RU2736786C1 - Strain "a/chicken/primorsk/419/2018 h9n2" of avian influenza virus of genus alphainfluenzavirus type influenza a virus subtype h9 for controlling antigenic and immunogenic activity of vaccines for avian influenza and for making biopreparations for diagnosis and specific prevention of avian influenza type a subtype h9 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and concerns a novel avian influenza virus strain type A subtype H9 family Orthomyxoviridae, genus Alphainfluenzavirus, deposited in FGBU "VNIIZZH" collection under registration number: N113 - dep / 19-3 - Collections of strains of microorganisms FGBU "VNIIZHZ". Presented strain reproduced in 10-day old embryonated SPF chicken hens, where it reaches the infectious titer of at least 10.13 lg EID₅₀/cm³. In the composition of the inactivated vaccine, the strain exhibits evident antigenic and immunogenic properties, inducing production of specific antibodies trapped in ELISA and HIR.

EFFECT: presented strain can be used for controlling antigenic and immunogenic activity of avian influenza vaccines and for making biopreparations for diagnosis and specific prevention of avian influenza type A subtype H9.

1 cl, 5 tbl, 5 ex, 1 dwg

Description

The invention relates to the field of veterinary virology and biotechnology, in particular to a new strain of the genus Alphainfluenzavirus of the type Influenza A virus subtype H9 and can be used to control the antigenic and immunogenic activity of vaccines against avian influenza of the subtype H9, as well as in the development and manufacture of diagnostic tools and specific prevention avian influenza subtype H9.

Avian influenza (AF) is an acute infectious viral disease of birds, characterized by damage to the digestive system, respiration, and high mortality. Different strains of avian influenza virus can cause 10 to 100% death among those infected and simultaneously affect several species of birds.

According to the international classification of the International Committee on Taxonomy of Viruses (ICTV), the release of Master Species List 2018b.v2, influenza viruses are assigned to the Orthomyxoviridae family (according to the ICTV classification - 00.046, according to the classification of The National Center for Biotechnology Information (NCBI) ID: 11308), including 7 genera: Alphainfluenzavirus (A), Betainfluenzavirus (B), Deltainfluenzavirus (D), Gammainfluenzavirus (C), Isavirus, Quaranjavirus, Thogovirus. The avian influenza virus belongs to the genus Alphainfluenzavirus (ID: 197911), the species Influenza A virus (ID 00.046.0.01.001).

The belonging of a strain of the influenza virus to a genus is determined by conservative elements of the genome, which set the antigenic characteristics of nucleocapsids.

Influenza virus virions have an outer lipoprotein membrane, often spherical in shape. The diameter of the spherical shapes is 60-180 nm. There are filamentous forms of virions up to 200-350 nm. Viral particles contain RNA - 1%, proteins - 70%, fats - 20%, carbohydrates - up to 8%.

Currently, avian influenza viruses (AIV) are divided into 16 subtypes for hemagglutinin (H1-H16) and 9 subtypes for neuraminidase (N1-N9) (13). Influenza viruses of all subtypes were isolated from birds with various combinations of hemagglutinin (HA) and neuraminidase (NA). However, in 2012 and 2013. data appeared on two new subtypes of hemagglutinin and neuraminidase - H17N10 and H18N11. In Guatemala (North America), several isolates of the H17N10 influenza virus subtype were isolated from bats (4), and in Peru (South America), an isolate of the H18N11 influenza virus was also isolated from a bat (14). According to the literature, no influenza virus of the indicated subtypes was isolated from birds.

Influenza viruses possess enormous ecological plasticity due to the high rates of evolution associated with the variability of their genome (11). The natural carriers of the avian influenza virus are wild waterfowl and seabirds, which are the reservoir of almost all influenza subtypes with the exception of H17N10 and H18N1, which were found in bats (5). The relationship between different subtypes of influenza viruses in wild birds does not significantly affect the state of the host population, but the introduction of the virus into unadapted populations of farm birds leads to severe epizootics with economic damage. First of all, this refers to the avian influenza virus of the H5, H7, and H9 subtypes (2, 16).

Influenza virus type A, subtype H9 (A / H9), a low-virulent influenza virus, was first isolated in 1966 from birds in the United States in Wisconsin. Since then, it has not only spread all over the world, but also significantly expanded the range of hosts (6, 7). It has been causing outbreaks in poultry since the mid-1990s. In mainland China, the A / H9 influenza virus was first isolated in 1994 and has become the most common influenza virus subtype among poultry in the last decade (12, 17). Although influenza A / H9 viruses are classified as low-virulent, their wide distribution among birds has led to large economic losses in poultry farming due to a significant decrease in egg production and high mortality caused by co-infection with other pathogens (6, 12).

In the Russian Federation, in February 2012, in one of the poultry farms in the Far Eastern region of the Russian Federation, an outbreak of respiratory disease with increased mortality caused by the avian influenza A / H9 virus was observed (2). During 2012-2017. periodically, antibodies to the H9 antigen from wild birds (Republic of Tyva) were detected. In 2017, antibodies were detected in the blood serum of wild birds from the Kirov region. Also in 2017-18. avian influenza virus was detected in wild migratory birds in the Tomsk region and in the Khabarovsk Territory. In 2018, at a poultry farm in the Primorsky Territory, in the Amur Region in wild ducks and in 2019 in the Chelyabinsk Region, the influenza A / H9 virus was detected. All these cases indicate the circulation of the H9 subtype influenza virus in the populations of domestic and wild birds in the Far East. It should be noted that Primorsky Krai borders directly on China, which is the leader in the number of outbreaks of avian influenza of the H9N2 subtype (2). At present, the circulation of the virus of the A / H9 subtype of all three genetic sublines (G1, Y280, Y439) belonging to the European lineage has been established in Russia.

Known strains of the avian influenza virus are used for the manufacture of diagnostic and vaccine preparations (3-12).

The known strain A / chicken / Israel / 215/2007 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (3).

The known strain A / turkey / Wisconsin / 1/1966 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (6).

The known strain A / Quail / Hong Kong / G1 / 1997 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (12).

The known strain A / Chicken / Beijing / 1/1994 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (12).

The known strain A / chicken / HongKong / Y439 / 1997 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (12).

The known strain A / chicken / Shanghai / F / 1998 (Ck / SH / F / 98) of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (9).

The known strain A / Chicken / Hong Kong / G9 / 1997 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (8).

The known strain A / Chicken / Shandong / 01/2008 of the avian influenza virus is used for the manufacture of diagnostic and vaccine preparations (18).

Thus, for the production of diagnostic and vaccine preparations, strains of the avian influenza virus belonging to the G1 lines (A / Quail / Hong Kong / G1 / 1997, A / chicken / Israel / 215/2007), Y280 (A / chicken / Shanghai / F / 1998) and Y439 (A / chicken / HongKong / Y439 / 1997). However, they all have several disadvantages. The bulk of virus strains was isolated more than ten years ago and antigenically differs from the currently existing variants.

The technical problem is to expand the arsenal of strains of avian influenza virus type A, subtype H9, possessing infectious, antigenic and immunogenic activity, retaining immunogenic properties after inactivation, thereby providing sensitive and highly specific diagnostics and vaccine preparations that create effective protection of poultry against a homologous virus.

This problem was solved by obtaining the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus, which is used to control the immunogenic and antigenic activity of vaccines and the manufacture of biological products for the diagnosis and specific prevention of avian influenza of the H9 subtype.

The viral isolate, which served as a source for obtaining the A / chicken / Primorsk / 419/2018 H9N2 strain, was isolated at FGBI ARRIAH in 2018 from samples received from a poultry farm in the Primorsky Territory of the Russian Federation. Due to virus isolation from the homogenate of internal organs from chickens, the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus was obtained.

The strain passed 3 passages on 10-day-old SPF-chicken embryos. Able to cause the death of chicken embryos within 48-72 hours of incubation.

The H9N2 subtype has been identified by polymerase chain reaction (PCR), nucleotide sequencing, RTGA (hemagglutination inhibition reaction) and RINA (neuraminidase inhibition reaction).

When conducting phylogenetic analysis of the nucleotide sequence of the HA gene fragment of the region 1414-1572 bp. it was found that the investigated isolate belongs to the Y-280 / G9-like genetic line, which is widespread in China and neighboring countries of Southeast Asia.

In terms of the HA gene fragment, the sequences of avian influenza viruses of the H9N2 subtype isolated in China (98.2-99.0% matches), as well as the sequences of avian influenza viruses of the H9N2 subtype, isolated in the territory of Southeast Asia (98- 99% match).

The hemagglutinin cleavage site contains basic amino acids and has an RSSR / GLF structure, which allows the virus to be characterized as potentially low-virulent.

Strain A / chicken / Primorsk / 419/2018 H9N2 of the HP virus type A, subtype H9N2, was deposited on April 10, 2019 in the Collection of microorganism strains of the Federal Center for Animal Health under registration number No. 113 - dep / 19-3 - KShM FGBI "ARRIAH".

The possibility of using the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus has been experimentally confirmed to control the immunogenic and antigenic activity of vaccines and for the preparation of biological products for specific prevention and diagnosis of avian influenza.

The essence of the invention is illustrated in a graphical representation (Fig. 1). FIG. 1 shows a dendrogram reflecting the phylogenetic relationship of the A / chicken / Primorsk / 419/2018 H9N2 strain with epizootic and vaccine strains of the H9 subtype avian influenza virus. The dendrogram is based on a comparison of the nucleotide sequences of the hemagglutinin gene region.

It was shown that the investigated strain A / chicken / Primorsk / 419/2018 H9N2 belongs to the extensive line of Y280 strains, first identified in Southeast Asia in 1997.

The essence of the invention is illustrated by the following sequence listing:

SEQ ID No. 1: represents the nucleotide sequence of the hemagglutinin gene fragment of strain A / chicken / Primorsk / 419/2018 H9N2;

SEQ ID NO: 2: represents the amino acid sequence of the hemagglutinin fragment of strain A / chicken / Primorsk / 419/2018 H9N2.

The studies were based on the determination of the primary structure of the hemagglutinin (HA) gene fragment of the test sample of the A / chicken / Primorsk / 419/2018 H9N2 strain with subsequent analysis of the phylogenetic relationship with other strains and isolates of the avian influenza virus of the H9N2 subtype. As a result of sequencing, the nucleotide (273 nt) and amino acid (91 aa) sequences of the HA gene fragment of the A / chicken / Primorsk / 419/2018 H9N2 strain were determined. The hemagglutinin cleavage site of the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus has an RSSR / GLF structure, which allows the virus to be characterized as potentially low-virulent.

The A / chicken / Primorsk / 419/2018 H9N2 avian influenza virus strain is characterized by the following traits and properties.

Morphological characteristics

The A / chicken / Primorsk / 419/2018 H9N2 avian influenza virus belongs to the Orthomyxoviridae family, the Alphainfluenzavirus genus, the Influenza A virus species, the H9N2 subtype and has morphological features characteristic of the type A influenza virus.

Influenza virus virions are enveloped, pleomorphic. The diameter of the spherical particles is about 100 nm; filamentary particles about 17 nm in diameter, 250 nm long or more. On the surface of the virion there are spike-like projections (up to 14 nm) formed by HA homotrimers. In type A viruses, NA molecules are located between the HA clusters.

The lipid membrane surrounds loop-shaped filaments of nucleocapsids with eight fragments of the viral genome, each of which is represented by a linear single-stranded antisense RNA.

The total genome length is about 13,600 nucleotides (nt). The largest fragment (No. 1) contains about 2350 bp; No. 2 - a little less than 2350 n.d .; No. 3, as a rule, - 2250 ND; No. 4 - 1780 n.d .; No. 5 - 1575 n.d .; No. 6 - 1420 n.d .; No. 7 - 1050 n.d .; No. 8 - 900 n.a.

Both ends of each genomic RNA strand contain

repeating segments, and the repeats of the 5 '-end region (in type A viruses they look like 5' -AGUAGAAACAAGG) are complementary to inverted repeats located at the 3 '-end. In addition, the 3 '-terminal regions of some fragments of the genome contain conservative stretches typical of viral strains that infect subjects of a particular biological species [ICTV-International Committee on Taxonomy of Viruses].

Antigenic properties

By its antigenic properties, the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus belongs to the Orthomyxoviridae family, the Alphainfluenzavirus genus, the Influenza A virus species, type A, the H9N2 subtype.

The hemagglutinating activity of the A / chicken / Primorsk / 419/2018 H9N2 strain is suppressed by a specific serum for avian influenza virus subtype H9N2 (IZSVe, Italy), which confirms that the A / chicken / Primorsk / 419/2018 H9N2 avian influenza virus belongs to the H9 subtype ... The specific serum titer was 6 log ₂ . The neuraminidase activity of the strain is suppressed by a specific serum to the avian influenza virus (AIV) of the H5N2 subtype (IZSVe, Italy).

Biotechnological properties

The inactivated virus induces antibodies that are detected by methods of inhibition of hemagglutinating activity (RTGA) and enzyme-linked immunosorbent assay (ELISA). So, 14 and 28 days after two-fold immunization of chickens with an inactivated vaccine from the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus in an inoculated volume of 1.0 cm ³ / head, the average titer of antibodies in the HA group, providing a hemagglutination inhibition reaction , was 3.2 log ₂ and 7.3 log ₂ , respectively; the titer of antibodies determined by ELISA was 3500 and 10262, respectively (table 5).

Geno- and chemotaxonomic characteristics

Strain A / chicken / Primorsk / 419/2018 H9N2 of avian influenza virus type A subtype H9 is an RNA-containing virus with a molecular weight of 250 × 10 ⁶ D.

The genome of the influenza virus is represented by single-stranded RNA of negative polarity in the form of 8 strands (segments) in a complex with a nucleoprotein (NP) protein.

Influenza virus virions have an outer lipoprotein membrane, often spherical in shape. The diameter of the spherical shapes is about 100 nm. Viral particles contain RNA - 1%, proteins - 70%, fats - 20%, carbohydrates - up to 8%.

Antigenic variability and taxonomic classification of the virus is based on the identification of two major surface proteins, hemagglutinin and neuraminidase.

Resistance to external factors

The strain A / chicken / Primorsk / 419/2018 H9N2 of the HP virus is sensitive to detergents, formaldehyde, beta-propiolactone, aziridine derivatives, and is rapidly inactivated upon heating (56 ° C), ultraviolet irradiation and at pH below 5.0.

Additional signs and properties

Immunogenic activity - immunogenic as part of an inactivated vaccine.

Reactogenicity - does not possess reactogenic properties. Immunization of chickens with a dose twice the inoculated dose does not cause visible clinical changes in the general condition of the bird, as well as local tissue damage at the injection site.

Pathogenic properties

Virulence - low (does not cause death of infected birds at a dose of 10 ^7.0 ELD ₅₀ ).

Contagiousness - contagious. Non-immune chickens become infected when they are kept together with infected ones.

The essence of the invention is illustrated by examples of its use, which do not limit the scope of the invention.

Example 1.

Determination of the subtype antigenic characteristic of the strain was carried out using a cross reaction of inhibition of hemagglutination (RTGA) and a reaction of inhibition of neuraminidase activity (RINA) with reference hyperimmune sera to antibodies of 16 H types of HP virus and with reference sera against avian influenza of subtypes N1-N2 for neuraminide. Samples of the viral suspension were preliminarily investigated in the hemagglutination reaction (RHA) to determine the hemagglutinating activity of the virus. The activity of the A / chicken / Primorsk / 419/2018 H9N2 AIV strain in the RHA was 9 log ₂ . As a negative control, we used normal blood serum of hens produced by FGBI "ARRIAH". The results are shown in Table 1 and Table 2.

In RTGA, the hemagglutinating activity of the test sample was suppressed by a specific serum for avian influenza virus subtype H9N2 (IZSVe, Italy), which confirms that the sample of the A / chicken / Primorsk / 419/2018 H9N2 avian influenza virus belongs to the H9 subtype. The serum titer was 6 log ₂ .

In RINA, the neuraminidase activity of the test sample was inhibited by a specific serum to AIV of the H5N2 subtype (IZSVe, Italy).

According to the results of serological studies, the A / chicken / Primorsk / 419/2018 H9N2 strain was assigned to the avian influenza virus subtype H9N2.

To identify the GP virus genome, total RNA was isolated from samples of avian tissue and extraembryonic fluid (EEF) of infected chicken embryos, which was used for staging reverse transcriptase polymerase chain reaction (RT-PCR). The primary indication of the M gene was performed by real-time RT-PCR (RT-PCR-RT) using primers M25F, M124R and a fluorescently labeled oligonucleotide probe M64. A fragment of the HA gene was amplified using classical RT-PCR. Sequencing was used to determine the nucleotide sequence of a fragment (273 nt) of the HA gene of the avian influenza A / chicken / Primorsk / 419/2018 H9N2 strain and the corresponding amino acid sequence (91 aa). It was found that, for the HA gene fragment to the avian influenza A / chicken / Primorsk / 419/2018 H9N2 strain, the sequences of avian influenza A viruses of the H9 subtype, isolated in the territory of Southeast Asia (98-99% matches), turned out to be the closest. The hemagglutinin cleavage site of the avian influenza A / chicken / Primorsk / 419/2018 H9N2 strain has an RSSR / GLF structure, which makes it possible to characterize the virus as potentially low-virulent.

Thus, on the basis of serological and molecular biological studies, it was established that the A / chicken / Primorsk / 419/2018 H9N2 strain belongs to the Orthomyxoviridae family, the Alphainfluenzavirus genus, the Influenza A virus species, type A, and the H9N2 subtype.

Example 2.

To use virus strains as control strains during infection in order to control the antigenic and immunogenic activity of vaccines, as well as for use as a diagnostic and industrial one, it is necessary to obtain a virus strain with high infectious activity. To determine infectious activity, a series of tenfold serial dilutions (from 10 ^-1 to 10 ^-10 ) was prepared from the viral material. Each dilution of the virus was inoculated into the allantoic cavity of four 10-day-old SPF-chicken embryos (EC) in a volume of 0.2 cm ³ . Four embryos of the control group were injected with PBS in the same way in a volume of 0.2 cm ³ . The specificity of death was confirmed in the reaction of hemagglutinating activity (HAA) by examining the extraembryonic fluid (EEF) from each infected embryo. The titer of the virus in the sample was determined by the Kerber method and expressed in units of EID ₅₀ / cm (Table 3).

Thus, the infectious (in vivo) activity of the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus with 10-fold titration in SPF-CE was 10.13 lg EID ₅₀ / cm ³ .

Example 3.

To use the strain to control antigenic and immunogenic activity, as well as to prepare biological products for the diagnosis and specific prevention of avian influenza of the H9 subtype, it is necessary to obtain a strain free from contamination by foreign viruses, bacteria and fungi. Confirmation of the absence of contamination with bacterial and fungal microflora was carried out in accordance with GOST 28085-89 “Biological preparations. Sterility control method ". The absence of mycoplasma contamination was carried out in accordance with the requirements of the State Pharmacopoeia XIV, volume 1, p. 1201-1222.

As a result of the research, it was established that the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus is not contaminated with bacteria, fungi and mycoplasmas. As a result of tests using the methods of polymerase chain reaction (PCR) and polymerase chain reaction with reverse transcription (RT-PCR), it was found that the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus is not contaminated with Newcastle disease (NB) viruses , infectious chicken bronchitis (IBC), infectious avian laryngotracheitis (ILT), infectious bursal disease (IBD), avian reovirus and contains only the genome of avian influenza virus type A subtype H9N2.

Example 4.

The pathogenic properties of the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus were studied on 40-day-old chickens. An egg cross bird was used in the experiment. The A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus was injected into chickens intramuscularly in a volume of 1.0 cm ³ with an infectious activity of 10 ⁷ EID ₅₀ / cm ³ . As a result of the experiment, no characteristic clinical signs of the disease (depression, ruffled plumage, refusal to feed, etc.) were observed in chickens, and the death of an infected bird was not noted.

Thus, the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus is low-virulent.

Example 5.

A vaccine was prepared to obtain immune blood sera to the A / chicken / Primorsk / 419/2018 H9N2 strain. The adjuvant Montanide ISA 70 (SEPPIC, France) was used to prepare the vaccine. To prepare the antigen, the extraembryonic fluid of infected SPF-CE with an infectious activity of 10.13 EID ₅₀ / cm ^{3 was used} , while the hemagglutinating activity was 6 log ₂ . Beta propiolactone (BPL) was used to inactivate the infectious properties of the avian influenza virus. For this, a working 10% solution was prepared from the initial solution of BPL in sterile demineralized water. In a vessel with vaccinated liquid, a working solution of the LTP was introduced to a final concentration of 0.05%. The vessel with the contents was incubated at a temperature of (18-26) ° C for 24 h with constant stirring on a shaker. After the end of the inactivation process, the viral material was placed in a household refrigerator (4-8) ° C, a 5.0 cm ³ sample of the material was previously taken from the vessel to conduct studies to determine the completeness of the virus inactivation and the sterility of the material. After confirming the completeness of inactivation, a series of laboratory vaccine samples was prepared. For this, 30 parts of the antigen with the given characteristics and 70 parts of the adjuvant were mixed. The emulsion was prepared on a 3000 rpm homogenizer. Chickens of 40 days of age were immunized intramuscularly in a grafted volume of 1.0 cm ³ per head. On the 14th and 28th days after vaccination, the immunized birds were bled and examined for the presence of specific antibodies to the avian influenza virus type A subtype H9 in ELISA and RTGA (Table 4, 5).

The research results indicate a pronounced antigenic activity of a laboratory sample of a vaccine based on the A / chicken / Primorsk / 419/2018 H9N2 strain of the avian influenza virus of the Alphainfluenzavirus genus Influenza A virus, subtype H9, because the presence of specific antibodies to influenza type A virus in ELISA and antihemagglutinins to influenza virus subtype H9N2 was established.

Sources of information taken into account in the preparation of the description of the invention to the application for the grant of a patent of the Russian Federation for the invention "Strain" A / chicken / Primorsk / 419/2018 H9N2 "of the avian influenza virus of the genus Alphainfluenzavirus of the species Influenza A virus subtype H9 to control antigenic and immunogenic activity of vaccines against avian influenza and for the manufacture of biological products for the diagnosis and specific prevention of avian influenza type A, subtype H9 ":

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2. Low pathogenic avian influenza caused by H9 virus subtype. Literature review. / Varkentin A.V., Volkov M.S., Irza V.N. - V .: Proceedings of the Federal Center for Animal Health, 2014, T. 12 (1). S. 41-53.

3. Antigenic Cartography of H9 Avian Influenza Virus and Its Application to Vaccine Selection / Y. Wang, I. Davidson, R. Fouchier, E. Spackman - Avian Diseases, 2015. Vol. 60 (1s). - P. 218-225.

4. Avian Influenza Virus. Springer Protocols / E. Spackman - L .: Methods in Molecular Biology, 2014. Vol. 1161 .-- P. 425.

5. A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA Tong SX, Li Y, Rivailler P, Conrardy C, Castillo DAA, et al. 2012. V 109. P. 4269-4274.

6. A Global Perspective on H9N2 Avian Influenza Virus / T. P. Peacock, J. James, J. E. Sealy and M. Iqbal. - Viruses, 2019. Vol. 11. - P. 620-628.

7. A novel genotype H9N2 influenza virus possessing human H5N1 internal genomes has been circulating in poultry in eastern China since 1998 / Zhang P., Tang Y., Liu X .. - J. Virol, 2009. Vol. 83. - P. 8428-8438.

8. Evolution and Molecular Epidemiology of H9N2 Influenza A Viruses from Quail in Southern China, 2000 to 2005./ KM Xu, KS Li, GJD Smith, JW Li, H. Tai, JX Zhang, RG Webster, JSM Peiris, H. Chen and Y. Guan. - Journal of Virology, 2007 / Vol. 81 (6), p. 2635-2645.

9. Evolution of H9N2 avian influenza virus in embryonated chicken eggs with or without homologous vaccine antibodies./ Jin H., Wang W., Yang X., Su H., Fan J., Zhu R., Wang S., Shi H ., Liu X. - Veterinary Research, 2018. Vol. 14 (71). P. 2-11.

10. Genetic characterization of HA gene of low pathogenic H9N2 influenza viruses isolated in Israel during 2006-2012 periods / I. Shkoda, N. Golender, [et al.]. - Virus Genes, 2013. Vol. 46. - P. 255-263.

11. Genome sequencing and phylogenetic analysis of three avian influenza H9N2 subtypes in Guangxi / Xie Z., Dong J., Tang X. [et al.]. - Virol. Sin. 2009. Vol. 24. - P. 2985-2988.

12. Genotypic evolution and epidemiological characteristics of H9N2 influenza virus in Shandong Province, China./ Li Y., Liu M., Sun Q., Zhang H., Zhang H., Jiang S., Liu S., Huang Y. - Poultry Science, 2019. Vol. 98 (9), P. 3288-3495.

13. Infection with avian influenza viruses / OIE. Terrestrial Animal Health Code, 2019. Chapter 10.4. - URL: https://www.oie.int/standard-setting/terrestrial-code/access-online.

14. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals / OIE. Avian influenza (infection with avian influenza viruses), 2018. Chapter 3.3.4. URL: https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.03.04_AI.pdf

15. New World Bats Harbor Diverse Influenza A Viruses. / Suxiang Tong, Xueyong Zhu, Yan Li, Mang Shi, Jing Zhang, et al. PLoS Pathog. 2013 V 9 (10) P. 1-12.

16. Phylogenetic analysis of influenza A viruses of H9 haemagglutinin subtype / Banks J., Speidel E.C., Harris P.A., Alexander D.J. - Avian Pathol., 2000. Vol. 29. - P. 353-360.

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18. The PA Subunit of the Influenza Virus Polymerase Complex Affects Replication and Airborne Transmission of the H9N2 Subtype Avian Influenza Virus. / Mengchan Hao, Shaojie Han, Dan Meng, Rong Li, Jing Lin, Meng Wang, Tong Zhou and Tongjie Ch .. -Viruses., 2019 V 11 (1), P. 2-12.

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Sequence listing

<110> Federal Governmental Budgetary Institution “Federal Center for Animal Health” (FGBI “ARRIAH”)

<120> Strain "A / chicken / Primorsk / 419/2018 H9N2" of the avian influenza virus of the genus Alphainfluenzavirus of the species Influenza A virus subtype H9 for the control of antigenic and immunogenic activity of vaccines against avian influenza and for the manufacture of biological products for the diagnosis and specific prevention of avian influenza type A subtype H9

<160> 2

<210> 1

<211> 273

<212> RNA

<213> avian influenza virus

<400> 1

AATCTAATAG CTCCATGGTA TGGATACATT CTTTCAGGAA AGAGCCATGG CAGAATTCTG 60

AAGACTGATT TAAAAAGGGG TAGCTGCACA GTGCAATGTC AGACAGAGAA AGGTGGCTTA 120

AACACAACAC TGCCATTCCA AAATGTAAGT AAGTATGCAT TTGGAAACTG CTCAAAATAC 180

ATTGGCATAA AGAGTCTCAA ACTTGCAGTT GGTCTGAGGA ATGTGCCTTC TAGATCTAGT 240

AGAGGACTAT TCGGGGCCAT AGCAGGGTTT ATA 273

<210> 2

<211> 91

<212> PRT

<213> avian influenza virus

<400> 2

Asn Leu Ile Ala Pro Trp Tyr Gly Tyr Ile Leu Ser Gly Lys Ser 15

His Gly Arg Ile Leu Lys Thr Asp Leu Lys Arg Gly Ser Cys Thr 30

Val Gln Cys Gln Thr Glu Lys Gly Gly Leu Asn Thr Thr Leu Pro 45

Phe Gln Asn Val Ser Lys Tyr Ala Phe Gly Asn Cys Ser Lys Tyr 60

Ile Gly Ile Lys Ser Leu Lys Leu Ala Val Gly Leu Arg Asn Val 75

Pro Ser Arg Ser Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 90

Ile 91

<---

Claims (1)

Avian influenza virus strain A / chicken / Primorsk / 419/2018 H9N2, family. Orthomyxoviridae, genus Alphainfluenzavirus of the species Influenza A virus, subtype H9, deposited in the collection of microorganism strains of the Federal Center for Animal Health Protection under registration number: No. 113 - dep / 19-3 - KShM FGBU "ARRIAH", to control the antigenic and immunogenic activity of vaccines against avian influenza and for the manufacture of biological products for the diagnosis and specific prevention of avian influenza type A, subtype H9.

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