AU2001270033A1

AU2001270033A1 - Nucleotide sequence of influenza A/Udorn/72 (H3N2) genome

Info

Publication number: AU2001270033A1
Application number: AU2001270033A
Authority: AU
Inventors: Jose M. Galarza; Theresa E. Latham
Original assignee: American Cyanamid Co
Current assignee: Wyeth Holdings LLC
Priority date: 2000-06-23
Filing date: 2001-06-21
Publication date: 2002-01-08
Also published as: CA2410284A1; BR0111819A; WO2002000884A2; KR20030013451A; IL153005A0; WO2002000884A3; CN1437612A; JP2004514415A; EP1292614A2; MXPA02012255A

Description

NUCELOTIDE SEQUENCE OF INFLUENZA A/Udorn/72 (H3N2) GENOME

Field Of The Invention

This invention relates to the nucleotide sequence of each of the segments of the Influenza virus strain A/Udorn/72 (H3N2) .

Background Of The Invention

The influenza viruses consist of subtypes designated A, B and C. Influenza A viruses possess a single negative strand RNA genome bf eight segments/ which encodes 10 polypeptides (proteins) that are required for the life cycle of the virus. The order of the proteins is as follows:

Segment : 1 2 3 4 5 6 7 8

Protein: PB2 PB1 PA HA NP NA Ml NS1

Spliced M2 NS2 Product:

Each of the eight RNA segments of the influenza subtype A complete genome is encapsidated with multiple subunits of the nucleocapsid protein (NP) and associated with a few molecules of the tri eric polymerase (PB1, PB2 and PA subunits) , thereby forming the ribonucleoprotein complex (RNP)

(Lamb, R.A., pages 1-87 of The Influenza Viruses, R.M. Krug, ed. (Plenum Press, 1989)). The NP protein is a structural and transcription/replication regulatory protein. Surrounding these structures is a layer of the matrix protein (Ml) , which is the major structural component of the virion and appears to serve as a nexus between the core and the viral envelope. This host cell-derived envelope is studded with the two major virally encoded surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) , which are antigenic determinants, and a much smaller amount of a nonglycosylated small protein M2 (Lamb 1989; Lamb, R.A., et al., Cell, 40, 627-633 (1985)). The M2 protein is a spliced gene product from segment #7 (which also encodes the Ml protein) which has ion channel activity; spliced genes are only present in infected cells. Similarly, the nonstructural NS2 protein is a spliced gene product from segment #8

(which also encodes the nonstructural NS1 protein) ; spliced genes are only present in infected cells. The HA glycoprotein is cleaved by a protease to form HA1 and HA2. Influenza viral infection is initiated by the attachment of the surface hemagglutinin to a sialic acid-containing cellular receptor. This first virus-cell interaction induces the uptake of the viral particle into the cell by receptor-mediated endocytosis. Under the low pH conditions of the endosome, the HA undergoes a conformational change that facilitates the interaction of the hydrophobic NH₂ terminal domain of HA2 and the endosomal membrane, resulting in membrane fusion and subsequent release of the core RNPs and matrix protein (Ml) into the cytosol. Disassociation of the RNPs and matrix proteins occurs in the cytosol before the RNPs are translocated to the nucleus where transcription and replication of the complete genome take place (Martin, K., and Helenius, A., Cell, 67, 117-130 (1991); Shapiro, G.I., et al . , J. Virology, 61, 764-773 (1987)) .

Following primary transcription, newly synthesized proteins initiate the replication of the viral genome which in turn increases transcription and protein synthesis. At this point of the virus life cycle, the surface glycoproteins HA and NA start to accumulate at discrete areas of the plasma membrane from where newly assembled virus will be released. Virus assembly is assumed to begin via some sort of interaction between the cytoplasmic and/or transmembrane domains of the four virally encoded structural proteins : the membrane anchored proteins (HA, NA and M2) , and the underlying matrix protein (Ml) , which in turn maintains a close association with the RNPs (Garoff, H., et al . , Microbiology and

Molecular Biology Reviews, 62, 1171-1190 (1998); Nayak, D.P., ASM News, 62, 411-414 (1996)). The contacts between matrix protein Ml and the RNP complexes, as well as the mechanism by which a complete set of eight RNPs gets incorporated into the mature virion particle, have not been well defined. Specific molecular contacts among the structural components are assumed to dictate how the process of morphogenesis initiates and progresses to the point of mature particle assembly and budding from the surface of the host cell.

There are a large number of influenza A virus subtypes and antigenic variants that continuously evolve by mutations introduced by the RNA polymerase during the replication of the viral genome. Mutations also occur by genetic reassortment between the segments of two different subtypes when they happen to infect the same cellular host. This biological characteristic of influenza A virus gives rise to new virus strains, which necessitates periodic updating of the immunogenic compositions against influenza. Therefore, the availability of a complete nucleotide sequence and a full length clone of one particular strain of influenza A virus is essential for the development of new and innovative immunogenic compositions against influenza. Thus, there is a need to determine the complete nucleotide sequence of an influenza A virus and to construct a full length clone containing that sequence .

In particular, the influenza A strain designated Influenza A/Udorn/72 (H3N2) was first isolated in 1972. The nucleotide sequences of the polymerase genes PB2, PB1 and PA have not been sequenced. The nucleotide sequences of the segments encoding HA, NP, Ml, M2, NS1 and NS2 have been sequenced. However, those sequences were obtained a number of years ago using instrumentation which may not have provided accurate sequences. Thus, there is a need to repeat the sequencing of those segments in order to provide updated, accurate sequences.

Summary Of The Invention

It is an object of this invention to determine the complete nucleotide sequence of the Influenza A/Udorn/72 (H3N2) viral strain and the spliced products of the segment #7 and #8 mRNAs. It is a further object of this invention to generate a clone of each of the eight genes and spliced messages. It is yet another object of this invention to determine the deduced amino acid sequences of all ten of the products of these genes and spliced messages.

These and other objects of this invention are achieved by the elucidation of isolated nucleic acid molecules constituting the nucleotide sequences of all of the segments of the Influenza A/Udorn/72 (H3N2) strain which together comprise the complete nucleotide sequence of that strain, as well as their spliced messages. All of these nucleotide sequences are presented in positive strand, antigenomic message sense. In particular, the complete nucleotide sequence consists of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13 and SEQ ID NO: 17, and biological equivalents thereof .

Alternatively, the complete nucleotide sequence includes a variant of the HA sequence, designated HA (PI) (SEQ ID NO:21), and consists of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO:21, and biological equivalents thereof.

In another embodiment of this invention, these isolated nucleic acid molecules are directed to individual isolated influenza A virus nucleic acid molecules which encode individual proteins and are selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 21 (the variant of SEQ ID NO: 7) , and biological equivalents thereof. In yet another embodiment of this invention, these individual isolated nucleic acid molecules encode proteins having amino acid sequences selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and biological equivalents thereof. In a further embodiment of this invention, there are provided individual isolated influenza A virus amino acid sequences, where said sequences are selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and biological equivalents thereof.

In a still further embodiment of this invention, there is provided an individual influenza A virus amino acid sequence, where said sequence has the sequence of SEQ ID NO: 8 or SEQ ID NO: 12. In still another embodiment of this invention, an isolated nucleic acid molecule having the nucleotide sequence of a segment of the Influenza A/Udorn/72 (H3N2) strain is used: (1) to design polymerase chain reaction (PCR) primers for use in a PCR assay to detect. the presence of the corresponding virus segment in a sample; or (2) to design and select peptides for use in an ELISA to detect the presence of the corresponding protein produced by that segment in a sample.

Detailed Description of the Invention

The nucleotide sequences and deduced amino acid sequences of several of the segments of the Influenza A/Udorn/72 (H3N2) strain have been published. Yuferov et al . reported the sequence of the HA gene of segment 4 (Yuferov, V.P., et al . , Proceedings of the Academy of Sciences of the USSR, 278, 738-742 (1984)). Buckler-White et al . reported the sequence of the NP gene of segment 5 (Buckler- White, A.J., and Murphy, B.R., Virology, 155, 345-355 (1986)) . Markoff et al . reported the sequence of the NA gene of segment 6 (Markoff, L., and Lai, C.J., Virology, 119, 288-297 (1982)). Lamb et al . in 1981 reported the sequence of the Ml gene and the M2 gene splice product of segment 7 (Ml in Lamb, R.A., and Lai, C.J., Virology, 112, 746-751 (1981) ("Lamb Virology 1981"), and M2 in Lamb, R.A., et al . , Proc. Natl. Acad. Sci. USA, 78, 4170-4174 (1981) ("Lamb PNAS 1981")). Lamb et al. in 1980 reported the sequence of the NS1 gene and the NS2 gene splice product of segment 8 (Lamb, R.A., and Lai, C.J., Cell, 21, 475- 485 (1980)

In order to resequence these segments, as well as to sequence those segments not previously elucidated, the Influenza A/Udorn/72 (H3N2) strain was grown in and purified from Madin-Darby canine kidney (MDCK) cells . The viral RNA was extracted from purified virions and amplified by RT-PCR using termini-specific primers. Gel purified genes were cloned into pGe T vectors (Pro ega) and sequenced using multiple sets of primers. The sequences of the termini were determined by 3' and 5" ligation and sequencing of the RT-PCR fragments crossing the. junction (Galarza, J.M., et al . , J. Virol., 70, 2360- 2368 (1996)). The M2 and NS2 genes are spliced products of the mRNA of segments seven and eight, respectively. These genes were recovered from mRNA purified from MDCK-Influenza A/Udorn-infected cells by RT-PCR using oligo-dT and gene-specific primers. The RT-PCR products were gel purified and cloned into pGemT vectors . All gene sequences were determined by using a fluorescence dye terminator with AmpliTaq DNA polymerase (Perkin-Elmer) and an Applied Biosystems ABI 377 DNA sequencer. This procedure was repeated with multiple clones for each fragment; the sequences obtained were consistent.

The complete genome of the Influenza A/Udorn/72 (H3N2) strain totaled 13628 nucleotides. This is the first description of the complete nucleotide sequence of the Influenza A/Udorn/72 (H3N2) strain.

The eight segments were sequenced in their coding regions, and their 5' and 3' non-coding regions, including regulatory sequences such as promoters, enhancers and polyadenylation signals. The segments, their number of nucleotides, their isolated nucleic acid molecule sequences (shown in positive strand, antigenomic, message sense, that is, in 5' to 3' orientation), their coding regions and amino acid translations are as follows: Segment #1: 2341 nucleotides, SEQ ID NO:l, coding region nucleotides 28-2304, encodes PB2, 759 amino acids (SEQ ID NO: 2) .

Segment #2: 2341 nucleotides, SEQ ID NO: 3, coding region nucleotides 25-2295, encodes PBl, 757 amino acids (SEQ ID NO: 4) .

Segment #3: 2233 nucleotides, SEQ ID NO: 5, coding region nucleotides 25-2172, encodes PA, 716 amino acids (SEQ ID NO: 6) .

Segment #4: 1765 nucleotides, SEQ ID NO: 7, coding region nucleotides 30-1727, encodes HA, 566 amino acids (SEQ ID NO: 8) .

SEQ ID NO:7 and SEQ ID NO: 8 differ from the published Yuferov et al. nucleotide and amino acid sequences for HA in several respects .

Yuferov SEQ ID NO: 7 or 8

Codon # Amino Acid # Codon # Amino Acid #

GAC Asp AAC Asn

81-83 18 81-83 18

GGT Gly GGG Gly

1101-1103 358 1101-1103 358

TAC Tyr TTC Phe

1485-1487 486 1485-1487 486

GAC Asp AAC Asn

1614-1616 529 1614-1616 529 Segment #5: 1565 nucleotides, SEQ ID NO: 9, coding region nucleotides 46-1539, encodes NP, 498 amino acids (SEQ ID NO:10).

SEQ ID NO: 9 and SEQ ID NO: 10 are identical to the published Buckler-White et al. nucleotide and amino acid sequences for NP.

Segment #6: 1466 nucleotides, SEQ ID NO: 11, coding region nucleotides 20-1426, encodes NA, 469 amino acids (SEQ ID NO: 12).

SEQ ID NO: 11 and SEQ ID NO: 12 differ from the published Markoff et al. nucleotide and amino acid sequences for NA in several respects.

Markoff SEQ ID NO: 11 or 12

Codon # Amino Acid # Codon # Amino Acid #

CAG Gin CTG Leu

104-106 29 104-106 29

GGA Gly GCA Ala

546-548 177 546-548 177

CAA Gin CAG Gin

695-697 226 695-697 226

ACA Thr GCA Ala

992-994 325 992-994 325

There are also several differences in the leader sequence upstream of the methionine at amino acid 1.

Segment #7: 1027 nucleotides, SEQ ID NO:13, coding region nucleotides 26-781, encodes Ml, 252 amino acids (SEQ ID NO: 14) . SEQ ID NO: 13 and SEQ ID NO: 14 are identical to the published Lamb Virology 1981 nucleotide and amino acid sequences for Ml.

Segment #7, spliced product: 322 nucleotides, SEQ ID NO: 15, coding region nucleotides 26-316, encodes M2, 97 amino acids (SEQ ID NO: 16) [first spliced amino acid at residue 10, nucleotides 53-55] .

SEQ ID NO: 15 and SEQ ID NO: 16 are identical to the published Lamb PNAS 1981 nucleotide and amino acid sequences for M2.

Segment #8: 890 nucleotides, SEQ ID NO: 17, coding region nucleotides 27-737, encodes NS1, 237 amino acids (SEQ ID NO: 18) .

SEQ ID NO: 17 and SEQ ID NO: 18 are identical to the published Lamb et al . 1980 nucleotide and amino acid sequences for NS1.

Segment #8, spliced product: 402 nucleotides, SEQ ID NO: 19, coding region nucleotides 27-389, encodes NS2, 121 amino acids (SEQ ID NO: 20) [first spliced amino acid at residue 11, nucleotides 57-59] .

SEQ ID NO: 19 and SEQ ID NO: 20 are identical to the published Lamb et al. 1980 nucleotide and amino acid sequences for NS2.

As mentioned above, the is also an alternate HA sequence, designated HA (PI) : Segment #4: 1764 nucleotides, SEQ ID NO: 21, coding region nucleotides 30-1727, encodes HA, 566 amino acids (SEQ ID NO:22).

Without being bound by theory, the HA (PI) sequence may incorporate minor mutations from the Influenza A/Udorn/72 (H3N2) strain which occurred over time. The HA (PI) sequence (SEQ ID NO:21) has 1764 nucleotides, which is one fewer than in the HA sequence (SEQ ID NO: 7), due to a one nucleotide deletion in the non-coding region of segment #4 (position 1756) .

The two HA sequences of this invention differ in their nucleotide and amino acid sequences as follows :

Nucleotide HA HA (PI) Amino Acid

Position Change

35 G A Silent

81 A G Asn to Asp

1103 G T Silent

1486 T A Phe to Tyr

1614 A G Asn to Asp

1756 T deletion Outside coding region

The HA (PI) sequence also differs slightly from the Yuferov et al. HA nucleotide sequence as follows :

Nucleotide HA (PI) Yuferov Amino Acid

Position Change

35 A G Silent

1756 deletion T Outside coding region

The nucleic acid sequences of the mRNAs of the segments, which are in genome, negative sense (that is, in 3 ' to 5' orientation) , are the complements of the positive strand, antigenomic, message sense sequences set forth above. In addition to the nucleotide sequences which encode the Influenza A/Udorn/72 (H3N2) viral proteins set forth above, the present invention further comprises isolated nucleic acid molecules containing individual influenza A virus nucleotide sequences which, by virtue of the redundancy of the genetic code, are biologically equivalent to those sequences which encode the viral proteins, that is, these other nucleotide sequences are characterized by nucleotide sequences which differ from those set forth herein, but which encode a protein having the same amino acid sequence as that encoded by any of the nucleotide sequences in SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID N0:7, SEQ ID NO: 11 and SEQ ID NO:21.

In particular, the invention contemplates those nucleotide sequences which are sufficiently duplicative of any of the sequences of SEQ ID N0:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 21, so as to permit hybridization therewith under standard high stringency Southern hybridization conditions, such as those described in Sambrook et al. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) .

This invention also comprises nucleotide sequences which encode amino acid sequences which differ from those of the Influenza A/Udorn/72 (H3N2) viral proteins, but which are biologically equivalent to those described for one of these viral proteins (SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6). Such amino acid sequences may be said to be biologically equivalent to any of the viral proteins if their sequences differ only by minor deletions from, insertions into or substitutions to the viral protein sequences, such that the tertiary configurations of the sequences are essentially unchanged from those of the viral proteins. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged (acidic) residue for another, such as aspartic acid for glutamic acid, or one positively charged (basic) residue for another, such as lysine for arginine or histidine, as well as changes based on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of structural and biological activity of the encoded products.

This invention further comprises the isolated amino acid sequences comprising the individual influenza A virus amino acid sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4 and SEQ ID NO: 6.

This invention still further comprises the individual isolated amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 12.

The fundamental biological properties of the Influenza A/Udorn/72 (H3N2) virus depend on its genome, which consists of eight segments. The segments of influenza virus consists of a single strand of negative polarity RNA, and each viral strain has its own specific nucleotide sequence. Complete differential identification between viral strains is, therefore, necessary to determine the nucleotide sequence of the virus. Since the entire specific nucleotide sequence, consisting of 13,628 bases, has been clearly determined as described herein, the virus can be identified at the genetic level, and the identification technique can therefore provide an absolute determination.

The sequences described herein are useful to provide an identification method comprising detecting a part of the nucleotide sequence of influenza viral antigenomic DNA using the polymerase chain reaction (PCR) method, as well as to generate peptides to detect an antigen produced by that DNA using an ELISA.

Having a completely sequenced and cloned genome of one particular influenza strain is advantageous for the research and development of new and innovative immunogenic compositions based on this genetic blueprint.

Defined mutations are introduced where desired into one or more of the segments whose sequences are set forth herein. One or more mutations are introduced by using site-directed mutagenesis. However, a mutant influenza virus cannot be directly generated from viral RNA, because neither the genomic viral RNA nor the antigenomic cDNA can serve as a direct template for synthesis. Instead, the viral RNA, after its encapsidation by NP, must be transcribed into positive-sense mRNA by the viral RNA polymerase complex.

Palese et al. (U.S. Patent 5,166,057, which is hereby incorporated by reference) described a reverse genetics helper virus-dependent system for the "rescue" of an influenza A virus segment. Briefly, a ribonucleoprotein (RNP) complex is generated by in vitro synthesis in the presence of the three polymerase proteins and NP. The RNP complex is then used to transfect eukaryotic cells. Subsequent infection with influenza A helper virus results in the generation of viruses possessing a gene derived from the cloned cDNA segment. A selection method is then used to separate the desired transfectants from the larger number of helper viruses .

A different system is used if an entire mutant influenza A strain is to be rescued. Using the system described by Neumann et al. (Neumann, G., et al., Proc. Natl. Acad. Sci. USA, 96, 9345-9350 (1999), which is hereby incorporated by reference) , a modified Influenza virus strain A/Udorn/72 (H3N2) is generated entirely from cloned cDNAs . This plasmid-based system does not require the use of helper virus infection. In summary, a cell line, such as human embryonic kidney cell line 293T, is transfected with eight plasmids, each encoding a viral RNA segment of the strain and flanked by a suitable RNA polymerase promoter and terminator, together with another four plasmids encoding the viral NP, PB2, PBl and PA proteins (which serve to synthesize RNPs intracellularly in vivo) . Yields are increased substantially by the addition of five further plasmids expressing the viral structural proteins HA, NA, Ml, M2 and NS2. In a variation of this system, only eight plasmids are required (Hoffmann, E., et al., Proc. Natl. Acad. Sci. USA, 97, 6108-6113 (2000), which is hereby incorporated by reference) . Each plasmid contains two promoters, a human RNA polymerase I (pol I) promoter and a human RNA polymerase II (pol II) promoter. After transfection of eukaryotic cells with the eight expression plasmids, the human pol I and II promoters each transcribe the plasmid template. This results in the synthesis of both viral mRNAs and vRNAs, ultimately leading to the generation of infectious influenza A virus. Because helper virus is not required in any of the 8, 12 or 17 plasmid transfection systems, the transfectant viruses are recovered without plaque purification. This system facilitates the production of live attenuated influenza A viruses for use in combating epidemics involving new HA or NA subtypes, where the corresponding segment of the Influenza A/Udorn/72 (H3N2) strain is mutated to match the sequence of the new subtypes. Predetermined mutations to the segment encoding HA or NA in the Influenza A/Udorn/72 (H3N2) strain which correspond to the sequence of a new circulating subtype are made by using site-directed mutagenesis. Such mutations are introduced by standard recombinant DNA methods into a DNA copy of the viral genome .

If desired, reassortant viruses are generated by substituting for the segment encoding the HA or NA of the Influenza A/Udorn/72 (H3N2) strain (or mutant thereof) the corresponding segment from a different influenza virus strain.

In another embodiment of this invention, an isolated nucleic acid molecule having the nucleotide sequence of a segment of the Influenza A/Udorn/72 (H3N2) strain is used to generate oligonucleotide probes (from either positive strand antigenomic message sense or negative strand complementary genomic sense) and to express peptides (from positive strand antigenomic message sense only) , which are used to detect the presence of a segment of that influenza A strain (or a mutant thereof) in samples of body fluids and tissues. The nucleotide sequences are used to design highly specific and sensitive diagnostic tests to detect the presence of the viral segment in a sample. PCR primers are synthesized with sequences based on the viral sequences described herein. The test sample is subjected to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to the nucleotide sequence described herein which have nucleotides which are distinct for a defined segment of that viral strain. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes.

ELISA tests are ■ used to detect the presence of a protein produced by a viral segment. Peptides are designed and selected to contain one or more distinct residues based on the viral sequences described herein. These peptides are then coupled to a hapten (e.g., keyhole limpet hemocyanin (KLH) and used to immunize animals (e.g., rabbits) for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies can then be used in a "capture ELISA" to detect a protein produced by that viral segment.

All references cited herein are hereby incorporated by reference. In order that this invention may be better understood, the following examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention. Examples

Example 1 Sequencing of the Influenza A/Udorn/72 (H3N2) Strain

The sequencing of each of the eight segments was carried out as follows:

RT-PCR Protocol

Influenza A/Udorn/72 (H3N2) was grown in Madin-Darby canine kidney (MDCK) cells and concentrated from clarified culture supernatant. Genomic viral RNA (vRNA) was prepared from lOOμl of virus stock using a RNAeasy extraction kit (Qiagen) .

The spliced products of mRNAs of segment numbers 7 and 8 (M2 and NS2 respectively) were isolated from total mRNA of influenza/Udorn-infected MDCK cells using an Oligotex mRNA kit (Qiagen) . Approximately lμg of either vRNA or mRNA was added to an eppendorf tube containing 2pmol of gene-specific primer and diethyl pyrocarbonate-treated water (to inhibit RNAses) to a final volume of 12μl. Oligo-dT primers were used to reverse transcribe the M2 and NS2 spliced messages. The RNA-pri er mixture was heated at 70 °C for 10 minutes and quickly chilled on ice. The following reagents were added to this tube: 4μl of 5X first strand buffer (250mM Tris-HCl pH 8.3, 375mM KCl, 15mM MgCl₂) , 2μl of 0.1M DTT, and Iμl of dNTP mix (10 mM each dATP, dTTP, dGTP, dCTP) and incubated at 42 °C for two minutes . Subsequently, lμl of Superscript II (Gibco) reverse transcriptase was added and re- incubated at 42 °C for an additional 50 minutes. The RT reaction was stopped by incubation at 70 °C for 15 minutes. PCR reactions were set up by adding 2μl of the previous RT to a mixture of the following reagents: 5OmM Tris-HCl (pH 8.0), 1.5mM gCl₂, 50mM KCl, lμM each primer, 0.2mM each dNTP and 2 units of Taq polymerase. DNA was amplified in a Perkin-Elmer 9600 PCR machine as follows: One cycle of denaturation at 94 °C for two minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 42 °C for 30 seconds, and • elongation at 72 °C for one minute. This was followed by one cycle of elongation at 72 °C for seven minutes. PCR-amplified DNA was analyzed on a 1% agarose gel and purified using a Qiagen purification kit.

Sequencing Protocol

Approximately 500ng of plasmid pGEM-T containing the corresponding DNA segment generated by RT-PCR was added to a 0.2ml tube containing 3.2pmol of sequencing primer and 8 μl of Terminator ready reaction mix (Perkin-Elmer) and placed into a Perkin- Elmer 9600 PCR machine. The reaction was amplified by 25 cycles at 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes. The products were then purified over a G50 Sephadex spin column, lyophilized, and resuspended in 3μl of 25mM EDTA (pH8.0) with 50 mg/ml Blue dextran loading dye. The samples were run on an ABI 377 automated sequencer and the sequence analyzed using Sequencher (sequence analysis program from Gene Codes Corporation) .

Determination of the nucleotide sequence of the 3 ' and 5' termini

Purified genomic vRNA (2.5μg) (see above) was treated with 10U of tobacco acid pyrophosphatase (Epicenter Technologies, Madison, Wis.) at 37 °C for 30 minutes to remove phosphate groups from the 5' terminus. Treated RNA was phenol-chloroform extracted and precipitated with ethanol. Subsequently, the RNA 5' and 3 ' termini were ligated with 50U of T4 RNA- ligase (Pharmacia Biotech.) at 37°C for one hour, extracted with phenol-chloroform and precipitated with ethanol . Ligated RNA was reverse transcribed and PCR in a single reaction with a GeneAmp Gold RNA PCR Reagent Kit ( PE Biosystems) using a forward and reverse pair of primers (see below) . Sequencing reactions were carried out as described above, using the primers specific for each RNA segment (Galarza, J. M., et al., 1996, J of Virol. 70:2360-2368).

Primers for sequencing influenza A/Udorn/72 (H3N2)

Primers in the forward direction are marked

"F" and are in antigenomic, plus sense. Primers in the reverse direction are marked "R" and are in genomic negative sense.

PB2 (Segment 1) Primers

Primer F/R Primer sequence bp spanned

• RT Primer

1107 F 5'-TATGGAAAGAATAAAAGAACTACGGAA-3' 27-53

(SEQ ID NO: 23)

• PCR Primers

1108 R 5'-TCGTTTTTAAACTATTCAACAT-3' 2307-2328

(SEQ ID NO: 24)

1107 F 5'-TATGGAAAGAATAAAAGAACTACGGAA-3' 27-53

(SEQ ID NO: 23) • Sequencing Primers

1101 F 5'-AGCAGGTCAATTATATTCAATATG-3' 1-24 (SEQ ID NO:25)

The primer sequence differs from the PB2 gene sequence as follows:

Position PB2 Gene 1101 F

5 A G

6 A G

7 A T

8 G C

9 C A

11 G T

12 G T

13 T A

14 C T 16 A T 18 T . C

20 T A

21 A T

22 T A 24 C G

1102 R 5'-AACAAGGTCGTTTTTAAACTATTC-3' 2312-2335

(SEQ ID NO: 26)

1103 F 5'-AGAACTCTATTCCAACAAATG-3' 1816-1836 (SEQ ID NO:27)

1104 R 5'-AATCGGATATTTCATTGCCAT-3' 178-198

(SEQ ID NO: 28; the primer sequence has a C at nucleotide 195, while the PB2 gene sequence has a T)

1105 F 5'-AGAACTCTATTCCAACAAATGAGGGATGTAGTT-3'

1816-1848 (SEQ ID NO: 29; the primer sequence has a G at nucleotide 1846, while the PB2 gene sequence has a C)

1106 R 5 ' - ATCGGATATTTCATTGCCATCATCCATTTCAT-3 '

166-198 (SEQ ID NO:30; the primer sequence has a C at nucleotide 195, while the PB2 gene sequence has a T)

1107 F 5'-TATGGAAAGAATAAAAGAACTACGGAA-3' 27-53

(SEQ ID NO: 23)

1108 R 5'-TCGTTTTTAAACTATTCAACAT-3' 2307-2328

(SEQ ID NO:24)

1109 F 5'-AGACGTGGTGTTGGTAATGAA-3' 2214-2234

(SEQ ID NO: 31)

1110 F 5'-CGAAGAGTTGACATAAACCCT-3' 454-474

(SEQ ID NO: 32)

1111 R 5'-TCATCCCTCATCCCCTCACAT-3' 1943-1963

(SEQ ID NO:33; the primer sequence has a C at nucleotide 1955, while the PB2 gene sequence has a G)

1112 R 5'-ATTTTCTGTTATCCTCTTGTCA-3' 204-224

(SEQ ID NO: 34; this primer has an extra nucleotide (a T) after nucleotide 220 compared to the actual PB2 gene sequence) 1113 F 5'-GTGGAGTCCGCTGTTTTGAG-3' 2083-2102

(SEQ ID NO: 35)

1114 F 5'-AGGATGGTGGACATTCTTAGG-3' 904-924

(SEQ ID NO: 36)

1115 R 5'-CCGATCAATGCTAACCACTAC-3' 1507-1527

(SEQ ID NO: 37)

1116 F 5'-AGCAAAAGCAGGTCAATTATATTCA-3' 1-25

(SEQ ID NO:38)

1117 R 5'-AGTAGAAACAAGGTCGTTTTTAAAC-3' 2317-2341 (SEQ ID NO:39)

• Primers used to determine terminal sequences

1104 R 5'-AATCGGATATTTCATTGCCAT-3' 178-198 (SEQ ID NO:28)

1109 F 5'-AGACGTGGTGTTGGTAATGAA-3' 2214-2234

(SEQ ID NO: 31)

1112 R 5'-ATTTTCTGTTATCCTCTTGTCA-3' 204-224

(SEQ ID NO:34)

1113 F 5'-GTGGAGTCCGCTGTTTTGAG-3' 2083-2102

(SEQ ID NO:35)

PBl (Segment 2) Primers

Primer F/R Primer sequence bp spanned

• RT primer 1215 F 5'-AGCAAAAGCAGGCAAACCAT-3' 1-20

(SEQ ID NO:40; the primer sequence has an A at nucleotide 4, while the PBl gene sequence has a G)

• PCR primers

1216 R 5'-AGTAGAAACAAGGCATTTTTT-3' 2321-2341 (SEQ ID NO: 41)

1215 F 5'-AGCAAAAGCAGGCAAACCAT-3' 1-20

(SEQ ID NO: 40)

• Sequencing primers

1201 F 5'-TCGAGCTGAAGAAGCTATGG-3' 1745-1764

(SEQ ID NO: 42; the primer sequence has a G at nucleotide 1752, while the PBl gene sequence has an A, and the primer sequence has an A at nucleotide 1761, while the PBl gene sequence has a G)

1202 R 5'-GTTCTGTTGACTGTGTCCAT-3^/ 142-161 (SEQ ID NO: 43)

1203 F 5'-TCGAGCTGAAGAAGCTATGGGAGCAGACCCGT-3'

1745-1776 (SEQ ID NO: 44) The primer sequence differs from the PBl gene sequence as follows: Position PBl Gene 1203 F

1752 A G

1761 G A

1770 A G

1776 C T

1204 R 5'-GTTCTGTTGACTGTGTCCATGGTGTATCC-3' 133-161

(SEQ ID NO: 45)

1205 F 5^/-AGCGA AGCAGGC AACCATTTGAATGGATGTCAA-3^,

1-35 (SEQ ID NO.-46)

1206 R 5'-ATTAAAAACAAGGCATTTTTTCATGAAGGAC-3'

2341-2311 (SEQ ID NO: 47; the primer sequence has an A at nucleotide 2337, while the PBl gene sequence has a G, and the primer sequence has a T at nucleotide 2340, while the PBl gene sequence has a G)

1207 F 5'-AATTTCCAGCATGGTGGAGGCCATGGTG-3' 2154-2181

(SEQ ID NO: 48)

1208 F 5'-GGGATCTTTGAAAACTCGTG-3' 325-344

(SEQ ID NO: 49)

1209 R 5'-CAGCATTGTTTACAGACTC-3' 1936-1954 (SEQ ID NO: 50)

1210 F 5'-ACCAAAGATGCAGAAAGAGG-3' 706-725

(SEQ ID NO: 51) 1211 R 5 ' -CTCATATTGATTCCGACTAA-3 ' 1438 - 1457

(SEQ ID NO: 52)

1212 R 5'-TGTCCACTTCCCTTTTTCTGA-3' 172-192 (SEQ ID NO: 53)

1213 F 5'-ATTTTTCCCCAGTAGTTCATAC-3' 2118-2139

(SEQ ID NO: 54)

1214 F 5' -ACGCTGTTGCAACTACACACTCCTG-3' 1997-2021

(SEQ ID NO: 55)

1215 F 5'-AGCAAAAGCAGGCAAACCAT-3' 1-20

(SEQ ID NO: 40)

1216 R 5'-AGTAGAAACAAGGCATTTTTT-3' 2321-2341

(SEQ ID NO: 41)

• Primers used to determine terminal sequences

1204 R 5' -GTTCTGTTGACTGTGTCCATGGTGTATCC-3' 133-161

(SEQ ID NO: 45)

1212 R 5' -TGTCCACTTCCCTTTTTCTGA-3' 172-192 (SEQ ID NO: 53)

1213 F 5'-ATTTTTCCCCAGTAGTTCATAC-3' 2118-2139

(SEQ ID NO : 54 )

1214 F 5 ' -ACGCTGTTGCAACTACACACTCCTG-3 ' 1997 -2021

(SEQ ID NO: 55)

PA (Segment 3) primers

Primer F/R Primer sequence bp spanned • RT Primer

1315 F 5'-AGCAAAAGCAGGTACTGATTCGAGA-3' 1-25 (SEQ ID NO: 56)

• PCR Primers

1315 F 5'-AGCAAAAGCAGGTACTGATTCGAGA-3' 1-25 (SEQ ID NO:56)

1316 R 5'-AGTAGAAACAAGGTACTTTTTTGGA-3' 2209-2233

(SEQ ID NO: 57)

• Sequencing primers

1301 F 5'-GAACCTGGAACCTTTGATCTT-3' 2053-2073

(SEQ ID NO: 58)

1302 R 5^/-ATTCACCACTG CCAGGCCAT-3^, 280-300

(SEQ ID NO: 59) The primer sequence differs from the PA gene sequence as follows :

Position PA Gene 1302 F

294 T C

297 T C

300 G

1303 F 5'-GAACCTGGAACCTTTGATCTTGAGGGGCTA-3'

2053-2082 (SEQ ID NO: 60; the primer sequence has an A at nucleotide 2075, while the PA gene sequence has a G) 1304 R 5'-ATTCACCACTGTCCAGGCCATTGACGCGTC-3'

271-300 (SEQ ID NO: 61) The primer sequence differs from the PA gene sequence as follows :

Position PA Gene 1304 F

274 G C

275 C G

276 G C

277 T A

294 T C

297 T C

300 G A

1305 F 5'-AGCAAAAGCAGGTAGTGATAG-3' 1-21

(SEQ ID NO: 62) The primer sequence differs from the PA gene sequence as follows:

Position PA Gene 1302 F

15 C G

20 T A

21 C G

1306 R 5'-AGTAGAAACAAGGTA-3' 2219-2233

(SEQ ID NO: 63)

1307 F 5'-AGCGAAAGCAGGTAGTGATTCGAGATGGA-3' 1-29

(SEQ ID NO: 64; the primer sequence has a G at nucleotide 4, while the PA gene sequence has an A)

1308 R 5' -AGTAGAAACAAGGTACTTTTTTGGACAG-3' 2206-2233

(SEQ ID NO: 65)

1309 R 5'-TCGATTTGTTGGAGTGACTGA-3' 1782-1802

(SEQ ID NO: 66)

1310 F 5'-GCCGAACTTCTCCTGCCTTGA-3' 684-704 (SEQ ID NO: 67)

1311 F 5'-GTATTCAATAGCCTGTATG-3' 1957-1975

(SEQ ID NO: 68)

1312 R 5'-GACTTCTCTCCTTGTCACTC-3' 386-405

(SEQ ID NO: 69)

1313 F 5'-ACGAGTCAGCTAAAGTGGGCA-3' 1111-1131

(SEQ ID NO:70)

1314 R 5 ' -GACACCTCTGCTGTGAAGTAA-3 ' 1356 - 1376

(SEQ ID NO: 71)

1315 F 5' -AGCGAAAGCAGGTAGTGATTCGAGA-3' 1-25 (SEQ ID NO:56)

1316 R 5' -AGTAGAAACAAGGTACTTTTTTGGA-3' 2209-2233

(SEQ ID NO: 57)

1317 F 5'-GGAGCTGAGAAACCGAAGTTT-3' 319-339

(SEQ ID NO: 72)

1318 R 5'-GACTTGGCCAATAAAGTCCTA-3' 1935-1955

(SEQ ID NO: 73) 1319 R 5'-GCCTGCGCATAGTTCCTGTGA-3' 644-664

(SEQ ID NO: 74)

1320 R 5'-TCTACCACTATTGACTCGCC-3' 196-215 (SEQ ID NO: 75)

• Primers used to determine terminal sequences

1301 F 5'-GAACCTGGAACCTTTGATCTT-3' 2053-2073 (SEQ ID NO: 58)

1303 F 5 ' -GAACCTGGAACCTTTGATCTTGAGGGGGCTA-3 '

2053-2082 (SEQ ID NO: 60)

1311 F 5 ' -GTATTCAATAGCCTGTATG-3 ' 1957 - 1975

(SEQ ID NO: 68)

1312 R 5'-GACTTCTCTCCTTGTCACTC-3' 386-405 (SEQ ID NO: 69)

1320 R 5'-TCTACCACTATTGACTCGCC-3' 196-215

(SEQ ID NO:75)

HA (Segment 4) Primers

Primer F/R Primer sequence bp spanned

• RT primer

1401 F 5' -AGCAAAAGCAGGGGATAATTCTA-3' 1-23

(SEQ ID NO:76)

• PCR primers

1401 F 5 ' -AGCAAAAGCAGGGGATAATTCTA-3 ' 1-23 (SEQ ID NO : 76 )

1402 R 5' -AGTAGAAACAAGGGTGTTTTTAA-3' 1743-1765

(SEQ ID NO:77)

• Sequencing Primers

1401 F 5' -AGCAAAAGCAGGGGATATTCTA-3' 1-23

(SEQ ID NO: 76)

1402 R 5' -AGTAGAAACAAGGGTGTTTTTAA-3' 1743-1765

(SEQ ID NO: 77)

1403 R 5'-ACAGTTTGTTCATTTCCGAG-3' 1400-1419 (SEQ ID NO:78)

1404 R 5'-ACTTCAGGGTGTTTTGCTTA-3' 1004-1023

(SEQ ID NO: 79)

1405 R 5'-GAACCCCCCAAATGTATAGT-3' 605-624

(SEQ ID NO: 80)

1406 R 5'-TGAGGAACTCTGAACCAGCT-3' 199-218

(SEQ ID NO: 81)

1407 F 5'-GTCACTAGTTGCCTCGTCAG-3' 404-423

(SEQ ID NO: 82)

1408 F 5'-CCGGGAGACATACTGGTAAT-3' 792-811 (SEQ ID NO: 83)

1409 F 5'-CAAATCAATGGGAAACTGAA-3' 1203-1222

(SEQ ID NO: 84)

1410 F 5'-TGAACTGAAGTCAGGATACA-3' 1592-1611

(SEQ ID NO: 85) • Primers used to determine terminal sequences

1405 R 5'-GAACCCCCCAAATGTATAGT-3' 605-624 (SEQ ID NO: 80)

1406 R 5'-TGAGGAACTCTGAACCAGCT-3' 199-218

(SEQ ID NO: 81)

1409 F 5'-CAAATCAATGGGAAACTGAA-3' 1203-1222

(SEQ ID NO: 84)

1410 F 5'-TGAACTGAAGTCAGGATACA-3' 1592-1611

(SEQ ID NO: 85)

NP (Segment 5) Primers

Primer F/R Primer sequence bp spanned

• RT Primer

1509 F 5' -AGCAAAAGCAGGGTTAATAATCAC-3' 1-24

(SEQ ID NO: 86)

• PCR Primers

1509 F 5' -AGCAAAAGCAGGGTTAATAATCAC-3' 1-24

(SEQ ID NO: 86)

1510 R 5 ' -AGTAGAAACAAGGGTATTTTTCCT-3 ' 1542 - 1565

(SEQ ID NO : 87 )

• Sequencing primers

1501 R 5'-TTGCACCTTCCATCATCCTT-3' 1380-1399 (SEQ ID NO : 88 )

1502 R 5'-GTCTATTCCCACTAAAGAGT-3' 932-951

(SEQ ID NO: 89)

1503 R 5'-TGCATCAGAGAGCACATCCT-3' 529-548

(SEQ ID NO: 90)

1504 F 5'-GAACTCGTCCTTTATGACAA-3' 364-383 (SEQ ID NO: 91)

1505 F 5'-AGAGCAATGGATCAAGT-3' 751-770

(SEQ ID NO: 92; there is a deletion of the three bases ATG at nucleotides 761- 763)

1506 F 5'-TACTATGGAATCAAGTACTC-3' 1161-1180

(SEQ ID NO: 93)

1507 R 5'-ATCAATCATCTTCCCGAC-3' 130-147

(SEQ ID NO: 94)

1508 F 5'-AGTGTCCTTCCGTGGGCG-3' 1410-1427

(SEQ ID NO: 95)

1509 F 5' -AGCAAAAGCAGGGTTAATAATCAC-3 ' 1-24

(SEQ ID NO: 86)

1510 R 5' -AGTAGAAACAAGGGTATTTTTCCT-3' 1542-1565 (SEQ ID NO: 87) ■

• Primers used to determine terminal sequences

1503 R 5'-TGCATCAGAGAGCACATCCT-3' 529-548 (SEQ ID NO:90) 1506 F 5'-TACTATGGAATCAAGTACTC-3' 1161-1180

(SEQ ID NO: 93)

NA (Segment 6) primers

Primer F/R Primer sequence bp spanned

• RT Primer

1601 F 5' -AGCAAAAGCAGGAGTGAAGATGA-3' 1-23

(SEQ ID NO:96)

• PCR primers

1601 F 5' -AGCAAAAGCAGGAGTGAAGATGA-3 ' 1-23

(SEQ ID NO: 96)

1602 R 5' -AGTAGAAACAAGGAGTTTTTTCTA-3 ' 1443 - 1466

(SEQ ID NO : 97 )

Sequencing primers

1601 F 5' -AGCAAAAGCAGGAGTGAAGATGA-3' 1-23

(SEQ ID NO: 96)

1602 R 5' -AGTAGAAACAAGGAGTTTTTTCTA-3 ' 1443-1466

(SEQ ID NO: 97)

1603 R 5'-TCGTTGTTTCTGGGTGTGTC-3' 989-1008

(SEQ ID NO: 98; the primer sequence has a T at nucleotide 992, while the NA gene sequence has an C)

1604 R 5'-TTATCATACCCAGTGACACA-3' 596-615

(SEQ ID NO: 99) 1605 R 5' -ATTATTATTGGTTCACACGG-3' 173-192

(SEQ ID NO:100)

1606 F 5'-TTATGTGTCATGCGATCCTG-3' 379-398

(SEQ ID NO: 101)

1607 F 5'-ATTGTTCATATTAGCCCATT-3' 803-822

(SEQ ID NO: 102)

1608 F 5' -ATAGGTCAGGTTATTCTGGT-3' 1224-1243

(SEQ ID NO: 103)

Primers used to determine terminal sequences

1608 F 5' -ATAGGTCAGGTTATTCTGGT-3' 1224-1243

(SEQ ID NO: 103)

1605 R 5' -ATTATTATTGGTTCACACGG-3' 173-192

(SEQ ID NO:100)

Ml (Segment 7) Primers

Primer F/R Primer sequence bp spanned

RT Primer

*1706 F 5'-AGCAAAAGCAGGTAG-3' 1-15

(SEQ ID NO: 104)

PCR primers

1707 R 5'-AGTAGAAACAAGGTA-3' 1013-1027

(SEQ ID NO: 105) 1706 F 5'-AGCAAAAGCAGGTAG-3' 1-15

(SEQ ID NO: 104)

*1701 R 5'-TTTACTCCAGCTCTATGCTGACAA-3' 985-1008

(SEQ ID NO: 106)

* These primers will amplify both the Ml and M2 genes.

• Sequencing primers

1701 R 5'-TTTACTCCAGCTCTATGCTGACAA-3' 985-1008

(SEQ ID NO:106)

1702 R 5'-GATCCAGCCATTTGCTCCAT-3' 590-609 (SEQ ID NO: 107)

1703 R 5' -GAGGTGACAGGATTGGTCTT-3' 169-188

(SEQ ID NO: 108)

1704 F 5'-CATGGACAGAGCAGTTAAAC-3' 301-320

(SEQ ID NO:109)

°1705F 5'-GCGAGTATCATTGGGATCTT-3' 801-820

(SEQ ID NO: 110)

o Common to Ml and M2 sequence.

• Primers used to determine terminal sequences

1705 F 5' -GCGAGTATCATTGGGATCTT-3' 801-820

(SEQ ID NO: 110)

1702 R 5'-GATCCAGCCATTTGCTCCAT-3' 590-609

(SEQ ID NO: 107)

1703 R 5' -GAGGTGACAGGATTGGTCTT-3 169-188 (SEQ ID NO : 108 )

Primers 1701, 1705 and 1706 are sufficient to sequence M2.

NSl (Segment 8) Primers

Primer F/R Primer sequence bp spanned

• RT Primer

*1801 F 5'-AGCAAAAGCAGGGTGACAAAGACA-3' 1-24

(SEQ ID NO:lll)

• PCR Primers

1801 F 5 ' -AGCAAAAGCAGGGTGACAAAGACA-3 ' 1-24

(SEQ ID NO: 111)

1802 R 5' -AGTAGAAACAAGGGTGTTTTTTAT-3' 867-890

(SEQ ID NO: 112)

*1803 R 5'-TTTTTTATCATTAAATAAGCTGAA-3' 851-874 (SEQ ID NO: 113)

* These primers will amplify both the NSl and NS2 genes .

• Sequencing Primers

°1801 F 5' -AGCAAAAGCAGGGTGACAAAGACA-3' 1-24

(SEQ ID NO: 111)

1802 R 5'-AGTAGAAACAAGGGTGTTTTTTAT-3' 867-890

(SEQ ID NO: 112) °1803 R 5'-TTTTTTATCATTAAATAAGCTGAA-3' 851-874

(SEQ ID NO: 113)

1804 R 5'-AATTGCATTTTTGACATCCT-3' 541-560

(SEQ ID NO: 114)

1805 R 5'-ATCTTCTCTACTATCTGCTT-3' 210-229

(SEQ ID NO: 115)

1806 F 5'-CAAGCAATCATGGATAAGAA-3' 387-406

(SEQ ID NO: 116)

°1807 F 5'-GGCGAGAACAGCTAGGTCAA-3' 692-711 (SEQ ID NO: 117)

o

Common to NSl and NS2 sequence

• Primers used to determine terminal sequences

1807 F 5'-GGCGAGAACAGCTAGGTCAA-3' 692-711

(SEQ ID NO: 117)

1804 R 5'-AATTGCATTTTTGACATCCT-3' 541-560 (SEQ ID NO: 114)

1806 F 5'-CAAGCAATCATGGATAAGAA-3' 387-406

(SEQ ID NO: 116)

1805 R 5'-ATCTTCTCTACTATCTGCTT-3' 210-229

(SEQ ID NO: 115)

Primers 1801, 1803 and 1807 are sufficient to sequence NS2. Example 2 PCR Assay to Detect a Segment of the Influenza A/Udorn/72 (H3N2) Strain

A PCR assay is used to detect the presence of a segment of the Influenza A/Udorn/72 (H3N2) strain. PCR primers are designed and selected based on homologies to the viral sequences described herein. The assay is conducted by subjecting the sample to reverse transcription of RNA, followed by PCR amplification of selected cDNA regions corresponding to the specific nucleotide sequence described herein. Amplified PCR products are identified on gels and their specificity confirmed by hybridization with specific nucleotide probes.

Example 3 ELISA to Detect an Antigen Produced by a Segment of the Influenza A/Udorn/72 (H3N2) Strain

An ELISA test is used to detect the presence of an antigen produced by a segment of the Influenza A/Udorn/72 (H3N2) strain. Peptides are designed and selected based on homologies to the viral sequences described herein. These peptides are then coupled to KLH and used to immunize rabbits for the production of monospecific polyclonal antibody. A selection of these polyclonal antibodies, or a combination of polyclonal and monoclonal antibodies is then used in a "capture ELISA" to detect the presence of a protein produced by that viral segment.

Claims

What is claimed is:

1. Isolated nucleic acid molecules which comprise the complete nucleotide sequence of the Influenza A/Udorn/72 (H3N2) strain in positive strand, antigenomic message sense, where said complete sequence consists of: (a) SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 17, or (b) SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 17 and SEQ ID NO: 21.

2. Isolated nucleic acid molecules of Claim 1 which comprise the complete nucleotide sequence of the Influenza A/Udorn/72 (H3N2) strain, where said complete sequence consists of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 17.

3. Isolated nucleic acid molecules of Claim 1 which comprise the complete nucleotide sequence of the Influenza A/Udorn/72 (H3N2) strain, where said complete sequence consists of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:17 and SEQ ID NO:21.

4. An isolated nucleic acid molecule comprising an individual influenza A virus nucleotide sequence in positive strand, antigenomic message sense, where said sequence is selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 21.

5. An isolated nucleic acid molecule of Claim 4, wherein the influenza A virus nucleotide sequence is SEQ ID NO:l.

6. An isolated nucleic acid molecule of Claim 4, wherein the influenza A virus nucleotide sequence is SEQ ID NO: 3.

7. An isolated nucleic acid molecule of Claim 4, wherein the influenza A virus nucleotide sequence is SEQ ID NO: 5.

8. An isolated nucleic acid molecule of Claim 4, wherein the influenza A virus nucleotide sequence is SEQ ID NO: 7.

9. An isolated nucleic acid molecule of Claim 4, wherein the influenza A virus nucleotide sequence is SEQ ID NO: 11.

10. An isolated nucleic acid molecule of Claim 4, wherein the influenza A virus nucleotide sequence is SEQ ID NO: 21.

11. An isolated nucleic acid molecule comprising an individual influenza A virus nucleotide sequence in positive strand, antigenomic message sense, where said sequence encodes a protein having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

12. An isolated nucleic acid molecule of Claim 11, wherein the influenza A virus nucleotide sequence encodes a protein whose sequence is SEQ ID NO: 2.

13. An isolated nucleic acid molecule of Claim 11, wherein the influenza A virus nucleotide sequence encodes a protein whose sequence is SEQ ID NO:4.

14. An isolated nucleic acid molecule of Claim 11, wherein the influenza A virus nucleotide sequence encodes a protein whose sequence is SEQ ID NO : 6.

15. An isolated amino acid sequence comprising an individual influenza A virus amino acid sequence, where said sequence is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

16. An isolated amino acid sequence of Claim 15, wherein the influenza A virus amino acid sequence is SEQ ID NO: 2.

17. An isolated amino acid sequence of Claim 15, wherein the influenza A virus amino acid sequence is SEQ ID NO: 4.

18. An isolated amino acid sequence of Claim 15, wherein the influenza A virus amino acid sequence is SEQ ID NO: 6.

19. An isolated amino acid sequence comprising an individual influenza A virus sequence, where said sequence is SEQ ID NO: 8.

20. An isolated amino acid sequence comprising an individual influenza A virus sequence, where said sequence is SEQ ID NO:12.