CA2339089C - Cold-adapted equine influenza viruses - Google Patents

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 viru s, 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 equin e 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 respons e in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recentl y 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 influenz a virus generated by cold-adaption.

Description

2 PCT/US99/185~3 COLD-ADAPTED EQUINE INFLUENZA VIRUSES FIELD OF THE INVENTION

The present invention relates to experimentally-generated cold-adapted equ~ne influenza viruses, and particularly to cold-adapted equine influenza viruses having additional phenotypes, such as attenuation, dominant interference, or temperature II
sensitivity. The invention also includes reassortant influenza A viruses which cont~in at least one genome segment from such an equine influenza virus, such that the reassoikant virus includes certain phenotypes of the donor equine influenza virus. The inventio'n 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 tq the use of these viruses in therapeutic compositions to protect animals from diseases caulsed by influenza viruses.
BACKGROUND OF THE INVENTION

Equine influenza virus has been recognized as a major respiratory pathogen in horses since about 1956. Disease symptoms caused by equine influenza virus can bel 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 yea rs.
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 canno 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 need for safe, effective therapeutic compositions to protect horses against equine influenza disease.

I'I

Production of therapeutic compositions comprising cold-adapted human influenza viruses is described, for example, in Maassab, et al., 1960, Nature 7,6I2614, and Maassab, e't 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 virug is temperature sensitive; that is, the virus does not grow well at certain higher, non-Penmissive temperatures at which the wild-type virus will grow and replicate.
Variolltts cold-adapted huinan influenza A viruses, produced by reassortment with existing cold-adapted human influenza A viruses, have been shown to elicit good immune respons~s 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. Patent Iolo 5,149,531, by Youngner, et al., issued September 22, 1992, the inventors of the presez~t invention further demonstrated that certain reassortant cold-adapted human influenza viruses also possess a dominant interference phenotype, i.e., they inhibit the growth of their corresponding parental wild-type strain, as well as heteralogous influenza A
viruses. U.S. Patent No. 4,683,137, by Coggins et al., issued July 28, 1987, andll U.S. Patent No. 4,693,893, by Campbell, issued September 15, 1987, disclose attenuat6d 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 ap significant danger of introducing into the environment a virus containing both human and equine influenza genes.
SUMMARY OF THE INVENTION

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 _

3 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.
In one embodiment, the invention is an isolated cold-adapted equine influenza virus that replicates in embryonated chicken eggs at a temperature ranging from about 26 C to about 30 C. The virus is produced using a method comprising (a) obtaining a wild-type, equine influenza virus, (b) passaging the wild-type equine influenza virus at progressively lower temperatures, and (c) selecting a virus that grows at the lowered temperature.
In another embodiment, the invention is a reassortant cold-adapted equine influenza A virus that grows at a temperature lower than about 34 C and replicates in embryonated chicken eggs at a temperature ranging from about 26 C to about 30 C.
The reassortant virus comprises: (a) at least one genome segment of a donor cold-adapted equine influenza virus generated by cold-adaptation using a method that includes: (i) obtaining a wild-type, equine influenza virus, (ii) passaging the wild-type equine influenza virus at progressively lower temperatures, and (iii) selecting a virus that grows at the lowered temperature; and (b) at least one genome segment of a recipient influenza A virus having an identifying phenotype selected from the group consisting of hemagglutinin activity and neuraminidase activity, and combinations thereof, wherein the influenza A virus genome segment confers at least one of the identifying phenotypes to the reassortant virus.
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.

{E5333764.DOC;1 }

3a A cold-adapted equine influenza virus is one that replicates in embryonated chicken eggs at a temperature ranging from about 26 C to about 30 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.
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 C to about 30 C, forms plaques in tissue culture cells at a permissive temperature of about 34 C, but does not form plaques in tissue culture cells at a non-permissive temperature of about 39 C.
In one embodiment, such a temperature sensitive virus comprises two mutations: a first mutation that inhibits plaque formation at a temperature of about 39 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 C.
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 C to about 30 C, forms plaques in tissue culture cells at a {E5333764.DOC;1 }

4 permissive temperature of about 34 C, but does not form plaques in tissue culture cells or express late viral proteins at a non-permissive temperature of about 37 C.
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.
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-2627, and progeny of such viruses. A progeny is a descendant virus arising from any of the above viruses identified by accession numbers.
Therapeutic compositions of the present invention include from about 105 TCID50 units to about 108 TCID50 units, and preferably about 2 x 106 TCID50 units, of a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention.
The present invention also includes a method to protect an animal from disease caused by an influenza A virus, which includes the administration 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.
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.
Another embodiment is a method to produce a reassortant influenza A virus through genetic reassortment of the genome segments of a donor cold-adapted equine {E5333764.DOC;1 }

WO 00/09702 PC11US99/185~3

-5-influenza virus of the present invention with the genome segments of a recipient influenza A vinis. Reassortant influenza A viruses of the present invention are prod' uced by a method that includes the steps of: (a) mixing the genome segments of a donor c'old-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 tl~e 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 reassortar,it 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.

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.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides experimentally-generated cold-adapted equine influenza viruses comprising certain defined phenotypes, which are disclosed herein. ~t 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-adapteo equine influenza viruses. As such, the terms "a" (or "an"), "one or more," and "at leasti, one" can be used iriterchangeably herein. It is also to be noted that the terms "comprising," "including," and "having" can be used interchangeably.
Furthermore, ar~
item "selected from the group consisting of' refers to one or more of the items in that group, including combinations thereof.
A cold-adapted equine influenza virus of the present invention is a virus that ha ~
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 11 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 PCT/US99/18~83

-6-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 rrus grown in tissue culture cells may be in a mixture with disrupted cells and tissue culiure medium.
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 Ithe ability of the virus to reproduce or "replicate" itself in a permissive host cell. As su~h, the terms, "growth of a vinis" and "replication of a virus" are used interchangeably ii herein. Growth or replication of a virus in a particular host cell can be demonstratedland measured by standard methods well-known to those skilled in the art of virology. Fo~
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 detectertd 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 hemagglutinin (HA) protein in the AF.
Naturally-occuning, i.,e., wild-type, equine influenza viruses replicate well at a temperature from about 34 C to about 39 T. For example, wild-type equine influenzal virus replicates in embryonated chicken eggs at a temperature of about 34 C, and replicates in tissue culture cells at a temperature from about 34 C to about 39 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 C. A preferred cold-adapted equine influenza virus of the present inventior~
replicates in embryonated chicken eggs at a temperature of about 28 C.
Another preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 26 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 C to about 30 C, i.e., at a range of

-7-temperatures at which a wild-type virus will grow poorly or not at all. It should be fi'oted 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 C, but also replicates in tissue culture cells at a temperature of about 34 T. As with wild-'type equine influenza viruses, cold-adap~ed 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 abo ut 34 C. Examples of suitable and preferred cold-adapted equine influenza viruses of the present invention are disclosed herein.

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 examph, 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 i~i comprising the influenza virus genome, which alter the genotype, i.e., the primary nucleotide se uence of those RNA se ents. As used herein, a "mutation"
is an cl ~
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, o~
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 ii 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 ___~

WO 00/09702 PCT/US99/185i3

-8- õ õ mutation,õ a ge:nome, a genotype," or a phenatype refer to one or more, or at le st one mutation, genome, genotype, or phenotype, respectively.
Additional, observable phenotypes in a cold-adapted equine influenza virus rtiay 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.
In one embodiment, a cold-adapted equine influenza virus of the present invention has a plienotype claaracterized by attenuation. A cold-adapted equine influenza virus is "attenuated," when administration of the virus to an equine influenz'a 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, a~n animal administered an attenuated, cold-adapted equine influenza virus of the present invention will display minimal or no, i.e., undetectable, clinical disease signs.
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 nol 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 li il

-9-about 30 C, preferably at about 28 C or about 26 C, and will form plaques in tisoue culture cells at a permissive temperature of about 34 C, but will not form plaques in tissue culture cells at a non-permissive temperature of about 39 C. Other tempera~ure sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30 C, preferably at a,bout 28 C or about 26 C, and will form plaques in tissue culture cells at a permissive temperature of about 34 C, but will not form plaques in tissue culture cells at a non-permissive temperature of about 37 C.
Certain cold-adapted equine influenza viruses of the present invention have dominant interference phenotype; that is, they dominate an irifection when co-infect~d 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/I/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.
Dominant interference of a cold-adapted equine influenza virus having a temperature sensitive phenotype can be measured by standard virological methods. Forl example, separate nionolayers 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 ~......-~..---~----__ ___.~~:.,~~

-In-temperaturc, whilc the wild-type virus is able to form plaques at both the pcrmissive and non-permissive temperatures. Thus it is possible to measure the growth of the wild-type virus irr 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-pctmissivc tcmperature of the wild-type virus in doubly infected cells.
Cold-adapted equine influenza viruses of the present invention are characterized primarily by one or more of the following identifying phenotypes: cold-adaptation, temperature sensittvity, dominant interference, and/or attenuation. As used herein, the phrasc "an equine influenza virus compnses the identifying phenotype(s) of cold-adaptation, tcmpcrature scnsitivity, dominant interference, and/or attenuation" refers to a virus having such a phenotype(s). Examples of such viruses include, hut are not limited to, LIV-I'821. identrt-ed hy accession No. ATCC VR-2(,25, ETV-P824, identified hy accession No. ATCC VR -zr,2a , and E:IV-MSV-+5, identifred by accession No.
ATCC
VR -2627 , as well as EIV-MSVO, EFV, MSV+l, EIV-MSV+2, EIV-MSV4-3, and EIV-I 5 MSV+.1. F'rociuction of such viruscs is described in the examples. For example, cold-adapted cqurnc influenza vrrus EiV-P821 is characterized by, i.e., has the identifying phenotypes of, (a) cold-adaptation, e.g., its ability to replicatc in embryonated chicken egg.s at a tempcraturc of about 26 C; (b) temperature sensitivity, e-g., its inability to form plaques in tissue culture cells and to express late genc products at a non-permissivc tcmperaturc of about 37 "C, and its irrability to form plaques in tissue culture cells and to synthesizc any viral proteins at a nort -permissive temperature of about 39 "C; (c) its attenuation upnn administration to an equine influenza virus-susceptihle animal; and (d) dorninant tnterference, e.g., its ability, when co-infected into a cell with a wild-type influcnza A virus, to interfere wrttt the growth of that wild-type virus.
Similarly, cold-adapted equtne influcnza virus EIV-P824 is charactenzed by (a) cold adaptation, e.g., its ahility to rcplicate in ernbtyonated chicken eggs at a temperature of about 28 "(';

(h) tcmpcranrre sensttrvity, e.I;., its inability to form plaqucs in tissue culture cetls at a rron-permissive temperaturc of about 39 "C; and (c) domittant intetfcrcnce, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with tlre growth of that wild-typc virus. Ln another example, cold-adapted equinc influenza vrnis E1V-MSV+S is charactenzed by (a) cold-adaptation, e.g., its ability to replicate in -11- ~
embryonated chicken eggs at a temperature of about 26 C; (b) temperature sensitiv~ty, e.g., its inability to form plaques in tissue culture cells at a non-permissive temperature of about 39 C; and (c) its attenuation upon administration to an equine influenza virus-susceptible animal.
In certain cases, the RNA segment upon which one or more mutations associ~ted 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 embodimen , one of the two mutations, localized by reassortment analysis as disclosed herein, inhibits, i.e., blocks or prevents, the ability of the virus to forrn plaques in tissue cultu~e cells at a non-pennissive temperature of about 39 C. This mutation co-segregates wil h 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 embodimertt, the second mutation inhibits all protein synthesis at a non-permissive temperature of about 39 C. As such, at the non-pennissive temperature, th''p virus genome is incapable of expressing any viral proteins. Examples of cold-adapted equine influenza viiruses possessing these characteristics are EIV-P821 and EN
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 IA. EIV-MSV+5 was derived by further serial passaging of EIV-P821, as described in Example I E.
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 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 C. An example of a cold-aciapted equine influenza virus possessing these characteristics is EN
P82I. This virus isolate replicates in embryonated chicken eggs at a temperature of about 26 C, and does not form plaques or express any viral proteins at a temperature of li about 39 C. Furtheirmore, EIV-P821 does not form plaques on MDCK cells at a non-permissive temperature of about 37 C, and at this temperature, late gene expression is ___- __~~---~--inhibttcd rn such a way that late proterns are not produced, i c., normal levels of NP
protern are synthcsized, reduccd or undetcctable levels of M 1 or I-iA
proteins are synthesrzetf, and crthanced levels of the polymerase proteins arc synthesized.
Sincc this phenotyPc is tyrificd by differential viral protcin synthesis, it is distinct from the protein synthcsis phenotype seen at a non-permissive remperanrre of about 39 C, which is typified hy the inhibition of synthesis of all viral proteins.
Pursuant tn 37 C'FR 1902 (a-c), cold-adapted equtne influenza i'in-ises, dcsiKnatc.d hercin as F.IV-PR21, and EIV-P824 were deposited with the American 'hype C'ulnrrc ('nllc.cticm (ATCC, IORO1 I Inivcrsity Roulevard, Manassas, VA 201 10-2209) undcr thc t3udapest T rcatv as AT'('C Accesstnn Nos ATY'(' VR-2625, and ATCt' VR-7624, respcctively, on .lulv 11, 1998 ('old-adapted equine influenza virus L-:IV-N4SVI S was deposited with the ATCC as ATCC Accession No AT('.C VR-2627 un Auvust 3, I998 I'ursuant to 37 ('FR 180(,, the deposits are made for a teim ul'at Icast thirty (30) ycars and at least five (5) vears after the most recertt reqrtest for thc furnishinEt (if a sample Of the deposit iras receiced hy the depositor~, Pnrsuant to 37 ('FR 1 SOR (a)(2), rrll restnctinns intpw~ed hy the depositrir nn the "rs-ailahility to the ptrhlic ik,ill he irrevoc.ahlv remove<I irpoti the grantine of th<' patent F'referrcd cold-adapted equtne influenza vrntscs of the present invention have the rdcntrfl'inf; phenutti7tcs of EIV-Pfi21, EIV-P824, and EIV-MSV+5. Particularly prefcrreri crild-adaptcd cqutne rnflucrtza viruses include FTV-P821, E1V-1'$24, ERI-MSV' ~;, and prof;cny of these viruses As us^d hercin, "progeny" are "offspnng," and as such can slightly altered phenotypcs cornpared to the parent virus, but retain iricntifytnF phenotypes nf thc parent vinis, for example, cold-adaptation, temperature scnsitrvtty, domrnartt interfcrence, or attcnuanon- Fnr exantple, cold-adapted equine rnflucnza virus F1V-MS\'+S is a"proFenv" of-cold-adaptec' cqutne influenza vtrIs EIV-PR' I"Prngcriy" also includc reassoriant influenza A viruses that cornprtse onc or more identtfvtng phenotypes of the donor parcnt vtrus.

Reassortant inffucnza A viruses of the present invention are produced by genetic rea~sonment of the penonic segments nf a ctonnr cold-adapted equine influcn7a virus of rhe prescnt invention with the penomc sct'me:irs of a recipient tnfluenza A
virus, and thcn sclectrnu a rcas~,onant x-irus that derives at least one of its cip-ht RNA geriome scgmcnts 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 interferencc. Preferably, reassortant influenza A viruses of the prescnt invention derive S at Ieast the attcnuation phenotype of the donor virus. Methods to isolate reassortant influenza vin-ses are well known to those skilled in the art of virology and are disclosed, for example, in Fields, ct al., 1996, Fields Viroloy,y, 3d ed., [_ippincott-Raven; and Palese, et al_, 1976, .L I'irol., 17, 876-884. Fields, et al., ibid. and Palese, et al., ibid.

A suitable donor equine influenza virus is a cold-adapted equine influenza vinis of the present invcntion, for example, E1V-P821, identified by accession No.
ATCC
VR-N,25, f:IV P924, identified by accession No. ATCC VR -z6?4 , or FIV-MSV-t5, identificci by accession N<t. ATCC VR -201.7 . A suitable recipient influenza A vints can he anothcr cquine influcnza virus, for example a Eurasran subtype 2 equine influenza virus such as A/equinc/Suffolk/89 (H3N8) or a subtype I equirie influenza virus such as A/Prague/I/56 (H7N7). A recipicnt influenza A virus can also he any influenza A virus capabie of forming a reassortant virus with a donor cold-adapted equinc influenza virus.
Examples of such influcnza A viruses include, but are not litniteci to, human influenza viruses such as A/Pucrto Rico/8/34 (H I N I), A/Hong Kong/156/97 (l I5N I), A/Sirigapore/l/57 (H2N2), and A/liong Kong/l/68 (H3N2); swine viruses such as A/Swine/lowa/l5/30 (11IN1); attd avian viruses such as A/mallard/New York/6750/78 (H2N2) and A/chickcn/Hong Kong/258/97 (H5NI). A reassortant virus of the present inventron can include anv combination of donor and recipient gene seginents, as long as the resulting rcassortant virus possesses at Icast one rdentifyinfi phenotype of the donor virus.

One example nf a reassortant vinis of the prescnt invention is a"fi + 2"
reassortant virtrs, in wliich the six "intemal gene segmertts," i.c., those comprising the NP, P13?, f'B 1, Pn, M, and NS genes, are derived front the donor cold-adapted equine influcnza virus genome, and the two "external gene scgmcnts," i.e., ttrose comprising the flA and NA genes, are derivecl from the recipient influenza A virus. A
resultant vints thus prnducecl has the attcnuated, cold-adapted, tentperature sensitive, and/or dominant interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.
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 morl specific mutations, associated with identified cold-adaptation, attenuation, temperatu~e sensitivity, or dominant interference phenotypes, are identified and are introduced back into a wild-type equine influenza virus strain using a reverse genetics approach. Rev 7' genetics entails using RNA polymerase complexes isolated from influenza virus-infeq 'ted cells to transcribe artificial influenza virus genome segments containing the mutation(I 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. Ac~d.
Sci. 87, 3802-3805; and in U.S. Patent No. 5,578,473, by Palese, et al., issued November 26, 1996. This approach allows one skilled in the art to produce additional cold-adapted equine influenza viiruses 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.
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 irivention 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.
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 virag'. 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 mutatilns, where those mutations have been identified to confer a certain identifying phenotypelon 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, sneezi~g, coughing, nasal discharge, rales, 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 manifestatior$s 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 li 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 Directigen0 FLU A test.
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.
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 II

WO 00/09702 PCT/[3S99/18~ 83 -lb-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 comprisin~ a cold-adapted equine influenza virus having a dominant interference phenotype may e effectively administered prior to subsequent exposure for a length of time correspon ~ ing 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-ada~,ted equine influenza virus having a dominant interference phenotype may be effectively II
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, ior example, up to about two days.
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.
Current vaccines available to protect horses against equine influenza virus disease are not effective in protecting young foals, most likely because they cannot II
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.l 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 youngi foals, as young as about 3 months of age, to protect against equine influenza disease without the induction of tolerance.
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 WO 00/09702 PCTlUS99/18 83 viruses of the present invention, one or more reassortant influenza A viru~es, influenza and/or one or more genetically-engineered equine influenza viruses of the present invention. Multivalent therapeutic compositions can include at least two cold-adapt~d equine influenza viruses, e.g.,-against North American subtype-2 virus isolates such'as A/equine/Kentucky/1/91 (HIN8), and Eurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); or one or more subtype-2 virus isolates and a subtype-~
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 vir ~'ts 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 pres~' nt invention can also contain one or more formulations to protect against one or more ot ~ er infectious agents in addition to influenza A virus. Such other infectious agents includ but not limited to: viruses; bacteria; fungi and fungal-related microorganisms; and parasites. Preferable multivalent therapeutic compositions include, but are not limited t, 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.
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 (Df buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of stabilizers include AI/A2 stabilizer, available from Diamond II
Animal Health, Des Moines, IA. 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 WO 00/09702 PCTiUS99/18583 buffers, stabilizers, etc., to which sterile water or saline can be added prior to salts, administration.
A therapeutic composition of the present invention may also include one or more adjuvants or carriers. Adjuvants are typically substances that enhance the immune II
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 piroduce an efficacious vaccine. Furthermore, in many cases known td 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.
Therapeutic compositions of the present invention include an amount of a col~l adapted equine influenza virus that is sufficient to protect an animal from challenge w~th 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 I 05 tissue culture infectious dose-50 (TCIDsa) units of virus to abo~t 108 TCIDso units of virus. As used herein, a"TCIDso unit" is amount of a virus which II
results in cytopathic effect in 50% of those cell cultures infected. Methods to measure and calculate TCIh-SO 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 TCID50 units to about 1' TCIDso 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 x 106 TCID5o units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention.

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 protoc~~ls 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.
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 m'Ly 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.
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 nebuiizer fitted over I!, the nose and mouth of the animal to be vaccinated.
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 II

WO 00109702 PCTNS99/185~3 the treated animal is resistant to the development of disease. In addition, whether efficacy of a theirapeutic composition of the present invention comprising a cold-ada'oted equine influenza virus having a dominant interference phenotype to ameliorate or re4uce disease symptoms in an animal previously inoculated or susceptible to inoculation w~th a virulent, wild-type equine influenza virus can be tested by screening for the reductior~ or absence of disease symptoms in the treated animal.
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 virus~ls that grow at a reduced temperature; (c) repeating the passaging and selection steps onelor 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.
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 viru~, 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 donoil equine influenza virus. Identifying phenotypes to select for include attenuation, cold-adaptation, temperature sensitivity, and dominant interference. Methods to screen for these phenotypes aree, 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.

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 "internai gene segments," i.e., those coding for the NP, PB2, PB l, PA, M, and NS genes, are derived i_~
~_ ~. . ,.~-_---WO 00/09702 PCT/US9911$5.0,3 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.
The present invention includes nucleic acid molecules isolated from equine influenza virus wild type strain A/equine/Kentucky/1/9I (H3N8), and cold-adapted equine influenza virus EIV-P821.
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 I
DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic aci' molecule has been purified.
The preserit 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., I
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 IDNO:7, SEQ IDNO: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, and/or a complement thereof.
Complements are defined as two single strands of nucleic acid in which the nucleotide 'i 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.
Preferred nucleic acid molecules encoding equine influenza M proteins are nei tM1o23, nei,vtIMIo23, nei,vt2MI023, neiNvtMz56, nei,vtiM75G, neiwQMn6, neica1MI023, neic,,M,p,3, neica,M756, and/or neic,-)M756, 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.

Preferrect nucleic acid molecules encoding equine influenza HA protems are neiti,tHA162, neiWtHA,69s, neica,HA1762, nei HA
ca2õ62, nei, ,,HA,e9s, and/or nei~aHA,e9s, the coding strands olF which are represented by SEQ ID NO:7, SEQ ID NO:9, SEQ
and/or SIEQ ID NO: 12.
Q ID

Preferred nucleic acid molecules encoding equine influenza PB2-N a nei PB2-N proteins e ~~ 124,, ne1,,,tPB2-N1z14i neica,PB2-N;241 neicazPB2-N
,Za,, neiCa,PB2-N1,,4 neicaz, and/or PB2-N 1214, the coding strands of which are represented by SEQ ID
NO:13, SE~
ID NO:15, SEQ TD NO:16, and/or SEQ ID NO:18. ~

Preferred nucleic acid molecules encoding equine influenza PB2-C proteins arI
neiwtlPB2-C1z33, nei PB2-C
w,z 1232, nei,~,PB2-Cõ9,, neic,,PB2-C1z32i nei,zPB2-C
nei PB2-C 'z"' ~~o ca, 194, the coding strands of which are represented by SEQ ID NO: 19, SEQ
NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25. ~

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:2d and/or SEQ ID NO:24 as well as nucleic acid molecules encoding such proteins.

Preferred ectuine influenza M proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei,"M,oz3, neiwt ,M1023, neiõtzMIozs, nei,,,M7s6, neiWtIM7s6, neiõtaMn6, neica,M nei M
,023, czz ,0,3, neic,,iVlsb, and/or nei WM756 =
Preferred equine iniluenza M proteins are PeiIMzSz, Peic,llVl,s,, and/or PeicazMzs, In one embodiment, a;preferred equine influenza M protein of the present invention is encoded by SEQ ID NO: l, 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.
Preferred eqtiine influenza HA proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei,,HA162, neiõrtHA1695, neica]HA16,, neic,,HA162, neiraiHA,69S, and/or neica2HA[695. Preferred equine influenza HA
proteins are P Pei,,,HAs6s, Pe:ic,]HAs6s, and/or Peica2HAs65. 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.

Preferred equine influenza PB2-N proteins of the present invention include proteins encoded by a nucieic acid molecule comprising nei,,PB2-NI241, nei,,PB2-N,2141 -23- ~
nei,8,PB2-N1241 nei,a2PB2=N1241, nei.,PB2-N,214 nei,~, and/or PB2-N1214.
Preferted equine influenza PB2-N proteins are P,"PB2-N404, P,a,PB2-Naoa, and/or PazPB2-Na0la= In one embodiment, a preferred equine influenza PB2-N protein of the present inventiln is encoded by SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 16, and/or SEQ ID NO:1 and, as such, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQ
iD
NO:17.
Preferreci equine influenza PB2-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei,,PB2-Ct233, nei,"2PB2- ~1232, nei,,PB2-C194, r,iei.,PB2-C,232, nei~~PB2-C123,, and/or nei,a,PB2-Ci94.
Preferred eqtine influenza PB2-N proteins are PJB2-C398I PcaIPB2-C398, and/or P,.2PB2-Ca98. In ond 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, iind, as such, has an amino acid sequence that includes SEQ ID
NO:Z,0 and/or SEQ ID NO:24.

Nucleic acid sequence SEQ ID NO: I represents the consensus sequence deduded from the coding strand of PC'R amplified nucleic acid molecules denoted herein as neiw, ,M,fl,3 and neiw,ZM1023, 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 neic,,M1023 and neica,M1023, the production of which is disclosed in the Examples. Nucleic acid sequente SEQ ID NO:7 repi-esents the deduced sequence of the coding strand of a PCR
amplified nucleic acid molecule denoted herein as nei,,,HA1762, the production of which is disclos fd 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 neica,HA1762 and neicaHA1762, 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 amplifed nucleic acid molecule denoted herein as neiwtPB2-N1241, 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 neir ,,PB2-Nt24, and nei,,,2PB2-N124,, 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 neiõrt,PB2-C1233, the production of which is disclosed in the exampl s.
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,,12PB2-C,232, th 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 neicatPB2-C1232, 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.
The present invention includes nucleic acid molecule comprising a cold-adaptell~i equine influenza virus encoding an M protein having an amino acid sequence comprising SEQ II) NO:5. Another embodiment of the present invention includes a nucleic acid moleciule comprising a cold-adapted equine influenza virus encoding an H
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 com risin a cold-ada ted e uine influenza p g p q virus encoding a PB2-C protein having an amino acid sequence comprising SEQ ID
NO:24.
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, and PB2-N, and proteins of the present invention.

Another embodiment of'the present invention is an antibody that selectively binds to an wild-type virus M, HA, PB2-N, PB2-C, PB2, 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, 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 and/or SEQ
ID NO:24.

WO 00/09702 PCTNS99/185~3 The following examples are provided for the purposes of illustration and are nl ot intended to limit the scope of the present invention.
Examule 1 This example discloses- the production and phenotypic characterization of seve~al cold-adapted equine influenza viruses of the present invention.
A. Parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8) (obtaine 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 exampl l~e , from Truslow Farrns, Chestertown, MD or from HyVac, Adel, IA, were inoculated with the parental equine influenza virus by injecting about 0.1 milliliter (ml) undiluted AF
containing approximately 106 plaque forming units (pfu) of virus into the allantoic cavi',ty 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 I, harvested AF was fi=ozen between passages. The AF was then used, either undiluted or II
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 C (passages 1-2) or about 30 C
and on subsequent passages, the incubation temperature was shifted down either to about 28 C,~
or to about 26 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 differeint limbs of the serial passage tree, A through E, as shown in Table 1.

WO 00/09702 PCTfUS99/1858~3 TABLE 1: Passage history of the limbs A through E.

Passage #

Temperature Limb A Limb B Limb C Limb D Limb E

28 C 30-33* 30-68* 30-33 30-69 26 C 9-65 34-69* 34-65 *= the infectious allantoic fluid was diluted I:1000 in these passages B. Virus isolates carried through the cold-adaptation procedure described in Ii 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 C), but no longer forms plaques at a higher, or non-permissive temperature (e.g., about 37 C or about 39 C), as follows. At each cold-adaptation passage, the AF was titered by plaque assay at about 34 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 fi well trays incubated at about 34 C and at about 39 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 C but did not grow at 39 C, divided by the total number of plaques, wasl 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).

~g.~.~..._---..~ .

TA13LE 2: Percent of isolated Clones that were temperature sensitive.
Percent Tcmperature Sensitivc Passagc# 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 1 00%

p52 57%
p62 100% 100%

p65 100%
p66 100% 88%
From the clonal isolates tested for temperature sensitivity, two were selected for I 5 fiuther study. Clone EIV-P821 was sclected from the 49th passage of limb B
and clone EIV-11824 was sclected from the 48th passage of limb C, as defined in Table I
Both of these vinis isolates were tctnpcrature sensitive, with plaquc formation of hnth isolates inhihited at a temperaturc of about 39 "C. At this temperature, protetn synthcsis was completcly inhibited by E1V-P821, hut EIV-P824 exhibited normal Icvels of protein svnthcsis. In additinn, plaque formation by ETV-P821 was inhibited at a tempefature of ahout 37 "C, and at this temperature, late gene expression was inhibited, i.e., nonnal levcls of NP protein were synthesized. reduccd or no M I or HA proteins were synthesized, and enhanced levels of the polymerase proteins were synthcsized_ The phenotype observed at 37 "C', being tvpified by diffcrential viral protcin synthesis, was dhstinct from the pmtetn synthesis phenotype seen at about 39 "C:, which was typified by the inhibition of synthesis of all viral proteins. Virus E1V-P821 has heen deposited with the Atnertcan 7vpe Culture Collection (ATCC) under Accession No. ATCC VR- 2625 and vrrus EIV-P824 has been depositcd with the ATCC under /\cccssion No. ATCC
VR- 2(,24 .
C. Funher characterization of the mutations in isolate EIV-P821 were carried out by reassortment analysis, as follows. Reassortment analysis in influenza viruses S allows one skilled in the art, under certain circumstanees, to correlate phenotypes of a given virus with putative mutations occurring on certain of the eight RNA
segments that comprise an influcnza A virus genome. This tcchnique is described, for example, in Palesc, et al_, ibid. A mixcd infection of EIV-PR21 and an avian influenza virus, A/mallardlNew Yorkl6750/78 was performed as follows. MDCK cells were co-infected with EIV-P821 at a multiplicity of infection (MOI) of 2 pfu/cell and A/rnallard/New York16750/78 at an MOI of either 2, 5, or 10 pfu/cell. The infected cells were incuhated at a temperature of ahout 34 C. The yields of the various co-infections were titered and -ndividual plaques were isolatcd at about 34 "C, and the resultant clonal isolates were characterized as to whether they were able to grow at about 39 C and about 37 C, and express their genes, i.e., synthesize viral proteins, at about 39 C, about 37 C, and about 34 C. Pt-otein synthesis was cvaluated by SDS-PAGE analysis of radiolabcled infected-cell lysates. 7hc IIA, NP and NS-1 proteins of the two parcnt viruses, each of which is encoded hy a scparate gcriomc segment, were distinguishable by SDS-PAGE
analysis, since these particular viral proteins, as derived from either the equine or the avian influenza virus, rnigrate at different apparent rnolecular weights. ln this way it was possible, at lcast for the IIA, NP, and NS-1 genes, to evaluate whether certain phenotypcs of the parent virus, e.g., the tcmpcrature sensitive and the protein synthesis phenotypes, co-segregate with the genome segments carrying these genes The results of the rcassortmcnt analyses investigating co-segregation of a) the mutation inhibiting plaque formation, i.e., thc inductiori ofCPE, at a non-permissive temperature of abnut 31) "C or b) the mutation inhibiting protein synthesis at a non-permissivc ternperature of about 39 C with cach of the EIV-P821 IfA, NP and NS- I protcins are shown in Tables 3 and 4, respcctively.

TABLE 3: Reassortment anal sis of the EIV-P821 39 C la ue fomnati Y p q on phenotype with avian influenza virus, A/mallard/New York/6750/78 Gene Virus ts+' ts z avian 26 13 HA.
equine 11 44 avian 37 8 NP
equine 0 49 avian 9 8 equine 12 20 number of clonal isolates able to induce CPE in tissue culture cells at a temperature of about 39 C.
2 number of clonal isolates inhibited in the ability to induce CPE in tissue culture cells at a temperature of about 39 C.

TABLE 4: Reassortment analysis of the EN-P821 39 C protein synthesis phenotYPe with avian influenza virus, A/mallard/New York/6750/78 Gene Virus ts+' ts2 avian 18 1 HA
equine 11 7 avian 34 5 NP
equine 7 8 NS-1 avian 10 4 equine 14 5 ~ number of cltOal isol~t~~which synthesize all viral proteins at a temperature o a out 2 number of clonal isolates inhibited in the ability to synthesize all viral proteins at a temperature of about 39 T.

The results demonstrated an association of the equine NP gene with a mutation causing the inability c-f EIV-P821 to form plaques at a non-permissive temperature of about 39 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 C. Thus, these data also demonstrated that e th plaque formation phenotype and the protein synthesis phenotype observed in virus EIVI
P821 were the result of separate mutations.
D. Studies were also conducted to determine if cold-adapted equine influenza viruses of the present invention have a dominant interference phenotype, that is, whethler they dominate in nnixed infection with the wild type parental virus A/Kentucky/i/91 (H3N8). The dominant interference phenotype of viruses EIV-P821 and EIV-P824 weie 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 infectd with either cold-adapted virus E1V-P821 or EIV-P824 at an MOI of 2, or simultaneousl¾y doubly infected with both the parental virus and one of the cold adapted viruses at an MOl of 2+2, all at a temperature of about 34 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 ctuplicate plaque assays performed at temperatures of about 34 C an d about 39 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 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 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 ifold. This assay therefore showed that cold-adapted equine influenza viruses EIV-P821 and EIV-P824 both exhibit the dominant interference phenotype.
E. Virus isolttte EIV-MSV+5 was derived from EIV-P82 1, as follows. EIV-P821 was passaged once in eggs, as described above, to produce a Master Seed Virus isolate, denoted hereiia as EIV-MSVO. EIV-MSVO 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 ETV-MSV+3, respectively. EN-MSV+3 was then subjected to two additional passages in MDC'K cclls, as follows. MDCK cells were grown in 150 cm' 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, I g/ml TPCK trypsin solution, 0.1215% 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 4 (for the second passage in MDCK
cells), and the viruses wcrc allowed to adsorb for 1 hour at about 34 C. The inoculum was removcd 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 "C for 21 hours. The virus-infected MDCK cells were harvested by shaking the flasks vigorously to disrupt the cell monolayer, resultinl; in virus isolates EIV-MSV+4 (the first passage in MDCK cells), and EIV-MSV+5 (the second passage in MDCK
cells).
Viruses F..N-MSVO and EIV-MSV+-5 were subjected to phenotypic analvsis, as described in section B above, to determine their ability to forrn plaques and synthesize viral proteins at temperatures of about 34 "C, about 37 "C, and about 39 "C.
Both EiV-MSVO and EN-MSV t 5 formed plaques in tissue culture cells at a temperature of about 34 "C, and nerther vinis isolate fonned plaqucs or exhibited detectable viral protein ~,ynthcsis at a tcmperature of about 39 "C. Virus FIV-MSVO had a similar temperature sensitive phrnntype as EIV-PR21 at a tcmperature of about 37 "C, i.e., it was intubited in plaque fonnatton, and latc gcne expression was inhibrted. However, EIV-MSV-+
5, 'S urihke its parent virus, EIV-Pf2I, did fonn plaques in tissue culture at a ternperarure of about 37 "C, and at this tcmperature, the virus synthesized normal amounts of all protrins. Virus EIV-MSV-+5 has been deposited with the ATCC under Accession No. ATCC VR- 2027 Exa le 2 Therapeutic compositions of the present invention were produced as follows.
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 x 105 pfu/ml in sterile PBS. Virus was inoculated into the allantoic cavity of the eggs as described in Example IA. Afte~ a 3-day incubation iin a humidified chamber at a temperature of about 34 C, AF
was harvested from the eggs according to the method described in Example IA. The harvested AF was mixed with a stabilizer solution, for example Al/A2 stabilizer , available from Diamond Animal Health, Des Moines, IA, at 25 % VN t stabilizer/AF II'.
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 fittedl with a swinging bucket rotor. The clarified fluid was distributed into I-mI
cryovials and was frozen at about -70 C. Virus stocks were titrated on MDCK cells by CPE
and plaque assay at abciut 34 C.
B. A large stock of EIV-P821 was propagated in MDCK cells as follows.
MDCK cells were grown in 150 cm2 tissue culture flasks in MEM tissue culture medium with Hanks Salts, containing 10% calf serum. The cells were then washed with sterile li 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 adsor for I hour at about 34 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 peir flask. The infected cells were incubated at about 34 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% VIV
(stabilizer/virus solution). The supernatants were distributed aseptically into cryovials and frozen at -70 C

e utic co ost it' C. Th rape mp ons 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, _...,.,.~. I--stock vials of EIV-P821 or EIV-MSV +5 were thawed and were diluted in an excipier~t 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 dilutiolp for administration to animals., - The vaccine compositions were held on ice prior to vaccinations. All itherapeutic compositions were titered on MDCK cells by standard methods just prior to vaccinations and wherever possible, an amount of the compositioil, treated identically to those administered to the animals, was titered after the vaccinatio lihs to ensure that the virus remained viable during the procedures.

Examule 3 A therapeutic composition comprising cold-adapted equine influenza virus EN~
P821 was tested for safety and its ability to replicate in three horses showing detectable li prior immunity to equine influenza virus as follows. EIV-P82 1, produced as described in Example lA, was grown in eggs as described in Example 2A and was formulated int Io a therapeutic composition comprising 107 pfu, EIV-P821/2ml BSA-MEM solution as described in Example 2C.

Three ponies having prior detectable hemagglutination inhibition (HAI) titers to equine influenza virus were inoculated with a therapeutic composition comprising EIV-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 niostril, I ml, per nostril.
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 I -I 1 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.

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 WO 00ro9702 rcz'/v 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 aniy abnormal reactions or overt clinical signs during the observation I
period.
To test for viral shedding in the animals, on days 0 through 11 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, o sterile Dacron polyester tipped applicators (available, e.g., from Hardwood Products C ., Guilford, ME) were inserted, together, into each nostril of the ponies. The swabs (foui total, two for each nostril) were broken off into a 15-mi conical centrifuge tube containing 2.5 ml of chilled transport medium comprising 5% glycerol, penicillin, streptomycin, neoniycin, 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 Exampde 1. The AF of the inoculated eggs was then tested for its ability to'I
cause hemagglutina.tion, 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 Flu A test, available from Becton-Dickinson (Cockeysville, MD).

Attempts to isolate EIV from the nasopharyngeal secretions of the three animals by egg inoculation vvere 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 ithat EIV-P821 was replicating in the seropositive ponies.
To test the antibody titers to EIV in the inoculated animals described in this example, as well as iin 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 infiibition (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.~ 1.

The HAI tiiters 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-P82 1.
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 5: HAI[ titers of vaccinated animals*
Animal HA.I Titer (days after vaccination) M

* HAI titers are expressed as the reciprocal of the highest dilution of serum which inhibited hemaggfutination of erythrocytes by a recent isolate of equine influenza virus.
Examl2le 4 This Exampl!e discloses an animal study to evaluate the safety and efficacy of a therapeutic composition comprising cold-adapted equine influenza virus E1V-P821.
A therapeutir, composition comprising cold-adapted equine influenza virus EIV-II
P821 was tested for attenuation, as well as its ability to protect horses from challenge with virulent equine influenza virus, as follows. EIV-P82 1, produced as described in Example 1, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10' pfu of virus/2mI 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' pfu of the EIV-P821 therapeutic composition, administered intranasally, using methods similar to those described in Example 3. One pony was given 10' pfu of the EIV-P821 therapeutic composition, administered orally, by injecting 6 ml of virus into the pharynx, using a 10-m1 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.
The vaccinated ponies were observed for approximately 30 minutes immediately following and at approximately four hours after vaccination for immediate type aliergic reactions, and the animals were further 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.
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 cliniical signs, such as those described in Example 3. None of the four vaccinated ponies iri this study exhibited any clinical signs during the observation period. This result demonstrated that cold-adapted equine influenza virus EN-exhibits the phenotype of attenuation.
To test for viiral shedding in the vaccinated animals, on days 0 through 11 following vaccinaticin, 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.
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 methiods, e.g., the Directigen Flu A test.

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 1 i 91 IN - - + + + + + + + + + -674 Oral - - - - - - - - - - - -I--~---To test the antibody titers to e uine influenza virus in q the vaccinated animals,blood was collected from the animals prior to vaccination and on days 7, 14, 21, and ~8 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.
The HAI titers of the four vaccinated ponies are shown in Table 7.
TABLE 7: HA,I titers after vaccination.

Animal HAI Titer (days after vaccination) IH Route 0 7 14 21 28 91 IN <10 <10 <10 <10 <10 673 IN 10 t0 10 20 20 674 Oral 20 40 40 40 40 Unlike the increase in HAI titer observed with the three animals described in tholl 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-P82 1.
Approximately four and one-half months after vaccine virus administration, all 8i ponies, i.e., the fourthat were vaccinated and the four non-vaccinated controls, were challenged by the following method. For each animal, 10' pfu of the virulent equine influenza virus strain A/equine/Kentucky/1/9I (H3N8) was suspended in 5 mI of water. I, 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.
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 feveir, 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 ~__-WO 00/09702 PCT/US99/18583 I'I

demonstrates that a therapeutic composition of the present invention can protect horses from equine influenza disease.
Example 5 This Exampie discloses an additional animal study to evaluate attenuation of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821, andll its ability to protect vaccinated horses from subsequent challenge with virulent equine influenza virus. Fuirthermore, this study evaluated the effect of exercise stress on the safety and efficacy of the therapeutic composition.

A therapeutic composition comprising cold-adapted equine influenza virus E1V-P821 was tested for safety and efficacy in horses, as follows. EIV-P82 1, produced as described in Example 1, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10' pfu virus/5ml 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'j two vaccinated groups and one unvaccinated control group. The ponies in group 2 were exercise stressed befare vaccination, while the ponies in vaccinate group 1 were held in aJ i stall.

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 - - Day90 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.

PCTlUS99118583 WO

TA.BLE 9: Exercise regimen for the ponies in Group 2.

Speed (m/sec) Time (min.) Incline ( ) 1.5 2 0 3.5 2 0 3.5 2 7 4.5t 2 7 5.5 t 2 7 6.5 t 2 7 7.5t 2 7 8.5t 2 7 3.5 2 7 _ 1.5 10 ot t Speed, in meters per second (m/sec) was increased for each animal every 2 minutes until the heart rate reached and maintained z200 beats per minute Groups I and 2 were given a therapeutic composition comprising 10' 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.
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.

To test for viral shedding in the vaccinated animals, before vaccination and on days I 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 I and 2, as shown in Table

10.

III

TABLE 10: Virus isolation after vaccination.

Animal Virus Isolation da s after vaccination) ( Y
Group ID Exercise 0 1 2 3 4 5 6 7 8 9 10 11 12 - - + + + + + - + + -16 - - + + + + + _ - - _ -', 1 17 No - - + + + + + + + - +

688 - - - - - + - + - - -7 - - - + + + + - - - - -2 435 Yes - - + + + + - - _ _ - - ' 907 - - - + - + + - - - _ - I
968 - - - - - + _ + - _ _ _ 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.l 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 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 12 <10 <10 <10 <10 <10 <10 80 320 320 640 16 <10 <10 20 20 <10 <10 20 160 320 320 17 <10 <10 10 10 10 10 80 160 160 160 165 <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 3 200 <10 20 80 80 40 Group Descrintion I Vaccination only 2 Vaccination and Exercise 3 Control On day 90 post vaccination, all 15 ponies were challenged with 10' pfu of equ'ine influenza virus strain A/equine/Kentucky/l/91 (H3N8) by the nebulizer method as described in Exaniple 4. Clinical observations, as described in Example 3, were performed on all animals three days before challenge and daily for I 1 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.
Thus, this e:xample 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 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 bincling 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 evaltiate whether the infectivity of therapeutic compositions of the present invention was adversely affected by growth for multiple passages in in vitro tissue culture.
EIV-P82 1, produced as described in Example I, was grown in eggs as described i in Example 2A or in IVIDCK cells as described in Example 2B. In each instance, the virus was passaged five times. ETV-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' pfu virus/2m1 BSA-MEM solution, as described in Example 2C, resulting in an egg-grown EN-P821 therapeuitic composition and an MDCK cell-grown EN-P821 therapeutic composition, respectively.
Eight ponies were used in this study. Serum from each of the animals was teste 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 EN-P821 therapeutic composition, and Group B received the MDCK-grown EN-P821 therapeutic composition, prepared as described in Example 2B. The therapeutic compositions were administered intranasally by the method described in Example 3.
The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day I 1 following vaccination for allergic reactions or clinical signs as described in Example 3. No aIlergiy:
reactions or overt clinical signs were observed in any of the animals.
1 S 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 12: HAI titers and virus isolation after vaccination.
HAI Titer DPV' Virus Isolation DPV' Group2 - ID 0 7 14. 21 28 0 1 2 3 4 5 6 7 8 9 10 11 31 <10 20 160 160 160 - EC - C EC EC C C EC - - -1 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 - - - -32 <10 <10 80 80 40 - EC - C- C - C - EC - -2 34 <10 20 160 160 160 - EC - C EC C EC C - - - -35 <10 <I0 80 80 40 - EC - C- C - C - EC - -42 < t 0< 10 80 80 40 -- - C - C EC EC - - - -E= Egg isolation posiitive; C=CPE isolation positive; - virus not detected by either of the methods 2 Group 1: Virus passaged 5X in MDCK cells; Group 2: Virus passaged 5X in Eggs Days Post-vaccination 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.
Exarr3ple 7 This example evaluated the minimum dose of a therapeutic composition comprising a cold-adapted equine influenza virus required to protect a horse from equinel, influenza virus infection.
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' pfu, which correlates to approximately 108 TCID50 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 II
protects a horse against virulent equine influenza virus challenge.

EIV-P82 1, produced as described in Example lA, was passaged and grown in MDCK cells as described in Example 2B and was formulated into a therapeutic composition comprising either 2 x 10', 2 x 105, 2 x 106, or 2 x 10' TCID50 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-mi dose of the indicated therapeutic composition, administered intranasally by methods similar to those described in I?xample 3.

TABLE 13: Vaccination protocol.

Group No. No. Animals Vaccine Dose, TCIDS Units 1 3 2 x 10' 2 4 2x106 3 4 2 x 105 4 4 2 x 104 5 4 control 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 fizrther, 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 abnornnai reactions or overt clinical signs from the vaccination.

Blood for serum analysis was collected 3 days before vaccination, on days 7, 14, 21, and 28 after vaccination, anci 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 th.: 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 14: HA.I titers post-vaccination and post-challenge, and challenge results.

Chall.
Dose in Animal Vaccination on Day 0, Challenge on Day 29 Sick No. TunIDS ID -1 7 14 21 28 35 42 +/-41 <10 <10 10 40 10 20 80 -I 2xi0' 42 40 40 40 40 40 <10 80 -II
200 <10 <10 80 40 160 40 40 -679 <10 10 40 40 40 20 20 -2 2x106 682 <10 <10 40 40 40 40 40 -R <10 10 40 20 160 40 40 -73 <10 <10 160 40 80 160 160 -3 2xI0s 712 <10 <10 20 20 40 40 20 -720 <10 20 80 40 80 80 160 -796 <10 <10 <10 <10 <10 10 80 +
75 <10 <10 <10 <10 <10 <10 160 +
4 2x104 724 <10 >10 <10 <10 <10 20 320 +
789 <10 10 320 160 320 320 320 -790 <10 <10 80 40 160 80 40 12 <10 <10 <10 20 20 40 40 -5 Control 22 10 20 40 10 160 40 640 -71 <10 <10 <10 <10 10 20 160 +
74 <10 <10 <10 <10 <10 <10 20 +
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 108 TCIDsQ units of challenge virus in a volume of 5 ml for each animal.
Clinical observations, as desci-ibed 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 I 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 HAl titers II~

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 10g TCIDso units, since, based on prior results, this level of challenge should have caused disease in all the control animals.
Nonetheless, the levels of seroconversion and the lack of clinical signs in the groups that receiveci a therapeutic composition comprising at least 2 x 106 TCID50 units of a cold-adapted equine influenza virus suggests that this amount was sufficient to protect a horse agaiinst equine influenza disease. Furthermore, a dose of 2x105 TCIDsp II
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 This example discloses an animal study to evaluate the duration of immunity of therapeutic composition comprising cold-adapted equine influenza virus EN-P821.
A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821, P821, produced as described in Example 1, was grown in eggs similarly to the procedurel, 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 Exaniple 2C. Thirty horses approximately l I 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 TCIDSo uniits of the Efl/-P821 therapeutic composition.
Vaccinations were performed on Day 0.
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, II''I
a distant examination for a period of at least 15 minutes was performed. This distant examination includecl observation for demeanor, behavior, coughing, sneezing, and nasal ', discharge. The examination on,X.7ay 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.

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 perforrned on Day 171 post-vaccination to monitor tl~e 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.
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 instantaneousl 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.
Challenges vvere 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 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.

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, Pennsylvania) 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.
All statistical methods' in this study were performed using SAS (SAS
Institute, Cary, NC), and P < 0.05 was considered to be statistically significant.
Beginning on DaV
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 (challenge day) to day 10 were included in the analysis; see Table 15.

TABLE 15: Effect of challenge on daily temperatures ( C) in vaccinated and control horses (least squares means).

Day post challenge Vaccinated (n=19) non-vaccinated (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 Table 15 shows that on days 2 through 8, vaccinated horses had lower temperatures (P <I
0.05) than the non-vaccinated control horses.
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 16: Clinical signs and scoring index.

Clinical Sign Description Score Coughing normal during observation period of 15 min 0 coughing once during observation I
cot.t in 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 Depressioii normal 0 depression resentt 1 tDepiression was assessed by subjective evaluation of individual animal behavior that included the following: failure to approach food rapidly, general lethargy, inappetence, and anorexia.

Each horse was scored for each of these categories. Additionally, submandibular lymp~
nodes were palpated to monitor for possible bacterial infection. In any case where there wa4s a different value recorded for a subjective clinical sign score from an observation on thlF
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 dispositior-, distant examinations were performed at 14, 18, and 21 days post-challenge. Data from days I through 10 post-challenge were included in the analysis.
These scores were summed on each day for each horse, and the vaccinates and controls wero compared using the 17Vilcoxon 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 meari clinical scores are stiown in Tables 17 and 18, respectively. Five days post-challenge, th mean rank of scores in the vaccinated horses was lower (P < 0.05) than in th~
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 th vaccinated horses than the non-vaccinated controls.

~II

TABLE 17: Effect of challenge on clinical sign scores in vaccinated and control horses (mean rank).

Day post challenge Vaccinated (n-19), Non-vaccinated (n-10), P-value mean rank* mean rank 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 I 1 dMs! 11.8 21.2 0.0051 *By Wilcoxon rank sum test.

TABLE 18: Effect of challenge on clinical sign scores in vaccinated and control horses (mean scores).

Day post challenge Vaccinated (n=19) Non-vaccinated (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 Nasopharyng,eal swabs were obtained on the day prior to challenge and on days I
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 numbei~
of days the chalIenge virus was shed was also lower (P < 0.05) in the vaccinated group a~l compared to the non-vaccinated controls.

TABLE 19: Percent of horses shedding virus per day post-challenge and mean numbtr of days of shedding per group.

Day post challenge Vaccinated (n-19) Non-vaccinated (n=10) 1 63.2 90 3 84.2 100 5 47.4 88.9*
6 10.5 77.8*
i' 5.3 20 average number of days shedding 4.1 5.6*
*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).

The scores froni clinical signs relevant to influenza and the objective temperaturle measurements both demonstrated a statistically significant reduction in the group of vaccinates when co:mpared to those from the control group; this is consistent with ari interpretation that the vaccine conferred significant protection from disease.

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 fo~
shedding on certain days post-challenge and the mean number of days of shedding pe~
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.
this study are co si with The results of n stent the interpretation that the vaccine safelyi, 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.l'I
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 a4 duration of clinical illness and a noticeable effect on the potential for viral shedding afterl exposure to a viruler-t strain of equine influenza. The finding that both vaccinates and controls were seronegative immediately prior to challenge at 6 months post-immunization II

p PCT/US99/185831I

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 durak~le protection.
Example 9 This Example cEiscloses 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.
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.
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 groupl contained a mixture of 2 vaccinates and three controls or vice versa. The challenge procedure was similax to that described in Example 8. Challenges were performed on Day 28 post-vaccination.
s rvations were rf rm for v c i a -4 Clinical ob e pe o ed the a c n tes and controls on Day and on Study Days 0 (before vaccination and up to 4 hours post-vaccination), I 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 Exampie 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).

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 20: Effi-,ct of challenge on daily temperatures ('C) in vaccinated and controll horses (least squares means).

Day post challenge Vaccinated (n=10) Non-vaccinated (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.

Data from days I 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 asi described in Example 9. In addition, these scores were summed across all days for eac&
horse, and compared in the same manner. Mean ranks are shown in Table 21. __ TABLE 21: Effect of challenge on clinical sign scores in vaccinated and control horks (mean rank).

Day post challenge Vaccinated (n=10) Non-vaccinated (n=10) P-value* ~
I 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 10 days 5.70 15.30 0.0003 *By Wilcoxon 2 sample test.

On day 4 post-challenge, the mean rank ofscores in the vaccinated horses was lowe r (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).
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 Youngiier et al., 1994, J. Clin. Microbiol. 32, 750-754. Serially diluted nasat samples were added to wells containing monolayers of. Madin Darby Canine Kidney I
(MDCK) cells. After incubation, wells were examined for the presence and degree o~
cytopathogenic effect. The quantity of virus in TCID50 units was calculated by thel~
Reed-Muench technique. The egg infectivity assay was performed as described in Examplell 1. The percent ofhoarses shedding challenge virus for each assay in each group is shown inI
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 I 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 22: Percent of horses shedding virus following challenge - cell culture assa ,y.

Day post challenge Vaccinated (n=10) Non-vaccinated (n=10) 2 0 70*
3 0 70*
4 20 100*
5 10 100*
6 20 100*
7 0 80*

average number of 0.5 5.5*
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 23: Percent of horsE:s shedding virus following challenge - egg infectivity assa~.
Daypost challenge Vaccinated (n=10) Non-vaccinated (n=10) 2 0 70*
3 10 70*
4 0 90*
5 10 70*
Ii 20 90*
7 0 50*

average number of 0.4 4.4*
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).

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 statisticallyl 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.
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 redu~e the potential for exposure of susceptible animals to the wild-type virus in an outbreak Qf influenza.
Overall, the results of this study show that the vaccine conferred protection again~t a heterologous cha:llenge by a member of the Eurasian lineage of equine influenza vinis strains.
Example 10 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.
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.

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, andi 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.
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 individuai horse on Day 36 post-vaccination.
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 24: Clinical sign observations: total scores, ranked by total score.

Group Halter Total Score"
Identit Days I to 9 post-challenge 1-1/accinate 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 "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.

The results of Table 24 show that the scores for vaccinates were between 0 and Z
which was signifcaritly lower than the score for controls, which were between 21 and 26.
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 25: Effect of Challenge on daily mean temperatures ( C) in vaccinated and control horses.

Day post control vaccinate difference challenge 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 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.
Nasopharyngeal swabs were collected an days 1 and 8, post-challenge, as described in Example 3. These samples were tested for shed virus by an egg infectivityl assay as described in Example 1. The results of the assay are shown in Table 26.
TABLE 26: Virus shedding post-challenge detected by egg infectivity.

Study day 35 37 3S 39 40 41 42 1 43 44 Days post-challenge -1 1 2 3 4 5 6 7 8 Group Identity Detection of virus* No. days No. positive per horse Vaccinates 15 0 2 0 3 3 0 2 1 0 5 Si2 0 0 3 3 2 2 0 0 0 4 No. horses positivie per 0 2 2 3 3 2 1 1 0 da Controls 07 0 3 3 3 3 3 3 1 0 7 15 No. horses positive per 0 4 4 4 4 4 4 4 0 da *Values refer to the number of eggs testing positive of 3 eggs tested per sample. For statistical analysis, al sample was considered positive for virus if at least I egg was positive per sample.

The results of Table 26 show that the number of horses positive per day was 20 higher for the controls than for the vaccinates. Additionally, control horses were positive for more days than vaccinates.
The scores from clinical signs relevant to influenza and the objective temperature I'I
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.
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 i 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.

Overall, the results of this study show that the vaccine safely conferred protectio 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.
Examl2le 11 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.
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, desiignated SEQ ID NO:26, and SEQ ID NO:27, respectively. A
nucleic acid molecule of 1023 nucleotides, denoted neiwtMI023, 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'TA cloning vector, available from Invitrogen, Carlsbad, CA, using standard procedures recommended by the manufacturer. The primers used were the T7 primer, designated by SEQ II) 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, CA. PCR products were prepared for sequencing using a PluSwlTMDye Terminator Cycle Sequencing Ready Reaction kit, a PtttsMTM dRhodamine Terminator Cycle Se uencin Ready Reaction kit, or a PtusMr"~ Bi D eT"' Terminator C cIe Se uencin q g g Y Y q g Ready Reaction kit, all available from PE Applied Biosystems, Foster City, CA, following the manufacturer's protocol. Specific PCR conditions used with the kit were a rapid ramp to 95 C, hold for 10 seconds followed by a rapid ramp to 50 C with a 5 second hold then a rapid ramp to 60 C with a 4 minutehold, 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-al, designated SEQ ID NO:31 and efM-s 1, designated SEQ ID NO:30 were used in a third reaction.

- ,, II

PCR products were purified by ethanol/magnesium chloride precipitation.
Automated sequencing of DNA samples was performed using an ABI PttisMT' Model 377 with XL
upgrade DNA Sequencer, available from PE Applied Biosystems.
Translation of SEQ ID NO: I indicates that nucleic acid molecule nei"'M1023 encodes a full-length equine influenza M protein of about 252 amino acids, referred to herein as PeiwM232, having amino acid sequence SEQ ID NO:2, assuming an open reading frame in wliich the initiation codon spans from nucleotide 25 through nucleotidle 28 of SEQ ID NO:1 and the termination codon spans from nucleotide 781 through nucleotide 783 of SEQ ID NO:I . The region encoding PeiõIM252, designated nei,,,M756, and having a coding strand comprising nucleotides 25 to 780 of SEQ ID NO:I, is represented by SEQ ID NO:3.
SEQ ID NO:1 and SEQ ID NO:3 represent the consensus sequence obtained from two wild type inucleic acid molecules, which differ in one nucleotide.
Nucleotide 663 of nei.1tM1023, i.e., nucleotide 649 of neiwtIM756, was adenine, while nucleotide 663 of nei,2M1023, i.e., niucleotide 649 of neil,12M7S6, 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 22! in PeiwtM252.
B. A nucleic acid molecule of 1023 nucleotides encoding a cold-adapted equine influenza virus M, denoted neic,tM1023, with a coding strand having a sequence designated SEQ ID NO:4 was produced by further PCR amplification and cloned into the pCR-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 neicatMtpZ3 encodes a full-length equine influenza M protein of about 252 amino acids, referred to herein as PeicatM252, 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 Peicai _M,52, designated neicM756, and having a at 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 __~s_ equine influenza M protein in the same manner resulted in molecules nei,,2M,023, identical to neiCa,MI023, and nei,,2M7S6, identical to nei,$,M7s6=
C. Comparison of the nucleic acid sequences of the coding strands of neiwtM,02:
(SEQ ID NO:I) and nei, .a,M1023 (SEQ ID NO:4) by DNA alignment reveals the followinl;
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,,,,M252 (SEQ
ID
NO:2) and Pei,,a,M252 (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 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.
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 neiõVtHA1762, 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 "'TA cloning vector as described in Example I lA. 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.

Translation of SEQ ID NO:7 indicates that nucleic acid molecule neiwtHA176Z
encodes a full-length equine influenza HA protein of about 565 amino acids, referred to herein as PeiwtHA565, 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 Peiõ,HA565, designated nei,,HA169s, and having a coding strand comprising nucleotides 30 to 1724 of SEQ ID
NO:7 is represented by SEQ ID NO:9.
B. A nucleic acid molecule of 1762 nucleotides encoding a cold-adapted equine influenza virus HA protein, denoted nei,a,HA,762, with a coding strand having a sequence designated SEQ ID NO:10 was produced as described in Example 1 IB. Plasmid DNA
purification and cycle sequencing were performed as described in Example 12, part A.
Translation of SEQ ID NO: 10 indicates that nucleic acid molecule neica,HA,762 encodes a full-length equine influenza HA protein of about 565 amino acids, referred to herein as Peica,HAs6s, 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,a,HAs6s, designated neic,,HA1695, and having a coding strand comprising nucleotides 30 to 1724 of SEQ ID
NO: 10, is represented by SEQ ID NO:12.
PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza HA protein in the same manner resulted in molecules nei,,HA17b2, identical to neica,HA1762, and neica2HA,69s, identical to neica,HA,69s.
C. Comparison of the nucleic acid sequences of the coding strands of neiHA176 , (SEQ ID NO:7) and nei,,,HAt762 (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 .a,HA56s (SEQ ID NO: 11) reveals the proteins Pei,,HAsbs (SEQ ID NO:8) and Pei, 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.
Exarpple 13 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:, 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,,,PB2-Nt241, with a coding strand having a nucleic acid sequence designated SEQ ID NO:13 was produced by fin-ther 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.

Translation of SEQ ID NO:13 indicates that nucleic acid molecule neiwPB2-N,241 encodes an N-terminal portion of influenza PB2 protein of about 404 amino acids, referred to herein as PW,,PB2-N404, 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,nPB2-N404, designated nei,,,PB2-N12,4, and having a coding strand comprising nucleotides 28 to 1239of SEQ ID NO:13 is represented by SEQ ID NO: 15.
B. A nucleic acid molecule of 1239 nucleotides encoding an N-terminal portiori of influenza PB2 cold-adapted equine influenza virus PB2-N protein, denoted nei.,PB2-Nt24,, 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.
Translation of SEQ ID NO:16 indicates that nucleic acid molecule neicaIPB2-N,241 encodes an N-terminal portion of equine influenza PB-2 protein of about amino acids, referred to herein as PcaIPB2-Naoa, 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 1D N0:16, and the last codon spans from nucleotide 1237 through nucleotide 1239. The region encoding Pca,PB2-N404, designated neica,PB2-N1z,4, and having a coding strand comprising nucleotides 28 to 1239 of SEQ
ID NO: 16, is represented by SEQ ID NO: 18.
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.PB2-N1241, identical to nei,a,PB2-N,241, and nei,.2PB2-N,2,4, identical to nei.,PB2-N,2,4.
C. Comparison of the nucleic acid sequences of the coding strands of neiwtPB2.=
N1241 (SEQ ID NO: 13) and neica,PB2-N124, (SEQ ID NO: 16) by DNA alignment reveals the following difference: a T to C base shift at base 370. Comparison of the amino aciclf sequences of proteins Põ,PB2-NQ(14 (SEQ ID NO: 14) and P,;,,PB2-N404 (SEQ ID
NO: 17) reveals the following difference: a Y to H shift at amino acid 124 relating to the a T to shift at base 370 in the DNA sequence.
Example 14 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.
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 amplif cation 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,,,,PB2-Ct233, 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 1 I B. Plasmid DNA was purified and sequenced as in Example I I A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance; efPB2-al, designated SEQ ID NO:40 and efPB2-sl, 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.

Translation of SEQ ID NO:19 indicates that nucleic acid molecule neiW,,PB2-C1233 encodes a C-terminal portion of influenza PB2 protein of about 398 amino aci&r,,õ
referred to herein as P,,,PB2-C398, having amino acid sequence SEQ ID NO:20, assumiiing an open reading frame having a first codon spans from nucleotide 3 through nucleotide. 5 and a terminatian 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,,,PB2-CJ98, designated nei,,,,LPB2-C194, and having a coding strand comprising nucleotides 3 to 1196 of SEQ ID
NO:19 ius represented by SEQ ID NO:2 1.
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,,,2PB2-N,232, with a coding strand with a sequence designated SEQ ID NO:22. nei,,,2PB2-N,232 is identical to nei,,,,,,PB2-C1233, expect that nei,,2PB2-Nt2321acks one nucleotide on the 5'-end. Translation of SEQ ID NO:22 indicates that nucleic acid molecule nei,,,,,PB2-C,Z33 also encodes P1z PB2-C39$ (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 neiõn2PB2-C1 44, is identical to SEQ ID NO:21.
B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminal portion oI' influenza PB2 cold-adapted equine influenza virus protein, denoted nei.,PB2-C123,, 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 pCRO-Blunt cloning vector was used.
Translation of SEQ ID NO:23 indicates that nucleic acid molecule nei,,,PB2-Ci232 encodes a C-terminal portion of equine influenza PB-2 protein of about 398 amino acids, referred to herein as Pc,,PB2-C398, 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 WO 00109702 PCTlUS99/18583 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,,.,PB2-C398s designated nei,,.,PB2-C1194, and having a coding strand comprising nucleotides 2 to 1195 of SEQ ID NO:23, is represented by SEQ ID NO:25.
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.2PB2-C1231, containing one less nucleotide at the 3'end than nei.,PB2-N,241; and nei,.2PB2-Nt214, identical to nei.,PB2-N,2,4=
C. Comparison of the nucleic acid sequences of the coding strands of neiõR,PBZ-C 1233 (SEQ ID NO:19) and nei,., PB2-C1232 (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 i:o A base shift at base 929 of SEQ ID NO:19. Comparison of the amino acid sequences o;f proteins P,,,PB2-C39S (SEQ ID NO:20) and P, ,.,PB2-398 (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.
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 LISTING

<110> The University of Pittsburgh, of the Commonwealth <120> COLD-ADAPTED EQUINE INFLUENZA VIRUSES

<130> HKZ-033CPPC
<140> not yet assigned <141> 1999-08-12 <150> 09/133,921 <151> 1998-08-13 <160> 43 <170> Patentln Ver. 2.0 <210> 1 <211> 1023 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (25) . . (780) <400> 1 gcaaaagcag gtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51 Met Ser Leu Leu Thr Glu Val Glu Thr tac gtt ctc tct atc gta cca tca ggc ccc ctc aaa gcc gag atc gcg 99 Tyr Val Leu Ser Iie Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala cag aga ctt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147 Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195 Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gag cga gga 243 Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac gga gat 291 Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga 339 Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg 90 95 , 100 105 gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tcc act 387 Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg gga act 435 Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483 Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca aca acc 531 Ile Ala Asp Ser G1n His Arg Ser His Arg Gln Met Val Thr Thr Thr aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt acc acg 579 Asn Pro Leu Ile Arg His Glu Asn Arg Met Vai Leu Ala Ser Thr Thr gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gca gca gag 627 Ala Lys Ala Met Giu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu gcc atg gag gtt gct agt aag gct agg cag atg gtr cag gca atg aga 675 Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Xaa Gln Ala Met Arg acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723 Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caa atg cag 771 Leu Glu Asn Leu Gin Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg 820 Arg Phe Lys cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880 tacgggttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940 cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000 gagtaaaaaa ctaccttgtt tct 1023 <210> 2 <211> 252 <212> PRT
<213> Equine influenza virus H3N8 <400> 2 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 .120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Xaa Gin Ala Met Arg Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys <210> 3 <211> 756 <212> DNA
<213> Equine influenza virus H3N8 <400> 3 atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcgtaccatc aggccccctc 60 aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120 gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180 ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300 aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360 actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420 gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480 tctcacaggc agatggtgac aacaaccaac ccattaatca gacatgaaaa cagaatggta 540 ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600 gaggccatgg aggttgctag taaggctagg cagatggtrc aggcaatgag aaccattggg 660 acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720 cagaaacgga tgggagtgca aatgcagcga ttcaag 756 <210> 4 <211> 1023 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (25)..(780) <400> 4 gcaaaagcag gtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51 Met Ser Leu Leu Thr Glu Val Glu Thr tac gtt ctc tct atc tta cca tca ggc ccc ctc aaa gcc gag atc gcg 99 Tyr Val Leu Ser Ile Leu Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala cag aga ctt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147 Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195 Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gag cga gga 243 Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac gga gat 291 Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga 339 Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tcc act 387 Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg gga act 435 Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483 Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca ata acc 531 Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Ile Thr aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt acc acg 579 Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gca gca gag 627 Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu gcc atg gag gtt gct agt aag gct agg cag atg gta cag gca atg aga 675 Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Val G1n Ala Met Arg acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723 Thr Ile Giy Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caa atg cag 771 Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg 820 Arg Phe Lys cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880 tacggcttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940 cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000 gagtaaaaaa ctaccttgtt tct 1023 <210> 5 <211> 252 <212> PRT
<213> Equine influenza virus H3NS
<400> 5 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Leu Pro 1 5 10 i5 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg G1u Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg Ser His Arg Gin Met Val Thr Ile Thr Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys <210> 6 <211> 756 <212> DNA
<213> Equine influenza virus H3N8 <400> 6 atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcttaccatc aggccccctc 60 aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120 gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180 ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300 aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360 actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420 gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480 tctcacaggc agatggtgac aataaccaac ccattaatca gacatgaaaa cagaatggta 540 ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600 gaggccatgg aggttgctag taaggctagg cagatggtac aggcaatgag aaccattggg 660 acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720 cagaaacgga tgggagtgca aatgcagcga ttcaag 756 <210> 7 <211> 1762 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (30)..(1724) <400> 7 agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata 53 Met Lys Thr Thr Ile Ile Leu Ile cca ctg acc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101 Pro Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn aca gcc aca tta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149 Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu gta aaa aca ata act gat gac caa att gag gtg aca aat gct act gaa 197 Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu tta gtt cag agc att tca ata ggg aaa ata tgc aac aac tca tat aga 245 Leu Val Gin Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg gtt cta gat gga aga aat tgc aca tta ata gat gca atg cta gga gac 293 Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttc ata gaa 341 Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu aga agc agc gct ttc agc agt tgc tac cca tat gac atc cct gac tat 389 Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttg gaa ttc aca 437 Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr gca gag gga ttc aca tgg aca ggt gtc act caa aac gga aga agt gga 485 Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly tcc tgc aaa agg gga tca gcc gat agt ttc ttt agc cga ctg aat tgg 533 Ser Cys Lys Arg Gly Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp cta aca gaa tct gga aac tct tac ccc aca ttg aat gtg aca atg cct 581 Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro 170 175 lso aac aat aaa aat ttc gac aaa cta tac atc tgg ggg att cat cac ccg 629 Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro agc tca aac aaa gag cag aca aaa ttg tac atc caa gaa tcg gga cga 677 Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gin Glu Ser Gly Arg _.-__...K.~~,,. ----_-_.~..~~----gta aca gtc tca aca aaa aga agt caa caa aca ata atc cct aac atc 725 Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile gga tct aga ccg cgg gtc agg ggt caa tca ggc agg ata agc ata tac 773 Gly Ser Arg Pro Arg Val Arg Gly Gln Ser Gly Arg Ile Ser Ile Tyr tgg acc att gta aaa cct gga gat atc cta atg ata aac agt aat ggc 821 Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Met Ile Asn Ser Asn Gly aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869 Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser tct gta atg aga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917 Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys att aca cca aat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965 Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val aac aaa gtt aca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013 Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr tta aag ctg gcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061 Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg gga atc ttt gga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp G1u Gly atg gtt gat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157 Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr gga caa gct gca gat eta aag agc act caa gca gcc atc gac cag atc 1205 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile aat gga aaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253 Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His caa ata gag aag gaa ttc tca gaa gta gaa ggg agg atc cag gac ttg 1301 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu gag aag tat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala gaa ttg ctg gtg gct cta aaa aat caa cat aca att gac tta aca gat 1397 Glu Leu Leu Val Ala Leu Lys Asn Gin His Thr Ile Asp Leu Thr Asp gca gaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445 Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu aac gcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493 Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys gat aat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr ata tac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589 Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637 Glu Leu Lys Ser G1y Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685 Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734 Ala Cys G1n Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile gatagttaaa aacacccttg tttctact 1762 <210> 8 <211> 565 <212> PRT

Wo 00/09702 PCT/US99/18583 <213> Equine influenza virus H3N8 <400> 8 Met Lys Thr Thr Ile Ile Leu Ile Pro Leu Thr His Trp Val Tyr Ser 1 5 10 = 15 Gin Asn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe G1n Tyr Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly Ser Cys Lys Arg Gly Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Giy Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gin Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Arg Val Arg Gly ~~,~.Q,,....~_______ Gin Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Vai Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Giy Trp Tyr Gly Phe Arg Tyr Gin Asn Ser Glu Gly Thr Giy Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg I1e Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Lys Asn Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 9 <211> 1695 <212> DNA
<213> Equine influenza virus H3N8 <400> 9 atgaagacaa ccattatttt gataccactg acccattggg tctacagtca aaacccaacc 60 agtggcaaca acacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120 gtaaaaacaa taactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180 atttcaatag ggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240 ttaatagatg caatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300 ctcttcatag aaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360 gcatcgctcc ggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420 acatggacag gtgtcactca aaacggaaga agtggatcct gcaaaagggg atcagccgat 480 agtttcttta gccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540 gtgacaatgc ctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600 agctcaaaca aagagcagac aaaattgtac atccaagaat cgggacgagt aacagtctca 660 acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgcg ggtcaggggt 720 caatcaggca ggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 780 -14- ~ -._.....,....,~,~~.-._.~

aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840 tctgtaatga gatcagatgc acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatcc ccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960 cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020 aagcaaatca gaggaatctt tggagcaata gcgggattca tagaaaacgg ctgggaagga 1080 atggttgatg ggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140 gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200 gaaaggacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggagg 1260 atccaggact tggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320 gaattgctgg tggctctaaa aaatcaacat acaattgact taacagatgc agaaatgaat 1380 aaattattcg agaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440 tgtttcaaga tataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500 tatgaccatt acatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560 gagttgaaat caggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620 ttaatttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680 tgcaacattt gcatt 1695 <210> 10 <211> 1762 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (30) .. (1724) <400> 10 agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata. 53 Met Lys Thr Thr Ile Ile Leu Ile cta ctg acc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101 Leu Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn aca gcc aca tta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149 Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu gta aaa aca ata act gat gac caa att gag gtg aca aat gct act gaa 197 Val Lys Thr Ile Thr Asp Asp Gin Ile Glu Val Thr Asn Ala Thr Glu tta gtt cag agc att tca ata ggg aaa ata tgc aac aac tca tat aga 245 Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg gtt cta gat gga aga aat tgc aca tta ata gat gca atg cta gga gac 293 Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttc ata gaa 341 Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu aga agc agc gct ttc agc agt tgc tac cca tat gac atc cct gac tat 389 Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttg gaa ttc aca 437 Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr gca gag gga ttc aca tgg aca ggt gtc act caa aac gga aga agt gga 485 Ala Giu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly tcc tgc aaa agg gaa tca gcc gat agt ttc ttt agc cga ctg aat tgg 533 Ser Cys Lys Arg Glu Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp cta aca gaa tct gga aac tct tac ccc aca ttg aat gtg aca atg cct 581 Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro aac aat aaa aat ttc gac aaa cta tac atc tgg ggg att cat cac ccg 629 Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro agc tca aac aaa gag cag aca aaa ttg tac atc caa gaa tca gga cga 677 ~~~

Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg gta aca gtc tca aca aaa aga agt caa caa aca ata atc cct aac atc 725 Val Thr Val Ser Thr Lys Arg Ser G1n Gin Thr Ile Ile Pro Asn Ile gga tct aga ccg tgg gtc agg ggt caa tca ggc agg ata agc ata tac 773 Gly Ser Arg Pro Trp Val Arg Gly Gin Ser Gly Arg Ile Ser Ile Tyr tgg acc att gta aaa cct gga gat atc cta acg ata aac agt aat ggc 821 Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Thr Ile Asn Ser Asn Gly aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869 Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser tct gta atg aga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917 Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys att aca cca aat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965 Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Vai aac aaa gtt aca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013 Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr tta aag ctg gcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061 Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg gga atc ttt gga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109 Gly I1e Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly atg gtt gat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157 Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Giu Gly Thr gga caa gct gca gat cta aag agc act caa gca gcc atc gac cag atc 1205 Gly Gin Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asp Gln Ile aat gga aaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253 Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His caa ata gag aag gaa ttc tca gaa gta gaa ggg aga atc cag gac ttg 1301 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gin Asp Leu gag aag tat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala gaa ttg ctg gtg get cta gaa aat caa cat aca att gac tta aca gat 1397 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp gca gaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445 Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg G1n Leu Arg Glu aac gcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493 Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys gat aat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr ata tac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589 Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe G1n Ile Lys Gly Val gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685 Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734 Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys I1e gatagttaaa aacacccttg tttctact 1762 <210> 11 <211> 565 <212> PRT
<213> Equine influenza virus H3N8 <400> 11 Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr Ala Giu Gly Phe Thr Trp Thr Gly Val Thr Gin Asn Gly Arg Ser Gly Ser Cys Lys Arg Glu Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly WO 00/09702 PCT/US99/1853.3 Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Thr Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 'Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys I3.e Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile 8er Phe Ala Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 12 <211> 1695 <212> DNA
<213> Equine influenza virus H3N8 <400> 12 atgaagacaa ccattatttt gatactactg acccattggg tctacagtca aaacccaacc 60 agtggcaaca acacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120 gtaaaaacaa taactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180 atttcaatag ggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240 ttaatagatg caatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300 ctcttcatag aaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360 gcatcgctcc ggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420 acatggacag gtgtcactca aaacggaaga agtggatcct gcaaaaggga atcagccgat 480 agtttcttta gccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540 gtgacaatgc ctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600 agctcaaaca aagagcagac aaaattgtac atccaagaat caggacgagt aacagtctca 660 acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgtg ggtcaggggt 720 caatcaggca ggataagcat atactggacc attgtaaaac ctggagatat cctaacgata 780 =21-___ aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840 tctgtaatga gatcagatgc acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatcc ccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960 cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020 aagcaaatca gaggaatctt tggagcaata gcgggattca tagaaaacgg' ctgggaagga 1080 atggttgatg ggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140 gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200 gaaaggacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggaga 1260 atccaggact tggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320 gaattgctgg tggctctaga aaatcaacat acaattgact taacagatgc agaaatgaat 1380 aaattattcg agaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440 tgtttcaaga tataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500 tatgaccatt acatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560 gagttgaaat caggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620 ttaatttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680 tgcaacattt gcatt 1695 <210> 13 <211> 1241 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (28)..(1239) <400> 13 agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp .~~..~

cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg G1n Glu Lys aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr acg agc aca att cat tat cca aaa gtc tac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438.
Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro G1y His Ala Asp ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gin Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Giy Gly Phe Thr ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Oly Ser Ser Val Lys Arg Glu Glu Glu Met Leu acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gin Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gin Ser att gct gaa gca ata att gta gcc atg gtg ttt tc 1241 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe <210> 14 <211> 404 <212> PRT
<213> Equine influenza virus H3N8 <400> 14 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gin Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Va1 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln Asp Vai Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu Leu G1n Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gin Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe <210> 15 <211> 1214 <212> DNA
<213> Equine influenza virus H3N8 <400> 15 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tctacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttc 1214 <210> 16 <211> 1241 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (28)..(1239) <400> 16 agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr acg agc aca att cat tat cca aaa gtc cac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe Glu Lys gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Giy Pro Val His Phe Arg Asn caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu Ala Gin Asp Val Ile Met Glu Val Val Phe Pro -2$-__ WO 00/09702 PCT/US99/1858:3 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser G1u Ser Gln Leu Thr I1e acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Giy Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys His Ser Thr Gin Ile Gly Gly Ile Arg Met Val Asp Ile ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly Asn Leu G1n Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu -Y.~.....______-WO 00/09702 PCT/US99/1858.4 gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser att gct gaa gca ata att gta gcc atg gtg ttt tc 1241 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe <210> 17 <211> 404 <212> PRT
<213> Equine influenza virus H3N8 <400> 17 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln Gly GZn Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala,Val Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe Glu Lys Vai Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln .30-Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu Leu Gin Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp Gin Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gin zle Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Giu Glu Gin Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Giy Tyr Glu Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe WO 00/09702 PCTlUS99/18583 <210> 18 <211> 1214 <212> DNA
<213> Equine influenza virus H3N8 <400> 18 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tccacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 .~--__..___~~ -_ ~.~.~

gccatggtgt tttc 1214 <210> 19 <211> 1233 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (3)..(1196) <400> 19 ta gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag 47 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys gca acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa 95 Ala Thr Arg Arg Leu Ile Gin Leu Ile Val Ser Gly Arg Asp Glu Gln tca att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat 143 Ser Ile Ala Glu Ala Zle Ile Val Ala Met Val Phe Ser Gln Glu Asp tgc atg ata aaa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca 191 Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala aat cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa 239 Asn Gin Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys gat gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat 287 Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn gtg atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag 335 Val Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu atg tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac 383 Met Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr tcc agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt 431 Ser Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val - 33 - -~_._---- -cgg gat caa agg gga aac ata cta ctg tcc'cct gaa gag gtc agt gaa 479 Arg Asp Gin Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu aca caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg 527 Thr Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met tgg gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg 575 Trp Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp atc atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc 623 Ile Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro aca atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc 671 Thr Met Leu Tyr Asn Lys Ile Glu Phe G1u Pro Phe Gln Ser Leu Val cct agg gcc acc aga agc caa tac a.gc ggt ttc gta aga acc ctg ttt 719 Pro Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe cag caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata 767 Gln Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gin Ile Ile aaa ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag 815 Lys Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln ttc tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt 863 Phe Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu gta aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg 911 Val Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg ctc aca gtc ctc gga aag gat gca ggt gcg ctt act gaa gac cca gat 9S9 Leu Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp gaa ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att 1007 Glu Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Giy Phe Leu Ile tta ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa 1055 Leu Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu ctg agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa 1103 Leu Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln ggg gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt 1151 Gly Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu act gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 1196 Thr Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn tagtgttgaa ttgtttaaaa acgaccttgt ttctact 1233 <210> 20 <211> 398 <212> PRT
<213> Equine influenza virus H3N8 <400> 20 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr Gln G1y Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val Lys Ile G1n Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Giy Glu Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys Arg I1e Arg Met Ala Ile Asn <210> 21 <211> 1194 <212> DNA
<213> Equine influenza virus H3N8 <400> 21 gaattcacaa tggtcggaag aagagcaaca gccattctca gaaaggcaac cagaagattg 60 attcaattga tagtaagtgg gagagatgaa caatcaattg ctgaagcaat aattgtagcc 120 atggtgtttt cgcaagaaga ttgcatgata aaagcagttc gaggcgattt gaacttcgtt 180 aatagagcaa atcagcgctt gaaccccatg catcaactct tgaggcattt ccaaaaagat 240 gcaaaagtgc ttttccagaa ttgggggatt gaacccatcg acaatgtgat gggaatgatt 300 ggaatattgc ctgacatgac cccaagcacc gagatgtcat tgagaggagt gagagtcagc 360 aaaatgggag tggatgagta ctccagcact gagagagtgg tggtgagcat tgaccgtttt 420 ttaagagttc gggatcaaag gggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480 caaggaacgg aaaagctgac aataatttat tcatcatcaa tgatgtggga gattaatggt 540 cccgaatcag tgttggtcaa tacttatcaa tggatcatca ggaactggga aattgtgaaa 600 attcaatggt cacaggatcc cacaatgtta tacaataaga tagaatttga gccattccag 660 tccctggtcc ctagggccac cagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720 caaatgcgag atgtacttgg aacatttgat actgctcaaa taataaaact cctccctttt 780 gccgctgctc ctccggaaca gagtaggatg cagttctctt ctttgactgt taatgtaaga 840 ggatcgggaa tgaggatact tgtaagaggc aattccccag tgttcaacta caataaagcc 900 actaagaggc tcacagtcct cggaaaggat gcaggtgcgc ttactgaaga cccagatgaa 960 ggtacggctg gagtagaatc tgctgttcta agagggtttc tcattttagg taaagaaaac 1020 aagagatatg gcccagcact aagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080 gctaatgtgc taattgggca aggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140 agcatactta ctgacagcca gacagcgacc aaaaggattc ggatggccat caat 1194 <210> 22 <211> 1232 <212> DNA
<213> Equine influenza virus H3NS
<400> 22 agaattcaca atggtcggaa gaagagcaac agccattctc agaaaggcaa ccagaagatt 60 gattcaattg atagtaagtg ggagagatga acaatcaatt gctgaagcaa taattgtagc 120 catggtgttt tcgcaagaag attgcatgat aaaagcagtt cgaggcgatt tgaacttcgt 180 taatagagca aatcagcgct tgaaccccat gcatcaactc ttgaggcatt tccaaaaaga 240 tgcaaaagtg cttttccaga attgggggat tgaacccatc gacaatgtga tgggaatgat 300 tggaatattg cctgacatga ccccaagcac cgagatgtca ttgagaggag tgagagtcag 360 caaaatggga gtggatgagt actccagcac tgagagagtg gtggtgagca ttgaccgttt 420 tttaagagtt cgggatcaaa ggggaaacat actactgtcc cctgaagagg tcagtgaaac 480 acaaggaacg gaaaagctga caataattta ttcatcatca atgatgtggg agattaatgg 540 tcccgaatca gtgttggtca atacttatca atggatcatc aggaactggg aaattgtgaa 600 aattcaatgg tcacaggatc ccacaatgtt atacaataag atagaatttg agccattcca 660 gtccctggtc cctagggcca ccagaagcca atacagcggt ttcgtaagaa ccctgtttca 720 gcaaatgcga gatgtacttg gaacatttga tactgctcaa ataataaaac tcctcccttt 780 tgccgctgct cctccggaac agagtaggat gcagttctct tctttgactg ttaatgtaag 840 aggatcggga atgaggatac ttgtaagagg caattcccca gtgttcaact acaataaagc 900 cactaagagg ctcacagtcc tcggaaagga tgcaggtgcg cttactgaag acccagatga 960 aggtacggct ggagtagaat ctgctgttct aagagggttt ctcattttag gtaaagaaaa 1020 caagagatat ggcccagcac taagcatcaa tgaactgagc aaacttgcaa aaggggagaa 1080 .

agctaatgtg ctaattgggc aaggggacgt ggtgttggta atgaaacgga aacgtgactc 1140 tagcatactt actgacagcc agacagcgac caaaaggatt cggatggcca tcaattagtg 1200 ttgaattgtt taaaaacgac cttgtttcta ct 1232 <210> 23 <211> 1232 <212> DNA
<213> Equine influenza virus H3N8 <220>
<221> CDS
<222> (2).:(1195) <400> 23 a gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 49 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 97 Thr Arg Arg Leu Ile G1n Leu Ile Val Ser Gly Arg Asp Glu Gln Ser att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 145 Ile Ala Glu Ala Ile I1e Val Ala Met Val Phe Ser Gln Glu Asp Cys atg ata caa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 193 Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 241 Gin Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 289 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 337 Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 385 Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser _~.
~_ agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 433 Ser Thr Glu Arg Val Val Val Ser Ile Asp'Arg Phe Leu Arg Val Arg gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 481 Asp Gin Arg G1y Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 529 Gin Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 577 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 625 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 673 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 721 Arg Ala Thr Arg Ser Gin Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 769 Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 817 Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 865 Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 913 Arg Gly Asn Ser Pro Vai Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu aca gtc ctc gga aaa gat gca ggt gcg ctt act gaa gac cca gat gaa 961 Thr Val Leu Gly Lys Asp Ala Giy Ala Leu Thr Glu Asp Pro Asp Glu ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 1009 Giy Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 1057 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 1105 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 1153 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 1195 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn tagtgttgaa ttgtttaaaa acgaccttgt ttctact 1232 <210> 24 <211> 398 <212> PRT
<213> Equine influenza virus H3N8 <400> 24 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gin Leu Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Vai Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr G1u Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val Lys Ile Gin Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln Gin Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn <210> 25 <211> 1194 <212> DNA
<213> Equine influenza virus 83N8 <400> 25 gaattcacaa tggtcggaag aagagcaaca gccattctca gaaaggcaac cagaagattg 60 attcaattga tagtaagtgg gagagatgaa caatcaattg ctgaagcaat aattgtagcc 120 atggtgtttt cgcaagaaga ttgcatgata caagcagttc gaggcgattt gaacttcgtt 180 aatagagcaa atcagcgctt gaaccccatg catcaactct tgaggcattt ccaaaaagat 240 gcaaaagtgc ttttccagaa ttgggggatt gaacccatcg acaatgtgat gggaatgatt 300 ggaatattgc ctgacatgac cccaagcacc gagatgtcat tgagaggagt gagagtcagc 360 aaaatgggag tggatgagta ctccagcact gagagagtgg tggtgagcat tgaccgtttt 420 ttaagagttc gggatcaaag gggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480 caaggaacgg aaaagctgac aataatttat tcatcatcaa tgatgtggga gattaatggt 540 cccgaatcag tgttggtcaa tacttatcaa tggatcatca ggaactggga aattgtgaaa 600 attcaatggt cacaggatcc cacaatgtta tacaataaga tagaatttga gccattccag 660 tccctggtcc ctagggccac cagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720 caaatgcgag atgtacttgg.aacatttgat actgctcaaa taataaaact cctccctttt 780 gccgctgctc ctccggaaca gagtaggatg cagttctctt ctttgactgt taatgtaaga 840 ggatcgggaa tgaggatact tgtaagaggc aattccccag tgttcaacta caataaagcc 900 actaagaggc tcacagtcct cggaaaagat gcaggtgcgc ttactgaaga cccagatgaa 960 ggtacggctg gagtagaatc tgctgttcta agagggtttc tcattttagg taaagaaaac 1020 aagagatatg gcccagcact aagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080 gctaatgtgc taattgggca aggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140 agcatactta ctgacagcca gacagcgacc aaaaggattc ggatggccat caat 1194 <210> 26 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 26 agcaaaagca ggtagatatt gaa 23 <210> 27 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 27 agtagaaaca aggtagtttt ttac 24 <210> 28 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 28 caggaaacag ctatgacc 18 -44- __..____.

<210> 29 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 29 taatacgact cactataggg 20 <210> 30 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 30 tggtgcacta gccagctg 18 <210> 31 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 31 ttgcctgtac catctgcc 18 <210> 32 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 32 agcaaaagca ggggatattt ctg 23 <210> 33 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 33 agtagaaaca agggtgtttt taa 23 <210> 34 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 34 gacatccctg actatg 16 <210> 35 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 35 gcatctgtta agtcaa 16 <210> 36 <211> 25 <212> DNA
<213> Artificial Sequence WO 00/09702 PCTlUS99/18583 <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 36 agcaaaagca ggtcaaatat attca 25 <210> 37 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 37 gaaaacacca tggctacaat tattgc 26 <210> 38 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 38 agaattcaca atggtcggaa gaagagc 27 <210> 39 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 39 agtagaaaca aggtcgtttt taaacaa 27 <210> 40 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 40 agccgtacct tcatctggg 19 <210> 41 <211> 19 <212> DNA
<213> Artificial. Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 41 agcactgaga gagtggtgg 19 <210> 42 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 42 gtaagaggca attccccag 19 <210> 43 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic Primer <400> 43 ,.~-~~.~.~~

cagcttttcc gttccttg Z$

Claims (80)

What is claimed:

1. An isolated cold-adapted equine influenza virus that replicates in embryonated chicken eggs at a temperature ranging from about 26°C to about 30°C, wherein said virus is produced using a method comprising:
(a) obtaining a wild-type, equine influenza virus;
(b) passaging said wild-type equine influenza virus at progressively lower temperatures; and (c) selecting a virus that grows at said temperature.

2. A reassortant cold-adapted equine influenza A virus that grows at a temperature lower than about 34°C and replicates in embryonated chicken eggs at a temperature ranging from about 26°C to about 30°C, said reassortant virus comprising:

(a) at least one genome segment of a donor cold-adapted equine influenza virus generated by cold-adaptation using a method comprising:
(i) obtaining a wild-type, equine influenza virus;
(ii) passaging said wild-type equine influenza virus at progressively lower temperatures; and (iii) selecting a virus that grows at said temperature;

wherein 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, and (b) at least one genome segment of a recipient influenza A virus having an identifying phenotype selected from the group consisting of hemagglutinin activity and neuraminidase activity, and combinations thereof; wherein said influenza A
virus genome segment confers at least one of said identifying phenotypes to said reassortant virus.

3. A therapeutic composition to protect an animal against disease caused by an influenza A virus, comprising said equine influenza virus as set forth in claims 1 or 2.

4. Use of a therapeutic composition as set forth in claim 3 for protecting an animal against disease caused by an influenza A virus.

5. A method to produce a cold-adapted equine influenza virus that grows at a temperature lower than about 34°C, wherein said virus replicates in embryonated chicken eggs at a temperature ranging from about 26°C to about 30°C, comprising the steps of:

a. passaging a wild-type equine influenza virus at a temperature lower than about 34°C; and b. selecting viruses that grow at a temperature lower than about 34°C.

6. A method to produce a reassortant cold-adapted equine influenza A

virus that grows at a temperature lower than about 34°C and replicates in embryonated chicken eggs at a temperature ranging from about 26°C to about 30°C, said reassortant 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 a reassortant virus comprising (a) 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, and (b) at least one phenotype of said recipient influenza A virus, wherein said phenotype is selected from the group consisting of hemagglutinin activity and neuraminidase activity, and combinations thereof.

7. A method to propagate a cold-adapted equine influenza virus as set forth in claim 1 or 2, comprising:

(a) obtaining said virus of claim 1 or 2;
(b) incubating said virus in either eggs or in tissue culture cells under conditions which allow said virus to replicate; and (c) isolating the progeny virus from said incubation step.

8. An isolated equine influenza nucleic acid molecule, wherein said equine influenza nucleic acid molecule is 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, and SEQ ID NO:25, and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of said nucleic acid sequences.

9. An isolated equine influenza nucleic acid molecule, wherein said equine influenza nucleic acid molecule encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ
ID NO:17, and SEQ ID NO:24.

10. An isolated equine influenza protein, wherein said equine influenza protein comprises an amino acid sequence selected from the group consisting of SEQ
ID NO:5, SEQ ID NO:11, SEQ ID NO:17, and SEQ ID NO:24.

11. The cold-adapted equine influenza virus as set forth in claim 1 wherein said cold-adapted equine influenza virus is attenuated.

12. The reassortant influenza A virus as set forth in claim 2 wherein said reassortant influenza A virus is attenuated.

13. The therapeutic composition as set forth in claim 3 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus is attenuated.

14. The use as set forth in claim 4 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus is attenuated.

15. The method as set forth in claims 5, 6 or 7 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus is attenuated.

16. The cold-adapted equine influenza virus as set forth in claims 1 or 11 wherein said cold-adapted equine influenza virus is temperature sensitive.

17. The reassortant influenza A virus as set forth in claims 2 or 12 wherein said reassortant influenza A virus is temperature sensitive.

18. The therapeutic composition as set forth in claims 3 or 13 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus is temperature sensitive.

19. The use as set forth in claims 4 or 14 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus is temperature sensitive.

20. The method as set forth in claims 5, 6, 7 or 15 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus is temperature sensitive.

21. The cold-adapted equine influenza virus as set forth in claims 1, 11 or 16 wherein said cold-adapted equine influenza virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 39 °C.

22. The reassortant influenza A virus as set forth in claims 2, 12 or 17 wherein said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 39 °C.

23. The therapeutic composition as set forth in claims 3, 13 or 18 wherein said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 39 °C.

24. The use as set forth in claims 4, 14 or 19 wherein said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 39 °C.

25. The method as set forth in claims 5, 6, 7, 15 or 20 wherein said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 39 °C.

26. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16 or 21 wherein said cold-adapted equine influenza virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 37 °C.

27. The reassortant influenza A virus as set forth in claims 2, 12, 17 or 22 wherein said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 37 °C.

28. The therapeutic composition as set forth in claims 3, 13, 18 or 23 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 37 °C.

29. The use as set forth in claims 4, 14, 19 or 24 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 37 °C.

30. The method as set forth in claims 5, 6, 7, 15, 20 or 25 wherein said cold-adapted equine influenza virus or said reassortant influenza A virus replicates in embryonated chicken eggs but does not form plaques in tissue culture cells at a temperature of about 37 °C.

31. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21 or 26 wherein a phenotype comprising a non-permissive temperature of about 39 °C is conferred on said cold-adapted equine influenza virus by at least two mutations in the genome of said virus, comprising a first mutation and a second mutation.

32. The reassortant influenza A virus as set forth in claims 2, 12, 17, 22 or 27 wherein a phenotype comprising a non-permissive temperature of about 39 °C is conferred on said reassortant influenza A virus by at least two mutations in the genome of said virus, comprising a first mutation and a second mutation.

33. The therapeutic composition as set forth in claims 3, 13, 18, 23 or 28 wherein a phenotype comprising a non-permissive temperature of about 39 °C is conferred on said cold-adapted equine influenza virus or said reassortant influenza A
virus by at least two mutations in the genome of said virus, comprising a first mutation and a second mutation.

34. The use as set forth in claims 4, 14, 19, 24 or 29 wherein a phenotype comprising a non-permissive temperature of about 39 °C is conferred on said cold-adapted equine influenza virus or said reassortant influenza A virus by at least two mutations in the genome of said virus, comprising a first mutation and a second mutation.

35. The method as set forth in claims 5, 6, 7, 15, 20, 25 or 30 wherein a phenotype comprising a non-permissive temperature of about 39 °C is conferred on said cold-adapted equine influenza virus or said reassortant influenza A virus by at least two mutations in the genome of said virus, comprising a first mutation and a second mutation.

36. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26 or 31 wherein said first mutation confers a phenotype comprising inhibition of plaque formation at a temperature of about 39 °C, and wherein said first mutation co-segregates with the segment of said genome comprising the nucleoprotein gene.

37. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31 or 36 wherein said second mutation confers a phenotype comprising inhibition of protein synthesis at a temperature of about 39 °C.

38. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36 or 37 further comprising at least one additional mutation, wherein said additional mutation confers a phenotype comprising a non-permissive temperature of about 37 °C, and wherein said phenotype is selected from the group consisting of inhibition of plaque formation at a temperature of about 37 °C and inhibition of the expression of late genes at a temperature of about 37 °C.

39. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37 or 38 wherein said cold-adapted equine influenza virus is producible by a method comprising the steps of:

a. passaging a wild-type equine influenza virus at a temperature lower than about 34°C; and b. selecting viruses that grow at a reduced temperature.

40. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28 or 33 wherein said cold-adapted equine influenza virus is producible by a method comprising the steps of:

a. passaging a wild-type equine influenza virus at a temperature lower than about 34°C; and b. selecting viruses that grow at a reduced temperature.

41. The use as set forth in claims 4, 14, 19, 24, 29 or 34 wherein said cold-adapted equine influenza virus is producible by a method comprising the steps of:

a. passaging a wild-type equine influenza virus at a temperature lower than about 34°C; and b. selecting viruses that grow at a reduced temperature.

42. The method as set forth in any one of claims 5, 6, 7, 15, 20, 25, 30 and 35 wherein said cold-adapted equine influenza virus is producible by a method comprising the steps of:

a. passaging a wild-type equine influenza virus at a temperature lower than about 34°C; and b. selecting viruses that grow at a reduced temperature.

43. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37, 38 or 39 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.

44. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37, 38, 39 or 43 wherein said passaging step is carried out in embryonated chicken eggs.

45. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37, 38, 39 or 44 wherein said cold-adapted equine influenza virus comprises a dominant interference phenotype.

46. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37, 38, 39, 44 or 45 wherein said cold-adapted equine influenza virus is derived from strain A/equine/Kentucky/1/91 (H3N8).

47. The reassortant influenza A virus as set forth in claims 2, 12, 17, 22, 27 or 32 wherein said genome segment is derived from strain A/equine/Kentucky/1/91 (H3N8).

48. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33 or 40 wherein said cold-adapted equine influenza virus or said genome segment is derived from strain A/equine/Kentucky/1/91 (H3N8).

49. The use as set forth in claims 4, 14, 19, 24, 29, 34 or 41 wherein said cold-adapted equine influenza virus or said genome segment is derived from strain A/equine/Kentucky/1/91 (H3N8).

50. The method as set forth in any one of claims 5, 6, 7, 15, 20, 25, 30, 35 and 42 wherein said cold-adapted equine influenza virus or said genome segment is derived from strain A/equine/Kentucky/1/91 (H3N8).

51. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37, 38, 39, 44, 45 or 46 wherein said cold-adapted equine influenza 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.

52. The reassortant influenza A virus as set forth in claims 2, 12, 17, 22, 27, 32 or 47 wherein said genome segment 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.

53. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40 or 48 wherein said cold-adapted equine influenza virus or said genome segment 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.

54. The use as set forth in claims 4, 14, 19, 24, 29, 34, 4l or 49 wherein said cold-adapted equine influenza virus or said genome segment 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.

55. The method as set forth in claims 5, 6, 7, 15, 20, 25, 30, 35, 42 or 50 wherein said cold-adapted equine influenza 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.

56. The cold-adapted equine influenza virus as set forth in claims 1, 11, 16, 21, 26, 31, 36, 37, 38, 39, 44, 45, 46 or 51 wherein said cold-adapted equine influenza virus is selected from the group consisting of:

(a) EIV-P821, identified by accession No. ATCC VR-2625;
(b) EIV-P824, identified by accession No. ATCC VR-2624;
(c) MSV+5, identified by accession No. ATCC VR-2627; and (d) a virus arising from cultures of said virus of (a), (b) or (c) in eggs or in tissue cultured cells.

57. The reassortant influenza A virus as set forth in claims 2, 12, 17, 22, 27, 32, 47 or 52 wherein said genome segment is selected from the group consisting of:

(a) EIV-P821, identified by accession No. ATCC VR-2625;
(b) EIV-P824, identified by accession No. ATCC VR-2624;
(c) MSV+5, identified by accession No. ATCC VR-2627; and (d) a virus arising from cultures of said virus of (a), (b) or (c) in eggs or in tissue cultured cells.

58. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48 or 53 wherein said cold-adapted equine influenza virus or said genome segment is selected from the group consisting of:

(a) EIV-P821, identified by accession No. ATCC VR-2625;
(b) EIV-P824, identified by accession No. ATCC VR-2624;
(c) MSV+5, identified by accession No. ATCC VR-2627; and (d) a virus arising from cultures of said virus of (a), (b) or (c) in eggs or in tissue cultured cells.

59. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49 or 54 wherein said cold-adapted equine influenza virus or said genome segment is selected from the group consisting of:

(a) EIV-P821, identified by accession No. ATCC VR-2625;
(b) EIV-P824, identified by accession No. ATCC VR-2624;
(c) MSV+5, identified by accession No. ATCC VR-2627; and (d) a virus arising from cultures of said virus of (a), (b) or (c) in eggs or in tissue cultured cells.

60. The method as set forth in claims 5, 6, 7, 15, 20, 25, 30, 35, 42, 50 or 55 wherein said cold-adapted equine influenza virus is selected from the group consisting of:

(a) EIV-P821, identified by accession No. ATCC VR-2625;
(b) EIV-P824, identified by accession No. ATCC VR-2624;
(c) MSV+5, identified by accession No. ATCC VR-2627; and (d) a virus arising from cultures of said virus of (a), (b) or (c) in eggs or in tissue cultured cells.

61. The reassortant influenza A virus as set forth in claims 2, 12, 17, 22, 27, 32, 47, 52 or 57 wherein said reassortant 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 (i) 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, and (ii) at least one phenotype of said recipient influenza A virus, wherein said phenotype is selected from the group consisting of hemagglutinin activity and neuraminidase activity, and combinations thereof.

62. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48, 53 or 58 wherein said reassortant 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 (a) 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, and (b) at least one phenotype of said recipient influenza A virus, wherein said phenotype is selected from the group consisting of hemagglutinin activity and neuraminidase activity, and combinations thereof.

63. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49, 54 or 59 wherein said reassortant 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 (a) 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, and (b) at least one phenotype of said recipient influenza A virus, wherein said phenotype is selected from the group consisting of hemagglutinin activity and neuraminidase activity, and combinations thereof.

64. The method as set forth in claims 5, 6, 7, 15, 20, 25, 30, 35, 42, 50, 55 or 60 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.

65. The reassortant influenza A virus as set forth in claims 2, 12, 17, 22, 27, 32, 47, 52, 57 or 61 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.

66. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48, 53, 58 or 62 wherein said animal is an equid.

67. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49, 54, 59 or 63 wherein said animal is an equid.

68. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48, 53, 58, 62 or 66 wherein said therapeutic composition is in a form suitable for administration to said animal by a route that will allow virus entry into mucosal cells of the upper respiratory tract.

69. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49, 54, 59, 63 or 67 wherein said therapeutic composition is in a form suitable for administration to said animal by a route that will allow virus entry into mucosal cells of the upper respiratory tract.

70. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48, 53, 58, 62, 66 or 68 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 in a form suitable for prophylactic administration to an equid, thereby eliciting an immune response against equine influenza virus in said equid.

71. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49, 54, 59, 63, 67 or 69 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 in a form suitable for prophylactic administration to an equid, thereby eliciting an immune response against equine influenza virus in said equid.

72. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48, 53, 58, 62, 66, 68 or 70 wherein said therapeutic composition comprises from about 10 5 TCID50 units to about 10 8 TCID50 units of said virus.

73. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49, 54, 59, 63, 67, 69 or 71 wherein said therapeutic composition comprises from about 10 5 units to about 10 8 TCID50 units of said virus.

74. The therapeutic composition as set forth in claims 3, 13, 18, 23, 28, 33, 40, 48, 53, 58, 62, 66, 68, 70 or 72 wherein said therapeutic composition further comprises an excipient.

75. The use as set forth in claims 4, 14, 19, 24, 29, 34, 41, 49, 54, 59, 63, 67, 69, 71 or 73 wherein said therapeutic composition further comprises an excipient.

76. The isolated equine influenza nucleic acid molecule as set forth in claim 8, wherein said nucleic acid molecule comprises a cold-adapted equine influenza virus 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, and SEQ ID NO:25.

77. The isolated equine influenza nucleic acid molecule as set forth in claims 8 or 76 wherein said nucleic acid molecule comprises a cold-adapted equine influenza virus encoding an M protein, said M protein having an amino acid sequence comprising SEQ ID NO:5.

78. The isolated equine influenza nucleic acid molecule as set forth in claims 8, 76 or 77 wherein said nucleic acid molecule comprises a cold-adapted equine influenza virus encoding an HA protein, said HA protein having an amino acid sequence comprising SEQ ID NO: 11.

79. The isolated equine influenza nucleic acid molecule as set forth in claims 8, 76, 77 or 78 wherein said nucleic acid molecule comprises a cold-adapted equine influenza virus encoding a PB2-N protein, said PB2-N protein having an amino acid sequence comprising SEQ ID NO: 17.

80. The isolated equine influenza nucleic acid molecule as set forth claims 8, 76, 77, 78 or 79 wherein said nucleic acid molecule comprises a cold-adapted equine influenza virus encoding a PB2-C protein, said PB2-C protein having an amino acid sequence comprising SEQ ID NO:24.