Patent application title: ABROGATING PROINFLAMMATORY CYTOKINE PRODUCTION DURING ONCOLYTIC REOVIRUS THERAPY
Inventors:
Matthew C. Coffey (Calgary, CA)
Bradley G. Thompson (Calgary, CA)
Hardev Pandha (Surrey, GB)
Assignees:
ONCOLYTICS BIOTECH INC.
IPC8 Class: AA61K3576FI
USPC Class:
424 932
Class name: Drug, bio-affecting and body treating compositions whole live micro-organism, cell, or virus containing genetically modified micro-organism, cell, or virus (e.g., transformed, fused, hybrid, etc.)
Publication date: 2013-09-19
Patent application number: 20130243732
Abstract:
Provided herein are methods for treating a proliferative disorder in a
subject comprising administering to the subject one or more reoviruses
and one or more agents that modulate expression or activity of
pro-inflammatory cytokines. For example, the agents may inhibit
expression or activity of pro-inflammatory cytokines.Claims:
1. A method for treating a proliferative disorder in a subject,
comprising the steps of: (a) administering to the subject a reovirus
comprising one or more modifications; and (b) administering to the
subject one or more agents that inhibit expression or activity of a
pro-inflammatory cytokine but does not inhibit the production of
neutralizing anti-reovirus antibodies (NARA).
2. The method of claim 1, wherein the pro-inflammatory cytokine is selected from the group consisting of IL-1I, IL-3, IL-6, IL-12, p70, IL-17, MIP-1I and RANTES.
3. The method of claim 1, wherein the one or more agents block innate and adaptive T-cell responses.
4. The method of claim 1, wherein the one or more agents have no effect on B-cell activity.
5. The method of claim 1, further comprising determining whether the proliferative disorder is a ras-mediated proliferative disorder.
6. The method of claim 1, further comprising selecting a subject with a ras-mediated proliferative disorder.
7. The method of claim 1, wherein the proliferative disorder is caused by a mutation in or dysregulation of PKR.
8. The method of claim 1, wherein the one or more modifications comprises a Leu at residue 979 of the lambda-3 polypeptide.
9. The method of claim 1, wherein the one or more modifications comprises a Lys at residue 198 of the sigma-3 polypeptide.
10. The method of claim 1, wherein the one or more modifications comprises an Asp at residue 73 of the mu-1 polypeptide.
11. The method of claim 1, wherein the one or more modifications comprises a Ser at residue 528 of the mu-2 polypeptide.
12. The method of claim 1, wherein the reovirus comprises a polypeptide selected from the group consisting of SEQ ID NO:18, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
13. The method of claim 1, wherein the reovirus comprises SEQ ID NO:18, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
14. The method of claim 13, wherein the reovirus further comprises SEQ ID NO:12.
15. The method of claim 1, wherein the reovirus comprises SEQ ID NOs: 11, 12, and 16-21.
16. The method of claim 15, wherein the reovirus further comprises SEQ ID NO:13 or SEQ ID NO:14.
17. The method of claim 15, wherein the reovirus further comprises SEQ ID NO:13 and SEQ ID NO:14.
18. The method of claim 1, wherein approximately 10.sup.3 to 10.sup.12 plaque forming units (PFU) of the reovirus is administered to the subject.
19. The method of claim 1, wherein approximately 5 to 1000 mg/m2 of the one or more agents that inhibits proinflammatory cytokines is administered to the subject.
20. The method of claim 1, wherein approximately 0.001-10,000 mg/kg body weight of the one or more agents that inhibits proinflammatory cytokines is administered to the subject.
21. The method of claim 1, wherein the one or more agents are platinum compounds.
22. The method of claim 21, wherein the platinum compounds are selected from the group consisting of cisplatin, carboplatin, oxaliplatin, or a combination thereof.
23. The method of claim 22, wherein approximately 175-200 mg/m2 of the cisplatin is administered to the subject.
24. The method of claim 22, wherein approximately 200-600 mg/m2 of the carboplatin is administered to the subject.
25. The method of claim 22, wherein 5 or 6 mg/mL minute (AUC) of the carboplatin is administered to the subject.
26. The method of claim 1, wherein the one or more agents that inhibit pro-inflammatory cytokines are administered at the same time, before or after the reovirus.
27. The method of claim 26, wherein the one or more agents that inhibits pro-inflammatory cytokines are administered at the same time as the reovirus.
28. The method of claim 26, wherein the one or more agents that inhibit pro-inflammatory cytokines is administered before the reovirus.
29. The method of claim 28, wherein the one or more agents are administered from 1 to 12 hours before the reovirus.
30. The method of claim 28, wherein the one or more agent are administered from 1 to 60 minutes before the reovirus.
31. The method of claim 1, wherein the reovirus is administered in multiple doses.
32. The method of claim 31, wherein the one or more agents that inhibit pro-inflammatory cytokines are administered once.
33. The method of claim 31, wherein the one or more agents that inhibits pro-inflammatory cytokines are administered in multiple doses.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser. No. 12/994,114, filed Nov. 22, 2010, which is national stage filing under 35 U.S.C. §371 of International Application No. PCT/CA2009/000721, filed May 27, 2009, which claims priority to U.S. Ser. No. 61/113,791, filed Nov. 12, 2008, and U.S. Ser. No. 61/056,292, filed May 27, 2008. These applications are incorporated by reference herein in their entireties.
BACKGROUND
[0002] Reovirus is a dsRNA virus with tropism to cancer cells having an activated Ras pathway. It has been demonstrated that administration of reovirus into tumor bearing animals results in generation of a robust anti-viral response mediated by both the humoral and cellular arms of the immune system. This anti-viral response can antagonize the oncolytic effectiveness of the therapeutic virus. As such, combinational use of immune suppressing agents to overcome this immune antagonism of reovirus oncolysis has been explored. It has been demonstrated that co-administration of agents that ablate the generation of neutralizing anti-reovirus antibodies (NARA) can result in morbidity in the test animals. The response in the test animals has been characterized by reovirus replication outside of the target tumor tissues, suggesting that humoral immunity serves a protective role in preventing reovirus infection of the host (Qiao et al., Clin. Cancer Res. 14(1):259-69 (2008)).
SUMMARY
[0003] Provided herein are methods for treating a proliferative disorder in a subject comprising administering to the subject one or more reoviruses and one or more agents that modulate expression or activity of pro-inflammatory cytokines For example, the agents may inhibit expression or activity of pro-inflammatory cytokines.
[0004] The details of one or more aspects are set forth in the accompanying drawings and description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIGS. 1A, 1B, 1C and 1D show reduced tumor growth and increased survival following reovirus/cisplatin combination therapy. C57Bl/6 (FIGS. 1A and 1C) and C3H (FIGS. 1B and 1D) mice bearing subcutaneous B16.F10 and K1735 tumors, respectively, were treated on days 1 and 4 with either reovirus alone via intratumoral (i.t.) injection (squares), cisplatin alone via intraperitoneal (i.p.) administration (triangles), or reovirus and cisplatin in combination (circles). Control treated mice (diamonds) received PBS. Tumors were measured on the days indicated and tumor volume expressed as tumor volume relative to volume at commencement of treatment (FIGS. 1A and 1B). Mice were euthanized when tumors exceeded 15 mm in any one dimension. Survival is expressed as Kaplan-Myer plots (FIGS. 1C and 1D).
[0006] FIG. 2 is a graph showing the neutralizing anti-reovirus antibody (NARA) response after no treatment (control) or treatment with reovirus, cisplatin or the combination of reovirus and cisplatin.
[0007] FIGS. 3A, 3B, 3C, 3D, 3E, 3F, and 3G are graphs showing pro-inflammatory cytokine response is abrogated by cisplatin. The response of IL-1I (FIG. 3A), IL-3 (FIG. 3B), IL-6 (FIG. 3C), IL-12 (FIG. 3D), IL-17 (FIG. 3E), MIP-1I (FIG. 3F) and RANTES (FIG. 3G) were measured after no treatment (control) or treatment with reovirus, cisplatin or the combination of reovirus and cisplatin.
DETAILED DESCRIPTION
[0008] As described previously (see, for example, U.S. Pat. Nos. 6,110,461; 6,136,307; 6,261,555; 6,344,195; 6,576,234; and 6,811,775), reoviruses use a host cell's Ras pathway machinery to downregulate double-stranded RNA-activated protein kinase (PKR) and thus replication in the cell. Based upon these discoveries, methods have been developed for using reoviruses to treat proliferative disorders. It has been demonstrated that reovirus therapy results in release of pro-inflammatory cytokines. The pro-inflammatory cytokines antagonize reovirus infection and reovirus spread into the tumor tissue. The protective function of the humoral arm of the immune system in preventing reovirus toxicity has been further suggested by the observation that while, athymic mice manifest no morbidity to reovirus infection, SClD mice and B-cell knock-out animals invariably die from reovirus infection.
[0009] Reovirus oncolysis can be enhanced in vitro by the use of cytotoxic agents. Surprisingly, as described herein, the combinational use of platinum compounds does not impact the production of NARA but has a profound effect on the production of pro-inflammatory cytokines including: IL-1I, IL-3, IL-6, IL-12 p70, IL-17, MIP-1I, and RANTES. Inhibition of pro-inflammatory cytokines by cisplatin prevents T-cell recognition of reovirus infected cells and allows virus replication to ensue without cellular immunity antagonism. However, cisplatin allows production of protective neutralizing anti-reovirus antibodies (NARA) NARA and its concomitant benefits (e.g., preventing reovirus toxicities in patients). The use of platinum compounds to selectively block both innate and adaptive T-cell responses, while having no effect on B-cell activity, has not previously been described.
[0010] Provided herein is a method of treating a proliferative disorder in a subject comprising administering a reovirus to the subject and administering to the subject an agent that modulates pro-inflammatory cytokines.
[0011] For example, the agent inhibits the expression or activity of the pro-inflammatory cytokines. As used herein, the term modulate refers to a change (positive or negative) of 10, 20, 30, 40, 50, 60, 70, 80, 90 percent or greater as compared to a control level. As used herein, control refers to a reference standard from an untreated sample or subject. By way of example, a control level is the level of expression or activity in a control sample in the absence of a stimulus. The control can be prior to, after recovery, or without the stimulus.
[0012] Optionally, the cytokine modulating agent blocks T-cell responses while having little to no effect on B-cell activity. Thus, the agent inhibits pro-inflammatory cytokines but does not inhibit or minimally inhibits production of NARA. Optionally, the agent is a platinum compound. Suitable platinum compounds include, but are not limited to, cisplatin, carboplatin, metaplatin and oxaliplatin.
[0013] Other agents that inhibit pro-inflammatory cytokines include, but are not limited to, TNF-I antibodies such as infliximab, CDP571, CDP870, and adalimumab; recombinant, human soluble p55 TNF receptors such as onercept; soluble TNF receptor and Fc fragment fusion proteins such as etanercept; pegylated Fab fragments of humanized antibody to TNF such as certolizumab pegol; chimeric antibodies to anti-I chain of IL-2 receptor such as basiliximab or daclizumab; IL-12p40 antibodies such as ABT-874; IL-6 receptor antibodies such as MRA or tocilizumab; IFN-K antibodies such as fontolizumab; antibodies that inhibit IL-1 binding to the IL-1 receptor such as AMG108; caspase-1 inhibitors that inhibit cytokine-release such as diarylsulphonylurene; IL-15 antibodies such as mepolizumab; IL-8 antibodies such as ABX-IL-8; IL-9 antibodies including IL-9 monoclonal antibodies; recombinant human IL-21 also referred to as 494C10; inhibitors of TNF-I, IL-115, IL-6 and granulocyte monocyte-colony stimulating factor expression such as biophylum sensitivum; NF-PB signaling blockers that inhibit pro-inflammatory cytokine expression such as simvastatin; and inhibitors of IL-6 expression and NF-PB activation such as (-)-epigallocatechin-3-gallate (EGCG).
[0014] Other agents include human recombinant lactoferrin, which inhibits cellular release of proinflammatory cytokines and prometastatic cytokines (including IL-6, IL-8, granulocyte macrophage colony-stimulating factor and TNF-α) Inhibitors of dendritic cell derived IL-12 and IL-18 such as rapamycin and sanglifehrin are also suitable for use in the provided methods. Rapamycin is an immunosuppressant that inhibits T cell mTOR kinase activation, and Sanglifehrin A is a cyclophilin-binding immunosuppressant that also inhibits IL-2 dependent T cell proliferation. Also suitable for use in the provided methods is dietary rutin colitis, which suppresses the induction of pro-inflammatory cytokines such as IL-1β, IL-6, and GM-CS.
[0015] Optionally, the method further includes selecting a subject with a proliferative disorder, wherein the subject is in need of inhibition of a pro-inflammatory cytokine response. For example, such a subject may include a subject with little response to reovirus alone or with a progressive resistance to reovirus therapy.
[0016] As used herein, the term proliferative disorder refers to any cellular disorder in which the cells proliferate more rapidly than normal tissue growth. A proliferative disorder includes, but is not limited to, neoplasms, which are also referred to as tumors. A neoplasm can include, but is not limited to, pancreatic cancer, breast cancer, brain cancer (e.g., glioblastoma), lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, neurofibromatosis 1, and leukemia. A neoplasm can be a solid neoplasm (e.g., sarcoma or carcinoma) or a cancerous growth affecting the hematopoietic system (e.g., lymphoma or leukemia). Other proliferative disorders include, but are not limited to, neurofibromatosis.
[0017] Generally, in proliferating disorders for which reovirus is used as a treatment, at least some of the proliferating cells associated with the disorder may have a mutation in which the Ras gene (or an element of the Ras signaling pathway) is activated, either directly (e.g., by an activating mutation in Ras) or indirectly (e.g., by activation of an upstream or downstream element in the Ras pathway). Activation of an upstream element in the Ras pathway includes, for example, transformation with epidermal growth factor receptor (EGFR) or Sos. See, for example, Wiessmuller and Wittinghofer, 1994, Cellular Signaling 6(3):247-267; and Barbacid, 1987, Ann. Rev. Biochem. 56, 779-827. Activation of a downstream element in the Ras pathway includes, for example, mutation within B-Raf. See, for example, Brose et al., 2002, Cancer Res. 62:6997-7000. A proliferative disorder that results, at least in part, by the activation of ras, an upstream element of ras, or an element in the ras signaling pathway is referred to herein as a ras-mediated proliferative disorder. In addition, the reovirus is useful for treating proliferative disorders caused by mutations or dysregulation of PKR. See, for example, Strong et al., 1998, EMBO J. 17:3351-62.
[0018] Optionally, the provided methods further comprise the step of selecting a subject with a ras-mediated proliferative disorder. Optionally, the provided methods comprise the step of determining whether the proliferative disorder is a ras-mediated proliferative disorder. Such methods for determining whether a proliferative disorder has a certain phenotype are known. See, for example, U.S. Pat. No. 7,306,902, which is incorporated herein by reference in its entirety.
[0019] As used herein, reovirus refers to any virus classified in the reovirus genus, whether naturally occurring, modified, or recombinant. Reoviruses are viruses with a double-stranded, segmented RNA genome. The virions measure 60-80 nm in diameter and possess two concentric capsid shells, each of which is icosahedral. The genome consists of double-stranded RNA in 10-12 discrete segments with a total genome size of 16-27 kbp. The individual RNA segments vary in size. Three distinct but related types of reoviruses have been recovered from many species. All three types share a common complement-fixing antigen.
[0020] The human reovirus includes three serotypes: type 1 (strain Lang or T1L), type 2 (strain Jones, T2J), and type 3 (strain Dearing or strain Abney, T3D). The three serotypes are easily identifiable on the basis of neutralization and hemagglutinin-inhibition assays. A reovirus according to this disclosure can be a type 3 mammalian orthoreovirus. Type 3 mammalian orthoreoviruses include, without limitation, Dearing and Abney strains (T3D or T3A, respectively). See, for example, ATCC Accession Nos. VR-232 and VR-824.
[0021] The reovirus may be naturally occurring or modified. The reovirus is naturally-occurring when it can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory. For example, the reovirus can be from a field source, that is, from a human who has been infected with the reovirus. The reovirus may also be selected or mutagenized for enhanced oncolytic activity.
[0022] The reovirus may be modified but still capable of lytically infecting a mammalian cell having an active ras pathway. The reovirus may be chemically or biochemically pretreated (e.g., by treatment with a protease, such as chymotrypsin or trypsin) prior to administration to the proliferating cells. Pretreatment with a protease removes the outer coat or capsid of the virus and may increase the infectivity of the virus. The reovirus may be coated in a liposome or micelle (Chandran and Nibert, J. of Virology 72(1):467-75 1998). For example, the virion may be treated with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents to generate a new infectious subviral particle (ISVP).
[0023] The reovirus may be a recombinant reovirus. For example, the recombinant reovirus can be a reassortant reovirus, which includes genomic segments from two or more genetically distinct reoviruses. Reassortment of reovirus genomic segments may occur following infection of a host organism with at least two genetically distinct reoviruses. Reassortment of viruses can be generated in cell culture, for example, by co-infection of permissive host cells with genetically distinct reoviruses. Accordingly, the provided methods include the use of a recombinant reovirus resulting from reassortment of genome segments from two or more genetically distinct reoviruses, including but not limited to, human reovirus, such as type 1 (e.g., strain Lang), type 2 (e.g., strain Jones), and type 3 (e.g., strain Dearing or strain Abney); non-human mammalian reoviruses; or avian reovirus. Recombinant reovirus can also be made by genetic engineering, chemically synthesized, or treatment with chemical or physical mutagens. Optionally, the provided methods include the use of recombinant reoviruses resulting from reassortment of genome segments from two or more genetically distinct reoviruses wherein at least one parental virus is genetically engineered, comprises one or more chemically synthesized genomic segment, has been treated with chemical or physical mutagens, or is itself the result of a recombination event. Optionally, the provided methods include the use of the recombinant reovirus that has undergone recombination in the presence of chemical mutagens, including but not limited to, dimethyl sulfate and ethidium bromide, or physical mutagens, including but not limited to, ultraviolet light and other forms of radiation.
[0024] Optionally, the provided methods include the use of reoviruses with mutations (including insertions, substitutions, deletions or duplications) in one or more genome segments. Such mutations can comprise additional genetic information as a result of recombination with a host cell genome or can comprise synthetic genes. For example, mutant reoviruses as described herein can contain a mutation that reduces or essentially eliminates expression of a sigma3 polypeptide or that results in the absence of a functional sigma3 polypeptide as described in U.S. Ser. No. 12/124,522, which is incorporated by reference herein in its entirety. A mutation that eliminates expression of a sigma3 polypeptide or that results in the absence of a functional sigma3 polypeptide can be in the nucleic acid encoding the sigma3 polypeptide (i.e., the S4 gene) or in a nucleic acid that encodes a polypeptide that regulates the expression or function of the sigma3 polypeptide.
[0025] As used herein, a mutation that reduces the expression of a sigma3 polypeptide refers to a mutation that results in a decrease in the amount of sigma3 polypeptides, compared to a reovirus expressing wild type levels of sigma3 polypeptide, of at least 30% (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, or 95%). As used herein, a mutation that essentially eliminates expression of a sigma3 polypeptide refers to a mutation that results in a decrease in the amount of sigma3 polypeptides, relative to the amount of sigma3 polypeptides produced by a wild type reovirus, of at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%). As used herein, a mutation that results in a decrease in or absence of a functional sigma3 polypeptide refers to a mutation that allows expression of the sigma3 polypeptide but that results in a sigma3 polypeptide that is not able to assemble or incorporate into the viral capsid. It would be understood that it may be desirable or necessary for sigma3 polypeptides to retain other functionalities (e.g., the ability to bind RNA) in order that the mutant reovirus retain the ability to propagate.
[0026] A mutation in a sigma3 polypeptide as described herein can result in a sigma3 polypeptide that is incorporated into the capsid at levels that are reduced relative to a sigma3 polypeptide that does not contain the mutation (e.g., a wild type sigma3 polypeptide). A mutation in a sigma3 polypeptide as described herein also can result in a sigma3 polypeptide that cannot be incorporated into a viral capsid. Without being bound by any particular mechanism, a sigma3 polypeptide may have reduced function or lack function due, for example, to an inability of the sigma3 polypeptide and the mu1 polypeptide to bind appropriately, or due to a conformational change that reduces or prohibits incorporation of the sigma3 polypeptide into the capsid.
[0027] In addition to a mutation that abolishes or reduces expression of the sigma3 polypeptide or that results in a non-functional or reduced-function sigma3 polypeptide, a mutant reovirus as described herein also can contain one or more further mutations (e.g., a second, third, or fourth mutation) in one of the other reovirus capsid polypeptides (e.g., mu1, lambda2, and/or sigma1). Reoviruses containing a mutation affecting the sigma3 polypeptide and, optionally, a further mutation in any or all of the other outer capsid proteins can be screened for the ability of such mutant reoviruses to infect and cause lysis of cells. For example, neoplastic cells that are resistant to lysis by wild type reovirus can be used to screen for effective mutant reoviruses described herein.
[0028] For example, a further mutation can reduce or essentially eliminate expression of a mu1 polypeptide or result in the absence of a functional mu1 polypeptide. The mu1 polypeptide, which is encoded by the M2 gene, is likely involved in cell penetration and may play a role in transcriptase activation. Each virion contains about 600 copies of mu1 polypeptides, which are present in the form of 1:1 complexes with sigma3 polypeptides. The mu1 polypeptide is myristolated on its N-terminus, and then the myristolated N-terminal 42 residues are cleaved off, resulting in a C-terminal fragment (mu1C). Additionally or alternatively, a further mutation can reduce or essentially eliminate expression of a lambda2 polypeptide or result in the absence of a functional lambda2 polypeptide, and/or a further mutation can reduce or essentially eliminate expression of a sigma1 polypeptide or result in the absence of a functional sigma1 polypeptide. The lambda2 polypeptide is encoded by the L2 gene and is involved in particle assembly, and exhibits guanylyltransferase and methyltransferase activity. The sigma1 polypeptide is encoded by the S1 gene and is involved in cell-attachment and serves as the viral hemagglutinin.
[0029] For example, the reovirus has a lambda-3 polypeptide having one or more amino acid modifications; a sigma-3 polypeptide having one or more amino acid modifications; a mu-1 polypeptide having one or more amino acid modifications; and/or a mu-2 polypeptide having one or more amino acid modifications, as described in U.S. Serial No. 12/046,095, which is incorporated by reference herein in its entirety. By way of example, the one or more amino acid modifications in the lambda-3 polypeptide are a Val at residue 214, an Ala at residue 267, a Thr at residue 557, a Lys at residue 755, a Met at residue 756, a Pro at residue 926, a Pro at residue 963, a Leu at residue 979, an Arg at residue 1045, a Val at residue 1071, or any combination thereof, numbered relative to GenBank Accession No. M24734.1. It is noted that, when the amino acid sequence is a Val at residue 214 or a Val at residue 1071, the amino acid sequence further includes at least one additional change in the amino acid sequence. Optionally, the lambda-3 polypeptide includes the sequence shown in SEQ ID NO:18. Further by way of example, the one or more amino acid modifications in the sigma-3 polypeptide are a Leu at residue 14, a Lys at residue 198, or any combination thereof, numbered relative to GenBank Accession No. K02739. It is noted that, when the amino acid sequence is a Leu at residue 14, the amino acid sequence further includes at least one additional change in the amino acid sequence. Optionally, the sigma-3 polypeptide includes the sequence shown in SEQ ID NO:14. Further by way of example, the one or more amino acid modifications in the mu-1 polypeptide is an Asp at residue 73 numbered relative to GenBank Accession No. M20161.1. Optionally, the mu-1 polypeptide includes the sequence shown in SEQ ID NO:16. Also by way of example, the amino acid modification mu-2 polypeptide is a Ser at residue 528 numbered relative to GenBank Accession No. AF461684.1. Optionally, the mu-1 polypeptide includes the sequence shown in SEQ ID NO:15. A reovirus as described herein having one or more modifications can further include a reovirus sigma-2 polypeptide. Such a sigma-2 polypeptide has a Cys at one or more of position 70, 127, 195, 241, 255, 294, 296, or 340, numbered relative to GenBank Accession No. NP 694684.1. Optionally, the sigma-2 polypeptide includes the sequence shown in SEQ ID NO:12.
[0030] Optionally, the reovirus has a L1 genome segment having one or more nucleic acid modifications; a S4 genome segment having one or more nucleic acid modifications; a M1 genome segment having one or more nucleic acid modifications; and/or a M2 genome segment having one or more nucleic acid modifications, as described in U.S. Ser. No. 12/046,095, which is incorporated by reference herein in its entirety. By way of example, the one or more nucleic acid modifications in the L1 genome segment are a T at position 660, a G at position 817, an A at position 1687, a G at position 2283, an ATG at positions 2284-2286, a C at position 2794, a C at position 2905, a C at position 2953, an A at position 3153, or a G at position 3231, numbered relative to GenBank Accession No. M24734.1. Optionally, the L1 genome segment includes the sequence shown in SEQ ID NO:8. Further by way of example, the one or more nucleic acid modifications in the S4 genome segment is an A at position 74 and an A at position 624, numbered relative to GenBank Accession No. K02739. Optionally, the S4 genome segment includes the sequence shown in SEQ ID NO:4. Further by way of example, the nucleic acid modification in the M2 genome segment can be a C at position 248, numbered relative to GenBank Accession No. M20161.1. In one embodiment, the M2 genome segment includes the sequence shown in SEQ ID NO:6. Also by way of example, the nucleic acid modification in the M1 genome segment is a T at position 1595, numbered relative to GenBank Accession No. AF461684.1. Optionally, the M1 genome segment includes the sequence shown in SEQ ID NO:5. A reovirus as described herein can include any modification or combination of modifications disclosed herein. Optionally, a reovirus as described herein includes genomic segments having the sequences shown in SEQ ID NOs:1-10 or the polypeptides shown in SEQ ID NOs:11, 12, and 16-21, and either or both SEQ ID NO:13 or 14. Optionally, a reovirus as disclosed herein is identified as IDAC Accession No. 190907-01.
[0031] A mutation or modification as referred to herein can be a substitution, insertion or deletion of one or more nucleotides. Point mutations include, for example, single nucleotide transitions (purine to purine or pyrimidine to pyrimidine) or transversions (purine to pyrimidine or vice versa) and single- or multiple-nucleotide deletions or insertions. A mutation in a nucleic acid can result in one or more conservative or non-conservative amino acid substitutions in the encoded polypeptide, which may result in conformational changes or loss or partial loss of function, a shift in the reading frame of translation (frame-shift) resulting in an entirely different polypeptide encoded from that point on, a premature stop codon resulting in a truncated polypeptide (truncation), or a mutation in a reovirus nucleic acid may not change the encoded polypeptide at all (silent or nonsense). See, for example, Johnson and Overington, 1993, J. Mol. Biol. 233:716-38; Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89:10915-19; and U.S. Pat. No. 4,554,101 for disclosure on conservative and non-conservative amino acid substitutions.
[0032] Mutations can be generated in the nucleic acid of a reovirus using any number of methods known in the art. For example, site directed mutagenesis can be used to modify a reovirus nucleic acid sequence. One of the most common methods of site-directed mutagenesis is oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, an oligonucleotide encoding the desired change(s) in sequence is annealed to one strand of the DNA of interest and serves as a primer for initiation of DNA synthesis. In this manner, the oligonucleotide containing the sequence change is incorporated into the newly synthesized strand. See, for example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488; Kunkel et al., 1987, Meth. Enzymol. 154:367; Lewis and Thompson, 1990, Nucl. Acids Res. 18:3439; Bohnsack, 1996, Meth. Mol. Biol. 57:1; Deng and Nickoloff, 1992, Anal. Biochem. 200:81; and Shimada, 1996, Meth. Mol. Biol. 57:157. Other methods are used routinely in the art to modify the sequence of a protein or polypeptide. For example, nucleic acids containing a mutation can be generated using PCR or chemical synthesis, or polypeptides having the desired change in amino acid sequence can be chemically synthesized. See, for example, Bang and Kent, 2005, Proc. Natl. Acad. Sci. USA 102:5014-9 and references therein.
[0033] Nucleic acids from reovirus particles can be isolated using standard commercially available nucleic acid methodology. See also, for example, Schiff et al., "Orthoreoviruses and Their Replication," Ch 52, in Fields Virology, Knipe and Howley, eds., 2006, Lippincott Williams and Wilkins. As used herein, isolated nucleic acids refer to nucleic acids that are separated from other nucleic acids with which they are usually associated. Thus, an isolated nucleic acid includes, without limitation, reoviral nucleic acid that is essentially free of non-reoviral (e.g., host cell) nucleic acid, or a reoviral genomic segment that is essentially free of nucleic acid corresponding to other genomic segments. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant or synthetic nucleic acid.
[0034] A mutant reovirus as described herein can be generated by reconstituting genome segments containing at least a mutation or modification using methods known in the art. See, for example, Schiff et al., "Orthoreoviruses and Their Replication," Ch 52, in Fields Virology, Knipe and Howley, eds., 2006, Lippincott Williams and Wilkins; Smith et al., 1969, Virology 39(4):791-810; and U.S. Pat. Nos. 7,186,542; 7,049,127; 6,808,916; and 6,528,305. A mutant reovirus also can be generated by expressing the reovirus genome segments using a plasmid-based reverse genetic system to produce an ISVP. See, for example, Kobayashi et al., 2007, Cell Host and Microbe 1:147-57. As used herein, a genetically-engineered or mutant ISVP is a mutant reovirus and refers to an ISVP generated from a reovirus carrying a genetically-engineered or a spontaneously generated mutation affecting at least the sigma3 polypeptide. The ISVPs described herein are stable and can be propagated as ISVPs for multiple (e.g., more than one, e.g., 2, 3, 4, 5, 10, 20, 50, or more) passages.
[0035] The mutant reoviruses described herein, produced via a genetically-engineered ISVP or via a plasmid-based reverse genetic system, can be cultured in, for example, human neoplastic cells or L929 mouse fibroblast cells. Mutant reoviruses disclosed herein can be cultured in cells that are only permissive to reovirus strains lacking the sigma3 polypeptide. Using such cell lines to passage the mutant reoviruses described herein can allow for selection of the mutants and also can be used to reduce or prevent reversions of the mutation(s).
[0036] The mutant reoviruses described herein, optionally, exhibit increased infectivity and/or decreased immunogenicity as compared to a non-mutant reovirus (e.g., a control reovirus) and can be selected on the basis of such traits. Increased infectivity can be evidenced by an increase in the range of neoplastic cells and/or the number of cells that are infected by a mutant reovirus compared to a reovirus that expresses a functional sigma3 polypeptide (e.g., an intact virion; e.g., a wild type reovirus). Decreased immunogenicity of mutant reoviruses can be evidenced by the inability of such mutant reoviruses to induce a significant immune response in the subject. The mutant reoviruses described herein also can be screened and selected for other desirable traits including, but not limited to, a faster rate of replication; a faster rate of packaging; the ability to induce apoptosis; the ability to affect lysis in and effectively kill human neoplastic cells lines; the ability to release effective tumor epitopes; interaction with standard chemotherapies; and an increased number of viral progeny. Additionally, mutant reoviruses can be selected for the ability to lytically infect a neoplastic cell (e.g., a mammalian cell having an active Ras pathway). See, for example, U.S. Pat. No. 7,052,832.
[0037] The reovirus is optionally a reovirus modified to reduce or eliminate an immune reaction to the reovirus. Such a modified reovirus is referred to herein as an immunoprotected reovirus. Such modifications include, but are not limited to, packaging of the reovirus in a liposome, a micelle, or other vehicle to mask the reovirus from the immune system. Alternatively, the outer capsid of the reovirus virion particle may be removed since the proteins present in the outer capsid are the major determinant of the host humoral and cellular responses.
[0038] Reoviruses can be purified using standard methodology. See, for example, Schiff et al., "Orthoreoviruses and Their Replication," Ch 52, in Fields Virology, Knipe and Howley, eds., 2006, Lippincott Williams and Wilkins; Smith et al., 1969, Virology 39(4):791-810; and U.S. Pat. Nos. 7,186,542; 7,049,127; 6,808,916; and 6,528,305. As used herein, purified mutant reoviruses refer to reoviruses that have been separated from cellular components that naturally accompany them. Typically, reoviruses are considered purified when they are at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the proteins and other cellular components with which they are naturally associated.
[0039] The herein provided reoviruses and agents can be administered in vitro or in vivo in a pharmaceutically acceptable carrier. Thus, pharmaceutical compositions that include a reovirus and/or agent that inhibits pro-inflammatory cytokines as described herein are provided. See, for example, U.S. Pat. No. 6,576,234 regarding reoviruses. In addition to one or more reoviruses and/or agents that inhibit pro-inflammatory cytokines, a pharmaceutical composition typically includes a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be a solid, semi-solid, or liquid material that can act as a vehicle, carrier or medium for the reovirus. Thus, compositions containing a reovirus and/or the provided agents can be in the form of tablets, pills, powders, lozenges, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
[0040] Optionally, the compositions containing a reovirus are suitable for infusion. For intravenous infusions, there are two types of fluids that are commonly used, crystalloids and colloids. Crystalloids are aqueous solutions of mineral salts or other water-soluble molecules. Colloids contain larger insoluble molecules, such as gelatin; blood itself is a colloid. The most commonly used crystalloid fluid is normal saline, a solution of sodium chloride at 0.9% concentration, which is close to the concentration in the blood (isotonic). Ringer's lactate or Ringer's acetate is another isotonic solution often used for large-volume fluid replacement. A solution of 5% dextrose in water, sometimes called D5W, is often used instead if the patient is at risk for having low blood sugar or high sodium.
[0041] Some examples of suitable carriers include phosphate-buffered saline or another physiologically acceptable buffer, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. A pharmaceutical composition additionally can include, without limitation, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. Pharmaceutical compositions can be formulated to provide quick, sustained or delayed release of a mutant reovirus after administration by employing procedures known in the art. In addition to the representative formulations described below, other suitable formulations for use in a pharmaceutical composition can be found in Remington: The Science and Practice of Pharmacy (21th ed.) ed. David B. Troy, Lippincott Williams & Wilkins, 2005. For preparing solid compositions such as tablets, a mutant reovirus can be mixed with a pharmaceutical carrier to form a solid composition. Optionally, tablets or pills can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
[0042] Liquid formulations that include a reovirus and/or other agents for oral administration or for injection generally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
[0043] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. These liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. Such compositions can be administered by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, orally or nasally, from devices which deliver the formulation in an appropriate manner.
[0044] Another formulation that is optionally employed in the methods of the present disclosure includes transdermal delivery devices (e.g., patches). Such transdermal patches may be used to provide continuous or discontinuous infusion of a mutant reovirus as described herein. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Pat. No. 5,023,252. Such patches can be constructed for continuous, pulsatile, or on-demand delivery of mutant reoviruses.
[0045] As described above, reoviruses and/or other agents are, if necessary, coated in a liposome or micelle to reduce or prevent an immune response in a mammal that has developed immunity toward a reovirus. Such compositions are referred to as immunoprotected reoviruses and/or agents. See, for example, U.S. Pat. Nos. 6,565,831 and 7,014,847. In addition, a mutant reovirus as disclosed herein (e.g., one that lacks or is deficient in sigma3 polypeptide or function) can be proteolytically treated with an enzyme to remove or partially remove any of the other outer capsid proteins present.
[0046] In the provided methods, the reovirus is administered in a manner so that it can ultimately contact the target tumor or tumor cells, for example, systemically. The route by which the reovirus is administered, as well as the formulation, carrier or vehicle, depends on the location as well as the type of the target cells. A wide variety of administration routes can be employed. For example, for a solid tumor that is accessible, the reovirus can be administered by injection directly to the tumor. For a hematopoietic tumor, for example, the reovirus can be administered intravenously or intravascularly. For tumors that are not easily accessible within the body, such as metastases, the reovirus is administered in a manner such that it can be transported systemically through the body of the mammal and thereby reach the tumor (e.g., intravenously or intramuscularly). Alternatively, the reovirus can be administered directly to a single solid tumor, where it then is carried systemically through the body to metastases. The reovirus can also be administered subcutaneously, intraperitoneally, intrathecally (e.g., for brain tumor), topically (e.g., for melanoma), orally (e.g., for oral or esophageal cancer), rectally (e.g., for colorectal cancer), vaginally (e.g., for cervical or vaginal cancer), nasally, by inhalation spray or by aerosol formulation (e.g., for lung cancer).
[0047] Optionally, the virus is administered continuously to a subject at least once per day or up to throughout the day on consecutive days, for a period of time. Thus, the virus is administered, for example, to subjects by means of intravenous administration in any pharmacologically acceptable solution, or as an infusion over a period of time. For example, the substance may be administered systemically by injection (e.g., IM or subcutaneously) or taken orally daily at least once per day, or administered by infusion in a manner that results in the daily delivery into the tissue or blood stream of the subject. When the virus is administered by infusion over a period of time, the period of time is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours, or any time between 1 and 24 hours, inclusive, or more. Optionally, the period of time is 5, 15, 30, 60, 90, 120, 150 or 180 minutes, or any time between 5 and 180 minutes, inclusive, or more. Thus, for example, the virus is administered by infusion for 60 minutes or about 60 minutes. Administrations can be repeated daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, 28 days or any number of days between 2 and 28 days, inclusive, or longer.
[0048] Therapeutic agents, such as the agents that inhibit production of pro-inflammatory cytokines, of the provided methods are also administered via a wide variety of administration routes. Thus, the agents are administered via any of several routes of administration, including, topically, orally, parenterally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization/inhalation, or by instillation via bronchoscopy. Optionally, the therapeutic agents are administered continuously in the manner set forth in the description above with respect to oncolytic viruses. Thus, for example, the agent is administered, for example, to subjects by means of intravenous administration in any pharmacologically acceptable solution, or as an infusion over a period of time. Optionally, the agents are administered locally at or near the site of the tumor. Alternatively, the agents are administered systemically. The agents that inhibit pro-inflammatory cytokines are administered in an amount sufficient (i.e., an effective amount) to inhibit one or more pro-inflammatory cytokines. By way of example, effective amounts of platinum compounds include from about 5 to 1000 mg/m2 of tumor volume, or any amount in between 5 and 1000 mg/m2, inclusive, or more. Thus, for example effective amounts of cisplatin include from about 175-200 mg/m2 and effective mounts for carboplatin include from about 100-600 mg/m2. Effective amounts of other agents range from 0.001-10,000 mg/kg body weight or any amount in between 0.001 and 10,000 mg/kg body weight, inclusive. Optionally, effective amounts of platinum compounds include approximately 2 to 7 mg/mL minute (AUC) as calculated by the Calvert formula. Optionally, effective amounts of platinum compounds include approximately 5 or 6 mg/mL minute (AUC) as calculated by the Calvert formula. Optionally, the platinum compounds are administered as an intravenous infusion over a period of 30 minutes.
[0049] The reovirus is administered in an amount that is sufficient to treat the proliferative disorder (e.g., an effective amount). A proliferative disorder is treated when administration of a reovirus to proliferating cells affects lysis (e.g., oncolysis) of the affected cells, resulting in a reduction in the number of abnormally proliferating cells, a reduction in the size of a neoplasm, and/or a reduction in or elimination of symptoms (e.g., pain) associated with the proliferating disorder. As used herein, the term oncolysis means at least 10% of the proliferating cells are lysed (e.g., at least about 20%, 30%, 40%, 50%, or 75% of the cells are lysed). The percentage of lysis can be determined, for example, by measuring the reduction in the size of a neoplasm or in the number of proliferating cells in a mammal, or by measuring the amount of lysis of cells in vitro (e.g., from a biopsy of the proliferating cells). An effective amount of a virus will be determined on an individual basis and may be based, at least in part, on the particular virus used; the individual's size, age, gender; and the size and other characteristics of the abnormally, proliferating cells. For example, for treatment of a human, approximately 103 to 1012 plaque forming units (PFU) of a virus are used, depending on the type, size and number of proliferating cells or neoplasms present. The effective amount can be, for example, from about 1.0 PFU/kg body weight to about 1015 PFU/kg body weight (e.g., from about 102 PFU/kg body weight to about 1013 PFU/kg body weight). Optionally, the effective amount is about 1×108 to about 1×1012 TCID50. Optionally, the effective amount is about 1×1010 TCID50.
[0050] By way of example, 5-6 mg/ml minute (AUC as calculated by the Calvert formula) of an agent that inhibits pro-inflammatory cytokines, such as carboplatin, is administered to the subject and 1×1010 TCID50 to 3×1010 TCID50 of a reovirus is administered to the subject. Optionally, the agent that inhibits pro-inflammatory cytokines is administered as a thirty minute to one hour intravenous infusion. Optionally, the reovirus is administered as a one hour intravenous infusion.
[0051] Optimal dosages of viruses and therapeutic agents and compositions comprising viruses and agents depend on a variety of factors. The exact amount required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the particular virus or vector used and its mode of administration. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the guidance provided herein.
[0052] Effective dosages and schedules for administering the compositions may be determined empirically. For example, animal models for a variety of proliferative disorders can be obtained from The Jackson Laboratory, 600 Main Street, Bar Harbor, Me. 04609 USA. Both direct (e.g., histology of tumors) and functional measurements (e.g., survival of a subject or size of a tumor) can be used to monitor response to therapies. These methods involve the sacrifice of representative animals to evaluate the population, increasing the animal numbers necessary for the experiments. Measurement of luciferase activity in the tumor provides an alternative method to evaluate tumor volume without animal sacrifice and allowing longitudinal population-based analysis of therapy.
[0053] The dosage ranges for the administration of compositions are those large enough to produce the desired effect in which the symptoms of the disease are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions and anaphylactic reactions. The dosage can be adjusted by the individual physician in the event of any counterindications.
[0054] Dosages vary and are administered in one or more dose administrations daily, for one or several days. The provided viruses and therapeutic agents are administered in a single dose or in multiple doses (e.g., two, three, four, six, or more doses). For example, where the administration is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Treatment may last from several days to several months or until diminution of the disease is achieved.
[0055] Combinations of the provided viruses and therapeutic agents are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term combination is used to refer to concomitant, simultaneous, or sequential administration of two or more agents. By way of example, the agent that inhibits proinflammatory cytokines is administered prior to or at the same time as the oncolytic virus. When one compound is administered prior to another compound, the first compound is administered minutes, hours, days, or weeks prior to administration of the second compound. For example, the first compound can be administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 36, 48, 60, or 72 hours, or any time between 1 and 72 hours, inclusive, prior to administration of a second compound. Optionally, the first compound is administered more than 72 hours prior to the second compound. By way of another example, the first compound can be administered at 1, 5, 15, 30, 60, 90, or 120 minutes, or any time between 1 and 120 minutes, inclusive, prior to administration of a second compound. Optionally, the first compound is administered at 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days, or any amount in between 1 and 28, inclusive, days prior to administration of the second compound. Optionally, the first compound is administered more than 28 days prior to the second compound. For example, the agent that inhibits proinflammatory cytokines is administered from about 1 to 8 hours prior to administration of the oncolytic virus. By way of another example, the agent that inhibits pro-inflammatory cytokines is administered at a time of about one hour prior to administration of the oncolytic virus.
[0056] By way of example, one cycle of treatment includes administering the agent that inhibits proinflammatory cytokines and the oncolytic virus includes on day 1. On days 2, 3, 4 and 5, only the oncolytic virus is administered to the subject. Optionally, the subject receives multiple cycles of treatment, for example, two, three, four, five or more cycles of treatment.
[0057] Reoviruses or a pharmaceutical composition comprising such reoviruses are optionally packaged into a kit. The kit also includes one or more agents or pharmaceutical compositions comprising such agents that inhibit pro-inflammatory cytokines It is contemplated that a kit, optionally, also includes one or more chemotherapeutic agents, one or more immunosuppressive agents, and/or one or more anti-antireovirus antibodies. A pharmaceutical composition can be formulated in a unit dosage form. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of a mutant reovirus calculated to produce the desired therapeutic effect in association with a suitable pharmaceutically acceptable carrier.
[0058] It is contemplated that the provided methods may be combined with other tumor therapies such as chemotherapy, radiotherapy, surgery, hormone therapy and/or immunotherapy. Thus, the oncolytic virus may be administered in conjunction with surgery or removal of the neoplasm. Therefore, provided herewith are methods for the treatment of a solid neoplasm comprising surgical removal of the neoplasm and administration of an oncolytic virus at or near to the site of the neoplasm.
[0059] It is further contemplated that the compositions in the provided methods are, optionally, administered in conjunction with or in addition to known anticancer compounds or chemotherapeutic agents. Chemotherapeutic agents are compounds which may inhibit the growth of tumors. Such agents, include, but are not limited to. 5-fluorouracil, mitomycin C, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracycline (Epirubicin and Doxurubicin), antibodies to receptors, such as herceptin, etopside, pregnasome, hormone therapies such as tamoxifen and anti-estrogens, interferons, aromatase inhibitors, progestational agents and LHRH analogs.
[0060] As used herein the terms treatment, treat, treating or ameliorating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction or amelioration in the severity of an established disease or condition or symptom of the disease or condition. For example, the method for treating cancer is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to control. Thus the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any percent reduction in between 10 and 100 as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition or symptoms of the disease or condition.
[0061] As used herein, references to decreasing, reducing, or inhibiting include a change of 10, 20, 30, 40, 50, 60, 70, 80, 90 percent or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.
[0062] As used herein, the term subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject with a disease or disorder. The term patient or subject includes human and veterinary subjects.
[0063] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if an inhibitor is disclosed and discussed and a number of modifications that can be made to a number of molecules including the inhibitor are discussed, each and every combination and permutation of the inhibitor, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
[0064] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.
[0065] A number of aspects have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, other aspects are within the scope of the claims.
EXAMPLES
Example 1
Anti-Tumor Activity of Reovirus and Cisplatin in Mouse Melanoma Model
[0066] Reovirus type 3 Dearing (RV) has demonstrated oncolytic activity in numerous in vitro systems, in vivo murine models and early clinical trials. To further these studies, the in vitro and in vivo oncolytic activity of RV in combination with cisplatin (CP), a pseudoalkylating chemotherapeutic which causes DNA cross-linking and is active in a wide range of cancers, was examined. The effect of RV and CP was assessed in vitro for synergistic tumor kill and mechanism of tumor death. A synergistic interaction (combination index value (CIV) of less than one) was observed between RV and CP (CIV: ED50, 0.42±0.03; ED75, 0.30±0.02; ED90, 0.24±0.01) on B16.F10 cells. Flow cytometric analysis showed a marked increase in apoptotic cells following combined exposure, compared to single agent exposure.
[0067] For in vivo evaluation, subcutaneous B16.F10 tumors in C57Bl/6 mice or K1735 tumors in C3H mice were treated with intratumoral (i.t.) RV and intraperitoneal (i.p.) CP either alone or in combination. Tumor volume was estimated thrice weekly. Tumors and organs were harvested post-treatment for viral retrieval and histology; serum samples were tested for cytokine production and induction of neutralising anti-reovirus antibody (NARA). FIGS. 1A, 1B, 1C and 1D show reduced tumor growth and increased survival following reovirus/cisplatin combination therapy. C57Bl/6 (FIGS. 1A and 1C) and C3H (FIGS. 1B and 1D) mice bearing subcutaneous B16.F10 and K1735 tumors respectively were treated on days 1 and 4 with either reovirus alone i.t. (squares), cisplatin alone i.p. (triangles), or reovirus and cisplatin in combination (circles). Control treated mice (diamonds) received PBS. Tumors were measured on the days indicated and tumor volume expressed as tumor volume relative to volume at commencement of treatment (FIGS. 1A and 1B). Mice were euthanized when tumors exceeded 15 mm in any one dimension. Survival is expressed as Kaplan-Myer plots (FIGS. 1C and 1D). These data show reduced tumor growth and extended median survival time was observed in mice treated with RV/CP combination therapy compare to single agent treatments (FIGS. 1A, 1B, 1C and 1D). Mean relative tumor volumes ±SD at day 12 were, control: all reached endpoint, RV alone: 8.92±6.94, CP alone: 9.87±2.80, RV plus CP: 3.86±2.24. Median survival (days) were, control: 6, RV: 12, CP: 8, combination of RV and CP: 17. Live virus was recovered from the tumors of all RV only treated animals and from the liver and heart of 1/6 mice. In contrast live virus was detected in only 50% of tumors from combination treated mice but in the liver of 4/6 mice. CP did not affect the neutralizing anti-reovirus antibody (NARA) response to RV (FIG. 2), but caused a marked attenuation of production of proinflammatory cytokines to RV when used in combination (FIGS. 3A, 3B, 3C, 3D, 3E, 3F, and 3G).
[0068] Taken together, these results show that the addition of chemotherapeutic agents can significantly enhance the anti-tumor efficacy of RV therapy. Furthermore, a reduction in viral inflammatory responses in vital organs by concomitant chemotherapy may allow more intensive dosing schedules to increase overall efficacy of the reovirus.
Example 2
Reovirus and Carboplatin Protocols for Humans
[0069] This is a study design of reovirus given intravenously with carboplatin every 3 weeks.
[0070] Carboplatin is administered as a 30 minute intravenous infusion at a dose calculated by the Calvert formula (AUC 5 mg/mL minute or 6 mg/mL minute with GFR measured by 51Cr EDTA). Reovirus is then administered as a 1 hour intravenous infusion at a dose of 1×1010 or 3×1010 TCID50.
[0071] On days 2 through 5, only reovirus will be administered, using the same dose and method as used on Day 1.
TABLE-US-00001 TABLE 2 Dosing Methods Carboplatin Dose Reovirus dose AUC mg/mL min (TCID50) Day 1 only Days 1-5 Method 1 5 1 × 1010 Method 2 5 3 × 1010 Method 3 6 1 × 1010 Method 4 6 3 × 1010
Sequence CWU
1
1
2111416DNAReovirus 1gctattggtc ggatggatcc tcgcctacgt gaagaagtag tacggctgat
aatcgcatta 60acgagtgata atggagcatc actgtcaaaa gggcttgaat caagggtctc
ggcgctcgag 120aagacgtctc aaatacactc tgatactatc ctccggatca cccagggact
cgatgatgca 180aacaaacgaa tcatcgctct tgagcaaagt cgggatgact tggttgcatc
agtcagtgat 240gctcaacttg caatctccag attggaaagc tctatcggag ccctccaaac
agttgtcaat 300ggacttgatt cgagtgttac ccagttgggt gctcgagtgg gacaacttga
gacaggactt 360gcagagctac gcgttgatca cgacaatctc gttgcgagag tggatactgc
agaacgtaac 420attggatcat tgaccactga gctatcaact ctgacgttac gagtaacatc
catacaagcg 480gatttcgaat ctaggatatc cacgttagag cgcacggcgg tcactagcgc
gggagctccc 540ctctcaatcc gtaataaccg tatgaccatg ggattaaatg atggactcac
gttgtcaggg 600aataatctcg ccatccgatt gccaggaaat acgggtctga atattcaaaa
tggtggactt 660cagtttcgat ttaatactga tcaattccag atagttaata ataacttgac
tctcaagacg 720actgtgtttg attctatcaa ctcaaggata ggcgcaactg agcaaagtta
cgtggcgtcg 780gcagtgactc ccttgagatt aaacagtagc acgaaggtgc tggatatgct
aatagacagt 840tcaacacttg aaattaattc tagtggacag ctaactgtta gatcgacatc
cccgaatttg 900aggtatccga tagctgatgt tagcggcggt atcggaatga gtccaaatta
taggtttagg 960cagagcatgt ggataggaat tgtctcctat tctggtagtg ggctgaattg
gagggtacag 1020gtgaactccg acatttttat tgtagatgat tacatacata tatgtcttcc
agcttttgac 1080ggtttctcta tagctgacgg tggagatcta tcgttgaact ttgttaccgg
attgttacca 1140ccgttactta caggagacac tgagcccgct tttcataatg acgtggtcac
atatggagca 1200cagactgtag ctatagggtt gtcgtcgggt ggtgcgcctc agtatatgag
taagaatctg 1260tgggtggagc agtggcagga tggagtactt cggttacgtg ttgagggggg
tggctcaatt 1320acgcactcaa acagtaagtg gcctgccatg accgtttcgt acccgcgtag
tttcacgtga 1380ggatcagacc accccgcggc actggggcat ttcatc
141621331DNAReovirus 2gctattcgct ggtcagttat ggctcgcgct
gcgttcctat tcaagactgt tgggtttggt 60ggtctgcaaa atgtgccaat taacgacgaa
ctatcttcac atctactccg agctggtaat 120tcaccatggc agttaacaca gtttttagac
tggataagcc ttgggagggg tttagctaca 180tcggctctcg ttccgacggc tgggtcaaga
tactatcaaa tgagttgcct tctaagtggc 240actctccaga ttccgttccg tcctaaccac
cgatggggag acattaggtt cttacgctta 300gtgtggtcag ctcctactct cgatggatta
gtcgtagctc caccacaagt tttggctcag 360cccgctttgc aagcacaggc agatcgagtg
tacgactgcg atgattatcc atttctagcg 420cgtgatccaa gattcaaaca tcgggtgtat
cagcaattga gtgctgtaac tctacttaac 480ttgacaggtt ttggcccgat ttcctacgtt
cgagtggatg aagatatgtg gagtggagat 540gtgaaccagc ttctcatgaa ctatttcggg
cacacgtttg cagagattgc atacacattg 600tgtcaagcct cggctaatag gccttgggaa
tatgacggta catatgctag gatgactcag 660attgtgttat ccttgttctg gctatcgtat
gtcggtgtaa ttcatcagca gaatacgtat 720cggacattct attttcagtg taatcggcga
ggtgacgccg ctgaggtgtg gattctttct 780tgttcgttga accattccgc acaaattaga
ccgggtaatc gtagcttatt cgttatgcca 840actagcccag attggaacat ggacgtcaat
ttgatcctga gttcaacgtt gacggggtgt 900ttgtgttcgg gttcacagct gccactgatt
gacaataatt cagtacctgc agtgtcgcgt 960aacatccatg gctggactgg tagagctggt
aaccaattgc atgggttcca ggtgagacga 1020atggtgactg aattttgtga caggttgaga
cgcgatggtg tcatgaccca agctcagcag 1080aatcaagttg aagcgttggc agatcagact
caacagttta agagggacaa gctcgaaacg 1140tgggcgagag aagacgatca atataatcag
gctcatccca actccacaat gttccgtacg 1200aaaccattta cgaatgcgca atggggacga
ggtaatacgg gggcgactag tgccgcgatt 1260gcagccctta tctgatcgtc ttggagtgag
ggggtccccc cacacacctc acgactgacc 1320acacattcat c
133131198DNAReovirus 3gctaaagtca
cgcctgtcgt cgtcactatg gcttcctcac tcagagctgc gatctccaag 60atcaagaggg
atgacgtcgg tcagcaagtt tgtcctaatt atgtcatgct gcggtcctct 120gtcacaacaa
aggtggtacg aaatgtggtt gagtatcaaa ttcgtacggg cggattcttt 180tcgtgcttag
ctatgctaag gccactccag tacgctaagc gtgagcgttt gcttggtcag 240aggaatctgg
aacgtatatc gactagggat atccttcaga ctcgtgattt acactcacta 300tgtatgccaa
ctcctgatgc gccaatgtct aatcatcaag catccaccat gagagagctg 360atttgcagtt
acttcaaggt cgatcatgcg gatgggttga aatatatacc catggatgag 420agatactctc
cgtcatcact tgccagattg tttaccatgg gcatggctgg gctgcacatt 480accactgagc
catcttataa gcgtgttccg attatgcact tagctgcgga cttggactgt 540atgacgctgg
ctctacctta catgattacg cttgatggtg atactgtggt tcctgtcgct 600ccaacactgt
cagcggaaca gcttctggac gacggactca aaggattagc atgcatggat 660atctcctatg
gatgtgaggt ggacgcgaat agccggccgg ctggtgatca gagtatggac 720tcttcacgct
gcatcaacga gttgtattgc gaggagacag cagaagccat ctgtgtgctt 780aagacatgcc
ttgtgttaaa ttgcatgcag tttaaacttg agatggatga cctagcacat 840aacgctgctg
agctggacaa gatacagatg atgataccct tcagtgagcg tgtttttagg 900atggcctcgt
cctttgcgac tattgatgcc cagtgtttta ggttttgcgt gatgatgaag 960gataaaaatc
tgaaaataga tatgcgtgaa acgacgagac tgtggactcg ttcagcatca 1020gatgattctg
tggccacgtc atctttaagt atttccctgg accggggtcg atgggtggcg 1080gctgacgcca
gtgatgctag actgctggtt tttccgattc gcgtgtaatg ggtgagtgag 1140ctgatgtggt
cgccaagaca tgtgccggtg tcttggtggt gggtgacgcc taatcatc
119841196DNAReovirus 4gctatttttg cctcttccca gacgttgtcg caatggaggt
gtgcttgccc aacggtcatc 60aggtcgtgga cttaattaac aacgcttttg aaggtcgtgt
atcaatctac agcgcgcaag 120agggatggga caaaacaatc tcagcacagc cagatatgat
ggtatgtggt ggcgccgtcg 180tttgcatgca ttgtctaggt gttgttggat ctctacaacg
caagctgaag catttgcctc 240accatagatg taatcaacag atccgtcatc aggattacgt
cgatgtacag ttcgcagacc 300gtgttactgc tcactggaag cggggtatgc tgtccttcgt
tgcgcagatg cacgagatga 360tgaatgacgt gtcgccagat gacctggatc gtgtgcgtac
tgagggaggt tcactagtgg 420agctgaaccg gcttcaggtt gacccaaatt caatgtttag
atcaatacac tcaagttgga 480cagatccttt gcaggtggtg gacgaccttg acactaagct
ggatcagtac tggacagcct 540taaacctgat gatcgactca tccgacttga tacccaactt
tatgatgaga gacccatcac 600acgcgttcaa tggtgtgaaa ctgaagggag atgctcgtca
aacccaattc tccaggactt 660ttgattcgag atcgagtttg gaatggggtg tgatggttta
tgattactct gagctggatc 720atgatccatc gaagggccgt gcttacagaa aggaattggt
gacgccagct cgagatttcg 780gtcactttgg attatcccat tattctaggg cgactacccc
aatccttgga aagatgccgg 840ccgtattctc aggaatgttg actgggaact gtaaaatgta
tccattcatt aaaggaacgg 900ctaagctgaa gacagtgcgc aagctagtgg aggcagtcaa
tcatgcttgg ggtgtcgaga 960agattagata tgctcttggg ccaggtggca tgacgggatg
gtacaatagg actatgcaac 1020aggcccccat tgtgctaact cctgctgctc tcacaatgtt
cccagatacc atcaagtttg 1080gggatttgaa ttatccagtg atgattggcg atccgatgat
tcttggctaa acacccccat 1140cttcacagcg ccgggcttga ccaacctggt gtgacgtggg
acaggcttca ttcatc 119652304DNAReovirus 5gctattcgcg gtcatggctt
acatcgcagt tcctgcggtg gtggattcac gttcgagtga 60ggctattgga ctgctagaat
cgtttggagt agacgctggg gctgacgcga atgacgtttc 120atatcaagat catgactatg
tgttggatca gttacagtac atgttagatg gatatgaggc 180tggtgacgtt atcgatgcac
tcgtccacaa gaattggtta catcactctg tctattgctt 240gttgccaccc aaaagtcaac
tattagagta ttggaaaagt aatccttcag cgataccgga 300caacgttgat cgtcggcttc
gtaaacgact aatgctaaag aaagatctca ggaaagatga 360tgaatacaat cagctagcgc
gtgctttcaa gatatcggat gtctacgcac ctctcatctc 420atccacgacg tcaccgatga
caatgataca gaacttgaat cgaggcgaga tcgtgtacac 480cacgacggac agggtaatag
gggctagaat cttgttatat gctcctagaa agtactatgc 540gtcaactctg tcatttacta
tgactaagtg catcattccg tttggtaaag aggtgggtcg 600tgttcctcac tctcgattta
atgttggcac atttccgtca attgctaccc cgaaatgttt 660tgtcatgagt ggggttgata
ttgagtccat cccaaatgaa tttatcaagt tgttttacca 720gcgcgtcaag agtgttcacg
ctaacatact aaatgacata tctcctcaga tcgtctctga 780catgataaac agaaagcgtc
tgcgcgttca tactccatca gatcgtcgag ccgcgcagtt 840gatgcatttg ccttaccatg
ttaaacgagg agcgtctcac gtcgacgttt acaaggtgga 900tgttgtagac atgttgttcg
aggtagtgga tgtggccgat gggttgcgca acgtatctag 960gaaactaact atgcataccg
ttcctgtatg tattcttgaa atgttgggta ttgagattgc 1020ggactattgc attcgtcaag
aggatggaat gctcacagat tggttcctac ttttaaccat 1080gctatctgat ggcttgactg
atagaaggac gcattgtcaa tacttgatta atccgtcaag 1140tgtgcctcct gatgtgatac
ttaacatctc aattactgga tttataaata gacatacaat 1200cgatgtcatg cctgacatat
atgacttcgt taaacccatt ggcgctgtgc tgcctaaggg 1260atcatttaaa tcaacaatta
tgagagttct tgattcaata tcaatattag gaatccaaat 1320catgccgcgc gcgcatgtag
ttgactcaga tgaggtgggc gagcaaatgg agcctacgtt 1380tgagcaggcg gttatggaga
tatacaaagg gattgctggc gttgactcgc tggatgatct 1440catcaagtgg gtgttgaact
cggatctcat tccgcatgat gacaggcttg gtcaattatt 1500tcaagcgttt ttgcctctcg
caaaggactt attagctcca atggccagaa agttttatga 1560taactcaatg agtgagggta
gattgctaac attctctcat gccgacagtg agttgctgaa 1620cgcaaattat tttggtcatt
tattgcgact aaaaatacca tatattacag aggttaatct 1680gatgattcgc aagaatcgtg
agggtggaga gctatttcag cttgtgttat cttatctata 1740taaaatgtat gctactagcg
cgcagcctaa atggtttgga tcattattgc gattgttaat 1800atgtccctgg ttacatatgg
agaaattaat aggagaagca gacccggcat ctacgtcggc 1860tgaaattggg tggcatatcc
ctcgtgaaca gctgatgcaa gatggatggt gtggatgtga 1920agacggattc attccctatg
ttagcatacg tgcgccaaga ctggttatag aggagttgat 1980ggagaagaac tggggccaat
atcatgccca agttattgtc actgatcagc ttgtcgtagg 2040cgaaccgcgg agggtatctg
ctaaggctgt gatcaagggt aaccacttac cagttaagtt 2100agtttcacga tttgcatgtt
tcacattgac ggcgaagtat gagatgaggc tttcgtgcgg 2160ccatagcact ggacgtggag
ctgcatacag tgcgagacta gctttccgat ctgacttggc 2220gtgatccgtg acatgcgtag
tgtgacacct gctcctaggt caatgggggt agggggcggg 2280ctaagactac gtacgcgctt
catc 230462204DNAReovirus
6ggctaatctg ctgaccgtta ctctgcaaag atggggaacg cttcctctat cgttcagacg
60atcaacgtca ctggagatgg caatgtattt aaaccatcag ctgaaacttc atctaccgct
120gtaccatcgt taagcttatc acctggaatg ctgaatcccg gaggggtacc atggattgct
180gttggagatg agacatctgt gacttcacca ggcgcattac gtcgaatgac gtcaaaggac
240atcccggaca cggcaataat caacacagac aattcatcag gcgccgtgcc aagcgaatca
300gccttggtgc cctacatcga tgagccgctg gtagtggtta cagagcatgc tattaccaac
360ttcaccaaag ctgagatggc acttgaattc aatcgtgagt tccttgacaa gatgcgtgtg
420ctgtcagtgt caccaaaata ttcggatctt ctgacctatg ttgactgcta cgtcggtgtg
480tctgctcgtc aggctttaaa caattttcag aaacaagtgc ctgtgattac acctactagg
540cagacgatgt atgtcgactc gatacaagcg gccttgaaag ctttagaaaa gtgggagatt
600gatctgagag tggctcaaac gttgctgcct acgaacgttc cgattggaga agtctcttgt
660ccaatgcagt cggtagtgaa actgctggat gatcagctgc cagatgacag cctgatacgg
720aggtatccca aggaagccgc cgtcgctttg gctaaacgaa acgggggaat acaatggatg
780gacgtatcag aaggcaccgt gatgaacgag gctgtcaacg ctgttgcagc tagtgcactg
840gcaccttcag catcagcccc acccttagaa gagaagtcaa agttaaccga acaagcgatg
900gatctcgtga ccgcggctga gcctgagata attgcctcac tcgcgccagt tcccgcaccc
960gtgtttgcca taccacctaa accagcagat tataatgtgc gtactctgag gatcgacgag
1020gccacttggc tgcgaatgat tccaaaatca atgaacacac cttttcaaat ccaggtgact
1080gataacacag gaactaattg gcatctcaat ttgagggggg ggactcgtgt agtgaatctg
1140gaccaaatcg ctccgatgcg gtttgtatta gatctagggg gaaagagtta taaagagacg
1200agctgggatc caaacggcaa gaaggtcgga ttcatcgttt ttcaatcgaa gataccattc
1260gaactttgga ctgctgcttc acagatcggt caagccacgg tggttaacta tgtccaacta
1320tacgctgaag acagctcatt taccgcgcag tctatcattg ctactacctc tttggcttat
1380aactatgagc ctgagcagtt gaataagact gaccctgaga tgaattatta tcttttggcg
1440acctttatag actcagccgc tataacgcca acgaatatga cacagcctga tgtttgggat
1500gccttgctga cgatgtcccc actatcagct ggcgaggtga cagtgaaggg tgcggtagtg
1560agtgaagtag tccctgcaga cttgataggt agctacactc cagaatccct aaacgcctca
1620cttccgaatg atgctgctag atgcatgatc gatagagctt cgaagatagc cgaagcaatc
1680aagattgatg atgatgctgg accagatgaa tattccccaa actctgtacc aattcaaggt
1740cagcttgcta tctcgcaact cgaaactgga tatggtgtgc gaatattcaa ccctaaaggg
1800atcctttcta aaattgcatc tagggcaatg caggctttca ttggtgaccc gagcacaatc
1860atcacgcagg cggcgccagt gttatcagac aagaataatt ggattgcatt ggcacaggga
1920gtgaaaacta gtctgcgtac taaaagtcta tcagcgggag tgaagactgc agtgagtaag
1980ctgagctcat ctgagtctat ccagaattgg actcaaggat tcttggataa agtgtcagcg
2040cattttccag caccaaagcc cgattgtccg actagcggag atagtggtga atcgtctaat
2100cgccgagtga agcgcgactc atacgcagga gtggtcaaac gtgggtacac acgttaggcc
2160gctcgccctg gtgacgcggg gttaagggat gcaggcaaat catc
220472241DNAReovirus 7gctaaagtga ccgtggtcat ggcttcattc aagggattct
ccgccaacac tgttccagtt 60tctaaggcca agcgtgacat atcatctctt gccgctactc
ctggacttcg ttcacaatcc 120ttcactccgt ctgtggatat gtctcaatcg cgtgaattcc
tcacaaaggc aattgagcaa 180gggtccatgt ctatacctta tcagcatgtg aatgtaccga
aagttgatcg taaagttgtt 240agcctggtag tgcgaccttt ctcttcaggt gctttctcta
tctctggagt gatttcgcca 300gcccatgcct atctactaga gtgtctaccc cagcttgagc
aggcgatggc ttttgttgct 360tcacctgagt ctttccaggc ttccgacgtc gcgaagcgct
ttgccataaa gccaggtatg 420agcctccagg atgccatcac tgcctttatt aactttgtgt
ccgcgatgct gaaaatgacg 480gtgactcgtc aaaactttga cgttattgtg gctgagatcg
agaggcttgc ttcaaccagc 540gtgtccgtca ggactgaaga agcgaaggtt gctgatgagg
agctaatgct attcgggtta 600gatcatagag ggccacagca gctggatgtt tctgacgcta
aagggataat gaaggctgct 660gatattcaga caactcatga tgtccatttg gcaccaggcg
ttggtaatat tgatcctgaa 720atctataacg aggggcggtt catgttcatg cagcacaagc
cacttgcggc ggatcaatcg 780tatttcacct tggagactgc ggattatttc aagatttatc
caacatacga tgaacatgat 840ggcaggatgg ctgaccaaaa gcagtcggga ttgatactgt
gtactaagga cgaggtattg 900gctgagcaaa ctatatttaa actggacgcc cctgatgaca
agactgttca tctgttggat 960cgcgatgacg accacgttgt tgccagattt actaaggtat
ttatagagga cgtggctccc 1020gggcatcatg ctgctcaaag atcgggacaa cgctctgtgc
ttgatgacct atatgcgaat 1080acgcaagtga tttccattac ttctgctgct ttaaagtggg
tggtcaagca cggcgtatct 1140gatggaatcg tgaacaggaa gaatgtcaaa gtgtgtgttg
gttttgaccc cctgtacacc 1200ttgtctacac ataacggggt gtccttatgt gccctgctga
tggacgaaaa actctctgtg 1260ctgaacagtg cgtgtcgtat gacgttacgc tcactcatga
agaccggacg cgacgttgat 1320gcacacagag cttttcagcg agtcctctct caaggataca
catcgctaat gtgctactat 1380catccttcac ggaagttggc atatggtgag gtgctctttc
tagaacgatc caatgacgtg 1440acagatggga tcaagcttca gttggacgca tctagacagt
gtcatgaatg tcctgtgttg 1500cagcagaaag tggttgagtt agagaaacag attattatgc
agaagtcaat ccagtcagac 1560cctaccccag tggcgctgca accattgttg tctcagttgc
gtgagttgtc tagtgaagtt 1620actaggctac agatggagtt gagtcgagct cagtccctga
atgctcagtt ggaggcggat 1680gtcaagtcag ctcaatcatg tagcttggat atgtatctga
gacaccacac ttgcattaat 1740ggtcatgcta aagaagatga attgcttgac gctgtgcgtg
tcgcgccgga tgtgaggaga 1800gaaatcatgg aaaagaggag tgaagtgaga caaggttggt
gcgaacgtat ttctaaggaa 1860gcagctgcca aatgtcaaac tgttattgat gacctgactt
tgatgaatgg aaagcaagca 1920caagagataa cagaattacg tgattcggct gaaaaatatg
agaaacagat tgcagagctg 1980gtgagtacca tcacccaaaa ccagataacg tatcagcaag
agctacaagc cttggtagcg 2040aaaaatgtgg aattggacgc gttgaatcag cgtcaggcta
agtctttgcg tattactccc 2100tctcttctat cagccactcc tatcgattca gttgatgatg
ttgctgactt aattgatttc 2160tctgttccaa ctgatgagtt gtaaataatc cgtgatgcag
tgttgcccta atcccttaag 2220ccttcccgac ccccattcat c
224183854DNAReovirus 8gctacacgtt ccacgacaat
gtcatccatg atactgactc agtttggacc gttcattgag 60agcatttcag gtatcactga
tcaatcgaat gacgtgtttg aagatgcagc aaaagcattc 120tctatgttta ctcgcagcga
tgtctacaag gcgctggatg aaataccttt ctctgatgat 180gcgatgcttc caatccctcc
aactatatat acgaaaccat ctcacgattc atattattac 240attgatgctc taaaccgtgt
gcgtcgcaaa acatatcagg gccctgatga cgtgtacgta 300cctaattgtt ctattgttga
attgctggag ccacatgaga ctctgacatc ttatgggcgg 360ttgtccgagg ccatcgagaa
tcgtgccaag gatggggaca gccaagccag aatcgccaca 420acgtatggta gaatcgctga
atctcaagct cgacagatta aggctccatt ggagaagttt 480gtgttggcac tattagtggc
cgaagcaggg gggtctttat atgatccagt tttgcagaag 540tatgatgaga ttccagatct
atcgcataat tgccctttat ggtgttttag agagatctgt 600cgtcacatat ctggtccatt
accagatcgg gcaccttatc tttacttatc tgcaggggtt 660ttctggttaa tgtcaccacg
aatgacgtct gcaatccctc cgctactatc cgatcttgtt 720aatttagcta ttttgcaaca
aactgcgggt ttagatccat cattagtgaa attgggagta 780cagatatgcc ttcatgcagc
agctagctca agttatgcat ggtttatctt aaagactaag 840tctatttttc ctcaaaacac
gttgcacagt atgtatgaat ctctagaagg gggatactgt 900cctaatcttg aatggttaga
gcctagatca gactataagt tcatgtacat gggagtcatg 960ccattgtccg ctaagtatgc
taggtcggcg ccgtccaatg ataagaaagc gcgggaactt 1020ggcgagaaat atggactgag
ctcagtcgtc ggtgagcttc gtaaacggac aaagacgtat 1080gttaaacatg actttgcttc
agtgaggtac attcgtgacg ctatggcatg tactagcggt 1140attttcttgg taagaacacc
caccgaaacg gtattgcaag aatatacgca gagtccggag 1200attaaggttc ccattcccca
gaaagactgg acaggcccaa taggtgaaat cagaattcta 1260aaagatacaa caagttccat
cgcgcgttac ttatatagaa catggtactt ggcagcggcg 1320agaatggcgg ctcaaccacg
tacgtgggat ccattgtttc aagcgattat gagatctcaa 1380tacgtgacag ctaggggtgg
atctggcgca gcactccgcg aatctttgta tgcaatcaat 1440gtgtcgttac ctgatttcaa
gggcttacca gtgaaggcag caactaagat attccaggcg 1500gcacaattag cgaacttgcc
gttctcccac acatcagtgg ctatactagc tgacacttca 1560atgggattgc gaaatcaggt
gcagaggcgg ccacgatcca ttatgccatt aaatgtgccc 1620cagcagcagg tttcggcgcc
ccatacattg acagcggatt acattaacta ccacatgaat 1680ctatcaacca cgtctggtag
tgcggtcatt gagaaggtga ttcctttagg tgtatacgct 1740tcgagccctc ctaaccagtc
gatcaacatt gacatatctg cgtgtgacgc tagtattact 1800tgggatttct ttctgtcagt
gattatggcg gctatacacg aaggtgtcgc tagtagctcc 1860attggaaaac catttatggg
ggttcctgca tccattgtaa atgatgagtc tgtcgttgga 1920gtgagagctg ctaggccgat
atcgggaatg cagaacatga ttcagcatct atcgaaacta 1980tataaacgtg gattttcata
tagagtaaac gattcttttt ctccaggtaa cgattttact 2040catatgacta ccactttccc
gtcaggttca acagccacct ctactgagca tactgctaat 2100aatagtacga tgatggaaac
tttcctgaca gtatggggac ccgaacatac tgacgaccct 2160gacgtcttac gtttaatgaa
gtctttaact attcaaagga attacgtatg tcaaggtgat 2220gatggattaa tgattatcga
tgggactact gctggtaagg tgaacagtga aactattcag 2280aagatgctag aattaatctc
aaaatatggt gaggaattcg gatggaaata tgacatagcg 2340tacgatggga ctgccgaata
cttaaagcta tacttcatat ttggctgtcg aattccaaat 2400cttagtcgcc atccaatcgt
ggggaaagaa cgggcgaatt cttcagcaga ggagccatgg 2460ccagcaattc tagatcagat
tatgggtgtc ttctttaatg gtgttcatga tgggttacag 2520tggcagcggt ggatacgtta
ttcatgggct ctatgctgtg ctttctcacg tcaaagaaca 2580atgattggtg agagcgtggg
ttaccttcaa tatcctatgt ggtcttttgt ctactgggga 2640ttaccactgg ttaaagcgtt
tgggtcagac ccatggatat tttcttggta catgcctact 2700ggagatctgg gaatgtatag
ttggattagc ttgatacgcc ctctgatgac aagatggatg 2760gtggctaatg gttacgtaac
tgacagatgc tcacccgtat tcgggaacgc agattatcgc 2820aggtgtttca atgaacttaa
actatatcaa ggttattata tggcacaatt gcccaggaat 2880cctaagaagt ctggacgagc
ggcccctcgg gaggtaagag aacaattcac tcaggcatta 2940tccgactatc tactgcaaaa
tccagagctg aagtcacgtg tgctacgtgg tcgtagtgag 3000tgggagaaat atggagcggg
gataattcac aatcctccgt cattattcga tgtgccccat 3060aaatggtatc agggtgcgca
agaggcagca atcgctacga gagaagagct ggcagaaatg 3120gatgagacat taatgcgcgc
tcgaaggcac agatattcga gcttttcaaa gttattagag 3180gcgtatctgc tcgtgaaatg
gcgaatgtgc gaggcccgcg aaccgtcggt ggatttgcga 3240ttaccattat gtgcgggtat
tgacccatta aactcagatc cttttctcaa gatggtaagc 3300gttggaccaa tgctccagag
tacgagaaag tactttgctc agacactatt catggcaaag 3360acggtgtcgg gtcttgacgt
taacgcgatt gatagcgcgt tattacgact gcgaacatta 3420ggtgctgata agaaagcatt
aacggcgcag ttattaatgg tggggcttca ggagtcagaa 3480gcggacgcat tggccgggaa
gataatgcta caggatgtga atactgtgca attagccaga 3540gtggttaact tagctgtgcc
agatacttgg atgtcgttag actttgactc tatgttcaaa 3600caccacgtca agctgcttcc
caaagatgga cgtcatctaa atactgatat tcctcctcga 3660atgggatggt tacgggccat
tttacgattc ttaggtgccg gaatggtaat gactgcgact 3720ggagttgctg tcgacatcta
tctggaggat atacatggcg gtggtcggtc acttggacag 3780agattcatga cttggatgcg
acaggaagga cggtcagcgt gagtctacca tgggtcgtgg 3840tgcgtcaact catc
385493916DNAReovirus
9gctaaatggc gcgatggcga acgtttgggg ggtgagactt gcagactcgt tatcttcacc
60cactattgag acacgaacgc gtcagtatac cttacacgat ctttgctcag acctagatgc
120taatccgggg agggaaccgt ggaaacctct gcgtaatcag cgtactaata atattgtggc
180tgtgcaatta ttcagaccat tgcagggttt agttttagat acccagcttt atggatttcc
240aggagcattt gatgactggg agcgattcat gagagagaag ctgcgtgtgc taaagtatga
300agtattgcgc atctatccaa tcagcaacta tagcaatgaa catgtcaacg tcttcgtggc
360caatgctttg gtgggcgctt tcctgtcgaa tcaagctttc tatgacctgc taccgttgtt
420gataattaat gacactatga ttggtgatct acttggcacg ggggcatcgc tatcacagtt
480ctttcaatct catggagatg tgctggaagt cgcagctggt cgtaagtatc tgcagatgga
540aaactactcc aacgatgacg atgatcctcc attatttgcg aaagacctgt cagattatgc
600taaagcattc tacagtgaca catatgaagt gttggacagg ttcttttgga cgcatgactc
660ttcagcgggg gtcttagtgc attatgataa gccaacgaat ggtcatcact atctgctggg
720tactttgact cagatggtca gtgcacctcc ttatattatt aacgctactg acgcaatgtt
780gcttgaatcc tgtctagaac agttctcagc taatgtgcgt gcgagacctg cgcaacccgt
840tacacgctta gaccaatgct atcatttaag atggggagca caatatgtag gagaagattc
900actgacatat cggttggggg tgttatcctt gctggctacc aatggatatc aattagctag
960accgattcca agacagttga cgaatcgatg gttgtcgagc tttgtgagtc aaattatgtc
1020tgacggcgtc aacgagactc cactgtggcc ccaagaaagg tatgtgcaga tcgcttatga
1080ttcaccatcc gttgttgatg gggctacgca atatggctat gtcaggaaga atcaactcag
1140actcggcatg agaatatcgg cgctgcaatc gctgagtgat acgccctcgc cggtacagtg
1200gcttccacaa tacaccatcg accaggcagc gatggacgaa ggcgatctga tggttagtcg
1260gcttacgcaa ctcccgttac gtcctgatta tggtaatatc tgggtcggcg atgcgctatc
1320ctattatgtg gactacaatc ggagtcatcg agtcgtgctt tcatcggaac ttcctcagct
1380tccggacaca tattttgatg gcgatgaaca gtatgggcgc agcctgttct cactagctcg
1440taagattggt gaccgctcgt tagtgaaaga tacggctgtc ttgaagcacg cttaccaagc
1500catcgatcca aatactggta aggagtatct gagatctcgg caatctgtcg catattttgg
1560tgcatcagcg ggtcattctg gtgccgacca gccgttagtc atagagccct ggattcaagg
1620gaaaatcagt ggtgtgccgc caccctcctc agtgcgacag ttcggctatg atgttgcccg
1680tggcgcgatc gtcgatctgg cgagaccatt tccttctgga gattatcaat ttgtctattc
1740ggatgttgac caggtggtcg atggccatga cgatctgagt atatcatctg gactggtgga
1800gagccttttg tcttcatgca tgcacgccac agcacccggg ggctcatttg ttgttaagat
1860aaattttccg actagacccg tatggcacta catcgaacag aagatcttgc ccaatattac
1920gtcatacatg ttgatcaagc ctttcgtcac caacaacgtc gaattgttct tcgtcgcttt
1980cggtgtgcat caacactcat cacttacttg gacatctgga gtgtacttct tcttggtgga
2040ccatttttat cgttatgaga ctttatctac gatctcacga caattgccgt cttttgggta
2100tgttgatgat gggtcttccg tgactggtat cgagacaatt agtattgaga accctggctt
2160ctcgaatatg acccaggccg ctcgcattgg tatctcagga ttgtgtgcta atgtaggtaa
2220cgcgcgtaag tccattgcca tttacgaatc tcatggggcc agagtattaa ctatcacatc
2280aaggagatct ccggcatcag ctagaagaaa gtctaggttg cgatatttgc cattaataga
2340ccctaggtcg ttagaggtac aggcgcgcac tattctgcca gctgatccag tgttatttga
2400aaacgtgagc ggagcgtcac cccatgtttg tctgacaatg atgtacaact tcgaagtgtc
2460gtcagcggta tatgatggag acgttgtgct agatcttggg acgggaccag aggctaaaat
2520ccttgaactg atacccgcaa cctctccagt cacatgcgtg gacatacggc ctacagcgca
2580gcctagtgga tgttggaacg ttcgtaccac gttccttgag ttagattatt tgagcgatgg
2640atggatcact ggggtgcgtg gggacatagt tacttgtatg ttatctttgg gggccgctgc
2700cgctggaaaa tcaatgactt ttgacgctgc gtttcagcaa ttaatcaaag tattatccaa
2760gagtacggct aatgttgtgc tggtgcaggt taactgccct acagacgtgg tgaggagcat
2820taagggctac ctagagatag attcgactaa caagaggtat aggttcccca aatttggtcg
2880agacgagccg tactctgaca tggatgcgct ggagaaaata tgtcgtaccg cctggccaaa
2940ctgctcaatt acctgggttc cattgtcata cgacttgcgg tggactagac tggcattatt
3000agagtccacg acattgagta gcgcgtcgat tagaattgct gagctgatgt ataaatacat
3060gcctattatg aggattgata ttcatggact acccatggaa aagcgaggta acttcatagt
3120ggggcagaac tgctcattag taatccctgg ttttaatgcg caggatgtct ttaactgtta
3180tttcaattcc gccctcgctt tctcgactga agatgtcaat gctgcgatga ttccccaagt
3240gtctgcgcag tttgatgcga ctaagggtga gtggacgttg gatatggtct tctccgacgc
3300aggaatctat accatgcagg ctctagtggg atctaatgct aatccagtct ctttgggttc
3360ctttgtagtt gattctccag atgtagatat aactgacgct tggccagctc agttagactt
3420tacgatcgcg ggaactgatg tcgatataac agttaatcct tattaccgtc tgatgacctt
3480tgtaaggatc gatggacagt ggcagattgc caatccagac aaatttcaat tcttttcgtc
3540ggcgtctggg acgttagtga tgaacgtcaa attagatatc gcagataaat atctactata
3600ctatatacga gatgtccagt ctcgagatgt tggcttttac attcagcatc cacttcaact
3660tttgaatacg atcacattgc caaccaacga ggaccttttt ctgagcgcac ctgacatgcg
3720agagtgggca gttaaggaaa gcggtaacac gatatgtata ctcaatagtc aagggtttgt
3780gctacctcaa gattgggatg tgttaacaga taccataagt tggtccccat cgatacccac
3840atacattgtg ccaccgggtg attatacctt gactcctctg taactcactg tccctcgtga
3900gcgcgcctaa ttcatc
3916103901DNAReovirus 10gctaatcgtc aggatgaagc ggattccaag gaagacaaag
ggcaaatcca gcggaaaggg 60caatgactca acagagagag cggacgatgg ctcgagccaa
ttaagagaca agcaaaacaa 120taaggctggc cccgccacta cggagcctgg cacatccaac
cgagagcaat acaaagctcg 180accaggtatt gcatctgtgc agagggccac tgaaagtgca
gaaatgccca tgaagaataa 240tgacgaaggg acgccagata agaaaggaaa tactaagggc
gacctagtta atgagcatag 300tgaggctaaa gacgaggcgg atgaagcgac gaagaagcag
gcaaaggata cagacaaaag 360taaagcgcaa gtcacatatt cagacactgg tatcaataat
gctaatgaac tgtcaagatc 420tgggaatgtg gataatgagg gtggaagtaa tcagaagccg
atgtctacca gaatagctga 480ggcaacgtct gctatagtgt cgaaacatcc tgcgcgtgtt
gggctgccac ctaccgctag 540cagtggtcat gggtatcagt gccatgtctg ttctgcagtc
ctgtttagtc ctttagacct 600agatgcccac gtcgcctcac atggtttgca tggtaacatg
acattaacat cgagtgatat 660ccagcgacat ataactgagt tcatcagctc atggcaaaat
catcctattg ttcaagtttc 720ggctgatgtc gaaaataaga aaactgctca attgcttcac
gctgacactc ctcgactcgt 780cacttgggat gctggtttgt gtacttcatt caaaatcgtc
ccgattgtgc cagctcaggt 840gccgcaggat gtactggcct atacgttttt cacctcttca
tacgctatcc aatcaccgtt 900tccagaggcg gcagtgtcta ggattgtggt gcatacgaga
tgggcatcta atgttgactt 960tgaccgagac tcgtctgtca tcatggcgcc acctacagaa
aacaatatcc atttgtttaa 1020acagttacta aatactgaaa ccctgtctgt aaggggggct
aatccgctaa tgttcagggc 1080gaatgtgttg catatgttgc tagagttcgt attagataac
ttgtatctga acagacatac 1140gggattctct caagaccaca cgccatttac tgagggtgct
aatttgcgtt cacttcctgg 1200ccccgatgct gagaaatggt actcgattat gtatccaacg
cgcatgggaa cgccgaatgt 1260atccaaaata tgtaatttcg tcgcctcttg tgtgcgaaat
cgggttggac ggtttgatcg 1320agcacagatg atgaacggag ctatgtcaga gtgggtggat
gtcttcgaga cttcagacgc 1380gctaaccgtc tccattcgag gtcgatggat ggctagacta
gctcgcatga acataaatcc 1440aacagagatc gaatgggcat tgactgaatg tgcacaagga
tatgtgactg tcacaagtcc 1500ttacgctcct agcgtaaata gattgatgcc ctatcgtatc
tccaacgctg agcggcaaat 1560atcacagata atcaggatca tgaacattgg caataacgcg
acggtgatac aacctgttct 1620gcaagatatt tcggtactcc ttcaacgcat atcaccactc
caaatagatc caactattat 1680ttccaacact atgtcaacag tctcggagtc tactactcag
accctcagcc ccgcgtcctc 1740aattttgggt aaactacgac caagcaactc agatttttct
agttttagag tcgcgttggc 1800tggatggctt tataatgggg ttgtgacgac ggtgattgat
gatagttcat atccaaaaga 1860cggcggcagc gtgacctcac ttgaaaatct gtgggatttc
ttcatccttg cgcttgctct 1920accactgaca actgacccct gtgcacctgt gaaagcattc
atgaccctag ccaacatgat 1980ggttggtttc gagacaatcc ctatggataa tcagatctat
actcaatcga gacgcgcgag 2040tgctttctca acgcctcaca cgtggccacg atgctttatg
aacatccagt taatttctcc 2100aatcgacgct cccatcttgc gacagtgggc tgaaattatt
catagatact ggcctaaccc 2160ttcacagatt cgttatggtg caccgaacgt tttcggctcg
gcaaatttgt tcactccacc 2220tgaggtgctg ttattgccaa tcgatcatca accagctaat
gtaacaacgc caacgctgga 2280cttcaccaat gagttaacta attggcgcgc tcgtgtctgt
gagcttatga agaatctcgt 2340tgataaccaa agatatcaac ctggatggac acaaagtcta
gtctcgtcaa tgcgcggaac 2400gctagacaaa ttgaagttga ttaaatcgat gacaccaatg
tatctgcaac agctggctcc 2460ggtagagtta gcagtgatag ctcccatgtt gccttttcca
cctttccagg tgccatacgt 2520ccgtctcgat cgtgacagag ttccaacaat ggttggagta
acacgacatt cacgagatac 2580tattactcag ccggcgctat cgctgtcgac aaccaatact
actgttggcg tgccactagc 2640tctagacgcg agggctatca ccgttgcgct gttgtcaggg
aaatatccgc cggatttggt 2700gacaaatgta tggtacgctg atgccattta cccaatgtat
gcagacacgg aggtgttctc 2760taatcttcag agagacatga ttacctgcga ggccgtgcag
acattagtga ctctggtggc 2820gcaaatatca gagacccagt atcctgtaga taggtatctt
gattggatcc catcactgag 2880agcatcggcg gcgacggcgg cgacatttgc tgagtgggtt
aatacttcaa tgaagacggc 2940gtttgatttg tctgatatgc tgttagagcc tctcctaagc
ggtgatccga ggatgactca 3000actagcgatt cagtatcagc agtacaatgg cagaacgttt
aatatcatac ctgaaatgcc 3060aggttcagta attgctgact gcgttcaatt aacagcagaa
gtctttaatc acgaatataa 3120cctgtttggg attgcgcggg gtgatatcat cattggccgt
gttcagtcga cacatttgtg 3180gtcaccgctg gctcctccac ctgacctggt gtttgatcgt
gatacccctg gtgttcacat 3240cttcggacga gattgccgta tatcgtttgg aatgaatggc
gccgcgccaa tgattagaga 3300tgagactgga ctgatggtgc cttttgaagg aaattggatt
ttcccactgg cgctttggca 3360aatgaataca cgatatttta atcaacagtt cgacgcgtgg
attaagacag gagagttgcg 3420aatccgcatt gagatgggcg cgtatccata tatgttgcat
tactatgatc cacgtcagta 3480cgctaatgca tggaatttaa catccgcctg gcttgaagaa
attacgccga cgagcatccc 3540atccgtgcct ttcatggtgc ccatttcaag tgatcatgac
atttcctctg ccccagctgt 3600ccaatatatc atttcaactg aatataatga tcggtctctg
ttctgcacta actcatcatc 3660tccccaaacc atcgctggac cagacaaaca cattccagtt
gagagatata acattctgac 3720caaccccgac gctccaccca cgcagataca actgcctgaa
gtcgttgact tgtacaacgt 3780cgtcacacgc tatgcgtatg agactccgcc tattaccgct
gttgttatgg gtgttccttg 3840atcctcatcc tcccaacagg tgctagagca ttgcgctcaa
tgctagttgg gccgattcat 3900c
390111455PRTReovirus 11Met Asp Pro Arg Leu Arg Glu
Glu Val Val Arg Leu Ile Ile Ala Leu1 5 10
15 Thr Ser Asp Asn Gly Ala Ser Leu Ser Lys Gly Leu
Glu Ser Arg Val 20 25 30
Ser Ala Leu Glu Lys Thr Ser Gln Ile His Ser Asp Thr Ile Leu Arg
35 40 45 Ile Thr Gln Gly
Leu Asp Asp Ala Asn Lys Arg Ile Ile Ala Leu Glu 50 55
60 Gln Ser Arg Asp Asp Leu Val Ala Ser
Val Ser Asp Ala Gln Leu Ala65 70 75
80 Ile Ser Arg Leu Glu Ser Ser Ile Gly Ala Leu Gln Thr Val
Val Asn 85 90 95
Gly Leu Asp Ser Ser Val Thr Gln Leu Gly Ala Arg Val Gly Gln Leu
100 105 110 Glu Thr Gly Leu Ala
Glu Leu Arg Val Asp His Asp Asn Leu Val Ala 115
120 125 Arg Val Asp Thr Ala Glu Arg Asn Ile
Gly Ser Leu Thr Thr Glu Leu 130 135
140 Ser Thr Leu Thr Leu Arg Val Thr Ser Ile Gln Ala Asp
Phe Glu Ser145 150 155
160 Arg Ile Ser Thr Leu Glu Arg Thr Ala Val Thr Ser Ala Gly Ala Pro
165 170 175 Leu Ser Ile Arg
Asn Asn Arg Met Thr Met Gly Leu Asn Asp Gly Leu 180
185 190 Thr Leu Ser Gly Asn Asn Leu Ala Ile
Arg Leu Pro Gly Asn Thr Gly 195 200
205 Leu Asn Ile Gln Asn Gly Gly Leu Gln Phe Arg Phe Asn Thr
Asp Gln 210 215 220
Phe Gln Ile Val Asn Asn Asn Leu Thr Leu Lys Thr Thr Val Phe Asp225
230 235 240 Ser Ile Asn Ser Arg
Ile Gly Ala Thr Glu Gln Ser Tyr Val Ala Ser 245
250 255 Ala Val Thr Pro Leu Arg Leu Asn Ser Ser
Thr Lys Val Leu Asp Met 260 265
270 Leu Ile Asp Ser Ser Thr Leu Glu Ile Asn Ser Ser Gly Gln Leu
Thr 275 280 285 Val
Arg Ser Thr Ser Pro Asn Leu Arg Tyr Pro Ile Ala Asp Val Ser 290
295 300 Gly Gly Ile Gly Met Ser
Pro Asn Tyr Arg Phe Arg Gln Ser Met Trp305 310
315 320 Ile Gly Ile Val Ser Tyr Ser Gly Ser Gly Leu
Asn Trp Arg Val Gln 325 330
335 Val Asn Ser Asp Ile Phe Ile Val Asp Asp Tyr Ile His Ile Cys Leu
340 345 350 Pro Ala Phe
Asp Gly Phe Ser Ile Ala Asp Gly Gly Asp Leu Ser Leu 355
360 365 Asn Phe Val Thr Gly Leu Leu Pro
Pro Leu Leu Thr Gly Asp Thr Glu 370 375
380 Pro Ala Phe His Asn Asp Val Val Thr Tyr Gly Ala Gln
Thr Val Ala385 390 395
400 Ile Gly Leu Ser Ser Gly Gly Ala Pro Gln Tyr Met Ser Lys Asn Leu
405 410 415 Trp Val Glu Gln
Trp Gln Asp Gly Val Leu Arg Leu Arg Val Glu Gly 420
425 430 Gly Gly Ser Ile Thr His Ser Asn Ser
Lys Trp Pro Ala Met Thr Val 435 440
445 Ser Tyr Pro Arg Ser Phe Thr 450 455
12418PRTReovirus 12Met Ala Arg Ala Ala Phe Leu Phe Lys Thr Val Gly Phe
Gly Gly Leu1 5 10 15
Gln Asn Val Pro Ile Asn Asp Glu Leu Ser Ser His Leu Leu Arg Ala
20 25 30 Gly Asn Ser Pro Trp
Gln Leu Thr Gln Phe Leu Asp Trp Ile Ser Leu 35 40
45 Gly Arg Gly Leu Ala Thr Ser Ala Leu Val
Pro Thr Ala Gly Ser Arg 50 55 60
Tyr Tyr Gln Met Ser Cys Leu Leu Ser Gly Thr Leu Gln Ile Pro
Phe65 70 75 80 Arg
Pro Asn His Arg Trp Gly Asp Ile Arg Phe Leu Arg Leu Val Trp
85 90 95 Ser Ala Pro Thr Leu Asp
Gly Leu Val Val Ala Pro Pro Gln Val Leu 100
105 110 Ala Gln Pro Ala Leu Gln Ala Gln Ala Asp
Arg Val Tyr Asp Cys Asp 115 120
125 Asp Tyr Pro Phe Leu Ala Arg Asp Pro Arg Phe Lys His Arg
Val Tyr 130 135 140
Gln Gln Leu Ser Ala Val Thr Leu Leu Asn Leu Thr Gly Phe Gly Pro145
150 155 160 Ile Ser Tyr Val Arg
Val Asp Glu Asp Met Trp Ser Gly Asp Val Asn 165
170 175 Gln Leu Leu Met Asn Tyr Phe Gly His Thr
Phe Ala Glu Ile Ala Tyr 180 185
190 Thr Leu Cys Gln Ala Ser Ala Asn Arg Pro Trp Glu Tyr Asp Gly
Thr 195 200 205 Tyr
Ala Arg Met Thr Gln Ile Val Leu Ser Leu Phe Trp Leu Ser Tyr 210
215 220 Val Gly Val Ile His Gln
Gln Asn Thr Tyr Arg Thr Phe Tyr Phe Gln225 230
235 240 Cys Asn Arg Arg Gly Asp Ala Ala Glu Val Trp
Ile Leu Ser Cys Ser 245 250
255 Leu Asn His Ser Ala Gln Ile Arg Pro Gly Asn Arg Ser Leu Phe Val
260 265 270 Met Pro Thr
Ser Pro Asp Trp Asn Met Asp Val Asn Leu Ile Leu Ser 275
280 285 Ser Thr Leu Thr Gly Cys Leu Cys
Ser Gly Ser Gln Leu Pro Leu Ile 290 295
300 Asp Asn Asn Ser Val Pro Ala Val Ser Arg Asn Ile His
Gly Trp Thr305 310 315
320 Gly Arg Ala Gly Asn Gln Leu His Gly Phe Gln Val Arg Arg Met Val
325 330 335 Thr Glu Phe Cys
Asp Arg Leu Arg Arg Asp Gly Val Met Thr Gln Ala 340
345 350 Gln Gln Asn Gln Val Glu Ala Leu Ala
Asp Gln Thr Gln Gln Phe Lys 355 360
365 Arg Asp Lys Leu Glu Thr Trp Ala Arg Glu Asp Asp Gln Tyr
Asn Gln 370 375 380
Ala His Pro Asn Ser Thr Met Phe Arg Thr Lys Pro Phe Thr Asn Ala385
390 395 400 Gln Trp Gly Arg Gly
Asn Thr Gly Ala Thr Ser Ala Ala Ile Ala Ala 405
410 415 Leu Ile 13254PRTReovirus 13Met Ala Ser
Ser Leu Arg Ala Ala Ile Ser Lys Ile Lys Arg Asp Asp1 5
10 15 Val Gly Gln Gln Val Cys Pro Asn
Tyr Val Met Leu Arg Ser Ser Val 20 25
30 Thr Thr Lys Val Val Arg Asn Val Val Glu Tyr Gln Ile
Arg Thr Gly 35 40 45
Gly Phe Phe Ser Cys Leu Ala Met Leu Arg Pro Leu Gln Tyr Ala Lys 50
55 60 Arg Glu Arg Leu Leu
Gly Gln Arg Asn Leu Glu Arg Ile Ser Thr Arg65 70
75 80 Asp Ile Leu Gln Thr Arg Asp Leu His Ser
Leu Cys Met Pro Thr Pro 85 90
95 Asp Ala Pro Met Ser Asn His Gln Ala Ser Thr Met Arg Glu Leu
Ile 100 105 110 Cys
Ser Tyr Phe Lys Val Asp His Ala Asp Gly Leu Lys Tyr Ile Pro 115
120 125 Met Asp Glu Arg Tyr Ser
Pro Ser Ser Leu Ala Arg Leu Phe Thr Met 130 135
140 Gly Met Ala Gly Leu His Ile Thr Thr Glu Pro
Ser Tyr Lys Arg Val145 150 155
160 Pro Ile Met His Leu Ala Ala Asp Leu Asp Cys Met Thr Leu Ala Leu
165 170 175 Pro Tyr Met
Ile Thr Leu Asp Gly Asp Thr Val Val Pro Val Ala Pro 180
185 190 Thr Leu Ser Ala Glu Gln Leu Leu
Asp Asp Gly Leu Lys Gly Leu Ala 195 200
205 Cys Met Asp Met Asp Val Arg Trp Thr Arg Ile Ala Gly
Arg Leu Val 210 215 220
Ile Arg Val Trp Thr Leu His Ala Ala Ser Thr Ser Cys Ile Ala Arg225
230 235 240 Arg Gln Gln Lys Pro
Ser Val Cys Leu Arg His Ala Leu Cys 245
250 14366PRTReovirus 14Met Ala Ser Ser Leu Arg Ala Ala
Ile Ser Lys Ile Lys Arg Asp Asp1 5 10
15 Val Gly Gln Gln Val Cys Pro Asn Tyr Val Met Leu Arg
Ser Ser Val 20 25 30
Thr Thr Lys Val Val Arg Asn Val Val Glu Tyr Gln Ile Arg Thr Gly
35 40 45 Gly Phe Phe Ser
Cys Leu Ala Met Leu Arg Pro Leu Gln Tyr Ala Lys 50 55
60 Arg Glu Arg Leu Leu Gly Gln Arg Asn
Leu Glu Arg Ile Ser Thr Arg65 70 75
80 Asp Ile Leu Gln Thr Arg Asp Leu His Ser Leu Cys Met Pro
Thr Pro 85 90 95
Asp Ala Pro Met Ser Asn His Gln Ala Ser Thr Met Arg Glu Leu Ile
100 105 110 Cys Ser Tyr Phe Lys
Val Asp His Ala Asp Gly Leu Lys Tyr Ile Pro 115
120 125 Met Asp Glu Arg Tyr Ser Pro Ser Ser
Leu Ala Arg Leu Phe Thr Met 130 135
140 Gly Met Ala Gly Leu His Ile Thr Thr Glu Pro Ser Tyr
Lys Arg Val145 150 155
160 Pro Ile Met His Leu Ala Ala Asp Leu Asp Cys Met Thr Leu Ala Leu
165 170 175 Pro Tyr Met Ile
Thr Leu Asp Gly Asp Thr Val Val Pro Val Ala Pro 180
185 190 Thr Leu Ser Ala Glu Gln Leu Leu Asp
Asp Gly Leu Lys Gly Leu Ala 195 200
205 Cys Met Asp Ile Ser Tyr Gly Cys Glu Val Asp Ala Asn Ser
Arg Pro 210 215 220
Ala Gly Asp Gln Ser Met Asp Ser Ser Arg Cys Ile Asn Glu Leu Tyr225
230 235 240 Cys Glu Glu Thr Ala
Glu Ala Ile Cys Val Leu Lys Thr Cys Leu Val 245
250 255 Leu Asn Cys Met Gln Phe Lys Leu Glu Met
Asp Asp Leu Ala His Asn 260 265
270 Ala Ala Glu Leu Asp Lys Ile Gln Met Met Ile Pro Phe Ser Glu
Arg 275 280 285 Val
Phe Arg Met Ala Ser Ser Phe Ala Thr Ile Asp Ala Gln Cys Phe 290
295 300 Arg Phe Cys Val Met Met
Lys Asp Lys Asn Leu Lys Ile Asp Met Arg305 310
315 320 Glu Thr Thr Arg Leu Trp Thr Arg Ser Ala Ser
Asp Asp Ser Val Ala 325 330
335 Thr Ser Ser Leu Ser Ile Ser Leu Asp Arg Gly Arg Trp Val Ala Ala
340 345 350 Asp Ala Ser
Asp Ala Arg Leu Leu Val Phe Pro Ile Arg Val 355
360 365 15365PRTReovirus 15Met Glu Val Cys Leu Pro
Asn Gly His Gln Val Val Asp Leu Ile Asn1 5
10 15 Asn Ala Phe Glu Gly Arg Val Ser Ile Tyr Ser
Ala Gln Glu Gly Trp 20 25 30
Asp Lys Thr Ile Ser Ala Gln Pro Asp Met Met Val Cys Gly Gly Ala
35 40 45 Val Val Cys
Met His Cys Leu Gly Val Val Gly Ser Leu Gln Arg Lys 50
55 60 Leu Lys His Leu Pro His His Arg
Cys Asn Gln Gln Ile Arg His Gln65 70 75
80 Asp Tyr Val Asp Val Gln Phe Ala Asp Arg Val Thr Ala
His Trp Lys 85 90 95
Arg Gly Met Leu Ser Phe Val Ala Gln Met His Glu Met Met Asn Asp
100 105 110 Val Ser Pro Asp Asp
Leu Asp Arg Val Arg Thr Glu Gly Gly Ser Leu 115
120 125 Val Glu Leu Asn Arg Leu Gln Val Asp
Pro Asn Ser Met Phe Arg Ser 130 135
140 Ile His Ser Ser Trp Thr Asp Pro Leu Gln Val Val Asp
Asp Leu Asp145 150 155
160 Thr Lys Leu Asp Gln Tyr Trp Thr Ala Leu Asn Leu Met Ile Asp Ser
165 170 175 Ser Asp Leu Ile
Pro Asn Phe Met Met Arg Asp Pro Ser His Ala Phe 180
185 190 Asn Gly Val Lys Leu Lys Gly Asp Ala
Arg Gln Thr Gln Phe Ser Arg 195 200
205 Thr Phe Asp Ser Arg Ser Ser Leu Glu Trp Gly Val Met Val
Tyr Asp 210 215 220
Tyr Ser Glu Leu Asp His Asp Pro Ser Lys Gly Arg Ala Tyr Arg Lys225
230 235 240 Glu Leu Val Thr Pro
Ala Arg Asp Phe Gly His Phe Gly Leu Ser His 245
250 255 Tyr Ser Arg Ala Thr Thr Pro Ile Leu Gly
Lys Met Pro Ala Val Phe 260 265
270 Ser Gly Met Leu Thr Gly Asn Cys Lys Met Tyr Pro Phe Ile Lys
Gly 275 280 285 Thr
Ala Lys Leu Lys Thr Val Arg Lys Leu Val Glu Ala Val Asn His 290
295 300 Ala Trp Gly Val Glu Lys
Ile Arg Tyr Ala Leu Gly Pro Gly Gly Met305 310
315 320 Thr Gly Trp Tyr Asn Arg Thr Met Gln Gln Ala
Pro Ile Val Leu Thr 325 330
335 Pro Ala Ala Leu Thr Met Phe Pro Asp Thr Ile Lys Phe Gly Asp Leu
340 345 350 Asn Tyr Pro
Val Met Ile Gly Asp Pro Met Ile Leu Gly 355 360
365 16736PRTReovirus 16Met Ala Tyr Ile Ala Val Pro Ala Val
Val Asp Ser Arg Ser Ser Glu1 5 10
15 Ala Ile Gly Leu Leu Glu Ser Phe Gly Val Asp Ala Gly Ala
Asp Ala 20 25 30
Asn Asp Val Ser Tyr Gln Asp His Asp Tyr Val Leu Asp Gln Leu Gln 35
40 45 Tyr Met Leu Asp Gly
Tyr Glu Ala Gly Asp Val Ile Asp Ala Leu Val 50 55
60 His Lys Asn Trp Leu His His Ser Val Tyr
Cys Leu Leu Pro Pro Lys65 70 75
80 Ser Gln Leu Leu Glu Tyr Trp Lys Ser Asn Pro Ser Ala Ile Pro
Asp 85 90 95 Asn
Val Asp Arg Arg Leu Arg Lys Arg Leu Met Leu Lys Lys Asp Leu
100 105 110 Arg Lys Asp Asp Glu
Tyr Asn Gln Leu Ala Arg Ala Phe Lys Ile Ser 115
120 125 Asp Val Tyr Ala Pro Leu Ile Ser Ser
Thr Thr Ser Pro Met Thr Met 130 135
140 Ile Gln Asn Leu Asn Arg Gly Glu Ile Val Tyr Thr Thr
Thr Asp Arg145 150 155
160 Val Ile Gly Ala Arg Ile Leu Leu Tyr Ala Pro Arg Lys Tyr Tyr Ala
165 170 175 Ser Thr Leu Ser
Phe Thr Met Thr Lys Cys Ile Ile Pro Phe Gly Lys 180
185 190 Glu Val Gly Arg Val Pro His Ser Arg
Phe Asn Val Gly Thr Phe Pro 195 200
205 Ser Ile Ala Thr Pro Lys Cys Phe Val Met Ser Gly Val Asp
Ile Glu 210 215 220
Ser Ile Pro Asn Glu Phe Ile Lys Leu Phe Tyr Gln Arg Val Lys Ser225
230 235 240 Val His Ala Asn Ile
Leu Asn Asp Ile Ser Pro Gln Ile Val Ser Asp 245
250 255 Met Ile Asn Arg Lys Arg Leu Arg Val His
Thr Pro Ser Asp Arg Arg 260 265
270 Ala Ala Gln Leu Met His Leu Pro Tyr His Val Lys Arg Gly Ala
Ser 275 280 285 His
Val Asp Val Tyr Lys Val Asp Val Val Asp Met Leu Phe Glu Val 290
295 300 Val Asp Val Ala Asp Gly
Leu Arg Asn Val Ser Arg Lys Leu Thr Met305 310
315 320 His Thr Val Pro Val Cys Ile Leu Glu Met Leu
Gly Ile Glu Ile Ala 325 330
335 Asp Tyr Cys Ile Arg Gln Glu Asp Gly Met Leu Thr Asp Trp Phe Leu
340 345 350 Leu Leu Thr
Met Leu Ser Asp Gly Leu Thr Asp Arg Arg Thr His Cys 355
360 365 Gln Tyr Leu Ile Asn Pro Ser Ser
Val Pro Pro Asp Val Ile Leu Asn 370 375
380 Ile Ser Ile Thr Gly Phe Ile Asn Arg His Thr Ile Asp
Val Met Pro385 390 395
400 Asp Ile Tyr Asp Phe Val Lys Pro Ile Gly Ala Val Leu Pro Lys Gly
405 410 415 Ser Phe Lys Ser
Thr Ile Met Arg Val Leu Asp Ser Ile Ser Ile Leu 420
425 430 Gly Ile Gln Ile Met Pro Arg Ala His
Val Val Asp Ser Asp Glu Val 435 440
445 Gly Glu Gln Met Glu Pro Thr Phe Glu Gln Ala Val Met Glu
Ile Tyr 450 455 460
Lys Gly Ile Ala Gly Val Asp Ser Leu Asp Asp Leu Ile Lys Trp Val465
470 475 480 Leu Asn Ser Asp Leu
Ile Pro His Asp Asp Arg Leu Gly Gln Leu Phe 485
490 495 Gln Ala Phe Leu Pro Leu Ala Lys Asp Leu
Leu Ala Pro Met Ala Arg 500 505
510 Lys Phe Tyr Asp Asn Ser Met Ser Glu Gly Arg Leu Leu Thr Phe
Ser 515 520 525 His
Ala Asp Ser Glu Leu Leu Asn Ala Asn Tyr Phe Gly His Leu Leu 530
535 540 Arg Leu Lys Ile Pro Tyr
Ile Thr Glu Val Asn Leu Met Ile Arg Lys545 550
555 560 Asn Arg Glu Gly Gly Glu Leu Phe Gln Leu Val
Leu Ser Tyr Leu Tyr 565 570
575 Lys Met Tyr Ala Thr Ser Ala Gln Pro Lys Trp Phe Gly Ser Leu Leu
580 585 590 Arg Leu Leu
Ile Cys Pro Trp Leu His Met Glu Lys Leu Ile Gly Glu 595
600 605 Ala Asp Pro Ala Ser Thr Ser Ala
Glu Ile Gly Trp His Ile Pro Arg 610 615
620 Glu Gln Leu Met Gln Asp Gly Trp Cys Gly Cys Glu Asp
Gly Phe Ile625 630 635
640 Pro Tyr Val Ser Ile Arg Ala Pro Arg Leu Val Ile Glu Glu Leu Met
645 650 655 Glu Lys Asn Trp
Gly Gln Tyr His Ala Gln Val Ile Val Thr Asp Gln 660
665 670 Leu Val Val Gly Glu Pro Arg Arg Val
Ser Ala Lys Ala Val Ile Lys 675 680
685 Gly Asn His Leu Pro Val Lys Leu Val Ser Arg Phe Ala Cys
Phe Thr 690 695 700
Leu Thr Ala Lys Tyr Glu Met Arg Leu Ser Cys Gly His Ser Thr Gly705
710 715 720 Arg Gly Ala Ala Tyr
Ser Ala Arg Leu Ala Phe Arg Ser Asp Leu Ala 725
730 735 17708PRTReovirus 17Met Gly Asn Ala Ser
Ser Ile Val Gln Thr Ile Asn Val Thr Gly Asp1 5
10 15 Gly Asn Val Phe Lys Pro Ser Ala Glu Thr
Ser Ser Thr Ala Val Pro 20 25
30 Ser Leu Ser Leu Ser Pro Gly Met Leu Asn Pro Gly Gly Val Pro
Trp 35 40 45 Ile
Ala Val Gly Asp Glu Thr Ser Val Thr Ser Pro Gly Ala Leu Arg 50
55 60 Arg Met Thr Ser Lys Asp
Ile Pro Asp Thr Ala Ile Ile Asn Thr Asp65 70
75 80 Asn Ser Ser Gly Ala Val Pro Ser Glu Ser Ala
Leu Val Pro Tyr Ile 85 90
95 Asp Glu Pro Leu Val Val Val Thr Glu His Ala Ile Thr Asn Phe Thr
100 105 110 Lys Ala Glu
Met Ala Leu Glu Phe Asn Arg Glu Phe Leu Asp Lys Met 115
120 125 Arg Val Leu Ser Val Ser Pro Lys
Tyr Ser Asp Leu Leu Thr Tyr Val 130 135
140 Asp Cys Tyr Val Gly Val Ser Ala Arg Gln Ala Leu Asn
Asn Phe Gln145 150 155
160 Lys Gln Val Pro Val Ile Thr Pro Thr Arg Gln Thr Met Tyr Val Asp
165 170 175 Ser Ile Gln Ala
Ala Leu Lys Ala Leu Glu Lys Trp Glu Ile Asp Leu 180
185 190 Arg Val Ala Gln Thr Leu Leu Pro Thr
Asn Val Pro Ile Gly Glu Val 195 200
205 Ser Cys Pro Met Gln Ser Val Val Lys Leu Leu Asp Asp Gln
Leu Pro 210 215 220
Asp Asp Ser Leu Ile Arg Arg Tyr Pro Lys Glu Ala Ala Val Ala Leu225
230 235 240 Ala Lys Arg Asn Gly
Gly Ile Gln Trp Met Asp Val Ser Glu Gly Thr 245
250 255 Val Met Asn Glu Ala Val Asn Ala Val Ala
Ala Ser Ala Leu Ala Pro 260 265
270 Ser Ala Ser Ala Pro Pro Leu Glu Glu Lys Ser Lys Leu Thr Glu
Gln 275 280 285 Ala
Met Asp Leu Val Thr Ala Ala Glu Pro Glu Ile Ile Ala Ser Leu 290
295 300 Ala Pro Val Pro Ala Pro
Val Phe Ala Ile Pro Pro Lys Pro Ala Asp305 310
315 320 Tyr Asn Val Arg Thr Leu Arg Ile Asp Glu Ala
Thr Trp Leu Arg Met 325 330
335 Ile Pro Lys Ser Met Asn Thr Pro Phe Gln Ile Gln Val Thr Asp Asn
340 345 350 Thr Gly Thr
Asn Trp His Leu Asn Leu Arg Gly Gly Thr Arg Val Val 355
360 365 Asn Leu Asp Gln Ile Ala Pro Met
Arg Phe Val Leu Asp Leu Gly Gly 370 375
380 Lys Ser Tyr Lys Glu Thr Ser Trp Asp Pro Asn Gly Lys
Lys Val Gly385 390 395
400 Phe Ile Val Phe Gln Ser Lys Ile Pro Phe Glu Leu Trp Thr Ala Ala
405 410 415 Ser Gln Ile Gly
Gln Ala Thr Val Val Asn Tyr Val Gln Leu Tyr Ala 420
425 430 Glu Asp Ser Ser Phe Thr Ala Gln Ser
Ile Ile Ala Thr Thr Ser Leu 435 440
445 Ala Tyr Asn Tyr Glu Pro Glu Gln Leu Asn Lys Thr Asp Pro
Glu Met 450 455 460
Asn Tyr Tyr Leu Leu Ala Thr Phe Ile Asp Ser Ala Ala Ile Thr Pro465
470 475 480 Thr Asn Met Thr Gln
Pro Asp Val Trp Asp Ala Leu Leu Thr Met Ser 485
490 495 Pro Leu Ser Ala Gly Glu Val Thr Val Lys
Gly Ala Val Val Ser Glu 500 505
510 Val Val Pro Ala Asp Leu Ile Gly Ser Tyr Thr Pro Glu Ser Leu
Asn 515 520 525 Ala
Ser Leu Pro Asn Asp Ala Ala Arg Cys Met Ile Asp Arg Ala Ser 530
535 540 Lys Ile Ala Glu Ala Ile
Lys Ile Asp Asp Asp Ala Gly Pro Asp Glu545 550
555 560 Tyr Ser Pro Asn Ser Val Pro Ile Gln Gly Gln
Leu Ala Ile Ser Gln 565 570
575 Leu Glu Thr Gly Tyr Gly Val Arg Ile Phe Asn Pro Lys Gly Ile Leu
580 585 590 Ser Lys Ile
Ala Ser Arg Ala Met Gln Ala Phe Ile Gly Asp Pro Ser 595
600 605 Thr Ile Ile Thr Gln Ala Ala Pro
Val Leu Ser Asp Lys Asn Asn Trp 610 615
620 Ile Ala Leu Ala Gln Gly Val Lys Thr Ser Leu Arg Thr
Lys Ser Leu625 630 635
640 Ser Ala Gly Val Lys Thr Ala Val Ser Lys Leu Ser Ser Ser Glu Ser
645 650 655 Ile Gln Asn Trp
Thr Gln Gly Phe Leu Asp Lys Val Ser Ala His Phe 660
665 670 Pro Ala Pro Lys Pro Asp Cys Pro Thr
Ser Gly Asp Ser Gly Glu Ser 675 680
685 Ser Asn Arg Arg Val Lys Arg Asp Ser Tyr Ala Gly Val Val
Lys Arg 690 695 700
Gly Tyr Thr Arg705 18721PRTReovirus 18Met Ala Ser Phe Lys Gly
Phe Ser Ala Asn Thr Val Pro Val Ser Lys1 5
10 15 Ala Lys Arg Asp Ile Ser Ser Leu Ala Ala Thr
Pro Gly Leu Arg Ser 20 25 30
Gln Ser Phe Thr Pro Ser Val Asp Met Ser Gln Ser Arg Glu Phe Leu
35 40 45 Thr Lys Ala
Ile Glu Gln Gly Ser Met Ser Ile Pro Tyr Gln His Val 50
55 60 Asn Val Pro Lys Val Asp Arg Lys
Val Val Ser Leu Val Val Arg Pro65 70 75
80 Phe Ser Ser Gly Ala Phe Ser Ile Ser Gly Val Ile Ser
Pro Ala His 85 90 95
Ala Tyr Leu Leu Glu Cys Leu Pro Gln Leu Glu Gln Ala Met Ala Phe
100 105 110 Val Ala Ser Pro Glu
Ser Phe Gln Ala Ser Asp Val Ala Lys Arg Phe 115
120 125 Ala Ile Lys Pro Gly Met Ser Leu Gln
Asp Ala Ile Thr Ala Phe Ile 130 135
140 Asn Phe Val Ser Ala Met Leu Lys Met Thr Val Thr Arg
Gln Asn Phe145 150 155
160 Asp Val Ile Val Ala Glu Ile Glu Arg Leu Ala Ser Thr Ser Val Ser
165 170 175 Val Arg Thr Glu
Glu Ala Lys Val Ala Asp Glu Glu Leu Met Leu Phe 180
185 190 Gly Leu Asp His Arg Gly Pro Gln Gln
Leu Asp Val Ser Asp Ala Lys 195 200
205 Gly Ile Met Lys Ala Ala Asp Ile Gln Thr Thr His Asp Val
His Leu 210 215 220
Ala Pro Gly Val Gly Asn Ile Asp Pro Glu Ile Tyr Asn Glu Gly Arg225
230 235 240 Phe Met Phe Met Gln
His Lys Pro Leu Ala Ala Asp Gln Ser Tyr Phe 245
250 255 Thr Leu Glu Thr Ala Asp Tyr Phe Lys Ile
Tyr Pro Thr Tyr Asp Glu 260 265
270 His Asp Gly Arg Met Ala Asp Gln Lys Gln Ser Gly Leu Ile Leu
Cys 275 280 285 Thr
Lys Asp Glu Val Leu Ala Glu Gln Thr Ile Phe Lys Leu Asp Ala 290
295 300 Pro Asp Asp Lys Thr Val
His Leu Leu Asp Arg Asp Asp Asp His Val305 310
315 320 Val Ala Arg Phe Thr Lys Val Phe Ile Glu Asp
Val Ala Pro Gly His 325 330
335 His Ala Ala Gln Arg Ser Gly Gln Arg Ser Val Leu Asp Asp Leu Tyr
340 345 350 Ala Asn Thr
Gln Val Ile Ser Ile Thr Ser Ala Ala Leu Lys Trp Val 355
360 365 Val Lys His Gly Val Ser Asp Gly
Ile Val Asn Arg Lys Asn Val Lys 370 375
380 Val Cys Val Gly Phe Asp Pro Leu Tyr Thr Leu Ser Thr
His Asn Gly385 390 395
400 Val Ser Leu Cys Ala Leu Leu Met Asp Glu Lys Leu Ser Val Leu Asn
405 410 415 Ser Ala Cys Arg
Met Thr Leu Arg Ser Leu Met Lys Thr Gly Arg Asp 420
425 430 Val Asp Ala His Arg Ala Phe Gln Arg
Val Leu Ser Gln Gly Tyr Thr 435 440
445 Ser Leu Met Cys Tyr Tyr His Pro Ser Arg Lys Leu Ala Tyr
Gly Glu 450 455 460
Val Leu Phe Leu Glu Arg Ser Asn Asp Val Thr Asp Gly Ile Lys Leu465
470 475 480 Gln Leu Asp Ala Ser
Arg Gln Cys His Glu Cys Pro Val Leu Gln Gln 485
490 495 Lys Val Val Glu Leu Glu Lys Gln Ile Ile
Met Gln Lys Ser Ile Gln 500 505
510 Ser Asp Pro Thr Pro Val Ala Leu Gln Pro Leu Leu Ser Gln Leu
Arg 515 520 525 Glu
Leu Ser Ser Glu Val Thr Arg Leu Gln Met Glu Leu Ser Arg Ala 530
535 540 Gln Ser Leu Asn Ala Gln
Leu Glu Ala Asp Val Lys Ser Ala Gln Ser545 550
555 560 Cys Ser Leu Asp Met Tyr Leu Arg His His Thr
Cys Ile Asn Gly His 565 570
575 Ala Lys Glu Asp Glu Leu Leu Asp Ala Val Arg Val Ala Pro Asp Val
580 585 590 Arg Arg Glu
Ile Met Glu Lys Arg Ser Glu Val Arg Gln Gly Trp Cys 595
600 605 Glu Arg Ile Ser Lys Glu Ala Ala
Ala Lys Cys Gln Thr Val Ile Asp 610 615
620 Asp Leu Thr Leu Met Asn Gly Lys Gln Ala Gln Glu Ile
Thr Glu Leu625 630 635
640 Arg Asp Ser Ala Glu Lys Tyr Glu Lys Gln Ile Ala Glu Leu Val Ser
645 650 655 Thr Ile Thr Gln
Asn Gln Ile Thr Tyr Gln Gln Glu Leu Gln Ala Leu 660
665 670 Val Ala Lys Asn Val Glu Leu Asp Ala
Leu Asn Gln Arg Gln Ala Lys 675 680
685 Ser Leu Arg Ile Thr Pro Ser Leu Leu Ser Ala Thr Pro Ile
Asp Ser 690 695 700
Val Asp Asp Val Ala Asp Leu Ile Asp Phe Ser Val Pro Thr Asp Glu705
710 715 720 Leu191267PRTReovirus
19Met Ser Ser Met Ile Leu Thr Gln Phe Gly Pro Phe Ile Glu Ser Ile1
5 10 15 Ser Gly Ile Thr
Asp Gln Ser Asn Asp Val Phe Glu Asp Ala Ala Lys 20
25 30 Ala Phe Ser Met Phe Thr Arg Ser Asp
Val Tyr Lys Ala Leu Asp Glu 35 40
45 Ile Pro Phe Ser Asp Asp Ala Met Leu Pro Ile Pro Pro Thr
Ile Tyr 50 55 60
Thr Lys Pro Ser His Asp Ser Tyr Tyr Tyr Ile Asp Ala Leu Asn Arg65
70 75 80 Val Arg Arg Lys Thr
Tyr Gln Gly Pro Asp Asp Val Tyr Val Pro Asn 85
90 95 Cys Ser Ile Val Glu Leu Leu Glu Pro His
Glu Thr Leu Thr Ser Tyr 100 105
110 Gly Arg Leu Ser Glu Ala Ile Glu Asn Arg Ala Lys Asp Gly Asp
Ser 115 120 125 Gln
Ala Arg Ile Ala Thr Thr Tyr Gly Arg Ile Ala Glu Ser Gln Ala 130
135 140 Arg Gln Ile Lys Ala Pro
Leu Glu Lys Phe Val Leu Ala Leu Leu Val145 150
155 160 Ala Glu Ala Gly Gly Ser Leu Tyr Asp Pro Val
Leu Gln Lys Tyr Asp 165 170
175 Glu Ile Pro Asp Leu Ser His Asn Cys Pro Leu Trp Cys Phe Arg Glu
180 185 190 Ile Cys Arg
His Ile Ser Gly Pro Leu Pro Asp Arg Ala Pro Tyr Leu 195
200 205 Tyr Leu Ser Ala Gly Val Phe Trp
Leu Met Ser Pro Arg Met Thr Ser 210 215
220 Ala Ile Pro Pro Leu Leu Ser Asp Leu Val Asn Leu Ala
Ile Leu Gln225 230 235
240 Gln Thr Ala Gly Leu Asp Pro Ser Leu Val Lys Leu Gly Val Gln Ile
245 250 255 Cys Leu His Ala
Ala Ala Ser Ser Ser Tyr Ala Trp Phe Ile Leu Lys 260
265 270 Thr Lys Ser Ile Phe Pro Gln Asn Thr
Leu His Ser Met Tyr Glu Ser 275 280
285 Leu Glu Gly Gly Tyr Cys Pro Asn Leu Glu Trp Leu Glu Pro
Arg Ser 290 295 300
Asp Tyr Lys Phe Met Tyr Met Gly Val Met Pro Leu Ser Ala Lys Tyr305
310 315 320 Ala Arg Ser Ala Pro
Ser Asn Asp Lys Lys Ala Arg Glu Leu Gly Glu 325
330 335 Lys Tyr Gly Leu Ser Ser Val Val Gly Glu
Leu Arg Lys Arg Thr Lys 340 345
350 Thr Tyr Val Lys His Asp Phe Ala Ser Val Arg Tyr Ile Arg Asp
Ala 355 360 365 Met
Ala Cys Thr Ser Gly Ile Phe Leu Val Arg Thr Pro Thr Glu Thr 370
375 380 Val Leu Gln Glu Tyr Thr
Gln Ser Pro Glu Ile Lys Val Pro Ile Pro385 390
395 400 Gln Lys Asp Trp Thr Gly Pro Ile Gly Glu Ile
Arg Ile Leu Lys Asp 405 410
415 Thr Thr Ser Ser Ile Ala Arg Tyr Leu Tyr Arg Thr Trp Tyr Leu Ala
420 425 430 Ala Ala Arg
Met Ala Ala Gln Pro Arg Thr Trp Asp Pro Leu Phe Gln 435
440 445 Ala Ile Met Arg Ser Gln Tyr Val
Thr Ala Arg Gly Gly Ser Gly Ala 450 455
460 Ala Leu Arg Glu Ser Leu Tyr Ala Ile Asn Val Ser Leu
Pro Asp Phe465 470 475
480 Lys Gly Leu Pro Val Lys Ala Ala Thr Lys Ile Phe Gln Ala Ala Gln
485 490 495 Leu Ala Asn Leu
Pro Phe Ser His Thr Ser Val Ala Ile Leu Ala Asp 500
505 510 Thr Ser Met Gly Leu Arg Asn Gln Val
Gln Arg Arg Pro Arg Ser Ile 515 520
525 Met Pro Leu Asn Val Pro Gln Gln Gln Val Ser Ala Pro His
Thr Leu 530 535 540
Thr Ala Asp Tyr Ile Asn Tyr His Met Asn Leu Ser Thr Thr Ser Gly545
550 555 560 Ser Ala Val Ile Glu
Lys Val Ile Pro Leu Gly Val Tyr Ala Ser Ser 565
570 575 Pro Pro Asn Gln Ser Ile Asn Ile Asp Ile
Ser Ala Cys Asp Ala Ser 580 585
590 Ile Thr Trp Asp Phe Phe Leu Ser Val Ile Met Ala Ala Ile His
Glu 595 600 605 Gly
Val Ala Ser Ser Ser Ile Gly Lys Pro Phe Met Gly Val Pro Ala 610
615 620 Ser Ile Val Asn Asp Glu
Ser Val Val Gly Val Arg Ala Ala Arg Pro625 630
635 640 Ile Ser Gly Met Gln Asn Met Ile Gln His Leu
Ser Lys Leu Tyr Lys 645 650
655 Arg Gly Phe Ser Tyr Arg Val Asn Asp Ser Phe Ser Pro Gly Asn Asp
660 665 670 Phe Thr His
Met Thr Thr Thr Phe Pro Ser Gly Ser Thr Ala Thr Ser 675
680 685 Thr Glu His Thr Ala Asn Asn Ser
Thr Met Met Glu Thr Phe Leu Thr 690 695
700 Val Trp Gly Pro Glu His Thr Asp Asp Pro Asp Val Leu
Arg Leu Met705 710 715
720 Lys Ser Leu Thr Ile Gln Arg Asn Tyr Val Cys Gln Gly Asp Asp Gly
725 730 735 Leu Met Ile Ile
Asp Gly Thr Thr Ala Gly Lys Val Asn Ser Glu Thr 740
745 750 Ile Gln Lys Met Leu Glu Leu Ile Ser
Lys Tyr Gly Glu Glu Phe Gly 755 760
765 Trp Lys Tyr Asp Ile Ala Tyr Asp Gly Thr Ala Glu Tyr Leu
Lys Leu 770 775 780
Tyr Phe Ile Phe Gly Cys Arg Ile Pro Asn Leu Ser Arg His Pro Ile785
790 795 800 Val Gly Lys Glu Arg
Ala Asn Ser Ser Ala Glu Glu Pro Trp Pro Ala 805
810 815 Ile Leu Asp Gln Ile Met Gly Val Phe Phe
Asn Gly Val His Asp Gly 820 825
830 Leu Gln Trp Gln Arg Trp Ile Arg Tyr Ser Trp Ala Leu Cys Cys
Ala 835 840 845 Phe
Ser Arg Gln Arg Thr Met Ile Gly Glu Ser Val Gly Tyr Leu Gln 850
855 860 Tyr Pro Met Trp Ser Phe
Val Tyr Trp Gly Leu Pro Leu Val Lys Ala865 870
875 880 Phe Gly Ser Asp Pro Trp Ile Phe Ser Trp Tyr
Met Pro Thr Gly Asp 885 890
895 Leu Gly Met Tyr Ser Trp Ile Ser Leu Ile Arg Pro Leu Met Thr Arg
900 905 910 Trp Met Val
Ala Asn Gly Tyr Val Thr Asp Arg Cys Ser Pro Val Phe 915
920 925 Gly Asn Ala Asp Tyr Arg Arg Cys
Phe Asn Glu Leu Lys Leu Tyr Gln 930 935
940 Gly Tyr Tyr Met Ala Gln Leu Pro Arg Asn Pro Lys Lys
Ser Gly Arg945 950 955
960 Ala Ala Pro Arg Glu Val Arg Glu Gln Phe Thr Gln Ala Leu Ser Asp
965 970 975 Tyr Leu Leu Gln
Asn Pro Glu Leu Lys Ser Arg Val Leu Arg Gly Arg 980
985 990 Ser Glu Trp Glu Lys Tyr Gly Ala Gly
Ile Ile His Asn Pro Pro Ser 995 1000
1005 Leu Phe Asp Val Pro His Lys Trp Tyr Gln Gly Ala Gln Glu
Ala Ala 1010 1015 1020
Ile Ala Thr Arg Glu Glu Leu Ala Glu Met Asp Glu Thr Leu Met Arg1025
1030 1035 1040 Ala Arg Arg His Arg
Tyr Ser Ser Phe Ser Lys Leu Leu Glu Ala Tyr 1045
1050 1055 Leu Leu Val Lys Trp Arg Met Cys Glu Ala
Arg Glu Pro Ser Val Asp 1060 1065
1070 Leu Arg Leu Pro Leu Cys Ala Gly Ile Asp Pro Leu Asn Ser Asp
Pro 1075 1080 1085 Phe
Leu Lys Met Val Ser Val Gly Pro Met Leu Gln Ser Thr Arg Lys 1090
1095 1100 Tyr Phe Ala Gln Thr Leu
Phe Met Ala Lys Thr Val Ser Gly Leu Asp1105 1110
1115 1120 Val Asn Ala Ile Asp Ser Ala Leu Leu Arg Leu
Arg Thr Leu Gly Ala 1125 1130
1135 Asp Lys Lys Ala Leu Thr Ala Gln Leu Leu Met Val Gly Leu Gln Glu
1140 1145 1150 Ser Glu Ala
Asp Ala Leu Ala Gly Lys Ile Met Leu Gln Asp Val Asn 1155
1160 1165 Thr Val Gln Leu Ala Arg Val Val
Asn Leu Ala Val Pro Asp Thr Trp 1170 1175
1180 Met Ser Leu Asp Phe Asp Ser Met Phe Lys His His Val
Lys Leu Leu1185 1190 1195
1200 Pro Lys Asp Gly Arg His Leu Asn Thr Asp Ile Pro Pro Arg Met Gly
1205 1210 1215 Trp Leu Arg Ala
Ile Leu Arg Phe Leu Gly Ala Gly Met Val Met Thr 1220
1225 1230 Ala Thr Gly Val Ala Val Asp Ile Tyr
Leu Glu Asp Ile His Gly Gly 1235 1240
1245 Gly Arg Ser Leu Gly Gln Arg Phe Met Thr Trp Met Arg Gln
Glu Gly 1250 1255 1260
Arg Ser Ala1265 201289PRTReovirus 20Met Ala Asn Val Trp Gly Val
Arg Leu Ala Asp Ser Leu Ser Ser Pro1 5 10
15 Thr Ile Glu Thr Arg Thr Arg Gln Tyr Thr Leu His
Asp Leu Cys Ser 20 25 30
Asp Leu Asp Ala Asn Pro Gly Arg Glu Pro Trp Lys Pro Leu Arg Asn
35 40 45 Gln Arg Thr Asn
Asn Ile Val Ala Val Gln Leu Phe Arg Pro Leu Gln 50 55
60 Gly Leu Val Leu Asp Thr Gln Leu Tyr
Gly Phe Pro Gly Ala Phe Asp65 70 75
80 Asp Trp Glu Arg Phe Met Arg Glu Lys Leu Arg Val Leu Lys
Tyr Glu 85 90 95
Val Leu Arg Ile Tyr Pro Ile Ser Asn Tyr Ser Asn Glu His Val Asn
100 105 110 Val Phe Val Ala Asn
Ala Leu Val Gly Ala Phe Leu Ser Asn Gln Ala 115
120 125 Phe Tyr Asp Leu Leu Pro Leu Leu Ile
Ile Asn Asp Thr Met Ile Gly 130 135
140 Asp Leu Leu Gly Thr Gly Ala Ser Leu Ser Gln Phe Phe
Gln Ser His145 150 155
160 Gly Asp Val Leu Glu Val Ala Ala Gly Arg Lys Tyr Leu Gln Met Glu
165 170 175 Asn Tyr Ser Asn
Asp Asp Asp Asp Pro Pro Leu Phe Ala Lys Asp Leu 180
185 190 Ser Asp Tyr Ala Lys Ala Phe Tyr Ser
Asp Thr Tyr Glu Val Leu Asp 195 200
205 Arg Phe Phe Trp Thr His Asp Ser Ser Ala Gly Val Leu Val
His Tyr 210 215 220
Asp Lys Pro Thr Asn Gly His His Tyr Leu Leu Gly Thr Leu Thr Gln225
230 235 240 Met Val Ser Ala Pro
Pro Tyr Ile Ile Asn Ala Thr Asp Ala Met Leu 245
250 255 Leu Glu Ser Cys Leu Glu Gln Phe Ser Ala
Asn Val Arg Ala Arg Pro 260 265
270 Ala Gln Pro Val Thr Arg Leu Asp Gln Cys Tyr His Leu Arg Trp
Gly 275 280 285 Ala
Gln Tyr Val Gly Glu Asp Ser Leu Thr Tyr Arg Leu Gly Val Leu 290
295 300 Ser Leu Leu Ala Thr Asn
Gly Tyr Gln Leu Ala Arg Pro Ile Pro Arg305 310
315 320 Gln Leu Thr Asn Arg Trp Leu Ser Ser Phe Val
Ser Gln Ile Met Ser 325 330
335 Asp Gly Val Asn Glu Thr Pro Leu Trp Pro Gln Glu Arg Tyr Val Gln
340 345 350 Ile Ala Tyr
Asp Ser Pro Ser Val Val Asp Gly Ala Thr Gln Tyr Gly 355
360 365 Tyr Val Arg Lys Asn Gln Leu Arg
Leu Gly Met Arg Ile Ser Ala Leu 370 375
380 Gln Ser Leu Ser Asp Thr Pro Ser Pro Val Gln Trp Leu
Pro Gln Tyr385 390 395
400 Thr Ile Asp Gln Ala Ala Met Asp Glu Gly Asp Leu Met Val Ser Arg
405 410 415 Leu Thr Gln Leu
Pro Leu Arg Pro Asp Tyr Gly Asn Ile Trp Val Gly 420
425 430 Asp Ala Leu Ser Tyr Tyr Val Asp Tyr
Asn Arg Ser His Arg Val Val 435 440
445 Leu Ser Ser Glu Leu Pro Gln Leu Pro Asp Thr Tyr Phe Asp
Gly Asp 450 455 460
Glu Gln Tyr Gly Arg Ser Leu Phe Ser Leu Ala Arg Lys Ile Gly Asp465
470 475 480 Arg Ser Leu Val Lys
Asp Thr Ala Val Leu Lys His Ala Tyr Gln Ala 485
490 495 Ile Asp Pro Asn Thr Gly Lys Glu Tyr Leu
Arg Ser Arg Gln Ser Val 500 505
510 Ala Tyr Phe Gly Ala Ser Ala Gly His Ser Gly Ala Asp Gln Pro
Leu 515 520 525 Val
Ile Glu Pro Trp Ile Gln Gly Lys Ile Ser Gly Val Pro Pro Pro 530
535 540 Ser Ser Val Arg Gln Phe
Gly Tyr Asp Val Ala Arg Gly Ala Ile Val545 550
555 560 Asp Leu Ala Arg Pro Phe Pro Ser Gly Asp Tyr
Gln Phe Val Tyr Ser 565 570
575 Asp Val Asp Gln Val Val Asp Gly His Asp Asp Leu Ser Ile Ser Ser
580 585 590 Gly Leu Val
Glu Ser Leu Leu Ser Ser Cys Met His Ala Thr Ala Pro 595
600 605 Gly Gly Ser Phe Val Val Lys Ile
Asn Phe Pro Thr Arg Pro Val Trp 610 615
620 His Tyr Ile Glu Gln Lys Ile Leu Pro Asn Ile Thr Ser
Tyr Met Leu625 630 635
640 Ile Lys Pro Phe Val Thr Asn Asn Val Glu Leu Phe Phe Val Ala Phe
645 650 655 Gly Val His Gln
His Ser Ser Leu Thr Trp Thr Ser Gly Val Tyr Phe 660
665 670 Phe Leu Val Asp His Phe Tyr Arg Tyr
Glu Thr Leu Ser Thr Ile Ser 675 680
685 Arg Gln Leu Pro Ser Phe Gly Tyr Val Asp Asp Gly Ser Ser
Val Thr 690 695 700
Gly Ile Glu Thr Ile Ser Ile Glu Asn Pro Gly Phe Ser Asn Met Thr705
710 715 720 Gln Ala Ala Arg Ile
Gly Ile Ser Gly Leu Cys Ala Asn Val Gly Asn 725
730 735 Ala Arg Lys Ser Ile Ala Ile Tyr Glu Ser
His Gly Ala Arg Val Leu 740 745
750 Thr Ile Thr Ser Arg Arg Ser Pro Ala Ser Ala Arg Arg Lys Ser
Arg 755 760 765 Leu
Arg Tyr Leu Pro Leu Ile Asp Pro Arg Ser Leu Glu Val Gln Ala 770
775 780 Arg Thr Ile Leu Pro Ala
Asp Pro Val Leu Phe Glu Asn Val Ser Gly785 790
795 800 Ala Ser Pro His Val Cys Leu Thr Met Met Tyr
Asn Phe Glu Val Ser 805 810
815 Ser Ala Val Tyr Asp Gly Asp Val Val Leu Asp Leu Gly Thr Gly Pro
820 825 830 Glu Ala Lys
Ile Leu Glu Leu Ile Pro Ala Thr Ser Pro Val Thr Cys 835
840 845 Val Asp Ile Arg Pro Thr Ala Gln
Pro Ser Gly Cys Trp Asn Val Arg 850 855
860 Thr Thr Phe Leu Glu Leu Asp Tyr Leu Ser Asp Gly Trp
Ile Thr Gly865 870 875
880 Val Arg Gly Asp Ile Val Thr Cys Met Leu Ser Leu Gly Ala Ala Ala
885 890 895 Ala Gly Lys Ser
Met Thr Phe Asp Ala Ala Phe Gln Gln Leu Ile Lys 900
905 910 Val Leu Ser Lys Ser Thr Ala Asn Val
Val Leu Val Gln Val Asn Cys 915 920
925 Pro Thr Asp Val Val Arg Ser Ile Lys Gly Tyr Leu Glu Ile
Asp Ser 930 935 940
Thr Asn Lys Arg Tyr Arg Phe Pro Lys Phe Gly Arg Asp Glu Pro Tyr945
950 955 960 Ser Asp Met Asp Ala
Leu Glu Lys Ile Cys Arg Thr Ala Trp Pro Asn 965
970 975 Cys Ser Ile Thr Trp Val Pro Leu Ser Tyr
Asp Leu Arg Trp Thr Arg 980 985
990 Leu Ala Leu Leu Glu Ser Thr Thr Leu Ser Ser Ala Ser Ile Arg
Ile 995 1000 1005 Ala
Glu Leu Met Tyr Lys Tyr Met Pro Ile Met Arg Ile Asp Ile His 1010
1015 1020 Gly Leu Pro Met Glu Lys
Arg Gly Asn Phe Ile Val Gly Gln Asn Cys1025 1030
1035 1040 Ser Leu Val Ile Pro Gly Phe Asn Ala Gln Asp
Val Phe Asn Cys Tyr 1045 1050
1055 Phe Asn Ser Ala Leu Ala Phe Ser Thr Glu Asp Val Asn Ala Ala Met
1060 1065 1070 Ile Pro Gln
Val Ser Ala Gln Phe Asp Ala Thr Lys Gly Glu Trp Thr 1075
1080 1085 Leu Asp Met Val Phe Ser Asp Ala
Gly Ile Tyr Thr Met Gln Ala Leu 1090 1095
1100 Val Gly Ser Asn Ala Asn Pro Val Ser Leu Gly Ser Phe
Val Val Asp1105 1110 1115
1120 Ser Pro Asp Val Asp Ile Thr Asp Ala Trp Pro Ala Gln Leu Asp Phe
1125 1130 1135 Thr Ile Ala Gly
Thr Asp Val Asp Ile Thr Val Asn Pro Tyr Tyr Arg 1140
1145 1150 Leu Met Thr Phe Val Arg Ile Asp Gly
Gln Trp Gln Ile Ala Asn Pro 1155 1160
1165 Asp Lys Phe Gln Phe Phe Ser Ser Ala Ser Gly Thr Leu Val
Met Asn 1170 1175 1180
Val Lys Leu Asp Ile Ala Asp Lys Tyr Leu Leu Tyr Tyr Ile Arg Asp1185
1190 1195 1200 Val Gln Ser Arg Asp
Val Gly Phe Tyr Ile Gln His Pro Leu Gln Leu 1205
1210 1215 Leu Asn Thr Ile Thr Leu Pro Thr Asn Glu
Asp Leu Phe Leu Ser Ala 1220 1225
1230 Pro Asp Met Arg Glu Trp Ala Val Lys Glu Ser Gly Asn Thr Ile
Cys 1235 1240 1245 Ile
Leu Asn Ser Gln Gly Phe Val Leu Pro Gln Asp Trp Asp Val Leu 1250
1255 1260 Thr Asp Thr Ile Ser Trp
Ser Pro Ser Ile Pro Thr Tyr Ile Val Pro1265 1270
1275 1280 Pro Gly Asp Tyr Thr Leu Thr Pro Leu
1285 211275PRTReovirus 21Met Lys Arg Ile Pro Arg
Lys Thr Lys Gly Lys Ser Ser Gly Lys Gly1 5
10 15 Asn Asp Ser Thr Glu Arg Ala Asp Asp Gly Ser
Ser Gln Leu Arg Asp 20 25 30
Lys Gln Asn Asn Lys Ala Gly Pro Ala Thr Thr Glu Pro Gly Thr Ser
35 40 45 Asn Arg Glu
Gln Tyr Lys Ala Arg Pro Gly Ile Ala Ser Val Gln Arg 50
55 60 Ala Thr Glu Ser Ala Glu Met Pro
Met Lys Asn Asn Asp Glu Gly Thr65 70 75
80 Pro Asp Lys Lys Gly Asn Thr Lys Gly Asp Leu Val Asn
Glu His Ser 85 90 95
Glu Ala Lys Asp Glu Ala Asp Glu Ala Thr Lys Lys Gln Ala Lys Asp
100 105 110 Thr Asp Lys Ser Lys
Ala Gln Val Thr Tyr Ser Asp Thr Gly Ile Asn 115
120 125 Asn Ala Asn Glu Leu Ser Arg Ser Gly
Asn Val Asp Asn Glu Gly Gly 130 135
140 Ser Asn Gln Lys Pro Met Ser Thr Arg Ile Ala Glu Ala
Thr Ser Ala145 150 155
160 Ile Val Ser Lys His Pro Ala Arg Val Gly Leu Pro Pro Thr Ala Ser
165 170 175 Ser Gly His Gly
Tyr Gln Cys His Val Cys Ser Ala Val Leu Phe Ser 180
185 190 Pro Leu Asp Leu Asp Ala His Val Ala
Ser His Gly Leu His Gly Asn 195 200
205 Met Thr Leu Thr Ser Ser Asp Ile Gln Arg His Ile Thr Glu
Phe Ile 210 215 220
Ser Ser Trp Gln Asn His Pro Ile Val Gln Val Ser Ala Asp Val Glu225
230 235 240 Asn Lys Lys Thr Ala
Gln Leu Leu His Ala Asp Thr Pro Arg Leu Val 245
250 255 Thr Trp Asp Ala Gly Leu Cys Thr Ser Phe
Lys Ile Val Pro Ile Val 260 265
270 Pro Ala Gln Val Pro Gln Asp Val Leu Ala Tyr Thr Phe Phe Thr
Ser 275 280 285 Ser
Tyr Ala Ile Gln Ser Pro Phe Pro Glu Ala Ala Val Ser Arg Ile 290
295 300 Val Val His Thr Arg Trp
Ala Ser Asn Val Asp Phe Asp Arg Asp Ser305 310
315 320 Ser Val Ile Met Ala Pro Pro Thr Glu Asn Asn
Ile His Leu Phe Lys 325 330
335 Gln Leu Leu Asn Thr Glu Thr Leu Ser Val Arg Gly Ala Asn Pro Leu
340 345 350 Met Phe Arg
Ala Asn Val Leu His Met Leu Leu Glu Phe Val Leu Asp 355
360 365 Asn Leu Tyr Leu Asn Arg His Thr
Gly Phe Ser Gln Asp His Thr Pro 370 375
380 Phe Thr Glu Gly Ala Asn Leu Arg Ser Leu Pro Gly Pro
Asp Ala Glu385 390 395
400 Lys Trp Tyr Ser Ile Met Tyr Pro Thr Arg Met Gly Thr Pro Asn Val
405 410 415 Ser Lys Ile Cys
Asn Phe Val Ala Ser Cys Val Arg Asn Arg Val Gly 420
425 430 Arg Phe Asp Arg Ala Gln Met Met Asn
Gly Ala Met Ser Glu Trp Val 435 440
445 Asp Val Phe Glu Thr Ser Asp Ala Leu Thr Val Ser Ile Arg
Gly Arg 450 455 460
Trp Met Ala Arg Leu Ala Arg Met Asn Ile Asn Pro Thr Glu Ile Glu465
470 475 480 Trp Ala Leu Thr Glu
Cys Ala Gln Gly Tyr Val Thr Val Thr Ser Pro 485
490 495 Tyr Ala Pro Ser Val Asn Arg Leu Met Pro
Tyr Arg Ile Ser Asn Ala 500 505
510 Glu Arg Gln Ile Ser Gln Ile Ile Arg Ile Met Asn Ile Gly Asn
Asn 515 520 525 Ala
Thr Val Ile Gln Pro Val Leu Gln Asp Ile Ser Val Leu Leu Gln 530
535 540 Arg Ile Ser Pro Leu Gln
Ile Asp Pro Thr Ile Ile Ser Asn Thr Met545 550
555 560 Ser Thr Val Ser Glu Ser Thr Thr Gln Thr Leu
Ser Pro Ala Ser Ser 565 570
575 Ile Leu Gly Lys Leu Arg Pro Ser Asn Ser Asp Phe Ser Ser Phe Arg
580 585 590 Val Ala Leu
Ala Gly Trp Leu Tyr Asn Gly Val Val Thr Thr Val Ile 595
600 605 Asp Asp Ser Ser Tyr Pro Lys Asp
Gly Gly Ser Val Thr Ser Leu Glu 610 615
620 Asn Leu Trp Asp Phe Phe Ile Leu Ala Leu Ala Leu Pro
Leu Thr Thr625 630 635
640 Asp Pro Cys Ala Pro Val Lys Ala Phe Met Thr Leu Ala Asn Met Met
645 650 655 Val Gly Phe Glu
Thr Ile Pro Met Asp Asn Gln Ile Tyr Thr Gln Ser 660
665 670 Arg Arg Ala Ser Ala Phe Ser Thr Pro
His Thr Trp Pro Arg Cys Phe 675 680
685 Met Asn Ile Gln Leu Ile Ser Pro Ile Asp Ala Pro Ile Leu
Arg Gln 690 695 700
Trp Ala Glu Ile Ile His Arg Tyr Trp Pro Asn Pro Ser Gln Ile Arg705
710 715 720 Tyr Gly Ala Pro Asn
Val Phe Gly Ser Ala Asn Leu Phe Thr Pro Pro 725
730 735 Glu Val Leu Leu Leu Pro Ile Asp His Gln
Pro Ala Asn Val Thr Thr 740 745
750 Pro Thr Leu Asp Phe Thr Asn Glu Leu Thr Asn Trp Arg Ala Arg
Val 755 760 765 Cys
Glu Leu Met Lys Asn Leu Val Asp Asn Gln Arg Tyr Gln Pro Gly 770
775 780 Trp Thr Gln Ser Leu Val
Ser Ser Met Arg Gly Thr Leu Asp Lys Leu785 790
795 800 Lys Leu Ile Lys Ser Met Thr Pro Met Tyr Leu
Gln Gln Leu Ala Pro 805 810
815 Val Glu Leu Ala Val Ile Ala Pro Met Leu Pro Phe Pro Pro Phe Gln
820 825 830 Val Pro Tyr
Val Arg Leu Asp Arg Asp Arg Val Pro Thr Met Val Gly 835
840 845 Val Thr Arg His Ser Arg Asp Thr
Ile Thr Gln Pro Ala Leu Ser Leu 850 855
860 Ser Thr Thr Asn Thr Thr Val Gly Val Pro Leu Ala Leu
Asp Ala Arg865 870 875
880 Ala Ile Thr Val Ala Leu Leu Ser Gly Lys Tyr Pro Pro Asp Leu Val
885 890 895 Thr Asn Val Trp
Tyr Ala Asp Ala Ile Tyr Pro Met Tyr Ala Asp Thr 900
905 910 Glu Val Phe Ser Asn Leu Gln Arg Asp
Met Ile Thr Cys Glu Ala Val 915 920
925 Gln Thr Leu Val Thr Leu Val Ala Gln Ile Ser Glu Thr Gln
Tyr Pro 930 935 940
Val Asp Arg Tyr Leu Asp Trp Ile Pro Ser Leu Arg Ala Ser Ala Ala945
950 955 960 Thr Ala Ala Thr Phe
Ala Glu Trp Val Asn Thr Ser Met Lys Thr Ala 965
970 975 Phe Asp Leu Ser Asp Met Leu Leu Glu Pro
Leu Leu Ser Gly Asp Pro 980 985
990 Arg Met Thr Gln Leu Ala Ile Gln Tyr Gln Gln Tyr Asn Gly Arg
Thr 995 1000 1005 Phe
Asn Ile Ile Pro Glu Met Pro Gly Ser Val Ile Ala Asp Cys Val 1010
1015 1020 Gln Leu Thr Ala Glu Val
Phe Asn His Glu Tyr Asn Leu Phe Gly Ile1025 1030
1035 1040 Ala Arg Gly Asp Ile Ile Ile Gly Arg Val Gln
Ser Thr His Leu Trp 1045 1050
1055 Ser Pro Leu Ala Pro Pro Pro Asp Leu Val Phe Asp Arg Asp Thr Pro
1060 1065 1070 Gly Val His
Ile Phe Gly Arg Asp Cys Arg Ile Ser Phe Gly Met Asn 1075
1080 1085 Gly Ala Ala Pro Met Ile Arg Asp
Glu Thr Gly Leu Met Val Pro Phe 1090 1095
1100 Glu Gly Asn Trp Ile Phe Pro Leu Ala Leu Trp Gln Met
Asn Thr Arg1105 1110 1115
1120 Tyr Phe Asn Gln Gln Phe Asp Ala Trp Ile Lys Thr Gly Glu Leu Arg
1125 1130 1135 Ile Arg Ile Glu
Met Gly Ala Tyr Pro Tyr Met Leu His Tyr Tyr Asp 1140
1145 1150 Pro Arg Gln Tyr Ala Asn Ala Trp Asn
Leu Thr Ser Ala Trp Leu Glu 1155 1160
1165 Glu Ile Thr Pro Thr Ser Ile Pro Ser Val Pro Phe Met Val
Pro Ile 1170 1175 1180
Ser Ser Asp His Asp Ile Ser Ser Ala Pro Ala Val Gln Tyr Ile Ile1185
1190 1195 1200 Ser Thr Glu Tyr Asn
Asp Arg Ser Leu Phe Cys Thr Asn Ser Ser Ser 1205
1210 1215 Pro Gln Thr Ile Ala Gly Pro Asp Lys His
Ile Pro Val Glu Arg Tyr 1220 1225
1230 Asn Ile Leu Thr Asn Pro Asp Ala Pro Pro Thr Gln Ile Gln Leu
Pro 1235 1240 1245 Glu
Val Val Asp Leu Tyr Asn Val Val Thr Arg Tyr Ala Tyr Glu Thr 1250
1255 1260 Pro Pro Ile Thr Ala Val
Val Met Gly Val Pro1265 1270 1275
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