Cold-adapted Equine Influenza Viruses

Abstract

The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions. Such methods include using a therapeutic composition as a vaccine to generate a protective immune response in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recently infected with a virulent virus, or is likely to be subsequently exposed to virulent virus in a few days whereby the therapeutic composition interferes with the growth of the virulent virus, even in the absence of immunity. The present invention also provides methods to produce cold-adapted equine influenza viruses, and reassortant influenza A viruses having at least one genome segment of an equine influenza virus generated by cold-adaptation.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. patent application Ser. No. 09/133,921, filed Aug. 13, 1998; and PCT/US99/18583, filed Aug. 12, 1999; each entitled COLD-ADAPTED EQUINE INFLUENZA VIRUSES. The patent applications referred to in this section are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to experimentally-generated cold-adapted equine influenza viruses, and particularly to cold-adapted equine influenza viruses having additional phenotypes, such as attenuation, dominant interference, or temperature sensitivity. The invention also includes reassortant influenza A viruses which contain at least one genome segment from such an equine influenza virus, such that the reassortant virus includes certain phenotypes of the donor equine influenza virus. The invention further includes genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise certain identifying phenotypes of a cold-adapted equine influenza virus of the present invention. The present invention also relates to the use of these viruses in therapeutic compositions to protect animals from diseases caused by influenza viruses.

BACKGROUND OF THE INVENTION

[0003] Equine influenza virus has been recognized as a major respiratory pathogen in horses since about 1956. Disease symptoms caused by equine influenza virus can be severe, and are often followed by secondary bacterial infections. Two subtypes of equine influenza virus are recognized, namely subtype-1, the prototype being A/Equine/Prague/1/56 (H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8). Presently, the predominant virus subtype is subtype-2, which has further diverged among Eurasian and North American isolates in recent years.

[0004] The currently licensed vaccine for equine influenza is an inactivated (killed) virus vaccine. This vaccine provides minimal, if any, protection for horses, and can produce undesirable side effects, for example, inflammatory reactions at the site of injection. See, e.g., Mumford, 1987, Equine Infectious Disease IV, 207-217, and Mumford, et al., 1993, Vaccine 11, 1172-1174. Furthermore, current modalities cannot be used in young foals, because they cannot overcome maternal immunity, and can induce tolerance in a younger animal. Based on the severity of disease, there remains a need for safe, effective therapeutic compositions to protect horses against equine influenza disease.

[0005] Production of therapeutic compositions comprising cold-adapted human influenza viruses is described, for example, in Maassab, et al., 1960, Nature 7, 612-614, and Maassab, et al., 1969, J. Immunol. 102, 728-732. Furthermore, these researchers noted that cold-adapted human influenza viruses, i.e., viruses that have been adapted to grow at lower than normal temperatures, tend to have a phenotype wherein the virus is temperature sensitive; that is, the virus does not grow well at certain higher, non-permissive temperatures at which the wild-type virus will grow and replicate. Various cold-adapted human influenza A viruses, produced by reassortment with existing cold-adapted human influenza A viruses, have been shown to elicit good immune responses in vaccinated individuals, and certain live attenuated cold-adapted reassortant human influenza A viruses have proven to protect humans against challenge with wild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol. 23, 73-76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep. 22, 1992, the inventors of the present invention further demonstrated that certain reassortant cold-adapted human influenza A viruses also possess a dominant interference phenotype, i.e., they inhibit the growth of their corresponding parental wild-type strain, as well as heterologous influenza A viruses.

[0006] U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987, and U.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, disclose attenuated therapeutic compositions produced by reassortment of wild-type equine influenza viruses with attenuated, cold-adapted human influenza A viruses. Although these therapeutic compositions appear to be generally safe and effective in horses, they pose a significant danger of introducing into the environment a virus containing both human and equine influenza genes.

SUMMARY OF THE INVENTION

[0007] The present invention provides experimentally-generated cold-adapted equine influenza viruses, reassortant influenza A viruses that comprise at least one genome segment of an equine influenza virus generated by cold-adaptation such that the equine influenza virus genome segment confers at least one identifying phenotype of a cold-adapted equine influenza virus on the reassortant virus, and genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise at least one identifying phenotype of a cold-adapted equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, dominant interference, and attenuation. The invention further provides a therapeutic composition to protect an animal against disease caused by an influenza A virus, where the therapeutic composition includes a cold-adapted equine influenza virus a reassortant influenza A virus, or a genetically-engineered equine influenza virus of the present invention. Also provided is a method to protect an animal from diseases caused by an influenza A virus which includes the administration of such a therapeutic composition. Also provided are methods to produce a cold-adapted equine influenza virus, and methods to produce a reassortant influenza A virus which comprises at least one genome segment of a cold-adapted equine influenza virus, where the equine influenza genome segment confers on the reassortant virus at least one identifying phenotype of the cold-adapted equine influenza virus.

[0008] A cold-adapted equine influenza virus is one that replicates in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C. Preferably, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention is attenuated, such that it will not cause disease in a healthy animal.

[0009] In one embodiment, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention is also temperature sensitive, such that the virus replicates in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C., forms plaques in tissue culture cells at a permissive temperature of about 34.degree. C., but does not form plaques in tissue culture cells at a non-permissive temperature of about 39.degree. C.

[0010] In one embodiment, such a temperature sensitive virus comprises two mutations: a first mutation that inhibits plaque formation at a temperature of about 39.degree. C., that mutation co-segregating with the genome segment that encodes the viral nucleoprotein gene; and a second mutation that inhibits all viral protein synthesis at a temperature of about 39.degree. C.

[0011] In another embodiment, a cold-adapted, temperature sensitive equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C., forms plaques in tissue culture cells at a permissive temperature of about 34.degree. C., but does not form plaques in tissue culture cells or express late viral proteins at a non-permissive temperature of about 37.degree. C.

[0012] Typically, a cold-adapted equine influenza virus of the present invention is produced by passaging a wild-type equine influenza virus one or more times, and then selecting viruses that stably grow and replicate at a reduced temperature. A cold-adapted equine influenza virus produced thereby includes, in certain embodiments, a dominant interference phenotype, that is, the virus, when co-infected with a parental equine influenza virus or heterologous wild-type influenza A virus, will inhibit the growth of that virus.

[0013] Examples of cold-adapted equine influenza viruses of the present invention include EIV-P821, identified by accession No. ATCC VR-2625; EIV-P824, identified by accession No. ATCC VR-2624; EIV-MSV+5, identified by accession No. ATCC VR-627; and progeny of such viruses.

[0014] Therapeutic compositions of the present invention include from about 10.sup.5 TCID.sub.50 units to about 10.sup.8 TCID.sub.50 units, and preferably about 2.times.10.sup.6 TCID.sub.50 units, of a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention.

[0015] The present invention also includes a method to protect an animal from disease caused by an influenza A virus, which includes the step of administering to the animal a therapeutic composition including a cold-adapted equine influenza virus, a reassortant influenza A virus, or a genetically-engineered equine influenza virus of the present invention. Preferred animals to protect include equids, with horses and ponies being particularly preferred.

[0016] Yet another embodiment of the present invention is a method to generate a cold-adapted equine influenza virus. The method includes the steps of passaging a wild-type equine influenza virus; and selecting viruses that grow at a reduced temperature. In one embodiment, the method includes repeating the passaging and selection steps one or more times, while progressively reducing the temperature. Passaging of equine influenza virus preferably takes place in embryonated chicken eggs.

[0017] Another embodiment is an method to produce a reassortant influenza A virus through genetic reassortment of the genome segments of a donor cold-adapted equine influenza virus of the present invention with the genome segments of a recipient influenza A virus. Reassortant influenza A viruses of the present invention are produced by a method that includes the steps of: (a) mixing the genome segments of a donor cold-adapted equine influenza virus with the genome segments of a recipient influenza A virus, and (b) selecting viruses which include at least one identifying phenotype of the donor equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, dominant interference, and attenuation. Preferably, such reassortant viruses at least include the attenuation phenotype of the donor virus. A typical reassortant virus will have the antigenicity of the recipient virus, that is, it will retain the hemagglutinin (HA) and neuraminidase (NA) phenotypes of the recipient virus.

[0018] The present invention further provides methods to propagate cold-adapted equine influenza viruses or reassortant influenza A viruses of the present invention. These methods include propagation in embryonated chicken eggs or in tissue culture cells.

[0019] The present invention also describes nucleic acid molecules encoding wild-type and cold-adapted equine influenza proteins M, HA, NS, PB2, PB2-N, PB2-C, PB1, PB1-N, PB 1-C, and PA-C. One embodiment of the present invention is an isolated equine nucleic acid molecule having a nucleic acid sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of such nucleic acid sequences. Another embodiment of the present invention is an isolated equine nucleic acid molecule that encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107. Another embodiment is an isolated equine influenza protein that comprises an amino acid sequence selected from a group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107. Also included in the present invention is a virus that include any of these nucleic acid molecules or proteins. In one embodiment, such a virus is equine influenza virus or a reassortant virus.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides experimentally-generated cold-adapted equine influenza viruses comprising certain defined phenotypes, which are disclosed herein. It is to be noted that the term "a" or "an" entity, refers to one or more of that entity; for example, "a cold-adapted equine influenza virus" can include one or more cold-adapted equine influenza viruses. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising," "including," and "having" can be used interchangeably. Furthermore, an item "selected from the group consisting of" refers to one or more of the items in that group, including combinations thereof.

[0021] A cold-adapted equine influenza virus of the present invention is a virus that has been generated in the laboratory, and as such, is not a virus as occurs in nature. Since the present invention also includes those viruses having the identifying phenotypes of such a cold-adapted equine influenza virus, an equine influenza virus isolated from a mixture of naturally-occurring viruses, i.e., removed from its natural milieu, but having the claimed phenotypes, is included in the present invention. A cold-adapted equine influenza virus of the present invention does not require any specific level of purity. For example, a cold-adapted equine influenza virus grown in embryonated chicken eggs may be in a mixture with the allantoic fluid (AF), and a cold-adapted equine influenza virus grown in tissue culture cells may be in a mixture with disrupted cells and tissue culture medium.

[0022] As used herein, an "equine influenza virus" is an influenza virus that infects and grows in equids, e.g., horses or ponies. As used herein, "growth" of a virus denotes the ability of the virus to reproduce or "replicate" itself in a permissive host cell. As such, the terms, "growth of a virus" and "replication of a virus" are used interchangeably herein. Growth or replication of a virus in a particular host cell can be demonstrated and measured by standard methods well-known to those skilled in the art of virology. For example, samples containing infectious virus, e.g., as contained in nasopharyngeal secretions from an infected horse, are tested for their ability to cause cytopathic effect (CPE), e.g., virus plaques, in tissue culture cells. Infectious virus may also be detected by inoculation of a sample into the allantoic cavity of embryonated chicken eggs, and then testing the AF of eggs thus inoculated for its ability to agglutinate red blood cells, i.e., cause hemagglutination, due to the presence of the influenza virus hemaglutinin (HA) protein in the AF.

[0023] Naturally-occurring, i.e., wild-type, equine influenza viruses replicate well at a temperature from about 34.degree. C. to about 39.degree. C. For example, wild-type equine influenza virus replicates in embryonated chicken eggs at a temperature of about 34.degree. C., and replicates in tissue culture cells at a temperature from about 34.degree. C. to about 39.degree. C. As used herein, a "cold-adapted" equine influenza virus is an equine influenza virus that has been adapted to grow at a temperature lower than the optimal growth temperature for equine influenza virus. One example of a cold-adapted equine influenza virus of the present invention is a virus that replicates in embryonated chicken eggs at a temperature of about 30.degree. C. A preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 28.degree. C. Another preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 26.degree. C. In general, preferred cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C., i.e., at a range of temperatures at which a wild-type virus will grow poorly or not at all. It should be noted that the ability of such viruses to replicate within that temperature range does not preclude their ability to also replicate at higher or lower temperatures. For example, one embodiment is a cold-adapted equine influenza virus that replicates in embryonated chicken eggs at a temperature of about 26.degree. C., but also replicates in tissue culture cells at a temperature of about 34.degree. C. As with wild-type equine influenza viruses, cold-adapted equine influenza viruses of the present invention generally form plaques in tissue culture cells, for example Madin Darby Canine Kidney Cells (MDCK) at a temperature of about 34.degree. C. Examples of suitable and preferred cold-adapted equine influenza viruses of the present invention are disclosed herein.

[0024] One embodiment of the present invention is a cold-adapted equine influenza virus that is produced by a method which includes passaging a wild-type equine influenza virus, and then selecting viruses that grow at a reduced temperature. Cold-adapted equine influenza viruses of the present invention can be produced, for example, by sequentially passaging a wild-type equine influenza virus in embryonated chicken eggs at progressively lower temperatures, thereby selecting for certain members of the virus mixture which stably replicate at the reduced temperature. An example of a passaging procedure is disclosed in detail in the Examples section. During the passaging procedure, one or more mutations appear in certain of the single-stranded RNA segments comprising the influenza virus genome, which alter the genotype, i.e., the primary nucleotide sequence of those RNA segments. As used herein, a "mutation" is an alteration of the primary nucleotide sequence of any given RNA segment making up an influenza virus genome. Examples of mutations include substitution of one or more nucleotides, deletion of one or more nucleotides, insertion of one or more nucleotides, or inversion of a stretch of two or more nucleotides. By selecting for those members of the virus mixture that stably replicate at a reduced temperature, a virus with a cold-adaptation phenotype is selected. As used herein, a "phenotype" is an observable or measurable characteristic of a biological entity such as a cell or a virus, where the observed characteristic is attributable to a specific genetic configuration of that biological entity, i.e., a certain genotype. As such, a cold-adaptation phenotype is the result of one or more mutations in the virus genome. As used herein, the terms "a mutation," "a genome," "a genotype," or "a phenotype" refer to one or more, or at least one mutation, genome, genotype, or phenotype, respectively.

[0025] Additional, observable phenotypes in a cold-adapted equine influenza virus may occur, and will generally be the result of one or more additional mutations in the genome of such a virus. For example, a cold-adapted equine influenza virus of the present invention may, in addition, be attenuated, exhibit dominant interference, and/or be temperature sensitive.

[0026] In one embodiment, a cold-adapted equine influenza virus of the present invention has a phenotype characterized by attenuation. A cold-adapted equine influenza virus is "attenuated," when administration of the virus to an equine influenza virus-susceptible animal results in reduced or absent clinical signs in that animal, compared to clinical signs observed in animals that are infected with wild-type equine influenza virus. For example, an animal infected with wild-type equine influenza virus will display fever, sneezing, coughing, depression, and nasal discharges. In contrast, an animal administered an attenuated, cold-adapted equine influenza virus of the present invention will display minimal or no, i.e., undetectable, clinical disease signs.

[0027] In another embodiment, a cold-adapted equine influenza virus of the present invention comprises a temperature sensitive phenotype. As used herein, a temperature sensitive cold-adapted equine influenza virus replicates at reduced temperatures, but no longer replicates or forms plaques in tissue culture cells at certain higher growth temperatures at which the wild-type virus will replicate and form plaques. While not being bound by theory, it is believed that replication of equine influenza viruses with a temperature sensitive phenotype is largely restricted to the cool passages of the upper respiratory tract, and does not replicate efficiently in the lower respiratory tract, where the virus is more prone to cause disease symptoms. A temperature at which a temperature sensitive virus will grow is referred to herein as a "permissive" temperature for that temperature sensitive virus, and a higher temperature at which the temperature sensitive virus will not grow, but at which a corresponding wild-type virus will grow, is referred to herein as a "non-permissive" temperature for that temperature sensitive virus. For example, certain temperature sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30.degree. C., preferably at about 28.degree. C. or about 26.degree. C., and will form plaques in tissue culture cells at a permissive temperature of about 34.degree. C., but will not form plaques in tissue culture cells at a non-permissive temperature of about 39.degree. C. Other temperature sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30.degree. C., preferably at about 28.degree. C. or about 26.degree. C., and will form plaques in tissue culture cells at a permissive temperature of about 34.degree. C., but will not form plaques in tissue culture cells at a non-permissive temperature of about 37.degree. C.

[0028] Certain cold-adapted equine influenza viruses of the present invention have a dominant interference phenotype; that is, they dominate an infection when co-infected into cells with another influenza A virus, thereby impairing the growth of that other virus. For example, when a cold-adapted equine influenza virus of the present invention, having a dominant interference phenotype, is co-infected into MDCK cells with the wild-type parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8), growth of the parental virus is impaired. Thus, in an animal that has recently been exposed to, or may be soon exposed to, a virulent influenza virus, i.e., an influenza virus that causes disease symptoms, administration of a therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype into the upper respiratory tract of that animal will impair the growth of the virulent virus, thereby ameliorating or reducing disease in that animal, even in the absence of an immune response to the virulent virus.

[0029] Dominant interference of a cold-adapted equine influenza virus having a temperature sensitive phenotype can be measured by standard virological methods. For example, separate monolayers of MDCK cells can be infected with (a) a virulent wild-type influenza A virus, (b) a temperature sensitive, cold-adapted equine influenza virus, and (c) both viruses in a co-infection, with all infections done at multiplicities of infection (MOI) of about 2 plaque forming units (pfu) per cell. After infection, the virus yields from the various infected cells are measured by duplicate plaque assays performed at the permissive temperature for the cold-adapted equine influenza virus and at the non-permissive temperature of that virus. A cold adapted equine influenza virus having a temperature sensitive phenotype is unable to form plaques at its non-permissive temperature, while the wild-type virus is able to form plaques at both the permissive and non-permissive temperatures. Thus it is possible to measure the growth of the wild-type virus in the presence of the cold adapted virus by comparing the virus yield at the non-permissive temperature of the cells singly infected with wild-type virus to the yield at the non-permissive temperature of the wild-type virus in doubly infected cells.

[0030] Cold-adapted equine influenza viruses of the present invention are characterized primarily by one or more of the following identifying phenotypes: cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation. As used herein, the phrase "an equine influenza virus comprises the identifying phenotype(s) of cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation" refers to a virus having such a phenotype(s). Examples of such viruses include, but are not limited to, EIV-P821, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, and EIV-MSV+5, identified by accession No. ATCC VR-2627, as well as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4. Production of such viruses is described in the examples. For example, cold-adapted equine influenza virus EIV-P821 is characterized by, i.e., has the identifying phenotypes of, (a) cold-adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 26.degree. C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells and to express late gene products at a non-permissive temperature of about 37.degree. C., and its inability to form plaques in tissue culture cells and to synthesize any viral proteins at a non-permissive temperature of about 39.degree. C.; (c) its attenuation upon administration to an equine influenza virus-susceptible animal; and (d) dominant interference, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with the growth of that wild-type virus. Similarly, cold-adapted equine influenza virus EIV-P824 is characterized by (a) cold adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 28.degree. C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells at a non-permissive temperature of about 39.degree. C.; and (c) dominant interference, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with the growth of that wild-type virus. In another example, cold-adapted equine influenza virus EIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 26.degree. C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells at a non-permissive temperature of about 39.degree. C.; and (c) its attenuation upon administration to an equine influenza virus-susceptible animal.

[0031] In certain cases, the RNA segment upon which one or more mutations associated with a certain phenotype occur may be determined through reassortment analysis by standard methods, as disclosed herein. In one embodiment, a cold-adapted equine influenza virus of the present invention comprises a temperature sensitive phenotype that correlates with at least two mutations in the genome of that virus. In this embodiment, one of the two mutations, localized by reassortment analysis as disclosed herein, inhibits, i.e., blocks or prevents, the ability of the virus to form plaques in tissue culture cells at a non-permissive temperature of about 39.degree. C. This mutation co-segregates with the segment of the equine influenza virus genome that encodes the nucleoprotein (NP) gene of the virus, i.e., the mutation is located on the same RNA segment as the NP gene. In this embodiment, the second mutation inhibits all protein synthesis at a non-permissive temperature of about 39.degree. C. As such, at the non-permissive temperature, the virus genome is incapable of expressing any viral proteins. Examples of cold-adapted equine influenza viruses possessing these characteristics are EIV-P821 and EIV MSV+5. EIV-P821 was generated by serial passaging of a wild-type equine influenza virus in embryonated chicken eggs by methods described in Example 1A. EIV-MSV+5 was derived by further serial passaging of EIV-P821, as described in Example 1E.

[0032] Furthermore, a cold-adapted, temperature sensitive equine influenza virus comprising the two mutations which inhibit plaque formation and viral protein synthesis at a non-permissive temperature of about 39.degree. C. can comprise one or more additional mutations, which inhibit the virus' ability to synthesize late gene products and to form plaques in tissue culture cells at a non-permissive temperature of about 37.degree. C. An example of a cold-adapted equine influenza virus possessing these characteristics is EIV-P821. This virus isolate replicates in embryonated chicken eggs at a temperature of about 26.degree. C., and does not form plaques or express any viral proteins at a temperature of about 39.degree. C. Furthermore, EIV-P821 does not form plaques on MDCK cells at a non-permissive temperature of about 37.degree. C., and at this temperature, late gene expression is inhibited in such a way that late proteins are not produced, i.e., normal levels of NP protein are synthesized, reduced or undetectable levels of M1 or HA proteins are synthesized, and enhanced levels of the polymerase proteins are synthesized. Since this phenotype is typified by differential viral protein synthesis, it is distinct from the protein synthesis phenotype seen at a non-permissive temperature of about 39.degree. C., which is typified by the inhibition of synthesis of all viral proteins.

[0033] Pursuant to 37 CFR .sctn. 1.802 (a-c), cold-adapted equine influenza viruses, designated herein as EIV-P821, an EIV-P824 were deposited with the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC Accession Nos. ATCC VR-2625, and ATCC VR-2624, respectively, on Jul. 11, 1998. Cold-adapted equine influenza virus EIV-MSV+5 was deposited with the ATCC as ATCC Accession No. ATCC VR-2627 on Aug. 3, 1998. Pursuant to 37 CFR.sctn. 1.806, the deposits are made for a term of at least thirty (30) years and at least five (5) years after the most recent request for the furnishing of a sample of the deposit was received by the depository. Pursuant to 37 CFR .sctn. 1.808 (a)(2), all restrictions imposed by the depositor on the availability to the public will be irrevocably removed upon the granting of the patent.

[0034] Preferred cold-adapted equine influenza viruses of the present invention have the identifying phenotypes of EIV-P821, EIV-P824, and EIV-MSV+5. Particularly preferred cold-adapted equine influenza viruses include EIV-P821, EIV-P824, EIV-MSV+5, and progeny of these viruses. As used herein, "progeny" are "offspring," and as such can slightly altered phenotypes compared to the parent virus, but retain identifying phenotypes of the parent virus, for example, cold-adaptation, temperature sensitivity, dominant interference, or attenuation. For example, cold-adapted equine influenza virus EIV-MSV+5 is a "progeny" of cold-adapted equine influenza virus EIV-P821. "Progeny" also include reassortant influenza A viruses that comprise one or more identifying phenotypes of the donor parent virus.

[0035] Reassortant influenza A viruses of the present invention are produced by genetic reassortment of the genome segments of a donor cold-adapted equine influenza virus of the present invention with the genome segments of a recipient influenza A virus, and then selecting a reassortant virus that derives at least one of its eight RNA genome segments from the donor virus, such that the reassortant virus acquires at least one identifying phenotype of the donor cold-adapted equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, attenuation, and dominant interference. Preferably, reassortant influenza A viruses of the present invention derive at least the attenuation phenotype of the donor virus. Methods to isolate reassortant influenza viruses are well known to those skilled in the art of virology and are disclosed, for example, in Fields, et al., 1996, Fields Virology, 3d ed., Lippincott-Raven; and Palese, et al., 1976, J. Virol., 17, 876-884. Fields, et al., ibid. and Palese, et al., ibid.

[0036] A suitable donor equine influenza virus is a cold-adapted equine influenza virus of the present invention, for example, EIV-P821, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, or EIV-MSV+5, identified by accession No. ATCC VR-2627. A suitable recipient influenza A virus can be another equine influenza virus, for example a Eurasian subtype 2 equine influenza virus such as A/equine/Suffolk/89 (H3N8) or a subtype 1 equine influenza virus such as A/Prague/1/56 (H7N7). A recipient influenza A virus can also be any influenza A virus capable of forming a reassortant virus with a donor cold-adapted equine influenza virus. Examples of such influenza A viruses include, but are not limited to, human influenza viruses such as A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1), A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses such as A/Swine/Iowa/15/30 (H1N1); and avian viruses such as A/mallard/New York/6750/78 (H2N2) and A/chicken/Hong Kong/258/97 (H5N1). A reassortant virus of the present invention can include any combination of donor and recipient gene segments, as long as the resulting reassortant virus possesses at least one identifying phenotype of the donor virus.

[0037] One example of a reassortant virus of the present invention is a "6+2" reassortant virus, in which the six "internal gene segments," i.e., those comprising the NP, PB2, PB1, PA, M, and NS genes, are derived from the donor cold-adapted equine influenza virus genome, and the two "external gene segments," i.e., those comprising the HA and NA genes, are derived from the recipient influenza A virus. A resultant virus thus produced has the attenuated, cold-adapted, temperature sensitive, and/or dominant interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.

[0038] In yet another embodiment, a cold-adapted equine influenza virus of the present invention can be produced through recombinant means. In this approach, one or more specific mutations, associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, are identified and are introduced back into a wild-type equine influenza virus strain using a reverse genetics approach. Reverse genetics entails using RNA polymerase complexes isolated from influenza virus-infected cells to transcribe artificial influenza virus genome segments containing the mutation(s), incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes. Reverse genetics methods for influenza viruses are described, for example, in Enami, et al., 1990, Proc. Natl. Acad. Sci. 87, 3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et al., issued Nov. 26, 1996. This approach allows one skilled in the art to produce additional cold-adapted equine influenza viruses of the present invention without the need to go through the lengthy cold-adaptation process, and the process of selecting mutants both in vitro and in vivo with the desired virus phenotype.

[0039] A cold-adapted equine influenza virus of the present invention may be propagated by standard virological methods well-known to those skilled in the art, examples of which are disclosed herein. For example, a cold-adapted equine influenza virus can be grown in embryonated chicken eggs or in eukaryotic tissue culture cells. Suitable continuous eukaryotic cell lines upon which to grow a cold-adapted equine influenza virus of the present invention include those that support growth of influenza viruses, for example, MDCK cells. Other suitable cells upon which to grow a cold-adapted equine influenza virus of the present invention include, but are not limited to, primary kidney cell cultures of monkey, calf, hamster or chicken.

[0040] In one embodiment, the present invention provides a therapeutic composition to protect an animal against disease caused by an influenza A virus, where the therapeutic composition includes either a cold-adapted equine influenza virus or a reassortant influenza A virus comprising at least one genome segment of an equine influenza virus generated by cold-adaptation, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus. In addition, a therapeutic composition of the present invention can include an equine influenza virus that has been genetically engineered to comprise one or more mutations, where those mutations have been identified to confer a certain identifying phenotype on a cold-adapted equine influenza virus of the present invention. As used herein, the phrase "disease caused by an influenza A virus" refers to the clinical manifestations observed in an animal which has been infected with a virulent influenza A virus. Examples of such clinical manifestations include, but are not limited to, fever, sneezing, coughing, nasal discharge, rates, anorexia and depression. In addition, the phrase "disease caused by an influenza A virus" is defined herein to include shedding of virulent virus by the infected animal. Verification that clinical manifestations observed in an animal correlate with infection by virulent equine influenza virus may be made by several methods, including the detection of a specific antibody and/or T-cell responses to equine influenza virus in the animal. Preferably, verification that clinical manifestations observed in an animal correlate with infection by a virulent influenza A virus is made by the isolation of the virus from the afflicted animal, for example, by swabbing the nasopharyngeal cavity of that animal for virus-containing secretions. Verification of virus isolation may be made by the detection of CPE in tissue culture cells inoculated with the isolated secretions, by inoculation of the isolated secretions into embryonated chicken eggs, where virus replication is detected by the ability of AF from the inoculated eggs to agglutinate erythrocytes, suggesting the presence of the influenza virus hemagglutinin protein, or by use of a commercially available diagnostic test, for example, the Directigen.RTM. FLU A test.

[0041] As used herein, the term "to protect" includes, for example, to prevent or to treat influenza A virus infection in the subject animal. As such, a therapeutic composition of the present invention can be used, for example, as a prophylactic vaccine to protect a subject animal from influenza disease by administering the therapeutic composition to that animal at some time prior to that animal's exposure to the virulent virus.

[0042] A therapeutic composition of the present invention, comprising a cold-adapted equine influenza virus having a dominant interference phenotype, can also be used to treat an animal that has been recently infected with virulent influenza A virus or is likely to be subsequently exposed in a few days, such that the therapeutic composition immediately interferes with the growth of the virulent virus, prior to the animal's production of antibodies to the virulent virus. A therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype may be effectively administered prior to subsequent exposure for a length of time corresponding to the approximate length of time that a cold-adapted equine influenza virus of the present invention will replicate in the upper respiratory tract of a treated animal, for example, up to about seven days. A therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype may be effectively administered following exposure to virulent equine influenza virus for a length of time corresponding to the time required for an infected animal to show disease symptoms, for example, up to about two days.

[0043] Therapeutic compositions of the present invention can be administered to any animal susceptible to influenza virus disease, for example, humans, swine, horses and other equids, aquatic birds, domestic and game fowl, seals, mink, and whales. Preferably, a therapeutic composition of the present invention is administered equids. Even more preferably, a therapeutic composition of the present invention is administered to a horse, to protect against equine influenza disease.

[0044] Current vaccines available to protect horses against equine influenza virus disease are not effective in protecting young foals, most likely because they cannot overcome the maternal antibody present in these young animals, and often, vaccination at an early age, for example 3 months of age, can lead to tolerance rather than immunity. In one embodiment, and in contrast to existing equine influenza virus vaccines, a therapeutic composition comprising a cold-adapted equine influenza virus of the present invention apparently can produce immunity in young animals. As such, a therapeutic composition of the present invention can be safely and effectively administered to young foals, as young as about 3 months of age, to protect against equine influenza disease without the induction of tolerance.

[0045] In one embodiment, a therapeutic composition of the present invention can be multivalent. For example, it can protect an animal from more than one strain of influenza A virus by providing a combination of one or more cold-adapted equine influenza viruses of the present invention, one or more reassortant influenza A viruses, and/or one or more genetically-engineered equine influenza viruses of the present invention. Multivalent therapeutic compositions can include at least two cold-adapted equine influenza viruses, e.g., against North American subtype-2 virus isolates such as A/equine/Kentucky/1/91 (H1N8), and Eurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); or one or more subtype-2 virus isolates and a subtype-1 virus isolate such as A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeutic composition of the present invention can include a cold-adapted equine influenza virus and a reassortant influenza A virus of the present invention, or two reassortant influenza A viruses of the present invention. A multivalent therapeutic composition of the present invention can also contain one or more formulations to protect against one or more other infectious agents in addition to influenza A virus. Such other infectious agents include, but not limited to: viruses; bacteria; fungi and fungal-related microorganisms; and parasites. Preferable multivalent therapeutic compositions include, but are not limited to, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention plus one or more compositions protective against one or more other infectious agents that afflict horses. Suitable infectious agents to protect against include, but are not limited to, equine infectious anemia virus, equine herpes virus, eastern, western, or Venezuelan equine encephalitis virus, tetanus, Streptococcus equi, and Ehrlichia resticii.

[0046] A therapeutic composition of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical or biological stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of stabilizers include A1/A2 stabilizer, available from Diamond Animal Health, Des Moines, Iowa. Standard formulations can either be liquids or solids which can be taken up in a suitable liquid as a suspension or solution for administration to an animal. In one embodiment, a non-liquid formulation may comprise the excipient salts, buffers, stabilizers, etc., to which sterile water or saline can be added prior to administration.

[0047] A therapeutic composition of the present invention may also include one or more adjuvants or carriers. Adjuvants are typically substances that enhance the immune response of an animal to a specific antigen, and carriers include those compounds that increase the half-life of a therapeutic composition in the treated animal. One advantage of a therapeutic composition comprising a cold-adapted equine influenza virus or a reassortant influenza A virus of the present invention is that adjuvants and carriers are not required to produce an efficacious vaccine. Furthermore, in many cases known to those skilled in the art, the advantages of a therapeutic composition of the present invention would be hindered by the use of some adjuvants or carriers. However, it should be noted that use of adjuvants or carriers is not precluded by the present invention.

[0048] Therapeutic compositions of the present invention include an amount of a cold-adapted equine influenza virus that is sufficient to protect an animal from challenge with virulent equine influenza virus. In one embodiment, a therapeutic composition of the present invention can include an amount of a cold-adapted equine influenza virus ranging from about 10.sup.5 tissue culture infectious dose-50 (TCID.sub.50) units of virus to about 10.sup.8 TCID.sub.50 units of virus. As used herein, a "TCID.sub.50 unit" is amount of a virus which results in cytopathic effect in 50% of those cell cultures infected. Methods to measure and calculate TCID.sub.50 are known to those skilled in the art and are available, for example, in Reed and Muench, 1938, Am. J. of Hyg. 27, 493-497. A preferred therapeutic composition of the present invention comprises from about 10.sup.6 TCID.sub.50 units to about 10.sup.7 TCID.sub.50 units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention. Even more preferred is a therapeutic composition comprising about 2.times.10.sup.6 TCID.sub.50 units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention.

[0049] The present invention also includes methods to protect an animal against disease caused by an influenza A virus comprising administering to the animal a therapeutic composition of the present invention. Preferred are those methods which protect an equid against disease caused by equine influenza virus, where those methods comprise administering to the equid a cold-adapted equine influenza virus. Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art, and examples are disclosed herein.

[0050] A preferable method to protect an animal against disease caused by an influenza A virus includes administering to that animal a single dose of a therapeutic composition comprising a cold-adapted equine influenza virus, a reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. The method of the present invention may also include administering subsequent, or booster doses of a therapeutic composition. Booster administrations can be given from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. Examples of suitable and preferred dosage schedules are disclosed in the Examples section.

[0051] A therapeutic composition of the present invention can be administered to an animal by a variety of means, such that the virus will enter and replicate in the mucosal cells in the upper respiratory tract of the treated animal. Such means include, but are not limited to, intranasal administration, oral administration, and intraocular administration. Since influenza viruses naturally infect the mucosa of the upper respiratory tract, a preferred method to administer a therapeutic composition of the present invention is by intranasal administration. Such administration may be accomplished by use of a syringe fitted with cannula, or by use of a nebulizer fitted over the nose and mouth of the animal to be vaccinated.

[0052] The efficacy of a therapeutic composition of the present invention to protect an animal against disease caused by influenza A virus can be tested in a variety of ways including, but not limited to, detection of antibodies by, for example, hemagglutination inhibition (HAI) tests, detection of cellular immunity within the treated animal, or challenge of the treated animal with virulent equine influenza virus to determine whether the treated animal is resistant to the development of disease. In addition, efficacy of a therapeutic composition of the present invention comprising a cold-adapted equine influenza virus having a dominant interference phenotype to ameliorate or reduce disease symptoms in an animal previously inoculated or susceptible to inoculation with a virulent, wild-type equine influenza virus can be tested by screening for the reduction or absence of disease symptoms in the treated animal.

[0053] The present invention also includes methods to produce a therapeutic composition of the present invention. Suitable and preferred methods for making a therapeutic composition of the present invention are disclosed herein. Pertinent steps involved in producing one type of therapeutic composition of the present invention, i.e., a cold-adapted equine influenza virus, include (a) passaging a wild-type equine influenza virus in vitro, for example, in embryonated chicken eggs; (b) selecting viruses that grow at a reduced temperature; (c) repeating the passaging and selection steps one or more times, at progressively lower temperatures, until virus populations are selected which stably grow at the desired lower temperature; and (d) mixing the resulting virus preparation with suitable excipients.

[0054] The pertinent steps involved in producing another type of therapeutic composition of the present invention, i.e., a reassortant influenza A virus having at least one genome segment of an equine influenza virus generated by adaptation, includes the steps of (a) mixing the genome segments of a donor cold-adapted equine influenza virus, which preferably also has the phenotypes of attenuation, temperature sensitivity, or dominant interference, with the genome segments of a recipient influenza A virus, and (b) selecting reassortant viruses that have at least one identifying phenotype of the donor equine influenza virus. Identifying phenotypes to select for include attenuation, cold-adaptation, temperature sensitivity, and dominant interference. Methods to screen for these phenotypes are well known to those skilled in the art, and are disclosed herein. It is preferable to screen for viruses that at least have the phenotype of attenuation.

[0055] Using this method to generate a reassortant influenza A virus having at least one genome segment of a equine influenza virus generated by cold-adaptation, one type of reassortant virus to select for is a "6+2" reassortant, where the six "internal gene segments," i.e., those coding for the NP, PB2, PB1, PA, M, and NS genes, are derived from the donor cold-adapted equine influenza virus genome, and the two "external gene segments," i.e., those coding for the HA and NA genes, are derived from the recipient influenza A virus. A resultant virus thus produced can have the cold-adapted, attenuated, temperature sensitive, and/or interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.

[0056] The present invention includes nucleic acid molecules isolated from equine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8), and cold-adapted equine influenza virus EIV-P821.

[0057] In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified.

[0058] The present invention includes nucleic acid molecules encoding wild-type and cold-adapted equine influenza virus proteins. Nucleic acid molecules of the present invention can be prepared by methods known to one skilled in the art. Proteins of the present invention can be prepared by methods known to one skilled in the art, i.e., recombinant DNA technology. Preferred nucleic acid molecules have coding strands comprising nucleic acid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:82, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and/or a complement thereof. Complements are defined as two single strands of nucleic acid in which the nucleotide sequence is such that they will hybridize as a result of base pairing throughout their full length. Given a nucleotide sequence, one of ordinary skill in the art can deduce the complement.

[0059] Preferred nucleic acid molecules encoding equine influenza M proteins are nei.sub.wtM.sub.1023, nei.sub.wt1M.sub.1023, nei.sub.wt2M.sub.1023, nei.sub.wtM.sub.756, nei.sub.wt1M.sub.756, nei.sub.wt2M.sub.756, nei.sub.ca1M.sub.1023, nei.sub.ca2M.sub.1023, nei.sub.ca1M.sub.756, and/or nei.sub.ca2M.sub.756, the coding strands of which are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6.

[0060] Preferred nucleic acid molecules encoding equine influenza HA proteins are nei.sub.wtHA.sub.1762, nei.sub.wtHA.sub.1695, nei.sub.ca1HA.sub.1762, nei.sub.ca2HA.sub.1762, nei.sub.ca1HA.sub.1695, and/or nei.sub.ca2HA.sub.1695, the coding strands of which are represented by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12.

[0061] Preferred nucleic acid molecules encoding equine influenza PB2-N proteins are nei.sub.wtPB2-N.sub.1241, nei.sub.wtPB2-N.sub.1214, nei.sub.ca1PB2-N.sub.1241, nei.sub.ca2PB2-N.sub.1241, nei.sub.ca1PB2-N.sub.1214 nei.sub.ca2, and/or PB2-N.sub.1214, the coding strands of which are represented by SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18.

[0062] Preferred nucleic acid molecules encoding equine influenza PB2-C proteins are nei.sub.wt1PB2-C.sub.1233, nei.sub.wt2PB2-C.sub.1232, nei.sub.wtPB2-C.sub.1194, nei.sub.ca1PB2-C.sub.1232, nei.sub.ca2PB2-C.sub.1231, and/or nei.sub.ca1PB2-C.sub.1194, the coding strands of which are represented by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25.

[0063] Preferred nucleic acid molecules encoding equine influenza PB2 proteins are nei.sub.wtPB2.sub.2341, nei.sub.wtPB2.sub.2277, nei.sub.ca1PB2.sub.2341, and/or nei.sub.ca1PB2.sub.2277, the coding strands of which are represented by SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49.

[0064] Preferred nucleic acid molecules encoding equine influenza NS proteins are nei.sub.wt1NS.sub.891, nei.sub.wt2NS.sub.891, nei.sub.wt1NS.sub.690, nei.sub.wt2NS.sub.690, nei.sub.wt3NS.sub.888, nei.sub.wt3NS.sub.690, nei.sub.wt4NS.sub.468, nei.sub.wt4NS.sub.293, nei.sub.ca1NS.sub.888, nei.sub.ca2NS.sub.888, nei.sub.ca1NS.sub.690, and/or nei.sub.ca2NS.sub.690 other coding strands of which are represented by SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQ ID NO:59.

[0065] Preferred nucleic acid molecules encoding equine influenza PB1-N proteins are nei.sub.wt1PB1-N.sub.1229, nei.sub.wt1PB1N.sub.1194, nei.sub.wt2PB1-N.sub.673, nei.sub.wt2PB1-N.sub.636, nei.sub.ca1PB1-N.sub.1225, nei.sub.ca1PB1-N.sub.1185, nei.sub.ca2PB1-N.sub.1221, and/or nei.sub.ca2PB1-N.sub.1185 the coding strands of which are represented by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71.

[0066] Preferred nucleic acid molecules encoding equine influenza PA-C proteins are nei.sub.wt1PA-C.sub.1228, nei.sub.wt1PA-C.sub.1164, nei.sub.wt2PA-C.sub.1223, nei.sub.wt2PA-C.sub.1164, nei.sub.ca1PA-C.sub.1233, nei.sub.ca2PA-C.sub.1233, nei.sub.ca1PA-C.sub.1170, and/or nei.sub.ca2PA-C.sub.1170 the coding strands of which are represented by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82.

[0067] Preferred nucleic acid molecules encoding equine influenza PB1-C proteins are nei.sub.wt1PB1-C.sub.1234, nei.sub.wt1PB1-C.sub.1188, nei.sub.wt2PB1-C.sub.1240, nei.sub.wt2PB1-C.sub.1188, nei.sub.ca1PB1-C.sub.1241, nei.sub.ca1PB1-C.sub.1188, nei.sub.ca2PB1-C.sub.1241 and/or nei.sub.ca2PB1-C.sub.1188, the coding strands of which are represented by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94 and/or SEQ ID NO:96.

[0068] Preferred nucleic acid molecules encoding equine influenza PB1 proteins are nei.sub.wtPB1.sub.2341, nei.sub.wtPB1.sub.2271, nei.sub.ca1PB1.sub.2341, nei.sub.ca1PB1.sub.2271, the coding strands of which are represented by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108.

[0069] The present invention includes proteins comprising SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO: 55, SEQ ID NO:58, SEQ ID NO: 63, SEQ ID NO:66, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO:86, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO: 107 as well as nucleic acid molecules encoding such proteins.

[0070] Preferred equine influenza M proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wtM.sub.1023, nei.sub.wt1M.sub.1023, nei.sub.wt2M.sub.1023, nei.sub.wtM.sub.756, nei.sub.wt1M.sub.756, nei.sub.wt2M.sub.756, nei.sub.ca1M.sub.1023, nei.sub.ca2M.sub.1023, nei.sub.ca1M.sub.756, and/or nei.sub.ca2M.sub.756. Preferred equine influenza M proteins are Pei.sub.wtM.sub.252, Pei.sub.ca1M.sub.252, and/or Pei.sub.ca2M.sub.252. In one embodiment, a preferred equine influenza M protein of the present invention is encoded by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6, and, as such, has an amino acid sequence that includes SEQ ID NO:2 and/or SEQ ID NO:5.

[0071] Preferred equine influenza HA proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wtHA.sub.1762, nei.sub.wtHA.sub.1695, nei.sub.ca1HA.sub.1762, nei.sub.ca2HA.sub.1762, nei.sub.ca1HA.sub.1695, and/or nei.sub.ca2HA.sub.1695. Preferred equine influenza HA proteins are P Pei.sub.wtHA.sub.565, Pei.sub.ca1HA.sub.565, and/or Pei.sub.ca2HA.sub.565. In one embodiment, a preferred equine influenza HA protein of the present invention is encoded by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12, and, as such, has an amino acid sequence that includes SEQ ID NO:8 and/or SEQ ID NO:11.

[0072] Preferred equine influenza PB2-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wtPB2-N.sub.1241, nei.sub.wtPB2-N.sub.1214, nei.sub.ca1PB2-N.sub.1241 nei.sub.ca2PB2-N.sub.1241, nei.sub.ca1PB2-N.sub.1214 nei.sub.ca2, and/or PB2-N.sub.1214. Preferred equine influenza PB2-N proteins are P.sub.wtPB2-N.sub.404, P.sub.ca1PB2-N.sub.404, and/or P.sub.ca2PB2-N.sub.404. In one embodiment, a preferred equine influenza PB2-N protein of the present invention is encoded by SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18, and, as such, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQ ID NO:17.

[0073] Preferred equine influenza PB2-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wt1PB2-C.sub.1233, nei.sub.wt2PB2-C.sub.1232, nei.sub.wtPB2-C.sub.1194, nei.sub.ca1PB2-C.sub.1232, nei.sub.ca2PB2-C.sub.1231, and/or nei.sub.ca1PB2-C.sub.1194. Preferred equine influenza PB2-N proteins are P.sub.wtPB2-C.sub.398, P.sub.ca1PB2-C.sub.398, and/or P.sub.ca2PB2-C.sub.398. In one embodiment, a preferred equine influenza PB2-C protein of the present invention is encoded by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25, and, as such, has an amino acid sequence that includes SEQ ID NO:20 and/or SEQ ID NO:24.

[0074] Preferred equine influenza PB2 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wtPB2.sub.2341, nei.sub.wtPB2.sub.2277, nei.sub.ca1PB2.sub.2341, and or nei.sub.ca1PB2.sub.2277. Preferred equine influenza PB2 proteins are Pei.sub.wtPB2.sub.759, and/or Pei.sub.ca1PB2.sub.759. In one embodiment, a preferred equine influenza PB2 protein of the present invention is encoded by SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49, and, as such, has an amino acid sequence that includes SEQ ID NO:45 and/or SEQ ID NO:48.

[0075] Preferred equine influenza NS proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wt1NS.sub.891, nei.sub.wt2NS.sub.891, nei.sub.wt1NS.sub.690, nei.sub.wt3NS.sub.888, nei.sub.wt4NS.sub.468, nei.sub.wt4NS.sub.293, nei.sub.ca1NS.sub.888, nei.sub.ca2NS.sub.888, and/or nei.sub.ca1NS.sub.690. Preferred equine influenza NS proteins are Pei.sub.wtNS.sub.230, Pei.sub.wt4NS.sub.97, and/or Pei.sub.ca1NS.sub.230. In one embodiment, a preferred equine influenza NS protein of the present invention is encoded by SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQ ID NO:59, and, as such, has an amino acid sequence that includes SEQ ID NO:51, SEQ ID NO:55 and/or SEQ ID NO:58.

[0076] Preferred equine influenza PB1-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wt1PB1-N.sub.1229, nei.sub.wt1PB1N.sub.1194, nei.sub.wt2PB1-N.sub.673, nei.sub.wt2PB1-N.sub.636, nei.sub.ca1PB21-N.sub.1225, nei.sub.ca1PB1-N.sub.1185, and/or nei.sub.ca2PB1-N.sub.1221. Preferred equine influenza PB1-N proteins are Pei.sub.wt1PB1-N.sub.398, P.sub.wt2PB1-N.sub.212, and/or P.sub.ca1PB1-N.sub.395. In one embodiment, a preferred equine influenza PB1-N protein of the present invention is encoded by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71, and, as such, has an amino acid sequence that includes SEQ ID NO:63, SEQ ID NO:66 and/or SEQ ID NO:69.

[0077] Preferred equine influenza PB1-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wt1PB1-C.sub.1234, nei.sub.wt1PB1-C.sub.1188, nei.sub.wt2PB1-C.sub.1240, nei.sub.wt2PB1-C.sub.1188, nei.sub.ca1PB1-C.sub.1241, nei.sub.ca1PB1-C.sub.1188, nei.sub.ca2PB1-C.sub.1241 and/or nei.sub.ca2PB1-C.sub.1188. Preferred equine influenza PB1-C proteins are Pei.sub.wt1PB1-C.sub.396, Pei.sub.wt2PB1-C.sub.396 Pei.sub.ca1PB1-C.sub.396, and/or Pei.sub.ca2PB1-C.sub.396. In one embodiment, a preferred equine influenza PB1-C protein of the present invention is encoded by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, and/or SEQ ID NO:96, and, as such, has an amino acid sequence that includes SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, and/or SEQ ID NO:95.

[0078] Preferred equine influenza PB1 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wtPB1.sub.2341, nei.sub.wtPB1.sub.2271, nei.sub.ca1PB1.sub.2341, nei.sub.ca1PB1.sub.2271. Preferred equine influenza PB1 proteins are Pei.sub.wtPB1.sub.757, and/or Pei.sub.ca1PB1.sub.757. In one embodiment, a preferred equine influenza PB1 protein of the present invention is encoded by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108, and, as such, has an amino acid sequence that includes SEQ ID NO:104 and/or SEQ ID NO:107.

[0079] Preferred equine influenza PA-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei.sub.wt1PA-C.sub.1228, nei.sub.wt1PA-C.sub.1164, nei.sub.wt2PA-C.sub.1223, nei.sub.ca1PA-C.sub.1233, nei.sub.ca2PA-C.sub.1233, and/or nei.sub.ca1PA-C.sub.1170. Preferred equine influenza PA-C proteins are Pei.sub.wt1PA-C.sub.388, and/or Pei.sub.ca1PA-C.sub.390. In one embodiment, a preferred equine influenza PA-C protein of the present invention is encoded by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82, and, as such, has an amino acid sequence that includes SEQ ID NO:77 and/or SEQ ID NO:81.

[0080] Nucleic acid sequence SEQ ID NO:1 represents the consensus sequence deduced from the coding strand of PCR amplified nucleic acid molecules denoted herein as nei.sub.wt1M.sub.1023 and nei.sub.wt2M.sub.1023, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei.sub.ca1M.sub.1023 and nei.sub.ca2M.sub.1023, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wtHA.sub.1762, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei.sub.ca1HA.sub.1762 and nei.sub.ca2HA.sub.1762, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:13 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wtPB2-N.sub.1241, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:16 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei.sub.ca1PB2-N.sub.1241 and nei.sub.ca2PB2-N.sub.1241, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:19 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt1PB2-C.sub.1233, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:22 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt2PB2-C.sub.1232, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:23 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca1PB2-C.sub.1232, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:44 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wtPB2.sub.2341 the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:47 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei.sub.ca1PB2.sub.2341 the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:50 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt1NS.sub.891 and nei.sub.wt2NS.sub.891 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:53 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt3NS.sub.888, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:54 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt4NS.sub.468, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:57 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca1NS.sub.888 and nei.sub.ca1NS.sub.887 the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:62 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei.sub.wt1PB1-N.sub.1229, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:65 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt2PB2-N.sub.673, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:68 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca1PB1-N.sub.1225, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:71 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca2PB1-N.sub.1221, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:76 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt1PA-C.sub.1228, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:79 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt2PA-C.sub.1223, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:80 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca1PA-C.sub.1233 and nei.sub.ca2PA-C.sub.1233 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:85 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca1PB1-C.sub.1234 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:88 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wt2PB1-C.sub.1240 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:91 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca1PB1-C.sub.1241 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:94 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.ca2PB1-C.sub.1241 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:103 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wtPB1.sub.2341 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:105 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.wtPB1.sub.2271 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:106 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.caPB1.sub.2341 the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:108 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei.sub.caPB1.sub.2271 the production of which is disclosed in the examples. Additional nucleic acid molecules, nucleic acid sequences, proteins and amino acid sequences are described in the Examples.

[0081] The present invention includes nucleic acid molecule comprising a cold-adapted equine influenza virus encoding an M protein having an amino acid sequence comprising SEQ ID NO:5. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding an HA protein having an amino acid sequence comprising SEQ ID NO:11. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-N protein having an amino acid sequence comprising SEQ ID NO:17. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-C protein having an amino acid sequence comprising SEQ ID NO:24. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB protein having an amino acid sequence comprising SEQ ID NO:48. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a NS protein having an amino acid sequence comprising SEQ ID NO:58. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-N protein having an amino acid sequence comprising SEQ ID NO:69. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PA-C protein having an amino acid sequence comprising SEQ ID NO:81. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-C protein having an amino acid sequence comprising SEQ ID NO:92. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1 protein having an amino acid sequence comprising SEQ ID NO:107.

[0082] It should be noted that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of the present invention and apparent amino acid sequences of M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 proteins of the present invention.

[0083] Another embodiment of the present invention is an antibody that selectively binds to an wild-type virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Another embodiment of the present invention is an antibody that selectively binds to a cold-adapted virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Preferred antibodies selectively bind to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO: 55, SEQ ID NO:58, SEQ ID NO: 63, SEQ ID NO:66, SEQ ID NO: 69, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO:86, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO: 107.

[0084] The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

EXAMPLE 1

[0085] This example discloses the production and phenotypic characterization of several cold-adapted equine influenza viruses of the present invention.

[0086] A. Parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8) (obtained from Tom Chambers, the University of Kentucky, Lexington, Ky.) was subjected to cold-adaptation in a foreign host species, i.e., embryonated chicken eggs, in the following manner. Embryonated, 10 or 11-day old chicken eggs, available, for example, from Truslow Farms, Chestertown, Md. or from HyVac, AdeI, IA, were inoculated with the parental equine influenza virus by injecting about 0.1 milliliter (ml) undiluted AF containing approximately 10.sup.6 plaque forming units (pfu) of virus into the allantoic cavity through a small hole punched in the shell of the egg. The holes in the eggs were sealed with nail polish and the eggs were incubated in a humidified incubator set at the appropriate temperature for three days. Following incubation, the eggs were candled and any non-viable eggs were discarded. AF was harvested from viable embryos by aseptically removing a portion of the egg shell, pulling aside the chorioallantoic membrane (CAM) with sterile forceps and removing the AF with a sterile pipette. The harvested AF was frozen between passages. The AF was then used, either undiluted or diluted 1:1000 in phosphate-buffered saline (PBS) as noted in Table 1, to inoculate a new set of eggs for a second passage, and so on. A total of 69 passages were completed. Earlier passages were done at either about 34.degree. C. (passages 1-2) or about 30.degree. C. and on subsequent passages, the incubation temperature was shifted down either to about 28.degree. C., or to about 26.degree. C. In order to increase the possibility of the selection of the desired phenotype of a stable, attenuated virus, the initial serial passage was expanded to included five different limbs of the serial passage tree, A through E, as shown in Table 1.

TABLE-US-00001 TABLE 1 Passage history of the limbs A through E. Passage # Temperature Limb A Limb B Limb C Limb D Limb E 34.degree. C. 1-2 1-2 1-2 1-2 1-2 30.degree. C. 3-8 3-29 3-29 3-29 3-29 28.degree. C. 30-33* 30-68* 30-33 30-69 26.degree. C. 9-65 34-69* 34-65 *= the infectious allantoic fluid was diluted 1:1000 in these passages

[0087] B. Virus isolates carried through the cold-adaptation procedure described in section A were tested for temperature sensitivity, i.e., a phenotype in which the cold-adapted virus grows at the lower, or permissive temperature (e.g., about 34.degree. C.), but no longer forms plaques at a higher, or non-permissive temperature (e.g., about 37.degree. C. or about 39.degree. C.), as follows. At each cold-adaptation passage, the AF was titered by plaque assay at about 34.degree. C. Periodically, individual plaques from the assay were clonally isolated by excision of the plaque area and placement of the excised agar plug in a 96-well tray containing a monolayer of MDCK cells. The 96-well trays were incubated overnight and the yield assayed for temperature sensitivity by CPE assay in duplicate 96-well trays incubated at about 34.degree. C. and at about 39.degree. C. The percent of the clones that scored as temperature sensitive mutants by this assay, i.e., the number of viral plaques that grew at 34.degree. C. but did not grow at 39.degree. C., divided by the total number of plaques, was calculated, and is shown in Table 2. Temperature sensitive isolates were then evaluated for protein synthesis at the non-permissive temperature by visualization of radiolabeled virus-synthesized proteins by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

TABLE-US-00002 TABLE 2 Percent of isolated Clones that were temperature sensitive. Percent Temperature Sensitive Passage# Limb A Limb B Limb C Limb D Limb E p36 56% 66% 0% 66% 54% p46 80% 60% 75% p47 80% p48 100% p49 100% 100% 50% p50 90% p51 100% p52 57% p62 100% 100% p65 100% p66 100% 88%

[0088] From the clonal isolates tested for temperature sensitivity, two were selected for further study. Clone EIV-P821 was selected from the 49th passage of limb B and clone EIV-P824 was selected from the 48th passage of limb C, as defined in Table 1. Both of these virus isolates were temperature sensitive, with plaque formation of both isolates inhibited at a temperature of about 39.degree. C. At this temperature, protein synthesis was completely inhibited by EIV-P821, but EIV-P824 exhibited normal levels of protein synthesis. In addition, plaque formation by EIV-P821 was inhibited at a temperature of about 37.degree. C., and at this temperature, late gene expression was inhibited, i.e., normal levels of NP protein were synthesized, reduced or no M1 or HA proteins were synthesized, and enhanced levels of the polymerase proteins were synthesized. The phenotype observed at 37.degree. C., being typified by differential viral protein synthesis, was distinct from the protein synthesis phenotype seen at about 39.degree. C., which was typified by the inhibition of synthesis of all viral proteins. Virus EIV-P821 has been deposited with the American Type Culture Collection (ATCC) under Accession No. ATCC VR-2625, and virus EIV-P824 has been deposited with the ATCC under Accession No. ATCC VR-2624.

[0089] C. Further characterization of the mutations in isolate EIV-P821 were carried out by reassortment analysis, as follows. Reassortment analysis in influenza viruses allows one skilled in the art, under certain circumstances, to correlate phenotypes of a given virus with putative mutations occurring on certain of the eight RNA segments that comprise an influenza A virus genome. This technique is described, for example, in Palese, et al., ibid. A mixed infection of EIV-P821 and an avian influenza virus, A/mallard/New York/6750/78 was performed as follows. MDCK cells were co-infected with EIV-P821 at a multiplicity of infection (MOI) of 2 pfu/cell and A/mallard/New York/6750/78 at an MOI of either 2, 5, or 10 pfu/cell. The infected cells were incubated at a temperature of about 34.degree. C. The yields of the various co-infections were titered and individual plaques were isolated at about 34.degree. C., and the resultant clonal isolates were characterized as to whether they were able to grow at about 39.degree. C. and about 37.degree. C., and express their genes, i.e., synthesize viral proteins, at about 39.degree. C., about 37.degree. C., and about 34.degree. C. Protein synthesis was evaluated by SDS-PAGE analysis of radiolabeled infected-cell lysates. The HA, NP and NS-1 proteins of the two parent viruses, each of which is encoded by a separate genome segment, were distinguishable by SDS-PAGE analysis, since these particular viral proteins, as derived from either the equine or the avian influenza virus, migrate at different apparent molecular weights. In this way it was possible, at least for the HA, NP, and NS-1 genes, to evaluate whether certain phenotypes of the parent virus, e.g., the temperature sensitive and the protein synthesis phenotypes, co-segregate with the genome segments carrying these genes. The results of the reassortment analyses investigating co-segregation of a) the mutation inhibiting plaque formation, i.e., the induction of CPE, at a non-permissive temperature of about 39.degree. C. or b) the mutation inhibiting protein synthesis at a non-permissive temperature of about 39.degree. C. with each of the EIV-P821 HA, NP and NS-1 proteins are shown in Tables 3 and 4, respectively.

TABLE-US-00003 TABLE 3 Reassortment analysis of the EIV-P821 39.degree. C. plaque formation phenotype with avian influenza virus, A/mallard/New York/6750/78 Gene Virus ts+.sup.1 ts-.sup.2 HA avian 26 13 equine 11 44 NP avian 37 8 equine 0 49 NS-1 avian 9 8 equine 12 20 .sup.1number of clonal isolates able to induce CPE in tissue culture cells at a temperature of about 39.degree. C. .sup.2number of clonal isolates inhibited in the ability to induce CPE in tissue culture cells at a temperature of about 39.degree. C.

TABLE-US-00004 TABLE 4 Reassortment analysis of the EIV-P821 39.degree. C. protein synthesis phenotype with avian influenza virus, A/mallard/New York/6750/78 Gene Virus ts+.sup.1 ts-.sup.2 HA avian 18 1 equine 11 7 NP avian 34 5 equine 7 8 NS-1 avian 10 4 equine 14 5 .sup.1number of clonal isolates which synthesize all viral proteins at a temperature of about 39.degree. C. .sup.2number of clonal isolates inhibited in the ability to synthesize all viral proteins at a temperature of about 39.degree. C.

[0090] The results demonstrated an association of the equine NP gene with a mutation causing the inability of EIV-P821 to form plaques at a non-permissive temperature of about 39.degree. C., but the results did not suggest an association of any of the HA, NP, or NS-1 genes with a mutation causing the inability of EIV-P821 to express viral proteins at a non-permissive temperature of about 39.degree. C. Thus, these data also demonstrated that the plaque formation phenotype and the protein synthesis phenotype observed in virus EIV-P821 were the result of separate mutations.

[0091] D. Studies were also conducted to determine if cold-adapted equine influenza viruses of the present invention have a dominant interference phenotype, that is, whether they dominate in mixed infection with the wild type parental virus A/Kentucky/1/91 (H3N8). The dominant interference phenotype of viruses EIV-P821 and EIV-P824 were evaluated in the following manner. Separate monolayers of MDCK cells were singly infected with the parental virus A/Kentucky/1/91 (H3N8) at an MOI of 2, singly infected with either cold-adapted virus EIV-P821 or EIV-P824 at an MOI of 2, or simultaneously doubly infected with both the parental virus and one of the cold adapted viruses at an MOI of 2+2, all at a temperature of about 34.degree. C. At 24 hours after infection, the media from the cultures were harvested and the virus yields from the various infected cells were measured by duplicate plaque assays performed at temperatures of about 34.degree. C. and about 39.degree. C. This assay took advantage of the fact that cold adapted equine influenza viruses EIV-P821 or EIV-P824 are temperature sensitive and are thus unable to form plaques at a non-permissive temperature of about 39.degree. C., while the parental virus is able to form plaques at both temperatures, thus making it possible to measure the growth of the parental virus in the presence of the cold adapted virus. Specifically, the dominant interference effect of the cold adapted virus on the growth of the parental virus was quantitated by comparing the virus yield at about 39.degree. C. of the cells singly infected with parental virus to the yield of the parental virus in doubly infected cells. EIV-P821, in mixed infection, was able to reduce the yield of the parental virus by approximately 200 fold, while EIV-P824, in mixed infection, reduced the yield of the parental virus by approximately 3200 fold. This assay therefore showed that cold-adapted equine influenza viruses EIV-P821 and EIV-P824 both exhibit the dominant interference phenotype.

[0092] E. Virus isolate EIV-MSV+5 was derived from EIV-P821, as follows. EIV-P821 was passaged once in eggs, as described above, to produce a Master Seed Virus isolate, denoted herein as EIV-MSV0. EIV-MSV0 was then subjected to passage three additional times in eggs, the virus isolates at the end of each passage being designated EIV-MSV+1, EIV-MSV+2, and EIV-MSV+3, respectively. EIV-MSV+3 was then subjected to two additional passages in MDCK cells, as follows. MDCK cells were grown in 150 cm.sup.2 tissue culture flasks in MEM tissue culture medium with Hanks Salts, containing 10% calf serum. The cells were then washed with sterile PBS and the growth medium was replaced with about 8 ml per flask of infection medium (tissue culture medium comprising MEM with Hanks Salts, 1 .mu.g/ml TPCK trypsin solution, 0.125% bovine serum albumin (BSA), and 10 mM HEPES buffer). MDCK cells were inoculated with AF containing virus EIV-MSV+3 (for the first passage in MDCK cells) or virus stock harvested from EIV-MSV+4 (for the second passage in MDCK cells), and the viruses were allowed to adsorb for 1 hour at about 34.degree. C. The inoculum was removed from the cell monolayers, the cells were washed again with PBS, and about 100 ml of infection medium was added per flask. The infected cells were incubated at about 34.degree. C. for 24 hours. The virus-infected MDCK cells were harvested by shaking the flasks vigorously to disrupt the cell monolayer, resulting in virus isolates EIV-MSV+4 (the first passage in MDCK cells), and EIV-MSV+5 (the second passage in MDCK cells).

[0093] Viruses EIV-MSV0 and EIV-MSV+5 were subjected to phenotypic analysis, as described in section B above, to determine their ability to form plaques and synthesize viral proteins at temperatures of about 34.degree. C., about 37.degree. C., and about 39.degree. C. Both EIV-MSV0 and EIV-MSV+5 formed plaques in tissue culture cells at a temperature of about 34.degree. C., and neither virus isolate formed plaques or exhibited detectable viral protein synthesis at a temperature of about 39.degree. C. Virus EIV-MSV0 had a similar temperature sensitive phenotype as EIV-P821 at a temperature of about 37.degree. C., i.e., it was inhibited in plaque formation, and late gene expression was inhibited. However, EIV-MSV+5, unlike its parent virus, EIV-P821, did form plaques in tissue culture at a temperature of about 37.degree. C., and at this temperature, the virus synthesized normal amounts of all proteins. Virus EIV-MSV+5 has been deposited with the ATCC under Accession No. ATCC VR-2627.

EXAMPLE 2

[0094] Therapeutic compositions of the present invention were produced as follows.

[0095] A. A large stock of EIV-P821 was propagated in eggs as follows, About 60 specific pathogen-free embryonated chicken eggs were candled and non-viable eggs were discarded. Stock virus was diluted to about 1.0.times.10.sup.5 pfu/ml in sterile PBS. Virus was inoculated into the allantoic cavity of the eggs as described in Example 1A. After a 3-day incubation in a humidified chamber at a temperature of about 34.degree. C., AF was harvested from the eggs according to the method described in Example 1A. The harvested AF was mixed with a stabilizer solution, for example A1/A2 stabilizer, available from Diamond Animal Health, Des Moines, Iowa, at 25% V/V (stabilizer/AF). The harvested AF was batched in a centrifuge tube and was clarified by centrifugation for 10 minutes at 1000 rpm in an IEC Centra-7R refrigerated table top centrifuge fitted with a swinging bucket rotor. The clarified fluid was distributed into 1-ml cryovials and was frozen at about -70.degree. C. Virus stocks were titrated on MDCK cells by CPE and plaque assay at about 34.degree. C.

[0096] B. A large stock of EIV-P821 was propagated in MDCK cells as follows. MDCK cells were grown in 150 cm.sup.2 tissue culture flasks in MEM tissue culture medium with Hanks Salts, containing 10% calf serum. The cells were then washed with sterile PBS and the growth medium was replaced with about 8 ml per flask of infection medium. The MDCK cells were inoculated with virus stock at an MOI ranging from about 0.5 pfu per cell to about 0.005 pfu per cell, and the viruses were allowed to adsorb for 1 hour at about 34.degree. C. The inoculum was removed from the cell monolayers, the cells were washed again with PBS, and about 100 ml of infection medium was added per flask. The infected cells were incubated at about 34.degree. C. for 24 hours. The virus-infected MDCK cells were harvested by shaking the flasks vigorously to disrupt the cell monolayer and stabilizer solution was added to the flasks at 25% V/V (stabilizer/virus solution). The supernatants were distributed aseptically into cryovials and frozen at -70.degree. C.

[0097] C. Therapeutic compositions comprising certain cold-adapted temperature sensitive equine influenza viruses of the present invention were formulated as follows. Just prior to vaccination procedures, such as those described in Examples 3-7 below, stock vials of EIV-P821 or EIV-MSV+5 were thawed and were diluted in an excipient comprising either water, PBS, or in MEM tissue culture medium with Hanks Salts, containing 0.125% bovine serum albumin (BSA-MEM solution) to the desired dilution for administration to animals. The vaccine compositions were held on ice prior to vaccinations. All therapeutic compositions were titered on MDCK cells by standard methods just prior to vaccinations and wherever possible, an amount of the composition, treated identically to those administered to the animals, was titered after the vaccinations to ensure that the virus remained viable during the procedures.

EXAMPLE 3

[0098] A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 was tested for safety and its ability to replicate in three horses showing detectable prior immunity to equine influenza virus as follows. EIV-P821, produced as described in Example 1A, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10.sup.7 pfu EIV-P821/2 ml BSA-MEM solution as described in Example 2C.

[0099] Three ponies having prior detectable hemagglutination inhibition (HAT) titers to equine influenza virus were inoculated with a therapeutic composition comprising EIV-P821 by the following method. Each pony was given a 2-ml dose of EIV-P821, administered intranasally using a syringe fitted with a blunt cannula long enough to reach past the false nostril, 1 ml per nostril.

[0100] The ponies were observed for approximately 30 minutes immediately following and at approximately four hours after vaccination for immediate type allergic reactions such as sneezing, salivation, labored or irregular breathing, shaking, anaphylaxis, or fever. The animals were further monitored on days 1-11 post-vaccination for delayed type allergic reactions, such as lethargy or anorexia. None of the three ponies in this study exhibited any allergic reactions from the vaccination.

[0101] The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day 11 following vaccination for clinical signs consistent with equine influenza. The ponies were observed for nasal discharge, ocular discharge, anorexia, disposition, heart rate, capillary refill time, respiratory rate, dyspnea, coughing, lung sounds, presence of toxic line on upper gum, and body temperature. In addition submandibular and parietal lymph nodes were palpated and any abnormalities were described. None of the three ponies in this study exhibited any abnormal reactions or overt clinical signs during the observation period.

[0102] To test for viral shedding in the animals, on days 0 through 111 following vaccination, nasopharyngeal swabs were collected from the ponies as described in Chambers, et al., 1995, Equine Practice, 17, 19-23. Chambers, et al., ibid. Briefly, two sterile Dacron polyester tipped applicators (available, e.g., from Hardwood Products Co., Guilford, Me.) were inserted, together, into each nostril of the ponies. The swabs (four total, two for each nostril) were broken off into a 15-ml conical centrifuge tube containing 2.5 ml of chilled transport medium comprising 5% glycerol, penicillin, streptomycin, neomycin, and gentamycin in PBS at physiological pH. Keeping the samples on wet ice, the swabs were aseptically wrung out into the medium and the nasopharyngeal samples were divided into two aliquots. One aliquot was used to attempt isolation of EIV by inoculation of embryonated eggs, using the method described in Example 1. The AF of the inoculated eggs was then tested for its ability to cause hemagglutination, by standard methods, indicating the presence of equine influenza virus in the AF. On days 2 and 3 post-vaccination, the other aliquots were tested for virus by the Directigen.RTM.V Flu A test, available from Becton-Dickinson (Cockeysville, Md.).

[0103] Attempts to isolate EIV from the nasopharyngeal secretions of the three animals by egg inoculation were unsuccessful. However on days 2 and 3, all animals tested positive for the presence of virus shedding using the Directigen Flu A test, consistent with the hypothesis that EIV-P821 was replicating in the seropositive ponies.

[0104] To test the antibody titers to EIV in the inoculated animals described in this example, as well as in the animals described in Examples 4-7, blood was collected from the animals prior to vaccination and on designated days post-vaccination. Serum was isolated and was treated either with trypsin/periodate or kaolin to block the nonspecific inhibitors of hemagglutination present in normal sera. Serum samples were tested for hemagglutination inhibition (HAI) titers against a recent EIV isolate by standard methods, described, for example in the "Supplemental assay method for conducting the hemagglutination inhibition assay for equine influenza virus antibody" (SAM 124), provided by the U.S.D.A. National Veterinary Services Laboratory under 9 CFR 113.2.

[0105] The HAI titers of the three ponies are shown in Table 5. As can be seen, regardless of the initial titer, the serum HAI titers increased at least four-fold in all three animals after vaccination with EIV-P821.

[0106] These data demonstrate that cold-adapted equine influenza virus EIV-P821 is safe and non-reactogenic in sero-positive ponies, and that these animals exhibited an increase in antibody titer to equine influenza virus, even though they had prior demonstrable titers.

TABLE-US-00005 TABLE 5 HAI titers of vaccinated animals* HAI Titer Animal (days after vaccination) ID 0 7 14 21 18 40 80 160 160 19 10 20 40 80 25 20 40 320 80 *HAI titers are expressed as the reciprocal of the highest dilution of serum which inhibited hemagglutination of erythrocytes by a recent isolate of equine influenza virus.

EXAMPLE 4

[0107] This Example discloses an animal study to evaluate the safety and efficacy of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821.

[0108] A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 was tested for attenuation, as well as its ability to protect horses from challenge with virulent equine influenza virus, as follows. EIV-P821, produced as described in Example 1, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10.sup.7 pfu of virus/2 ml water, as described in Example 2C. Eight EIV-seronegative ponies were used in this study. Three of the eight ponies were vaccinated with a 2-ml dose comprising 10.sup.7 pfu of the EIV-P821 therapeutic composition, administered intranasally, using methods similar to those described in Example 3. One pony was given 10.sup.7 pfu of the EIV-P821 therapeutic composition, administered orally, by injecting 6 ml of virus into the pharynx, using a 10-ml syringe which was adapted to create a fine spray by the following method. The protruding "seat" for the attachment of needles was sealed off using modeling clay and its cap was left in place. About 10 holes were punched through the bottom of the syringe, i.e., surrounding the "seat," using a 25-gauge needle. The syringe was placed into the interdental space and the virus was forcefully injected into the back of the mouth. The remaining four ponies were held as non-vaccinated controls.

[0109] The vaccinated ponies were observed for approximately 30 minutes immediately following and at approximately four hours after vaccination for immediate type allergic reactions, and the animals were farther monitored on days 1-11 post-vaccination for delayed type allergic reactions, both as described in Example 3. None of the four vaccinated ponies in this study exhibited any abnormal reactions from the vaccination.

[0110] The ponies were observed daily, at approximately the same time each day, starting two days before virus vaccination and continuing through day 11 following vaccination for clinical signs, such as those described in Example 3. None of the four vaccinated ponies in this study exhibited any clinical signs during the observation period. This result demonstrated that cold-adapted equine influenza virus EIV-P821 exhibits the phenotype of attenuation.

[0111] To test for viral shedding in the vaccinated animals, on days 0 through 11 following vaccination, nasopharyngeal swabs were collected from the ponies as described in Example 3. The nasopharyngeal samples were tested for virus in embryonated chicken eggs according to the method described in Example 3.

[0112] As shown in Table 6, virus was isolated from only one vaccinated animal using the egg method. However, as noted in Example 3, the lack of isolation by this method does not preclude the fact that virus replication is taking place, since replication may be detected by more sensitive methods, e.g., the Directigen Flu A test.

TABLE-US-00006 TABLE 6 Virus isolation in eggs after vaccination. Animal Virus Isolation (days after vaccination) ID Route 0 1 2 3 4 5 6 7 8 9 10 11 91 IN - - + + + + + + + + + - 666 IN - - - - - - - - - - - - 673 IN - - - - - - - - - - - - 674 Oral - - - - - - - - - - - -

[0113] To test the antibody titers to equine influenza virus in the vaccinated animals, blood was collected from the animals prior to vaccination and on days 7, 14, 21, and 28 post-vaccination. Serum samples were isolated and were tested for hemagglutination inhibition (HAI) titers against a recent EIV isolate according to the methods described in Example 3.

[0114] The HAI titers of the four vaccinated ponies are shown in Table 7.

TABLE-US-00007 TABLE 7 HAI titers after vaccination. Animal HAI Titer (days after vaccination) ID Route 0 7 14 21 28 91 IN <10 <10 <10 <10 <10 666 IN 10 10 10 20 20 673 IN 10 10 10 20 20 674 Oral 20 40 40 40 40

[0115] Unlike the increase in HAI titer observed with the three animals described in the study in Example 3, the animals in this study did not exhibit a significant increase, i.e., greater than four-fold, in HAI titer following vaccination with EIV-P821.

[0116] Approximately four and one-half months after vaccine virus administration, all 8 ponies, i.e., the four that were vaccinated and the four non-vaccinated controls, were challenged by the following method. For each animal, 10.sup.7 pfu of the virulent equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) was suspended in 5 ml of water. A mask was connected to a nebulizer, and the mask was placed over the animal's muzzle, including the nostrils. Five (5) ml was nebulized for each animal, using settings such that it took 5-10 minutes to deliver the full 5 ml. Clinical observations, as described in Example 3, were performed on all animals three days before challenge and daily for 11 days after challenge.

[0117] Despite the fact that the vaccinated animals did not exhibit marked increases in their HAI titers to equine influenza virus, all four vaccinated animals were protected against equine influenza virus challenge. None of the vaccinated animals showed overt clinical signs or fever, although one of the animals had a minor wheeze for two days. On the other hand, all four non-vaccinated ponies shed virus and developed clinical signs and fever typical of equine influenza virus infection. Thus, this example demonstrates that a therapeutic composition of the present invention can protect horses from equine influenza disease.

EXAMPLE 5

[0118] This Example discloses an additional animal study to evaluate attenuation of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821, and its ability to protect vaccinated horses from subsequent challenge with virulent equine influenza virus. Furthermore, this study evaluated the effect of exercise stress on the safety and efficacy of the therapeutic composition.

[0119] A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 was tested for safety and efficacy in horses, as follows. EIV-P821, produced as described in Example 1, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10.sup.7 pfu virus/5 ml water, as described in Example 2C. Fifteen ponies were used in this study. The ponies were randomly assigned to three groups of five animals each, as shown in Table 8, there being two vaccinated groups and one unvaccinated control group. The ponies in group 2 were exercise stressed before vaccination, while the ponies in vaccinate group 1 were held in a stall.

TABLE-US-00008 TABLE 8 Vaccination/challenge protocol. Group No. Ponies Exercise Vaccine Challenge 1 5 -- Day 0 Day 90 2 5 Days -4 to 0 Day 0 Day 90 3 5 -- -- Day 90

[0120] The ponies in group 2 were subjected to exercise stress on a treadmill prior to vaccination, as follows. The ponies were acclimated to the use of the treadmill by 6 hours of treadmill use at a walk only. The actual exercise stress involved a daily exercise regimen starting 4 days before and ending on the day of vaccination (immediately prior to vaccination). The treadmill exercise regimen is shown in Table 9.

TABLE-US-00009 TABLE 9 Exercise regimen for the ponies in Group 2. Speed (m/sec) Time (min.) Incline (.degree.) 1.5 2 0 3.5 2 0 3.5 2 7 4.5.dagger. 2 7 5.5.dagger. 2 7 6.5.dagger. 2 7 7.5.dagger. 2 7 8.5.dagger. 2 7 3.5 2 7 1.5 10 0.dagger. .dagger.Speed, in meters per second (m/sec) was increased for each animal every 2 minutes until the heart rate reached and maintained .gtoreq.200 beats per minute

[0121] Groups 1 and 2 were given a therapeutic composition comprising 10.sup.7 pfu of EIV-P821, by the nebulization method described for the challenge described in Example 4. None of the vaccinated ponies in this study exhibited any immediate or delayed allergic reactions from the vaccination.

[0122] The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day 11 following vaccination for clinical signs, such as those described in Example 3. None of the vaccinated ponies in this study exhibited any overt clinical signs during the observation period.

[0123] To test for viral shedding in the vaccinated animals, before vaccination and on days 1 through 11 following vaccination, nasopharyngeal swabs were collected from the ponies as described in Example 3. The nasopharyngeal samples were tested for virus in embryonated chicken eggs according to the method described in Example 3. Virus was isolated from the vaccinated animals, i.e., Groups 1 and 2, as shown in Table 10.

TABLE-US-00010 TABLE 10 Virus isolation after vaccination. Animal Virus Isolation (days after vaccination) Group ID Exercise 0 1 2 3 4 5 6 7 8 9 10 11 1 12 No - - + + + + + - + + - - 16 - - + + + + + - - - - - 17 - - + + + + + + + - + - 165 - - - - - - - - - - - - 688 - - - - - + - + - - - - 2 7 Yes - - - + + + + - - - - - 44 - - - - - - - - - - - - 435 - - + + + + - - - - - - 907 - - - + - + + - - - - - 968 - - - - - + - + - - - -

[0124] To test the antibody titers to equine influenza virus in the vaccinated animals, blood was collected prior to vaccination and on days 7, 14, 21, and 28 post-vaccination. Serum samples were isolated and were tested for HAI titers against a recent EIV isolate according to the methods described in Example 3. These titers are shown in Table 11.

TABLE-US-00011 TABLE 11 HAI titers after vaccination and after challenge on day 90. Animal Day Post-vaccination Group ID -1 7 14 21 28 91 105 112 119 126 1 12 <10 <10 <10 <10 <10 <10 80 320 320 640 1 16 <10 <10 20 20 <10 <10 20 160 320 320 1 17 <10 <10 10 10 10 10 80 160 160 160 1 165 <10 <10 10 10 10 10 80 80 80 80 1 688 <10 <10 20 20 20 20 20 20 20 40 2 7 <10 <10 10 10 <10 <10 20 80 80 40 2 44 <10 <10 20 20 20 10 80 320 320 320 2 435 <10 <10 20 20 10 <10 20 80 80 80 2 907 <10 <10 10 10 20 10 10 40 80 80 2 968 <10 <10 <10 <10 <10 <10 40 160 160 160 3 2 <10 80 640 640 320 3 56 <10 80 320 320 320 3 196 <10 20 160 80 80 3 198 10 40 160 320 320 3 200 <10 20 80 80 40 Group Description 1 Vaccination only 2 Vaccination and Exercise 3 Control

[0125] On day 90 post vaccination, all 15 ponies were challenged with 10.sup.7 pfu of equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by the nebulizer method as described in Example 4. Clinical observations, as described in Example 3, were performed on all animals three days before challenge and daily for 11 days after challenge. There were no overt clinical signs observed in any of the vaccinated ponies. Four of the five non-vaccinated ponies developed fever and clinical signs typical of equine influenza virus infection.

[0126] Thus, this example demonstrates that a therapeutic composition of the present invention protects horses against equine influenza disease, even if the animals are stressed prior to vaccination.

EXAMPLE 6

[0127] This Example compared the infectivities of therapeutic compositions of the present invention grown in eggs and grown in tissue culture cells. From a production standpoint, there is an advantage to growing therapeutic compositions of the present invention in tissue culture rather than in embryonated chicken eggs. Equine influenza virus, however, does not grow to as high a titer in cells as in eggs. In addition, the hemagglutinin of the virus requires an extracellular proteolytic cleavage by trypsin-like proteases for infectivity. Since serum contains trypsin inhibitors, virus grown in cell culture must be propagated in serum-free medium that contains trypsin in order to be infectious. It is well known by those skilled in the art that such conditions are less than optimal for the viability of tissue culture cells. In addition, these growth conditions may select for virus with altered binding affinity for equine cells, which may affect viral infectivity since the virus needs to bind efficiently to the animal's nasal mucosa to replicate and to stimulate immunity. Thus, the objective of the study disclosed in this example was to evaluate whether the infectivity of therapeutic compositions of the present invention was adversely affected by growth for multiple passages in vitro tissue culture.

[0128] EIV-P821, produced as described in Example 1, was grown in eggs as described in Example 2A or in MDCK cells as described in Example 2B. In each instance, the virus was passaged five times. EIV-P821 was tested for its cold-adaptation and temperature sensitive phenotypes after each passage. The egg and cell-passaged virus preparations were formulated into therapeutic compositions comprising 10.sup.7 pfu virus/2 ml BSA-MEM solution, as described in Example 2C, resulting in an egg-grown EIV-P821 therapeutic composition and an MDCK cell-grown EIV-P821 therapeutic composition, respectively.

[0129] Eight ponies were used in this study. Serum from each of the animals was tested for HAI titers to equine influenza virus prior to the study. The animals were randomly assigned into one of two groups of four ponies each. Group A received the egg-grown EIV-P821 therapeutic composition, and Group B received the MDCK-grown EIV-P821 therapeutic composition, prepared as described in Example 2B. The therapeutic compositions were administered intranasally by the method described in Example 3.

[0130] The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day 11 following vaccination for allergic reactions or clinical signs as described in Example 3. No allergic reactions or overt clinical signs were observed in any of the animals.

[0131] Nasopharyngeal swabs were collected before vaccination and daily for 11 days after vaccination. The presence of virus material in the nasal swabs was determined by the detection of CPE on MDCK cells infected as described in Example 1, or by inoculation into eggs and examination of the ability of the infected AF to cause hemagglutination, as described in Example 3. The material was tested for the presence of virus only, and not for titer of virus in the sample. Virus isolation results are listed in Table 12. Blood was collected and serum samples from days 0, 7, 14, 21 and 28 after vaccination were tested for hemagglutination inhibition antibody titer against a recent isolate. HAI titers are also listed in Table 12.

TABLE-US-00012 TABLE 12 HAI titers and virus isolation after vaccination. HAI Titer (DPV.sup.3) Virus Isolation.sup.1 (DPV.sup.3) Group.sup.2 ID 0 7 14 21 28 0 1 2 3 4 5 6 7 8 9 10 11 1 31 <10 20 160 160 160 -- EC -- C EC EC C C EC -- -- -- 37 <10 40 160 160 160 -- EC C C EC C C C -- -- -- -- 40 <10 20 80 160 80 -- EC EC C -- C EC C -- EC EC -- 41 <10 40 160 160 80 -- EC EC C EC C EC EC -- -- -- -- 2 32 <10 <10 80 80 40 -- EC -- C -- C -- C -- EC -- -- 34 <10 20 160 160 160 -- EC -- C EC C EC C -- -- -- -- 35 <10 <10 80 80 40 -- EC -- C -- C -- C -- EC -- -- 42 <10 <10 80 80 40 -- -- -- C -- C EC EC -- -- -- -- .sup.1E = Egg isolation positive; C = CPE isolation positive; -- = virus not detected by either of the methods .sup.2Group 1: Virus passaged 5X in MDCK cells; Group 2: Virus passaged 5X in Eggs .sup.3Days Post-vaccination

[0132] The results in Table 12 show that there were no significant differences in infectivity or immunogenicity between the egg-grown and MDCK-grown EIV-P821 therapeutic compositions.

EXAMPLE 7

[0133] This example evaluated the minimum dose of a therapeutic composition comprising a cold-adapted equine influenza virus required to protect a horse from equine influenza virus infection.

[0134] The animal studies disclosed in Examples 3-6 indicated that a therapeutic composition of the present invention was efficacious and safe. In those studies, a dose of 10.sup.7 pfu, which correlates to approximately 10.sup.8 TCID.sub.50 units, was used. However, from the standpoints of cost and safety, it is advantageous to use the minimum virus titer that will protect a horse from disease caused by equine influenza virus. In this study, ponies were vaccinated with four different doses of a therapeutic composition comprising a cold-adapted equine influenza virus to determine the minimum dose which protects a horse against virulent equine influenza virus challenge.

[0135] EIV-P821, produced as described in Example 1A, was passaged and grown in MDCK cells as described in Example 2B and was formulated into a therapeutic composition comprising either 2.times.10.sup.4, 2.times.10.sup.5, 2.times.10.sup.6, or 2.times.10.sup.7 TCID.sub.50 units/1 ml BSA-MEM solution as described in Example 2C. Nineteen horses of various ages and breeds were used for this study. The horses were assigned to four vaccine groups, one group of three horses and three groups of four horses, and one control group of four horses (see Table 13). Each of the ponies in the vaccine groups were given a 1-ml dose of the indicated therapeutic composition, administered intranasally by methods similar to those described in Example 3.

TABLE-US-00013 TABLE 13 Vaccination protocol. Vaccine Dose, Group No. No. Animals TCID.sub.50 Units 1 3 2 .times. 10.sup.7 2 4 2 .times. 10.sup.6 3 4 2 .times. 10.sup.5 4 4 2 .times. 10.sup.4 5 4 control

[0136] The ponies were observed for approximately 30 minutes immediately following and at approximately four hours after vaccination for immediate type reactions, and the animals were further monitored on days 1-11 post-vaccination for delayed type reactions, both as described in Example 3. None of the vaccinated ponies in this study exhibited any abnormal reactions or overt clinical signs from the vaccination.

[0137] Blood for serum analysis was collected 3 days before vaccination, on days 7, 14, 21, and 28 after vaccination, and after challenge on Days 35 and 42. Serum samples were tested for HAI titers against a recent EIV isolate according to the methods described in Example 3. These titers are shown in Table 14. Prior to challenge on day 29, 2 of the 3 animals in group 1, 4 of the 4 animals in group 2, 3 of the 4 animals in group 3, and 2 of the 4 animals in group 4 showed at least 4-fold increases in HAI titers after vaccination. In addition, 2 of the 4 control horses also exhibited increases in HAI titers. One interpretation for this result is that the control horses were exposed to vaccine virus transmitted from the vaccinated horses, since all the horses in this study were housed in the same barn.

TABLE-US-00014 TABLE 14 HAI titers post-vaccination and post-challenge, and challenge results. Dose Chall. in TCID.sub.50 Animal Vaccination on Day 0, Challenge on Day 29 Sick No. units ID -1 7 14 21 28 35 42 +/- 1 2 .times. 10.sup.7 41 <10 <10 10 40 10 20 80 - 42 40 40 40 40 40 <10 80 - 200 <10 <10 80 40 160 40 40 - 2 2 .times. 10.sup.6 679 <10 10 40 40 40 20 20 - 682 <10 <10 40 40 40 40 40 - 795 20 80 160 160 320 320 640 - R <10 10 40 20 160 40 40 - 3 2 .times. 10.sup.5 73 <10 <10 160 40 80 160 160 - 712 <10 <10 20 20 40 40 20 - 720 <10 20 80 40 80 80 160 - 796 <10 <10 <10 <10 <10 10 80 + 4 2 .times. 10.sup.4 75 <10 <10 <10 <10 <10 <10 160 + 724 <10 >10 <10 <10 <10 20 320 + 789 <10 10 320 160 320 320 320 - 790 <10 <10 80 40 160 80 40 5 Control 12 <10 <10 <10 20 20 40 40 - 22 10 20 40 10 160 40 640 - 71 <10 <10 <10 <10 10 20 160 + 74 <10 <10 <10 <10 <10 <10 20 +

[0138] On day 29 post vaccination, all 19 ponies were challenged with equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by the nebulizer method as described in Example 4. The challenge dose was prospectively calculated to contain about 10.sup.8 TCID.sub.50 units of challenge virus in a volume of 5 ml for each animal. Clinical observations, as described in Example 3, were monitored beginning two days before challenge, the day of challenge, and for 11 days following challenge. As shown in Table 14, no animals in groups 1 or 2 exhibited clinical signs indicative of equine influenza disease, and only one out of four animals in group 3 became sick. Two out of four animals in group 4 became sick, and only two of the four control animals became sick. The results in Table 14 suggest a correlation between seroconversion and protection from disease, since, for example, the two control animals showing increased HAI titers during the vaccination period did not show clinical signs of equine influenza disease following challenge. Another interpretation, however, was that the actual titer of the challenge virus may have been less than the calculated amount of 10.sup.8 TCID.sub.50 units, since, based on prior results, this level of challenge should have caused disease in all the control animals.

[0139] Nonetheless, the levels of seroconversion and the lack of clinical signs in the groups that received a therapeutic composition comprising at least 2.times.10.sup.6 TCID.sub.50 units of a cold-adapted equine influenza virus suggests that this amount was sufficient to protect a horse against equine influenza disease. Furthermore, a dose of 2.times.10.sup.5 TCID.sub.50 units induced seroconversion and gave clinical protection from challenge in 3 out of 4 horses, and thus even this amount may be sufficient to confer significant protection in horses against equine influenza disease.

EXAMPLE 8

[0140] This example discloses an animal study to evaluate the duration of immunity of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821.

[0141] A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821, produced as described in Example 1, was grown in eggs similarly to the procedure described in Example 2A, was expanded by passage in MDCK cells similarly to the procedure described in Example 2B, and was formulated into a therapeutic composition as described in Example 2C. Thirty horses approximately 11 to 12 months of age were used for this study. Nineteen of the horses were each vaccinated intranasally into one nostril using a syringe with a delivery device tip attached to the end, with a 1.0 ml dose comprising 6 logs of TCID.sub.50 units of the EIV-P821 therapeutic composition. Vaccinations were performed on Day 0.

[0142] The horses were observed on Day 0 (before vaccination and up to 4 hours post-vaccination) and on Study Days 1, 2, 3, 7, 15, and 169 post-vaccination. On these days, a distant examination for a period of at least 15 minutes was performed. This distant examination included observation for demeanor, behavior, coughing, sneezing, and nasal discharge. The examination on Day 169 also served to confirm that the horses were in a condition of health suitable for transport to the challenge site which was located approximately 360 miles from the vaccination site.

[0143] The animals were acclimated to the challenge site and were observed approximately daily by a veterinarian or animal technician for evidence of disease. A general physical examination was performed on Day 171 post-vaccination to monitor the following: demeanor, behavior, coughing, sneezing, and nasal discharge. From Days 172 to 177, similar observations as well as rectal temperature were recorded, according to the judgment of the attending veterinarian for any individual horse with abnormal clinical presentation.

[0144] No vaccinated horses showed any adverse reactions post-vaccination. One vaccinate was found dead about two months after vaccination. This horse showed no evidence of adverse reaction when observed for at least one month after vaccination. Although no cause of death could be firmly established, the death was not instantaneous and was considered to be consistent with possible contributing factors such as colic, bone fracture, or severe worm burden. Since there was no other evidence for any adverse reactions post-vaccination in any other vaccinates, it is highly unlikely that the vaccine contributed to any adverse reaction in this case.

[0145] Challenges were performed on Day 181 post-vaccination. The following wild-type isolate of equine influenza virus previously shown to cause disease in horses was used as the challenge virus: A/equine/2/Kentucky/91. Prior to infection of each challenge group, the challenge material was rapidly thawed at approximately 37.degree. C. The virus was diluted with phosphate-buffered saline to a total volume of approximately 21 ml. The diluted material was stored chilled on ice until immediately before inoculation. Before inoculation and at the end of nebulization for each challenge group, a sample of diluted challenge virus was collected for pre- and post-inoculation virus titer confirmation. Vaccinates and controls were randomly assigned to 4 challenge groups of 6 horses each and one challenge group of 5 horses so that each challenge group contained a mixture of 4 vaccinates and 2 controls or 3 vaccinates and 2 controls.

[0146] Challenge virus in aerosol form was delivered through a tube inserted through a small opening centrally in the plastic ceiling with an ultrasonic nebulizer (e.g., DeVilbiss Model 099HD, DeVilbiss Healthcare Inc., Somerset, Pa.) for a period of approximately 10 minutes. The horses remained in the chamber for a further period of approximately 30 minutes after the nebulization had been completed (total exposure time, approximately 40 minutes). At that time, the plastic was removed to vent the chamber, and the horses were released and returned to their pen. The challenge procedure was repeated for each group.

[0147] All statistical methods in this study were performed using SAS (SAS Institute, Cary, N.C.), and P<0.05 was considered to be statistically significant. Beginning on Day 178 post-vaccination (three days prior to challenge) through Day 191 (day 10 post-challenge), the horses were observed daily by both distant and individual examinations. Rectal temperatures were measured at these times. Data from day 0 (challenge day) to day 10 were included in the analysis; see Table 15.

TABLE-US-00015 TABLE 15 Effect of challenge on daily temperatures (.degree. C.) in vaccinated and control horses (least squares means). Day post Vaccinated non-vaccinated challenge (n = 19) (n = 10) P-value 0 100.7 100.8 0.8434 1 100.5 100.4 0.7934 2 103.4 104.9 0.0024 3 101.8 103.9 0.0001 4 101.5 103.2 0.0002 5 101.7 103.8 0.0001 6 101.3 103.6 0.0001 7 100.7 102.3 0.0007 8 100.5 101.4 0.0379 9 100.1 100.3 0.7416 10 100.3 100.5 0.7416 pooled SEM* 0.27 0.38 *Standard error of the mean

[0148] Table 15 shows that on days 2 through 8, vaccinated horses had lower temperatures (P<0.05) than the non-vaccinated control horses.

[0149] The distant examination consisted of a period of 20 minutes where the following observations were made: coughing, nasal discharge, respiration, and depression. Scoring criteria are shown in Table 16.

TABLE-US-00016 TABLE 16 Clinical signs and scoring index. Clinical Sign Description Score Coughing normal during observation period of 15 min 0 coughing once during observation 1 coughing twice or more during observation 2 Nasal discharge normal 0 abnormal, serous 1 abnormal, mucopurulent 2 abnormal, profuse 3 Respiration normal 0 abnormal (dyspnea, tachypnea) 1 Depression normal 0 depression present.sup..dagger. 1 .sup..dagger.Depression was assessed by subjective evaluation of individual animal behavior that included the following: failure to approach food rapidly, general lethargy, inappetence, and anorexia.

[0150] Each horse was scored for each of these categories. Additionally, submandibular lymph nodes were palpated to monitor for possible bacterial infection. In any case where there was a different value recorded for a subjective clinical sign score from an observation on the same day at the distant versus the individual examination, the greater score was used in the compilation and analysis of results. For purposes of assessing the health of the horses prior to final disposition, distant examinations were performed at 14, 18, and 21 days post-challenge. Data from days 1 through 10 post-challenge were included in the analysis. These scores were summed on each day for each horse, and the vaccinates and controls were compared using the Wilcoxon rank sums test. In addition, these scores were summed across all days for each horse, and compared in the same manner. The mean ranks and mean clinical scores are shown in Tables 17 and 18, respectively. Five days post-challenge, the mean rank of scores in the vaccinated horses was lower (P<0.05) than in the non-vaccinated control horses; and this effect continued on days 6, 7, 8, 9, and 10 (P<0.05). The cumulative rank over the entire test period was also lower (P<0.05) in the vaccinated horses than the non-vaccinated controls.

TABLE-US-00017 TABLE 17 Effect of challenge on clinical sign scores in vaccinated and control horses (mean rank). Vaccinated Non-vaccinated Day post (n = 19), (n = 10), challenge mean rank* mean rank P-value 0 13.6 17.6 0.1853 1 16.4 12.4 0.2015 2 15.1 14.9 0.9812 3 13.3 18.3 0.1331 4 13.5 17.9 0.1721 5 12.4 19.9 0.0237 6 12.7 19.4 0.0425 7 12.1 20.6 0.0074 8 12.6 19.6 0.0312 9 13.1 18.7 0.0729 10 12.3 20.1 0.0135 total over 11.8 21.2 0.0051 11 days *By Wilcoxon rank sum test.

TABLE-US-00018 TABLE 18 Effect of challenge on clinical sign scores in vaccinated and control horses (mean scores). Day post Vaccinated Non-vaccinated challenge (n = 19) (n = 10) 0 1.2 1.6 1 1.5 0.9 2 2.4 2.5 3 3.2 4.1 4 3.4 4.3 5 3.2 4.7 6 3.4 4.8 7 3.3 4.7 8 3.2 4.5 9 3.2 3.9 10 2.4 3.4

[0151] Nasopharyngeal swabs were obtained on the day prior to challenge and on days 1 to 8 post-challenge, as described in Example 3, and tested for shed virus by cell culture assay. The percent of horses shedding challenge virus in each group is shown in Table 19. The percent of horses shedding the challenge virus in the vaccinated group was lower (P<0.05) on days 5 and 6 post-challenge than in the non-vaccinated controls. The mean number of days the challenge virus was shed was also lower (P<0.05) in the vaccinated group as compared to the non-vaccinated controls.

TABLE-US-00019 TABLE 19 Percent of horses shedding virus per day post-challenge and mean number of days of shedding per group. Day post Vaccinated Non-vaccinated challenge (n = 19) (n = 10) -1 0 0 1 63.2 90 2 100 100 3 84.2 100 4 100 100 5 47.4 88.9* 6 10.5 77.8* 7 5.3 20 8 0 0 average number of 4.1 5.6* days shedding *Within a time point, vaccinates different from non-vaccinates (P < 0.05) by either Fisher's exact test (percent data) or Wilcoxon rank sums test (days shedding).

[0152] The scores from clinical signs relevant to influenza and the objective temperature measurements both demonstrated a statistically significant reduction in the group of vaccinates when compared to those from the control group; this is consistent with an interpretation that the vaccine conferred significant protection from disease.

[0153] The ability of horses to shed influenza virus post-challenge was also significantly reduced in vaccinates as compared to controls in both the incidence of horses positive for shedding on certain days post-challenge and the mean number of days of shedding per horse. This decreased shedding by vaccinates is important in that it should serve to reduce the potential for exposure of susceptible animals to the wild-type virus in an outbreak of influenza.

[0154] The results of this study are consistent with the interpretation that the vaccine safely conferred protection for 6 months from clinical disease caused by equine influenza and reduced the potential for the spread of naturally occurring virulent equine influenza virus. While the degree of protection from disease was not complete (13 out of 19 vaccinates were protected, while 10/10 controls were sick), there was a clear reduction in the severity and duration of clinical illness and a noticeable effect on the potential for viral shedding after exposure to a virulent strain of equine influenza. The finding that both vaccinates and controls were seronegative immediately prior to challenge at 6 months post-immunization suggests that immunity mediated by something other than serum antibody may be of primary importance in the ability of this vaccine to confer measurable and durable protection.

EXAMPLE 9

[0155] This Example discloses an animal study to evaluate the ability of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 to aid in the prevention of disease following exposure to a heterologous strain of equine influenza virus.

[0156] The heterologous strain tested was A/equine/2/Saskatoon/90, described genetically as a Eurasian strain (obtained from Hugh Townsend, University of Saskatchewan). Twenty female Percheron horses approximately 15 months of age (at the time of vaccination) were used for the efficacy study. The horses were assigned to two groups, one group of 10 to be vaccinated and another group of 10 to serve as non-vaccinated controls. On day 0, the vaccinate group was vaccinated in the manner described in Example 8.

[0157] The challenge material, i.e. equine flu strain A/equine/2/Saskatoon/90 [H3N8] was prepared similarly to the preparation in Example 8. Vaccinates and controls were randomly assigned to 4 challenge groups of 5 horses each such that each challenge group contained a mixture of 2 vaccinates and three controls or vice versa. The challenge procedure was similar to that described in Example 8. Challenges were performed on Day 28 post-vaccination.

[0158] Clinical observations were performed for the vaccinates and controls on Day -4 and on Study Days 0 (before vaccination and up to 4 hours post-vaccination), 1 to 7, 12, 15 to 17, 19 to 23, 25 to 38, and 42. For days on which clinical observations were performed during Days -4 to 42, clinical observations including rectal temperature were recorded according to the judgment of the attending veterinarian for any individual horse with abnormal clinical presentation. Horses were scored using the same criteria as in Example 8 (Table 15). Distant examinations were performed on these days as described in Example 8. On Day 20 and from Days 25 to 38, the horses were also observed by both distant and individual examinations (also performed as described in Example 8).

[0159] Rectal temperatures were measured daily beginning 3 days prior to challenge, and continuing until 10 days post-challenge. Day 0 is the day relative to challenge. Data from days 0 through 10 were included in the analysis. Statistical methods and criteria were identical to those used in Example 8. On days 2, 5 and 7, vaccinated horses had statistically significant lower body temperatures than the non-vaccinated control horses (Table 20).

TABLE-US-00020 TABLE 20 Effect of challenge on daily temperatures (.degree. C.) in vaccinated and control horses (least squares means). Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) P-value 0 99.9 99.8 0.9098 1 100.5 100.3 0.4282 2 101.0 102.8 0.0001 3 100.7 100.6 0.7554 4 101.0 101.3 0.4119 5 100.8 102.1 0.0004 6 100.4 100.4 0.9774 7 100.3 101.1 0.0325 8 100.6 100.7 0.8651 9 100.5 100.6 0.8874 10 100.5 100.1 0.2465 Standard error of the mean = 0.249.

[0160] Data from days 1 through 10 post-challenge were included in the analysis. These scores were summed on each day for each horse, and the vaccinates and controls were compared using the Wilcoxon rank sums test. All statistical methods were performed as described in Example 9. In addition, these scores were summed across all days for each horse, and compared in the same manner. Mean ranks are shown in Table 21.

TABLE-US-00021 TABLE 21 Effect of challenge on clinical sign scores in vaccinated and control horses (mean rank). Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) P-value* 1 8.85 12.15 0.1741 2 8.80 12.20 0.1932 3 8.90 12.10 0.2027 4 7.60 13.40 0.0225 5 6.90 14.10 0.0053 6 7.00 14.00 0.0059 7 6.90 14.10 0.0053 8 7.60 13.40 0.0251 9 6.90 14.10 0.0048 10 6.10 14.90 0.0006 total over 5.70 15.30 0.0003 10 days *By Wilcoxon 2 sample test.

[0161] On day 4 post-challenge, the mean rank of scores in the vaccinated horses was lower (P<0.05) than the non-vaccinated control horses, and this effect continued throughout the remainder of the study (P<0.05). The cumulative rank over the entire test period was also lower in the vaccinated horses than the non-vaccinated controls (P<0.05).

[0162] Nasopharyngeal swabs were collected on days 1 and 8 post-challenge, as described in Example 3. The nasal samples were analyzed for the presence of virus by cell inoculation with virus detection by cytopathogenic effect (CPE) or by egg inoculation with virus detection by hemagglutination (HA). The cell-culture assay was performed as generally described by Youngner et al., 1994, J. Clin. Microbiol. 32, 750-754. Serially diluted nasal samples were added to wells containing monolayers of Madin Darby Canine Kidney (MDCK) cells. After incubation, wells were examined for the presence and degree of cytopathogenic effect. The quantity of virus in TCID.sub.50 units was calculated by the Reed-Muench technique. The egg infectivity assay was performed as described in Example 1. The percent of horses shedding challenge virus for each assay in each group is shown in Tables 22 and 23. The percent of horses shedding the challenge virus in the vaccinated group was lower (P<0.05) on days 2 through 7 post-challenge by either method. No differences were seen on days 1 or 8 post-challenge. The number of days the challenge virus was shed was also lower (P<0.05) in the vaccinated group as compared to the non-vaccinated controls; see Tables 22 and 23.

TABLE-US-00022 TABLE 22 Percent of horses shedding virus following challenge - cell culture assay. Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) 1 0 0 2 0 70* 3 0 70* 4 20 100* 5 10 100* 6 20 100* 7 0 80* 8 0 30 average 0.5 5.5* number of days shedding *Within a time point, vaccinates different from non-vaccinates, P < 0.05 by either Fisher's exact test (percent data) or Wilcoxon 2 sample test (days shedding)

TABLE-US-00023 TABLE 23 Percent of horses shedding virus following challenge - egg infectivity assay. Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) 1 0 0 2 0 70* 3 10 70* 4 0 90* 5 10 70* 6 20 90* 7 0 50* 8 0 0 average 0.4 4.4* number of days shedding *Within a time point, vaccinates different from non-vaccinates, P < 0.05 by either Fisher's exact test (percent data) or Wilcoxon 2 sample test (days shedding).

[0163] The extent (severity and duration) of clinical signs of influenza among vaccinates was substantially reduced relative to the controls. The scores from clinical signs relevant to influenza and the objective temperature measurements both demonstrated a statistically significant reduction in the group of vaccinates when compared to those from the control group; indicating that the vaccine conferred significant protection from disease by the heterologous strain.

[0164] The ability of horses to shed influenza virus post-challenge was also significantly reduced in vaccinates as opposed to controls in both the incidence of horses positive for shedding on certain days post-challenge and the mean number of days of shedding per horse. This decreased shedding by vaccinates is important in that it should serve to reduce the potential for exposure of susceptible animals to the wild-type virus in an outbreak of influenza.

[0165] Overall, the results of this study show that the vaccine conferred protection against a heterologous challenge by a member of the Eurasian lineage of equine influenza virus strains.

EXAMPLE 10

[0166] This Example discloses an animal study to evaluate the ability of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 to aid in the prevention of disease following exposure to a heterologous strain of equine influenza virus.

[0167] The heterologous strain tested was A/equine/2/Kentucky/98 [H3N8] (obtained from Tom Chambers, University of Kentucky). Eight ponies aged 5 to 7 months were used for this efficacy study. The horses were assigned to two groups, one group of 4 to be vaccinated and another group of 4 to serve as non-vaccinated controls. Ponies were vaccinated as described in Example 8, on Day 0.

[0168] Clinical observations were performed for the vaccinates on Study Day 0 (before vaccination and at 4 hours post-vaccination), as well as on Days 1 to 8, 23, 30 to 50, and 57 post-vaccination. Controls were observed clinically on Days 29 to 50 and 57. The observations were performed and scored as described in Example 8.

[0169] The challenge material i.e. equine flu strain from Kentucky/98, was prepared by passing the isolated virus two times in eggs. The inoculum for each horse was prepared by thawing 0.5 ml of the virus, then diluting in 4.5 ml of sterile phosphate-buffered saline. The inoculum was administered by nebulization using a mask for each individual horse on Day 36 post-vaccination.

[0170] The clinical observation scores were summed on each day for each horse, and horses were ranked according to the cumulative total score from days 1 to 9 post-challenge. Theses results are shown in Table 24.

TABLE-US-00024 TABLE 24 Clinical sign observations: total scores, ranked by total score. Total Score.sup.# Halter Days 1 to 9 Group Identity post-challenge 1-Vaccinate 50 0 1-Vaccinate 52 0 1-Vaccinate 55 1 1-Vaccinate 15 2 2-Control 61 21 2-Control 20 25 2-Control 7 26 2-Control 13 26 .sup.#Total scores represent the sum of daily scores (where daily scores equal the sum of scores for coughing, nasal discharge, respiration, and depression) and are ranked from the lowest (least severe) to highest (most severe) scores.

[0171] The results of Table 24 show that the scores for vaccinates were between 0 and 2, which was significantly lower than the score for controls, which were between 21 and 26.

[0172] Rectal temperatures were measured daily beginning 6 days prior to challenge, and continuing until 9 days post-challenge. Day 0 is the day relative to challenge. Data from days 0 through 9 were included in the analysis. These results are shown in Table 25.

TABLE-US-00025 TABLE 25 Effect of Challenge on daily mean temperatures (.degree. C.) in vaccinated and control horses. Day post challenge control vaccinate difference 0 99.7 99.5 0.2 1 100.0 99.6 0.4 2 103.9 100.2 3.7 3 99.8 99.2 0.6 4 99.6 99.1 0.5 5 99.8 99.3 0.5 6 99.6 99.3 0.3 7 99.3 99.0 0.3 8 99.7 99.6 0.1 9 99.5 99.1 0.4

[0173] The temperatures of the control horses were higher than the temperatures of the vaccinated horses on all days. The temperature in control horses was significantly higher on day 2.

[0174] Nasopharyngeal swabs were collected on days 1 and 8, post-challenge, as described in Example 3. These samples were tested for shed virus by an egg infectivity assay as described in Example 1. The results of the assay are shown in Table 26.

TABLE-US-00026 TABLE 26 Virus shedding post-challenge detected by egg infectivity. Study day No. 35 37 38 39 40 41 42 43 44 days Days post-challenge positive Identity -1 1 2 3 4 5 6 7 8 per Group No. Detection of virus* horse Vaccinates 15 0 2 0 3 3 0 2 1 0 5 50 0 0 0 0 0 1 0 0 0 1 52 0 0 3 3 2 2 0 0 0 4 55 0 2 3 1 3 0 0 0 0 4 No. horses positive 0 2 2 3 3 2 1 1 0 per day Controls 07 0 3 3 3 3 3 3 1 0 7 13 0 3 3 3 3 3 3 1 0 7 20 0 2 3 3 3 3 3 1 0 7 61 0 3 3 3 3 3 3 2 0 7 No. horses positive 0 4 4 4 4 4 4 4 0 per day *Values refer to the number of eggs testing positive of 3 eggs tested per sample. For statistical analysis, a sample was considered positive for virus if at least 1 egg was positive per sample.

[0175] The results of Table 26 show that the number of horses positive per day was higher for the controls than for the vaccinates. Additionally, control horses were positive for more days than vaccinates.

[0176] The scores from clinical signs relevant to influenza and the objective temperature measurements both demonstrated significant differences in the group of vaccinates when compared to the control group; this shows that the vaccine conferred significant protection from disease caused by the heterologous strain Kentucky/98.

[0177] The ability of horses to shed influenza virus post-challenge was also significantly reduced in vaccinates as opposed to controls in the mean number of days of shedding per horse. This decreased shedding by vaccinates is important in that it should serve to reduce the potential for exposure of susceptible animals to the wild-type virus in an outbreak of influenza.

[0178] Overall, the results of this study show that the vaccine safely conferred protection to a heterologous challenge by a recent and clinically relevant isolate. When the results of this study are viewed in the light of the protection previously demonstrated against heterologous challenge with a Eurasian strain (Example 9), there is clear evidence to support the assertion that this modified live vaccine can confer protection against heterologous as well as homologous equine influenza infection.

EXAMPLE 11

[0179] This example describes the cloning and sequencing of equine influenza M (matrix) protein nucleic acid molecules for wild type and cold-adapted equine influenza viruses.

[0180] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus M protein, were produced as follows. A PCR product containing an equine M gene was produced by PCR amplification from equine influenza virus DNA, and primers w584 and w585, designated SEQ ID NO:26, and SEQ ID NO:27, respectively. A nucleic acid molecule of 1023 nucleotides, denoted nei.sub.wtM.sub.1023, with a coding strand having a nucleic acid sequence designated SEQ ID NO:1 was produced by further PCR amplification using the above described PCR product as a template and cloned into pCR 2.1.RTM.TA cloning vector, available from Invitrogen, Carlsbad, Calif., using standard procedures recommended by the manufacturer. The primers used were the T7 primer, designated by SEQ ID NO:29 and the REV primer, designated by SEQ ID NO:28. Plasmid DNA was purified using a mini-prep method available from Qiagen, Valencia, Calif. PCR products were prepared for sequencing using a PRISM.TM. Dye Terminator Cycle Sequencing Ready Reaction kit, a PRISM.TM. dRhodamine Terminator Cycle Sequencing Ready Reaction kit, or a PRISM.TM. BigDye.TM. Terminator Cycle Sequencing Ready Reaction kit, all available from PE Applied Biosystems, Foster City, Calif., following the manufacturer's protocol. Specific PCR conditions used with the kit were a rapid ramp to 95.degree. C., hold for 10 seconds followed by a rapid ramp to 50.degree. C. with a 5 second hold then a rapid ramp to 60.degree. C. with a 4 minute hold, repeating for 25 cycles. Different sets of primers were used in different reactions: T7 and REV were used in one reaction; w584 and w585 were used in a second reaction; and efM-a1, designated SEQ ID NO:31 and efM-s1, designated SEQ ID NO:30 were used in a third reaction. PCR products were purified by ethanol/magnesium chloride precipitation. Automated sequencing of DNA samples was performed using an ABI PRISM.TM. Model 377 with XL upgrade DNA Sequencer, available from PE Applied Biosystems.

[0181] Translation of SEQ ID NO:1 indicates that nucleic acid molecule nei.sub.wtM.sub.1023 encodes a full-length equine influenza M protein of about 252 amino acids, referred to herein as Pei.sub.wtM.sub.252, having amino acid sequence SEQ ID NO:2, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 28 of SEQ ID NO:1 and the termination codon spans from nucleotide 781 through nucleotide 783 of SEQ ID NO:1. The region encoding Pei.sub.wtM.sub.252, designated nei.sub.wtM.sub.756, and having a coding strand comprising nucleotides 25 to 780 of SEQ ID NO:1, is represented by SEQ ID NO:3.

[0182] SEQ ID NO:1 and SEQ ID NO:3 represent the consensus sequence obtained from two wild type nucleic acid molecules, which differ in one nucleotide. Nucleotide 663 of nei.sub.wt1M.sub.1023, i.e., nucleotide 649 of nei.sub.wt1M.sub.756, was adenine, while nucleotide 663 of nei.sub.wt2M.sub.1023, i.e., nucleotide 649 of nei.sub.wt2M.sub.756, was guanine. Translation of these sequences does not result in an amino acid change at the corresponding amino acid; both translate to valine at residue 221 in Pei.sub.wtM.sub.252.

[0183] B. A nucleic acid molecule of 1023 nucleotides encoding a cold-adapted equine influenza virus M, denoted nei.sub.ca1M.sub.1023, with a coding strand having a sequence designated SEQ ID NO:4 was produced by further PCR amplification and cloned into the pCR.RTM.-Blunt cloning vector available from Invitrogen, using conditions recommended by the manufacturer, and primers T7 and REV. Plasmid DNA purification and cycle sequencing were performed as described in Example 11, part A. Translation of SEQ ID NO:4 indicates that nucleic acid molecule nei.sub.ca1M.sub.1023 encodes a full-length equine influenza M protein of about 252 amino acids, referred to herein as Pei.sub.ca1M252, having amino acid sequence SEQ ID NO:5, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 28 of SEQ ID NO:4 and the termination codon spans from nucleotide 781 through nucleotide 783 of SEQ ID NO:4. The region encoding Pei.sub.ca1M.sub.252, designated nei.sub.ca1M.sub.756, and having a coding strand comprising nucleotides 25 to 780 of SEQ ID NO:4, is represented by SEQ ID NO:6. PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza M protein in the same manner resulted in molecules nei.sub.ca2M.sub.1023, identical to nei.sub.ca1M.sub.1023, and nei.sub.ca2M.sub.756, identical to nei.sub.ca1M.sub.756.

[0184] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtM.sub.1023 (SEQ ID NO:1) and nei.sub.ca1M.sub.1023 (SEQ ID NO:4) by DNA alignment reveals the following differences: a G to T shift at base 67, a C to T shift at base 527, and a G to C shift at base 886. Comparison of the amino acid sequences of proteins Pei.sub.wtM.sub.252 (SEQ ID NO:2) and Pei.sub.ca1M.sub.252 (SEQ ID NO:5) reveals the following differences: a V to L shift at amino acid 23 relating to the G to T shift at base 67 in the DNA sequences; and a T to I shift at amino acid 187 relating to the C to T shift at base 527 in the DNA sequences.

EXAMPLE 12

[0185] This example describes the cloning and sequencing of equine influenza HA (hemagglutinin) protein nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

[0186] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus HA proteins were produced as follows. A PCR product containing an equine HA gene was produced by PCR amplification from equine influenza virus DNA and primers w578 and w579, designated SEQ ID NO:32 and SEQ ID NO:33, respectively. A nucleic acid molecule of 1762 nucleotides encoding a wild-type HA protein, denoted nei.sub.wtHA.sub.1762, with a coding strand having a nucleic acid sequence designated SEQ ID NO:7 was produced by further PCR amplification using the above-described PCR product as a template and cloned into pCR 2.1.RTM.TA cloning vector as described in Example 11A. Plasmid DNA was purified and sequenced as in Example 11A, except that primers used in the sequencing kits were either T7 and REV in one case, or HA-1, designated SEQ ID NO:34, and HA-2, designated SEQ ID NO:35, in a second case.

[0187] Translation of SEQ ID NO:7 indicates that nucleic acid molecule nei.sub.wtHA.sub.1762 encodes a full-length equine influenza HA protein of about 565 amino acids, referred to herein as Pei.sub.wtHA.sub.565, having amino acid sequence SEQ ID NO:8, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 33 of SEQ ID NO:7 and the termination codon spans from nucleotide 1725 through nucleotide 1727 of SEQ ID NO:7. The region encoding Pei.sub.wtHA.sub.565, designated nei.sub.wtHA.sub.1695, and having a coding strand comprising nucleotides 30 to 1724 of SEQ ID NO:7 is represented by SEQ ID NO:9.

[0188] B. A nucleic acid molecule of 1762 nucleotides encoding a cold-adapted equine influenza virus HA protein, denoted nei.sub.ca1HA.sub.1762, with a coding strand having a sequence designated SEQ ID NO:10 was produced as described in Example 11B. Plasmid DNA purification and cycle sequencing were performed as described in Example 12, part A.

[0189] Translation of SEQ ID NO: 10 indicates that nucleic acid molecule nei.sub.ca1HA.sub.1762 encodes a full-length equine influenza HA protein of about 565 amino acids, referred to herein as Pei.sub.ca1HA.sub.565, having amino acid sequence SEQ ID NO:11, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 33 of SEQ ID NO:10 and the termination codon spans from nucleotide 1725 through nucleotide 1727 of SEQ ID NO:10. The region encoding Pei.sub.ca1HA.sub.565, designated nei.sub.ca1HA.sub.1695, and having a coding strand comprising nucleotides 30 to 1724 of SEQ ID NO:10, is represented by SEQ ID NO:12.

[0190] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza HA protein in the same manner resulted in molecules nei.sub.ca2HA.sub.1762, identical to nei.sub.ca1HA.sub.1762, and nei.sub.ca2HA.sub.1695, identical to nei.sub.ca1HA.sub.1695.

[0191] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtHA.sub.1762 (SEQ ID NO:7) and nei.sub.ca1HA.sub.1762 (SEQ ID NO:10) by DNA alignment reveals the following differences: a C to T shift at base 55, a G to A shift at base 499, a G to A shift at base 671, a C to T shift at base 738, a T to C shift at base 805, a G to A shift at base 1289, and an A to G shift at base 1368. Comparison of the amino acid sequences of proteins Pei.sub.wtHA.sub.565 (SEQ ID NO:8) and Pei.sub.ca1HA.sub.565 (SEQ ID NO:11) reveals the following differences: a P to L shift at amino acid 18 relating to the C to T shift at base 55 in the DNA sequences; a G to E shift at amino acid 166 relating to the G to A shift at base 499 in the DNA sequences; an R to W shift at amino acid 246 relating to the C to T shift at base 738 in the DNA sequences; an M to T shift at amino acid 268 relating to the T to C shift at base 805 in the DNA sequences; a K to E shift at amino acid 456 relating to the A to G shift at base 1368 in the DNA sequences. There is no change of the serine (S) at residue 223 relating to the G to A shift at base 671 in the DNA sequences, nor is there a change of the arginine (R) at residue 429 relating to the G to A shift at base 1289 in the DNA sequences.

EXAMPLE 13

[0192] This example describes the cloning and sequencing of equine influenza PB2 protein (RNA-directed RNA polymerase) nucleic acid molecules corresponding to the N-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

[0193] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2-N proteins were produced as follows. A PCR product containing an N-terminal portion of the equine PB2 gene was produced by PCR amplification from equine influenza virus DNA, and primers w570 and w571, designated SEQ ID NO:36 and SEQ ID NO:37, respectively. A nucleic acid molecule of 1241 nucleotides encoding a wild type PB2-N protein, denoted nei.sub.wtPB2-N.sub.1241, with a coding strand having a nucleic acid sequence designated SEQ ID NO:13 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11B, except that only T7 and REV primers were used in the sequencing kits.

[0194] Translation of SEQ ID NO:13 indicates that nucleic acid molecule nei.sub.wtPB2-N.sub.1241 encodes an N-terminal portion of influenza PB2 protein of about 404 amino acids, referred to herein as P.sub.wtPB2-N.sub.404, having amino acid sequence SEQ ID NO:14, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:13, and the last codon spans from nucleotide 1237 through nucleotide 1239. The region encoding P.sub.wtPB2-N.sub.404, designated nei.sub.wtPB2-N.sub.1214, and having a coding strand comprising nucleotides 28 to 1239 of SEQ ID NO:13 is represented by SEQ ID NO:15.

[0195] B. A nucleic acid molecule of 1239 nucleotides encoding an N-terminal portion of influenza PB2 cold-adapted equine influenza virus PB2-N protein, denoted nei.sub.ca1PB2-N.sub.1241, with a coding strand having a sequence designated SEQ ID NO:16 was produced, and sequenced as described in as in Example 12, part A.

[0196] Translation of SEQ ID NO:16 indicates that nucleic acid molecule nei.sub.ca1PB2-N.sub.1241 encodes an N-terminal portion of equine influenza PB-2 protein of about 404 amino acids, referred to herein as P.sub.ca1PB2-N.sub.404, having amino acid sequence SEQ ID NO:17, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:16, and the last codon spans from nucleotide 1237 through nucleotide 1239. The region encoding P.sub.ca1PB2-N.sub.404, designated nei.sub.ca1PB2-N.sub.1214, and having a coding strand comprising nucleotides 28 to 1239 of SEQ ID NO:16, is represented by SEQ ID NO:18.

[0197] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB2-N protein in the same manner resulted in molecules nei.sub.ca2PB2-N.sub.1241, identical to nei.sub.ca1PB2-N.sub.1241, and nei.sub.ca2PB2-N.sub.1214, identical to nei.sub.ca1PB2-N.sub.1214.

[0198] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtPB2-N.sub.1241 (SEQ ID NO:13) and nei.sub.ca1PB2-N.sub.1241 (SEQ ID NO:16) by DNA alignment reveals the following difference: a T to C base shift at base 370. Comparison of the amino acid sequences of proteins P.sub.wtPB2-N.sub.404 (SEQ ID NO:14) and P.sub.ca1PB2-N.sub.404 (SEQ ID NO:17) reveals the following difference: a Y to H shift at amino acid 124 relating to the a T to C shift at base 370 in the DNA sequence.

EXAMPLE 14

[0199] This example describes the cloning and sequencing of equine influenza PB2 protein (RNA-directed RNA polymerase) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

[0200] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2-C proteins were produced as follows. A PCR product containing the C-terminal portion of the equine PB2 gene was produced by PCR amplification using from equine influenza virus DNA and primers w572 and w573, designated SEQ ID NO:38 and SEQ ID NO:39, respectively. A nucleic acid molecule of 1233 nucleotides encoding a wild type PB2-C protein, denoted nei.sub.wtPB2-C.sub.1233, with a coding strand having a nucleic acid sequence designated SEQ ID NO:19 was produced by further PCR amplification using the above-described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance; efPB2-a1, designated SEQ ID NO:40 and efPB2-s1, designated SEQ ID NO:41 were used in another instance, and efPB2-a2, designated SEQ ID NO:42 and efPB2-s2, designated SEQ ID NO:43 were used in another instance.

[0201] Translation of SEQ ID NO:19 indicates that nucleic acid molecule nei.sub.wt1PB2-C.sub.1233 encodes a C-terminal portion of influenza PB2 protein of about 398 amino acids, referred to herein as P.sub.wtPB2-C.sub.398, having amino acid sequence SEQ ID NO:20, assuming an open reading frame having a first codon spans from nucleotide 3 through nucleotide 5 and a termination codon which spans from nucleotide 1197 through nucleotide 1199 of SEQ ID NO:19. Because SEQ ID NO:19 is only a partial gene sequence, it does not contain an initiation codon. The region encoding P.sub.wtPB2-C.sub.398, designated nei.sub.wtPB2-C.sub.1194, and having a coding strand comprising nucleotides 3 to 1196 of SEQ ID NO:19 is represented by SEQ ID NO:21.

[0202] PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB2-N protein in the same manner resulted in a nucleic acid molecule of 1232 nucleotides denoted nei.sub.wt2PB2-N.sub.1232, with a coding strand with a sequence designated SEQ ID NO:22. nei.sub.wt2PB2-N.sub.1232 is identical to nei.sub.wt1PB2-C.sub.1233, expect that nei.sub.wt2PB2-N.sub.1232 lacks one nucleotide on the 5'-end. Translation of SEQ ID NO:22 indicates that nucleic acid molecule nei.sub.wt1PB2-C.sub.1233 also encodes P.sub.wtPB2-C.sub.398 (SEQ ID NO:20), assuming an open reading frame having a first codon which spans from nucleotide 2 through nucleotide 4 and a termination codon spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:22. Because SEQ ID NO:22 is only a partial gene sequence, it does not contain an initiation codon. The nucleic acid molecule having a coding strand comprising nucleotides 2 to 1195 of SEQ ID NO:22, denoted nei.sub.wt2PB2-C.sub.1194, is identical to SEQ ID NO:21.

[0203] B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminal portion of influenza PB2 cold-adapted equine influenza virus protein, denoted nei.sub.ca1PB2-C.sub.1232, and having a coding strand having a sequence designated SEQ ID NO:23 was produced as described in as in Example 14, part A, except that the pCR.RTM.-Blunt cloning vector was used.

[0204] Translation of SEQ ID NO:23 indicates that nucleic acid molecule nei.sub.ca1PB2-C.sub.1232 encodes a C-terminal portion of equine influenza PB-2 protein of about 398 amino acids, referred to herein as P.sub.ca1PB2-C.sub.398, having amino acid sequence SEQ ID NO:24, assuming an open reading frame having a first codon which spans from nucleotide 2 through nucleotide 4 and a termination codon spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:23. Because SEQ ID NO:23 is only a partial gene sequence, it does not contain an initiation codon. The region encoding P.sub.ca1PB2-C.sub.398, designated nei.sub.ca1PB2-C1194, and having a coding strand comprising nucleotides 2 to 1195 of SEQ ID NO:23, is represented by SEQ ID NO:25.

[0205] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB2-C protein in the same manner resulted in molecules nei.sub.ca2PB2-C.sub.1231, containing one less nucleotide at the 3'end than nei.sub.ca1PB2-N.sub.1241; and nei.sub.ca2PB2-N.sub.1214, identical to nei.sub.ca1PB2-N.sub.1214.

[0206] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wt1PB2-C.sub.1233 (SEQ ID NO:19) and nei.sub.ca1PB2-C.sub.1232 (SEQ ID NO:23) by DNA alignment reveals the following differences: an A to C base shift at base 153 of SEQ ID NO:19, and a G to A base shift at base 929 of SEQ ID NO:19. Comparison of the amino acid sequences of proteins P.sub.wtPB2-C.sub.398 (SEQ ID NO:20) and P.sub.ca1PB2-.sub.398 (SEQ ID NO:24) reveals the following difference: a K to Q shift at amino acid 51 when relating to the an A to C base shift at base 153 in the DNA sequences. There is no amino acid shift resulting from the G to A base shift at base 929.

EXAMPLE 15

[0207] This example describes the cloning and sequencing of equine influenza PB2 protein (RNA-directed RNA polymerase) nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

[0208] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2 proteins were produced as follows. The wild type or cold-adapted equine influenza genes were cloned in two fragments, the N-terminal portion was produced as in Example 13 and the C-terminal portion of the gene was produced as in Example 14.

[0209] The DNA sequence for the wild type equine influenza PB2 gene was generated by combining the consensus sequences for the wild type PB2-N protein, denoted nei.sub.wtPB2-N.sub.1241 (SEQ ID NO: 13) with the gene fragments for the wild type PB2-C protein, denoted nei.sub.wt1PB2-C.sub.1233 (SEQ ID NO:19) and nei.sub.wt2PB2-C.sub.1232 (SEQ ID NO: 22). The result of combining the consensus sequences from the N-terminal and C-terminal portions of the PB2 wild type influenza virus yielded a complete DNA sequence denoted nei.sub.wtPB2.sub.2341 (SEQ ID NO:44). Translation of SEQ ID NO:44 indicates that the nucleic acid molecule nei.sub.wtPB2.sub.2341 encodes a full length equine influenza PB2 protein of about 759 amino acids referred to herein as Pei.sub.wtPB2.sub.759, having amino acid sequence SEQ ID NO: 45, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO: 44 and the termination codon spans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO: 44. The region encoding Pei.sub.wtPB2.sub.759, designated nei.sub.wtPB2.sub.2277, and having a coding strand comprising nucleotides 28 to 2304 of SEQ ID NO: 44, is SEQ ID NO: 46.

[0210] B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equine influenza virus PB2, denoted nei.sub.ca1PB2.sub.2341, with a sequence denoted SEQ ID NO: 47 was produced by combining the sequences for the N-terminal and C-terminal portions of the PB2 cold-adapted equine influenza gene. The clones for the N-terminal sequences are denoted nei.sub.ca1PB2-N.sub.1241 and nei.sub.ca2PB2-N.sub.1241 which are identical and are represented by SEQ ID NO:16. The clones for the C-terminal sequences are denoted nei.sub.ca1PB2-C.sub.1232 and nei.sub.ca2PB2-C.sub.1231, represented by SEQ ID NO:23.

[0211] Translation of SEQ ID NO:47 indicates that nucleic acid molecule nei.sub.ca1PB2.sub.2341 encodes a full-length equine influenza PB2 protein of about 759 amino acids, referred to herein as Pei.sub.ca1PB2.sub.759 having amino acid sequence SEQ ID NO:48, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO: 47 and the termination codon spans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO:47. The region encoding Pei.sub.ca1PB2.sub.759 designated nei.sub.ca1PB2.sub.2277 and having a coding strand comprising nucleotides 28 to 2304 of SEQ ID NO:49.

[0212] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtPB.sub.2341 (SEQ ID NO:44) and nei.sub.ca1PB2.sub.2341 (SEQ ID NO:47) by DNA alignment reveals the following differences: a T to C base shift at base 370, an A to C base shift at base 1261, and a G to A base shift at base 2037. Comparison of the amino acid sequences of proteins Pei.sub.wtPB2.sub.759 (SEQ ID NO:45) and Pei.sub.ca1PB2.sub.759 (SEQ ID NO:48) reveals the following differences: a Y to H shift at amino acid 124 relating to the a T to C shift at base 370 in the DNA sequence, a K to Q shift at amino acid 421 relating to the A to C shift at base 1261 in the DNA sequence. The third nucleotide shift at base 2037 does not result in an amino acid shift at amino.

EXAMPLE 16

[0213] This example describes the cloning and sequencing of equine influenza NS (nonstructural) protein nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

[0214] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus NS proteins were produced as follows. A PCR product containing an equine NS gene was produced by PCR amplification from equine influenza virus DNA and primers w586 and w587, designated SEQ ID NO:59 and SEQ ID NO:60, respectively. A nucleic acid molecule of 891 nucleotides encoding a wild-type NS protein, denoted nei.sub.wtNS.sub.891, with a coding strand having a nucleic acid sequence designated SEQ ID NO:50 was produced by further PCR amplification using the above-described PCR product as a template and cloned into pCR 2.1.RTM.TA cloning vector as described in Example 11A. Plasmid DNA was purified and sequenced as in Example 11A, except that primers used in the sequencing kits were only T7 and REV were used in the sequencing kits.

[0215] Translation of SEQ ID NO:50 indicates that nucleic acid molecule nei.sub.wt1NS.sub.891 encodes a full-length equine influenza NS protein of about 230 amino acids, referred to herein as Pei.sub.wt1NS.sub.230, having amino acid sequence SEQ ID NO:51, assuming an open reading frame in which the initiation codon spans from nucleotide 27 through nucleotide 29 of SEQ ID NO:50 and the termination codon spans from nucleotide 717 through nucleotide 719 of SEQ ID NO:50. The region encoding Pei.sub.wt1NS.sub.230, designated nei.sub.wt1NS.sub.690, and having a coding strand comprising nucleotides 27 to 716 of SEQ ID NO:50 is represented by SEQ ID NO:52.

[0216] PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza NS protein in the same manner resulted in molecules nei.sub.wt2NS.sub.891, identical to nei.sub.wt1NS.sub.891 in the coding region; i.e. nei.sub.wt2NS.sub.690, is identical to nei.sub.wt1NS.sub.690. nei.sub.wt2NS.sub.891 differs from nei.sub.wt1NS.sub.891 in one nucleotide at base 827 (G to A) which is 111 bases downstream from the stop codon. PCR amplification of a third nucleic acid encoding a wild type equine influenza NS protein in the same manner resulted in a nucleic acid molecule of 888 nucleotides denoted nei.sub.wt3NS.sub.888, with a coding strand with a nucleic acid sequence designated SEQ ID NO: 53. nei.sub.wt3NS.sub.888 is identical to nei.sub.wt1NS.sub.891, except that nei.sub.wt3NS.sub.888, lacks two nucleotides on the 5' end and one nucleotide on the 3' end. Translation of SEQ ID NO:53 indicates that nucleic acid molecule nei.sub.wt3NS.sub.888 also encodes Pei.sub.wt1NS.sub.230 (SEQ ID NO:51), assuming an open reading frame having an initiation codon which spans from nucleotide 25 through nucleotide 27 of SEQ ID NO: 53 and a termination codon which spans from nucleotide 715 through nucleotide 717 of SEQ ID NO:53. The nucleic acid molecule having a coding strand comprising nucleotides 25 to 714 of SEQ ID 53, denoted nei.sub.wt3NS.sub.690, is identical to SEQ ID NO:52.

[0217] PCR amplification of a fourth nucleic acid of 468 nucleotides encoding a C-terminal portion of the wild type equine influenza NS protein, denoted nei.sub.wt4NS.sub.468 and having a coding sequence designated SEQ ID NO:54 was produced. Translation of SEQ ID NO:54 indicates that nucleic acid molecule nei.sub.wt4NS.sub.468 encodes a C-terminal portion of equine influenza NS protein of about 97 amino acids, referred to herein as Pei.sub.wt4NS.sub.97, having amino acid sequence SEQ ID NO:55, assuming an open reading frame having a first codon which spans from nucleotide 3 to 5 of SEQ ID NO: 54, and a termination codon spans from nucleotide 294 through 296 of SEQ ID NO:54. Because SEQ ID NO:54 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei.sub.wt4NS.sub.97, designated nei.sub.wt4NS.sub.293, and having a coding strand comprising nucleotides 1 to 293 of SEQ ID NO:54, is represented by SEQ ID NO:56.

[0218] B. A nucleic acid molecule of 888 nucleotides encoding a cold-adapted equine influenza virus NS protein, denoted nei.sub.ca1NS.sub.888 with a coding strand having a sequence designated SEQ ID NO:57 was produced and sequenced as described in Example 16, part A.

[0219] Translation of SEQ ID NO:57 indicates that nucleic acid molecule nei.sub.ca1NS.sub.888 encodes a full-length equine influenza NS protein of about 230 amino acids, referred to herein as Pei.sub.ca1NS.sub.230, having amino acid sequence SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 27 through nucleotide 29 of SEQ ID NO:57 and the termination codon spans from nucleotide 717 through nucleotide 719 of SEQ ID NO:57. The region encoding Pei.sub.ca1NS.sub.230, designated nei.sub.ca1NS.sub.690, and having a coding strand comprising nucleotides 27 to 716 of SEQ ID NO:57, is represented by SEQ ID NO:59.

[0220] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza NS protein in the same manner resulted in molecules nei.sub.ca2NS.sub.887, containing one less nucleotide at the 3' end than nei.sub.ca1NS.sub.888; the coding region nei.sub.ca2NS.sub.690 is identical to nei.sub.ca1NS.sub.690.

[0221] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtNS.sub.891 (SEQ ID NO:50) and nei.sub.ca1NS.sub.888 (SEQ ID NO:57) by DNA alignment reveals the following difference: an A to G shift at base 827 which is 111 bases downstream from the stop codon. The 3' fragment encoding nei.sub.wt4NS.sub.468 (SEQ ID NO:54) has one shift T to C found at base 633 relative to the full-length consensus sequence. Comparison of the amino acid sequences of proteins Pei.sub.wtNS.sub.230 (SEQ ID NO:51) and Pei.sub.ca1NS.sub.230 (SEQ ID NO:58) reveals that there are no differences between amino acid sequences of the wild type and cold-adapted proteins.

EXAMPLE 17

[0222] This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase 1) nucleic acid molecules corresponding to the N-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

[0223] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1-N proteins were produced as follows. A PCR product containing an N-terminal portion of the equine PB1 gene was produced by PCR amplification from equine influenza virus DNA, and primers T7 and REV. A nucleic acid molecule of 1229 nucleotides encoding a wild type PB1-N protein, denoted nei.sub.wt1PB1-N.sub.1229, with a coding strand having a nucleic acid sequence designated SEQ ID NO:62 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11B, except that only T7 and REV primers were used in the sequencing kits.

[0224] Translation of SEQ ID NO:62 indicates that nucleic acid molecule nei.sub.wt1PB1-N.sub.1229 encodes an N-terminal portion of influenza PB1 protein of about 398 amino acids, referred to herein as Pei.sub.wt1PB1-N.sub.398, having amino acid sequence SEQ ID NO:63, assuming an open reading frame in which the initiation codon spans from nucleotide 36 through nucleotide 38 of SEQ ID NO:62, and the last codon spans from nucleotide 1227 through nucleotide 1229 of SEQ ID NO:62. The region encoding Pei.sub.wt1PB1-N.sub.398, designated nei.sub.wt1PB1-N.sub.1194, and having a coding strand comprising nucleotides 36 to 1229 of SEQ ID NO:62 is represented by SEQ ID NO:64.

[0225] PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB1-N protein in the same manner resulted in a nucleic acid molecule of 673 nucleotides denoted nei.sub.wt2PB1-N.sub.673, with a coding strand with a sequence designated SEQ ID NO:65. Translation of SEQ ID NO:65 indicates that nucleic acid molecule nei.sub.wt2PB1-N.sub.673 encodes Pei.sub.wt2PB1-N.sub.212 (SEQ ID NO:66), assuming an open reading frame having an initiation codon which spans from nucleotide 36 through nucleotide 38 of SEQ ID NO:65 and a last codon which spans from nucleotide 671 through nucleotide 673 of SEQ ID NO:65. Because SEQ ID NO:65 is only a partial gene sequence, it does not contain a stop codon. The nucleic acid molecule having a coding strand comprising nucleotides 36 to 671 of SEQ ID NO:65, denoted nei.sub.wt2PB1-N.sub.636, is designated SEQ ID NO:67.

[0226] B. A nucleic acid molecule of 1225 nucleotides encoding an N-terminal portion of influenza PB1 cold-adapted equine influenza virus PB1-N protein, denoted nei.sub.ca1PB1-N.sub.1225, with a coding strand having a sequence designated SEQ ID NO:68 was produced, and sequenced as described in as in Example 17, part A.

[0227] Translation of SEQ ID NO:68 indicates that nucleic acid molecule nei.sub.ca1PB1-N.sub.1225 encodes an N-terminal portion of equine influenza PB-1 protein of about 395 amino acids, referred to herein as Pei.sub.ca1PB1-N.sub.395, having amino acid sequence SEQ ID NO:69, assuming an open reading frame in which the initiation codon spans from nucleotide 34 through nucleotide 36 of SEQ ID NO:68, and a last codon which spans from nucleotide 1216 through nucleotide 1218 of SEQ ID NO:68. The region encoding Pei.sub.ca1PB1-N.sub.395, designated nei.sub.ca1PB1-N.sub.1185, and having a coding strand comprising nucleotides 34 to 1218 of SEQ ID NO:68, is represented by SEQ ID NO:70.

[0228] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB1-N protein in the same manner resulted in molecules nei.sub.ca2PB1-N.sub.1221, designated SEQ ID NO:71, containing four less nucleotides at the 5' end than nei.sub.ca1PB1-N.sub.1225; the coding region nei.sub.ca2PB1-N.sub.1185, is identical to nei.sub.ca1PB1-N.sub.1185.

[0229] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtPB1-N.sub.1229 (SEQ ID NO:62) and nei.sub.ca1PB1-N.sub.1225 (SEQ ID NO:68) by DNA alignment reveals no differences in the coding regions. Comparison of the amino acid sequences of proteins Pei.sub.wtPB1-N.sub.395 (SEQ ID NO:63) and Pei.sub.ca1PB1-N.sub.395 (SEQ ID NO:69) also reveals no differences.

EXAMPLE 18

[0230] This example describes the cloning and sequencing of equine influenza PB 1 protein (RNA-directed RNA polymerase 1) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

[0231] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1-C proteins were produced as follows. A PCR product containing an C-terminal portion of the equine PB1 gene was produced by PCR amplification from equine influenza virus DNA, and primer w569 designated SEQ ID NO:102. A nucleic acid molecule of 1234 nucleotides encoding a wild type PB1-C protein, denoted nei.sub.wt1PB1-C.sub.1234, with a coding strand having a nucleic acid sequence designated SEQ ID NO:85 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11A, except that different primers were used in the sequencing kits. T7, REV, w569, efPB1-a1, designated SEQ ID NO:97, efPB1-a2, designated SEQ ID NO:98, efPB1-s1, designated SEQ ID NO: 99, efPB1-s2, designated SEQ ID NO: 100, and efPB1-s3, designated SEQ ID NO:101 were used in one instance, T7, REV, efPB1-a1, efPB1-a2, efPB1-s1, efPB1-s2, and efPB1-s3 were used in another instance and T7 and REV were used in another instance.

[0232] Translation of SEQ ID NO:85 indicates that nucleic acid molecule nei.sub.wt1PB1-C.sub.1234 encodes an C-terminal portion of influenza PB1 protein of about 396 amino acids, referred to herein as Pei.sub.wt1PB1-C.sub.396, having amino acid sequence SEQ ID NO:86, assuming an open reading frame in which the first codon spans from nucleotide 1 through nucleotide 3 of SEQ ID NO:85 and a termination codon which spans from nucleotide 1189 through nucleotide 1191 of SEQ ID NO:85. Because SEQ ID NO:85 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei.sub.wt1PB1-C.sub.396, designated nei.sub.wt1PB1-C.sub.1188, and having a coding strand comprising nucleotides 1 to 1188 of SEQ ID NO:85 is represented by SEQ ID NO:87.

[0233] PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB 1-C protein in the same manner resulted in a nucleic acid molecule of 1240 nucleotides denoted nei.sub.wt2PB1-C.sub.1240, with a coding strand with a sequence designated SEQ ID NO:88. Translation of SEQ ID NO:88 indicates that nucleic acid molecule nei.sub.wt2PB1-N.sub.1240 encodes a molecule designated Pei.sub.wt2PB1-C.sub.396 (SEQ ID NO: 89) which differs from Pei.sub.wt1PB1-C.sub.396 (SEQ ID NO:85) in one nucleotide. Nucleotide 382 of nei.sub.wt1PB1-C.sub.1234, i.e. nucleotide 382 of nei.sub.wt1PB1-C.sub.1188 was A, while nucleotide 389 of nei.sub.wt2PB1-C.sub.1240, i.e. nucleotide 382 of nei.sub.wt2PB1-C.sub.1188 was T. Translation of nei.sub.wt2PB1-C.sub.1240 results in an amino acid change of T to S.

[0234] B. A nucleic acid molecule of 1241 nucleotides encoding an C-terminal portion of influenza PB1 cold-adapted equine influenza virus PB 1-C protein, denoted nei.sub.ca1PB1-C.sub.1241, with a coding strand having a sequence designated SEQ ID NO:91 was produced, and sequenced as described in as in Example 18, part A.

[0235] Translation of SEQ ID NO:91 indicates that nucleic acid molecule nei.sub.ca1PB1-C.sub.1241 encodes an C-terminal portion of equine influenza PB-1 protein of about 396 amino acids, referred to herein as Pei.sub.ca1PB1-C.sub.396, having amino acid sequence SEQ ID NO:92, assuming an open reading frame in which the first codon spans from nucleotide 8 through nucleotide 10 of SEQ ID NO:91 and a termination codon that spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:91. Because SEQ ID NO:91 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei.sub.ca1PB1-C.sub.396, designated nei.sub.ca1PB1-C.sub.1188, and having a coding strand comprising nucleotides 8 to 1195 of SEQ ID NO:91, is represented by SEQ ID NO:93.

[0236] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB1-C protein in the same manner resulted in a nucleic acid molecule of 1241 nucleotides denoted nei.sub.ca2PB1-C.sub.1241, with a coding strand with a sequence designated SEQ ID NO:94. Translation of SEQ ID NO:94 indicates that nucleic acid molecule nei.sub.ca2PB1-C.sub.1241 encodes a molecule designated Pei.sub.ca2PB1-C.sub.396 (SEQ ID NO: 95) which differs from Pei.sub.ca1PB1-C.sub.396(SEQ ID NO:92) in one nucleotide. Nucleotide 1044 of nei.sub.ca1PB1-C.sub.1241, i.e. nucleotide 1037 of nei.sub.ca1PB1-N.sub.1188 was A, while nucleotide 1044 of nei.sub.ca2PB1-C.sub.1241, i.e. nucleotide 1037 of nei.sub.ca2PB1-C.sub.1188 was G. Translation of nei.sub.ca2PB1-C.sub.1241 results in an amino acid change of R to K.

[0237] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wt1PB1-C.sub.1234 (SEQ ID NO:85) and nei.sub.ca1PB1-C.sub.1241 (SEQ ID NO:91) by DNA alignment reveals the following differences: a C to T shift at base 600 of SEQ ID NO:85, and a T to A shift at base 603 of SEQ ID NO:85. Comparison of the amino acid sequences of proteins Pei.sub.wt1PB1-C.sub.396 (SEQ ID NO:86) and Pei.sub.ca1PB1-N.sub.396 (SEQ ID NO:92) reveals the following difference: a H to Q amino acid shift 203 when relating to the T to A base shift at base 603 in the DNA sequences. There is no amino acid shift resulting from the C to T base shift at base 600.

EXAMPLE 19

[0238] This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase) nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

[0239] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB 1 proteins were produced as follows. The wild type or cold-adapted equine influenza genes were cloned in two fragments, the N-terminal portion was produced as in Example 17 and the C-terminal portion of the gene was produced as in Example 18.

[0240] The DNA sequence for the wild type equine influenza PB1 gene was generated by combining the sequences for the wild type PB1-N protein, nei.sub.wtPB1-N.sub.1229 (SEQ ID NO:62) and nei.sub.wt2PB1-N.sub.673 (SEQ ID NO: 65) with the gene fragments for the wild type PB1-C protein, denoted nei.sub.wt1PB1-C.sub.1234 (SEQ ID NO:85) and nei.sub.wt2PB1-C.sub.1240 (SEQ ID NO: 88). The result of combining the N-terminal and C-terminal portions of the PB1 wild type influenza virus yielded a complete DNA sequence of 2341 nucleotides denoted nei.sub.wtPB1.sub.2341 (SEQ ID NO:103). Translation of SEQ ID NO:103 indicates that the nucleic acid molecule nei.sub.wtPB2.sub.2341 encodes a full length equine influenza PB1 protein of about 757 amino acids referred to herein as Pei.sub.wtPB1.sub.757, having amino acid sequence SEQ ID NO:104, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 27 of SEQ ID NO: 103 and the termination codon spans from nucleotide 2293 through nucleotide 2295 of SEQ ID NO: 103. The region encoding Pei.sub.wtPB1.sub.757 designated nei.sub.wtPB1.sub.2271, and having a coding strand comprising nucleotides 25 to 2292 of SEQ ID NO: 103, is SEQ ID NO: 105.

[0241] B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equine influenza virus PB1, denoted nei.sub.ca1PB1.sub.2341, with a sequence denoted SEQ ID NO: 106 was produced by combining the sequences for the N-terminal and C-terminal portions of the PB1 cold-adapted equine influenza gene. The clones for the N-terminal sequences are denoted nei.sub.ca1PB1-N.sub.1225 (SEQ ID NO: 68) and nei.sub.ca2PB1-N.sub.1221 (SEQ ID NO: 71). The clones for the C-terminal sequences are denoted nei.sub.ca1PB1-C.sub.1241 (SEQ ID NO:91) and nei.sub.ca2PB1-C.sub.1241, (SEQ ID NO: 94).

[0242] Translation of SEQ ID NO:106 indicates that nucleic acid molecule nei.sub.ca1PB1.sub.2341 encodes a full-length equine influenza PB1 protein of about 757 amino acids, referred to herein as Pei.sub.ca1PB1.sub.757 having amino acid sequence SEQ ID NO:107, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 27 SEQ ID NO: 106 and the termination codon spans from nucleotide 2296 through nucleotide 2298 of SEQ ID NO:106. The region encoding Pei.sub.ca1PB1.sub.757 designated nei.sub.ca1PB1.sub.2271 and having a coding strand comprising nucleotides 25 to 2295 of SEQ ID NO:108.

[0243] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wtPB1.sub.2341 (SEQ ID NO:103) and nei.sub.ca1PB1.sub.2341 (SEQ ID NO:106) by DNA alignment reveals the following differences: a C to T base shift at base 1683, and a T to A base shift at base 1686. Comparison of the amino acid sequences of proteins Pei.sub.wtPB1.sub.757 (SEQ ID NO:104) and Pei.sub.ca1PB1.sub.757 (SEQ ID NO:107) reveals the following differences: no shift in base C at amino acid 561 relating to the C to T shift at base 1683, and a H to Q shift at amino acid 562 relating to the a T to A shift at base 1683 in the DNA sequence.

EXAMPLE 20

[0244] This example describes the cloning and sequencing of equine influenza PA protein (RNA polymerase A) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

[0245] A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PA-C proteins were produced as follows. A PCR product containing the C-terminal portion of the equine PA gene was produced by PCR amplification using from equine influenza virus DNA and primers C+PA and C-PA, designated SEQ ID NO:83 and SEQ ID NO:84 respectively. A nucleic acid molecule of 1228 nucleotides encoding a wild type PA-C protein, denoted nei.sub.wt1PA-C.sub.1228, with a coding strand having a nucleic acid sequence designated SEQ ID NO:76 was produced by further PCR amplification using the above-described PCR product as a template and cloned as described in Example 1B. Plasmid DNA was purified and sequenced as in Example 11A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance; PAC-1, designated SEQ ID NO:72, PAC-2, designated SEQ ID NO:73, PAC-3, designated SEQ ID NO:74, PAC-4, designated SEQ ID NO: 75, T7 and REV were used in another instance; and PAC-1, PAC-2, T7 and REV were used in another instance.

[0246] Translation of SEQ ID NO:76 indicates that nucleic acid molecule nei.sub.wt1PA-C.sub.1228 encodes a C-terminal portion of influenza PA protein of about 388 amino acids, referred to herein as Pei.sub.wt1PA-C.sub.388, having amino acid sequence SEQ ID NO:77, assuming an open reading frame having a first codon spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:76 and a termination codon which spans from nucleotide 1167 through nucleotide 1169 of SEQ ID NO:76. Because SEQ ID NO:76 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei.sub.wt1PA-C.sub.388, designated nei.sub.wt1PA-C.sub.1164, and having a coding strand comprising nucleotides 3 to 1166 of SEQ ID NO:76 is represented by SEQ ID NO:78.

[0247] PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PA-C protein in the same manner resulted in a nucleic acid molecule of 1223 nucleotides denoted nei.sub.wt2PA-C.sub.1223, with a coding strand with a sequence designated SEQ ID NO:79. nei.sub.wt2PA-C.sub.1223 is identical to nei.sub.wt1PA-C.sub.1228, with the exception of a T to C base shift at base 753 and that nei.sub.wt2PA-C.sub.1223 lacks five nucleotides on the 3'-end. Translation of SEQ ID NO:79 indicates that nucleic acid molecule nei.sub.wt2PA-C.sub.1223 also encodes Pei.sub.wt1PA-C.sub.388 (SEQ ID NO:77), assuming an open reading frame having a first codon which spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:79 and a termination codon which spans from nucleotide 1167 through nucleotide 1169 of SEQ ID NO:79. Because SEQ ID NO:79 is only a partial gene sequence, it does not contain an initiation codon. The nucleic acid molecule having a coding strand comprising nucleotides 3 to 1166 of SEQ ID NO:79, denoted nei.sub.wt2PA-C.sub.1223, is identical to SEQ ID NO 78.

[0248] B. A nucleic acid molecule of 1233 nucleotides encoding a C-terminal portion of influenza PA-C cold-adapted equine influenza virus protein, denoted nei.sub.ca1PA-C.sub.1233, and having a coding strand having a sequence designated SEQ ID NO:80 was produced as described in as in Example 20, part A, except that the pCR.RTM.-Blunt cloning vector was used.

[0249] Translation of SEQ ID NO:80 indicates that nucleic acid molecule nei.sub.ca1PA-C.sub.1233 encodes a C-terminal portion of equine influenza PA protein of about 390 amino acids, referred to herein as Pei.sub.ca1PA-C.sub.390, having amino acid sequence SEQ ID NO:81, assuming an open reading frame having a first codon which spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:80 and a termination codon which spans from nucleotide 1173 through nucleotide 1175 of SEQ ID NO:80. Because SEQ ID NO:80 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei.sub.ca1PA-C.sub.390, designated nei.sub.ca1PA-C.sub.1170, and having a coding strand comprising nucleotides 3 to 1172 of SEQ ID NO:80, is represented by SEQ ID NO:82.

[0250] PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PA-C protein in the same manner resulted in molecule nei.sub.ca2PA-C.sub.1233, containing one A to G base shift at base 953 as compared to nei.sub.ca1PA-C.sub.1233; this base shift does not result in an amino acid change so Pei.sub.ca2PA-C.sub.390, is identical to Pei.sub.ca1PA-C.sub.390 (SEQ ID NO: 81.)

[0251] C. Comparison of the nucleic acid sequences of the coding strands of nei.sub.wt1PA-C.sub.1228 (SEQ ID NO:76) and nei.sub.ca1PA-C.sub.1233 (SEQ ID NO:80) by DNA alignment reveals the following difference: an C to T base shift at base 753 of SEQ ID NO:80. Comparison of the amino acid sequences of proteins Pei.sub.wt1PA-C.sub.388 (SEQ ID NO:77) and Pei.sub.ca1PA-.sub.390 (SEQ ID NO:81) reveals the following difference: a W to R shift at amino acid 251 when relating to the C to T base shift at base 753 in the DNA sequences.

[0252] While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Sequence CWU 1

1

10811023DNAEquine influenza virus H3N8CDS(25)..(780)misc_feature(663)..(663)At nucleotide 663, r = a or g At amino acid residue 213, Xaa = Val 1gcaaaagcag gtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51Met Ser Leu Leu Thr Glu Val Glu Thr1 5tac gtt ctc tct atc gta cca tca ggc ccc ctc aaa gcc gag atc gcg 99Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala10 15 20 25cag aga ctt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala30 35 40ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys45 50 55ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gag cga gga 243Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly60 65 70ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac gga gat 291Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp75 80 85cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga 339Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg90 95 100 105gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tcc act 387Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr110 115 120ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg gga act 435Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr125 130 135gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln140 145 150atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca aca acc 531Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Thr Thr155 160 165aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt acc acg 579Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr170 175 180 185gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gca gca gag 627Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu190 195 200gcc atg gag gtt gct agt aag gct agg cag atg gtr cag gca atg aga 675Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Xaa Gln Ala Met Arg205 210 215acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu220 225 230ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caa atg cag 771Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln235 240 245cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg 820Arg Phe Lys250cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880tacgggttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000gagtaaaaaa ctaccttgtt tct 10232252PRTEquine influenza virus H3N8misc_feature(213)..(213)The 'Xaa' at location 213 stands for Val. 2Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro1 5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu165 170 175Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Lys195 200 205Ala Arg Gln Met Xaa Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser210 215 220Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245 2503756DNAEquine influenza virus H3N8 3atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcgtaccatc aggccccctc 60aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480tctcacaggc agatggtgac aacaaccaac ccattaatca gacatgaaaa cagaatggta 540ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600gaggccatgg aggttgctag taaggctagg cagatggtrc aggcaatgag aaccattggg 660acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720cagaaacgga tgggagtgca aatgcagcga ttcaag 75641023DNAEquine influenza virus H3N8CDS(25)..(780) 4gcaaaagcag gtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51Met Ser Leu Leu Thr Glu Val Glu Thr1 5tac gtt ctc tct atc tta cca tca ggc ccc ctc aaa gcc gag atc gcg 99Tyr Val Leu Ser Ile Leu Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala10 15 20 25cag aga ctt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala30 35 40ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys45 50 55ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gag cga gga 243Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly60 65 70ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac gga gat 291Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp75 80 85cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga 339Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg90 95 100 105gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tcc act 387Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr110 115 120ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg gga act 435Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr125 130 135gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln140 145 150atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca ata acc 531Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Ile Thr155 160 165aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt acc acg 579Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr170 175 180 185gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gca gca gag 627Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu190 195 200gcc atg gag gtt gct agt aag gct agg cag atg gta cag gca atg aga 675Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Val Gln Ala Met Arg205 210 215acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu220 225 230ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caa atg cag 771Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln235 240 245cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg 820Arg Phe Lys250cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880tacggcttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000gagtaaaaaa ctaccttgtt tct 10235252PRTEquine influenza virus H3N8 5Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Leu Pro1 5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala100 105 110Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val Thr Ile Thr Asn Pro Leu Ile Arg His Glu165 170 175Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Lys195 200 205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser210 215 220Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys245 2506756DNAEquine influenza virus H3N8 6atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcttaccatc aggccccctc 60aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480tctcacaggc agatggtgac aataaccaac ccattaatca gacatgaaaa cagaatggta 540ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600gaggccatgg aggttgctag taaggctagg cagatggtac aggcaatgag aaccattggg 660acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720cagaaacgga tgggagtgca aatgcagcga ttcaag 75671762DNAEquine influenza virus H3N8CDS(30)..(1724) 7agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata 53Met Lys Thr Thr Ile Ile Leu Ile1 5cca ctg acc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101Pro Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn10 15 20aca gcc aca tta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu25 30 35 40gta aaa aca ata act gat gac caa att gag gtg aca aat gct act gaa 197Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu45 50 55tta gtt cag agc att tca ata ggg aaa ata tgc aac aac tca tat aga 245Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg60 65 70gtt cta gat gga aga aat tgc aca tta ata gat gca atg cta gga gac 293Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp75 80 85ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttc ata gaa 341Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu90 95 100aga agc agc gct ttc agc agt tgc tac cca tat gac atc cct gac tat 389Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr105 110 115 120gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttg gaa ttc aca 437Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr125 130 135gca gag gga ttc aca tgg aca ggt gtc act caa aac gga aga agt gga 485Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly140 145 150tcc tgc aaa agg gga tca gcc gat agt ttc ttt agc cga ctg aat tgg 533Ser Cys Lys Arg Gly Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp155 160 165cta aca gaa tct gga aac tct tac ccc aca ttg aat gtg aca atg cct 581Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro170 175 180aac aat aaa aat ttc gac aaa cta tac atc tgg ggg att cat cac ccg 629Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro185 190 195 200agc tca aac aaa gag cag aca aaa ttg tac atc caa gaa tcg gga cga 677Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg205 210 215gta aca gtc tca aca aaa aga agt caa caa aca ata atc cct aac atc 725Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile220 225 230gga tct aga ccg cgg gtc agg ggt caa tca ggc agg ata agc ata tac 773Gly Ser Arg Pro Arg Val Arg Gly Gln Ser Gly Arg Ile Ser Ile Tyr235 240 245tgg acc att gta aaa cct gga gat atc cta atg ata aac agt aat ggc 821Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Met Ile Asn Ser Asn Gly250 255 260aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser265 270 275 280tct gta atg aga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys285 290 295att aca cca aat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val300 305 310aac aaa gtt aca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr315 320 325tta aag ctg gcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg330 335 340gga atc ttt gga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly345 350 355 360atg gtt gat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr365 370 375gga caa gct gca gat cta aag agc act caa gca gcc atc gac cag atc 1205Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile380 385 390aat gga aaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His395 400 405caa ata gag aag gaa ttc tca gaa gta gaa ggg agg atc cag gac ttg 1301Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu410 415 420gag aag tat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala425 430 435 440gaa ttg ctg gtg gct cta aaa aat caa cat aca att gac tta aca gat 1397Glu Leu Leu Val Ala Leu Lys Asn Gln His Thr Ile Asp Leu Thr Asp445 450 455gca gaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu460 465 470aac gcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys475 480 485gat aat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr490 495 500ata tac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val505 510 515

520gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala525 530 535ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp540 545 550gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile555 560 565gatagttaaa aacacccttg tttctact 17628565PRTEquine influenza virus H3N8 8Met Lys Thr Thr Ile Ile Leu Ile Pro Leu Thr His Trp Val Tyr Ser1 5 10 15Gln Asn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His20 25 30His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln35 40 45Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Ile Gly50 55 60Lys Ile Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr65 70 75 80Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe Gln Tyr85 90 95Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys100 105 110Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala115 120 125Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly130 135 140Val Thr Gln Asn Gly Arg Ser Gly Ser Cys Lys Arg Gly Ser Ala Asp145 150 155 160Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr165 170 175Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu180 185 190Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys195 200 205Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser210 215 220Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Arg Val Arg Gly225 230 235 240Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp245 250 255Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr260 265 270Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro275 280 285Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro290 295 300Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys305 310 315 320Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg325 330 335Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly340 345 350Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe355 360 365Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser370 375 380Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile385 390 395 400Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu405 410 415Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys420 425 430Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Lys Asn435 440 445Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu450 455 460Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly465 470 475 480Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile485 490 495Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn500 505 510Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp515 520 525Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val530 535 540Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg545 550 555 560Cys Asn Ile Cys Ile56591695DNAEquine influenza virus H3N8 9atgaagacaa ccattatttt gataccactg acccattggg tctacagtca aaacccaacc 60agtggcaaca acacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120gtaaaaacaa taactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180atttcaatag ggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240ttaatagatg caatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300ctcttcatag aaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360gcatcgctcc ggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420acatggacag gtgtcactca aaacggaaga agtggatcct gcaaaagggg atcagccgat 480agtttcttta gccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540gtgacaatgc ctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600agctcaaaca aagagcagac aaaattgtac atccaagaat cgggacgagt aacagtctca 660acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgcg ggtcaggggt 720caatcaggca ggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 780aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840tctgtaatga gatcagatgc acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900ggaagcatcc ccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020aagcaaatca gaggaatctt tggagcaata gcgggattca tagaaaacgg ctgggaagga 1080atggttgatg ggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200gaaaggacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggagg 1260atccaggact tggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320gaattgctgg tggctctaaa aaatcaacat acaattgact taacagatgc agaaatgaat 1380aaattattcg agaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440tgtttcaaga tataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500tatgaccatt acatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560gagttgaaat caggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620ttaatttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680tgcaacattt gcatt 1695101762DNAEquine influenza virus H3N8CDS(30)..(1724) 10agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata 53Met Lys Thr Thr Ile Ile Leu Ile1 5cta ctg acc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101Leu Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn10 15 20aca gcc aca tta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu25 30 35 40gta aaa aca ata act gat gac caa att gag gtg aca aat gct act gaa 197Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu45 50 55tta gtt cag agc att tca ata ggg aaa ata tgc aac aac tca tat aga 245Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg60 65 70gtt cta gat gga aga aat tgc aca tta ata gat gca atg cta gga gac 293Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp75 80 85ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttc ata gaa 341Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu90 95 100aga agc agc gct ttc agc agt tgc tac cca tat gac atc cct gac tat 389Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr105 110 115 120gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttg gaa ttc aca 437Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr125 130 135gca gag gga ttc aca tgg aca ggt gtc act caa aac gga aga agt gga 485Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly140 145 150tcc tgc aaa agg gaa tca gcc gat agt ttc ttt agc cga ctg aat tgg 533Ser Cys Lys Arg Glu Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp155 160 165cta aca gaa tct gga aac tct tac ccc aca ttg aat gtg aca atg cct 581Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro170 175 180aac aat aaa aat ttc gac aaa cta tac atc tgg ggg att cat cac ccg 629Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro185 190 195 200agc tca aac aaa gag cag aca aaa ttg tac atc caa gaa tca gga cga 677Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg205 210 215gta aca gtc tca aca aaa aga agt caa caa aca ata atc cct aac atc 725Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile220 225 230gga tct aga ccg tgg gtc agg ggt caa tca ggc agg ata agc ata tac 773Gly Ser Arg Pro Trp Val Arg Gly Gln Ser Gly Arg Ile Ser Ile Tyr235 240 245tgg acc att gta aaa cct gga gat atc cta acg ata aac agt aat ggc 821Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Thr Ile Asn Ser Asn Gly250 255 260aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser265 270 275 280tct gta atg aga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys285 290 295att aca cca aat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val300 305 310aac aaa gtt aca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr315 320 325tta aag ctg gcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg330 335 340gga atc ttt gga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly345 350 355 360atg gtt gat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr365 370 375gga caa gct gca gat cta aag agc act caa gca gcc atc gac cag atc 1205Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile380 385 390aat gga aaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His395 400 405caa ata gag aag gaa ttc tca gaa gta gaa ggg aga atc cag gac ttg 1301Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu410 415 420gag aag tat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala425 430 435 440gaa ttg ctg gtg gct cta gaa aat caa cat aca att gac tta aca gat 1397Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp445 450 455gca gaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu460 465 470aac gcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys475 480 485gat aat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr490 495 500ata tac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val505 510 515 520gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala525 530 535ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp540 545 550gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile555 560 565gatagttaaa aacacccttg tttctact 176211565PRTEquine influenza virus H3N8 11Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Val Tyr Ser1 5 10 15Gln Asn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His20 25 30His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln35 40 45Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Ile Gly50 55 60Lys Ile Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr65 70 75 80Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe Gln Tyr85 90 95Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys100 105 110Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala115 120 125Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly130 135 140Val Thr Gln Asn Gly Arg Ser Gly Ser Cys Lys Arg Glu Ser Ala Asp145 150 155 160Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr165 170 175Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu180 185 190Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys195 200 205Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser210 215 220Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly225 230 235 240Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp245 250 255Ile Leu Thr Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr260 265 270Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro275 280 285Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro290 295 300Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys305 310 315 320Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg325 330 335Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly340 345 350Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe355 360 365Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser370 375 380Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile385 390 395 400Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu405 410 415Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys420 425 430Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn435 440 445Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu450 455 460Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly465 470 475 480Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile485 490 495Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn500 505 510Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp515 520 525Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val530 535 540Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg545 550 555 560Cys Asn Ile Cys Ile565121695DNAEquine influenza virus H3N8 12atgaagacaa ccattatttt gatactactg acccattggg tctacagtca aaacccaacc 60agtggcaaca acacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120gtaaaaacaa taactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180atttcaatag ggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240ttaatagatg caatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300ctcttcatag aaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360gcatcgctcc ggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420acatggacag gtgtcactca aaacggaaga agtggatcct gcaaaaggga atcagccgat 480agtttcttta gccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540gtgacaatgc ctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600agctcaaaca aagagcagac aaaattgtac atccaagaat caggacgagt aacagtctca 660acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgtg ggtcaggggt 720caatcaggca ggataagcat atactggacc attgtaaaac ctggagatat cctaacgata 780aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840tctgtaatga gatcagatgc

acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900ggaagcatcc ccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020aagcaaatca gaggaatctt tggagcaata gcgggattca tagaaaacgg ctgggaagga 1080atggttgatg ggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200gaaaggacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggaga 1260atccaggact tggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320gaattgctgg tggctctaga aaatcaacat acaattgact taacagatgc agaaatgaat 1380aaattattcg agaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440tgtttcaaga tataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500tatgaccatt acatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560gagttgaaat caggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620ttaatttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680tgcaacattt gcatt 1695131241DNAEquine influenza virus H3N8CDS(28)..(1239) 13agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54Met Glu Arg Ile Lys Glu Leu Arg Asp1 5cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val10 15 20 25gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys30 35 40aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile45 50 55aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln60 65 70ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val75 80 85atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr90 95 100 105acg agc aca att cat tat cca aaa gtc tac aaa act tat ttt gaa aaa 390Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys110 115 120gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn125 130 135caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp140 145 150ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro155 160 165aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile170 175 180 185acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu190 195 200atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe205 210 215ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His220 225 230ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu235 240 245gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn250 255 260 265ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu270 275 280gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile285 290 295ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala300 305 310gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr315 320 325ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu330 335 340 345acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu350 355 360gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala365 370 375acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser380 385 390att gct gaa gca ata att gta gcc atg gtg ttt tc 1241Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe395 40014404PRTEquine influenza virus H3N8 14Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met50 55 60Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro100 105 110Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe151214DNAEquine influenza virus H3N8 15atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300aggaatggac caacaacgag cacaattcat tatccaaaag tctacaaaac ttattttgaa 360aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200gccatggtgt tttc 1214161241DNAEquine influenza virus H3N8CDS(28)..(1239) 16agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54Met Glu Arg Ile Lys Glu Leu Arg Asp1 5cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val10 15 20 25gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys30 35 40aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile45 50 55aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln60 65 70ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val75 80 85atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr90 95 100 105acg agc aca att cat tat cca aaa gtc cac aaa act tat ttt gaa aaa 390Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe Glu Lys110 115 120gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn125 130 135caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp140 145 150ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro155 160 165aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile170 175 180 185acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu190 195 200atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe205 210 215ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His220 225 230ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu235 240 245gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn250 255 260 265ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu270 275 280gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile285 290 295ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala300 305 310gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr315 320 325ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu330 335 340 345acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu350 355 360gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala365 370 375acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser380 385 390att gct gaa gca ata att gta gcc atg gtg ttt tc 1241Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe395 40017404PRTEquine influenza virus H3N8 17Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met50 55 60Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro100 105 110Lys Val His Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe181214DNAEquine influenza virus H3N8 18atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300aggaatggac caacaacgag cacaattcat tatccaaaag tccacaaaac ttattttgaa 360aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa

agaagcacaa 480gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200gccatggtgt tttc 1214191233DNAEquine influenza virus H3N8CDS(3)..(1196) 19ta gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag 47Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys1 5 10 15gca acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa 95Ala Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln20 25 30tca att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat 143Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp35 40 45tgc atg ata aaa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca 191Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala50 55 60aat cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa 239Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys65 70 75gat gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat 287Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn80 85 90 95gtg atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag 335Val Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu100 105 110atg tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac 383Met Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr115 120 125tcc agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt 431Ser Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val130 135 140cgg gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa 479Arg Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu145 150 155aca caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg 527Thr Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met160 165 170 175tgg gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg 575Trp Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp180 185 190atc atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc 623Ile Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro195 200 205aca atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc 671Thr Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val210 215 220cct agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt 719Pro Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe225 230 235cag caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata 767Gln Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile240 245 250 255aaa ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag 815Lys Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln260 265 270ttc tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt 863Phe Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu275 280 285gta aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg 911Val Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg290 295 300ctc aca gtc ctc gga aag gat gca ggt gcg ctt act gaa gac cca gat 959Leu Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp305 310 315gaa ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att 1007Glu Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile320 325 330 335tta ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa 1055Leu Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu340 345 350ctg agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa 1103Leu Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln355 360 365ggg gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt 1151Gly Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu370 375 380act gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 1196Thr Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn385 390 395tagtgttgaa ttgtttaaaa acgaccttgt ttctact 123320398PRTEquine influenza virus H3N8 20Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala1 5 10 15Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser20 25 30Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys35 40 45Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn50 55 60Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp65 70 75 80Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val85 90 95Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met100 105 110Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser115 120 125Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg130 135 140Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr145 150 155 160Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp165 170 175Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile180 185 190Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr195 200 205Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro210 215 220Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln225 230 235 240Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys245 250 255Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe260 265 270Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val275 280 285Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu290 295 300Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu305 310 315 320Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu325 330 335Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu340 345 350Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly355 360 365Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr370 375 380Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn385 390 395211194DNAEquine influenza virus H3N8 21gaattcacaa tggtcggaag aagagcaaca gccattctca gaaaggcaac cagaagattg 60attcaattga tagtaagtgg gagagatgaa caatcaattg ctgaagcaat aattgtagcc 120atggtgtttt cgcaagaaga ttgcatgata aaagcagttc gaggcgattt gaacttcgtt 180aatagagcaa atcagcgctt gaaccccatg catcaactct tgaggcattt ccaaaaagat 240gcaaaagtgc ttttccagaa ttgggggatt gaacccatcg acaatgtgat gggaatgatt 300ggaatattgc ctgacatgac cccaagcacc gagatgtcat tgagaggagt gagagtcagc 360aaaatgggag tggatgagta ctccagcact gagagagtgg tggtgagcat tgaccgtttt 420ttaagagttc gggatcaaag gggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480caaggaacgg aaaagctgac aataatttat tcatcatcaa tgatgtggga gattaatggt 540cccgaatcag tgttggtcaa tacttatcaa tggatcatca ggaactggga aattgtgaaa 600attcaatggt cacaggatcc cacaatgtta tacaataaga tagaatttga gccattccag 660tccctggtcc ctagggccac cagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720caaatgcgag atgtacttgg aacatttgat actgctcaaa taataaaact cctccctttt 780gccgctgctc ctccggaaca gagtaggatg cagttctctt ctttgactgt taatgtaaga 840ggatcgggaa tgaggatact tgtaagaggc aattccccag tgttcaacta caataaagcc 900actaagaggc tcacagtcct cggaaaggat gcaggtgcgc ttactgaaga cccagatgaa 960ggtacggctg gagtagaatc tgctgttcta agagggtttc tcattttagg taaagaaaac 1020aagagatatg gcccagcact aagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080gctaatgtgc taattgggca aggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140agcatactta ctgacagcca gacagcgacc aaaaggattc ggatggccat caat 1194221232DNAEquine influenza virus H3N8 22agaattcaca atggtcggaa gaagagcaac agccattctc agaaaggcaa ccagaagatt 60gattcaattg atagtaagtg ggagagatga acaatcaatt gctgaagcaa taattgtagc 120catggtgttt tcgcaagaag attgcatgat aaaagcagtt cgaggcgatt tgaacttcgt 180taatagagca aatcagcgct tgaaccccat gcatcaactc ttgaggcatt tccaaaaaga 240tgcaaaagtg cttttccaga attgggggat tgaacccatc gacaatgtga tgggaatgat 300tggaatattg cctgacatga ccccaagcac cgagatgtca ttgagaggag tgagagtcag 360caaaatggga gtggatgagt actccagcac tgagagagtg gtggtgagca ttgaccgttt 420tttaagagtt cgggatcaaa ggggaaacat actactgtcc cctgaagagg tcagtgaaac 480acaaggaacg gaaaagctga caataattta ttcatcatca atgatgtggg agattaatgg 540tcccgaatca gtgttggtca atacttatca atggatcatc aggaactggg aaattgtgaa 600aattcaatgg tcacaggatc ccacaatgtt atacaataag atagaatttg agccattcca 660gtccctggtc cctagggcca ccagaagcca atacagcggt ttcgtaagaa ccctgtttca 720gcaaatgcga gatgtacttg gaacatttga tactgctcaa ataataaaac tcctcccttt 780tgccgctgct cctccggaac agagtaggat gcagttctct tctttgactg ttaatgtaag 840aggatcggga atgaggatac ttgtaagagg caattcccca gtgttcaact acaataaagc 900cactaagagg ctcacagtcc tcggaaagga tgcaggtgcg cttactgaag acccagatga 960aggtacggct ggagtagaat ctgctgttct aagagggttt ctcattttag gtaaagaaaa 1020caagagatat ggcccagcac taagcatcaa tgaactgagc aaacttgcaa aaggggagaa 1080agctaatgtg ctaattgggc aaggggacgt ggtgttggta atgaaacgga aacgtgactc 1140tagcatactt actgacagcc agacagcgac caaaaggatt cggatggcca tcaattagtg 1200ttgaattgtt taaaaacgac cttgtttcta ct 1232231232DNAEquine influenza virus H3N8CDS(2)..(1195) 23a gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 49Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala1 5 10 15acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 97Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser20 25 30att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 145Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys35 40 45atg ata caa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 193Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn50 55 60cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 241Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp65 70 75 80gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 289Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val85 90 95atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 337Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met100 105 110tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 385Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser115 120 125agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 433Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg130 135 140gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 481Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr145 150 155 160caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 529Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp165 170 175gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 577Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile180 185 190atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 625Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr195 200 205atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 673Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro210 215 220agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 721Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln225 230 235 240caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 769Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys245 250 255ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 817Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe260 265 270tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 865Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val275 280 285aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 913Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu290 295 300aca gtc ctc gga aaa gat gca ggt gcg ctt act gaa gac cca gat gaa 961Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu305 310 315 320ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 1009Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu325 330 335ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 1057Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu340 345 350agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 1105Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly355 360 365gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 1153Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr370 375 380gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 1195Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn385 390 395tagtgttgaa ttgtttaaaa acgaccttgt ttctact 123224398PRTEquine influenza virus H3N8 24Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala1 5 10 15Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser20 25 30Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys35 40 45Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn50 55 60Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp65 70 75 80Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val85 90 95Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met100 105 110Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser115 120 125Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg130 135 140Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr145 150 155 160Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp165 170 175Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile180 185 190Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr195 200 205Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro210 215 220Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln225 230 235 240Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys245 250 255Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe260 265 270Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val275 280 285Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu290 295

300Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu305 310 315 320Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu325 330 335Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu340 345 350Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly355 360 365Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr370 375 380Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn385 390 395251194DNAEquine influenza virus H3N8 25gaattcacaa tggtcggaag aagagcaaca gccattctca gaaaggcaac cagaagattg 60attcaattga tagtaagtgg gagagatgaa caatcaattg ctgaagcaat aattgtagcc 120atggtgtttt cgcaagaaga ttgcatgata caagcagttc gaggcgattt gaacttcgtt 180aatagagcaa atcagcgctt gaaccccatg catcaactct tgaggcattt ccaaaaagat 240gcaaaagtgc ttttccagaa ttgggggatt gaacccatcg acaatgtgat gggaatgatt 300ggaatattgc ctgacatgac cccaagcacc gagatgtcat tgagaggagt gagagtcagc 360aaaatgggag tggatgagta ctccagcact gagagagtgg tggtgagcat tgaccgtttt 420ttaagagttc gggatcaaag gggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480caaggaacgg aaaagctgac aataatttat tcatcatcaa tgatgtggga gattaatggt 540cccgaatcag tgttggtcaa tacttatcaa tggatcatca ggaactggga aattgtgaaa 600attcaatggt cacaggatcc cacaatgtta tacaataaga tagaatttga gccattccag 660tccctggtcc ctagggccac cagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720caaatgcgag atgtacttgg aacatttgat actgctcaaa taataaaact cctccctttt 780gccgctgctc ctccggaaca gagtaggatg cagttctctt ctttgactgt taatgtaaga 840ggatcgggaa tgaggatact tgtaagaggc aattccccag tgttcaacta caataaagcc 900actaagaggc tcacagtcct cggaaaagat gcaggtgcgc ttactgaaga cccagatgaa 960ggtacggctg gagtagaatc tgctgttcta agagggtttc tcattttagg taaagaaaac 1020aagagatatg gcccagcact aagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080gctaatgtgc taattgggca aggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140agcatactta ctgacagcca gacagcgacc aaaaggattc ggatggccat caat 11942623DNAArtificialSynthetic Primer 26agcaaaagca ggtagatatt gaa 232724DNAArtificialSynthetic Primer 27agtagaaaca aggtagtttt ttac 242818DNAArtificialSynthetic Primer 28caggaaacag ctatgacc 182920DNAArtificialSynthetic Primer 29taatacgact cactataggg 203018DNAArtificialSynthetic Primer 30tggtgcacta gccagctg 183118DNAArtificialSynthetic Primer 31ttgcctgtac catctgcc 183223DNAArtificialSynthetic Primer 32agcaaaagca ggggatattt ctg 233323DNAArtificialSynthetic Primer 33agtagaaaca agggtgtttt taa 233416DNAArtificialSynthetic Primer 34gacatccctg actatg 163516DNAArtificialSynthetic Primer 35gcatctgtta agtcaa 163625DNAArtificialSynthetic Primer 36agcaaaagca ggtcaaatat attca 253726DNAArtificialSynthetic Primer 37gaaaacacca tggctacaat tattgc 263827DNAArtificialSynthetic Primer 38agaattcaca atggtcggaa gaagagc 273927DNAArtificialSynthetic Primer 39agtagaaaca aggtcgtttt taaacaa 274019DNAArtificialSynthetic Primer 40agccgtacct tcatctggg 194119DNAArtificialSynthetic Primer 41agcactgaga gagtggtgg 194219DNAArtificialSynthetic Primer 42gtaagaggca attccccag 194318DNAArtificialSynthetic Primer 43cagcttttcc gttccttg 18442341DNAEquine influenza virus H3N8CDS(28)..(2304) 44agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54Met Glu Arg Ile Lys Glu Leu Arg Asp1 5cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val10 15 20 25gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys30 35 40aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile45 50 55aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln60 65 70ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val75 80 85atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr90 95 100 105acg agc aca att cat tat cca aaa gtc tac aaa act tat ttt gaa aaa 390Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys110 115 120gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn125 130 135caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp140 145 150ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro155 160 165aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile170 175 180 185acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu190 195 200atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe205 210 215ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His220 225 230ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu235 240 245gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn250 255 260 265ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu270 275 280gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile285 290 295ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala300 305 310gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr315 320 325ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu330 335 340 345acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu350 355 360gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala365 370 375acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser380 385 390att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 1254Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys395 400 405atg ata aaa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 1302Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn410 415 420 425cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 1350Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp430 435 440gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 1398Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val445 450 455atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 1446Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met460 465 470tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 1494Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser475 480 485agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 1542Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg490 495 500 505gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 1590Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr510 515 520caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 1638Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp525 530 535gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 1686Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile540 545 550atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 1734Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr555 560 565atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 1782Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro570 575 580 585agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 1830Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln590 595 600caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 1878Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys605 610 615ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 1926Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe620 625 630tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 1974Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val635 640 645aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 2022Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu650 655 660 665aca gtc ctc gga aag gat gca ggt gcg ctt act gaa gac cca gat gaa 2070Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu670 675 680ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 2118Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu685 690 695ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 2166Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu700 705 710agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 2214Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly715 720 725gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 2262Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr730 735 740 745gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 2304Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn750 755tagtgttgaa ttgtttaaaa acgaccttgt ttctact 234145759PRTEquine influenza virus H3N8 45Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met50 55 60Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro100 105 110Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn435 440 445Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr515 520 525Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val545 550 555 560Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly595 600 605Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala610 615 620Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe645 650 655Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala660 665 670Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750Arg Ile Arg Met Ala Ile Asn755462277DNAEquine influenza virus H3N8 46atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg

cgagatacta 60acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300aggaatggac caacaacgag cacaattcat tatccaaaag tctacaaaac ttattttgaa 360aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200gccatggtgt tttcgcaaga agattgcatg ataaaagcag ttcgaggcga tttgaacttc 1260gttaatagag caaatcagcg cttgaacccc atgcatcaac tcttgaggca tttccaaaaa 1320gatgcaaaag tgcttttcca gaattggggg attgaaccca tcgacaatgt gatgggaatg 1380attggaatat tgcctgacat gaccccaagc accgagatgt cattgagagg agtgagagtc 1440agcaaaatgg gagtggatga gtactccagc actgagagag tggtggtgag cattgaccgt 1500tttttaagag ttcgggatca aaggggaaac atactactgt cccctgaaga ggtcagtgaa 1560acacaaggaa cggaaaagct gacaataatt tattcatcat caatgatgtg ggagattaat 1620ggtcccgaat cagtgttggt caatacttat caatggatca tcaggaactg ggaaattgtg 1680aaaattcaat ggtcacagga tcccacaatg ttatacaata agatagaatt tgagccattc 1740cagtccctgg tccctagggc caccagaagc caatacagcg gtttcgtaag aaccctgttt 1800cagcaaatgc gagatgtact tggaacattt gatactgctc aaataataaa actcctccct 1860tttgccgctg ctcctccgga acagagtagg atgcagttct cttctttgac tgttaatgta 1920agaggatcgg gaatgaggat acttgtaaga ggcaattccc cagtgttcaa ctacaataaa 1980gccactaaga ggctcacagt cctcggaaag gatgcaggtg cgcttactga agacccagat 2040gaaggtacgg ctggagtaga atctgctgtt ctaagagggt ttctcatttt aggtaaagaa 2100aacaagagat atggcccagc actaagcatc aatgaactga gcaaacttgc aaaaggggag 2160aaagctaatg tgctaattgg gcaaggggac gtggtgttgg taatgaaacg gaaacgtgac 2220tctagcatac ttactgacag ccagacagcg accaaaagga ttcggatggc catcaat 2277472341DNAEquine influenza virus H3N8CDS(28)..(2304) 47agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54Met Glu Arg Ile Lys Glu Leu Arg Asp1 5cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val10 15 20 25gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys30 35 40aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile45 50 55aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln60 65 70ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val75 80 85atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr90 95 100 105acg agc aca att cat tat cca aaa gtc cac aaa act tat ttt gaa aaa 390Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe Glu Lys110 115 120gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn125 130 135caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp140 145 150ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro155 160 165aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile170 175 180 185acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu190 195 200atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe205 210 215ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His220 225 230ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu235 240 245gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn250 255 260 265ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu270 275 280gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile285 290 295ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala300 305 310gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr315 320 325ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu330 335 340 345acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu350 355 360gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala365 370 375acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser380 385 390att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 1254Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys395 400 405atg ata caa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 1302Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn410 415 420 425cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 1350Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp430 435 440gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 1398Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val445 450 455atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 1446Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met460 465 470tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 1494Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser475 480 485agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 1542Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg490 495 500 505gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 1590Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr510 515 520caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 1638Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp525 530 535gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 1686Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile540 545 550atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 1734Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr555 560 565atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 1782Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro570 575 580 585agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 1830Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln590 595 600caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 1878Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys605 610 615ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 1926Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe620 625 630tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 1974Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val635 640 645aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 2022Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu650 655 660 665aca gtc ctc gga aaa gat gca ggt gcg ctt act gaa gac cca gat gaa 2070Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu670 675 680ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 2118Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu685 690 695ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 2166Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu700 705 710agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 2214Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly715 720 725gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 2262Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr730 735 740 745gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 2304Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn750 755tagtgttgaa ttgtttaaaa acgaccttgt ttctact 234148759PRTEquine influenza virus H3N8 48Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met50 55 60Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro100 105 110Lys Val His Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg130 135 140Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu180 185 190Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr210 215 220Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val260 265 270Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln275 280 285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser325 330 335Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Gln Ala Val Arg Gly405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn435 440 445Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu450 455 460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu500 505 510Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr515 520 525Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val545 550 555 560Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly595 600 605Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala610 615 620Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe645 650 655Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala660 665 670Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr690 695 700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys740 745 750Arg Ile Arg Met Ala Ile Asn755492277DNAEquine influenza virus H3N8 49atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300aggaatggac caacaacgag cacaattcat tatccaaaag tccacaaaac ttattttgaa 360aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200gccatggtgt tttcgcaaga

agattgcatg atacaagcag ttcgaggcga tttgaacttc 1260gttaatagag caaatcagcg cttgaacccc atgcatcaac tcttgaggca tttccaaaaa 1320gatgcaaaag tgcttttcca gaattggggg attgaaccca tcgacaatgt gatgggaatg 1380attggaatat tgcctgacat gaccccaagc accgagatgt cattgagagg agtgagagtc 1440agcaaaatgg gagtggatga gtactccagc actgagagag tggtggtgag cattgaccgt 1500tttttaagag ttcgggatca aaggggaaac atactactgt cccctgaaga ggtcagtgaa 1560acacaaggaa cggaaaagct gacaataatt tattcatcat caatgatgtg ggagattaat 1620ggtcccgaat cagtgttggt caatacttat caatggatca tcaggaactg ggaaattgtg 1680aaaattcaat ggtcacagga tcccacaatg ttatacaata agatagaatt tgagccattc 1740cagtccctgg tccctagggc caccagaagc caatacagcg gtttcgtaag aaccctgttt 1800cagcaaatgc gagatgtact tggaacattt gatactgctc aaataataaa actcctccct 1860tttgccgctg ctcctccgga acagagtagg atgcagttct cttctttgac tgttaatgta 1920agaggatcgg gaatgaggat acttgtaaga ggcaattccc cagtgttcaa ctacaataaa 1980gccactaaga ggctcacagt cctcggaaaa gatgcaggtg cgcttactga agacccagat 2040gaaggtacgg ctggagtaga atctgctgtt ctaagagggt ttctcatttt aggtaaagaa 2100aacaagagat atggcccagc actaagcatc aatgaactga gcaaacttgc aaaaggggag 2160aaagctaatg tgctaattgg gcaaggggac gtggtgttgg taatgaaacg gaaacgtgac 2220tctagcatac ttactgacag ccagacagcg accaaaagga ttcggatggc catcaat 227750891DNAEquine influenza virus H3N8CDS(27)..(716) 50agcaaaagca gggtgacaaa aacata atg gat tcc aac act gtg tca agc ttt 53Met Asp Ser Asn Thr Val Ser Ser Phe1 5cag gta gac tgt ttt ctt tgg cat gtc cgc aaa cga ttt gca gac caa 101Gln Val Asp Cys Phe Leu Trp His Val Arg Lys Arg Phe Ala Asp Gln10 15 20 25gaa ctg ggt gat gcc cca ttc ctt gac cgg ctt cgc cga gac cag aag 149Glu Leu Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys30 35 40tcc cta aaa gga aga ggt agc act ctt ggt ctg gac atc gaa aca gcc 197Ser Leu Lys Gly Arg Gly Ser Thr Leu Gly Leu Asp Ile Glu Thr Ala45 50 55act cgt gca gga aag cag ata gtg gag cag att ctg gaa gag gaa tca 245Thr Arg Ala Gly Lys Gln Ile Val Glu Gln Ile Leu Glu Glu Glu Ser60 65 70gat gag gca ctt aaa atg acc att gcc tct gtt cct gct tca cgc tac 293Asp Glu Ala Leu Lys Met Thr Ile Ala Ser Val Pro Ala Ser Arg Tyr75 80 85tta act gac atg act ctt gat gag atg tca aga gac tgg ttc atg ctc 341Leu Thr Asp Met Thr Leu Asp Glu Met Ser Arg Asp Trp Phe Met Leu90 95 100 105atg ccc aag cag aaa gta aca ggc tcc cta tgt ata aga atg gac cag 389Met Pro Lys Gln Lys Val Thr Gly Ser Leu Cys Ile Arg Met Asp Gln110 115 120gca atc atg gat aag aac atc ata ctt aaa gca aac ttt agt gtg att 437Ala Ile Met Asp Lys Asn Ile Ile Leu Lys Ala Asn Phe Ser Val Ile125 130 135ttc gaa agg ctg gag aca cta ata cta ctt aga gcc ttc acc gaa gaa 485Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala Phe Thr Glu Glu140 145 150gga gca gtc gtt ggc gaa att tca cca ttg cct tct ctt cca gga cat 533Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser Leu Pro Gly His155 160 165act aat gag gat gtc aaa aat gca att ggg gtc ctc atc gga gga ctt 581Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu Ile Gly Gly Leu170 175 180 185aaa tgg aat gat aat acg gtt aga atc tct gaa act cta cag aga ttc 629Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu Thr Leu Gln Arg Phe190 195 200gct tgg aga agc agt cat gag aat ggg aga cct tca ttc cct cca aag 677Ala Trp Arg Ser Ser His Glu Asn Gly Arg Pro Ser Phe Pro Pro Lys205 210 215cag aaa cga aaa atg gag aga aca att gag cca gaa gtt tgaagaaata 726Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro Glu Val220 225 230agatggttga ttgaagaagt gcgacataga ttgaaaaata cagaaaatag ttttgaacaa 786ataacattta tgcaagcctt acaactattg cttgaagtag gacaagagat aagaactttc 846tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc tacta 89151230PRTEquine influenza virus H3N8 51Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile50 55 60Val Glu Gln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Met Thr65 70 75 80Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp85 90 95Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr100 105 110Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile115 120 125Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile145 150 155 160Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val180 185 190Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu195 200 205Asn Gly Arg Pro Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg210 215 220Thr Ile Glu Pro Glu Val225 23052690DNAEquine influenza virus H3N8 52atggattcca acactgtgtc aagctttcag gtagactgtt ttctttggca tgtccgcaaa 60cgatttgcag accaagaact gggtgatgcc ccattccttg accggcttcg ccgagaccag 120aagtccctaa aaggaagagg tagcactctt ggtctggaca tcgaaacagc cactcgtgca 180ggaaagcaga tagtggagca gattctggaa gaggaatcag atgaggcact taaaatgacc 240attgcctctg ttcctgcttc acgctactta actgacatga ctcttgatga gatgtcaaga 300gactggttca tgctcatgcc caagcagaaa gtaacaggct ccctatgtat aagaatggac 360caggcaatca tggataagaa catcatactt aaagcaaact ttagtgtgat tttcgaaagg 420ctggagacac taatactact tagagccttc accgaagaag gagcagtcgt tggcgaaatt 480tcaccattgc cttctcttcc aggacatact aatgaggatg tcaaaaatgc aattggggtc 540ctcatcggag gacttaaatg gaatgataat acggttagaa tctctgaaac tctacagaga 600ttcgcttgga gaagcagtca tgagaatggg agaccttcat tccctccaaa gcagaaacga 660aaaatggaga gaacaattga gccagaagtt 69053888DNAEquine influenza virus H3N8 53caaaagcagg gtgacaaaaa catgatggat tccaacactg tgtcaagctt tcaggtagac 60tgttttcttt ggcatgtccg caaacgattt gcagaccaag aactgggtga tgccccattc 120cttgaccggc ttcgccgaga ccagaagtcc ctaaaaggaa gaggtagcac tcttggtctg 180gacatcgaaa cagccactcg tgcaggaaag cagatagtgg agcagattct ggaagaggaa 240tcagatgagg cacttaaaat gaccattgcc tctgttcctg cttcacgcta cttaactgac 300atgactcttg atgagatgtc aagagactgg ttcatgctca tgcccaagca gaaagtaaca 360ggctccctat gtataagaat ggaccaggca atcatggata agaacatcat acttaaagca 420aactttagtg tgattttcga aaggctggag acactaatac tacttagagc cttcaccgaa 480gaaggagcag tcgttggcga aatttcacca ttgccttctc ttccaggaca tactaatgag 540gatgtcaaaa atgcaattgg ggtcctcatc ggaggactta aatggaatga taatacggtt 600agaatctctg aaactctaca gagattcgct tggagaagca gtcatgagaa tgggagacct 660tcattccctc caaagcagaa acgaaaaatg gagagaacaa ttgagccaga agtttgaaga 720aataagatgg ttgattgaag aagtgcgaca tagattgaaa aatacagaaa atagttttga 780acaaataaca tttatgcaag ccttacaact attgcttgaa gtagaacaag agataagaac 840tttctcgttt cagcttattt aatgataaaa aacacccttg tttctact 88854468DNAEquine influenza virus H3N8CDS(3)..(293) 54ac ttt agt gtg att ttc gaa agg ctg gag aca cta ata cta ctt aga 47Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg1 5 10 15gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att tca cca ttg cct 95Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro20 25 30tct ctt cca gga cat act aat gag gat gtc aaa aat gca att ggg gtc 143Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val35 40 45ctc atc gga gga ctt aaa tgg aat gat aat acg gtt aga atc tct gaa 191Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu50 55 60act cta cag aga ttc gct cgg aga agc agt cat gag aat ggg aga cct 239Thr Leu Gln Arg Phe Ala Arg Arg Ser Ser His Glu Asn Gly Arg Pro65 70 75tca ttc cct cca aag cag aaa cga aaa atg gag aga aca att gag cca 287Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro80 85 90 95gaa gtt tgaagaaata agatggttga ttgaagaagt gcgacataga ttgaaaaata 343Glu Valcagaaaatag ttttgaacaa ataacattta tgcaagcctt acaactattg cttgaagtag 403aacaagagat aagaactttc tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc 463tacta 4685597PRTEquine influenza virus H3N8 55Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala1 5 10 15Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser20 25 30Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu35 40 45Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu Thr50 55 60Leu Gln Arg Phe Ala Arg Arg Ser Ser His Glu Asn Gly Arg Pro Ser65 70 75 80Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro Glu85 90 95Val56293DNAEquine influenza virus H3N8 56actttagtgt gattttcgaa aggctggaga cactaatact acttagagcc ttcaccgaag 60aaggagcagt cgttggcgaa atttcaccat tgccttctct tccaggacat actaatgagg 120atgtcaaaaa tgcaattggg gtcctcatcg gaggacttaa atggaatgat aatacggtta 180gaatctctga aactctacag agattcgctc ggagaagcag tcatgagaat gggagacctt 240cattccctcc aaagcagaaa cgaaaaatgg agagaacaat tgagccagaa gtt 29357888DNAEquine influenza virus H3N8CDS(27)..(716) 57agcaaaagca gggtgacaaa aacata atg gat tcc aac act gtg tca agc ttt 53Met Asp Ser Asn Thr Val Ser Ser Phe1 5cag gta gac tgt ttt ctt tgg cat gtc cgc aaa cga ttt gca gac caa 101Gln Val Asp Cys Phe Leu Trp His Val Arg Lys Arg Phe Ala Asp Gln10 15 20 25gaa ctg ggt gat gcc cca ttc ctt gac cgg ctt cgc cga gac cag aag 149Glu Leu Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys30 35 40tcc cta aaa gga aga ggt agc act ctt ggt ctg gac atc gaa aca gcc 197Ser Leu Lys Gly Arg Gly Ser Thr Leu Gly Leu Asp Ile Glu Thr Ala45 50 55act cgt gca gga aag cag ata gtg gag cag att ctg gaa gag gaa tca 245Thr Arg Ala Gly Lys Gln Ile Val Glu Gln Ile Leu Glu Glu Glu Ser60 65 70gat gag gca ctt aaa atg acc att gcc tct gtt cct gct tca cgc tac 293Asp Glu Ala Leu Lys Met Thr Ile Ala Ser Val Pro Ala Ser Arg Tyr75 80 85tta act gac atg act ctt gat gag atg tca aga gac tgg ttc atg ctc 341Leu Thr Asp Met Thr Leu Asp Glu Met Ser Arg Asp Trp Phe Met Leu90 95 100 105atg ccc aag cag aaa gta aca ggc tcc cta tgt ata aga atg gac cag 389Met Pro Lys Gln Lys Val Thr Gly Ser Leu Cys Ile Arg Met Asp Gln110 115 120gca atc atg gat aag aac atc ata ctt aaa gca aac ttt agt gtg att 437Ala Ile Met Asp Lys Asn Ile Ile Leu Lys Ala Asn Phe Ser Val Ile125 130 135ttc gaa agg ctg gag aca cta ata cta ctt aga gcc ttc acc gaa gaa 485Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala Phe Thr Glu Glu140 145 150gga gca gtc gtt ggc gaa att tca cca ttg cct tct ctt cca gga cat 533Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser Leu Pro Gly His155 160 165act aat gag gat gtc aaa aat gca att ggg gtc ctc atc gga gga ctt 581Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu Ile Gly Gly Leu170 175 180 185aaa tgg aat gat aat acg gtt aga atc tct gaa act cta cag aga ttc 629Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu Thr Leu Gln Arg Phe190 195 200gct tgg aga agc agt cat gag aat ggg aga cct tca ttc cct cca aag 677Ala Trp Arg Ser Ser His Glu Asn Gly Arg Pro Ser Phe Pro Pro Lys205 210 215cag aaa cga aaa atg gag aga aca att gag cca gaa gtt tgaagaaata 726Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro Glu Val220 225 230agatggttga ttgaagaagt gcgacataga ttgaaaaata cagaaaatag ttttgaacaa 786ataacattta tgcaagcctt acaactattg cttgaagtag aacaagagat aagaactttc 846tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc ta 88858230PRTEquine influenza virus H3N8 58Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile50 55 60Val Glu Gln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Met Thr65 70 75 80Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp85 90 95Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr100 105 110Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile115 120 125Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile145 150 155 160Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn165 170 175Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val180 185 190Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu195 200 205Asn Gly Arg Pro Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg210 215 220Thr Ile Glu Pro Glu Val225 23059690DNAEquine influenza virus H3N8 59atggattcca acactgtgtc aagctttcag gtagactgtt ttctttggca tgtccgcaaa 60cgatttgcag accaagaact gggtgatgcc ccattccttg accggcttcg ccgagaccag 120aagtccctaa aaggaagagg tagcactctt ggtctggaca tcgaaacagc cactcgtgca 180ggaaagcaga tagtggagca gattctggaa gaggaatcag atgaggcact taaaatgacc 240attgcctctg ttcctgcttc acgctactta actgacatga ctcttgatga gatgtcaaga 300gactggttca tgctcatgcc caagcagaaa gtaacaggct ccctatgtat aagaatggac 360caggcaatca tggataagaa catcatactt aaagcaaact ttagtgtgat tttcgaaagg 420ctggagacac taatactact tagagccttc accgaagaag gagcagtcgt tggcgaaatt 480tcaccattgc cttctcttcc aggacatact aatgaggatg tcaaaaatgc aattggggtc 540ctcatcggag gacttaaatg gaatgataat acggttagaa tctctgaaac tctacagaga 600ttcgcttgga gaagcagtca tgagaatggg agaccttcat tccctccaaa gcagaaacga 660aaaatggaga gaacaattga gccagaagtt 6906021DNAArtificialSynthetic Primer 60agcaaagcag gtgacaaaaa c 216119DNAArtificialSynthetic Primer 61agtagaaaca agggtgttt 19621229DNAEquine influenza virus H3N8CDS(36)..(1229) 62gaattcggct tagcaaaagc aggcaaacta tttga atg gat gtc aat ccg act 53Met Asp Val Asn Pro Thr1 5cta ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc 101Leu Leu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe10 15 20cct tat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac 149Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr25 30 35acc atg gat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa 197Thr Met Asp Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys40 45 50tgg aca aca aac act gag att gga gca cca caa ctt aat cca atc gat 245Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp55 60 65 70gga ccg ctt cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat 293Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp75 80 85tgt gta ttg gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc 341Cys Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile90 95 100ttt gaa aat tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga 389Phe Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg105 110 115gtg gac aaa cta aca caa ggc cga caa act tac gat tgg acc ttg aat 437Val Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn120 125 130agg aat caa cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc 485Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe135 140 145 150aga tca aat gat ctg act tcc agt gag tca ggg aga tta atg gac ttc 533Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe155 160 165ctc aaa gat gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca 581Leu Lys Asp Val Met Glu Ser Met Asn Lys Glu

Glu Met Glu Ile Thr170 175 180aca cac ttc caa cgg aag aga aga gta aga gac aac atg aca aag aga 629Thr His Phe Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg185 190 195atg gtg aca cag aga acc ata ggg aag aaa aaa caa cga tta aac aga 677Met Val Thr Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg200 205 210aag agc tat ctg atc agg gca tta acc tta aac aca atg acc aag gac 725Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp215 220 225 230gct gag aga ggg aaa ttg aaa cga cga gca att gca acc cca gga atg 773Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met235 240 245cag ata aga ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata 821Gln Ile Arg Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile250 255 260tgt gaa aag ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa 869Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys265 270 275aag gcc aaa ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa 917Lys Ala Lys Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln280 285 290gac act gaa ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat 965Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn295 300 305 310gaa aat cag aac cca cgc atg ttc ctg gca atg atc aca tac ata act 1013Glu Asn Gln Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr315 320 325aga aac cag cca gaa tgg ttc aga aat gtt cta agc att gca ccg att 1061Arg Asn Gln Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile330 335 340atg ttc tca aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa 1109Met Phe Ser Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu345 350 355agc aaa agt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca 1157Ser Lys Ser Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala360 365 370agc att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag 1205Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu375 380 385 390aag ata cga cca caa gcc gaa ttc 1229Lys Ile Arg Pro Gln Ala Glu Phe39563398PRTEquine influenza virus H3N8 63Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser145 150 155 160Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser370 375 380Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Gln Ala Glu Phe385 390 395641194DNAEquine influenza virus H3N8 64atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacaagccga attc 119465673DNAEquine influenza virus H3N8CDS(36)..(671) 65gaattcggct tagcaaaagc aggcaaacta tttga atg gat gtc aat ccg act 53Met Asp Val Asn Pro Thr1 5cta ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc 101Leu Leu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe10 15 20cct tat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac 149Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr25 30 35acc atg gat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa 197Thr Met Asp Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys40 45 50tgg aca aca aac act gag att gga gca cca caa ctt aat cca atc gat 245Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp55 60 65 70gga ccg ctt cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat 293Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp75 80 85tgt gta ttg gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc 341Cys Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile90 95 100ttt gaa aat tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga 389Phe Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg105 110 115gtg gac aaa cta aca caa ggc cga caa act tac gat tgg acc ttg aat 437Val Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn120 125 130agg aat caa cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc 485Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe135 140 145 150aga tca aat gat ctg act tcc agt gag tca ggg aga tta atg gac ttc 533Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe155 160 165ctc aaa gat gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca 581Leu Lys Asp Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr170 175 180aca cac ttc caa cgg aag aga aga gta aga gac aac atg aca aag aga 629Thr His Phe Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg185 190 195atg gtg aca cag aga acc ata ggg aag aaa aaa caa cga tta aa 673Met Val Thr Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu200 205 21066212PRTEquine influenza virus H3N8 66Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser145 150 155 160Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu21067636DNAEquine influenza virus H3N8 67atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgatta 636681225DNAEquine influenza virus H3N8CDS(34)..(1218) 68gaattcagga gcaaaagcag gcaaactatt tga atg gat gtc aat ccg act cta 54Met Asp Val Asn Pro Thr Leu1 5ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct 102Leu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro10 15 20tat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc 150Tyr Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr25 30 35atg gat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg 198Met Asp Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp40 45 50 55aca aca aac act gag att gga gca cca caa ctt aat cca atc gat gga 246Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly60 65 70ccg ctt cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt 294Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys75 80 85gta ttg gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt 342Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe90 95 100gaa aat tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg 390Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val105 110 115gac aaa cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg 438Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg120 125 130 135aat caa cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga 486Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg140 145 150tca aat gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc 534Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu155 160 165aaa gat gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca 582Lys Asp Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr170 175 180cac ttc caa cgg aag aga aga gta aga gac aac atg aca aag aga atg 630His Phe Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met185 190 195gtg aca cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aag 678Val Thr Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys200 205 210 215agc tat ctg atc agg gca tta acc tta aac aca atg acc aag gac gct 726Ser Tyr Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala220 225 230gag aga ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cag 774Glu Arg Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln235 240 245ata aga ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgt 822Ile Arg Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys250 255 260gaa aag ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa aag 870Glu Lys Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys265 270 275gcc aaa ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa gac 918Ala Lys Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp280 285 290 295act gaa ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat gaa 966Thr Glu Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu300 305 310aat cag aac cca cgc atg ttc ctg gca atg atc aca tac ata act aga 1014Asn Gln Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Arg315 320 325aac cag cca gaa tgg ttc aga aat gtt cta agc att gca ccg att atg 1062Asn Gln Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile Met330 335 340ttc tca aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa agc 1110Phe Ser Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser345 350 355aaa agt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 1158Lys Ser Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser360 365 370 375att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 1206Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys380 385 390ata cga cca ccc tgaattc 1225Ile Arg Pro Pro39569395PRTEquine influenza virus H3N8 69Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser145 150 155 160Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275

280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser370 375 380Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Pro385 390 395701185DNAEquine influenza virus H3N8 70atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac caccc 1185711221DNAEquine influenza virus H3N8 71gaattcagga aagcaggcaa actatttgaa tggatgtcaa tccgactcta ctcttcttaa 60aggtgccagc gcaaaatgct ataagcacaa cattccctta tactggagat cctccctaca 120gtcatggaac agggacagga tacaccatgg atactgtcaa cagaacacat caatactcag 180aaaaggggaa atggacaaca aacactgaga ttggagcacc acaacttaat ccaatcgatg 240gaccgcttcc tgaagacaat gaaccaagtg ggtacgccca aacagattgt gtattggaag 300caatggcttt ccttgaagaa tcccatcccg gaatctttga aaattcgtgt cttgaaacaa 360tggaggtggt tcagcagaca agagtggaca aactaacaca aggccgacaa acttacgatt 420ggaccttgaa taggaatcaa cctgccgcaa cagcacttgc taatacaatt gaagtgttca 480gatcaaatga tctgacttcc agtgagtcag ggagattaat ggacttcctc aaagatgtca 540tggagtccat gaacaaggaa gaaatggaaa taacaacaca cttccaacgg aagagaagag 600taagagacaa catgacaaag agaatggtga cacagagaac catagggaag aaaaaacaac 660gattaaacag aaagagctat ctgatcaggg cattaacctt aaacacaatg accaaggacg 720ctgagagagg gaaattgaaa cgacgagcaa ttgcaacccc aggaatgcag ataagagggt 780ttgtatattt tgttgaaaca ttagcccgaa gaatatgtga aaagcttgaa caatcaggat 840tgccagttgg cggtaatgag aaaaaggcca aactggctaa tgtcgtcaga aaaatgatga 900ctaattccca agacactgaa ctctccttca ccatcactgg ggacaatacc aaatggaatg 960aaaatcagaa cccacgcatg ttcctggcaa tgatcacata cataactaga aaccagccag 1020aatggttcag aaatgttcta agcattgcac cgattatgtt ctcaaataaa atggcaagac 1080tggggaaagg atatatgttt gaaagcaaaa gtatgaaatt gagaactcaa ataccagcag 1140aaatgctcgc aagcattgat ctgaaatatt tcaatgattc aacaaaaaag aaaattgaga 1200agatacgacc accctgaatt c 12217218DNAArtificialSynthetic Primer 72gcaaatgcag gaccaaag 187318DNAArtificialSynthetic Primer 73gactgaggac tcagcttc 187419DNAArtificialSynthetic Primer 74caatatcctc cccaatttc 197519DNAArtificialSynthetic Primer 75ggaaggtttg caggacctt 19761228DNAEquine influenza virus H3N8CDS(3)..(1166) 76gg ggc ggg tac cca aac tat ctc caa gct tgg aag caa gta tta gca 47Gly Gly Tyr Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu Ala1 5 10 15gaa cta caa gac ctt gag aac gaa gaa aag acc cct aag acc aag aat 95Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys Asn20 25 30atg aaa aaa aca agc caa ttg aaa tgg gca ctc ggt gaa aat atg gca 143Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Ala35 40 45cca gag aaa gtg gat ttt gag gat tgt aaa gac atc aat gat ttg aaa 191Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu Lys50 55 60cag tat gac agt gat gag cca gaa aca agg tct ctt gca agt tgg att 239Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp Ile65 70 75caa agt gag ttc aac aaa gct tgt gag ctg aca gat tca agc tgg ata 287Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp Ile80 85 90 95gag ctc gat gaa att ggg gag gat att gcc cca ata gaa tac att gcg 335Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile Ala100 105 110agc atg agg aga aat tat ttt act gct gag gtt tcc cat tgt aga gca 383Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala115 120 125aca gaa tat ata atg aag gga gtg tac atc aac act gct cta ctc aat 431Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn130 135 140gca tcc tgt gct gcg atg gat gaa ttc caa tta att ccg atg ata agc 479Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile Ser145 150 155aaa tgc agg acc aaa gaa ggg aga agg aag aca aat tta tat gga ttc 527Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe160 165 170 175ata ata aag gga agg tcc cat tta agg aat gat acc gac gtg gta aac 575Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val Asn180 185 190ttt gta agt atg gaa ttt tct ctc act gat cca aga ttt gag cca cat 623Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu Pro His195 200 205aaa tgg gaa aaa tac tgc gtt cta gaa att gga gac atg ctc cta agg 671Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg210 215 220act gct gta ggt caa gtg tca aga ccc atg ttt ttg tat gta agg aca 719Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val Arg Thr225 230 235aat gga acc tct aaa att aaa atg aaa cgg gga atg gaa atg aga cgc 767Asn Gly Thr Ser Lys Ile Lys Met Lys Arg Gly Met Glu Met Arg Arg240 245 250 255tgc ctc ctt cag tct ctg caa cag att gaa agc atg atc gaa gct gag 815Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu260 265 270tcc tca gtc aaa gaa aag gac atg acc aaa gaa ttc ttt gag aac aaa 863Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn Lys275 280 285tca gag aca tgg cct ata gga gag tcc ccc aaa gga gtg gaa gag ggc 911Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu Gly290 295 300tca atc ggg aag gtt tgc agg acc tta tta gca aaa tct gtg ttt aac 959Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn305 310 315agt ttg tat gca tct cca caa ctg gaa ggg ttt tca gct gaa tct agg 1007Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg320 325 330 335aaa tta ctt ctc att gtt cag gcc ctt agg gat aac ctg gaa cct gga 1055Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly340 345 350acc ttt gat att ggg ggg tta tat gaa tca att gag gag tgc ctg att 1103Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu Ile355 360 365aat gat ccc tgg gtt ttg ctc aat gca tct tgg ttc aac tcc ttc ctt 1151Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu370 375 380aca cat gca ctg aag tagttgtagc aatgctacta tttgctatcc atactgtcca 1206Thr His Ala Leu Lys385aaaaagtact cgagccccca ag 122877388PRTEquine influenza virus H3N8 77Gly Gly Tyr Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu Ala Glu1 5 10 15Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys Asn Met20 25 30Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Ala Pro35 40 45Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu Lys Gln50 55 60Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp Ile Gln65 70 75 80Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp Ile Glu85 90 95Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile Ala Ser100 105 110Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala Thr115 120 125Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn Ala130 135 140Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile Ser Lys145 150 155 160Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe Ile165 170 175Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val Asn Phe180 185 190Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu Pro His Lys195 200 205Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg Thr210 215 220Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val Arg Thr Asn225 230 235 240Gly Thr Ser Lys Ile Lys Met Lys Arg Gly Met Glu Met Arg Arg Cys245 250 255Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu Ser260 265 270Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn Lys Ser275 280 285Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu Gly Ser290 295 300Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn Ser305 310 315 320Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg Lys325 330 335Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly Thr340 345 350Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu Ile Asn355 360 365Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu Thr370 375 380His Ala Leu Lys385781164DNAEquine influenza virus H3N8 78ggcgggtacc caaactatct ccaagcttgg aagcaagtat tagcagaact acaagacctt 60gagaacgaag aaaagacccc taagaccaag aatatgaaaa aaacaagcca attgaaatgg 120gcactcggtg aaaatatggc accagagaaa gtggattttg aggattgtaa agacatcaat 180gatttgaaac agtatgacag tgatgagcca gaaacaaggt ctcttgcaag ttggattcaa 240agtgagttca acaaagcttg tgagctgaca gattcaagct ggatagagct cgatgaaatt 300ggggaggata ttgccccaat agaatacatt gcgagcatga ggagaaatta ttttactgct 360gaggtttccc attgtagagc aacagaatat ataatgaagg gagtgtacat caacactgct 420ctactcaatg catcctgtgc tgcgatggat gaattccaat taattccgat gataagcaaa 480tgcaggacca aagaagggag aaggaagaca aatttatatg gattcataat aaagggaagg 540tcccatttaa ggaatgatac cgacgtggta aactttgtaa gtatggaatt ttctctcact 600gatccaagat ttgagccaca taaatgggaa aaatactgcg ttctagaaat tggagacatg 660ctcctaagga ctgctgtagg tcaagtgtca agacccatgt ttttgtatgt aaggacaaat 720ggaacctcta aaattaaaat gaaacgggga atggaaatga gacgctgcct ccttcagtct 780ctgcaacaga ttgaaagcat gatcgaagct gagtcctcag tcaaagaaaa ggacatgacc 840aaagaattct ttgagaacaa atcagagaca tggcctatag gagagtcccc caaaggagtg 900gaagagggct caatcgggaa ggtttgcagg accttattag caaaatctgt gtttaacagt 960ttgtatgcat ctccacaact ggaagggttt tcagctgaat ctaggaaatt acttctcatt 1020gttcaggccc ttagggataa cctggaacct ggaacctttg atattggggg gttatatgaa 1080tcaattgagg agtgcctgat taatgatccc tgggttttgc tcaatgcatc ttggttcaac 1140tccttcctta cacatgcact gaag 1164791223DNAEquine influenza virus H3N8 79ggggcgggta cccaaactat ctccaagctt ggaagcaagt attagcagaa ctacaagacc 60ttgagaacga agaaaagacc cctaagacca agaatatgaa aaaaacaagc caattgaaat 120gggcactcgg tgaaaatatg gcaccagaga aagtggattt tgaggattgt aaagacatca 180atgatttgaa acagtatgac agtgatgagc cagaaacaag gtctcttgca agttggattc 240aaagtgagtt caacaaagct tgtgagctga cagattcaag ctggatagag ctcgatgaaa 300ttggggagga tattgcccca atagaataca ttgcgagcat gaggagaaat tattttactg 360ctgaggtttc ccattgtaga gcaacagaat atataatgaa gggagtgtac atcaacactg 420ctctactcaa tgcatcctgt gctgcgatgg atgaattcca attaattccg atgataagca 480aatgcaggac caaagaaggg agaaggaaga caaatttata tggattcata ataaagggaa 540ggtcccattt aaggaatgat accgacgtgg taaactttgt aagtatggaa ttttctctca 600ctgatccaag atttgagcca cataaatggg aaaaatactg cgttctagaa attggagaca 660tgctcctaag gactgctgta ggtcaagtgt caagacccat gtttttgtat gtaaggacaa 720atggaacctc taaaattaaa atgaaatggg gaatggaaat gagacgctgc ctccttcagt 780ctctgcaaca gattgaaagc atgatcgaag ctgagtcctc agtcaaagaa aaggacatga 840ccaaagaatt ctttgagaac aaatcagaga catggcctat aggagagtcc cccaaaggag 900tggaagaggg ctcaatcggg aaggtttgca ggaccttatt agcaaaatct gtgtttaaca 960gtttgtatgc atctccacaa ctggaagggt tttcagctga atctaggaaa ttacttctca 1020ttgttcaggc ccttagggat aacctggaac ctggaacctt tgatattggg gggttatatg 1080aatcaattga ggagtgcctg attaatgatc cctgggtttt gctcaatgca tcttggttca 1140actccttcct tacacatgca ctgaagtagt tgtagcaatg ctactatttg ctatccatac 1200tgtccaaaaa agtactcgag ccc 1223801233DNAEquine influenza virus H3N8CDS(3)..(1172) 80at gaa aag ggt ata aac cca aac tat ctc caa gct tgg aag caa gta 47Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val1 5 10 15tta gca gaa cta caa gac ctt gag aac gaa gaa aag acc cct aag acc 95Leu Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr20 25 30aag aat atg aaa aaa aca agc caa ttg aaa tgg gca ctc ggt gaa aat 143Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn35 40 45atg gca cca gag aaa gtg gat ttt gag gat tgt aaa gac atc aat gat 191Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp50 55 60ttg aaa cag tat gac agt gat gag cca gaa aca agg tct ctt gca agt 239Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser65 70 75tgg att caa agt gag ttc aac aaa gct tgt gag ctg aca gat tca agc 287Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser80 85 90 95tgg ata gag ctc gat gaa att ggg gag gat att gcc cca ata gaa tac 335Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr100 105 110att gcg agc atg agg aga aat tat ttt act gct gag gtt tcc cat tgt 383Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys115 120 125aga gca aca gaa tat ata atg aag gga gtg tac atc aac act gct cta 431Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu130 135 140ctc aat gca tcc tgt gct gcg atg gat gaa ttc caa tta att ccg atg 479Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met145 150 155ata agc aaa tgc agg acc aaa gaa ggg aga agg aag aca aat tta tat 527Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr160 165 170 175gga ttc ata ata aag gga agg tcc cat tta agg aat gat acc gac gtg 575Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val180 185 190gta aac ttt gta agt atg gaa ttt tct ctc act gat cca aga ttt gag 623Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu195 200 205cca cat aaa tgg gaa aaa tac tgc gtt cta gaa att gga gac atg ctc 671Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu210 215 220cta agg act gct gta ggt caa gtg tca aga ccc atg ttt ttg tat gta 719Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val225 230 235agg aca aat gga acc tct aaa att aaa atg aaa tgg gga atg gaa atg 767Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met240 245 250 255aga cgc tgc ctc ctt cag tct ctg caa cag att gaa agc atg atc gaa 815Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu260 265 270gct gag tcc tca gtc aaa gaa aag gac atg acc aaa gaa ttc ttt gag 863Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu275 280 285aac aaa tca gag aca tgg cct ata gga gag tcc ccc aaa gga gtg gaa 911Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu290 295 300gag ggc tca atc ggg aag gtt tgc agg acc tta tta gca aaa tct gtg 959Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu Leu

Ala Lys Ser Val305 310 315ttt aac agt ttg tat gca tct cca caa ctg gaa ggg ttt tca gct gaa 1007Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu320 325 330 335tct agg aaa tta ctt ctc att gtt cag gcc ctt agg gat aac ctg gaa 1055Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu340 345 350cct gga acc ttt gat att ggg ggg tta tat gaa tca att gag gag tgc 1103Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys355 360 365ctg att aat gat ccc tgg gtt ttg ctc aat gca tct tgg ttc aac tcc 1151Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser370 375 380ttc ctt aca cat gca ctg aag tagttgtagc aatgctacta tttgctatcc 1202Phe Leu Thr His Ala Leu Lys385 390atactgtcca aaaaagtacc ttgtttctac t 123381390PRTEquine influenza virus H3N8 81Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu1 5 10 15Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys20 25 30Asn Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met35 40 45Ala Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu50 55 60Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp65 70 75 80Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp85 90 95Ile Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile100 105 110Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg115 120 125Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu130 135 140Asn Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile145 150 155 160Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly165 170 175Phe Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val180 185 190Asn Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu Pro195 200 205His Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu210 215 220Arg Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val Arg225 230 235 240Thr Asn Gly Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met Arg245 250 255Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala260 265 270Glu Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn275 280 285Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu290 295 300Gly Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe305 310 315 320Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser325 330 335Arg Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro340 345 350Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu355 360 365Ile Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe370 375 380Leu Thr His Ala Leu Lys385 390821170DNAEquine influenza virus H3N8 82gaaaagggta taaacccaaa ctatctccaa gcttggaagc aagtattagc agaactacaa 60gaccttgaga acgaagaaaa gacccctaag accaagaata tgaaaaaaac aagccaattg 120aaatgggcac tcggtgaaaa tatggcacca gagaaagtgg attttgagga ttgtaaagac 180atcaatgatt tgaaacagta tgacagtgat gagccagaaa caaggtctct tgcaagttgg 240attcaaagtg agttcaacaa agcttgtgag ctgacagatt caagctggat agagctcgat 300gaaattgggg aggatattgc cccaatagaa tacattgcga gcatgaggag aaattatttt 360actgctgagg tttcccattg tagagcaaca gaatatataa tgaagggagt gtacatcaac 420actgctctac tcaatgcatc ctgtgctgcg atggatgaat tccaattaat tccgatgata 480agcaaatgca ggaccaaaga agggagaagg aagacaaatt tatatggatt cataataaag 540ggaaggtccc atttaaggaa tgataccgac gtggtaaact ttgtaagtat ggaattttct 600ctcactgatc caagatttga gccacataaa tgggaaaaat actgcgttct agaaattgga 660gacatgctcc taaggactgc tgtaggtcaa gtgtcaagac ccatgttttt gtatgtaagg 720acaaatggaa cctctaaaat taaaatgaaa tggggaatgg aaatgagacg ctgcctcctt 780cagtctctgc aacagattga aagcatgatc gaagctgagt cctcagtcaa agaaaaggac 840atgaccaaag aattctttga gaacaaatca gagacatggc ctataggaga gtcccccaaa 900ggagtggaag agggctcaat cgggaaggtt tgcaggacct tattagcaaa atctgtgttt 960aacagtttgt atgcatctcc acaactggaa gggttttcag ctgaatctag gaaattactt 1020ctcattgttc aggcccttag ggataacctg gaacctggaa cctttgatat tggggggtta 1080tatgaatcaa ttgaggagtg cctgattaat gatccctggg ttttgctcaa tgcatcttgg 1140ttcaactcct tccttacaca tgcactgaag 11708325DNAArtificialSynthetic Primer 83ggggcgggta cccaaactat ctcca 258427DNAArtificialSynthetic Primer 84gggggctcga gtactttttt ggacagt 27851234DNAEquine influenza virus H3N8CDS(1)..(1188) 85atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc att gat 48Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp1 5 10 15ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag ata cga 96Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg20 25 30cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atg atg 144Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met35 40 45gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac 192Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn50 55 60ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg 240Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu65 70 75 80caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa 288Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu85 90 95gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc 336Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val100 105 110ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc aca 384Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr115 120 125ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc 432Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe130 135 140agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca 480Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala145 150 155 160gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aat aat 528Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn165 170 175gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc att aag 576Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag ata caa 624Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln195 200 205acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cga tca 672Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser210 215 220aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aac atc 720Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile225 230 235 240aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg atg gat 768Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp245 250 255gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc gtt agc 816Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser260 265 270cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct gcg cat 864His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His275 280 285ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca cac tct 912Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser290 295 300tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa agg gga 960Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly305 310 315 320ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg ttt gaa 1008Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu325 330 335aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att tct agt 1056Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser340 345 350atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga att gac 1104Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp355 360 365ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc atg aag 1152Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys370 375 380atc tgt tcc acc att gaa gag ctc aga cgg caa aaa tagtgaattt 1198Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395agcttgatct tcgtgaaaaa atgccttgtt tctact 123486396PRTEquine influenza virus H3N8 86Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp1 5 10 15Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg20 25 30Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met35 40 45Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn50 55 60Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu65 70 75 80Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu85 90 95Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val100 105 110Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr115 120 125Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe130 135 140Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala145 150 155 160Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn165 170 175Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln195 200 205Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser210 215 220Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile225 230 235 240Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp245 250 255Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser260 265 270His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His275 280 285Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser290 295 300Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly305 310 315 320Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu325 330 335Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser340 345 350Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp355 360 365Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys370 375 380Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395871188DNAEquine influenza virus H3N8 87atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgc 600catagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188881240DNAEquine influenza virus H3N8CDS(8)..(1195) 88caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 49Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser1 5 10att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys15 20 25 30ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met35 40 45atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile50 55 60tta aac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp65 70 75ggt ctg caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn80 85 90cat gaa gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa 337His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys95 100 105 110ctg gtc ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr115 120 125ggc tca ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433Gly Ser Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala130 135 140aat ttc agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu145 150 155tct gca gac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile160 165 170aat aat gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe175 180 185 190att aag gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag 625Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln195 200 205ata caa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr210 215 220cga tca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr225 230 235aac atc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu240 245 250atg gat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe255 260 265 270gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865Val Ser His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro275 280 285gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913Ala His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr290 295 300cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961His Ser Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln305 310 315agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu320 325 330ttt gaa aaa ttc ttc ccc agc agc tca tac aga

aga cca gtc gga att 1057Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile335 340 345 350tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg355 360 365att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc 1153Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile370 375 380atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt ctact 124089396PRTEquine influenza virus H3N8 89Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp1 5 10 15Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg20 25 30Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met35 40 45Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn50 55 60Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu65 70 75 80Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu85 90 95Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val100 105 110Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Ser115 120 125Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe130 135 140Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala145 150 155 160Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn165 170 175Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln195 200 205Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser210 215 220Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile225 230 235 240Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp245 250 255Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser260 265 270His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His275 280 285Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser290 295 300Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly305 310 315 320Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu325 330 335Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser340 345 350Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp355 360 365Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys370 375 380Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395901188DNAEquine influenza virus H3N8 90atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360tcctacataa atagaaccgg ctcattcgaa ttcacaagct ttttctaccg gtatggtttt 420gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgc 600catagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188911241DNAEquine influenza virus H3N8CDS(8)..(1195) 91caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 49Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser1 5 10att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys15 20 25 30ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met35 40 45atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile50 55 60tta aac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp65 70 75ggt ctg caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn80 85 90cat gaa gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa 337His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys95 100 105 110ctg gtc ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr115 120 125ggc aca ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433Gly Thr Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala130 135 140aat ttc agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu145 150 155tct gca gac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile160 165 170aat aat gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe175 180 185 190att aag gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag 625Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln195 200 205ata caa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr210 215 220cga tca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr225 230 235aac atc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu240 245 250atg gat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe255 260 265 270gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865Val Ser His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro275 280 285gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913Ala His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr290 295 300cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961His Ser Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln305 310 315agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu320 325 330ttt gaa aaa ttc ttc ccc agc agc tca tac aga aaa cca gtc gga att 1057Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Lys Pro Val Gly Ile335 340 345 350tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg355 360 365att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc 1153Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile370 375 380atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt tctact 124192396PRTEquine influenza virus H3N8 92Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp1 5 10 15Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg20 25 30Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met35 40 45Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn50 55 60Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu65 70 75 80Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu85 90 95Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val100 105 110Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr115 120 125Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe130 135 140Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala145 150 155 160Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn165 170 175Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln195 200 205Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser210 215 220Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile225 230 235 240Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp245 250 255Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser260 265 270His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His275 280 285Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser290 295 300Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly305 310 315 320Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu325 330 335Lys Phe Phe Pro Ser Ser Ser Tyr Arg Lys Pro Val Gly Ile Ser Ser340 345 350Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp355 360 365Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys370 375 380Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395931188DNAEquine influenza virus H3N8 93atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgt 600caaagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020agcagctcat acagaaaacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188941241DNAEquine influenza virus H3N8CDS(8)..(1195) 94caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 49Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser1 5 10att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys15 20 25 30ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met35 40 45atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile50 55 60tta aac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp65 70 75ggt ctg caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn80 85 90cat gaa gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa 337His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys95 100 105 110ctg gtc ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr115 120 125ggc aca ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433Gly Thr Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala130 135 140aat ttc agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu145 150 155tct gca gac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile160 165 170aat aat gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe175 180 185 190att aag gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag 625Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln195 200 205ata caa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr210 215 220cga tca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr225 230 235aac atc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu240 245 250atg gat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe255 260 265 270gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865Val Ser His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro275 280 285gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913Ala His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr290 295 300cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961His Ser Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln305 310 315agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu320 325 330ttt gaa aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att 1057Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile335 340 345 350tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg355 360 365att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc

1153Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile370 375 380atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt tctact 124195396PRTEquine influenza virus H3N8 95Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp1 5 10 15Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg20 25 30Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met35 40 45Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn50 55 60Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu65 70 75 80Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu85 90 95Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val100 105 110Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr115 120 125Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe130 135 140Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala145 150 155 160Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn165 170 175Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys180 185 190Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln195 200 205Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser210 215 220Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile225 230 235 240Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp245 250 255Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser260 265 270His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His275 280 285Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser290 295 300Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly305 310 315 320Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu325 330 335Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser340 345 350Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp355 360 365Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys370 375 380Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys385 390 395961188DNAEquine influenza virus H3N8 96atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgt 600caaagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 11889720DNAArtificialSynthetic Primer 97taaatagaac cggcacattc 209817DNAArtificialSynthetic Primer 98caaagaaatt gaatcag 179918DNAArtificialSynthetic Primer 99caagcattac tactgcac 1810019DNAArtificialSynthetic Primer 100agtctgttcc cacagtttc 1910120DNAArtificialSynthetic Primer 101gaattcgaat gtgccggttc 2010220DNAArtificialSynthetic Primer 102aaaacaagga ttttttcacg 201032341DNAEquine influenza virus H3N8CDS(25)..(2295)misc_feature(1489)..(1489)At nucleotide 1489, w = a or t/u At amino acid residue 489, Xaa = Thr or Ser 103agcaaaagca ggcaaactat ttga atg gat gtc aat ccg act cta ctc ttc 51Met Asp Val Asn Pro Thr Leu Leu Phe1 5tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct tat act 99Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr10 15 20 25gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc atg gat 147Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp30 35 40act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg aca aca 195Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr45 50 55aac act gag att gga gca cca caa ctt aat cca atc gat gga ccg ctt 243Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu60 65 70cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt gta ttg 291Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu75 80 85gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt gaa aat 339Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn90 95 100 105tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg gac aaa 387Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Lys110 115 120cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg aat caa 435Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln125 130 135cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga tca aat 483Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn140 145 150gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc aaa gat 531Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu Lys Asp155 160 165gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca cac ttc 579Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr His Phe170 175 180 185caa cgg aag aga aga gta aga gac aac atg aca aag aga atg gtg aca 627Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met Val Thr190 195 200cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aag agc tat 675Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys Ser Tyr205 210 215ctg atc agg gca tta acc tta aac aca atg acc aag gac gct gag aga 723Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala Glu Arg220 225 230ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cag ata aga 771Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln Ile Arg235 240 245ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgt gaa aag 819Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys Glu Lys250 255 260 265ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa aag gcc aaa 867Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys Ala Lys270 275 280ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa gac act gaa 915Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp Thr Glu285 290 295ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat gaa aat cag 963Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn Gln300 305 310aac cca cgc atg ttc ctg gca atg atc aca tac ata act aga aac cag 1011Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Arg Asn Gln315 320 325cca gaa tgg ttc aga aat gtt cta agc att gca ccg att atg ttc tca 1059Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile Met Phe Ser330 335 340 345aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa agc aaa agt 1107Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys Ser350 355 360atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc att gat 1155Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp365 370 375ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag ata cga 1203Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg380 385 390cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atg atg 1251Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met395 400 405gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac 1299Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn410 415 420 425ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg 1347Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu430 435 440caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa 1395Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu445 450 455gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc 1443Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val460 465 470ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc wca 1491Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Xaa475 480 485ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc 1539Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe490 495 500 505agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca 1587Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala510 515 520gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aat aat 1635Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn525 530 535gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc att aag 1683Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys540 545 550gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag ata caa 1731Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln555 560 565acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cga tca 1779Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser570 575 580 585aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aac atc 1827Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile590 595 600aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg atg gat 1875Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp605 610 615gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc gtt agc 1923Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser620 625 630cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct gcg cat 1971His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His635 640 645ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca cac tct 2019Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser650 655 660 665tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa agg gga 2067Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly670 675 680ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg ttt gaa 2115Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu685 690 695aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att tct agt 2163Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser700 705 710atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga att gac 2211Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp715 720 725ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc atg aag 2259Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys730 735 740 745atc tgt tcc acc att gaa gag ctc aga cgg caa aaa tagtgaattt 2305Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys750 755agcttgatct tcgtgaaaaa atgccttgtt tctact 2341104757PRTEquine influenza virus H3N8misc_feature(489)..(489)The 'Xaa' at location 489 stands for Thr, or Ser. 104Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser145 150 155 160Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser370 375 380Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser405 410 415Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr420 425 430Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala435 440 445Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Arg Thr Gly Xaa Phe Glu Phe Thr Ser Phe Phe485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Met Thr515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala530 535 540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg

Ser Phe Glu Leu565 570 575Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val625 630 635 640Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys725 730 735Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu740 745 750Leu Arg Arg Gln Lys7551052271DNAEquine influenza virus H3N8 105atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacttctggt cgatgggact 1200gcttcactga gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtactaggt 1260gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320ctgcaatcat ccgatgattt tgctttgata gtgaatgcgc ctaatcatga aggaatacag 1380gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440aagtcctaca taaatagaac cggcwcattc gaattcacaa gctttttcta ccggtatggt 1500tttgtcgcca atttcagcat ggagctaccc agttttgggg tttccgggat aaatgaatct 1560gcagacatga gcattggaat gacagttatc aaaaacaaca tgataaataa tgatctcggt 1620cccgccacgg cacaaatggc actccaactc ttcattaagg attatcggta cacataccgg 1680tgccatagag gcgataccca gatacaaacc agaagatcct ttgagttgaa gaaactgtgg 1740gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatacaac 1800atcagaaacc tacacatccc ggaagtctgt ttgaaatggg agctgatgga tgaagattat 1860aaagggaggc tatgtaatcc attgaatcct ttcgttagcc acaaagaaat tgaatcagtg 1920aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980gcaacaacac actcttggat ccccaagagg aaccggtcca tattgaacac aagtcaaagg 2040ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100cccagcagct catacagaag accagtcgga atttctagta tggttgaggc catggtgtcc 2160agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa a 22711062341DNAEquine influenza virus H3N8CDS(25)..(2295)misc_feature(2144)..(2144)At nucleotide 2144, r = a or g At amino acid residue 707, Xaa = Arg or Lys 106agcaaaagca ggcaaactat ttga atg gat gtc aat ccg act cta ctc ttc 51Met Asp Val Asn Pro Thr Leu Leu Phe1 5tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct tat act 99Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr10 15 20 25gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc atg gat 147Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp30 35 40act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg aca aca 195Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr45 50 55aac act gag att gga gca cca caa ctt aat cca atc gat gga ccg ctt 243Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu60 65 70cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt gta ttg 291Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu75 80 85gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt gaa aat 339Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn90 95 100 105tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg gac aaa 387Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Lys110 115 120cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg aat caa 435Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln125 130 135cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga tca aat 483Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn140 145 150gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc aaa gat 531Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu Lys Asp155 160 165gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca cac ttc 579Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr His Phe170 175 180 185caa cgg aag aga aga gta aga gac aac atg aca aag aga atg gtg aca 627Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met Val Thr190 195 200cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aag agc tat 675Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys Ser Tyr205 210 215ctg atc agg gca tta acc tta aac aca atg acc aag gac gct gag aga 723Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala Glu Arg220 225 230ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cag ata aga 771Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln Ile Arg235 240 245ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgt gaa aag 819Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys Glu Lys250 255 260 265ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa aag gcc aaa 867Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys Ala Lys270 275 280ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa gac act gaa 915Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp Thr Glu285 290 295ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat gaa aat cag 963Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn Gln300 305 310aac cca cgc atg ttc ctg gca atg atc aca tac ata act aga aac cag 1011Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Arg Asn Gln315 320 325cca gaa tgg ttc aga aat gtt cta agc att gca ccg att atg ttc tca 1059Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile Met Phe Ser330 335 340 345aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa agc aaa agt 1107Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys Ser350 355 360atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc att gat 1155Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp365 370 375ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag ata cga 1203Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg380 385 390cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atg atg 1251Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met395 400 405gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac 1299Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn410 415 420 425ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg 1347Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu430 435 440caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa 1395Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu445 450 455gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc 1443Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val460 465 470ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc aca 1491Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr475 480 485ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc 1539Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe490 495 500 505agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca 1587Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala510 515 520gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aat aat 1635Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn525 530 535gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc att aag 1683Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys540 545 550gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag ata caa 1731Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln555 560 565acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cga tca 1779Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser570 575 580 585aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aac atc 1827Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile590 595 600aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg atg gat 1875Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp605 610 615gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc gtt agc 1923Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser620 625 630cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct gcg cat 1971His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His635 640 645ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca cac tct 2019Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser650 655 660 665tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa agg gga 2067Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly670 675 680ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg ttt gaa 2115Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu685 690 695aaa ttc ttc ccc agc agc tca tac aga ara cca gtc gga att tct agt 2163Lys Phe Phe Pro Ser Ser Ser Tyr Arg Xaa Pro Val Gly Ile Ser Ser700 705 710atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga att gac 2211Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp715 720 725ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc atg aag 2259Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys730 735 740 745atc tgt tcc acc att gaa gag ctc aga cgg caa aaa tagtgaattt 2305Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys750 755agcttgatct tcgtgaaaaa atgccttgtt tctact 2341107757PRTEquine influenza virus H3N8misc_feature(707)..(707)The 'Xaa' at location 707 stands for Arg, or Lys. 107Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu100 105 110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser145 150 155 160Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys165 170 175Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg180 185 190Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys195 200 205Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu245 250 255Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly290 295 300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly340 345 350Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser370 375 380Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser405 410 415Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr420 425 430Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala435 440 445Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Met Thr515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala530 535 540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys Gln Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu565 570 575Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val625 630 635 640Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser690 695 700Tyr Arg Xaa Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys725 730 735Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu740

745 750Leu Arg Arg Gln Lys7551082271DNAEquine influenza virus H3N8 108atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacttctggt cgatgggact 1200gcttcactga gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtactaggt 1260gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320ctgcaatcat ccgatgattt tgctttgata gtgaatgcgc ctaatcatga aggaatacag 1380gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440aagtcctaca taaatagaac cggcacattc gaattcacaa gctttttcta ccggtatggt 1500tttgtcgcca atttcagcat ggagctaccc agttttgggg tttccgggat aaatgaatct 1560gcagacatga gcattggaat gacagttatc aaaaacaaca tgataaataa tgatctcggt 1620cccgccacgg cacaaatggc actccaactc ttcattaagg attatcggta cacataccgg 1680tgtcaaagag gcgataccca gatacaaacc agaagatcct ttgagttgaa gaaactgtgg 1740gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatacaac 1800atcagaaacc tacacatccc ggaagtctgt ttgaaatggg agctgatgga tgaagattat 1860aaagggaggc tatgtaatcc attgaatcct ttcgttagcc acaaagaaat tgaatcagtg 1920aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980gcaacaacac actcttggat ccccaagagg aaccggtcca tattgaacac aagtcaaagg 2040ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100cccagcagct catacagaar accagtcgga atttctagta tggttgaggc catggtgtcc 2160agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa a 2271

Claims

1. A cold-adapted equine influenza virus.

2. The virus of claim 1, wherein said virus replicates in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C.

3. The virus of claim 1, wherein said virus is attenuated.

4. The virus of claim 1, wherein said virus is temperature sensitive.

5. The virus of claim 1, wherein said virus replicates in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C., but does not form plaques in tissue culture cells at a temperature of about 39.degree. C.

6. The virus of claim 1, wherein said virus replicates in embryonated chicken eggs at a temperature ranging from about 26.degree. C. to about 30.degree. C., but does not form plaques in tissue culture cells at a temperature of about 37.degree. C.

7. The virus of claim 1, wherein a phenotype comprising a non-permissive temperature of about 39.degree. C. is conferred on said virus by at least two mutations in the genome of said virus, comprising a first mutation and a second mutation.

8. The virus of claim 7, wherein said first mutation confers a phenotype on said virus comprising inhibition of plaque formation at a temperature of about 39.degree. C., and wherein said first mutation co-segregates with the segment of said genome comprising the nucleoprotein gene of said virus.

9. The virus of claim 7, wherein said second mutation confers a phenotype on said virus comprising inhibition of protein synthesis of said virus at a temperature of about 39.degree. C.

10. The virus of claim 7, further comprising at least one additional mutation, wherein said additional mutation confers a phenotype comprising a non-permissive temperature of about 37.degree. C. on said virus, and wherein said phenotype is selected from the group consisting of inhibition of plaque formation at a temperature of about 37.degree. C. and inhibition of the expression of the late genes of said virus at a temperature of about 37.degree. C.

11. The virus of claim 1, wherein said virus is produced by a method comprising the steps of: a. passaging a wild-type equine influenza virus; and b. selecting viruses that grow at a reduced temperature.

12. The virus of claim 11, wherein said virus is produced by a method further comprising repetition of said passaging and selection steps one or more times, wherein said reduced temperature is made progressively lower.

13. The virus of claim 11, wherein said passaging step is carried out in embryonated chicken eggs.

14. The virus of claim 11, wherein said virus comprises a dominant interference phenotype.

15. The virus of claim 1, wherein said virus is derived from strain A/equine/Kentucky/1/91 (H3N8).

16. The virus of claim 1, wherein said virus comprises the identifying phenotypes of a virus selected from the group consisting of: EIV-P821, identified by accession No. ATCC VR-2625 EIV-P824, identified by accession No. ATCC VR-2624 and MSV+5, identified by accession No. ATCC VR-2627.

17. The virus of claim 1, wherein said virus is selected from the group consisting of: EIV-P821, identified by accession No. ATCC VR-2625; EIV-P824, identified by accession No. ATCC VR-2624; MSV+5, identified by accession No. ATCC VR-2627; and progeny of any of said viruses having any of said accession numbers.

18. A reassortant influenza A virus comprising at least one genome segment of an equine influenza virus generated by cold-adaptation, said equine influenza virus having an identifying phenotype selected from the group consisting of cold-adaptation, temperature sensitivity, dominant interference, and attenuation, wherein said equine influenza virus genome segment confers at least one of said identifying phenotypes to said reassortant virus.

19. The reassortant influenza A virus of claim 18, wherein said virus is produced by a method comprising the steps of: a. mixing the genome segments of a donor cold-adapted equine influenza virus with the genome segments of a recipient influenza A virus; and b. selecting a reassortant virus comprising at least one phenotype of said donor equine influenza virus, wherein said phenotype is selected from the group consisting of cold-adaptation, temperature sensitivity, dominant interference, and attenuation.

20. The reassortant influenza A virus of claim 18, wherein said recipient influenza A virus comprises hemagglutinin and neuraminidase phenotypes different than those of said donor equine influenza virus, and wherein said reassortant virus comprises the hemagglutinin and neuraminidase phenotypes of said recipient virus.

21. A therapeutic composition to protect an animal against disease caused by an influenza A virus, comprising a virus selected from the group consisting of: (a) a cold-adapted equine influenza virus; and (b) a reassortant influenza A virus comprising at least one genome segment of an equine influenza virus generated by cold-adaptation, said equine influenza virus having an identifying phenotype selected from the group consisting of cold-adaptation, temperature sensitivity, dominant interference, and attenuation, wherein said equine influenza virus genome segment confers at least one of said identifying phenotypes to said reassortant virus.

22. The therapeutic composition of claim 21, wherein said therapeutic composition comprises a cold-adapted equine influenza virus, wherein said disease is caused by equine influenza virus, and wherein said therapeutic composition is administered prophylactically to an equid, thereby eliciting an immune response against equine influenza virus in said equid.

23. The therapeutic composition of claim 21, wherein said therapeutic composition comprises from about 10.sup.5 TCID.sub.50 units to about 10.sup.8 TCID.sub.50 units of said virus.

24. The therapeutic composition of claim 21, further comprising an excipient.

25. A method to protect an animal against disease caused by an influenza A virus comprising administering to said animal a therapeutic composition comprising a virus selected from the group consisting of: (a) a cold-adapted equine influenza virus; and (b) a reassortant influenza A virus comprising at least one genome segment of an equine influenza virus generated by cold-adaptation, said equine influenza virus having an identifying phenotype selected from the group consisting of cold-adaptation, temperature sensitivity, dominant interference, and attenuation, wherein said equine influenza virus genome segment confers at least one of said identifying phenotypes to said reassortant virus.

26. The method of claim 25, wherein said animal is an equid.

27. The method of claim 25, wherein said therapeutic composition comprises a cold-adapted equine influenza virus, wherein said disease is caused by equine influenza virus, and wherein said therapeutic composition is administered prophylactically to an equid, thereby eliciting an immune response against equine influenza virus in said equid.

28. The method of claim 25, wherein said therapeutic composition is administered to said animal by a route that will allow virus entry into mucosal cells of the upper respiratory tract.

29. A method to produce a cold-adapted equine influenza virus comprising the steps of: a. passaging a wild-type equine influenza virus; and b. selecting viruses that grow at a reduced temperature.

30. The method of claim 29, wherein said cold-adapted equine influenza virus is produced by a method further comprising repetition of said passaging and selection steps one or more times, wherein said reduced temperature is made progressively lower.

31. A method to produce a reassortant influenza A virus having at least one genome segment of an equine influenza virus generated by cold-adaptation, said equine influenza virus having an identifying phenotype selected from the group consisting of cold-adaptation, temperature sensitivity, dominant interference, and attenuation, comprising the steps of: a. mixing the genome segments of a donor cold-adapted equine influenza virus with the genome segments of a recipient influenza A virus; and b. selecting reassortant a virus comprising at least one phenotype of said donor equine influenza virus, wherein said phenotype is selected from the group consisting of cold-adaptation, temperature sensitivity, dominant interference, and attenuation.

32. The method of claim 31, wherein said recipient influenza A virus comprises hemagglutinin and neuraminidase phenotypes different than those of said donor equine influenza virus, and wherein said reassortant virus comprises the hemagglutinin and neuraminidase phenotypes of said recipient virus.

33. A method to propagate a cold-adapted equine influenza virus comprising a method selected from the group consisting of propagating said virus in eggs and propagating said virus in tissue culture cells.

34. An isolated equine influenza nucleic acid molecule selected from the group consisting of: a. an isolated nucleic acid molecule that encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107; and b. an isolated nucleic acid molecule fully complementary to a nucleic acid molecule of (a).

35. The nucleic acid molecule of claim 34, wherein said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of said nucleic acid sequences.

36. A nucleic acid molecule of claim 34, wherein said nucleic acid molecule encodes a protein.

37. A nucleic acid molecule of claim 34, wherein said nucleic acid molecule is selected from the group consisting of Pei.sub.ca1M.sub.252, Pei.sub.ca1HA.sub.565, Pei.sub.ca1PB2-N.sub.404, Pei.sub.ca1PB2-C.sub.398, Pei.sub.ca1PB2.sub.759, Pei.sub.ca1NS.sub.230, Pei.sub.ca1PB1-N.sub.395, Pei.sub.ca1PA-C.sub.390 Pei.sub.ca1PB1-C.sub.396, Pei.sub.ca2PB1-C.sub.396 and Pei.sub.ca1PB1.sub.757.

38. An isolated equine influenza protein that comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107.

39. The protein of claim 38, wherein said protein is encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108.

40. An isolated virus comprising a nucleic acid molecule that encodes a protein selected from the group consisting SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107.

41. The virus of claim 40, wherein said virus comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108.

42. The virus of claim 40, wherein said virus is selected from the group consisting of equine influenza virus and a reassortant influenza virus.

43. The virus of claim 1, wherein said virus comprises a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:106 and SEQ ID NO:108.

44. The virus of claim 1, wherein said virus encodes a protein selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:107.

45. The virus of claim 18, wherein said virus comprises a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:106 and SEQ ID NO:108.

46. The virus of claim 18, wherein said virus encodes a protein selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:107.