# Patent

## Title
Cold-adapted equine influenza viruses

## Abstract

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

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a 35 U.S.C. 371 of International PCT Application No. PCT/US01/05048, filed Feb. 16, 2001; which is a continuation-in-part of U.S. patent application Ser. No. 09/506,286, filed Feb. 16, 2000, now issued as U.S. Pat. No. 6,482,414 B1; all entitled “COLD-ADAPTED EQUINE INFLUENZA VIRUSES” and incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to nucleic acids and proteins of experimentally-generated cold-adapted equine influenza viruses, and particularly to cold-adapted equine influenza viruses having additional phenotypes, such as attenuation, dominant interference, or temperature sensitivity. The invention also includes reassortant influenza A viruses which contain at least one genome segment from such an equine influenza virus, such that the reassortant virus includes certain phenotypes of the donor equine influenza virus. The invention further includes genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise certain identifying phenotypes of a cold-adapted equine influenza virus of the present invention. The present invention also relates to the use of these viruses in therapeutic compositions to protect animals from diseases caused by influenza viruses.
BACKGROUND OF THE INVENTION
Equine influenza virus has been recognized as a major respiratory pathogen in horses since about 1956. Disease symptoms caused by equine influenza virus can be severe, and are often followed by secondary bacterial infections. Two subtypes of equine influenza virus are recognized, namely subtype-1, the prototype being A/Equine/Prague/1/56 (H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8). Presently, the predominant virus subtype is subtype-2, which has further diverged among Eurasian and North American isolates in recent years. The currently licensed vaccine for equine influenza is an inactivated (killed) virus vaccine. This vaccine provides minimal, if any, protection for horses, and can produce undesirable side effects, for example, inflammatory reactions at the site of injection. See, e.g., Mumford, 1987, Equine Infectious Disease IV, 207–217, and Mumford, et al., 1993, Vaccine 11, 1172–1174. Furthermore, current modalities cannot be used in young foals, because they cannot overcome maternal immunity, and can induce tolerance in a younger animal. Based on the severity of disease, there remains a need for safe, effective therapeutic compositions to protect horses against equine influenza disease.
Production of therapeutic compositions comprising cold-adapted human influenza viruses is described, for example, in Maassab, et al., 1960, Nature 7,612–614, and Maassab, et al., 1969, J. Immunol. 102, 728–732. Furthermore, these researchers noted that cold-adapted human influenza viruses, i.e., viruses that have been adapted to grow at lower than normal temperatures, tend to have a phenotype wherein the virus is temperature sensitive; that is, the virus does not grow well at certain higher, non-permissive temperatures at which the wild-type virus will grow and replicate. Various cold-adapted human influenza A viruses, produced by reassortment with existing cold-adapted human influenza A viruses, have been shown to elicit good immune responses in vaccinated individuals, and certain live attenuated cold-adapted reassortant human influenza A viruses have proven to protect humans against challenge with wild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol. 23, 73–76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep. 22, 1992, the inventors of the present invention further demonstrated that certain reassortant cold-adapted human influenza A viruses also possess a dominant interference phenotype, i.e., they inhibit the growth of their corresponding parental wild-type strain, as well as heterologous influenza A viruses.
U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987, and U.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, disclose attenuated therapeutic compositions produced by reassortment of wild-type equine influenza viruses with attenuated, cold-adapted human influenza A viruses. Although these therapeutic compositions appear to be generally safe and effective in horses, they pose a significant danger of introducing into the environment a virus containing both human and equine influenza genes.
SUMMARY OF THE INVENTION
The present invention provides nucleic acids and proteins of experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses.
Examples of cold-adapted equine influenza viruses of the present invention include EIV-P821, identified by accession No. ATCC VR-2625; EIV-P824, identified by accession No. ATCC VR-2624; EIV-MSV+5, identified by accession No. ATCC VR-627; and progeny of such viruses. Cold-adapted equine influenza viruses of the invention, and their methods of making, are disclosed in related U.S. Pat. No. 6,177,082, by Dowling et al., issued Jan. 23, 2001; and WO 00/09702, by Dowling et al., published Feb. 24, 2000, both of which are incorporated herein by reference in their entirety.
The present invention also describes nucleic acid molecules encoding wild-type and cold-adapted equine influenza proteins PB2, NS, PB1, PA and NA. One embodiment of the present invention is an isolated equine nucleic acid molecule having a nucleic acid sequence selected from a group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:19, SEQ ED NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100 and SEQ ID NO:102 and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of such nucleic acid sequences. Another embodiment of the present invention is an isolated equine nucleic acid molecule that encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103. Another embodiment is an isolated equine influenza protein that comprises an amino acid sequence selected from a group consisting of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103. Also included in the present invention is a virus that include any of these nucleic acid molecules or proteins. In one embodiment, such a virus is equine influenza virus or a reassortant virus.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides nucleic acids and proteins of experimentally-generated cold-adapted equine influenza viruses comprising certain defined phenotypes, which are disclosed herein. It is to be noted that the term “a” or “an” entity, refers to one or more of that entity; for example, “a cold-adapted equine influenza virus” can include one or more cold-adapted equine influenza viruses. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising,” “including,” and “having” can be used interchangeably. Furthermore, an item “selected from the group consisting of” refers to one or more of the items in that group, including combinations thereof.
A cold-adapted equine influenza virus of the present invention is a virus that has been generated in the laboratory, and as such, is not a virus as occurs in nature. Since the present invention also includes those viruses having the identifying phenotypes of such a cold-adapted equine influenza virus, an equine influenza virus isolated from a mixture of naturally-occurring viruses, i.e., removed from its natural milieu, but having the claimed phenotypes, is included in the present invention. A cold-adapted equine influenza virus of the present invention does not require any specific level of purity. For example, a cold-adapted equine influenza virus grown in embryonated chicken eggs may be in a mixture with the allantoic fluid (AF), and a cold-adapted equine influenza virus grown in tissue culture cells may be in a mixture with disrupted cells and tissue culture medium.
As used herein, an “equine influenza virus” is an influenza virus that infects and grows in equids, e.g., horses or ponies. As used herein, “growth” of a virus denotes the ability of the virus to reproduce or “replicate” itself in a permissive host cell. As such, the terms, “growth of a virus” and “replication of a virus” are used interchangeably herein. Growth or replication of a virus in a particular host cell can be demonstrated and measured by standard methods well-known to those skilled in the art of virology. For example, samples containing infectious virus, e.g., as contained in nasopharyngeal secretions from an infected horse, are tested for their ability to cause cytopathic effect (CPE), e.g., virus plaques, in tissue culture cells. Infectious virus may also be detected by inoculation of a sample into the allantoic cavity of embryonated chicken eggs, and then testing the AF of eggs thus inoculated for its ability to agglutinate red blood cells, i.e., cause hemagglutination, due to the presence of the influenza virus hemagglutinin (HA) protein in the AF.
Cold-adapted equine influenza viruses of the present invention are characterized primarily by one or more of the following identifying phenotypes: cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation. As used herein, the phrase “an equine influenza virus comprises the identifying phenotype(s) of cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation” refers to a virus having such a phenotype(s). Examples of such viruses include, but are not limited to, EIV-P821, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, and EIV-MSV+5, identified by accession No. ATCC VR-2627, as well as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4.
Pursuant to 37 CFR § 1.802 (a–c), cold-adapted equine influenza viruses, designated herein as EIV-P821, an EIV-P824 were deposited with the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC Accession Nos. ATCC VR-2625, and ATCC VR-2624, respectively, on Jul. 11, 1998. Cold-adapted equine influenza virus EIV-MSV+5 was deposited with the ATCC as ATCC Accession No. ATCC VR-2627 on Aug. 3, 1998. Pursuant to 37 CFR§ 1.806, the deposits are made for a term of at least thirty (30) years and at least five (5) years after the most recent request for the furnishing of a sample of the deposit was received by the depository. Pursuant to 37 CFR § 1.808 (a)(2), all restrictions imposed by the depositor on the availability to the public will be irrevocably removed upon the granting of the patent.
The present invention includes nucleic acid molecules isolated from equine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8), and cold-adapted equine influenza virus EIV-P821.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified.
The present invention includes nucleic acid molecules encoding wild-type and cold-adapted equine influenza virus proteins. Nucleic acid molecules of the present invention can be prepared by methods known to one skilled in the art. Proteins of the present invention can be prepared by methods known to one skilled in the art, i.e., recombinant DNA technology. Preferred nucleic acid molecules have coding strands comprising nucleic acid sequences SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100 and SEQ ID NO:102 and/or a complement thereof. Complements are defined as two single strands of nucleic acid in which the nucleotide sequence is such that they will hybridize as a result of base pairing throughout their full length. Given a nucleotide sequence, one of ordinary skill in the art can deduce the complement.
Preferred nucleic acid molecules encoding equine influenza PB2 proteins are neiwtPB22341, neiwtPB22277, neica1PB22341, and/or neica1PB22277, the coding strands of which are represented by SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, and/or SEQ ID NO:29.
Preferred nucleic acid molecules encoding equine influenza NS proteins are neiwt1NS891, neiwt1NS690, neiwt3NS888, neiwt4NS468, neiwt4NS293, neica1NS888, and/or neica1NS690, the coding strands of which are represented by SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39 and/or SEQ ID NO:41.
Preferred nucleic acid molecules encoding equine influenza PB1-N proteins are neiwt1PB1-N1229, neiwt1PB1-N1194, neiwt2PB1-N673, neiwt2PB1-N636, neica1PB1-N1225, neica1PB1-N1185, neica2PB1-N1221, and/or neica2PB1-N1185, the coding strands of which are represented by SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, and/or SEQ ID NO:51.
Preferred nucleic acid molecules encoding equine influenza PB1-C proteins are neiwt1PB1-C1234, neiwt1PB1-C1188, neiwt2PB1-C1240, neica1PB1-C1241, neica1PB1-C1188, and/or neica2PB1-C1241, the coding strands of which are represented by SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, and/or SEQ ID NO:94.
Preferred nucleic acid molecules encoding equine influenza PB1 proteins are neiwtPB12341, neiwtPB12271, neica1PB12341, neica1PB12271, the coding strands of which are represented by SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, and/or SEQ ID NO:73.
Preferred nucleic acid molecules encoding equine influenza PA-C proteins are neiwt1PA-C1228, neiwt1PA-C1164, neiwt2PA-C1223, neica1PA-C1233, and neica1PA-C1170, the coding strands of which are represented by SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, and/or SEQ ID NO:86.
Preferred nucleic acid molecules encoding equine influenza PA-N proteins are neiwtPA-N1216, neiwtPA-N1193, neicaPA-N1217, and neicaPA-N1193, the coding strands of which are represented by SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97 and SEQ ID NO:98.
Preferred nucleic acid molecules encoding equine influenza PA proteins are neiwtPA2148 and neicaPA2148, the coding strands of which are represented by SEQ ID NO:100 and SEQ ID NO:102.
Preferred nucleic acid molecules encoding equine influenza NA proteins are neicaNA1478 and neicaNA1410, the coding strands of which are represented by SEQ ID NO:87 and SEQ ID NO:89.
The present invention includes proteins comprising SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103 as well as nucleic acid molecules encoding such proteins.
Preferred equine influenza PB2-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPB2-N1241, neiwtPB2-N1214, neica1PB2-N1241, neica1PB2-N1214 neica2, and/or PB2-N1214. Preferred equine influenza PB2-N proteins are PwtPB2-N404, Pca1PB2-N404, and/or Pca2PB2-N404. In one embodiment, a preferred equine influenza PB2-N protein of the present invention is encoded by SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:8, and/or SEQ ID NO:10, and, as such, has an amino acid sequence that includes SEQ ID NO:6 and/or SEQ ID NO:9.
Preferred equine influenza PB2-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PB2C-1233, neiwt2PB2-C1232, neiwtPB2-C1194, neica1PB2-C1232, and/or neica1PB2-C1194. In one embodiment, a preferred equine influenza PB2-C protein of the present invention is encoded by SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:21, and/or SEQ ID NO:23, and, as such, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQ ID NO:22.
Preferred equine influenza PB2 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPB22341, neiwtPB22277, neica1PB22341, and or neica1PB22277. In one embodiment, a preferred equine influenza PB2 protein of the present invention is encoded by SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, and/or SEQ ID NO:29, and, as such, has an amino acid sequence that includes SEQ ID NO:25 and/or SEQ ID NO:28.
Preferred equine influenza NS proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1NS891, neiwt1NS690, neiwt3NS888, neiwt4NS468, neiwt4NS293, neica1NS888, and/or neica1NS690. Preferred equine influenza NS proteins are PeiwtNS230, Peiwt4NS97, and/or Peica1NS230. In one embodiment, a preferred equine influenza NS protein of the present invention is encoded by SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39 and/or SEQ ID NO:41, and, as such, has an amino acid sequence that includes SEQ ID NO:33, SEQ ID NO:37 and/or SEQ ID NO:40.
Preferred equine influenza PB1-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PB1-N1229, neiwt1PB1-N1194, neiwt2PB1-N673, neiwt2PB1-N636, neica1PB21-N1225, neica1PB1-N1185, and/or neica2PB1-N1221. Preferred equine influenza PB1-N proteins are Peiwt1PB1-N398, Pwt2PB1-N212 and/or Pca1PB1-N395. In one embodiment, a preferred equine influenza PB1-N protein of the present invention is encoded by SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, and/or SEQ ID NO:51, and, as such, has an amino acid sequence that includes SEQ ID NO:43, SEQ ID NO:46 and/or SEQ ID NO:49.
Preferred equine influenza PB1-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PB1-C1234, neiwt1PB1-C1188, neiwt2PB1-C1240, neica1PB1-C1241, neica1PB1-C1188, and/or neica2PB1-C1241. Preferred equine influenza PB1-C proteins are Peiwt1PB1-C396, Peiwt2PB1-C396 Peica1PB1-C396, and/or Peica2PB1-C396. In one embodiment, a preferred equine influenza PB1-C protein of the present invention is encoded by SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, and/or SEQ ID NO:66, and, as such, has an amino acid sequence that includes SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, and/or SEQ ID NO:67.
Preferred equine influenza PB1 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPB12341, neiwtPB12271, neica1PB12341, neica1PB12271. Preferred equine influenza PB1 proteins are PeiwtPB1757, and/or Peica1PB1757. In one embodiment, a preferred equine influenza PB1 protein of the present invention is encoded by SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, and/or SEQ ID NO:73, and, as such, has an amino acid sequence that includes SEQ ID NO:69 and/or SEQ ID NO:72.
Preferred equine influenza PA-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwt1PA-C1228, neiwt1PA-C1164, neiwt2PA-C1223, neica1PA-C1233, and/or neica1PA-C1170. Preferred equine influenza PA-C proteins are Peiwt1PA-C388, and/or Peica1PA-C390. In one embodiment, a preferred equine influenza PA-C protein of the present invention is encoded by SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, and/or SEQ ID NO:86, and, as such, has an amino acid sequence that includes SEQ ID NO:77 and/or SEQ ID NO:85.
Preferred equine influenza PA-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPA-N1216, neiwtPA-N1193, neicaPA-N1217 and neicaPA-N1193. Preferred equine influenza PA-N proteins are PeiwtPA-N397 and/or PeicaPA-N397. In one embodiment, a preferred equine influenza PA-N protein of the present invention is encoded by SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, and/or SEQ ID NO:98, and, as such, has an amino acid sequence that includes SEQ ID NO:96 and/or SEQ ID NO:99.
Preferred equine influenza PA proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neiwtPA1248 and neicaPA1248. Preferred equine influenza PA proteins are PeiwtPA716 and/or PeicaPA716. In one embodiment, a preferred equine influenza PA protein of the present invention is encoded by SEQ ID NO:100 and/or SEQ ID NO:102, and, as such, has an amino acid sequence that includes SEQ ID NO:101 and/or SEQ ID NO:103.
Preferred equine influenza NA proteins of the present invention include proteins encoded by a nucleic acid molecule comprising neicaNA1478 and neicaNA1410. A preferred equine influenza NA protein is PeicaNA470. In one embodiment, a preferred equine influenza NA protein of the present invention is encoded by SEQ ID NO:87 and/or SEQ ID NO:89, and, as such, has an amino acid sequence that includes SEQ ID NO:88.
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:9. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-C protein having an amino acid sequence comprising SEQ ID NO:22. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2 protein having an amino acid sequence comprising SEQ ID NO:28. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a NS protein having an amino acid sequence comprising SEQ ID NO:40. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-N protein having an amino acid sequence comprising SEQ ID NO:49. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PA-C protein having an amino acid sequence comprising SEQ ID NO:85. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-C protein having an amino acid sequence comprising SEQ ID NO:64 and/or SEQ ID NO:67. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1 protein having an amino acid sequence comprising SEQ ID NO:72. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PA-N protein having an amino acid sequence comprising SEQ ID NO:99. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PA protein having an amino acid sequence comprising SEQ ID NO:103. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a NA protein having an amino acid sequence comprising SEQ ID NO:88. 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 PB2-N, PB2-C, PB2, NS, PB1-N, PB1-C, PB1, PA-C, PA-N, PA, and NA proteins of the present invention.
Another embodiment of the present invention is an antibody that selectively binds to an wild-type virus PB2-N, PB2-C, PB2, NS, PB1-N, PB1-C, PB1, PA-C, PA-N, PA and NA protein of the present invention. Another embodiment of the present invention is an antibody that selectively binds to a cold-adapted virus PB2-N, PB2-C, PB2, NS, PB1-N, PB1-C, PB1, PA-C, PA-N, PA and NA protein of the present invention. Preferred antibodies selectively bind to SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
EXAMPLE 1
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 a 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 neiwtPB2-N1241, with a coding strand having a nucleic acid sequence designated SEQ ID NO:13 was produced by further PCR amplification using the above described PCR product as a template and cloned into pCR 2.1®TA cloning vector, available from Invitrogen, Carlsbad, Calif., using standard procedures recommended by the manufacturer. The primers used were the T7 primer, designated by SEQ ID NO:29 and the REV primer, designated by SEQ ID NO:28. Plasmid DNA was purified using a mini-prep method available from Qiagen, Valencia, Calif. PCR products were prepared for sequencing using a PRISMTM Dye Terminator Cycle Sequencing Ready Reaction kit, a PRISMTM dRhodamine Terminator Cycle Sequencing Ready Reaction kit, or a PRISMTM BigDyeTM Terminator Cycle Sequencing Ready Reaction kit, all available from PE Applied Biosystems, Foster City, Calif., following the manufacturer's protocol. Specific PCR conditions used with the kit were a rapid ramp to 95° 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 minute hold, repeating for 25 cycles. T7 and REV primers were used in one reaction. PCR products were purified by ethanol/magnesium chloride precipitation. Automated sequencing of DNA samples was performed using an ABI PRISMTM Model 377 with XL upgrade DNA Sequencer, available from PE Applied Biosystems.
Translation of SEQ ID NO:13 indicates that nucleic acid molecule neiwtPB2-N1241 encodes a N-terminal portion of influenza PB2 protein of about 404 amino acids, referred to herein as PwtPB2-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 PwtPB2-N404, designated neiwtPB2-N1214, and having a coding strand comprising nucleotides 28 to 1239 of SEQ ID NO:13 is represented by SEQ ID NO:15.
B. A nucleic acid molecule of 1239 nucleotides encoding a N-terminal portion of influenza PB2 cold-adapted equine influenza virus PB2-N protein, denoted neica1PB2-N1241, with a coding strand having a sequence designated SEQ ID NO:16 was produced, and sequenced as described in part A.
Translation of SEQ ID NO:16 indicates that nucleic acid molecule neica1PB2-N1241 encodes a—terminal portion of equine influenza PB-2 protein of about 404 amino acids, referred to herein as Pca1PB2-N404, having amino acid sequence SEQ ID NO:17, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:16, and the last codon spans from nucleotide 1237 through nucleotide 1239. The region encoding Pca1PB2-N404, designated neica1PB2-N1214, 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 neica2PB2-N1241, identical to neica1PB2-N1241, and neica2PB2-N1214, identical to neica1PB2-N1214.
C. Comparison of the nucleic acid sequences of the coding strands of neiwtPB2-N1241 (SEQ ID NO:13) and neica1PB2-N1241 (SEQ ID NO:16) by DNA alignment reveals the following difference: a T to C base shift at base 370. Comparison of the amino acid sequences of proteins PwtPB2-N404 (SEQ ID NO:14) and Pca1PB2-N404 (SEQ ID NO:17) reveals the following difference: a Y to H shift at amino acid 124 relating to the a T to C shift at base 370 in the DNA sequence.
EXAMPLE 2
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 amplification using from equine influenza virus DNA and primers w572 and w573, designated SEQ ID NO:38 and SEQ ID NO:39, respectively. A nucleic acid molecule of 1233 nucleotides encoding a wild type PB2-C protein, denoted neiwtPB2-C1233, 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 1B. Plasmid DNA was purified and sequenced as in Example 1A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance; efPB2-a1, designated SEQ ID NO:40 and efPB2-s1, designated SEQ ID NO:41 were used in another instance, and efPB2-a2, designated SEQ ID NO:42 and efPB2-s2, designated SEQ ID NO:43 were used in another instance.
Translation of SEQ ID NO:19 indicates that nucleic acid molecule neiwt1PB2-C1233 encodes a C-terminal portion of influenza PB2 protein of about 398 amino acids, referred to herein as PwtPB2-C398, having amino acid sequence SEQ ID NO:20, assuming an open reading frame having a first codon spans from nucleotide 3 through nucleotide 5 and a termination codon which spans from nucleotide 1197 through nucleotide 1199 of SEQ ID NO:19. Because SEQ ID NO:19 is only a partial gene sequence, it does not contain an initiation codon. The region encoding PwtPB2-C398, designated neiwtPB2-C1194, and having a coding strand comprising nucleotides 3 to 1196 of SEQ ID NO:19 is represented by SEQ ID NO:21.
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 neiwt2PB2-N1232 with a coding strand with a sequence designated SEQ ID NO:22. neiwt2PB2-N1232 is identical to neiwt1PB2-C1233, expect that neiwt2PB2-N1232 lacks one nucleotide on the 5′-end. Translation of SEQ ID NO:22 indicates that nucleic acid molecule neiwt1PB2-C1233 also encodes PwtPB2-C398 (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 neiwt2PB2-C1194, is identical to SEQ ID NO:21.
B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminal portion of influenza PB2 cold-adapted equine influenza virus protein, denoted neica1PB2-C1232, and having a coding strand having a sequence designated SEQ ID NO:23 was produced as described in part A, except that the pCR®-Blunt cloning vector was used.
Translation of SEQ ID NO:23 indicates that nucleic acid molecule neica1PB2-C1232 encodes a C-terminal portion of equine influenza PB-2 protein of about 398 amino acids, referred to herein as Pca1PB2-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 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 Pca1PB2-C398, designated neica1PB2-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 neica2PB2-C1231, containing one less nucleotide at the 3′end than neica1PB2-N1241; and neica2PB2-N1214, identical to neica1PB2-N1214.
C. Comparison of the nucleic acid sequences of the coding strands of neiwt1PB2-C1233 (SEQ ID NO:19) and neica1PB2-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 to A base shift at base 929 of SEQ ID NO:19. Comparison of the amino acid sequences of proteins PwtPB2-C398 (SEQ ID NO:20) and Pca1PB2-398 (SEQ ID NO:24) reveals the following difference: a K to Q shift at amino acid 51 when relating to the an A to C base shift at base 153 in the DNA sequences. There is no amino acid shift resulting from the G to A base shift at base 929.
EXAMPLE 3
This example describes the cloning and sequencing of equine influenza PB2 protein (RNA-directed RNA polymerase) nucleic acid molecules for wild type or cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2 proteins were produced as follows. The wild type or cold-adapted equine influenza genes were cloned in two fragments, the N-terminal portion was produced as in Example 1 and the C-terminal portion of the gene was produced as in Example 2. The DNA sequence for the wild type equine influenza PB2 gene was generated by combining the consensus sequences for the wild type PB2-N protein, denoted neiwtPB2-N1241, (SEQ ID NO:13) with the gene fragments for the wild type PB2-C protein, denoted neiwt1PB2-C1233 (SEQ ID NO:19) and neiwt2PB2-C1232 (SEQ ID NO:22). The result of combining the consensus sequences from the N-terminal and C-terminal portions of the PB2 wild type influenza virus yielded a complete DNA sequence denoted neiwtPB22341 (SEQ ID NO:44). Translation of SEQ ID NO:44 indicates that the nucleic acid molecule neiwtPB22341 encodes a full length equine influenza PB2 protein of about 759 amino acids referred to herein as PeiwtPB2759, having amino acid sequence SEQ ID NO: 45, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO: 44 and the termination codon spans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO: 44. The region encoding PeiwtPB2759, designated neiwtPB22277, and having a coding strand comprising nucleotides 28 to 2304 of SEQ ID NO: 44, is SEQ ID NO: 46.
B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equine influenza virus PB2, denoted neica1PB22341, with a sequence denoted SEQ ID NO: 47 was produced by combining the sequences for the N-terminal and C-terminal portions of the PB2 cold-adapted equine influenza gene. The clones for the N-terminal sequences are denoted neica1PB2-N1241, and neica2PB2-N1241, which are identical and are represented by SEQ ID NO:16. The clones for the C-terminal sequences are denoted neica1PB2-C1232 and neica2PB2-C1231, represented by SEQ ID NO:23.
Translation of SEQ ID NO:47 indicates that nucleic acid molecule neica1PB22341 encodes a full-length equine influenza PB2 protein of about 759 amino acids, referred to herein as Peica1PB2759, having amino acid sequence SEQ ID NO:48, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO: 47 and the termination codon spans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO:47. The region encoding Peica1PB2759 designated neica1PB22277 and having a coding strand comprising nucleotides 28 to 2304 of SEQ ID NO:49.
C. Comparison of the nucleic acid sequences of the coding strands of neiwtPB2341 (SEQ ID NO:44) and neica1PB22341 (SEQ ID NO:47) by DNA alignment reveals the following differences: a T to C base shift at base 370, a A to C base shift at base 1261, and a G to A base shift at base 2037. Comparison of the amino acid sequences of proteins PeiwtPB2759 (SEQ ID NO:45) and Peica1PB2759 (SEQ ID NO:48) reveals the following differences: a Y to H shift at amino acid 124 relating to the a T to C shift at base 370 in the DNA sequence, a K to Q shift at amino acid 421 relating to the A to C shift at base 1261 in the DNA sequence. The third nucleotide shift at base 2037 does not result in an amino acid shift.
EXAMPLE 4
This example describes the cloning and sequencing of equine influenza NS (nonstructural) protein nucleic acid molecules for wild type or cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus NS proteins were produced as follows. A PCR product containing an equine NS gene was produced by PCR amplification from equine influenza virus DNA and primers w586 and w587, designated SEQ ID NO:59 and SEQ ID NO:60, respectively. A nucleic acid molecule of 891 nucleotides encoding a wild-type NS protein, denoted neiwtNS891, with a coding strand having a nucleic acid sequence designated SEQ ID NO:50 was produced by further PCR amplification using the above-described PCR product as a template and cloned into pCR 2.1®TA cloning vector as described in Example 1A. Plasmid DNA was purified and sequenced as in Example 1A, except that primers used in the sequencing kits were only T7 and REV.
Translation of SEQ ID NO:50 indicates that nucleic acid molecule neiwt1NS891 encodes a full-length equine influenza NS protein of about 230 amino acids, referred to herein as Peiwt1NS230, having amino acid sequence SEQ ID NO:51, assuming an open reading frame in which the initiation codon spans from nucleotide 27 through nucleotide 29 of SEQ ID NO:50 and the termination codon spans from nucleotide 717 through nucleotide 719 of SEQ ID NO:50. The region encoding Peiwt1NS230, designated neiwt1NS690, and having a coding strand comprising nucleotides 27 to 716 of SEQ ID NO:50 is represented by SEQ ID NO:52.
PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza NS protein in the same manner resulted in molecules neiwt2NS891, identical to neiwt1NS891, in the coding region, i.e. neiwt2NS690, is identical to neiwt1NS690. neiwt2NS891 differs from neiwt1NS891 in one nucleotide at base 827 (G to A) which is 111 bases downstream from the stop codon. PCR amplification of a third nucleic acid encoding a wild type equine influenza NS protein in the same manner resulted in a nucleic acid molecule of 888 nucleotides denoted neiwt3NS888, with a coding strand with a nucleic acid sequence designated SEQ ID NO: 53. neiwt3NS888 is identical to neiwt1NS891, except that neiwt3NS888, lacks two nucleotides on the 5′ end and one nucleotide on the 3′ end. Translation of SEQ ID NO:53 indicates that nucleic acid molecule neiwt3NS888 also encodes Peiwt1NS230 (SEQ ID NO:51), assuming an open reading frame having an initiation codon which spans from nucleotide 25 through nucleotide 27 of SEQ ID NO:53 and a termination codon which spans from nucleotide 715 through nucleotide 717 of SEQ ID NO:53. The nucleic acid molecule having a coding strand comprising nucleotides 25 to 714 of SEQ ID 53, denoted neiwt3NS690, is identical to SEQ ID NO:52.
PCR amplification of a fourth nucleic acid of 468 nucleotides encoding a C-terminal portion of the wild type equine influenza NS protein, denoted neiwt4NS468 and having a coding sequence designated SEQ ID NO:54 was produced. Translation of SEQ ID NO:54 indicates that nucleic acid molecule neiwt4NS468 encodes a C-terminal portion of equine influenza NS protein of about 97 amino acids, referred to herein as Peiwt4NS97, having amino acid sequence SEQ ID NO:55, assuming an open reading frame having a first codon which spans from nucleotide 3 to 5 of SEQ ID NO: 54, and a termination codon spans from nucleotide 294 through 296 of SEQ ID NO:54. Because SEQ ID NO:54 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Peiwt4NS97, designated neiwt4NS293, and having a coding strand comprising nucleotides 1 to 293 of SEQ ID NO:54, is represented by SEQ ID NO: 56.
B. A nucleic acid molecule of 888 nucleotides encoding a cold-adapted equine influenza virus NS protein, denoted neica1NS888, with a coding strand having a sequence designated SEQ ID NO:57 was produced and sequenced as described in part A.
Translation of SEQ ID NO:57 indicates that nucleic acid molecule neica1NS888 encodes a full-length equine influenza NS protein of about 230 amino acids, referred to herein as Peica1NS230, having amino acid sequence SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 27 through nucleotide 29 of SEQ ID NO:57 and the termination codon spans from nucleotide 717 through nucleotide 719 of SEQ ID NO:57. The region encoding Peica1NS230, designated neica1NS690, and having a coding strand comprising nucleotides 27 to 716 of SEQ ID NO:57, is represented by SEQ ID NO:59.
PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza NS protein in the same manner resulted in molecules neica2NS887, containing one less nucleotide at the 3′ end than neica1NS888; the coding region neica2NS690 is identical to neica1NS690.
C. Comparison of the nucleic acid sequences of the coding strands of neiwtNS891 (SEQ ID NO:50) and neica1NS888 (SEQ ID NO:57) by DNA alignment reveals the following difference: a A to G shift at base 827 which is 111 bases downstream from the stop codon. The 3′ fragment encoding neiwt4NS468 (SEQ ID NO:54) has one shift T to C found at base 633 relative to the full-length consensus sequence. Comparison of the amino acid sequences of proteins PeiwtNS230 (SEQ ID NO:51) and Peica1NS230 (SEQ ID NO:58) reveals that there are no differences between amino acid sequences of the wild type and cold-adapted proteins.
EXAMPLE 5
This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase 1) nucleic acid molecules corresponding to the N-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1-N proteins were produced as follows. A PCR product containing a N-terminal portion of the equine PB1 gene was produced by PCR amplification from equine influenza virus DNA, and primers T7 and REV. A nucleic acid molecule of 1229 nucleotides encoding a wild type PB1-N protein, denoted neiwt1PB1-N1229, with a coding strand having a nucleic acid sequence designated SEQ ID NO:62 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 1B. Plasmid DNA was purified and sequenced as in Example 1B, except that only T7 and REV primers were used in the sequencing kits.
Translation of SEQ ID NO:62 indicates that nucleic acid molecule neiwt1PB1-N1229 encodes a N-terminal portion of influenza PB1 protein of about 398 amino acids, referred to herein as Peiwt1PB1-N398, having amino acid sequence SEQ ID NO:63, assuming an open reading frame in which the initiation codon spans from nucleotide 36 through nucleotide 38 of SEQ ID NO:62, and the last codon spans from nucleotide 1227 through nucleotide 1229 of SEQ ID NO:62. The region encoding Peiwt1PB1-N398, designated neiwt1PB1-N1194, and having a coding strand comprising nucleotides 36 to 1229 of SEQ ID NO:62 is represented by SEQ ID NO:64.
PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB1-N protein in the same manner resulted in a nucleic acid molecule of 673 nucleotides denoted neiwt2PB1-N673, with a coding strand with a sequence designated SEQ ID NO:65. Translation of SEQ ID NO:65 indicates that nucleic acid molecule neiwt2PB1-N673 encodes Peiwt2PB1-N212 (SEQ ID NO:66), assuming an open reading frame having an initiation codon which spans from nucleotide 36 through nucleotide 38 of SEQ ID NO:65 and a last codon which spans from nucleotide 671 through nucleotide 673 of SEQ ID NO:65. Because SEQ ID NO:65 is only a partial gene sequence, it does not contain a stop codon. The nucleic acid molecule having a coding strand comprising nucleotides 36 to 671 of SEQ ID NO:65, denoted neiwt2PB1-N636, is designated SEQ ID NO:67.
B. A nucleic acid molecule of 1225 nucleotides encoding a N-terminal portion of influenza PB1 cold-adapted equine influenza virus PB1-N protein, denoted neica1PB1-N1225, with a coding strand having a sequence designated SEQ ID NO:68 was produced, and sequenced as described in part A.
Translation of SEQ ID NO:68 indicates that nucleic acid molecule neica1PB1-N1225 encodes a N-terminal portion of equine influenza PB-1 protein of about 395 amino acids, referred to herein as Peica1PB1-N395, having amino acid sequence SEQ ID NO:69, assuming an open reading frame in which the initiation codon spans from nucleotide 34 through nucleotide 36 of SEQ ID NO:68, and a last codon which spans from nucleotide 1216 through nucleotide 1218 of SEQ ID NO:68. The region encoding Peica1PB1-N395, designated neica1PB1-N1185, and having a coding strand comprising nucleotides 34 to 1218 of SEQ ID NO:68, is represented by SEQ ID NO:70.
PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB1-N protein in the same manner resulted in molecules neica2PB1-N1221, designated SEQ ID NO:71, containing four less nucleotides at the 5′ end than neica1PB1-N1225; the coding region neica2PB1-N1185, is identical to neica1PB1-N1185.
C. Comparison of the nucleic acid sequences of the coding strands of neiwtPB1-N1229 (SEQ ID NO:62) and neica1PB1-N1225 (SEQ ID NO:68) by DNA alignment reveals no differences in the coding regions. Comparison of the amino acid sequences of proteins PeiwtPB1-N395 (SEQ ID NO:63) and Peica1PB1-N395 (SEQ ID NO:69) also reveals no differences.
EXAMPLE 6
This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase 1) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1-C proteins were produced as follows. A PCR product containing an C-terminal portion of the equine PB1 gene was produced by PCR amplification from equine influenza virus DNA, and primer w569 designated SEQ ID NO:102. A nucleic acid molecule of 1234 nucleotides encoding a wild type PB1-C protein, denoted neiwt1PB1-C1234, with a coding strand having a nucleic acid sequence designated SEQ ID NO:85 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 1B. Plasmid DNA was purified and sequenced as in Example 1A, except that different primers were used in the sequencing kits. T7, REV, w569, efPB1-a1, designated SEQ ID NO:97, efPB1-a2, designated SEQ. ID NO:98, efPB1-s1, designated SEQ ID NO: 99, efPB1-s2, designated SEQ ID NO: 100, and efPB1-s3, designated SEQ ID NO:101 were used in one instance, T7, REV, efPB1-a1, efPB1-a2, efPB1-s1, efPB1-s2, and efPB1-s3 were used in another instance and T7 and REV were used in another instance.
Translation of SEQ ID NO:85 indicates that nucleic acid molecule neiwt1PB1-C1234 encodes an C-terminal portion of influenza PB1 protein of about 396 amino acids, referred to herein as Peiwt1PB1-C396, having amino acid sequence SEQ ID NO:86, assuming an open reading frame in which the first codon spans from nucleotide 1 through nucleotide 3 of SEQ ID NO:85 and a termination codon which spans from nucleotide 1189 through nucleotide 1191 of SEQ ID NO:85. Because SEQ ID NO:85 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Peiwt1PB1-C396, designated neiwt1PB1-C1188, and having a coding strand comprising nucleotides 1 to 1188 of SEQ ID NO:85 is represented by SEQ ID NO:87.
PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB1-C protein in the same manner resulted in a nucleic acid molecule of 1240 nucleotides denoted neiwt2PB1-C1240, with a coding strand with a sequence designated SEQ ID NO:88. Translation of SEQ ID NO:88 indicates that nucleic acid molecule neiwt2PB1-N1240 encodes a molecule designated Peiwt2PB1-C396 (SEQ ID NO: 89) which differs from Peiwt1PB1-C396 (SEQ ID NO:85) in one nucleotide. Nucleotide 382 of neiwt1PB1-C1234, i.e. nucleotide 382 of neiwt1PB1-C1188 was A, while nucleotide 389 of neiwt2PB1-C1240, i.e. nucleotide 382 of neiwt2PB1-C1188 was T. Translation of neiwt2PB1-C1240 results in an amino acid change of T to S.
B. A nucleic acid molecule of 1241 nucleotides encoding an C-terminal portion of influenza PB1 cold-adapted equine influenza virus PB1-C protein, denoted neica1PB1-C1241, with a coding strand having a sequence designated SEQ ID NO:91 was produced, and sequenced as described in part A.
Translation of SEQ ID NO:91 indicates that nucleic acid molecule neica1PB1-C1241 encodes an C-terminal portion of equine influenza PB-1 protein of about 396 amino acids, referred to herein as Peica1PB1-C396, having amino acid sequence SEQ ID NO:92, assuming an open reading frame in which the first codon spans from nucleotide 8 through nucleotide 10 of SEQ ID NO:91 and a termination codon that spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:91. Because SEQ ID NO:91 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Peica1PB1-C396, designated neica1PB1-C1188, and having a coding strand comprising nucleotides 8 to 1195 of SEQ ID NO:91, is represented by SEQ ID NO:93.
PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB1-C protein in the same manner resulted in a nucleic acid molecule of 1241 nucleotides denoted neica2PB1-C1241, with a coding strand with a sequence designated SEQ ID NO:94. Translation of SEQ ID NO:94 indicates that nucleic acid molecule neica2PB1-C1241 encodes a molecule designated Peica2PB1-C396 (SEQ ID NO:95) which differs from Peica1PB1-C396 (SEQ ID NO:92) in one nucleotide. Nucleotide 1044 of neica1PB1-C1241, i.e. nucleotide 1037 of neica1PB1-N1188 was A, while nucleotide 1044 of neica2PB1-C1241, i.e. nucleotide 1037 of neica2PB1-C1188 was G. Translation of neica2PB1-C1241 results in an amino acid change of R to K.
C. Comparison of the nucleic acid sequences of the coding strands of neiwt1PB1-C1234 (SEQ ID NO:85) and neica1PB1-C1241 (SEQ ID NO:91) by DNA alignment reveals the following differences: a C to T shift at base 600 of SEQ ID NO:85, and a T to A shift at base 603 of SEQ ID NO:85. Comparison of the amino acid sequences of proteins Peiwt1PB1-C396 (SEQ ID NO:86) and Peica1PB1-N396 (SEQ ID NO:92) reveals the following difference: a H to Q amino acid shift 203 when relating to the T to A base shift at base 603 in the DNA sequences. There is no amino acid shift resulting from the C to T base shift at base 600.
EXAMPLE 7
This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase) nucleic acid molecules for wild type or cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1 proteins were produced as follows. The wild type or cold-adapted equine influenza genes were cloned in two fragments, the N-terminal portion was produced as in Example 5 and the C-terminal portion of the gene was produced as in Example 6.
The DNA sequence for the wild type equine influenza PB1 gene was generated by combining the sequences for the wild type PB1-N protein, neiwt1PB1-N1229 (SEQ ID NO:62) and neiwt2PB1-N673 (SEQ ID NO:65) with the gene fragments for the wild type PB1-C protein, denoted neiwt1PB1-C1234 (SEQ ID NO:85) and neiwt2PB1-C1240 (SEQ ID NO: 88). The result of combining the N-terminal and C-terminal portions of the PB1 wild type influenza virus yielded a complete DNA sequence of 2341 nucleotides denoted neiwtPB12341 (SEQ ID NO:103). Translation of SEQ ID NO:103 indicates that the nucleic acid molecule neiwtPB22341 encodes a full length equine influenza PB1 protein of about 757 amino acids referred to herein as PeiwtPB1757, having amino acid sequence SEQ ID NO:104, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 27 of SEQ ID NO: 103 and the termination codon spans from nucleotide 2293 through nucleotide 2295 of SEQ ID NO: 103. The region encoding, PeiwtPB1757 designated neiwtPB12271, and having a coding strand comprising nucleotides 25 to 2292 of SEQ ID NO: 103, is SEQ ID NO:105.
B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equine influenza virus PB1, denoted neica1PB12341, with a sequence denoted SEQ ID NO: 106 was produced by combining the sequences for the N-terminal and C-terminal portions of the PB1 cold-adapted equine influenza gene. The clones for the N-terminal sequences are denoted neica1PB1-N1225 (SEQ ID NO: 68) and neica2PB1-N1221 (SEQ ID NO: 71). The clones for the C-terminal sequences are denoted neica1PB1-C1241 (SEQ ID NO:91) and neica2PB1-C1241, (SEQ ID NO: 94).
Translation of SEQ ID NO:106 indicates that nucleic acid molecule neica1PB12341 encodes a fill-length equine influenza PB1 protein of about 757 amino acids, referred to herein as Peica1PB1757, having amino acid sequence SEQ ID NO:107, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 27 SEQ ID NO: 106 and the termination codon spans from nucleotide 2296 through nucleotide 2298 of SEQ ID NO:106. The region encoding Peica1PB1757 designated neica1PB12271 and having a coding strand comprising nucleotides 25 to 2295 of SEQ ID NO:108.
C. Comparison of the nucleic acid sequences of the coding strands of neiwtPB12341 (SEQ ID NO:103) and neica1PB12341 (SEQ ID NO:106) by DNA alignment reveals the following differences: a C to T base shift at base 1683, and a T to A base shift at base 1686. Comparison of the amino acid sequences of proteins PeiwtPB1757 (SEQ ID NO:104) and Peica1PB1757 (SEQ ID NO:107) reveals the following differences: no shift in base C at amino acid 561 relating to the C to T shift at base 1683, and a H to Q shift at amino acid 562 relating to the a T to A shift at base 1683 in the DNA sequence.
EXAMPLE 8
This example describes the cloning and sequencing of equine influenza PA protein ( RNA polymerase A) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PA-C proteins were produced as follows. A PCR product containing the C-terminal portion of the equine PA gene was produced by PCR amplification using from equine influenza virus DNA and primers C+PA and C−PA, designated SEQ ID NO:83 and SEQ ID NO:84 respectively. A nucleic acid molecule of 1228 nucleotides encoding a wild type PA-C protein, denoted neiwt1PA-C1228, with a coding strand having a nucleic acid sequence designated SEQ ID NO:76 was produced by further PCR amplification using the above-described PCR product as a template and cloned as described in Example 1B. Plasmid DNA was purified and sequenced as in Example 1A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance; PAC-1,designated SEQ ID NO:72, PAC-2, designated SEQ ID NO:73, PAC-3, designated SEQ ID NO:74, PAC-4, designated SEQ ID NO: 75, T7 and REV were used in another instance; and PAC-1, PAC-2, T7 and REV were used in another instance.
Translation of SEQ ID NO:76 indicates that nucleic acid molecule neiwt1PA-C1228 encodes a C-terminal portion of influenza PA protein of about 388 amino acids, referred to herein as Peiwt1PA-C388, having amino acid sequence SEQ ID NO:77, assuming an open reading frame having a first codon spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:76 and a termination codon which spans from nucleotide 1167 through nucleotide 1169 of SEQ ID NO:76. Because SEQ ID NO:76 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Peiwt1PA-C388, designated neiwt1PA-C1164, and having a coding strand comprising nucleotides 3 to 1166 of SEQ ID NO:76 is represented by SEQ ID NO:78.
PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PA-C protein in the same manner resulted in a nucleic acid molecule of 1223 nucleotides denoted neiwt2PA-C1223, with a coding strand with a sequence designated SEQ ID NO:79. neiwt2PA-C1223 is identical to neiwt1PA-C1228, with the exception of a T to C base shift at base 753 and that neiwt2PA-C1223 lacks five nucleotides on the 3′-end. Translation of SEQ ID NO:79 indicates that nucleic acid molecule neiwt2PA-C1223 also encodes Peiwt1PA-C388 (SEQ ID NO:77), assuming an open reading frame having a first codon which spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:79 and a termination codon which spans from nucleotide 1167 through nucleotide 1169 of SEQ ID NO:79. Because SEQ ID NO:79 is only a partial gene sequence, it does not contain an initiation codon. The nucleic acid molecule having a coding strand comprising nucleotides 3 to 1166 of SEQ ID NO:79, denoted neiwt2PA-C1223, is identical to SEQ ID NO 78.
B. A nucleic acid molecule of 1233 nucleotides encoding a C-terminal portion of influenza PA-C cold-adapted equine influenza virus protein, denoted neica1PA-C1233, and having a coding strand having a sequence designated SEQ ID NO:80 was produced as described in part A, except that the pCR®-Blunt cloning vector was used.
Translation of SEQ ID NO:80 indicates that nucleic acid molecule neica1PA-C1233 encodes a C-terminal portion of equine influenza PA protein of about 390 amino acids, referred to herein as Peica1PA-C390, having amino acid sequence SEQ ID NO:81, assuming an open reading frame having a first codon which spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:80 and a termination codon which spans from nucleotide 1173 through nucleotide 1175 of SEQ ID NO:80. Because SEQ ID NO:80 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Peica1PA-C390, designated neica1PA-C1170, and having a coding strand comprising nucleotides 3 to 1172 of SEQ ID NO:80, is represented by SEQ ID NO:82.
PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PA-C protein in the same manner resulted in molecule neica2PA-C1233, containing one A to G base shift at base 953 as compared to neica1PA-C1233; this base shift does not result in an amino acid change so Peica2PA-C390, is identical to Peica1PA-C390 (SEQ ID NO:81.)
C. Comparison of the nucleic acid sequences of the coding strands of neiwt1PA-C1228 (SEQ ID NO:76) and neica1PA-C1233 (SEQ ID NO:80) by DNA alignment reveals the following difference: an C to T base shift at base 753 of SEQ ID NO:80. Comparison of the amino acid sequences of proteins Peiwt1PA-C388 (SEQ ID NO:77) and Peica1PA-390 (SEQ ID NO:81) reveals the following difference: a W to R shift at amino acid 251 when relating to the C to T base shift at base 753 in the DNA sequences.
EXAMPLE 9
This example describes the cloning and sequencing of equine influenza PA protein 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 PA-N proteins were produced as follows. A PCR product containing the N-terminal portion of the equine influenza PA gene was produced using equine influenza virus DNA and primers REV (SEQ ID NO:1), T7 (SEQ ID NO:2), PAN-2 (SEQ ID NO:90), PAN-3 (SEQ ID NO:91), PAN-4 (SEQ ID NO:92), and PAN-5 (SEQ ID NO:93). Plasmid DNA was purified and sequenced as in Example 1.
A nucleic acid molecule of 1216 nucleotides encoding a wild type PA-N protein, denoted neiwtPA-N1216, with a coding strand having a nucleic acid sequence designated SEQ ID NO:X5.
Translation of SEQ ID NO:X5 indicates that nucleic acid molecule neiwtPA-N1216 encodes a N-terminal portion of influenza PA protein of about 397 amino acids, referred to herein as PeiwtPA-N397, having amino acid sequence SEQ ID NO:X7, assuming an open reading frame from the starting methionine (M) through glutamic acid (E) at amino acid 397. The region encoding PeiwtPA-N397 designated neiwtPA-N1193 and having a coding strand comprising nucleotides 24 to 1214 of SEQ ID NO:X5, is herein designated SEQ ID NO:X6.
B. A nucleic acid molecule of 1217 nucleotides encoding an N-terminal portion of influenza PA-N cold-adapted equine influenza virus protein, denoted neicaPA-N1217, with a coding strand having a nucleic acid sequence designated SEQ ID NO:X8.
Translation of SEQ ID NO:X9 indicates that nucleic acid molecule neicaPA-N1217 encodes a N-terminal portion of influenza PA protein of about 397 amino acids, referred to herein as PeicaPA-N397, having amino acid sequence SEQ ID NO:X10, assuming an open reading frame from the starting methionine (M) through glutamic acid (E) at amino acid 397. The region encoding PeicaPA-N397, designated neicaPA-N1193 and having a coding strand comprising nucleotides 25 to 1215 of SEQ ID NO:X8, is designated herein as SEQ ID NO:X9.
C. Comparison of the amino acid sequences of proteins PeiwtPA-N397 (SEQ ID NO:X7) and PeicaPA-N397 (SEQ ID NO:X10) reveals the following differences: a glutamic acid (E) to lysine (K) at amino acid 59 and an alanine (A) to threonine (T) at amino acid 156.
EXAMPLE 10
This example describes the cloning and sequencing of equine influenza PA protein nucleic acid molecules, for wild type or cold-adapted equine influenza viruses.
A. The nucleic acid sequences encoding wild type or cold-adapted equine influenza virus PA proteins were compiled as follows. The open reading frame DNA sequence is 2148 nucleotides long for both stains. There is a 214 nucleotide overlap from the C-terminal and N-terminal fragments generated in Examples 8 and 9, respectively. The open reading frame sequences encode the RNA Polymerase A, otherwise known as RNA Polymerase 2 gene, which has 716 amino acid residues from the starting methionine (M) through lysine (K) at amino acid 716.
A nucleic acid molecule of 2148 nucleotides encoding the wild type protein is designed neiwtPA2148, and has the coding strand of nucleic acid sequence SEQ ID NO:X11.
Translation of SEQ ID NO:X11 indicates that nucleic acid molecule neiwtPA2148 encodes the equine influenza virus PA protein of about 716 amino acids, referred to herein as PeiwtPA716, having amino acid sequence SEQ ID NO:X12.
B. A nucleic acid molecule of 2148 nucleotides encoding the cold-adapted protein is designated neicaPA2148, and has the coding strand of nucleic acid sequence SEQ ID NO:X13.
Translation of SEQ ID NO:X13 indicates that nucleic acid molecule neicaPA2148 encodes a protein of about 716 amino acids, referred to herein as PEIcaPA716, having amino acid sequence SEQ ID NO:X14.
C. Comparison of the wild type and cold-adapted virus sequences reveals three discrepancies: a G to A base shift at base 175, base 466, and base 870. The base change at base 175 results in a glutamic acid (E) to lysine (K) change at amino acid 59. The base change at 466 results in an alanine (A) to threonine (T) change at amino acid 156. The base change at 870 does not result in an amino acid change at amino acid 290.
EXAMPLE 11
This example describes the cloning and sequencing of equine influenza Neuraminidase (NA) protein nucleic acid molecules for cold-adapted equine influenza viruses.
A. Nucleic acid molecules encoding cold-adapted equine influenza virus NA proteins were produced as follows. A PCR product was produced by PCR amplification using equine influenza virus DNA and primers M13 reverse primer (REV) and T7 primer (T7), designated SEQ ID NO:1 and SEQ ID NO:2, respectively. A nucleic acid molecule of 1478 molecules encoding the cold-adapted NA protein, denoted neicaNA1478 with a coding strand having a nucleic acid sequence designated SEQ ID NO:87 was produced.
Translation of SEQ ID NO:87 indicates that nucleic acid molecule neicaNA1478 encodes a full-length protein of 470 amino acids, referred to herein as PeicaNA470, having amino acid sequence SEQ ID NO:88, assuming an open reading frame in which the initiation codons spans from nucleotide 29 through 31 of SEQ ID NO:87 and the termination codon spans from nucleotide 1439 through 1441 of SEQ ID NO:87. The region encoding PeicaNA470, designated neicaNA1412, and having a coding strand comprising nucleotide 29 to 1441 of SEQ ID NO:87, is represented by SEQ ID NO:89.
EXAMPLE 12
This example describes the cloning and sequencing of the N-terminal and C-terminal regions of the equine influenza nucleoprotein (NP) nucleic acid molecules for wild type and cold adapted equine influenza virus.
A. Nucleic acid molecules encoding wild type and cold adapted equine influenza virus NP proteins were produced as follows. Wild-type strains 1 and 2 were cloned into pCR2.1 (Invitrogen), and cold-adapted strains 1 and 2 were cloned into pCR-Blunt cloning vector (Invitrogen). All strains were sequenced using M13 REV and T7 primers, SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
B. N-Terminal Regions
A nucleic acid molecule of 738 nucleotides encoding the N-terminus of the wild-type strain 1 NP protein, denoted neiwt1 NP-N738, with a coding strand having a nucleic acid sequence designated SEQ ID NO:104, was produced. Translation of SEQ ID NO:104 indicates that nucleic acid molecule neiwt1 NP-N738 encodes the N-terminal 246 amino acids of NP protein, designated Peiwt1 NP-N246, having amino acid sequence SEQ ID NO:105.
A nucleic acid molecule of 693 nucleotides encoding the N-terminus of the wild-type strain 1 NP protein, denoted neiwt1 NP-N693, with a coding strand having a nucleic acid sequence designated SEQ ID NO:106 was produced. Translation of SEQ ID NO:106 indicates that nucleic acid molecule neiwt1 NP-N693 encodes the N-terminal 231 amino acids of NP protein, designated Peiwt1 NP-N231, having amino acid sequence SEQ ID NO:107.
A nucleic acid molecule of 738 nucleotides encoding the N-terminus of the wild-type strain 2 NP protein, denoted neiwt2 NP-N738, with a coding strand having a nucleic acid sequence designated SEQ ID NO:108, was produced. Translation of SEQ ID NO:108 indicates that nucleic acid molecule neiwt2 NP-N738 encodes the N-terminal 246 amino acids of NP protein, designated Peiwt2 NP-N246, having amino acid sequence SEQ ID NO:109.
A nucleic acid molecule of 693 nucleotides encoding the N-terminus of the wild-type strain 2 NP protein, denoted neiwt2 NP-N693, with a coding strand having a nucleic acid sequence designated SEQ ID NO:110, was produced. Translation of SEQ ID NO:110 indicates that nucleic acid molecule neiwt2 NP-N693 encodes the N-terminal 231 amino acids of NP protein, designated Peiwt2 NP-N231, having amino acid sequence SEQ ID NO:111.
An N-terminal protein fragment of 245 amino acids of NP protein, designated Peica1 NP-N245, having amino acid sequence SEQ ID NO:112, was generated.
A nucleic acid molecule of 690 nucleotides encoding the N-terminus of the cold-adapted strain 1 NP protein, denoted neica1 NP-N690, with a coding strand having a nucleic acid sequence designated SEQ ID NO:113, was produced. Translation of SEQ ID NO:113 indicates that nucleic acid molecule neica1 NP-N690 encodes the N-terminal 230 amino acids of NP protein, designated Peica1 NP-N230, having amino acid sequence SEQ ID NO:114.
A nucleic acid molecule of 735 nucleotides encoding the N-terminus of the cold-adapted strain 2 Np protein, denoted neica2 NP-N735, with a coding strand having a nucleic acid sequence designated SEQ ID NO:115 was produced. Translation of SEQ ID NO:115 indicates that nucleic acid molecule neica2 NP-N735 encodes the N-terminal 245 amino acids of NP protein, designated Peica2 NP-N245, having amino acid sequence SEQ ID NO:116.
A nucleic acid molecule of 690 nucleotides encoding the N-terminus of the cold-adapted strain 2, denoted neica2 NP-N690, with a coding strand having a nucleic acid sequence designated SEQ ID NO:117, was produced. Translation of SEQ ID NO:117 indicates that nucleic acid molecule neica2 NP-N690 encodes the N-terminal 230 amino acids of NP protein, designated Peica2 NP-N230, having amino acid sequence SEQ ID NO:118.
C. C-Terminal Regions
A nucleic acid molecule of 679 nucleotides encoding the C-terminal of the wild type strain 1, denoted neiwt1 NP-C679, with a coding strand having a nucleic acid sequence designated SEQ ID NO:119, was produced. Translation of SEQ ID NO:119 indicates that nucleic acid molecule neiwt1 NP-C679 encodes the C-terminal 226 amino acids of NP protein, designated Peiwt1 NP-C226, having amino acid sequence SEQ ID NO:120.
A nucleic acid molecule of 656 nucleotides encoding the C-terminal of the wild type strain 1, denoted neiwt1 NP-C656, with a coding strand having a nucleic acid sequence designated SEQ ID NO:121, was produced. Translation of SEQ ID NO:121 indicates that nucleic acid molecule neiwt1 NP-C656 encodes the C-terminal 218 amino acids of NP protein, designated Peiwt1 NP-C218, having amino acid sequence SEQ ID NO:122.
A nucleic acid molecule of 679 nucleotides encoding the C-terminal of the wild type strain 1, denoted neiwt2 NP-C679, with a coding strand having a nucleic acid sequence designated SEQ ID NO:123, was produced. Translation of SEQ ID NO:123 indicates that nucleic acid molecule neiwt2 NP-C679 encodes the C-terminal 226 amino acids of NP protein, designated Peiwt2 NP-C226, having amino acid sequence SEQ ID NO:124.
A nucleic acid molecule of 656 nucleotides encoding the C-terminal of the wild type strain 2, denoted neiwt2 NP-C656, with a coding strand having a nucleic acid sequence designated SEQ ID NO:125, was produced. Translation of SEQ ID NO:125 indicates that nucleic acid molecule neiwt2 NP-C656 encodes the C-terminal 218 amino acids of NP protein, designated Peiwt2 NP-C218, having amino acid sequence SEQ ID NO:126.
A nucleic acid molecule of 656 nucleotides encoding the C-terminal of the cold-adapted strain 1, denoted neica1 NP-C665, with a coding strand having a nucleic acid sequence designated SEQ ID NO:127, was produced. Translation of SEQ ID NO:127 indicates that nucleic acid molecule neica1 NP-C665 encodes the C-terminal 222 amino acids of NP protein, designated Peica1 NP-C222, having amino acid sequence SEQ ID NO:128.
A nucleic acid molecule of 642 nucleotides encoding the C-terminal of the cold-adapted strain 1, denoted neica1 NP-C642, with a coding strand having a nucleic acid sequence designated SEQ ID NO:129 was produced. Translation of SEQ ID NO:129 indicates that nucleic acid molecule neica1 NP-C642 encodes the C-terminal 214 amino acids of NP protein, designated Peica1 NP-C214, having amino acid sequence SEQ ID NO:130.
D. Comparisons
Consensus sequences for the 5′ and 3′ ends of the NP gene were generated, but there is an approximately 148 base pair gap in the data that prevents the formation of a contiguous sequence when compared to protein and DNA data currently in the GenBank databases (a nucleoprotein from GenBank named flanpg; Gorman et al, J. Virol. 64, 1487 (1990)). The flanpg sequences were used to compare the 5′ and 3′ fragments of the present NP genes to get relative positions that are used to designate position for the various differences in the bases between the Wild Type and the ca strains. The flanpg sequence allows for the approximately 148 base pairs that are missing between the 5′ and 3′ fragments needed to construct the entire gene.
The DNA codes for the N-terminal 735–738 bases and the C-terminal 665–679 bases with a gap of approximately 149–166 bases between the two fragments of the Wild Type and cold-adapted (ca) strains, respectively. There are seven discrepancies between the wild type and ca strains. The ca strains have discrepancies at base 146 (G to T) and at base 228 (A to G). At base 492, the wt1 only has a discrepancy versus the other strains of (A to C). At base 541, the ca1 only has a discrepancy versus the other strains of (C to A). There is a base discrepancy at position 645 in the DNA (G to A). At base 670, the ca strains have a discrepancy (G to A). At base 1019, the wt1 only has a discrepancy versus the other strains of (G to A). There are no other points of discrepancy between the full-length Wild Type and ca strains.
The full-length sequence is proposed to consist of 1497 bases, however there is no overlap between the 5′ and 3′ ends of the DNA sequences generated. The sequences listed are for the 5′ end and the 3′ end of the open reading frame of the gene. The gene is missing approximately 148 base pairs between these two fragment sequences that would generate the complete gene sequence.
A comparison of all the Wild Type and ca strain clones (wt1, wt2, ca1, and ca2) by amino acid translation for the protein sequence coding only for the open reading frame (ORF) from the 5′ and 3′ DNA fragments was compared to the GenBank nucleoprotein noted above. The DNA codes for the N-terminal 230 amino acids and the C-terminal 214 amino acids with a gap of approximately 55 amino acids between the two fragments. There are seven discrepancies between the ca and Wild Type strains. The ca strains have a discrepancy (G to V) at amino acid 34, and at amino acid 61 (I to M). At amino acid 149, the wt1 only has a discrepancy versus the other strains of (Q to H). At amino acid 166, ca1 only has a discrepancy versus the other strains of (L to M). Although there is a base discrepancy at position 600 in the DNA sequence, there is no change in the corresponding amino acid sequence. At amino acid 209, the ca strains have a discrepancy (G to S). At amino acid 325, the wt1 only has a discrepancy versus the other strains of R to K). There are no other points of discrepancy between the fill-length Wild Type and ca strains.
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.

## Claims

1. An isolated equine influenza protein that comprises the amino acid sequence of SEQ ID NO:28.

2. A composition comprising the isolated equine influenza protein of claim 1 and an excipient.