RU2767359C1 - Strain "a/chicken/chelyabinsk/314-1/2020 h9n2" of avian influenza virus of genus alphainfluenzavirus of species influenza a virus subtype h9 for control of antigenic and immunogenic activity of vaccines against avian influenza and for production of biopreparations for diagnosis and specific prevention of avian influenza type a subtype h9 - Google Patents

Abstract

FIELD: veterinary science; biotechnology.

SUBSTANCE: invention refers to veterinary virology and biotechnology and concerns a novel strain of influenza type A subtype H9 of Orthomyxoviridae family, genus Alphainfluenzavirus, deposited in the collection of FGBI "ARRIAH" under registration number: No. 333 — dep/20-121 — SCMS FGBU "ARRIAH". Proposed strain is reproduced in 11-day-old chicken embryos of SPF chickens, where it reaches an infectious titre of not less than 8.5 lg EID₅₀/cm³. In the composition of the inactivated vaccine, the strain has pronounced antigenic and immunogenic properties, inducing production of specific antibodies trapped in ELISA and RTGA.

EFFECT: disclosed strain can be used to control antigenic and immunogenic activity of avian influenza vaccines, as well as for preparing biopreparations for diagnosis and specific prevention of avian influenza type A subtype H9.

1 cl, 5 tbl, 1 dwg, 5 ex

Description

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

Avian influenza is an acute, highly contagious viral disease of domestic, synanthropic and wild birds, characterized by septicemia, manifested by depression, edema, damage to the respiratory and digestive organs [3].

The causative agent is an RNA-containing virus belonging to the Orthomyxoviridae family (according to the classification of the International Committee on Taxonomy of Viruses (ICTV) - 00.046), the genus Alphainfluenzavirus (ID: 197911 no classification of The National Center for Biotechnology Information (NCBI), to the species Influenza A virus (ID 00.046.0.01.001).

To date, avian influenza is caused only by influenza A virus, which is subdivided into subtypes based on antigenic differences between two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Currently, 18 hemagglutinating (H1-H18) and 11 neuraminidase subtypes (N1-N11) of influenza A viruses are known [11].

Low pathogenicity avian influenza viruses of the H9N2 subtype show almost equally low pathogenicity under experimental conditions when tested by IVPI, but in the field they often show moderate to high morbidity and mortality [13].

The ability of these viruses to exchange genetic material increases the likelihood of the evolution and emergence of viruses with unknown properties, possibly with a high pandemic potential [2].

In connection with the possibility of human infection with the H9N2 avian influenza virus, the World Health Organization (WHO) pays close attention to it along with the H5, H7 avian influenza virus subtypes, is working on the antigenic and genetic characterization of the virus in order to develop specific prevention tools in order to be prepared for possible pandemics [9, 16].

Known strains of avian influenza virus isolated in the territory of the Russian Federation, used for the manufacture of diagnostic and vaccine preparations.

Known strain "NOVOSIBIRSK" avian influenza virus Influenzae virus avicum to control the immunogenic and antigenic activity of vaccines and the manufacture of biological products for the diagnosis and specific prevention of avian influenza [5]. This is an industrial strain of avian influenza virus type A, subtype H5N1.

A strain A/goose/Kalmykia/813/16 H5N8 of the avian influenza virus Influenza virus avicum type A subtype H5 is known 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 H5 [6] .

A strain A/chiken/Kostroma/3175/17 H5N2 of the avian influenza virus subtype H5N2 Infuenza A virus of the genus Alphainfluenzavirus is known to control the antigenic and immunogenic activity of avian influenza vaccines and to manufacture antigen-containing diagnosticums [7].

Known strain "A/chicken/Primorsk/419/2018 H9N2" avian influenza virus (analog), isolated in Primorsky Krai. The strain is designed 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 [8]. However, this strain has the following disadvantages: the strain belongs to the Y280 genetic line, whose representatives are widespread in China and Southeast Asia, but based on the literature data, H9N2 viruses of the G1 genetic line are no less widespread [11]. Isolates of this genetic line have been isolated in many countries of the Middle East, including Egypt, Iran, Israel, Saudi Arabia, and the United Arab Emirates [12, 17]. The virus has also been routinely isolated in Iraq, Jordan, Kuwait, Lebanon, and Oman [14,15]. In the Russian Federation for the period 2012-2020, G1 genetic lineage viruses were detected in the Amur Region (A/chicken/Amursky/03/12, A/chicken/Amur_Russia/17/2018), Chelyabinsk Region (A/chicken/Chelyabinsk/30/ 2019), Novosibirsk region (A/chicken/Siberia/03/2018) and Trans-Baikal Territory (A/chicken/Zabaikal/2137/2019) [1, 4].

The technical problem is to expand the arsenal of current strains of avian influenza virus type A subtype H9 circulating in the Russian Federation and possessing infectious, antigenic and immunogenic activity, retaining immunogenic properties after inactivation, thereby ensuring the production of sensitive and highly specific diagnosticums and vaccine preparations that create effective protection of poultry against homologous virus.

The closest to the proposed invention in terms of essential features is the vaccine strain A/chicken/Israel/215/2007 H9N2 (Ck/215), belonging to the G1 genetic line and used in Israel in the manufacture of a vaccine against low pathogenic avian influenza virus subtype H9 [10]. However, this strain has a number of disadvantages. The strain was isolated more than ten years ago and antigenically differs from the currently existing variants, and one of the most important factors in choosing an effective strain for the manufacture of biological products for diagnostics and specific prevention, as well as for controlling the antigenic and immunogenic activity of vaccines against avian influenza viruses is the antigenic correspondence between the hemagglutinin protein of the vaccine and the strain circulating in the field. The A/chicken/Israel/215/2007 H9N2 strain is antigenically different from the isolates circulating in the Russian Federation.

This problem was solved by obtaining the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus, which can be used to control the immunogenic and antigenic activity of vaccines and the manufacture of biological preparations for the diagnosis and specific prevention of H9 subtype avian influenza.

The virus isolate, which served as a source for obtaining the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain, was isolated at the FGBI ARRIAH in 2020 from samples received from a poultry farm in the Chelyabinsk region of the Russian Federation.

As a result of virus isolation from the homogenate of the internal organs of chickens, the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" of the avian influenza virus was obtained.

The strain passed 3 passages on 11-day-old SPF-chicken embryos. Able to cause death of chicken embryos within 48-72 hours of incubation.

The H9N2 subtype was identified by the polymerase chain reaction (PCR), nucleotide sequencing, RTGA (hemagglutination inhibition test) and RINA (neuraminidase activity inhibition test).

When conducting a phylogenetic analysis of the nucleotide sequence of the HA gene fragment of the region 1414-1573 bp. It was found that the studied isolate "A/chicken/Chelyabinsk/314-1/2020 H9N2" of the avian influenza virus belongs to the G1 genetic line, which is widespread in the Middle East.

According to the HA gene fragment, the sequences of H9N2 subtype avian influenza viruses isolated in Israel (98.2-99.0% coincidence), as well as the sequences of H9N2 subtype avian influenza viruses isolated in the Middle East (98-99% coincidences).

The hemagglutinin cleavage site contains basic amino acids and has the RSSR/GLF structure, which makes it possible to characterize the virus as potentially low virulent.

The strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" of avian influenza virus type A, subtype H9N2, was deposited on December 11, 2020 in the State Collection of Microorganism Strains of the Federal Center for Animal Health under registration number No. 333 - deposit /20-121-GKSHM FGBI "ARRIAH".

The possibility of using the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus to control the immunogenic and antigenic activity of vaccines and to prepare biological preparations for the specific prevention and diagnosis of avian influenza has been experimentally confirmed.

The essence of the invention is illustrated in the graphic image (Fig. 1). In FIG. Figure 1 shows a dendrogram reflecting the phylogenetic relationships of the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain with epizootic and vaccine strains of 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 studied strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" belongs to an extensive line of G1 strains identified in the Middle East.

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 the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2";

SEQ ID No. 2: represents the amino acid sequence of the hemagglutinin fragment of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2".

The studies were based on determining the primary structure of the hemagglutinin (HA) gene fragment of the test sample of the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain, followed by analysis of phylogenetic relationships with other strains and isolates of the H9N2 subtype avian influenza virus. As a result of sequencing, the nucleotide (360 n.a.) and amino acid (119 a.a.) sequences of the HA gene fragment of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" were determined.

The A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus is characterized by the following features and properties.

Morphological characteristic

The A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of avian influenza virus belongs to the Orthomyxoviridae family, Alphainfluenzavirus genus, Influenza A virus species, H9N2 subtype, and has morphological features characteristic of type A influenza virus.

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

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 length of the genome is about 13,600 nucleotides (nt). The largest fragment (No. 1) contains about 2350 a.i.; No. 2 - a little less than 2350 acting; No. 3, as a rule, - 2250 acting; No. 4 - 1780 acting; No. 5 - 1575 acting; No. 6 -1420 acting; No. 7 - 1050 acting; No. 8 - 900 N.O.

At both ends of each genomic RNA strand, repeating segments are contained, and the repeats of the 5'-terminal region (in type A viruses they have the form 5'-AGUAGAAACAAGG) are complementary to the inverted repeats located at the 3'-end. In addition, the 3'-terminal regions of some genome fragments contain conservative segments typical of viral strains that infect subjects of a certain biological species [ICTV-International Committee on Taxonomy of Viruses].

Antigenic properties

According to its antigenic properties, the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" of the avian influenza virus belongs to the family Orthomyxoviridae, genus Alphainfluenzavirus, species Influenza A virus, type A, subtype H9N2.

The hemagglutinating activity of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" is suppressed by specific serum to the avian influenza virus subtype H9N2 ("IZSVe", Italy), which confirms the belonging of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" avian influenza virus subtype H9. The specific serum titer was 6 log ₂ . The neuraminidase activity of the strain is inhibited by specific serum to the avian influenza virus (AIV) subtype H5N2 (IZSVe, Italy).

Biotechnological properties

The inactivated virus induces antibodies detected by the hemagglutination inhibition test (HITA). So, 14 days after a single immunization of chickens with an inactivated vaccine from the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus in an inoculation volume ^of 0.5 cm inhibition of hemagglutination, amounted to 3.8 log ₂ (table 5).

Geno- and chemotaxonomic characteristics

The A/chicken/Chelyabinsky314-1/2020 H9N2 strain 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 complex with a nucleoprotein (NP) protein.

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

The 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/Chelyabinsk/314-1/2020 H9N2" of the HP virus is sensitive to detergents, formaldehyde, beta-propiolactone, aziridine derivatives, and is quickly inactivated upon heating (56°C), ultraviolet irradiation and at pH below 5.0.

Additional features 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 vaccination dose does not cause visible clinical changes in the general condition of the bird, as well as local tissue lesions at the injection site.

Pathogenic properties

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

Contagious - contagious. Non-immune chickens become infected when 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 characteristics of the strain was carried out using a cross-reaction of hemagglutination inhibition (HAI) and the reaction of inhibition of neuraminidase activity (RINA) with reference hyperimmune sera to antibodies of 16 H-types of avian influenza virus and with reference sera against avian influenza subtypes N1-N2 for neuraminidase. Samples of the viral suspension were preliminarily examined in the hemagglutination test (HHA) to determine the hemagglutination activity of the virus. The activity of the avian influenza virus strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" in the RHA was 8 log ₂ . As a negative control, we used normal blood serum of chickens produced by the Federal State Budgetary Institution "ARRIAH". The results are presented in table 1 and table 2.

In RTGA, the hemagglutination activity of the test sample was suppressed by a specific serum for the H9N2 subtype avian influenza virus (IZSVe, Italy), which confirms that the sample of the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus belongs to the H9 subtype. Serum titer was 6 log ₂ .

In the RINA, the neuraminidase activity of the test sample was suppressed by a specific serum for the H5N2 subtype avian influenza virus (IZSVe, Italy).

According to the results of serological studies, the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain was assigned to the H9N2 subtype avian influenza virus.

Also, for the subtype of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2", molecular biological studies of extraembryonic fluid (EEF) obtained from chicken embryos free from specific pathogenic microflora (SPF-CE) infected with this strain were performed. To do this, total RNA was isolated, which was used to set up the reverse transcriptase polymerase chain reaction (RT-PCR). Primary indication of the M gene was performed by real-time RT-PCR (RT-PCR-RT) using primers M25F, M124R and fluorescently labeled oligonucleotide probe M64. A fragment of the HA gene was amplified using classical RT-PCR. The nucleotide sequence of the fragment (360 bp) of the HA gene of the avian influenza virus strain A/chicken/Chelyabinsk/314-1/2020 H9N2 and the corresponding amino acid sequence (119 bp) were determined by sequencing. It was found that, according to the HA gene fragment, the sequences of H9 subtype avian influenza A viruses previously isolated in the Middle East (98-99% coincidences).

Thus, based on serological and molecular biological studies, the strain “A/chicken/Chelyabinsk/314-1/2020 H9N2” was established to belong to the family Orthomyxoviridae, genus Alphainfluenzavirus, species Influenza A virus, type A, subtype H9N2.

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 production one, it is necessary to obtain a virus strain with high infectious activity. To determine the infectious activity of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" of the avian influenza virus, a series of ten-fold 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 11-day-old SPF-chicken embryos (CE) 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 hemagglutination activity test (HA) by examining the extraembryonic fluid (EEF) from each infected embryo. The titer of the virus in the sample was determined by the method of Kerber and expressed in units of EID ₅₀ /cm ³ (table 3).

Thus, the infectious (in vivo) activity of the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" of the avian influenza virus during 10-fold titration in SPF-CE was 8.5 lg EID ₅₀ /cm ³ .

Example 3

To use the strain to control antigenic and immunogenic activity, as well as to prepare biopreparations for the diagnosis and specific prevention of H9 subtype avian influenza, it is necessary to obtain a strain free from contamination by foreign viruses, bacteria and fungi. Confirmation of the absence of contamination by bacterial and fungal microflora was carried out in accordance with GOST 28085 “Biological medicinal products for veterinary use. Method of bacteriological control of sterility. The absence of contamination with mycoplasmas was carried out in accordance with the requirements of the Global Fund XIV, volume 1, p. 1201-1222.

As a result of the research, it was found that the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus is not contaminated with bacteria, fungi and mycoplasmas. As a result of tests using polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR), it was found that the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of avian influenza virus was not contaminated with Newcastle influenza viruses. diseases (NB), infectious bronchitis of chickens (IBK), infectious avian laryngotracheitis (ILT), infectious bursal disease (IBD), avian reovirus and contains only the genome of the avian influenza virus type A subtype H9N2.

Example 4

The pathogenic properties of the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of avian influenza virus were studied in 30-day-old chickens. An egg cross bird was used in the experiment. The A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of avian influenza virus was injected intramuscularly into chickens in a volume of 0.5 cm ³ with an infectious activity of 106 EID50/cm ³ . As a result, typical clinical signs of the disease were observed in chickens (depression, ruffled plumage, refusal to feed, etc.), and no death of the infected bird was noted.

Thus, the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus is low virulent.

Example 5

To obtain immune blood sera against the strain "A/chicken/Chelyabinsk/314-1/2020 H9N2", a vaccine was prepared containing this strain as the main component (the proposed invention). The adjuvant Montanide ISA 70 (SEPPIC, France) was used to manufacture the vaccine. To prepare the antigen, the extraembryonic fluid of SPF-CE infected with the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain with an infectious activity of 8.5 EID ₅₀ /cm ³ was used, while the hemagglutination activity was 6 log ₂ . Beta-propiolactone (BPL) was used to inactivate the infectious properties of the avian influenza virus. For this purpose, a 10% working solution in sterile demineralized water was prepared from the initial LPL solution. The working solution of BPL was added to the vessel with the virus-containing liquid 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 refrigerator (4-8)°C; a sample of the material in a volume of 5.0 cm ³ 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 samples of the vaccine was prepared. To do this, 30 parts of the antigen with the desired characteristics and 70 parts of the adjuvant were mixed. The content of antigenic material and oil adjuvant in the inoculation dose of the vaccine in the ratio of 30:70 is optimal, as it ensures tolerant presentation of the antigen in the body of the immunized bird. The emulsion was obtained on a homogenizer at 3000 rpm. Immunization of 30-day-old chickens was carried out intramuscularly in a grafting volume of 0.5 cm ³ per head. On days 14 and 42 after vaccination, blood was taken from immunized birds 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 results of the studies indicate a pronounced antigenic activity of the laboratory sample of the vaccine based on the A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of the avian influenza virus of the genus Alphainfluenzavirus of the species Influenza A virus subtype H9, because the presence of specific antibodies to the influenza virus subtype H9N2 in ELISA and antihemagglutinins to the avian influenza virus subtype H9N2 in RTHA were established.

Thus, the claimed strain "A/chicken/Chelyabinsk/314-1/2020 H9N2" of the avian influenza virus of the genus Alphainfluenzavirus of the species Influenza A virus subtype H9 is necessary to create diagnostic preparations used in typing new viral variants, assess the activity of antiviral drugs and the effectiveness of vaccines , as well as for the creation of biological products for the specific prevention of avian influenza type A subtype H9.

Sources of information taken into account when compiling the description of the invention to the application for the issuance of a patent of the Russian Federation for the invention "Strain "A/chicken/Chelyabinsk/314-1/2020 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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Claims (1)

A/chicken/Chelyabinsk/314-1/2020 H9N2 strain of avian influenza virus of the family Orthomyxoviridae, genus Alphainfluenzavirus of the species Influenza A virus subtype H9, deposited in the Collection of Microorganism Strains of the Federal State Budgetary Institution “ARRIAH” under registration number: No. 333 - dep / 20- 121-GKSHM FGBI "ARRIAH" for the manufacture of biological products for the specific prevention of avian influenza type A subtype H9.

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