KR20210056185A - H9N2 recombinant influenza virus, a composition for preparing the same, and a vaccine composition containing the same - Google Patents

Abstract

Provided are H9N2 recombinant influenza virus with low-virulence for a chicken embryo and high-proliferation embryonated chicken eggs, a composition for preparing the same, a cell transfected therewith, a vaccine composition, and a composition for diagnosing avian influenza virus.

Description

The H9N2 recombinant influenza virus, a composition for its preparation, and a vaccine composition containing the same {H9N2 recombinant influenza virus, a composition for preparing the same, and a vaccine composition containing the same}

It relates to a H9N2 recombinant influenza virus, a composition for its preparation, and a vaccine composition comprising the same.

The influenza virus belongs to Orthomyxoviridae and is a virus having 8 negative single-stranded RNA segments as its genome. Hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins 1 and 2 (matrix 1 & 2: M1 and M2, respectively) from 8 RNA fragments , Polymerase units A, B1 and B2 (polymerase subunits A, B1 & B2: PA, PB1, and PB2, respectively), and nonstructural proteins 1 & 2 (nonstructural proteins 1 &2; NS1 and NS2, respectively) are made. In addition, recently, PA-X and PB1-F2 proteins have been made from PA and PB1 genes, respectively, and are known to play an important role in viral pathogenicity.

Avian influenza viruses are classified into low pathogenic avian influenza and highly pathogenic avian influenza according to their pathogenicity to chickens. In Korea, low pathogenic H9N2 avian influenza A/chicken/Korea/01310/2001(H9N2)(01310) was passaged 20 times in embryonated eggs, and a vaccine inactivated (A/chicken/Korea/01310_CE20/2001(H9N2)) was used. Yes (Korean Patent Publication No. 2007-0005867). In the process of passage 20 times, the proliferation of embryonic eggs in the allantoic fluid was increased, but the pathogenicity to the fetus was very high, so the serious mortality rate increased within 48 hours after inoculation, and there is a problem that the virus cannot proliferate in embryonic eggs for a long time. However, it is not known which viral genes are associated with the pathogenicity of 01310-E20 virus to embryonated embryos.

Therefore, it is necessary to prepare a recombinant avian influenza virus with low pathogenicity and high proliferation of fetal embryos by identifying genes related to the pathogenicity of the 01310-E20 virus.

H9N2 recombinant influenza virus is provided.

It provides a composition for producing H9N2 recombinant influenza virus or a production method using the same.

It provides a cell transformed with a composition for producing H9N2 recombinant influenza virus.

It provides a vaccine composition comprising the H9N2 recombinant influenza virus.

One aspect is the nonstructural protein (NS) of the influenza virus 0028-E20 strain; And

Influenza virus 01310-E20 strain of polymerase B1 (polymerase subunit B1: PB1), polymerase B2 (polymerase subunit B2: PB2), polymerase A (polymerase subunit A: PA), hemagglutinin (HA) protein It provides a H9N2 recombinant influenza virus comprising one or more proteins selected from the group consisting of, nucleoprotein (NP), neuraminidase (NA), and matrix protein (matrix: M).

Influenza virus is a virus of the family Orthomyxoviridae and can cause flu or a flu. The influenza virus H9N2 strain is also called avian influenza virus subtype H9N2.

Influenza virus 0028-E20 strain may be H9N2 subtype avian influenza virus SNU0028 [A/chicken/Korea/SNU0028/2000 (H9N2)] (Korean Patent No. 00708593). The 0028-E20 strain may be a virus obtained by subculturing SNU0028 [A/chicken/Korea/SNU0028/2000 (H9N2)] in a chicken embryo for 20 days. The 0028-E20 strain is a virus deposited as KCTC 10866BP. It is a safe vaccine week because embryonated eggs are highly productive and low pathogenic H9N2 subtype avian influenza virus (AIV), which has no fetal pathogenicity, high productivity, and does not have mutations related to pathogenicity to mammals. I can.

Nonstructural proteins (NS) are homodimeric RNA-binding proteins required for viral replication. The non-structural protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 of Korean Patent No. 00808593 or encoded by the nucleic acid sequence of SEQ ID NO: 5 or 7. The non-structural protein may be a polypeptide comprising the amino acid sequence of Uniprot A9YCZ0 or A9YCZ1. The non-structural protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Influenza virus 01310-E20 strain may be a domestic low pathogenic avian influenza virus A/chicken/Korea/01310/2001 (H9N2) (hereinafter referred to as '01310') (Korean Patent No. 0790801). The 01310 strain is a domestic isolate isolated as in Example 1 of Korean Patent No. 0790801 and has low pathogenicity. However, the 01310 strain, although low pathogenic, is a pathogenic virus, so it cannot be deposited with a depository institution, and is currently being stored in the Bird Disease Division of the Agriculture, Forestry and Fisheries Quarantine and Inspection Headquarters. The 01310-E20 strain may be A/chicken/Korea/01310_CE20/2001 (H9N2) virus passaged 20 times in embryonated eggs.

Polymerase B1 (polymerase subunit B1: PB1) is one of the subunits of the RNA-dependent RNA polymerase of influenza virus. The polymerase B1 protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

Polymerase B2 (polymerase subunit B2: PB2) is one of the subunits of the RNA-dependent RNA polymerase of the influenza virus. Polymerase B2 protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

Polymerase A (polymerase subunit A: PA) is one of the subunits of the RNA-dependent RNA polymerase of the influenza virus. The polymerase A protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

Hemagglutinin (HA) protein is also called hemagglutination protein or hemagglutinin. The hemagglutinin may be a polypeptide comprising the amino acid sequence of Uniprot I6QRZ5. The hemagglutinin may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 6.

Nucleoprotein (NP) is a structural protein that encapsulates negative strand viral RNA. The nucleocapsid may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

Neuraminidase (NA) refers to an enzyme that hydrolyzes neuraminic acid to separate sialic acid. The neuraminidase may be required when the virus enters or exits the host's cells. The neuraminidase may be neuraminidase derived from influenza virus 01310-E20 strain. The neuraminidase may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 of Korean Patent No. 0790801. The neuraminidase may be a polypeptide comprising the amino acid sequence of Uniprot I6RJA2. The neuraminidase may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

Matrix protein (M) is a structural protein that connects the viral core and the viral envelope. The matrix protein may be a polypeptide comprising the amino acid sequence of Uniprot I6QXX2 or I6RB09. The matrix protein may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 9.

The H9N2 recombinant influenza virus is one or more selected from the group consisting of polymerase B1, polymerase B2, polymerase A, hemagglutinin protein, nucleocapsid, neuraminidase, and matrix protein of influenza virus 01310-E20 strain. It may contain proteins. The H9N2 recombinant influenza virus may include polymerase B1, polymerase B2, polymerase A, hemagglutinin protein, nucleocapsid, neuraminidase, and matrix protein of influenza virus 01310-E20 strain.

The H9N2 recombinant influenza virus may be deposited under the accession number KCTC13269BP (2017.05.17).

The H9N2 recombinant influenza virus may be a low pathogenic to pathogenic and highly proliferative virus in fetal fetuses. The H9N2 recombinant influenza virus is not only not pathogenic to fetuses, but is highly proliferative, so it is very useful for producing a safe and productive influenza virus vaccine.

Another aspect is a polynucleotide encoding a non-structural protein of the influenza virus 0028-E20 strain; And

Polynucleotide encoding one or more proteins selected from the group consisting of polymerase B1, polymerase B2, polymerase A, hemagglutinin protein, nucleocapsid, neuraminidase, and matrix protein of the influenza virus 01310-E20 strain It provides a composition for producing a recombinant H9N2 influenza virus comprising a.

Influenza virus 0028-E20 strain, non-structural protein, influenza virus 01310-E20 strain, polymerase B1, polymerase B2, polymerase A, hemagglutinin protein, nucleocapsid, neuraminidase, matrix protein, and H9N2 Recombinant influenza virus is as described above.

The polynucleotide comprises a nucleic acid sequence encoding any one of the non-structural protein, polymerase B1, polymerase B2, polymerase A, hemagglutinin protein, nucleocapsid, neuraminidase, and matrix protein. It can be a polynucleotide. The polynucleotide may be changed according to codon usage.

The polynucleotide may be included in a vector. The vector may be an expression vector. The vector may include regulatory regions (eg, promoters, enhancers, and silencers) necessary for expression in animal cells. The polynucleotide may be operably linked to a regulatory region. The vector may further include an origin of replication, a polyA sequence, a multiple cloning site, a selection marker, and the like.

The vector may be a plasmid vector, a cosmid vector, a bacteriophage vector, or a viral vector. The viral vector may be an adenovirus vector, a retroviral vector, or an adeno-associated virus vector.

Another aspect provides a method for producing a recombinant H9N2 influenza virus comprising the step of transforming the H9N2 recombinant influenza virus into the cell by incubating the H9N2 recombinant influenza virus production composition and cells according to one aspect.

The cell may be a cell capable of producing a recombinant virus. For example, the cells are 293T, MDCK, Vero, DF1, PK15, and ST1 cells. The normal cell may be an algal animal cell. The cells may be embryonic eggs of chicks or fetal kidney cells.

The step of incubating the H9N2 recombinant influenza virus preparation composition and cells may include adding the H9N2 recombinant influenza virus to the allantoic fluid of embryonated eggs and culturing the allantoic fluid.

The transformation may be a nucleic acid of the composition for producing H9N2 recombinant influenza virus is introduced into the cell. Transformation can be carried out by methods known in the art. For example, transformation can be performed by transduction, transfection, microinjection, lipofection, or electroporation.

The method may further comprise the step of isolating the H9N2 recombinant influenza virus from the transformed cells.

Another aspect provides a cell transformed with the composition for producing H9N2 recombinant influenza virus according to one aspect.

The composition and transformation for preparing H9N2 recombinant influenza virus are as described above.

The cell may be a cell capable of producing a recombinant virus. For example, the cells are 293T, MDCK, Vero, DF1, PK15, and ST1 cells. The normal cell may be an algal animal cell. The cells may be embryonic eggs of chicks or fetal kidney cells.

Another aspect provides a vaccine composition comprising the H9N2 recombinant influenza virus according to one aspect.

The H9N2 recombinant influenza virus is as described above.

The vaccine composition may be for preventing flu or bird flu. The vaccine composition may be for administration to birds (eg, chickens, ducks) or mammals (eg, humans, pigs, dogs, cats, horses, cows, sheep, mice, camels). The prevention refers to any action of inhibiting or delaying the onset of diseases (eg, flu, avian venom) caused by influenza virus by administration of the vaccine composition.

The vaccine composition may contain an effective amount of H9N2 recombinant influenza virus. The term “effective amount” refers to an amount sufficient to exhibit the effect of prophylaxis or treatment when administered to an individual in need thereof. The effective amount can be appropriately selected by a person skilled in the art according to the cell or individual to be selected. The severity of the disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors, including drugs used in combination or concurrent with the composition used, and other fields of medicine. It can be determined according to factors well-known in the. The vaccine composition may be administered in an amount including ^{about 1x10 7} to 1x10 ¹¹ , 1x10 ⁸ to 5x10 ¹⁰ , 5x10 ⁸ to 2x10 ^{10 viral particles.}

The vaccine composition may be a dead poison vaccine or a live poison vaccine composition.

The vaccine composition may contain a pharmaceutically acceptable carrier. The carrier is used in the sense of including an excipient, diluent or adjuvant. The carrier is, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl blood. It may be selected from the group consisting of rolidone, water, physiological saline, a buffer such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

The vaccine composition may be administered by a parenteral route (eg, intravascular, intravenous, intraarterial, intramuscular or subcutaneous, etc.), orally, nasal, rectal, transdermal, or by inhalation via aerosol. The dosage of the vaccine composition may be, for example, about 1x10 ⁷ to 1x10 ¹¹ , 1x10 ⁸ to 5x10 ¹⁰ , 5x10 ⁸ to 2x10 ¹⁰ viral particles. The administration may be administered once a day, 2 to 24 times a day, 1 to 6 times a week, 1 to 3 times a month, or 1 to 12 times a year.

The vaccine composition may be formulated as an oral dosage form (eg, powder, tablet, capsule, syrup, pill, or granule), or a parenteral formulation (eg, an injection). In addition, the composition may be prepared in a systemic formulation or a topical formulation.

The vaccine composition may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple.

Another aspect provides a method of preventing flu or bird flu, comprising administering to an individual a vaccine composition according to one aspect.

The individual may be a bird (eg, chicken, duck) or a mammal (eg, human, pig, dog, mouse).

The dosage of the vaccine composition varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. The vaccine composition may be administered in an amount including ^{about 1x10 7} to 1x10 ¹¹ , 1x10 ⁸ to 5x10 ¹⁰ , 5x10 ⁸ to 2x10 ^{10 viral particles.} The administration may be administered once a day, 2 to 24 times a day, 1 to 6 times a week, 1 to 3 times a month, or 1 to 12 times a year.

Another aspect provides a composition for diagnosing avian influenza virus comprising an antisera against H9N2 recombinant influenza virus according to one aspect.

Antisera is also called immune serum, and refers to serum that has antibodies specific to antigens from outside. Serological diagnosis is possible by using antiserum for diagnostic purposes.

The recombinant H9N2 influenza virus with low pathogenicity and high proliferation of fetal embryos can be usefully utilized as vaccines with high proliferation of fetal and embryonic embryos.

1 shows a schematic diagram of the recombinant viruses r01310-E20 and r01310-E20-NS (0028).

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Construction and characterization of a recombinant virus (r01310-E20-NS(0028)) having 7 genes of 01310-E20 and 0028 NS genome segment

1-1. Recombinant virus production

For the production of recombinant influenza virus, Hoffmann's reverse genetics vector system (Korean Patent No. 0862758) was used.

Specifically, the Hoffman vector in which the PA, NP, M, HA and NA gene segments of the 01310-E20 virus were cloned, and the Hoffman vector in which the 3'terminal 4 nucleotide of the 3'end of the PB1 genome was replaced with'U', and the KBNP-0028 NS genome A Hoffman vector (Korean Patent No. 1426407) plasmid in which the fragment was cloned was prepared.

DMEM (GIBCO BRL) medium containing 5% (v/v) FBS was added to a 6-well cell culture vessel, and ^{2×10 6} cells/2 ml of 293T cells (Bio Resource Center, KCTC) were added to each well. Cells were attached by incubation for about 3 to 4 hours at 37° C. and 5% CO _2. After removing the medium, 2 ml of Opti-MEM medium (Invitrogen Co. USA) was added.

All eight of the prepared plasmids were put in an amount of 300 ng each in one 1.5 ml tube, and Opti-MEM medium was added to a final 25 μl. In another 1.5 ml tube, 6 µl of Plus reagent (Invitrogen Co. USA) and 69 µl of Opti-MEM medium were added and mixed, and then added to a 1.5 ml tube containing the plasmid for mixing, and the mixture was mixed at room temperature. Incubated for about 15 minutes at.

4 µl of lipofectamine (Invitrogen Co) and 96 µl of Opti-MEM were mixed, added to the tube with the plasmid, and incubated for 15 minutes. The reaction product was added to each well containing 293T cells by 100 μl. Cells were cultured at 37° C. and 5% CO ₂ for 20 hours, and then 10 μg of trypsin (2.5 μg/4 μl) was added per well. After 24 hours, the supernatant was harvested, and 100 µl of the harvested stock solution was inoculated into 10 to 11 day old SPF embryonated eggs (Sunrise Co., NY) by the allantoic route.

The inoculated embryonated eggs were cultured at 37° C. for 3 days, and the allantoic fluid was harvested to check whether hemagglutination. All inoculated embryos were positive for hemagglutination. The recombinant virus was named r01310-E20 or r01310-E20-NS (0028), and a schematic diagram of the virus is shown in FIG. 1. r01310-E20-NS(0028) was deposited with the Biotechnology Research Institute's Biological Resource Center on May 17, 2017, and was given the deposit number KCTC13269BP.

The hemagglutination titer of this recombinant virus was measured, diluted 100 times, and the virus (E2) grown in embryonated eggs by the same method was stored at -70°C.

1-2. Virus titer measurement

The proliferation titer (50% embryo infection dose, EID ₅₀ /ml) of the recombinant viruses of Example 1-1 in fetuses was measured. Specifically, each of the recombinant viruses ^{was diluted 10 -1} to 10 ^-9 with a phosphate buffer solution, and inoculated to 5 SPF embryos of 10 to 11 days old for each dilution factor by 100 µl by the allantoic route. After incubation for 3 days, the allantoic fluid was harvested. Hemagglutination was confirmed with chicken red blood cells, and virus titer (EID ₅₀ / ㎖) was measured according to the calculation formula of the Reed-Muench method. The calculated viral titers (EID ₅₀ /ml (log 10)) are shown in Table 1.

Recombinant virus EID ₅₀ /ml (log10) r01310-E20 9.16±0.2 r01310-E20-NS(0028) 9.36±0.2

As shown in Table 1, the viral titer of r01310-E20 was 10 ^9.16 , and that of r01310-E20-NS (0028) was 10 ^9.36 EID ₅₀ /ml. Therefore, r01310-E20-NS(0028) showed similar fetal proliferation compared to r01310-E20.

1-3. Fetal pathogenicity measurement

Fetal pathogenicity of the recombinant virus was measured. Specifically, the recombinant virus of Example 1-1 ^{was diluted with a phosphate buffer solution to 10 2} EID ₅₀ /0.1 ml each, and 10 days-old SPF embryonated eggs were inoculated into the allantoic cavity. Thereafter, the time at which the fetus dies was measured, and the mean death time (MDT) was measured. The measured average killing time is shown in Table 2.

Recombinant virus MDT (Mean Death Time, h) r01310-E20 48.8±0.2 r01310-E20-NS(0028) 94.4±0.2

As shown in Table 2, the MDT of the r01310-E20_NS(0028) virus was significantly longer than that of the r01310-E20 virus. Therefore, it was confirmed that the r01310-E20_NS(0028) virus was significantly less pathogenic to fetuses.

Example 2. Fetal low pathogenic r01310-E20-NS (0028) Recombinant avian influenza virus productivity evaluation

In order to investigate the productivity improvement of r01310-E20-NS(0028) compared to r01310-E20 virus, 13 of the two recombinant viruses were inoculated into 10-day-old SPF embryos at a dose of 10 ² EID _{50 /0.1 ml, respectively.} The r01310-E20 virus obtained the virus from the allantoic fluid at 36 hours before the poultry died, and the r01310-E20-NS (0028) obtained the virus from the allantoic fluid after 72 hours. The amount of the two recombinant viruses was compared by comparing the hemagglutination titer in the total amount of the obtained allantoic fluid. The total hemagglutination titers in allantoic fluid of r01310-E20 and r01310-E20-NS (0028) are shown in Tables 3 and 4, respectively.

r01310-E20 Allantoic fluid (ml) Hemagglutination titer Total hemagglutination titer in allantoic fluid One 12.6 2 ⁹ 6,451 2 12.5 2 ⁸ 3,200 3 11.75 2 ⁹ 6,016 4 12 2 ⁸ 3,072 5 13 2 ⁸ 3,328 6 9.75 2 ⁸ 2,496 6(2) 3 2 ⁶ 192 7 11 2 ⁸ 2,816 8 11 2 ⁸ 2,816 9 9.75 2 ⁸ 2,496 10 13 2 ⁸ 3,328 11 11.8 2 ⁹ 6,042 12 12 2 ⁸ 3,072 13 11.5 2 ⁸ 2,944 gun 154.65 48,269

r01310-E20-NS(0028) Allantoic fluid (ml) Hemagglutination titer Total hemagglutination titer in allantoic fluid One 11.25 2 ⁹ 5,760 2 12.5 2 ⁹ 6,400 3 14.25 2 ⁹ 7,296 4 14 2 ⁹ 7,168 5 15 2 ⁸ 3,840 6 13 2 ⁹ 6,656 7 14.75 2 ⁹ 7,552 8 13 2 ¹⁰ 13,312 9 11.75 2 ⁸ 3,008 10 11.5 2 ⁹ 5,888 11 13.75 2 ⁹ 7,040 12 13.75 2 ⁸ 3,520 13 14.5 2 ⁹ 7,424 gun 173 84,864

As shown in Tables 3 and 4, when inoculated with r01310E20_NS(0028), both the amount of allantoic fluid and hemagglutination titer were higher than those inoculated with r01310-E20. Therefore, it was confirmed that r01310E20_NS(0028) has higher productivity in the production of vaccine strain virus than r01310E20.

Biological Resource Center KCTC13269BP 20170517

<110> SEOUL NATIONAL UNIVERSITY R&DB Foundation <120> H9N2 recombinant influenza virus, a composition for preparing the same, and a vaccine composition containing the same <130> PN129940KR <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 280 <212> PRT <213> Artificial Sequence <220> <223> Nonstructural protein of influenza virus 0028-E20 strain <400> 1 Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60 Val Glu Gln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Val Thr 65 70 75 80 Val Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Glu 85 90 95 Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Ala 100 105 110 Gly Ser Leu Cys Ile Lys Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125 Thr Leu Lys Ala Asn Phe Ser Val Ile Phe Asn Arg Leu Glu Thr Leu 130 135 140 Val Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asp Glu Asp Val Lys Asn 165 170 175 Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val 180 185 190 Arg Val Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser Asp Glu 195 200 205 Asn Gly Gly Pro Ser Leu Ser Pro Lys Gln Lys Arg Lys Met Ala Arg 210 215 220 Thr Ile Glu Pro Glu Val *** Arg Asn Lys Val Ala Asp Arg Arg Asn 225 230 235 240 Thr Thr *** Ile Lys Asp Tyr Arg Glu Gln Leu Arg Thr Asp Asn Val 245 250 255 Tyr Ala Ser Leu Thr Thr Ile Ala *** Ser Gly Ala Arg Asp Lys Asn 260 265 270 Phe Leu Val Ser Ala Tyr Leu Xaa 275 280 <210> 2 <211> 230 <212> PRT <213> Artificial Sequence <220> <223> Nonstructural protein of influenza virus 0028-E20 strain <400> 2 Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60 Val Glu Gln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Val Thr 65 70 75 80 Val Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Glu 85 90 95 Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Ala 100 105 110 Gly Ser Leu Cys Ile Lys Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125 Thr Leu Lys Ala Asn Phe Ser Val Ile Phe Asn Arg Leu Glu Thr Leu 130 135 140 Val Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asp Glu Asp Val Lys Asn 165 170 175 Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val 180 185 190 Arg Val Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser Asp Glu 195 200 205 Asn Gly Gly Pro Ser Leu Ser Pro Lys Gln Lys Arg Lys Met Ala Arg 210 215 220 Thr Ile Glu Pro Glu Val 225 230 <210> 3 <211> 757 <212> PRT <213> Artificial Sequence <220> <223> Polymerase subunit B1 of influenza virus 01310_E20 strain <400> 3 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Thr 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser 145 150 155 160 Gly Arg Leu Ile Asp Leu Leu Lys Asp Val Met Glu Ser Met Asp Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Val Val Glu 245 250 255 Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Asn Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Asn Ile Asp Leu Lys Tyr Phe Asn Glu Ser 370 375 380 Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr 385 390 395 400 Ala Ala Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser 405 410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr Thr 420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445 Leu Ile Val Asn Ala Leu Asp His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510 Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr 515 520 525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu 610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val 625 630 635 640 Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650 655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg 660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met 675 680 685 Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 Lys Glu Glu Phe Ala Glu Ile Met Lys Thr Cys Ser Thr Ile Glu Glu 740 745 750 Leu Arg Arg Gln Lys 755 <210> 4 <211> 759 <212> PRT <213> Artificial Sequence <220> <223> Polymerase subunit B2 of influenza virus 01310_E20 strain <400> 4 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60 Glu Ile Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Lys Val Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Ile His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Arg Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Gly Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Ile Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Lys Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Leu Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly 405 410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 435 440 445 Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu 450 455 460 Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu 500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr 515 520 525 Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser 530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Val Glu 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ala Arg Gly Gln Tyr 580 585 590 Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly 595 600 605 Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620 Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala 660 665 670 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser 675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr 690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys 740 745 750 Arg Ile Arg Met Ala Ile Asn 755 <210> 5 <211> 716 <212> PRT <213> Artificial Sequence <220> <223> Polymerase subunit A of influenza virus 01310_E20 strain <400> 5 Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu 1 5 10 15 Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asp Pro Lys Ile Glu Thr 20 25 30 Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45 Ser Asp Phe His Phe Ile Asp Glu Arg Gly Glu Ser Ile Ile Val Glu 50 55 60 Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu 65 70 75 80 Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn 85 90 95 Thr Thr Gly Val Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr 100 105 110 Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His 115 120 125 Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His 130 135 140 Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp 145 150 155 160 Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe 165 170 175 Thr Ile Arg Gln Glu Met Ala Gly Arg Gly Leu Trp Asp Ser Phe Arg 180 185 190 Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr 195 200 205 Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser 210 215 220 Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly 225 230 235 240 Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg 245 250 255 Ile Glu Pro Phe Leu Arg Thr Thr Pro Arg Pro Ile Arg Leu Pro Asp 260 265 270 Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu 275 280 285 Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu 290 295 300 Tyr Asp Ala Val Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro 305 310 315 320 Asn Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu 325 330 335 Ala Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu His Glu Glu 340 345 350 Lys Ile Pro Lys Thr Lys Asn Leu Lys Lys Thr Ser Gln Leu Lys Trp 355 360 365 Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys 370 375 380 Lys Asp Ile Ser Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ser 385 390 395 400 Arg Ser Leu Ala Ser Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu 405 410 415 Leu Thr Asp Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val 420 425 430 Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala 435 440 445 Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr 450 455 460 Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe 465 470 475 480 Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg 485 490 495 Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg 500 505 510 Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr 515 520 525 Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu 530 535 540 Ile Gly Asp Met Leu Leu Arg Thr Ala Ile Gly Gln Val Ser Arg Pro 545 550 555 560 Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys 565 570 575 Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile 580 585 590 Glu Ser Met Ile Glu Ala Glu Ser Ser Ile Lys Glu Lys Asp Met Thr 595 600 605 Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser 610 615 620 Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu 625 630 635 640 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu 645 650 655 Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu 660 665 670 Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu 675 680 685 Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala 690 695 700 Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys 705 710 715 <210> 6 <211> 560 <212> PRT <213> Artificial Sequence <220> <223> Hemagglutinin of influenza virus 01310_E20 strain <400> 6 Met Glu Ile Ile Ala Leu Ile Ala Ile Leu Val Val Thr Gly Thr Ser 1 5 10 15 Asp Ala Asp Lys Ile Cys Ile Gly Tyr Gln Ser Thr Asn Ser Thr Glu 20 25 30 Thr Val Asp Thr Leu Val Glu Asn Asn Val Pro Val Thr His Thr Lys 35 40 45 Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Asn Leu 50 55 60 Gly His Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Val Tyr 65 70 75 80 Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Lys Glu Trp Ser Tyr 85 90 95 Ile Val Glu Arg Ser Ser Ala Val Asn Gly Met Cys Tyr Pro Gly Arg 100 105 110 Val Glu Asn Leu Glu Glu Leu Arg Ser Phe Phe Ser Ser Ala Arg Ser 115 120 125 Tyr Lys Arg Leu Leu Leu Phe Pro Asp Arg Thr Trp Asn Val Thr Phe 130 135 140 Asn Gly Thr Ser Lys Ala Cys Ser Gly Ser Phe Tyr Arg Ser Met Arg 145 150 155 160 Trp Leu Thr His Lys Asn Asn Ser Tyr Pro Ile Gln Asp Ala Gln Tyr 165 170 175 Thr Asn Asp Trp Gly Lys Asn Ile Leu Phe Met Trp Gly Ile His His 180 185 190 Pro Pro Thr Asp Thr Glu Gln Met Asn Leu Tyr Lys Lys Ala Asp Thr 195 200 205 Thr Thr Ser Ile Thr Thr Glu Asp Ile Asn Arg Thr Phe Lys Pro Gly 210 215 220 Ile Gly Pro Arg Pro Leu Val Asn Gly Gln Gln Gly Arg Ile Asp Tyr 225 230 235 240 Tyr Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Ile Arg Ser Asn 245 250 255 Gly Asn Leu Ile Ala Pro Trp Tyr Gly His Ile Leu Ser Gly Glu Ser 260 265 270 His Gly Arg Ile Leu Lys Thr Asp Leu Asn Ser Gly Asn Cys Ile Ile 275 280 285 Gln Cys Gln Thr Glu Lys Gly Gly Leu Asn Thr Thr Leu Pro Phe Gln 290 295 300 Asn Val Ser Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr Val Gly Val 305 310 315 320 Lys Ser Leu Lys Leu Ala Val Gly Leu Arg Asn Val Pro Ala Thr Ser 325 330 335 Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp 340 345 350 Pro Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln 355 360 365 Gly Val Gly Ile Ala Ala Asp Lys Glu Ser Thr Gln Glu Ala Val Asp 370 375 380 Lys Ile Thr Ser Lys Val Asn Asn Ile Ile Asp Lys Met Asn Lys Gln 385 390 395 400 Tyr Glu Ile Ile Asp His Glu Phe Ser Glu Ile Glu Ala Arg Leu Asn 405 410 415 Met Ile Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Ile Trp Ala Tyr 420 425 430 Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Asp 435 440 445 His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu 450 455 460 Gly Ser Asn Ala Ile Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His 465 470 475 480 Lys Cys Asp Asp Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asp 485 490 495 Arg Leu Lys Tyr Lys Glu Glu Ser Lys Leu Glu Arg Gln Lys Ile Glu 500 505 510 Gly Val Lys Leu Glu Ser Glu Glu Thr Tyr Lys Ile Leu Thr Ile Tyr 515 520 525 Ser Thr Val Ala Ser Ser Leu Val Leu Ala Ile Gly Leu Ala Ala Phe 530 535 540 Met Phe Trp Ala Met Ser Asn Gly Ser Cys Arg Cys Asn Ile Cys Ile 545 550 555 560 <210> 7 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> Nucleoprotein of influenza virus 01310_E20 strain <400> 7 Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly 1 5 10 15 Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met 20 25 30 Val Gly Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45 Leu Ser Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu 50 55 60 Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu 65 70 75 80 Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile 85 90 95 Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp 100 105 110 Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp 115 120 125 Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn 130 135 140 Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175 Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190 Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg 195 200 205 Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn 210 215 220 Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Val Asp 225 230 235 240 Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu 245 250 255 Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His 260 265 270 Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly 275 280 285 Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe 290 295 300 Arg Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu 305 310 315 320 Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala 325 330 335 Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Ala Arg Val 340 345 350 Val Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn 355 360 365 Glu Asn Thr Glu Thr Met Asp Ser Ser Thr Leu Glu Leu Arg Ser Arg 370 375 380 Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg 385 390 395 400 Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg 405 410 415 Asn Leu Pro Phe Glu Lys Ala Thr Ile Met Ala Ala Phe Thr Gly Asn 420 425 430 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met 435 440 445 Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe 450 455 460 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp 465 470 475 480 Met Ser Lys Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 495 Asp Asn <210> 8 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Neuraminidase of influenza virus 01310_E20 strain <400> 8 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr 1 5 10 15 Ile Ala Thr Val Cys Phe Leu Met Gln Ile Ala Ile Leu Ala Thr Thr 20 25 30 Val Thr Leu His Phe Lys Gln Asn Glu Cys Ser Ile Pro Ser Asn Asn 35 40 45 Gln Ala Val Pro Cys Asp Ile Glu Lys Glu Leu Cys Pro Lys Val Val 50 55 60 Glu Tyr Arg Ser Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly Phe Ala 65 70 75 80 Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Ser Ile 85 90 95 Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Ser Ser Asn Lys Cys Tyr 100 105 110 Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Lys His Ser Asn 115 120 125 Gly Thr Ile His Asp Arg Ile Ser His Arg Thr Leu Leu Met Asn Glu 130 135 140 Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile Ala Trp 145 150 155 160 Ser Ser Ser Ser Cys His Asp Gly Arg Ala Trp Leu His Val Cys Val 165 170 175 Thr Gly Asp Asp Arg Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Val 180 185 190 Leu Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg Thr Gln 195 200 205 Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr 210 215 220 Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe Ile Lys 225 230 235 240 Glu Gly Lys Ile Val His Ile Ser Gln Leu Ser Gly Ser Ala Gln His 245 250 255 Ile Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Asp Val Arg Cys Val 260 265 270 Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Ile Asp Ile Asn 275 280 285 Met Ala Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly Leu Val 290 295 300 Gly Asp Thr Pro Arg Asn Asp Asp Ser Ser Ser Asn Ser Asn Cys Lys 305 310 315 320 Asp Pro Asn Asn Glu Arg Gly Asn Pro Gly Val Lys Gly Trp Ala Phe 325 330 335 Asp Tyr Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Lys Asp Ser 340 345 350 Arg Ser Gly Tyr Glu Thr Phe Arg Val Ile Gly Gly Trp Thr Thr Ala 355 360 365 Asn Ser Lys Ser Gln Val Asn Arg Gln Val Ile Val Asp Asn Asn Asn 370 375 380 Trp Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile 385 390 395 400 Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Gln Glu Thr 405 410 415 Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser 420 425 430 Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile Asn Phe 435 440 445 Met Pro Ile 450 <210> 9 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> Matrix protein of influenza virus 01310_E20 strain <400> 9 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Ala Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln 195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Ile Gln Arg Phe Lys *** Thr Ser Arg 245 250 255 Tyr Cys Arg Lys Tyr His Trp Asp Leu Ala Leu Asp Ile Val Asp Ser 260 265 270 *** Ser Ser Phe Leu Gln Met His Leu Ser Ser Pro *** Ile Arg Phe 275 280 285 Glu Lys Arg Ala Phe Tyr Gly Arg Ser Ala *** Val Tyr Glu Gly Arg 290 295 300 Val Ser Thr Gly Thr Ala Glu Cys Cys Gly Cys *** Arg *** Ser Phe 305 310 315 320 Cys Gln His Arg Ala Gly Val Xaa 325

Claims (8)

Nonstructural protein (NS) of influenza virus 0028-E20 strain; And
Influenza virus 01310-E20 strain of polymerase B1 (polymerase subunit B1: PB1), polymerase B2 (polymerase subunit B2: PB2), polymerase A (polymerase subunit A: PA), hemagglutinin (HA) protein H9N2 recombinant influenza virus comprising one or more proteins selected from the group consisting of, nucleoprotein (NP), neuraminidase (NA), and matrix protein (matrix: M). The H9N2 recombinant influenza virus according to claim 1, which is a virus deposited with accession number KCTC13269BP. A polynucleotide encoding a non-structural protein of the influenza virus 0028-E20 strain; And
Polynucleotide encoding one or more proteins selected from the group consisting of polymerase B1, polymerase B2, polymerase A, hemagglutinin protein, nucleocapsid, neuraminidase, and matrix protein of the influenza virus 01310-E20 strain A composition for preparing H9N2 recombinant influenza virus comprising a. The composition of claim 3, wherein the polynucleotide is contained in a vector. Cells transformed with the composition for producing H9N2 recombinant influenza virus of claim 3. The cell of claim 5, wherein the cell is selected from the group consisting of 293T, MDCK, Vero, DF1, PK15, and ST1 cells. A vaccine composition comprising the H9N2 recombinant influenza virus of claim 1. A composition for diagnosing avian influenza virus comprising antisera against the H9N2 recombinant influenza virus of claim 1.

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