagcaacat
acacatataa aacctggaat tacaacaatc tgcaacgggt 7920tgtcgaacct gccttagctt
acctcccata tgctacaagt gccttgaagt tgttcacacc 7980cacaagatta gagagtgtgg
tcatactcag ttctacaatt tacaagacat acctctctat 8040aaggaagggt aagagtgacg
gcttgttagg tacaggcata agtgcagcca tggagatctt 8100aaaccaaaac ccaatctcag
taggtatatc tgtgatgctg ggggtaggtg ccatcgccgc 8160ccataatgca atagaatcta
gtgaacagaa aagaactttg ctgatgaagg tctttgtaaa 8220aaacttctta gaccaagcag
caacagatga gctagtcaaa gagaaccctg aaaaaataat 8280catggctcta tttgaagcag
tccagaccat aggaaacccc ctaagactca tctaccatct 8340gtacggggtg tactataagg
ggtgggaagc aaaagaactc gcagagaaaa ctgctggccg 8400caacttattc acattgatca
tgtttgaggc ctttgagctt ttaggtatgg actcagaagg 8460aaagataaga aacttgtcag
gcaactacat actggactta atcttcaact tgcataataa 8520attaaacaag gggctcaaaa
aactagtcct tgggtgggct cctgcacctt tgagctgtga 8580ttggacacca agtgatgaga
gaataagcct acctcataac aactacttaa gggtagaaac 8640caggtgtcct tgtggctatg
agatgaaggc aataaaaaat gttgctggta aattgacaaa 8700agttgaagaa aaggggtcct
tcctatgcag gaatagatta gggagaggac ctccaaactt 8760caaagtaaca aagttctatg
atgataactt gatagaagtc aagccagtag ctaggctaga 8820aggccaggtg gacctctatt
acaagggagt aacagctaag ttagactaca acaatgggaa 8880agtactgtta gctaccaaca
agtgggaggt ggaccacgct ttcctgacca gactagtaaa 8940gaagcacaca gggataggtt
ttaaaggtgc atatttgggt gaccgaccag accatcaaga 9000tcttgtcgat agagattgtg
caactataac gaagaactca gtacagttcc taaaaatgaa 9060gaagggttgc gctttcacat
atgacctaac aatctctaac cttgtcaggc ttattgaact 9120agtccataag aataatttac
aagaaagaga gatccctacc gtgacagtaa ctacttggct 9180tgcatattct tttgtcaatg
aagacctggg gactatcaag cctgtattgg gggagaaagt 9240catcccagaa ccccccgagg
agttgagtct ccaacccacc gtgagactag tcaccactga 9300aacagcaata accataacag
gggaggctga agtgatgacg acagggatca caccagtggt 9360agagatgaaa gaagaacctc
agctggacca ccagtcaact accctaaagg tagggttgaa 9420ggaaggggaa tatccagggc
caggagttaa ccctaaccat ttagcagagg tgatagatga 9480gaaagatgac aggccttttg
tcctaatcat cggtaacaaa ggttctacct cgaacagagc 9540aagaacggcc aagaatatac
ggctgtacaa aggaaacaac ccaagagaga tcagggatct 9600gatgagccaa ggaagaatat
tgacggttgc tctaaaagag ttggacccgg aattaaaaga 9660attagtagat tacaagggga
cctttctcaa tagggaagct ttagaagccc taagcttagg 9720taagccaatc aagaggaaaa
ccacaacagc aatgatcagg aggttaatag agccagaggt 9780tgaggaggaa ctaccagatt
ggttccaagc ggaagaaccc ctatttttgg aagcaaaaat 9840acagaatgac ttataccacc
taattggcag tgtagatagt ataaaaagca aagcaaagga 9900attaggggcc acagataaca
caaagatagt gaaggaagtt ggggctagga cctatacgat 9960gaaattgagc agctggagca
cacaagttac aaaaaaacag atgagtctag cccctctctt 10020tgaagagctg ttattaaagt
gccctccatg tagtaaaatt tcaaagggac atatggtgtc 10080agcataccaa ctggctcaag
gaaactggga acccctcggg tgtggggtct atatgggaac 10140cataccagct aggcgtctca
agatccaccc ttatgaggct taccttaaac tcaaagagct 10200ggtggaagtt gaatcttcga
gggccactgc aaaagaatcc atcataagag aacataacac 10260ctggatcctg cggaaggtga
gacatgaagg gaacctaaga accaaatcaa tgatcaaccc 10320tgggaaaata tcagatcagc
tatgcagaga tggacacaaa agaaacatat ataataagat 10380cataggctca acaatggcct
ctgctggtat taggctggag aaactgccag tagtccgagc 10440ccaaactgac acaaccagtt
tccaccaagc cataagagaa aaaattgata aaacagaaaa 10500caagcagacc cctgaattgc
atgaagaact aatgaaggtc ttcgactgct taaagatccc 10560agagctgaag gaatcgtatg
atgaagtttc atgggaacaa ttagaagccg ggataaaccg 10620taagggtgca gcaggctatc
tagagagcaa gaacataggg gaagtcctag acacagagaa 10680acacatagta gagcagctga
tcaaggatct gaggaagggg aagaagatta ggtactatga 10740aacagccatc cccaagaatg
agaagagaga cgtcagcgac gactgggaag ccggagagtt 10800cgttgatgaa aagaaaccaa
gagtaatcca gtacccggac gccaaggtga gactggccat 10860tacaaaagtg atgtacaaat
gggtaaagca aaaaccagtg gtgatacccg gctatgaagg 10920taaaacacct ctatttgaca
tattcaacaa agtgaagaag gaatgggatt cattccagga 10980ccccgtagca gtgagctttg
acaccaaagc gtgggataca caagtcacca gtagagacct 11040aatgttgata aaggatatcc
agaaatatta tttcaagaga agtatacaca aatttttaga 11100tacaataaca gaacacatgg
tggaggtacc tgtcattaca gcagacggtg aagtttacat 11160aaggaatggt cagaggggta
gtggccaacc cgacacaagt gctggtaata gtatgttgaa 11220tgtcctaacc atgatatatg
ctttctgtaa aagtacaggc ataccttaca ggggattcag 11280cagagtggca agaatccatg
tgtgtggtga tgatggcttt ttgataacag agagaggact 11340gggactgaaa ttctctgaga
agggtatgca gatattacat gaggccggga agccccagaa 11400aataactgaa ggggacaaaa
tgaaagtggc atacagattc gaggacatag agttttgttc 11460ccatactccc gtgccagtca
gatgggcaga taacaccagt agttacatgg cagggaggag 11520cacagccact atactagcta
agatggcaac caggctggat tccagcggag agaggggtag 11580cacagcttat gagaaggccg
tagccttcag cttccttttg atgtactcat ggaatcccgt 11640agttagaagg atctgcttac
tggtgttgtc acagtttcca gaaatatccc catccaaaaa 11700cacaatatac tactaccaag
gggatcccat agctgcgtac agagaagtga tagggaaaca 11760gctgtgtgaa ctgaaaagaa
caggatttga gaagctggct ggtctgaatt tgagtatgac 11820cactctaggc atctggacaa
aacatactag taaaagacta atccaagcct gtgtagaaat 11880aggtaagaga gaaggtacct
ggttagttaa tgctgacaga ctgattgcag gaaagactgg 11940gaagttttac atcccaagca
ctggtgtcac tctgttggga aaacactatg aggaaattaa 12000cttaaagcaa aaggcggcac
aaccgccgat agagggggtt gacagatata agttgggccc 12060catagttaat gttatcttga
gaaggctgag ggtgatgctg atgacagttg ccagcggaag 12120ctggtgaatc cgtccggagc
gtcgtgccct cactcaaggt ttttaattgt aaatattgta 12180aatagacagc taagatattt
attgtagttg gatagtaatg cagtgatagt aaatacccca 12240atttaacact acctccaatg
cactaagcac tttagctgtg tgaggttaac tcgacgtcca 12300cggttggact agggaagacc
tctaacagcc cc 123321111840DNAArtificial
SequenceXIKE-C-NdN BVDV-Sequence 11gtatacgaga ttagctaaag aactcgtata
tggattggac gtcaacaaat ttttaattgg 60caacgtaggg aaccttcccc tcagcgaagg
ccgaaaagag gctagccatg cccttagtag 120gactagcaaa agtaggggac tagcggtagc
agtgagttcg ttggatggcc gaacccctga 180gtacagggga gtcgtcaatg gttcgacact
ccattagtcg aggagtctcg agatgccatg 240tggacgaggg catgcccacg gcacatctta
acccatgcgg gggttgcatg ggtgaaagcg 300ctattcgtgg cgttatggac acagcctgat
agggtgtagc agagacctgc tattccgcta 360gtaaaaactc tgctgtacat ggcacatgga
gttgttttcc gatgaaggga gcaagggtgc 420tacaagtaag aagcagccta agccagatag
gatagaaaaa ggtaagatga aaatagcccc 480aaaagagaca gaaaaagatt gcaaaaccag
accccccgac gcgactatag tagtagaagg 540ggttaagtac caggtgaaga aaaaaggaaa
ggtaagggga aaaaatactc aagatgggtt 600atatcacaac aagaataagc cccctgaatc
aagaaaaaaa ttggaaaagg cactgctggc 660ttgggccatc ttagcagcgg tcctgcttca
gctggtaaca ggagagaata tcacccagtg 720gaacttgatg gacaacggca ccgagggaat
acagcaagcg atgttcctaa gaggggtgaa 780caggagtcta ttaggaattt ggccagagaa
aatttgcacc ggagtaccaa ctcacttagc 840aacagactat gagcttaaag agatagtggg
gatgatggac gcgagtgaga agaccaacta 900cacgtgttgc aggttgcaaa gacatgagtg
gaataaacat ggttggtgta actggtttca 960tatagaaccg tggatatggt tgatgaacaa
aacccaaaac aacctgacag aagggcaacc 1020gcttagggag tgtgctgtga cttgtaggta
tgacaaggaa acagaattga acatcgtgac 1080acaggctagg gacagaccta caactctgac
aggttgcaag aaaggcaaga atttctcttt 1140cgcaggtgtt atactggatg ggccctgtaa
ctttaaagta tcggttgaag atgtgctgtt 1200caaggagcac gattgcggca acatgctgca
agagaccgcg atacagctac tcgatggggc 1260aaccaacacc attgagggag caagggtagg
gacggccaag ttgacaacct ggttagggaa 1320gcaattaggg atccttggta agaagttgga
gaacaaaagc aaagcatggt ttggtgcaca 1380tgcagcaagt ccatactgcg gagtggagag
gaagatcggt tacgtatggt atacaaaaaa 1440ctgcactcca gcttgccttc caagaaacac
tagaataata ggccccggga aatttgatac 1500caacgccgaa gatggaaaaa tactccatga
gatggggggg cacctctcag aatttgtcct 1560attgtccttg gtggttctgt ctgactttgc
cccggaaacc gcgagcgtca tctacttggt 1620tctacatttt gcgatcccgc aaagccacgt
tgatgtagac acatgcgaca agaaccagct 1680gaatttaacg gtagcaacca cagtagcaga
ggtcatacca gggacagtgt ggaacctagg 1740gaagtatgtc tgcataagac cagactggtg
gccatatgag acgacgacag tcttcgtcat 1800agaggaagca gggcaagtaa tcaaattgat
gctaagggcc atcagagact taactaggat 1860atggaatgct gccactacca cagctttctt
aatcttttta gtaaaagcac tgaggggaca 1920actaatccaa gggctattgt ggctgatgct
aataacagga gcacagggct tccctgaatg 1980caaagagggc ttccaatatg ccatatctaa
agacaggaaa atggggttat tggggccaga 2040gagcttaact acaacatggc acctccccac
caaaaaaata gtggattcca tggtgcatgt 2100atggtgtgaa ggaaaagact tgaaaatatt
aaaaatgtgc acaaaggaag agaggtatct 2160agtggctgtg cacgagagag ccttatcaac
cagtgccgag tttatgcaga tcagtgatgg 2220gacaataggc ccagacgtga tagatatgcc
tgatgacttt gagtttggac tctgcccttg 2280tgactcaaaa ccagtgataa agggcaaatt
taatgccagc ttactgaatg gaccagcttt 2340ccagatggta tgcccacagg ggtggactgg
tacaatagaa tgcaccctag cgaaccaaga 2400caccttggac acaactgtca ttaggacata
tagaagaact accccatttc agcggagaaa 2460atggtgtacc tatgaaaaaa taatagggga
agatatctat gaatgcattc taggtggaaa 2520ctggacatgc ataaccggtg accatagcag
gttgaaagac ggacctatca agaagtgtaa 2580gtggtgtggc catgacttcg tcaactcaga
ggggctacca cactacccaa taggcaagtg 2640catgctcatc aacgagagtg ggtacaggta
tgtagatgac acctcttgcg ataggggtgg 2700tgtagccata gttccatctg gcaccgtaaa
gtgtagaata ggtaacgtca cggtgcaagt 2760tatcgctact aacaatgatc tgggacccat
gccttgcagc ccagctgaag tgatagcaag 2820tgaaggacca gtggaaaaga ctgcatgcac
attcaactat tcaaggactc tacctaataa 2880gtattatgag ccaagggacc ggtacttcca
acaatacatg ttaaaagggg agtggcaata 2940ttggttcgac ctggattctg tagaccacca
caaagactac ttctcagagt tcataatcat 3000agcagtggtc gccttgttgg gtggtaagta
cgtactgtgg ctcttgataa catacacaat 3060actgtctgag cagatggcta tgggtgctgg
agtgaatact gaagagatag tcatgatagg 3120caatttgctg acagacagtg atattgaggt
tgtggtttat ttccttcttc tgtacttaat 3180agttaaagag gaactggcga ggaaatggat
tatactggta taccacatcc ttgtagccaa 3240ccctatgaaa acaattgggg tcgtcttact
aatgctaggg ggagtggtga aggccagcag 3300aatcaatgct gatgaccaaa gtgctatgga
cccatgcttt cttctcgtga caggcgtagt 3360ggctgttttg atgatcgcta gaagagaacc
tgccacatta ccactgattg tagcattgct 3420agcaataaga acatcaggat tcctactgcc
cgctagcatt gatgtaactg tagcagtagt 3480attaattgta cttttgttgg ctagctacat
aacagactac tttagatata aaaagtggct 3540tcaactctta tttagtctga tagctggtat
ctttattata aggagcttaa aacatatcaa 3600ccagatggag gtaccagaaa tatctatgcc
aagttggaga cctctagctc tggtcctttt 3660ctatataaca tctacagcaa taaccactaa
ttgggacatt gacttagcag gcttcctgct 3720gcaatgggcg ccagcagtga tcatgatggc
taccatgtgg gcagactttt tgactctgat 3780catagtcctg cccagttacg agttatctaa
gctttacttc ctaaagaacg tcaggacaga 3840cgtggaaaag aactggctcg gcaaagtgaa
atacagacag atcagttcag tttatgacat 3900ctgtgacagt gaggaagcag tgtacctatt
tccatcaagg cataagagtg gaagcaggcc 3960agatttcata ttaccttttt tgaaagccgt
gttaataagc tgcatcagca gccaatggca 4020agtggtttac atttcttacc taatactgga
aattacatac tatatgcaca ggaaaatcat 4080agatgaggtg tcaggaggag caaattttct
atcaagactc atagcagcca tcatagaatt 4140aaattgggcc atagatgatg aggaatgtaa
aggactgaag aaactgtatc tcttgtcagg 4200gagagcgaag aatttgatag ttaaacataa
ggtaagaaat gaagccgtcc acagatggtt 4260tggtgaggag gaaatatacg gggcacccaa
ggtgatcact atcataaaag ctagtaccct 4320aagtaaaaac aggcactgca taatctgcac
gatctgtgaa gggaaagaat ggaatggagc 4380caactgccca aagtgtggaa gacaaggaaa
gcccataaca tgtggaatga cactcgcaga 4440ctttgaggag aaacattaca aaaagatatt
tataagagaa gaatcttctt gtcctgtgcc 4500ttttgatcct tcttgccatt gtaattattt
tcgccacgat gggcctttca ggaaagagta 4560taagggttac gtccaataca cagccagagg
acaactcttt ctgaggaacc taccaattct 4620agcgacgaag atgaagctat taatggtggg
aaacctcggc gcagaaattg gcgacctgga 4680acatctagga tgggtactga gagggccagc
cgtgtgcaaa aaaattacca accatgagaa 4740gtgccacgta aacatcatgg ataagctaac
tgcatttttt ggaatcatgc ctagaggcac 4800gacccctagg gcacctgtga ggttccccac
agcactacta aaagtgagaa gggggctaga 4860gacgggatgg gcttacacgc accaaggagg
gatcagctcg gtagaccatg tcacagccgg 4920aaaggattta ctagtgtgtg acagtatggg
caggaccagg gttgtctgtc atagtaacaa 4980taagatgact gatgagactg agtatggcat
caagaccgac tcagggtgtc ccgaaggtgc 5040gaggtgttac gtgctaaacc cagaagctgt
taacatttct ggcacaaaag gagctatggt 5100acacctccag aaaacggggg gggagttcac
atgtgtcact gcctcaggga ccccggcttt 5160cttcgatctg aaaaatctaa aaggctggtc
cgggctacca atttttgaag catccagtgg 5220cagggtggtt ggtagggtga aagtcggcaa
gaatgaggat tccaagccca ccaaactaat 5280gagcggaatc cagacagtgt ctaagaacca
gacagaccta gcggacatcg taaaaaaatt 5340gactagtatg aacagaggag agttcaaaca
gataacatta gccactgggg caggaaaaac 5400tacggaactg ccaaggtccg tcatagagga
gatagggagg cacaaaaggg tcttagtcct 5460gataccattg agagcagcag cagagtcagt
gtatcagtat atgagagtga agtacccaag 5520tatatctttc aatttgagaa taggagatat
gaaggaaggt gacatggcca ctggtatcac 5580ctacgcctca tatgggtact tttgtcagct
tcctcagccc aaactgagag ctgccatggt 5640agagtactca tatatattct tagatgagta
ccactgtgct acacccgagc aattagcaat 5700aattggaaag atacacaggt ttgctgaaaa
tcttagagtg gtagcaatga cagcaacccc 5760agctggaacg gtcacaacga ctggtcagaa
acaccctata gaggagttca tagccccaga 5820ggtgatgaaa ggtgaagatc taggtagtga
atacttggat attgcagggt tgaagatacc 5880gactgaagag atgaaaggca acatgctcgt
gttcgcgcca actaggaaca tggcagtaga 5940aacagctaag aaattgaagg ctaagggata
caactctgga tactattaca gtggggaaaa 6000cccagagaac ttgagggtgg taacctcgca
atccccgtat gtggtagtag ccaccaatgc 6060catagagtca ggtgtgacat taccagactt
agacacagtt gtagacactg gactaaagtg 6120tgagaagagg gtgaggattt cttcaaaaat
gcccttcatt gtaacaggac ttaagagaat 6180ggcagtcaca atcggagagc aagcccagcg
caggggtaga gtaggaagag tcaagccagg 6240taggtactat aggagtcaag aaacagcttc
agggtcaaaa gattaccatt acgacctact 6300gcaagcccag aggtacggaa tagaagatgg
aattaatgta acaaagtcat tcagggagat 6360gaactatgat tggagccttt acgaagagga
cagcttgatg ataactcaac tcgaggtcct 6420taacaacctc cttatatcag aagacctgcc
tgccgcagtg aagaacatca tggcccggac 6480cgatcaccca gaacccatac aactggccta
taacagttat gaaaaccaaa ttccagtgct 6540gttcccaaag atcaaaaatg gtgaggtgac
agacagttat gagaattaca catatctcaa 6600tgcaagaaaa ttaggagagg acgtgccggc
atatgtgtac gccacagagg atgaggatct 6660agcagtggat cttctgggta tggattggcc
ggacccaggc aaccaacagg tggtagagac 6720agggagggca ttaaaacaag taactggctt
atccacagca gaaaacgccc tcttgatagc 6780cctattcggc tacgtcgggt accagacact
ttcaaaaagg cacataccca tgattactga 6840catctataca cttgaagacc acaggcttga
ggacacaacc cacctccagt ttgccccaaa 6900cgctataagg accgacggca aggactcaga
gttgaaggaa ttagctgtgg gagaccttga 6960taaatatgtg gacgcactgg tagactactc
caaacaaggg atgaaattca tcaaagtcca 7020agctgaaaag gtcagagact cccagtctac
gaaggaaggc ttgcaaacca ttaaggagta 7080tgtggataag tttatacaat cactaacaga
gaataaggag gagatcatca ggtatggact 7140atggggagtt cacacggcac tctacaaaag
cttggcagcg agactggggc atgaaacagc 7200ttttgcaact ttagtggtaa aatggttggc
ttttgggggc gaaacggtat ctgctcacat 7260caagcaagta gcagttgatc tagtagtata
ttatatcatc aacaaaccat cttttcctgg 7320agatacagag acccaacaag aggggaggaa
gtttgtggct agtcttttta tatctgcact 7380agcaacatac acatataaaa cctggaatta
caacaatctg caacgggttg tcgaacctgc 7440cttagcttac ctcccatatg ctacaagtgc
cttgaagttg ttcacaccca caagattaga 7500gagtgtggtc atactcagtt ctacaattta
caagacatac ctctctataa ggaagggtaa 7560gagtgacggc ttgttaggta caggcataag
tgcagccatg gagatcttaa accaaaaccc 7620aatctcagta ggtatatctg tgatgctggg
ggtaggtgcc atcgccgccc ataatgcaat 7680agaatctagt gaacagaaaa gaactttgct
gatgaaggtc tttgtaaaaa acttcttaga 7740ccaagcagca acagatgagc tagtcaaaga
gaaccctgaa aaaataatca tggctctatt 7800tgaagcagtc cagaccatag gaaaccccct
aagactcatc taccatctgt acggggtgta 7860ctataagggg tgggaagcaa aagaactcgc
agagaaaact gctggccgca acttattcac 7920attgatcatg tttgaggcct ttgagctttt
aggtatggac tcagaaggaa agataagaaa 7980cttgtcaggc aactacatac tggacttaat
cttcaacttg cataataaat taaacaaggg 8040gctcaaaaaa ctagtccttg ggtgggctcc
tgcacctttg agctgtgatt ggacaccaag 8100tgatgagaga ataagcctac ctcataacaa
ctacttaagg gtagaaacca ggtgtccttg 8160tggctatgag atgaaggcaa taaaaaatgt
tgctggtaaa ttgacaaaag ttgaagaaaa 8220ggggtccttc ctatgcagga atagattagg
gagaggacct ccaaacttca aagtaacaaa 8280gttctatgat gataacttga tagaagtcaa
gccagtagct aggctagaag gccaggtgga 8340cctctattac aagggagtaa cagctaagtt
agactacaac aatgggaaag tactgttagc 8400taccaacaag tgggaggtgg accacgcttt
cctgaccaga ctagtaaaga agcacacagg 8460gataggtttt aaaggtgcat atttgggtga
ccgaccagac catcaagatc ttgtcgatag 8520agattgtgca actataacga agaactcagt
acagttccta aaaatgaaga agggttgcgc 8580tttcacatat gacctaacaa tctctaacct
tgtcaggctt attgaactag tccataagaa 8640taatttacaa gaaagagaga tccctaccgt
gacagtaact acttggcttg catattcttt 8700tgtcaatgaa gacctgggga ctatcaagcc
tgtattgggg gagaaagtca tcccagaacc 8760ccccgaggag ttgagtctcc aacccaccgt
gagactagtc accactgaaa cagcaataac 8820cataacaggg gaggctgaag tgatgacgac
agggatcaca ccagtggtag agatgaaaga 8880agaacctcag ctggaccacc agtcaactac
cctaaaggta gggttgaagg aaggggaata 8940tccagggcca ggagttaacc ctaaccattt
agcagaggtg atagatgaga aagatgacag 9000gccttttgtc ctaatcatcg gtaacaaagg
ttctacctcg aacagagcaa gaacggccaa 9060gaatatacgg ctgtacaaag gaaacaaccc
aagagagatc agggatctga tgagccaagg 9120aagaatattg acggttgctc taaaagagtt
ggacccggaa ttaaaagaat tagtagatta 9180caaggggacc tttctcaata gggaagcttt
agaagcccta agcttaggta agccaatcaa 9240gaggaaaacc acaacagcaa tgatcaggag
gttaatagag ccagaggttg aggaggaact 9300accagattgg ttccaagcgg aagaacccct
atttttggaa gcaaaaatac agaatgactt 9360ataccaccta attggcagtg tagatagtat
aaaaagcaaa gcaaaggaat taggggccac 9420agataacaca aagatagtga aggaagttgg
ggctaggacc tatacgatga aattgagcag 9480ctggagcaca caagttacaa aaaaacagat
gagtctagcc cctctctttg aagagctgtt 9540attaaagtgc cctccatgta gtaaaatttc
aaagggacat atggtgtcag cataccaact 9600ggctcaagga aactgggaac ccctcgggtg
tggggtctat atgggaacca taccagctag 9660gcgtctcaag atccaccctt atgaggctta
ccttaaactc aaagagctgg tggaagttga 9720atcttcgagg gccactgcaa aagaatccat
cataagagaa cataacacct ggatcctgcg 9780gaaggtgaga catgaaggga acctaagaac
caaatcaatg atcaaccctg ggaaaatatc 9840agatcagcta tgcagagatg gacacaaaag
aaacatatat aataagatca taggctcaac 9900aatggcctct gctggtatta ggctggagaa
actgccagta gtccgagccc aaactgacac 9960aaccagtttc caccaagcca taagagaaaa
aattgataaa acagaaaaca agcagacccc 10020tgaattgcat gaagaactaa tgaaggtctt
cgactgctta aagatcccag agctgaagga 10080atcgtatgat gaagtttcat gggaacaatt
agaagccggg ataaaccgta agggtgcagc 10140aggctatcta gagagcaaga acatagggga
agtcctagac acagagaaac acatagtaga 10200gcagctgatc aaggatctga ggaaggggaa
gaagattagg tactatgaaa cagccatccc 10260caagaatgag aagagagacg tcagcgacga
ctgggaagcc ggagagttcg ttgatgaaaa 10320gaaaccaaga gtaatccagt acccggacgc
caaggtgaga ctggccatta caaaagtgat 10380gtacaaatgg gtaaagcaaa aaccagtggt
gatacccggc tatgaaggta aaacacctct 10440atttgacata ttcaacaaag tgaagaagga
atgggattca ttccaggacc ccgtagcagt 10500gagctttgac accaaagcgt gggatacaca
agtcaccagt agagacctaa tgttgataaa 10560ggatatccag aaatattatt tcaagagaag
tatacacaaa tttttagata caataacaga 10620acacatggtg gaggtacctg tcattacagc
agacggtgaa gtttacataa ggaatggtca 10680gaggggtagt ggccaacccg acacaagtgc
tggtaatagt atgttgaatg tcctaaccat 10740gatatatgct ttctgtaaaa gtacaggcat
accttacagg ggattcagca gagtggcaag 10800aatccatgtg tgtggtgatg atggcttttt
gataacagag agaggactgg gactgaaatt 10860ctctgagaag ggtatgcaga tattacatga
ggccgggaag ccccagaaaa taactgaagg 10920ggacaaaatg aaagtggcat acagattcga
ggacatagag ttttgttccc atactcccgt 10980gccagtcaga tgggcagata acaccagtag
ttacatggca gggaggagca cagccactat 11040actagctaag atggcaacca ggctggattc
cagcggagag aggggtagca cagcttatga 11100gaaggccgta gccttcagct tccttttgat
gtactcatgg aatcccgtag ttagaaggat 11160ctgcttactg gtgttgtcac agtttccaga
aatatcccca tccaaaaaca caatatacta 11220ctaccaaggg gatcccatag ctgcgtacag
agaagtgata gggaaacagc tgtgtgaact 11280gaaaagaaca ggatttgaga agctggctgg
tctgaatttg agtatgacca ctctaggcat 11340ctggacaaaa catactagta aaagactaat
ccaagcctgt gtagaaatag gtaagagaga 11400aggtacctgg ttagttaatg ctgacagact
gattgcagga aagactggga agttttacat 11460cccaagcact ggtgtcactc tgttgggaaa
acactatgag gaaattaact taaagcaaaa 11520ggcggcacaa ccgccgatag agggggttga
cagatataag ttgggcccca tagttaatgt 11580tatcttgaga aggctgaggg tgatgctgat
gacagttgcc agcggaagct ggtgaatccg 11640tccggagcgt cgtgccctca ctcaaggttt
ttaattgtaa atattgtaaa tagacagcta 11700agatatttat tgtagttgga tagtaatgca
gtgatagtaa ataccccaat ttaacactac 11760ctccaatgca ctaagcactt tagctgtgtg
aggttaactc gacgtccacg gttggactag 11820ggaagacctc taacagcccc
11840123749PRTArtificial
SequenceXIKE-C-NdN 12Met Glu Leu Phe Ser Asp Glu Gly Ser Lys Gly Ala Thr
Ser Lys Lys 1 5 10 15
Gln Pro Lys Pro Asp Arg Ile Glu Lys Gly Lys Met Lys Ile Ala Pro
20 25 30 Lys Glu Thr Glu
Lys Asp Cys Lys Thr Arg Pro Pro Asp Ala Thr Ile 35
40 45 Val Val Glu Gly Val Lys Tyr Gln Val
Lys Lys Lys Gly Lys Val Arg 50 55
60 Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn
Lys Pro Pro 65 70 75
80 Glu Ser Arg Lys Lys Leu Glu Lys Ala Leu Leu Ala Trp Ala Ile Leu
85 90 95 Ala Ala Val Leu
Leu Gln Leu Val Thr Gly Glu Asn Ile Thr Gln Trp 100
105 110 Asn Leu Met Asp Asn Gly Thr Glu Gly
Ile Gln Gln Ala Met Phe Leu 115 120
125 Arg Gly Val Asn Arg Ser Leu Leu Gly Ile Trp Pro Glu Lys
Ile Cys 130 135 140
Thr Gly Val Pro Thr His Leu Ala Thr Asp Tyr Glu Leu Lys Glu Ile 145
150 155 160 Val Gly Met Met Asp
Ala Ser Glu Lys Thr Asn Tyr Thr Cys Cys Arg 165
170 175 Leu Gln Arg His Glu Trp Asn Lys His Gly
Trp Cys Asn Trp Phe His 180 185
190 Ile Glu Pro Trp Ile Trp Leu Met Asn Lys Thr Gln Asn Asn Leu
Thr 195 200 205 Glu
Gly Gln Pro Leu Arg Glu Cys Ala Val Thr Cys Arg Tyr Asp Lys 210
215 220 Glu Thr Glu Leu Asn Ile
Val Thr Gln Ala Arg Asp Arg Pro Thr Thr 225 230
235 240 Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser
Phe Ala Gly Val Ile 245 250
255 Leu Asp Gly Pro Cys Asn Phe Lys Val Ser Val Glu Asp Val Leu Phe
260 265 270 Lys Glu
His Asp Cys Gly Asn Met Leu Gln Glu Thr Ala Ile Gln Leu 275
280 285 Leu Asp Gly Ala Thr Asn Thr
Ile Glu Gly Ala Arg Val Gly Thr Ala 290 295
300 Lys Leu Thr Thr Trp Leu Gly Lys Gln Leu Gly Ile
Leu Gly Lys Lys 305 310 315
320 Leu Glu Asn Lys Ser Lys Ala Trp Phe Gly Ala His Ala Ala Ser Pro
325 330 335 Tyr Cys Gly
Val Glu Arg Lys Ile Gly Tyr Val Trp Tyr Thr Lys Asn 340
345 350 Cys Thr Pro Ala Cys Leu Pro Arg
Asn Thr Arg Ile Ile Gly Pro Gly 355 360
365 Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His
Glu Met Gly 370 375 380
Gly His Leu Ser Glu Phe Val Leu Leu Ser Leu Val Val Leu Ser Asp 385
390 395 400 Phe Ala Pro Glu
Thr Ala Ser Val Ile Tyr Leu Val Leu His Phe Ala 405
410 415 Ile Pro Gln Ser His Val Asp Val Asp
Thr Cys Asp Lys Asn Gln Leu 420 425
430 Asn Leu Thr Val Ala Thr Thr Val Ala Glu Val Ile Pro Gly
Thr Val 435 440 445
Trp Asn Leu Gly Lys Tyr Val Cys Ile Arg Pro Asp Trp Trp Pro Tyr 450
455 460 Glu Thr Thr Thr Val
Phe Val Ile Glu Glu Ala Gly Gln Val Ile Lys 465 470
475 480 Leu Met Leu Arg Ala Ile Arg Asp Leu Thr
Arg Ile Trp Asn Ala Ala 485 490
495 Thr Thr Thr Ala Phe Leu Ile Phe Leu Val Lys Ala Leu Arg Gly
Gln 500 505 510 Leu
Ile Gln Gly Leu Leu Trp Leu Met Leu Ile Thr Gly Ala Gln Gly 515
520 525 Phe Pro Glu Cys Lys Glu
Gly Phe Gln Tyr Ala Ile Ser Lys Asp Arg 530 535
540 Lys Met Gly Leu Leu Gly Pro Glu Ser Leu Thr
Thr Thr Trp His Leu 545 550 555
560 Pro Thr Lys Lys Ile Val Asp Ser Met Val His Val Trp Cys Glu Gly
565 570 575 Lys Asp
Leu Lys Ile Leu Lys Met Cys Thr Lys Glu Glu Arg Tyr Leu 580
585 590 Val Ala Val His Glu Arg Ala
Leu Ser Thr Ser Ala Glu Phe Met Gln 595 600
605 Ile Ser Asp Gly Thr Ile Gly Pro Asp Val Ile Asp
Met Pro Asp Asp 610 615 620
Phe Glu Phe Gly Leu Cys Pro Cys Asp Ser Lys Pro Val Ile Lys Gly 625
630 635 640 Lys Phe Asn
Ala Ser Leu Leu Asn Gly Pro Ala Phe Gln Met Val Cys 645
650 655 Pro Gln Gly Trp Thr Gly Thr Ile
Glu Cys Thr Leu Ala Asn Gln Asp 660 665
670 Thr Leu Asp Thr Thr Val Ile Arg Thr Tyr Arg Arg Thr
Thr Pro Phe 675 680 685
Gln Arg Arg Lys Trp Cys Thr Tyr Glu Lys Ile Ile Gly Glu Asp Ile 690
695 700 Tyr Glu Cys Ile
Leu Gly Gly Asn Trp Thr Cys Ile Thr Gly Asp His 705 710
715 720 Ser Arg Leu Lys Asp Gly Pro Ile Lys
Lys Cys Lys Trp Cys Gly His 725 730
735 Asp Phe Val Asn Ser Glu Gly Leu Pro His Tyr Pro Ile Gly
Lys Cys 740 745 750
Met Leu Ile Asn Glu Ser Gly Tyr Arg Tyr Val Asp Asp Thr Ser Cys
755 760 765 Asp Arg Gly Gly
Val Ala Ile Val Pro Ser Gly Thr Val Lys Cys Arg 770
775 780 Ile Gly Asn Val Thr Val Gln Val
Ile Ala Thr Asn Asn Asp Leu Gly 785 790
795 800 Pro Met Pro Cys Ser Pro Ala Glu Val Ile Ala Ser
Glu Gly Pro Val 805 810
815 Glu Lys Thr Ala Cys Thr Phe Asn Tyr Ser Arg Thr Leu Pro Asn Lys
820 825 830 Tyr Tyr Glu
Pro Arg Asp Arg Tyr Phe Gln Gln Tyr Met Leu Lys Gly 835
840 845 Glu Trp Gln Tyr Trp Phe Asp Leu
Asp Ser Val Asp His His Lys Asp 850 855
860 Tyr Phe Ser Glu Phe Ile Ile Ile Ala Val Val Ala Leu
Leu Gly Gly 865 870 875
880 Lys Tyr Val Leu Trp Leu Leu Ile Thr Tyr Thr Ile Leu Ser Glu Gln
885 890 895 Met Ala Met Gly
Ala Gly Val Asn Thr Glu Glu Ile Val Met Ile Gly 900
905 910 Asn Leu Leu Thr Asp Ser Asp Ile Glu
Val Val Val Tyr Phe Leu Leu 915 920
925 Leu Tyr Leu Ile Val Lys Glu Glu Leu Ala Arg Lys Trp Ile
Ile Leu 930 935 940
Val Tyr His Ile Leu Val Ala Asn Pro Met Lys Thr Ile Gly Val Val 945
950 955 960 Leu Leu Met Leu Gly
Gly Val Val Lys Ala Ser Arg Ile Asn Ala Asp 965
970 975 Asp Gln Ser Ala Met Asp Pro Cys Phe Leu
Leu Val Thr Gly Val Val 980 985
990 Ala Val Leu Met Ile Ala Arg Arg Glu Pro Ala Thr Leu Pro
Leu Ile 995 1000 1005
Val Ala Leu Leu Ala Ile Arg Thr Ser Gly Phe Leu Leu Pro Ala 1010
1015 1020 Ser Ile Asp Val Thr
Val Ala Val Val Leu Ile Val Leu Leu Leu 1025 1030
1035 Ala Ser Tyr Ile Thr Asp Tyr Phe Arg Tyr
Lys Lys Trp Leu Gln 1040 1045 1050
Leu Leu Phe Ser Leu Ile Ala Gly Ile Phe Ile Ile Arg Ser Leu
1055 1060 1065 Lys His
Ile Asn Gln Met Glu Val Pro Glu Ile Ser Met Pro Ser 1070
1075 1080 Trp Arg Pro Leu Ala Leu Val
Leu Phe Tyr Ile Thr Ser Thr Ala 1085 1090
1095 Ile Thr Thr Asn Trp Asp Ile Asp Leu Ala Gly Phe
Leu Leu Gln 1100 1105 1110
Trp Ala Pro Ala Val Ile Met Met Ala Thr Met Trp Ala Asp Phe 1115
1120 1125 Leu Thr Leu Ile Ile
Val Leu Pro Ser Tyr Glu Leu Ser Lys Leu 1130 1135
1140 Tyr Phe Leu Lys Asn Val Arg Thr Asp Val
Glu Lys Asn Trp Leu 1145 1150 1155
Gly Lys Val Lys Tyr Arg Gln Ile Ser Ser Val Tyr Asp Ile Cys
1160 1165 1170 Asp Ser
Glu Glu Ala Val Tyr Leu Phe Pro Ser Arg His Lys Ser 1175
1180 1185 Gly Ser Arg Pro Asp Phe Ile
Leu Pro Phe Leu Lys Ala Val Leu 1190 1195
1200 Ile Ser Cys Ile Ser Ser Gln Trp Gln Val Val Tyr
Ile Ser Tyr 1205 1210 1215
Leu Ile Leu Glu Ile Thr Tyr Tyr Met His Arg Lys Ile Ile Asp 1220
1225 1230 Glu Val Ser Gly Gly
Ala Asn Phe Leu Ser Arg Leu Ile Ala Ala 1235 1240
1245 Ile Ile Glu Leu Asn Trp Ala Ile Asp Asp
Glu Glu Cys Lys Gly 1250 1255 1260
Leu Lys Lys Leu Tyr Leu Leu Ser Gly Arg Ala Lys Asn Leu Ile
1265 1270 1275 Val Lys
His Lys Val Arg Asn Glu Ala Val His Arg Trp Phe Gly 1280
1285 1290 Glu Glu Glu Ile Tyr Gly Ala
Pro Lys Val Ile Thr Ile Ile Lys 1295 1300
1305 Ala Ser Thr Leu Ser Lys Asn Arg His Cys Ile Ile
Cys Thr Ile 1310 1315 1320
Cys Glu Gly Lys Glu Trp Asn Gly Ala Asn Cys Pro Lys Cys Gly 1325
1330 1335 Arg Gln Gly Lys Pro
Ile Thr Cys Gly Met Thr Leu Ala Asp Phe 1340 1345
1350 Glu Glu Lys His Tyr Lys Lys Ile Phe Ile
Arg Glu Glu Ser Ser 1355 1360 1365
Cys Pro Val Pro Phe Asp Pro Ser Cys His Cys Asn Tyr Phe Arg
1370 1375 1380 His Asp
Gly Pro Phe Arg Lys Glu Tyr Lys Gly Tyr Val Gln Tyr 1385
1390 1395 Thr Ala Arg Gly Gln Leu Phe
Leu Arg Asn Leu Pro Ile Leu Ala 1400 1405
1410 Thr Lys Met Lys Leu Leu Met Val Gly Asn Leu Gly
Ala Glu Ile 1415 1420 1425
Gly Asp Leu Glu His Leu Gly Trp Val Leu Arg Gly Pro Ala Val 1430
1435 1440 Cys Lys Lys Ile Thr
Asn His Glu Lys Cys His Val Asn Ile Met 1445 1450
1455 Asp Lys Leu Thr Ala Phe Phe Gly Ile Met
Pro Arg Gly Thr Thr 1460 1465 1470
Pro Arg Ala Pro Val Arg Phe Pro Thr Ala Leu Leu Lys Val Arg
1475 1480 1485 Arg Gly
Leu Glu Thr Gly Trp Ala Tyr Thr His Gln Gly Gly Ile 1490
1495 1500 Ser Ser Val Asp His Val Thr
Ala Gly Lys Asp Leu Leu Val Cys 1505 1510
1515 Asp Ser Met Gly Arg Thr Arg Val Val Cys His Ser
Asn Asn Lys 1520 1525 1530
Met Thr Asp Glu Thr Glu Tyr Gly Ile Lys Thr Asp Ser Gly Cys 1535
1540 1545 Pro Glu Gly Ala Arg
Cys Tyr Val Leu Asn Pro Glu Ala Val Asn 1550 1555
1560 Ile Ser Gly Thr Lys Gly Ala Met Val His
Leu Gln Lys Thr Gly 1565 1570 1575
Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe
1580 1585 1590 Asp Leu
Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu 1595
1600 1605 Ala Ser Ser Gly Arg Val Val
Gly Arg Val Lys Val Gly Lys Asn 1610 1615
1620 Glu Asp Ser Lys Pro Thr Lys Leu Met Ser Gly Ile
Gln Thr Val 1625 1630 1635
Ser Lys Asn Gln Thr Asp Leu Ala Asp Ile Val Lys Lys Leu Thr 1640
1645 1650 Ser Met Asn Arg Gly
Glu Phe Lys Gln Ile Thr Leu Ala Thr Gly 1655 1660
1665 Ala Gly Lys Thr Thr Glu Leu Pro Arg Ser
Val Ile Glu Glu Ile 1670 1675 1680
Gly Arg His Lys Arg Val Leu Val Leu Ile Pro Leu Arg Ala Ala
1685 1690 1695 Ala Glu
Ser Val Tyr Gln Tyr Met Arg Val Lys Tyr Pro Ser Ile 1700
1705 1710 Ser Phe Asn Leu Arg Ile Gly
Asp Met Lys Glu Gly Asp Met Ala 1715 1720
1725 Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr Phe Cys
Gln Leu Pro 1730 1735 1740
Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr Ser Tyr Ile Phe 1745
1750 1755 Leu Asp Glu Tyr His
Cys Ala Thr Pro Glu Gln Leu Ala Ile Ile 1760 1765
1770 Gly Lys Ile His Arg Phe Ala Glu Asn Leu
Arg Val Val Ala Met 1775 1780 1785
Thr Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys His
1790 1795 1800 Pro Ile
Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp 1805
1810 1815 Leu Gly Ser Glu Tyr Leu Asp
Ile Ala Gly Leu Lys Ile Pro Thr 1820 1825
1830 Glu Glu Met Lys Gly Asn Met Leu Val Phe Ala Pro
Thr Arg Asn 1835 1840 1845
Met Ala Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn 1850
1855 1860 Ser Gly Tyr Tyr Tyr
Ser Gly Glu Asn Pro Glu Asn Leu Arg Val 1865 1870
1875 Val Thr Ser Gln Ser Pro Tyr Val Val Val
Ala Thr Asn Ala Ile 1880 1885 1890
Glu Ser Gly Val Thr Leu Pro Asp Leu Asp Thr Val Val Asp Thr
1895 1900 1905 Gly Leu
Lys Cys Glu Lys Arg Val Arg Ile Ser Ser Lys Met Pro 1910
1915 1920 Phe Ile Val Thr Gly Leu Lys
Arg Met Ala Val Thr Ile Gly Glu 1925 1930
1935 Gln Ala Gln Arg Arg Gly Arg Val Gly Arg Val Lys
Pro Gly Arg 1940 1945 1950
Tyr Tyr Arg Ser Gln Glu Thr Ala Ser Gly Ser Lys Asp Tyr His 1955
1960 1965 Tyr Asp Leu Leu Gln
Ala Gln Arg Tyr Gly Ile Glu Asp Gly Ile 1970 1975
1980 Asn Val Thr Lys Ser Phe Arg Glu Met Asn
Tyr Asp Trp Ser Leu 1985 1990 1995
Tyr Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu Val Leu Asn
2000 2005 2010 Asn Leu
Leu Ile Ser Glu Asp Leu Pro Ala Ala Val Lys Asn Ile 2015
2020 2025 Met Ala Arg Thr Asp His Pro
Glu Pro Ile Gln Leu Ala Tyr Asn 2030 2035
2040 Ser Tyr Glu Asn Gln Ile Pro Val Leu Phe Pro Lys
Ile Lys Asn 2045 2050 2055
Gly Glu Val Thr Asp Ser Tyr Glu Asn Tyr Thr Tyr Leu Asn Ala 2060
2065 2070 Arg Lys Leu Gly Glu
Asp Val Pro Ala Tyr Val Tyr Ala Thr Glu 2075 2080
2085 Asp Glu Asp Leu Ala Val Asp Leu Leu Gly
Met Asp Trp Pro Asp 2090 2095 2100
Pro Gly Asn Gln Gln Val Val Glu Thr Gly Arg Ala Leu Lys Gln
2105 2110 2115 Val Thr
Gly Leu Ser Thr Ala Glu Asn Ala Leu Leu Ile Ala Leu 2120
2125 2130 Phe Gly Tyr Val Gly Tyr Gln
Thr Leu Ser Lys Arg His Ile Pro 2135 2140
2145 Met Ile Thr Asp Ile Tyr Thr Leu Glu Asp His Arg
Leu Glu Asp 2150 2155 2160
Thr Thr His Leu Gln Phe Ala Pro Asn Ala Ile Arg Thr Asp Gly 2165
2170 2175 Lys Asp Ser Glu Leu
Lys Glu Leu Ala Val Gly Asp Leu Asp Lys 2180 2185
2190 Tyr Val Asp Ala Leu Val Asp Tyr Ser Lys
Gln Gly Met Lys Phe 2195 2200 2205
Ile Lys Val Gln Ala Glu Lys Val Arg Asp Ser Gln Ser Thr Lys
2210 2215 2220 Glu Gly
Leu Gln Thr Ile Lys Glu Tyr Val Asp Lys Phe Ile Gln 2225
2230 2235 Ser Leu Thr Glu Asn Lys Glu
Glu Ile Ile Arg Tyr Gly Leu Trp 2240 2245
2250 Gly Val His Thr Ala Leu Tyr Lys Ser Leu Ala Ala
Arg Leu Gly 2255 2260 2265
His Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala Phe 2270
2275 2280 Gly Gly Glu Thr Val
Ser Ala His Ile Lys Gln Val Ala Val Asp 2285 2290
2295 Leu Val Val Tyr Tyr Ile Ile Asn Lys Pro
Ser Phe Pro Gly Asp 2300 2305 2310
Thr Glu Thr Gln Gln Glu Gly Arg Arg Phe Val Ala Ser Leu Phe
2315 2320 2325 Ile Ser
Ala Leu Ala Thr Tyr Thr Tyr Lys Thr Trp Asn Tyr Asn 2330
2335 2340 Asn Leu Gln Arg Val Val Glu
Pro Ala Leu Ala Tyr Leu Pro Tyr 2345 2350
2355 Ala Thr Ser Ala Leu Lys Leu Phe Thr Pro Thr Arg
Leu Glu Ser 2360 2365 2370
Val Val Ile Leu Ser Ser Thr Ile Tyr Lys Thr Tyr Leu Ser Ile 2375
2380 2385 Arg Lys Gly Lys Ser
Asp Gly Leu Leu Gly Thr Gly Ile Ser Ala 2390 2395
2400 Ala Met Glu Ile Leu Asn Gln Asn Pro Ile
Ser Val Gly Ile Ser 2405 2410 2415
Val Met Leu Gly Val Gly Ala Ile Ala Ala His Asn Ala Ile Glu
2420 2425 2430 Ser Ser
Glu Gln Lys Arg Thr Leu Leu Met Lys Val Phe Val Lys 2435
2440 2445 Asn Phe Leu Asp Gln Ala Ala
Thr Asp Glu Leu Val Lys Glu Asn 2450 2455
2460 Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala Val
Gln Thr Ile 2465 2470 2475
Gly Asn Pro Leu Arg Leu Ile Tyr His Leu Tyr Gly Val Tyr Tyr 2480
2485 2490 Lys Gly Trp Glu Ala
Lys Glu Leu Ala Glu Lys Thr Ala Gly Arg 2495 2500
2505 Asn Leu Phe Thr Leu Ile Met Phe Glu Ala
Phe Glu Leu Leu Gly 2510 2515 2520
Met Asp Ser Glu Gly Lys Ile Arg Asn Leu Ser Gly Asn Tyr Ile
2525 2530 2535 Leu Asp
Leu Ile Phe Asn Leu His Asn Lys Leu Asn Lys Gly Leu 2540
2545 2550 Lys Lys Leu Val Leu Gly Trp
Ala Pro Ala Pro Leu Ser Cys Asp 2555 2560
2565 Trp Thr Pro Ser Asp Glu Arg Ile Ser Leu Pro His
Asn Asn Tyr 2570 2575 2580
Leu Arg Val Glu Thr Arg Cys Pro Cys Gly Tyr Glu Met Lys Ala 2585
2590 2595 Ile Lys Asn Val Ala
Gly Lys Leu Thr Lys Val Glu Glu Lys Gly 2600 2605
2610 Ser Phe Leu Cys Arg Asn Arg Leu Gly Arg
Gly Pro Pro Asn Phe 2615 2620 2625
Lys Val Thr Lys Phe Tyr Asp Asp Asn Leu Ile Glu Val Lys Pro
2630 2635 2640 Val Ala
Arg Leu Glu Gly Gln Val Asp Leu Tyr Tyr Lys Gly Val 2645
2650 2655 Thr Ala Lys Leu Asp Tyr Asn
Asn Gly Lys Val Leu Leu Ala Thr 2660 2665
2670 Asn Lys Trp Glu Val Asp His Ala Phe Leu Thr Arg
Leu Val Lys 2675 2680 2685
Lys His Thr Gly Ile Gly Phe Lys Gly Ala Tyr Leu Gly Asp Arg 2690
2695 2700 Pro Asp His Gln Asp
Leu Val Asp Arg Asp Cys Ala Thr Ile Thr 2705 2710
2715 Lys Asn Ser Val Gln Phe Leu Lys Met Lys
Lys Gly Cys Ala Phe 2720 2725 2730
Thr Tyr Asp Leu Thr Ile Ser Asn Leu Val Arg Leu Ile Glu Leu
2735 2740 2745 Val His
Lys Asn Asn Leu Gln Glu Arg Glu Ile Pro Thr Val Thr 2750
2755 2760 Val Thr Thr Trp Leu Ala Tyr
Ser Phe Val Asn Glu Asp Leu Gly 2765 2770
2775 Thr Ile Lys Pro Val Leu Gly Glu Lys Val Ile Pro
Glu Pro Pro 2780 2785 2790
Glu Glu Leu Ser Leu Gln Pro Thr Val Arg Leu Val Thr Thr Glu 2795
2800 2805 Thr Ala Ile Thr Ile
Thr Gly Glu Ala Glu Val Met Thr Thr Gly 2810 2815
2820 Ile Thr Pro Val Val Glu Met Lys Glu Glu
Pro Gln Leu Asp His 2825 2830 2835
Gln Ser Thr Thr Leu Lys Val Gly Leu Lys Glu Gly Glu Tyr Pro
2840 2845 2850 Gly Pro
Gly Val Asn Pro Asn His Leu Ala Glu Val Ile Asp Glu 2855
2860 2865 Lys Asp Asp Arg Pro Phe Val
Leu Ile Ile Gly Asn Lys Gly Ser 2870 2875
2880 Thr Ser Asn Arg Ala Arg Thr Ala Lys Asn Ile Arg
Leu Tyr Lys 2885 2890 2895
Gly Asn Asn Pro Arg Glu Ile Arg Asp Leu Met Ser Gln Gly Arg 2900
2905 2910 Ile Leu Thr Val Ala
Leu Lys Glu Leu Asp Pro Glu Leu Lys Glu 2915 2920
2925 Leu Val Asp Tyr Lys Gly Thr Phe Leu Asn
Arg Glu Ala Leu Glu 2930 2935 2940
Ala Leu Ser Leu Gly Lys Pro Ile Lys Arg Lys Thr Thr Thr Ala
2945 2950 2955 Met Ile
Arg Arg Leu Ile Glu Pro Glu Val Glu Glu Glu Leu Pro 2960
2965 2970 Asp Trp Phe Gln Ala Glu Glu
Pro Leu Phe Leu Glu Ala Lys Ile 2975 2980
2985 Gln Asn Asp Leu Tyr His Leu Ile Gly Ser Val Asp
Ser Ile Lys 2990 2995 3000
Ser Lys Ala Lys Glu Leu Gly Ala Thr Asp Asn Thr Lys Ile Val 3005
3010 3015 Lys Glu Val Gly Ala
Arg Thr Tyr Thr Met Lys Leu Ser Ser Trp 3020 3025
3030 Ser Thr Gln Val Thr Lys Lys Gln Met Ser
Leu Ala Pro Leu Phe 3035 3040 3045
Glu Glu Leu Leu Leu Lys Cys Pro Pro Cys Ser Lys Ile Ser Lys
3050 3055 3060 Gly His
Met Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Glu 3065
3070 3075 Pro Leu Gly Cys Gly Val Tyr
Met Gly Thr Ile Pro Ala Arg Arg 3080 3085
3090 Leu Lys Ile His Pro Tyr Glu Ala Tyr Leu Lys Leu
Lys Glu Leu 3095 3100 3105
Val Glu Val Glu Ser Ser Arg Ala Thr Ala Lys Glu Ser Ile Ile 3110
3115 3120 Arg Glu His Asn Thr
Trp Ile Leu Arg Lys Val Arg His Glu Gly 3125 3130
3135 Asn Leu Arg Thr Lys Ser Met Ile Asn Pro
Gly Lys Ile Ser Asp 3140 3145 3150
Gln Leu Cys Arg Asp Gly His Lys Arg Asn Ile Tyr Asn Lys Ile
3155 3160 3165 Ile Gly
Ser Thr Met Ala Ser Ala Gly Ile Arg Leu Glu Lys Leu 3170
3175 3180 Pro Val Val Arg Ala Gln Thr
Asp Thr Thr Ser Phe His Gln Ala 3185 3190
3195 Ile Arg Glu Lys Ile Asp Lys Thr Glu Asn Lys Gln
Thr Pro Glu 3200 3205 3210
Leu His Glu Glu Leu Met Lys Val Phe Asp Cys Leu Lys Ile Pro 3215
3220 3225 Glu Leu Lys Glu Ser
Tyr Asp Glu Val Ser Trp Glu Gln Leu Glu 3230 3235
3240 Ala Gly Ile Asn Arg Lys Gly Ala Ala Gly
Tyr Leu Glu Ser Lys 3245 3250 3255
Asn Ile Gly Glu Val Leu Asp Thr Glu Lys His Ile Val Glu Gln
3260 3265 3270 Leu Ile
Lys Asp Leu Arg Lys Gly Lys Lys Ile Arg Tyr Tyr Glu 3275
3280 3285 Thr Ala Ile Pro Lys Asn Glu
Lys Arg Asp Val Ser Asp Asp Trp 3290 3295
3300 Glu Ala Gly Glu Phe Val Asp Glu Lys Lys Pro Arg
Val Ile Gln 3305 3310 3315
Tyr Pro Asp Ala Lys Val Arg Leu Ala Ile Thr Lys Val Met Tyr 3320
3325 3330 Lys Trp Val Lys Gln
Lys Pro Val Val Ile Pro Gly Tyr Glu Gly 3335 3340
3345 Lys Thr Pro Leu Phe Asp Ile Phe Asn Lys
Val Lys Lys Glu Trp 3350 3355 3360
Asp Ser Phe Gln Asp Pro Val Ala Val Ser Phe Asp Thr Lys Ala
3365 3370 3375 Trp Asp
Thr Gln Val Thr Ser Arg Asp Leu Met Leu Ile Lys Asp 3380
3385 3390 Ile Gln Lys Tyr Tyr Phe Lys
Arg Ser Ile His Lys Phe Leu Asp 3395 3400
3405 Thr Ile Thr Glu His Met Val Glu Val Pro Val Ile
Thr Ala Asp 3410 3415 3420
Gly Glu Val Tyr Ile Arg Asn Gly Gln Arg Gly Ser Gly Gln Pro 3425
3430 3435 Asp Thr Ser Ala Gly
Asn Ser Met Leu Asn Val Leu Thr Met Ile 3440 3445
3450 Tyr Ala Phe Cys Lys Ser Thr Gly Ile Pro
Tyr Arg Gly Phe Ser 3455 3460 3465
Arg Val Ala Arg Ile His Val Cys Gly Asp Asp Gly Phe Leu Ile
3470 3475 3480 Thr Glu
Arg Gly Leu Gly Leu Lys Phe Ser Glu Lys Gly Met Gln 3485
3490 3495 Ile Leu His Glu Ala Gly Lys
Pro Gln Lys Ile Thr Glu Gly Asp 3500 3505
3510 Lys Met Lys Val Ala Tyr Arg Phe Glu Asp Ile Glu
Phe Cys Ser 3515 3520 3525
His Thr Pro Val Pro Val Arg Trp Ala Asp Asn Thr Ser Ser Tyr 3530
3535 3540 Met Ala Gly Arg Ser
Thr Ala Thr Ile Leu Ala Lys Met Ala Thr 3545 3550
3555 Arg Leu Asp Ser Ser Gly Glu Arg Gly Ser
Thr Ala Tyr Glu Lys 3560 3565 3570
Ala Val Ala Phe Ser Phe Leu Leu Met Tyr Ser Trp Asn Pro Val
3575 3580 3585 Val Arg
Arg Ile Cys Leu Leu Val Leu Ser Gln Phe Pro Glu Ile 3590
3595 3600 Ser Pro Ser Lys Asn Thr Ile
Tyr Tyr Tyr Gln Gly Asp Pro Ile 3605 3610
3615 Ala Ala Tyr Arg Glu Val Ile Gly Lys Gln Leu Cys
Glu Leu Lys 3620 3625 3630
Arg Thr Gly Phe Glu Lys Leu Ala Gly Leu Asn Leu Ser Met Thr 3635
3640 3645 Thr Leu Gly Ile Trp
Thr Lys His Thr Ser Lys Arg Leu Ile Gln 3650 3655
3660 Ala Cys Val Glu Ile Gly Lys Arg Glu Gly
Thr Trp Leu Val Asn 3665 3670 3675
Ala Asp Arg Leu Ile Ala Gly Lys Thr Gly Lys Phe Tyr Ile Pro
3680 3685 3690 Ser Thr
Gly Val Thr Leu Leu Gly Lys His Tyr Glu Glu Ile Asn 3695
3700 3705 Leu Lys Gln Lys Ala Ala Gln
Pro Pro Ile Glu Gly Val Asp Arg 3710 3715
3720 Tyr Lys Leu Gly Pro Ile Val Asn Val Ile Leu Arg
Arg Leu Arg 3725 3730 3735
Val Met Leu Met Thr Val Ala Ser Gly Ser Trp 3740
3745 1313PRTBovine Viral Diarrhea
VirusMISC_FEATURE(1)..(13)Conserved protein sequence within glycoprotein
Erns 13Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys Thr Gly 1
5 10 1420PRTBovine Viral Diarrhea
VirusMISC_FEATURE(1)..(20)Conserved protein sequence within glycoprotein
Erns 14Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Phe
His 1 5 10 15 Ile
Glu Pro Trp 20 1511PRTBovine Viral Diarrhea
VirusMISC_FEATURE(1)..(11)Conserved protein sequence within glycoprotein
Erns 15Ser Leu His Gly Ile Trp Pro Glu Lys Ile Cys 1 5
10 1612PRTBovine Viral Diarrhea
VirusMISC_FEATURE(1)..(12)Conserved protein sequence within glycoprotein
Erns 16Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp 1
5 10
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