KR20220138290A - H9N2 Strain Genetic Recombinant Low Pathogenic Avian Influenza A Virus, Manufacturing Method and Vaccine Composition Thereof Using H9N2 Avian Influenza Virus Belonging to Y280 Lineage - Google Patents

Abstract

The present invention relates to a genetic recombinant H9N2 subtype avian influenza A virus using low pathogenic H9N2 subtype avian influenza A virus belonging to a Y280 lineage, a preparation method thereof, and a vaccine composition including the same as an active ingredient. According to the present invention, a genetic recombinant vaccine having an effect of defending against a low pathogenic H9N2 serum-type avian influenza virus is prepared, thereby contributing to disease control through the effective defending effect in case of occurrence of low pathogenic H9 subtype avian influenza introduced from the overseas or developed domestically. In addition, a human-friendly gene in an HA receptor-binding domain of the avian influenza virus in the vaccine composition is replaced by the gene of HA generally contained in the low pathogenic H9N2 subtype avian influenza virus such that the vaccine composition has low risk to the human body and excellent safety and defending capability in chickens.

Description

TECHNICAL FIELD [0002] H9N2 Strain Genetic Recombinant Low Pathogenic Avian Influenza A Virus, Manufacturing Method and Vaccine Composition Thereof Using H9N2 Avian Influenza Virus Belonging to Y280 Lineage}

The present invention relates to a vaccine prepared using a H9N2 serotype low pathogenic avian influenza virus strain containing a human infectious gene to prevent the H9N2 serotype low pathogenic avian influenza virus.

Influenza virus (Influenza Virus) is an RNA virus belonging to the family Orthomyxoviridae, and serotypes are divided into A, B, and C types. Among them, types B and C have been confirmed to be infected only in humans, and type A has been confirmed to be infected not only in various kinds of poultry and wild birds, but also in humans, horses, pigs, and other mammals.

Avian Influenza Virus (AIV) is a virus belonging to type A, and HA is 16 according to two proteins in the virus outer membrane, Hemagglutinin (HA) and Neuraminidase (NA) genes. The type and NA are divided into 9 types, and there are 144 types of serotypes (16 types of HA, 9 types of NA), and the feature is that there is no cross-protection between different serotypes. HA serves to attach the virus to somatic cells, and NA allows the virus to penetrate into the cell.

The low pathogenic H9N2 avian influenza virus is characterized by clinical symptoms such as respiratory symptoms and decreased egg production in poultry, and may cause economic damage such as death in some individuals. Low pathogenic H9N2 avian influenza virus is currently occurring worldwide, and can be classified into North American family and Eurasian family. Among them, the Eurasian series is divided into BJ94, G1, Y439, and Y280 series. BJ94 is mainly popular in Southeast Asia, G1 is in the Middle East and North Africa, and Y439 series is mainly popular in Europe and Southeast Asia including Korea. Among preventive vaccines, live virus vaccines are almost impossible to develop due to the characteristics of viruses that are easily mutated.

More specifically, the H9N2 type was first isolated from turkeys in the United States in 1966, and in the case of Asia, it is prevalent in poultry in many countries, so the H9N2 type vaccine is mainly used in Middle Eastern countries such as China, Iran, Pakistan and Jordan, including Korea. have. Cases of chicken-derived H9N2 virus infection in pigs and humans have been reported, and it was confirmed that some H9N2 viruses efficiently re-isolated from mice and killed mice without an acclimatization step (Guo et al., 2000; Choi et al., 2004). ). In the case of China, H9N2 vaccine has been used since early 1998, and at least 20 commercial vaccines are used as most of the inactivated oil vaccines. has been reported to have

In Korea, H9N2 type avian influenza virus spread to farms after H9N2 was isolated in 1996, causing economic damage. Recently, the low pathogenic H9N2 virus has been isolated only in the traditional market. There were 100 cases in '15 and 149 cases in '16, but it decreased sharply from '17. Influenza), it was estimated that the detection rate decreased due to strengthening quarantine policies such as prohibition of livestock trade in traditional markets.

However, the vaccine strain currently in use (Y439 series, 01310) is a virus isolated in 2001 and shows a difference of about 10% in HA gene homology with the recent domestic pandemic. . Moreover, in June 2020, a new Y280-series low pathogenic H9N2 avian influenza virus was isolated from the domestic traditional market, and has been continuously isolated since then. , 01310) and about 20% HA gene homology difference. Due to this, if the low pathogenic H9N2 avian influenza virus of the Y280 family is introduced into domestic poultry farms, a lot of economic damage is expected, so it is urgent to develop a vaccine against it.

Patent Publication No. 10-2010-0047940 (published on May 11, 2010)

The present inventors made intensive research efforts to develop a novel recombinant vaccine against H9N2 low pathogenic avian influenza A virus. As a result, an attenuated H9N2-type recombinant avian influenza A virus (rgHS314 vaccine strain) was prepared by substituting the human-friendly amino acid of the H9N2-type low pathogenic avian influenza A virus, and by verifying its high stability, immunity and vaccine efficacy, the present invention The invention was completed.

Accordingly, it is an object of the present invention to provide a method for producing an H9N2-type recombinant avian influenza A virus (rgHS314 vaccine strain).

Another object of the present invention is to provide a recombinant H9N2-type avian influenza A virus.

Another object of the present invention is to provide a vaccine composition against H9 serotype avian influenza A virus.

In order to achieve the above object, the present inventors developed the present invention using the H9N2 serotype low pathogenic avian influenza virus strain (A/chicken/Korea/LBM314/2020(H9N2)) isolated in June 2020 in Korea.

Low pathogenicity Y280 family showing high protective ability against H9N2 type using H9N2 serotype low pathogenic avian influenza A virus isolated from low pathogenic avian influenza virus strain in 2020 (origin virus; A/chicken/Korea/LBM314/2020(H9N2)) The present invention was achieved by selecting a vaccine strain containing the HA gene and preparing a vaccine using the same.

According to one aspect of the present invention, the present invention provides a method for producing a recombinant H9N2-type avian influenza A virus comprising the steps of:

(a) H9N2-type low pathogenic avian influenza A virus-derived HA (hemagglutinin) gene consisting of the nucleotide sequence of SEQ ID NO: 5 and H9N2-type low pathogenic avian influenza A virus-derived NA (neuraminidase) gene consisting of the nucleotide sequence of SEQ ID NO: 3 into vectors, respectively cloning to prepare a recombinant plasmid;

(b) Nucleotide sequences encoding PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) derived from low pathogenic avian influenza A virus cloning each into a vector to prepare a recombinant plasmid;

(c) transfecting the recombinant plasmid of steps (a) and (b) into a packaging cell; and

(d) obtaining H9N2-type recombinant avian influenza A virus from the culture supernatant of the packaging cells.

As used herein, the term 'avian influenza' refers to an acute infectious disease of birds caused by avian influenza virus infection. The causative agent of the avian influenza is influenza A virus.

As used herein, the term 'influenza A virus' is a virus that causes a highly contagious respiratory disease commonly called 'flu' that occurs in animals and humans, and has a fragmented genome consisting of single-stranded negative RNA fragments. It refers to an enveloped RNA virus.

As used herein, the term 'recombinant influenza A virus' refers to a virus in which RNA fragments of influenza A virus are recombined using genetic recombination technology. For example, viruses containing genetic material produced by the combination of RNA fragments of at least two donor viruses (H9N2 serotype low pathogenic influenza A virus and H1N1 serotype low pathogenic influenza A virus).

As used herein, the term 'Highly Pathogenic Avian Influenza A virus (HPAI)' refers to (i) when inoculated into 8 susceptible chickens 4-8 weeks old and 6 or more (75%) die within 10 days; (ii) Intravenous pathogenicity (IVPI, intravenous inoculation of the virus into 4 to 8-week-old susceptible chickens of 10 animals and check the clinical symptoms and mortality of chickens every 24 hours for 10 days, respiratory symptoms, depression , diarrhea, integument or cyanosis, edema of the head, and the severity of neurological symptoms were recorded as scores and the sum of the sum of symptoms multiplied by the symptom value divided by 100) is 1.2 or higher; (iii) In the case of low pathogenicity H5 or H7 type in chickens, it means that the amino acid sequence of the HA protein segment is determined to be similar to high pathogenicity.

In the present specification, the term 'Low Pathogenic Avian Influenza A Virus (LPAI)' refers to less than 2 numbers (25%) within 10 days after inoculating 8 4-8 week old susceptible chickens, rather than highly pathogenic influenza A virus. ) in case of death; (ii) an intravenous pathogenicity index of less than 1.2; (iii) In the case of low pathogenicity H5 or H7 type in chickens, it means a case in which the amino acid sequence of the HA protein segment is determined to be different from that of high pathogenicity.

As used herein, the term 'H9N2-type avian influenza A virus' refers to an influenza A virus in which the antigenic property of the HA protein, which is the surface protein of the influenza A virus, is H9 type, and the antigenic property of the NA protein is N2 type.

A method for producing the H9N2-type recombinant avian influenza A virus of the present invention will be described step-by-step.

Step (a): Preparation of recombinant plasmids containing HA and NA of low pathogenic avian influenza A virus, respectively

First, a recombinant plasmid was obtained by cloning HA (hemagglutinin) derived from H9N2-type low pathogenic influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 5 and NA (neuraminidase) derived from H9N2-type low pathogenic influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 3, respectively, into a vector. manufacture

In the present specification, the term 'nucleotide sequence' is construed to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence in addition to the above-mentioned sequence. The substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to the maximum correspondence, and the aligned sequence is analyzed using an algorithm commonly used in the art. means a nucleotide sequence that exhibits homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al. , Nuc. Acids Res. 16:10881-90 (1988); Huang et al. , Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al. , Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. , J. Mol. Biol. 215:403-10(1990)) is accessible from the National Center for Biological Information (NCBI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLAST is accessible through the BLAST page of the NCBI website. A method for comparing sequence homology using this program can be found on the BLAST help page of the NCBI website.

The term 'vector' as used herein is a linear or circular DNA molecule composed of a fragment encoding a polypeptide of interest operably linked to additional fragments that serve for transcription of the vector. Such additional fragments include promoter and terminator sequences. A vector also includes one or more origins of replication, one or more selectable markers, and the like. Vectors are generally derived from plasmid or viral DNA, or contain elements of both.

In one embodiment of the present invention, the vector is a pHW2000 vector (see Hoffmann E et al. Proc Natl Acad Sci USA 2000 23;97(11):6108-13).

The recombinant plasmid of step (a) includes HA consisting of the nucleotide sequence of SEQ ID NO: 5 and NA consisting of the nucleotide sequence of SEQ ID NO: 3 derived from H9N2-type low pathogenic avian influenza A virus.

In one embodiment of the present invention, the recombinant plasmid of step (a) is composed of a first recombinant plasmid containing HA and a second recombinant plasmid containing NA.

Step (b): Preparation of recombinant plasmids containing PB2, PB1, PA, NP, M and NS of low pathogenic influenza A virus, respectively

Next, PB2, PB1, PA, NP, M and NS derived from low pathogenic influenza A virus were cloned into vectors, respectively, to prepare a recombinant plasmid.

The recombinant plasmid of step (b) contains PB2, PB1, PA, NP, M and NS derived from a low pathogenic influenza A virus, respectively.

In one embodiment of the present invention, the recombinant plasmid is a third recombinant plasmid comprising PB2, a fourth recombinant plasmid comprising PB1, a fifth recombinant plasmid comprising PA, a sixth recombinant plasmid comprising NP, M It consists of a 7th recombinant plasmid containing and an 8th recombinant plasmid containing NS.

Step (c): Transfection into packaging cells

Next, the first to eighth recombinant plasmids of steps (a) and (b) are transfected into packaging cells.

As used herein, the term 'packaging cell' refers to a cell capable of producing a recombinant virus through a non-viral transfection method. For example, the packaging cell is a recombinant H9N2-type influenza A in which the HA, NA, PB2, PB1, PA, NP, M and NS proteins expressed from the transfected first to eighth recombinant plasmids are assembled. virus can be produced.

Step (d): Obtaining H9N2-type recombinant influenza A virus

Finally, the culture supernatant of the packaging cells is centrifuged to obtain an H9N2-type recombinant avian influenza A virus (rgHS314 (H9N2)).

As used herein, the term rgHS314 (H9N2) refers to a virus obtained by the method for producing the H9N2-type recombinant avian influenza A virus according to the present invention.

In one embodiment of the present invention, the H9N2-type low pathogenic avian influenza A virus of step (a) is A/chicken/Korea/LBM314/2020 (H9N2).

According to the nomenclature of avian influenza virus [subtype/origin host/separation region/isolation order/separation year (HA type, NA type)], the A/chicken/Korea/LBM314/2020(H9N2) is an influenza A virus. As a host, it is a virus isolated in 2020 in Korea, which means that it has serotypes of H9 and N2.

In a specific embodiment of the present invention, the H9N2 low pathogenic avian influenza A virus of step (a) is A/chicken/Korea/LBM314/2020 (H9N2) belonging to the Y280 family.

In the present specification, "Y280 family" is defined by the expert group of WHO / OIE / FAO from the criteria for classifying the group of H9 influenza based on the phylogenetic characterization and sequence homology of the HA gene of H9 type influenza. Among the series, it means a series belonging to the Eurasian series (refer to FIG. 1).

The existing vaccine strain currently in use for H9N2 avian influenza A virus (01310, indicated in red in FIG. 1) and the H9N2 type isolated from the traditional market by 2018 are the Y439 series, but were separated from the traditional market in 2020 and until recently. The H9N2 type, which is continuously in vogue, is the Y280 series (see FIG. 1).

In one embodiment of the present invention, the protein of HA derived from H9N2 low pathogenic avian influenza A virus in step (a) is amino acid Leu-Met- located at positions 234 to 236 of HA consisting of the amino acid sequence of SEQ ID NO: 2 Gly(LMG) is substituted with Gln-Gln-Gly(QQG).

LMG is located at positions 234 to 236 of SEQ ID NO: 2, and as a result of replacing LMG with QQG, QQG is located at positions 234 to 236 of SEQ ID NO: 6.

H3 numbering (H3 numbering) is the numbering by arranging the gene sequence based on the H3 type of the amino acid position number of HA. is used as Specifically, leucine (Leu) located at position 234 in H9 is located at position 226 in H3 based on A/Aichi/2/1968 (H3N2), and is generally described as L226.

In the present invention, amino acids Leu-Met-Gly (LMG) located at positions 234 to 236 of HA consisting of the amino acid sequence of SEQ ID NO: 2 are located at positions 226 to 228 based on H3 numbering. Amino acid position numbers 234 to 236 of HA described in the claims of the present invention are described as amino acid position numbers 226 to 228 of HA based on H3 numbering in the detailed description and examples of the present invention.

The H9N2-type low pathogenic avian influenza A virus of the present invention contains Leu-Met-Gly, a human-friendly amino acid, at 226 to 228 of the HA protein consisting of the amino acid sequence of SEQ ID NO: 2, so it may be harmful to the human body, so it is safe for this To develop a vaccine strain, the human-friendly amino acid Leu-Met-Gly (LMG) is substituted with Gln-Gln-Gly (QQG).

Influenza A virus HA is a surface glycoprotein, and when the influenza A virus is introduced into a cell, it plays a role of interacting with a specific receptor on the cell surface. The HA is composed of HA1 and HA2 subunits, and the receptor-binding site (RBS) motif of the HA1 plays an important role in cell receptor specificity and determines the host range of the virus. The RBS of the HA1 binds to N-acetylneuraminic acid (sialic acid, SA) on the surface of the target cell. In the HA, when the amino acid at position 226 is glutamine (Q226), it has an affinity for bird sialic acid α2-3SA, and when the amino acid at position 226 is leucine (L226), it has affinity for sialic acid α2-6SA of mammals or humans There is Mars.

As used herein, the term 'human-friendly amino acid' or 'human-infected amino acid' refers to the amino acid of HA of avian influenza A virus that imparts the property that the avian influenza A virus exhibits high affinity to human sialic acid receptor, The 'human-friendly gene' refers to a gene encoding such human-friendly amino acids.

In another embodiment of the present invention, the human-friendly amino acid of the present invention is Leu (L226) located at position 226 of HA. In another embodiment of the present invention, the human-friendly amino acid of the present invention is Leu-Met (LM) located at positions 226 and 227 of HA.

The present invention also attenuates H9N2 low pathogenic avian influenza A virus by substituting QQG for human-friendly amino acid positions 226 to 228 LMG of HA of H9N2 low pathogenic avian influenza A virus.

In one embodiment of the present invention, in the HA gene from H9N2 low pathogenic avian influenza A virus in step (a), nucleotides TTGATG located at 700 to 705 of HA consisting of the nucleotide sequence of SEQ ID NO: 1 are CAACAA it will be substituted

Nucleotides TTGATG are located at positions 700 to 705 of SEQ ID NO: 1, and as a result of the substitution of TTGATG with CAACAA, nucleotides CAACAA are located at positions 700 to 705 of SEQ ID NO: 5.

The H9N2-type low pathogenic avian influenza A virus of the present invention has TTGATG, a human affinity gene, at No. 700 to No. 705 of HA consisting of the nucleotide sequence of SEQ ID NO: 1, which can be harmful to the human body. The human affinity gene TTGATG is substituted with CAACAA.

The present invention attenuates H9N2 low pathogenic avian influenza A virus by substituting CAACAA for the human affinity gene TTGATG of HA of H9N2 low pathogenic avian influenza A virus.

In one embodiment of the present invention, the low pathogenic influenza A virus of step (b) is A/PR/8/34 (H1N1).

In one embodiment of the present invention, PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and Nucleotide sequences encoding non-structural protein (NS) consist of the nucleotide sequences of SEQ ID NOs: 7 to 12, respectively.

A recombinant plasmid is prepared by cloning PB2, PB1, PA, NP, M and NS derived from a low pathogenic influenza A virus, each consisting of the nucleotide sequences of SEQ ID NOs: 7 to 12 of the low pathogenic influenza A virus, into a vector.

In one embodiment of the present invention, the recombinant plasmid is a third recombinant plasmid comprising PB2 consisting of the nucleotide sequence of SEQ ID NO: 7, a fourth recombinant plasmid comprising PB1 consisting of the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9 A fifth recombinant plasmid comprising PA consisting of the nucleotide sequence of Consists of an eighth recombinant plasmid comprising a NS consisting of a nucleotide sequence of 12.

In one embodiment of the present invention, the packaging cells of step (c) are one or more cells selected from the group consisting of 293T, MDCK, Vero, DF1, PK15, and ST1 cells. In a specific embodiment of the present invention, the packaging cells of step (c) are 293T cells.

In another embodiment of the present invention, in the method for producing the H9N2-type recombinant influenza A virus of the present invention, the culture supernatant of step (d) is inoculated into specific pathogen free (SPF) hatched eggs, and the virus It may further comprise the step of obtaining.

In another embodiment of the present invention, the H9N2-type recombinant avian influenza A virus strain of the present invention can be obtained by a production method comprising the following steps: from A/chicken/Korea/LBM314/2020 (H9N2) strain A first step of amplifying the HA and NA genes substituted with human-friendly amino acids; a second step of transforming by recombination of the gene of step 1; a third step of amplifying the PB2, PB1, PA, NP, M and NS genes from the low pathogenic avian influenza A virus A/PR/8/34 (H1N1) strain; a fourth step of transfecting the plasmid obtained in steps 2 and 3 into 293T cells; and a fifth step of inoculating the culture supernatant into specific pathogen free (SPF) hatched eggs.

According to another aspect of the present invention, the present invention provides an H9N2-type recombinant avian influenza A virus comprising a negative-sense ssRNA of the following genes:

(i) HA (hemagglutinin) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 5;

(ii) NA (neuraminidase) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 3; and

(iii) PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) genes derived from low pathogenic influenza A virus.

In one embodiment of the present invention, the amino acid Leu-Met-Gly located at positions 234 to 236 of HA consisting of the amino acid sequence of SEQ ID NO: 2 is Gln-Gln of the HA protein derived from the H9N2-type low pathogenic avian influenza A virus. It is substituted with -Gly.

In one embodiment of the present invention, in the HA gene derived from the H9N2-type low pathogenic avian influenza A virus, nucleotides TTGATG located at positions 700 to 705 of HA consisting of the nucleotide sequence of SEQ ID NO: 1 are substituted with CAACAA.

In one embodiment of the present invention, the low pathogenic influenza A virus-derived PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) ) The gene consists of the nucleotide sequence of SEQ ID NOs: 7 to 12, respectively.

Since the H9N2-type recombinant influenza A virus of the present invention is prepared by the method for producing the H9N2-type recombinant influenza A virus, which is another aspect of the present invention, overlapping contents are cited in order to avoid excessive complexity of the present specification, omit the description.

According to another aspect of the present invention, the present invention provides a vaccine composition against H9 serotype avian influenza A virus, comprising the H9N2 recombinant avian influenza A virus comprising a negative-sense ssRNA of the following gene:

(i) HA (hemagglutinin) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 5;

(ii) NA (neuraminidase) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 3; and

(iii) PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) genes derived from low pathogenic influenza A virus.

In one embodiment of the present invention, the H9 serotype avian influenza A virus comprises a Y280 family HA gene.

In one embodiment of the present invention, the low pathogenic influenza A virus-derived PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) ) The gene consists of the nucleotide sequence of SEQ ID NOs: 7 to 12, respectively.

As used herein, the term 'vaccine composition' refers to a composition that positively affects the immune response of a subject. The vaccine composition provides a subject with an enhanced systemic or local immune response elicited by a cellular immune response, eg, a Cytotoxic T Lymphocyte (CTL) or a humoral immune response, eg, an antibody.

The subject of the vaccine composition of the present invention includes, but is not limited to, birds, humans, dogs, horses, pigs, cats, and the like.

The vaccine composition of the present invention is a vaccine for animal prevention of H9N2 serotype low pathogenic avian influenza A virus. In a specific embodiment of the present invention, the subject of the vaccine composition is poultry and wild birds including chickens, ducks, quails, turkeys, and geese.

The vaccine composition of the present invention contains a pharmaceutically effective amount of the recombinant H9N2-type avian influenza A virus (rgHS314(H9N2)) of the present invention.

In one embodiment of the present invention, the vaccine composition of the present invention may further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve prevention, alleviation or therapeutic efficacy for a disease or pathological condition caused by a virus. Pharmaceutically acceptable carriers that may be included in the composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid. calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it is not The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The vaccine composition of the present invention may contain other components, such as stabilizers, excipients, other pharmaceutically acceptable compounds or any other antigen or portion thereof. Vaccines may be in the form of freeze-dried preparations or suspensions, all of which are common in the field of vaccine production.

The vaccine composition of the present invention may be administered to a subject in need thereof in a pharmaceutically effective amount.

A suitable dosage of the vaccine composition of the present invention varies depending on factors such as formulation method, administration mode, subject's age, weight, sex, morbidity, food, administration time, administration route, excretion rate, and response sensitivity. can be decided On the other hand, the dosage of the vaccine composition of the present invention is preferably 0.001-100 mg/kg (body weight) per day.

The dosage form of the vaccine composition of the present invention may be in the form of an enteric coating unit, inoculation for intraperitoneal, intramuscular or subcutaneous administration, aerosol spray, oral or intranasal use. It is also possible to administer in drinking water or as edible pellets.

The vaccine composition of the present invention may be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes.

The vaccine composition of the present invention may be administered parenterally. Parenteral administration of the present invention refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion. For parenteral administration of the vaccine of the present invention, it is necessary to prepare a unit dose dosage form by mixing substances that are nontoxic to the receptor and compatible with other preparation ingredients at a pharmaceutically acceptable concentration and dosage under desired purity.

The formulation of the vaccine of the present invention can be applied in any formulation, and the prepared formulation can be used for oral, injection, and application. The formulation is most preferably formulated for injection.

The vaccine composition of the present invention can also be delivered as a single vaccine by expressing heterologous antigens and immune modulatory molecules such as cytokines in the same recombinant, and as a "cocktail" comprising two or more viral vectors carrying different foreign genes or adjuvants. can be administered.

In one embodiment of the present invention, the vaccine composition of the present invention may include an adjuvant. As used herein, the term "adjuvant (adjuvant)" generally refers to any substance that increases a humoral or cellular immune response to an antigen (eg, alum, Freund's complete adjuvant, Freund's incomplete adjuvant, LPS) , poly IC, poly AU, etc.). The adjuvant of the present invention includes alum, complete Freund's adjuvant, incomplete Freund's adjuvant, LPS, poly IC, poly AU, lipofectin (Lipofectin), pyrotaxi (lipotaxi), CaPO4, DEAE dex Minerals such as trans, polybrene, saponins, aluminum hydroxide, gels, lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions Surface-active substances, including but not limited to dinitrophenol.

In one embodiment of the present invention, the H9N2-type recombinant avian influenza A virus in the vaccine composition of the present invention is inactivated.

The inactivation is a method known in the art, for example, formalin, ultraviolet light, heat, BEI (Bianry Ethyleneimine), beta-propiolactone (Beta-Propiolactone) to the virus can be achieved by treatment.

The vaccine composition of the present invention may be prepared by adding an oil adjuvant to the inactivated H9N2-type recombinant avian influenza A virus and mixing them to obtain an inactivated oil vaccine or a dead poison vaccine.

In another embodiment of the present invention, the vaccine composition of the present invention is a dead poison vaccine using the H9N2 serotype recombinant avian influenza A virus strain of the present invention.

The dead-venom vaccine production method is the attenuated recombinant H9N2 serotype virus strain rgHS314 (H9N2) strain (depository institution: KVCC accession number: VR210004) according to the present invention in a specific pathogen-free (SPF) incubated egg for 12 hours to 72 hours. a first step of culturing for an hour or 50 to 60 hours to obtain an allantoic fluid; And after adding 0.01% to 0.1% formalin to the allantoic fluid obtained in the first step, treatment at 0°C to 10°C, preferably at 4°C, for 6 hours to 24 hours, preferably for 12 hours and a second step of inactivation.

In a specific embodiment of the present invention, the first step of the production method for the dead poison vaccine is to obtain an allantoic fluid by culturing the rgHS314 (H9N2) strain in an egg hatched without a specific pathogen for 48 hours. In another specific embodiment of the present invention, the second step of the production method of the dead poison vaccine is a step of inactivating at 4° C. for 12 hours after adding 0.01% formalin to the allantoic fluid obtained in the first step.

The vaccine composition of the present invention can be used for the prevention of H9 serotype avian influenza A virus. The vaccine composition of the present invention can also be used for the prevention of H9 serotype avian influenza A virus belonging to the Y280 family. The vaccine composition of the present invention can also be used for prophylaxis of H9N2 serotype avian influenza A virus. The vaccine composition of the present invention can also be used for the prevention of H9N2 serotype avian influenza A virus belonging to the Y280 family. As used herein, the term “prevention” refers to the prevention or protective treatment of a disease or disease state.

Since the vaccine composition for the H9 serotype avian influenza A virus of the present invention includes the H9N2 recombinant avian influenza A virus, which is another aspect of the present invention, overlapping contents are cited in order to avoid excessive complexity of the present specification. and the description thereof is omitted.

A brief description of the sequence

SEQ ID NO: 1 is the nucleotide sequence of HA of A/chicken/Korea/LBM314/2020 (H9N2).

SEQ ID NO: 2 is the amino acid sequence of HA of A/chicken/Korea/LBM314/2020 (H9N2).

SEQ ID NO: 3 is the nucleotide sequence of the NA of A/chicken/Korea/LBM314/2020 (H9N2).

SEQ ID NO: 4 is the amino acid sequence of the NA of A/chicken/Korea/LBM314/2020 (H9N2).

SEQ ID NO: 5 is the nucleotide sequence of HA of rgHS314 (H9N2).

SEQ ID NO: 6 is the amino acid sequence of HA of rgHS314 (H9N2).

SEQ ID NO: 7 is the nucleotide sequence of PB2 of A/PR/8/34 (H1N1).

SEQ ID NO: 8 is the nucleotide sequence of PB1 of A/PR/8/34 (H1N1).

SEQ ID NO: 9 is the nucleotide sequence of PA of A/PR/8/34 (H1N1).

SEQ ID NO: 10 is the nucleotide sequence of the NP of A/PR/8/34 (H1N1).

SEQ ID NO: 11 is the nucleotide sequence of M of A/PR/8/34 (H1N1).

SEQ ID NO: 12 is the nucleotide sequence of NS of A/PR/8/34 (H1N1).

SEQ ID NOs: 13 to 30 are primer sequences for HA, NA, PB2, PB1, PA, NP, M and NS genes used for production of H9N2-type recombinant avian influenza A virus.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing a recombinant H9N2-type avian influenza A virus.

(b) The present invention provides a recombinant H9N2-type avian influenza A virus.

(c) The present invention provides a vaccine composition against the H9 serotype avian influenza A virus belonging to the Y280 family, including the H9N2 gene recombinant avian influenza A virus.

(d) when the vaccine composition of the present invention is used, the protective effect against H9N2 low pathogenic avian influenza A virus is excellent, It can contribute to control, and in the vaccine composition, the human affinity gene of the HA receptor-binding site of the avian influenza virus is substituted with the HA gene possessed by the general H9N2-type low pathogenic avian influenza A virus. It has excellent safety and defense properties.

1 shows a gene tree for hemagglutinin (HA) of H9N2-type low pathogenic avian influenza A virus.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (vol/vol) %.

Example

Example 1. LBM314/H9N2 virus isolation and identification method

'20.6.23. As part of the AI regular inspection on the day, the throat and feces of native chickens raised at a traditional market in Hwasun, Jeollanam-do, South Korea were collected as samples during the inspection of poultry and mammals in the traditional market. The M (matrix protein) gene of avian influenza virus was confirmed through genetic test (rRT-PCR), and the virus was propagated through egg inoculation. The virus was isolated from the allantoic fluid of 10-day-old SPF eggs after incubation at 37° C. for 60-72 hours. H9N2-type low pathogenic avian influenza A virus belonging to the Y280 family (A/chicken/Korea/LBM314/2020(H9N2)) was confirmed through the above genetic test.

As summarized in Table 1 below, the nucleotide sequence of HA (hemagglutinin) of the A/chicken/Korea/LBM314/2020 (H9N2) influenza virus is shown in SEQ ID NO: 1, and the amino acid sequence of HA is shown in SEQ ID NO: 2 It was. In addition, the nucleotide sequence and amino acid sequence of NA (Neuraminidase) of the A/chicken/Korea/LBM314/2020 (H9N2) influenza virus are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

virus strain gene division SEQ ID NO: A/chicken/Korea/LBM314/2020(H9N2) HA nucleic acid One A/chicken/Korea/LBM314/2020(H9N2) HA amino acid 2 A/chicken/Korea/LBM314/2020(H9N2) NA nucleic acid 3 A/chicken/Korea/LBM314/2020(H9N2) NA amino acid 4

Example 2. Vaccine Preparation

2-1. Low pathogenicity HA human infection gene (H9) substitution

Low pathogenicity H9N2 avian influenza has a human affinity gene (LMG: TTGATGGGA) at positions 226-228 of the HA protein (based on H3 numbering), so it can be harmful to the human body. Accordingly, the present inventors developed a vaccine strain in which the human affinity gene LMG at positions 226-228 of the HA protein was substituted with QQG in order to develop a safer vaccine strain.

RNA was extracted from the A/chicken/Korea/LBM314/2020 (H9N2) influenza virus using an RNA extraction kit (iNtRON, Korea). In order to remove the human affinity gene TTG (L226) of the HA part, two segments were amplified using the overlapping primers shown in Table 2 below, and then the HA gene was used through PCR using these two segments. was amplified (PCR conditions: 94°C for 30 seconds, 57°C for 1 minute, 72°C for 4 minutes/35 times), and was used as the material for the following experiment.

name base sequence SEQ ID NO: HA1F TCCG AAG TTG GGG GGG ATG GAG GCA GTA 13 Sub HA F TCA ACG GTC AAC AAG GAA GAA TTA ATT ATT ATT G 14 Sub HA R TAA TTC TTC CTT GTT GAC CGT TGA CAA GAG 15 HA R GGGC CGC CGG GTT ATT TTA TAT ACA AATGTTGC 16

2-2. Gene NA (N2) amplification of the low pathogenic H9N2 strain

After making cDNA by performing an experiment in the same manner as in Example 2-1, amplification of NA (N2) by PCR using the primers at the ends shown in Table 3 below (PCR conditions: 94°C for 30 seconds, 57°C 1 minute, 72° C. 2 minutes/35 times), and was used as the material for the following experiment.

name base sequence SEQ ID NO: N2-1F TCCG AAG TTG GGG GGG AGCAAAAGCAGGAG 17 N2-1413R GGGC CGC CGG GTT ATT AGTAGAAACAAGGAGT 18

2-3. Vector cloning of the low pathogenic H9N2 strain

The genes HA and NA amplified by PCR in Examples 2-1 and 2-2 were conjugated to pHW2000 vector with In-Fusion HD Cloning kit (Takara, USA), and then E. coli (stella competent cells) After transformation using LB broth (Bio Science, Korea), culture amplified at 37 ° C. in a shaking incubator for 18 hours, and purified with Plasmid DNA purification Kit (iNtRON, Korea) to HA and NA genes got

2-4. A/PR/8/34 (H1N1) Caution Cloning

In A/PR/8/34 (H1N1) virus (ATCC, USA), experiments were performed in the same manner as in Examples 2-1 to 2-3, and PB2, PB1, PA, NP, M, NS genes were identified in the table below. After amplification through PCR (PCR conditions: 94°C 30 sec, 57°C 1 min, 72°C 4 min/35 times) using the specific primers shown in 4, the pHW2000 vector was added to the In-Fusion HD Cloning kit (Takara, USA). ), then transformed using E. coli (stella competent cells), and then cultured for 18 hours in a shaking incubator at 37°C using LB broth (Bioscience, Korea), followed by plasmid amplification. PB2, PB1, PA, NP, M and NS genes were obtained by purification with a DNA purification Kit (iNtRON, Korea).

name base sequence SEQ ID NO: Phw-PB2-1F TCCG AAG TTG GGG GGG AGCGAAAGCAGGTC 19 Phw-PB2-2341R GGGC CGC CGG GTT ATT AGTAGAAACAAGGTCG 20 Phw-PB1-1F TCCG AAG TTG GGG GGG AGCGAAAGCAGGCA 21 Phw-PB1-2341R GGGC CGC CGG GTT ATT AGTAGAAACAAGGCAT 22 Phw-PA-1F TCCG AAG TTG GGG GGG AGCGAAAGCAGGTA 23 Phw-PA-2233R GGGC CGC CGG GTT ATT AGTAGAAACAAGGTAC 24 Phw-NP-1F TCCG AAG TTG GGG GGG AGCAAAAGCAGGGT 25 Phw-NP-1565R GGGC CGC CGG GTT ATT AGTAGAAACAAGGGTA 26 Phw-M-1F TCCG AAG TTG GGG GGG AGCAAAAGCAGGTA 27 Phw-M-1027R GGGC CGC CGG GTT ATT AGTAGAAACAAGGTAG 28 Phw-NS-1F TCCG AAG TTG GGG GGG AGCAAAAGCAGGGT 29 Phw-NS-890R GGGC CGC CGG GTT ATT AGTAGAAACAAGGGTG 30

As summarized in Table 5 below, the nucleotide sequences of PB2, PB1, PA, NP, M and NS plasmids of the A/PR/8/34 (H1N1) virus obtained above are shown in SEQ ID NOs: 7 to 12, respectively. .

virus strain gene division SEQ ID NO: A/PR/8/34 (H1N1) PB2 nucleic acid 7 A/PR/8/34 (H1N1) PB1 nucleic acid 8 A/PR/8/34 (H1N1) PA nucleic acid 9 A/PR/8/34 (H1N1) NP nucleic acid 10 A/PR/8/34 (H1N1) M nucleic acid 11 A/PR/8/34 (H1N1) NS nucleic acid 12

2-5. Preparation of attenuated recombinant H9N2 serotype strain

After culturing 293T cells at 37° C., 5% CO ₂ , 0.5 μl of each of the plasmids obtained in Examples 2-3 and 2-4 were used in each of Lipofectamine LTX and Plus Reagent (Lipofectamin LTX and Plus Reagent, Invitrogen). , USA) was inoculated (transfection). 48 hours after inoculation, the supernatant was inoculated into 10-day-old specific pathogen free (SPF) hatched eggs to obtain an attenuated recombinant H9N2 serotype virus strain (hereinafter referred to as rgHS314 (H9N2)).

As summarized in Table 6 below, the nucleotide sequence and amino acid sequence of HA of rgHS314 (H9N2) are shown in SEQ ID NOs: 5 and 6, respectively.

virus strain gene division SEQ ID NO: rgHS314 (H9N2) HA nucleic acid 5 rgHS314 (H9N2) HA amino acid 6

Example 3. Preparation method of dead poison vaccine

After inoculation of the rgHS314 (H9N2) strain obtained in Example 2 (deposit institution: Korea Veterinary Culture Collection, KVCC, deposit date: January 26, 2021, accession number: VR210004) into 100 10-day-old SPF hatched eggs Incubated for 48 hours in a 37 ℃ incubator. After refrigeration of hatched eggs cultured for 48 hours at 4° C. for 6 hours, allantoic fluid was obtained. After the obtained allantoic fluid was concentrated to 1/10, the concentrated virus was inactivated with 0.01% formalin at 4°C for 12 hours.

Experimental example

Experimental Example 1. Verification of substitution of human-friendly amino acid gene in HA of rgHS314 (H9N2)

In order to verify whether the HA gene is substituted for rgHS314 (H9N2) obtained in Example 2, an experiment was performed as follows.

1-1. Verification by genetic analysis

From the rgHS314 (H9N2) vaccine strain, the HA gene was amplified by PCR in the experimental method of Example 2-1 and cloned into the pHW2000 vector, followed by Applied Biosystems using the BigDye Terminator v3.1 Cycle Sequencing Kit (Bionics, Korea). After obtaining the nucleotide sequence with 3730XL DNA Analyzer, the human affinity gene was confirmed using the CLC program (USA).

As a result of the experiment, it was confirmed that the gene LMG of the rgHS314 vaccine strain HA was substituted with QQG as shown in Table 7 below.

virus strain division base sequence A/chicken/Korea/LBM314/2020(H9N2) HA nucleic acid 5'-GTCAACGGT TTGATG GGAAGAATT-3' amino acid N-terminal-VNG LM GRI-C-terminal rgHS314 (H9N2) HA nucleic acid 5'-GTCAACGGT CAACAA GGAAGAATT-3' amino acid N-terminal-VNG QQ GRI-C-terminal

Experimental Example 2. Validation of safety and immunity of rgHS314 vaccine

In order to confirm the safety and immunity of the vaccine obtained in Example 3 against the H9N2 serotype low pathogenic avian influenza virus, an experiment was performed using SPF chickens as follows.

2-1. Vaccine safety test

As it is a vaccine developed to use SPF chickens as an actual target animal, it was used for the efficacy test of the low pathogenic H9N2 avian influenza vaccine. 1 ml (500 ul/dose: amount of antigen and excipient mixed) rgHS314 (H9N2) avian influenza vaccine 2 weeks after inoculation with the biceps muscle of 10 6-week-old SPF chickens, serum was obtained and antibody titer was measured It was verified by hemagglutination inhibition (HI). Ten SPF chickens used as controls were inoculated with physiological saline to compare efficacy.

As a result of the experiment, as shown in Table 8 below, all of the 10 vaccinated SPF chickens exhibited a hemagglutination inhibitory (HI) titer of ²⁷ or higher, and it was confirmed that they did not show special clinical symptoms such as death or weight loss. could

No. vaccinated object control HI titer Number of deaths/inoculations HI titer Number of deaths/inoculations One 12 0/10 <1 0/10 2 12 <1 3 10 <1 4 12 <1 5 11 <1 6 11 <1 7 12 <1 8 12 <1 9 12 <1 10 11 <1

Experimental Example 3. Efficacy verification of rgHS314 (H9N2) avian influenza vaccine strain

In order to verify the efficacy of the rgHS314 (H9N2) vaccine antigen obtained in Example 3, an experiment was performed as follows.

3-1. H9N2 attack vaccination

10 6-week-old SPF chickens were inoculated with rgHS314(H9N2) avian influenza vaccine antigen by 0.5 ml each in the biceps muscle of the right leg 3 weeks after inoculation, 10 ⁶ Egg Infectious dose (EID ₅₀ ) with 10 unvaccinated controls was challenged by nasal inoculation to determine survival and clinical symptoms for 14 days.

As a result of the experiment, both the challenge-vaccinated vaccine group and the non-vaccine control group survived for 14 days and no mortality occurred, but clinical symptoms such as diarrhea were observed on the 3rd and 5th days in the non-vaccine control group.

3-2. Validation of histological viral titer

According to the experimental method of Experimental Example 3-1, 10 ⁶ Egg Infectious dose (EID ₅₀ ) was inoculated through the nose, and then on the 1st, 3rd, 5th, and 7th days (dpi, days post-immunization) from the experimental group and the control group using a cotton swab. (swab) was performed to determine the virus titer (log ₁₀ EID ₅₀ /0.1ml). The results are shown in Table 9.

As a result of the experiment, as shown in Table 9, in the experimental group inoculated with the rgHS314(H9N2) vaccine antigen, the low pathogenic H9N2 avian influenza virus was not detected in the Oropharyngeal (OP) swab sample, whereas the control group was in the pharyngeal (OP) swab sample. It was confirmed that high titer virus was detected.

vaccine
challenge 1dpi 3dpi 5dpi 7dpi OP CL OP CL OP CL OP CL vaccine Y280 0/8(-)** 0/8 (-) 0/8(-)* 0/8 (-) 0/8 (-) 0/8 (-) 0/8 (-) 0/8 (-) Non-vaccinated control group Y280 7/8 (3.6) 0/8 (-) 5/8 (1.6) 0/8 (-) 2/8 (1.5) 1/8 (1.5) 0/8 (-) 1/8 (1.5)

dpi: days post-immunization; OP: Oropharyngeal; CL: cloacal. Virus shedding/viability (viral titer, log ₁₀ EID ₅₀ /0.1ml), *p value < 0.05, **p value < 0.01 There is a statistically significant difference.

In addition, the virus titer was measured in parenchymal organs such as Trachea, Lung, Cecal Tonsil, Kidney, and Spleen through an autopsy on the 5th day. The experimental results are shown in Table 10.

As shown in Table 10, the vaccine group did not detect the low pathogenic H9N2 avian influenza virus in the cecumal tonsils, whereas the non-vaccine control group could confirm that the virus was detected in the cecal tonsils.

Vaccine challenge Trachea Lung Cecal Tonsil Kidney Spleen vaccine Y280 0/8 (-) 0/8 (-) 0/8(-)* 0/8 (-) 0/8 (-) Non-vaccine control group Y280 3/8 (1.5) 2/8 (1.4) 4/8 (1.7) 2/8 (2.4) 0/8 (-)

There is a statistically significant difference between the number of virus shedding/viability (viral titer, log ₁₀ EID ₅₀ /0.1ml), *p value < 0.05, **p value < 0.01.

3-3. Comparison of efficacy between the existing vaccine (01310) and the rgHS314 vaccine candidate

The existing vaccine strain (01310) marked with a red square in FIG. 1 and the H9N2 influenza virus isolated from the traditional market by 2018 are of the Y439 family, but the H9N2 influenza virus that has been isolated from the traditional market in 2020 and continues to be prevalent until recently is the Y280 series.

In order to compare the efficacy of the existing vaccine (01310) of the H9N2-type Y439 series and the new vaccine candidate strain (rgHS314) of the present invention, which is the H9N2-type Y280 series, according to the experimental methods of Experimental Examples 3-1 and 3-2, 10 ⁶ On the 5th day (5 dpi) after EID ₅₀ challenge inoculation, organs (Trachea, Tra), lung (Lung), cecal tonsil (Cecal Tonsil, CT), kidney (Kidney, Kid), spleen ( Virus titers (log ₁₀ EID ₅₀ /0.1 ml) were measured in the parenchyma of Spleen, SPL). The results are shown in Table 11 below. The number of virus shedding animals relative to the number of live animals for each organ was expressed as the number of virus shedding/viable animals, and the virus titer was indicated in parentheses.

Group No. of chicken Challenge
(titer) Virus replication (EID ₅₀ /0.1ml) at 5dpi Tra Lung CT Kid SPL Existing vaccines
(01310) 8 Y280
(6.0) 2/8
(2.8) 0/8 ^* 2/8
(1.4) 0/8 0/8 Control 5 2/5
(2.0) 3/5
(1.4) 2/5
(1.6) 0/5 1/5
(1.2) New vaccine candidate strain (rgHS314) 8 0/8 0/8 0/8 ^* 0/8 0/8 Control 8 3/8
(1.5) 2/8
(1.4) 4/8
(1.7) 2/8
(2.4) 0/8

In the case of LPAI H9N2 vaccine efficacy evaluation, the efficacy of the vaccine is recognized when it is suppressed by 80% compared to the non-vaccine control group in the cecal tonsil (CT). As a result of the experiment, as shown in Table 11 above, the existing vaccine (01310 strain) did not have the effect of inhibiting virus isolation compared to the non-vaccine control group in CT, but the currently developed new vaccine candidate strain (rgHS314) of the present invention was compared to the non-vaccine control group. A statistically significant effect of virus isolation inhibition (* P value < 0.05) is recognized. In other organs (trachea, Tra), the effect of the current vaccine in suppressing virus isolation compared to the non-vaccine control group is not recognized.

<110> REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) <120> H9N2 Strain Genetic Recombinant Low Pathogenic Avian Influenza A Virus, Manufacturing Method and Vaccine Composition Thereof Using H9N2 Avian Influenza Virus Belonging to Y280 Lineage <130> PN210057 <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 1683 <212> RNA <213> Influenza A virus <400> 1 atggaggcag tatcactaat aactatacta gtagtggcaa cagtaagcaa tgcagataag 60 atctgcatcg gctatcaatc aacaaactcc acggaaactg tggacacact aacagaaaac 120 aatgtccctg taacacatgc caaagaactg ctccacacag agcataatgg gatgctgtgt 180 gcaacaagct tgggacaccc tcttattcta gacacctgca ccattgaagg gctaatttat 240 ggcaatcctt cttgtgatcc attgctggga ggaaaagaat ggtcctatat cgtcgagagg 300 ccatcagccg ttaacggatt atgttatccc gggaatgtag aaaatctaga agagctaaga 360 ttacttttta gttcttctag gtcttatcaa aggatccaga tctttccaga cacaatctgg 420 aatgtgtctt acagtgggac aagcaaagca tgctcggatt cattctacag aagcatgaga 480 tggttgactc aaaagaacaa cgcttaccct acccaagatg ctcaatacac aaataatcaa 540 ggaaagaaca ttct tttcat gtggggtata aatcatccac ccactgatgc tgcgcagaca 600 aatctgtaca ccagaactga cacaacaaca agtgtggcaa cagaggaaat gaatagggtc 660 tttaaaccat tgataggacc aaggcctctt gtcaacggtt tgatgggaag aattaattat 720 tattggtcgg tattgaaacc gggccaaacg ctgcggataa aatctgatgg gaatctaata 780 gctccatggt atggacacat cctttcagga gagagccacg gaagaatcct aaagactgac 840 ttaaaaatgg gtagctgcac agtgcagtgt caaacagaga aaggtggctt aaacacaaca 900 ttgcccttcc aaaatgtaag taagtatgca tttggaaact gctcaaagta cattggcgta 960 aagagtctca aacttgcagt tggtctgagg aatgtgcctt ctagatctag cagaggacta 1020 ttcggggcca tagcaggatt tatagaggga ggttggtcag gactggttgc tggttggtat 1080 gggttccaac attcaaatga ccaaggggtt ggtatggcag cagatagaga ttccacccaa 1140 aaggcagttg ataaaataac atccaaagtg aataatatag tcgacaaaat gaacaagcag 1200 tatgaaatca ttgatcatga attcagtgag gtagaaacta ggcttaacat gatcaatgat 1260 aaggttgatg atcaaatcca agatatatgg gcatataatg cagaattgct agttctgctc 1320 gaaaaccaga aaacactcga tgaacatgac gctaatgtga acaatctata taataaagtg 1380 aagagggcgt tgggttccaa tgcagt ggaa gatgggagag gatgtttcga gctataccac 1440 aaatgtgata accattgcat ggagacaatt cggaatggga cctacaacag gaggaagtat 1500 caagaggaat caaaattaga aagacagaaa atagaggggg tcaagctgga atctgaagaa 1560 acttacaaaa tcctcaccat ttattcgact gtcgcctcat ctcttgtgat tgcaatgggg 1620 tttgctgcct ttttgttctg ggccatgtcc aacgggtcct gcagatgcaa catttgtata 1680 taa 1683 <210> 2 <211> 560 <212> PRT <213> Influenza A virus <400> 2 Met Glu Ala Val Ser Leu Ile Thr Ile Leu Val Val Ala Thr Val Ser 1 5 10 15 Asn Ala Asp Lys Ile Cys Ile Gly Tyr Gln Ser Thr Asn Ser Thr Glu 20 25 30 Thr Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Ala Lys 35 40 45 Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu 50 55 60 Gly His Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Ile Tyr 65 70 75 80 Gly Asn Pro Ser Cys Asp Pro Leu Leu Gly Gly Lys Glu Trp Ser Tyr 85 90 95 Ile Val Glu Arg Pro Ser Ala Val Asn Gly Leu Cys Tyr Pro Gly Asn 100 105 110 Val Glu Asn Leu Glu Glu Leu Arg Leu Leu Phe Ser Ser Ser Arg Ser 115 120 125 Tyr Gln Arg Ile Gln Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr 130 135 140 Ser Gly Thr Ser Lys Ala Cys Ser Asp Ser Phe Tyr Arg Ser Met Arg 145 150 155 160 Trp Leu Thr Gln Lys Asn Asn Ala Tyr Pro Thr Gln Asp Ala Gln Tyr 165 170 175 Thr Asn Asn Gln Gly Lys Asn Ile Leu Phe Met Trp Gly Ile Asn His 180 185 190 Pro Pro Thr Asp Ala Ala Gln Thr Asn Leu Tyr Thr Arg Thr Asp Thr 195 200 205 Thr Thr Ser Val Ala Thr Glu Glu Met Asn Arg Val Phe Lys Pro Leu 210 215 220 Ile Gly Pro Arg Pro Leu Val Asn Gly Leu Met Gly Arg Ile Asn Tyr 225 230 235 240 Tyr Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Ile Lys Ser Asp 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 Lys Met Gly Ser Cys Thr Val 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 Ser Lys Tyr Ile Gly Val 305 310 315 320 Lys Ser Leu Lys Leu Ala Val Gly Leu Arg Asn Val Pro Ser Arg Ser 325 330 335 Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp 340 345 350 Ser Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln 355 360 365 Gly Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Val Asp 370 375 380 Lys Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln 385 390 395 400 Tyr Glu Ile Ile Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn 405 410 415 Met Ile Asn Asp Lys Val 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 Glu 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 Val Glu Asp Gly Arg Gly Cys Phe Glu Leu Tyr His 465 470 475 480 Lys Cys Asp Asn His Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn 485 490 495 Arg Arg Lys Tyr Gln 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 Ile Ala Met Gly Phe Ala Ala Phe 530 535 540 Leu Phe Trp Ala Met Ser Asn Gly Ser Cys Arg Cys Asn Ile Cys Ile 545 550 555 560 <210> 3 <211> 1401 <212> RNA <213> Influenza A virus <400> 3 atgaatccaa atcagaagat aacagcaatt ggctctgttt ctctaatcat tgcgataata aca tacctgttggcgacattaa cat tacctgatta aca 60 tactgcttcct gaatgcagta acccatcgaa taatcaggtg gtgccatgtg aaccgatcat aatagagagg 180 aacacagtgc atttgaatag cactaccata gagagggaaa tttgtcctaa ggtagcagaa 240 tataaaaatt ggttaaaacc acaatgccta attacagggt tcgccccttt ctcaaaggac 300 aactcaatta ggctctctgc aagtggggat gtctgggtaa caagagaacc ttatgtctca 360 tgcagtccag acaaatgtta tcaatttgca cttgggcagg gaaccaccct gaagaacaag 420 cactcaaatg gcactacacg cgatagaacc ccttacagaa ctcttttaat gaatgaatta 480 ggtgtcccgt ttcatttagg aaccaaacaa gtgtgcatag catggtctag ttcaagctgt 540 tatgatggga aagcatggtt acacgtttgt gttactgggg atgatcaaaa tgctactgct 600 agtatcatct atgatgggat gctcgttgac agtattggat catggtccaa aaacatcctc 660 agaactcagg agtcagaatg tgttttgcatt gtatggaactt gtatggaactt 720 ggaagtgcat caggagttgc cgacactaga gtattattca taagagaagg gaaaattgta 780 aatattagac cgttgtcagg aagtgctcag cacgttgagg aatgctcctg ttatccccga 840 tatcctgaaa ttagatgtgt ttgcagagac aattggaagg gctccaatag gcccattgta 900 tatataaata tggctgatta tagcattgaa tccagttatg tgtgctcggg acttgttggc 960 gacacaccaa gaaatgatga tagctccagc agcagcaact gcagagaccc taataacgaa 1020 agaggggccc caggagtgaa agggtgggct tttgacgacg ggaatgatgt ttggatggga 1080 cggacaatca aaaatggttc gcgctcaggt tatgagactt ttagggtcat aaatggttgg 1140 actgtggcta attcaaagtc acagataaat aggcaagtca tagttgacag tgacgactgg 1200 tctgggtatt ccggcatctt ctctgttgag ggcagagaat gcatcaacag gtgtttttat 1260 gtggagttga taagagggag accacaggaa cccagagtgt ggtggacatc aaatagcatc 1320 attgtattct gtggaacctc aggtacatat ggaacaggct catggcctga tggagcgaat 1380 atcaacttca tgtcaatata a 1401 <210> 4 <211> 466 <212> PRT <213> Influenza A virus <400> 4 Met Asn Pro Asn Gln Lys Ile Thr Ala Ile Gly Ser Val Ser Leu Ile 1 5 10 15 Ile Ala Ile Ile Cys Leu Leu Met Gln Ile Al a Ile Leu Thr Thr Thr 20 25 30 Met Thr Leu His Phe Gly Gln Lys Glu Cys Ser Asn Pro Ser Asn Asn 35 40 45 Gln Val Val Pro Cys Glu Pro Ile Ile Ile Ile Glu Arg Asn Thr Val His 50 55 60 Leu Asn Ser Thr Thr Ile Glu Arg Glu Ile Cys Pro Lys Val Ala Glu 65 70 75 80 Tyr Lys Asn Trp Leu Lys Pro Gln Cys Leu Ile Thr Gly Phe Ala Pro 85 90 95 Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Ser Gly Asp Val Trp 100 105 110 Val Thr Arg Glu Pro Tyr Val Ser Cys Ser Pro Asp Lys Cys Tyr Gln 115 120 125 Phe Ala Leu Gly Gln Gly Thr Thr Leu Lys Asn Lys His Ser Asn Gly 130 135 140 Thr Thr Arg Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu 145 150 155 160 Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile Ala Trp Ser 165 170 175 Ser Ser Ser Cys Tyr Asp Gly Lys Ala Trp Leu His Val Cys Val Thr 180 185 190 Gly As p Asp Gln Asn Ala Thr Ala Ser Ile Ile Tyr Asp Gly Met Leu 195 200 205 Val Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg Thr Gln Glu 210 215 220 Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Ala Val Val Met Thr Asp 225 230 235 240 Gly Ser Ala Ser Gly Val Ala Asp Thr Arg Val Leu Phe Ile Arg Glu 245 250 255 Gly Lys Ile Val Asn Ile Arg Pro Leu Ser Gly Ser Ala Gln His Val 260 265 270 Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Glu Ile Arg Cys Val Cys 275 280 285 Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Tyr Ile Asn Met 290 295 300 Ala Asp Tyr Ser Ile Glu Ser Ser Tyr Val Cys Ser Gly Leu Val Gly 305 310 315 320 Asp Thr Pro Arg Asn Asp Asp Ser Ser Ser Ser Ser Asn Cys Arg Asp 325 330 335 Pro Asn Asn Glu Arg Gly Ala Pro Gly Val Lys Gly Trp Ala Phe Asp 340 345 350 Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Lys Asn Gly Ser Arg 355 360 365 Ser Gly Tyr Glu Thr Phe Arg Val Ile Asn Gly Trp Thr Val Ala Asn 370 375 380 Ser Lys Ser Gln Ile Asn Arg Gln Val Ile Val Asp Ser Asp Asp Trp 385 390 395 400 Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Arg Glu Cys Ile Asn 405 410 415 Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Gln Glu Pro Arg 420 425 430 Val Trp Trp Thr Ser Asn Ser Ile Ile Val Phe Cys Gly Thr Ser Gly 435 440 445 Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile Asn Phe Met 450 455 460 Ser Ile 465 <210> 5 <211> 1683 <212> RNA <213> Artificial Sequence <220> <223> rgHS314 HA nt <400> 5 atggaggca g tatcactaat aactatacta gtagtggcaa cagtaagcaa tgcagataag 60 atctgcatcg gctatcaatc aacaaactcc acggaaactg tggacacact aacagaaaac 120 aatgtccctg taacacatgc caaagaactg ctccacacag agcataatgg gatgctgtgt 180 gcaacaagct tgggacaccc tcttattcta gacacctgca ccattgaagg gctaatttat 240 ggcaatcctt cttgtgatcc attgctggga ggaaaagaat ggtcctatat cgtcgagagg 300 ccatcagccg ttaacggatt atgttatccc gggaatgtag aaaatctaga agagctaaga 360 ttacttttta gttcttctag gtcttatcaa aggatccaga tctttccaga cacaatctgg 420 aatgtgtctt acagtgggac aagcaaagca tgctcggatt cattctacag aagcatgaga 480 tggttgactc aaaagaacaa cgcttaccct acccaagatg ctcaatacac aaataatcaa 540 ggaaagaaca ttcttttcat gtggggtata aatcatccac ccactgatgc tgcgcagaca 600 aatctgtaca ccagaactga cacaacaaca agtgtggcaa cagaggaaat gaatagggtc 660 tttaaaccat tgataggacc aaggcctctt gtcaacggtc aacaaggaag aattaattat 720 tattggtcgg tattgaaacc gggccaaacg ctgcggataa aatctgatgg gaatctaata 780 gctccatggt atggacacat cctttcagga gagagccacg gaagaatcct aaagactgac 840 ttaaaaatgg gtagctgcac agtgcagt gt caaacagaga aaggtggctt aaacacaaca 900 ttgcccttcc aaaatgtaag taagtatgca tttggaaact gctcaaagta cattggcgta 960 aagagtctca aacttgcagt tggtctgagg aatgtgcctt ctagatctag cagaggacta 1020 ttcggggcca tagcaggatt tatagaggga ggttggtcag gactggttgc tggttggtat 1080 gggttccaac attcaaatga ccaaggggtt ggtatggcag cagatagaga ttccacccaa 1140 aaggcagttg ataaaataac atccaaagtg aataatatag tcgacaaaat gaacaagcag 1200 tatgaaatca ttgatcatga attcagtgag gtagaaacta ggcttaacat gatcaatgat 1260 aaggttgatg atcaaatcca agatatatgg gcatataatg cagaattgct agttctgctc 1320 gaaaaccaga aaacactcga tgaacatgac gctaatgtga acaatctata taataaagtg 1380 aagagggcgt tgggttccaa tgcagtggaa gatgggagag gatgtttcga gctataccac 1440 aaatgtgata accattgcat ggagacaatt cggaatggga cctacaacag gaggaagtat 1500 caagaggaat caaaattaga aagacagaaa atagaggggg tcaagctgga atctgaagaa 1560 acttacaaaa tcctcaccat ttattcgact gtcgcctcat ctcttgtgat tgcaatgggg 1620 tttgctgcct ttttgttctg ggccatgtcc aacgggtcct gcagatgcaa catttgtata 1680 taa 1683 <210 > 6 <211> 560 <212> PRT < 213> Artificial Sequence <220> <223> rgHS314 HA aa <400> 6 Met Glu Ala Val Ser Leu Ile Thr Ile Leu Val Val Ala Thr Val Ser 1 5 10 15 Asn Ala Asp Lys Ile Cys Ile Gly Tyr Gln Ser Thr Asn Ser Thr Glu 20 25 30 Thr Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Ala Lys 35 40 45 Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu 50 55 60 Gly His Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Ile Tyr 65 70 75 80 Gly Asn Pro Ser Cys Asp Pro Leu Leu Gly Gly Lys Glu Trp Ser Tyr 85 90 95 Ile Val Glu Arg Pro Ser Ala Val Asn Gly Leu Cys Tyr Pro Gly Asn 100 105 110 Val Glu Asn Leu Glu Glu Leu Arg Leu Leu Phe Ser Ser Ser Ser Arg Ser 115 120 125 Tyr Gln Arg Ile Gln Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr 130 135 140 Ser Gly Thr Ser Lys Ala Cys Ser Asp Ser Phe Tyr Arg Ser Met Arg 145 150 155 160 Trp Leu Thr Gln Lys Asn Asn Ala T yr Pro Thr Gln Asp Ala Gln Tyr 165 170 175 Thr Asn Asn Gln Gly Lys Asn Ile Leu Phe Met Trp Gly Ile Asn His 180 185 190 Pro Pro Thr Asp Ala Ala Gln Thr Asn Leu Tyr Thr Arg Thr Asp Thr 195 200 205 Thr Thr Ser Val Ala Thr Glu Glu Met Asn Arg Val Phe Lys Pro Leu 210 215 220 Ile Gly Pro Arg Pro Leu Val Asn Gly Gln Gln Gly Arg Ile Asn Tyr 225 230 235 240 Tyr Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Ile Lys Ser Asp 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 Lys Met Gly Ser Cys Thr Val 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 S er Lys Tyr Ile Gly Val 305 310 315 320 Lys Ser Leu Lys Leu Ala Val Gly Leu Arg Asn Val Pro Ser Arg Ser 325 330 335 Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Gly Trp 340 345 350 Ser Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln 355 360 365 Gly Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Val Asp 370 375 380 Lys Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln 385 390 395 400 Tyr Glu Ile Ile Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn 405 410 415 Met Ile Asn Asp Lys Val 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 Glu 435 440 445 His Asp Ala Asn Val Asn Asn L eu Tyr Asn Lys Val Lys Arg Ala Leu 450 455 460 Gly Ser Asn Ala Val Glu Asp Gly Arg Gly Cys Phe Glu Leu Tyr His 465 470 475 480 Lys Cys Asp Asn His Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn 485 490 495 Arg Arg Lys Tyr Gln 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 Ile Ala Met Gly Phe Ala Ala Phe 530 535 540 Leu Phe Trp Ala Met Ser Asn Gly Ser Cys Arg Cys Asn Ile Cys Ile 545 550 555 560 <210> 7 <211> 2313 <212> RNA <213> Influenza A virus <400> 7 tcaattatat tcaatatgga aagaataaaa gaactaagaa atctaatgtc gcagtctcgc 60 acccgcgaga tactcacaaa aaccaccgtg gaccatatgg ccataatcaa gaagtacaca 120 tcaggaagac aggagaagaa cccagcactt aggatgaaat ggatgatggc aatgaaatat 180 ccaattacag cagacaagag gataacggaa atgattcctg agagaaatga gcaaggacaa 240 actttatgga gtaaaatgaa tgatgccgga tcagaccgag tgatggtatc accactggct 300 gtgacatggt ggaataggaa tggaccaa ta acaaatacag ttcattatcc aaaaatctac 360 aaaacttatt ttgaaagagt cgaaaggcta aagcatggaa cctttggccc tgtccatttt 420 agaaaccaag tcaaaatacg tcggagagtt gacataaatc ctggtcatgc agatctcagt 480 gccaaggagg cacaggatgt aatcatggaa gttgttttcc ctaacgaagt gggagccagg 540 atactaacat cggaatcgca actaacgata accaaagaga agaaagaaga actccaggat 600 tgcaaaattt ctcctttgat ggttgcatac atgttggaga gagaactggt ccgcaaaacg 660 agattcctcc cagtggctgg tggaacaagc agtgtgtaca ttgaagtgtt gcatttgact 720 caaggaacat gctgggaaca gatgtatact ccaggagggg aagtgaggaa tgatgatgtt 780 gatcaaagct tgattattgc tgctaggaac atagtgagaa gagctgcagt atcagcagat 840 ccactagcat ctttattgga gatgtgccac agcacacaga ttggtggaat taggatggta 900 gacatcctta ggcagaaccc aacagaagag caagccgtgg atatatgcaa ggctgcaatg 960 ggactgagaa ttagctcatc cttcagtttt ggtggattca catttaagag aacaagcgga 1020 tcatcagtca agagagagga agaggtgctt acgggcaatc ttcaaacatt gaagataaga 1080 gtgcatgagg gatatgaaga gttcacaatg gttgggagaa gagcaacagc catactcaga 1140 aaagcaacca ggagattgat tcagctgata gtgagtggga gag acgaaca gtcgattgcc 1200 gaagcaataa ttgtggccat ggtattttca caagaggatt gtatgataaa agcagtcaga 1260 ggtgatctga atttcgtcaa tagggcgaat cagcgattga atcctatgca tcaactttta 1320 agacattttc agaaggatgc gaaagtgctt tttcaaaatt ggggagttga acctatcgac 1380 aatgtgatgg gaatgattgg gatattgccc gacatgactc caagcatcga gatgtcaatg 1440 agaggagtga gaatcagcaa aatgggtgta gatgagtact ccagcacgga gagggtagtg 1500 gtgagcattg accgtttttt gagaatccgg gaccaacgag gaaatgtact actgtctccc 1560 gaggaggtca gtgaaacaca gggaacagag aaactgacaa taacttactc atcgtcaatg 1620 atgtgggaga ttaatggtcc tgaatcagtg ttggtcaata cctatcaatg gatcatcaga 1680 aactgggaaa ctgttaaaat tcagtggtcc cagaacccta caatgctata caataaaatg 1740 gaatttgaac catttcagtc tttagtacct aaggccatta gaggccaata cagtgggttt 1800 gtaagaactc tgttccaaca aatgagggat gtgcttggga catttgatac cgcacagata 1860 ataaaacttc ttcccttcgc agccgctcca ccaaagcaaa gtagaatgca gttctcctca 1920 tttactgtga atgtgagggg atcaggaatg agaatacttg taaggggcaa ttctcctgta 1980 ttcaactata acaaggccac gaagagactc acagttctcg gaaaggatg c tggcacttta 2040 actgaagacc cagatgaagg cacagctgga gtggagtccg ctgttctgag gggattcctc 2100 attctgggca aagaggacaa gagatatggg ccagcactaa gcatcaatga actgagcaac 2160 cttgcgaaag gagagaaggc taatgtgcta attgggcaag gagacgtggt gttggtaatg 2220 aaacggaaac gggactctag catacttact gacagccaga cagcgaccaa aagaattcgg 2280 atggccatca attagtgtcg aatagtttaa aaa 2313 <210> 8 <211> 2313 <212> RNA <213 > Influenza A virus <400> 8 gcaaaccatt tgaatggatg tcaatccgac cttacttttc ttaaaagtgc cagcacaaaa 60 tgctataagc acaactttcc cttatactgg agaccctcct tacagccatg ggacaggaac 120 aggatacacc atggatactg tcaacaggac acatcagtac tcagaaaagg gaagatggac 180 aacaaacacc gaaactggag caccgcaact caacccgatt gatgggccac tgccagaaga 240 caatgaacca agtggttatg cccaaacaga ttgtgtattg gaggcgatgg ctttccttga 300 ggaatcccat cctggtattt ttgaaaactc gtgtattgaa acgatggagg ttgttcagca 360 aacacgagta gacaagctga cacaaggccg acagacctat gactggactc taaatagaaa 420 ccaacctgct gcaacagcat tggccaacac aatagaagtg ttcagatcaa atggcctcac 480 ggccaatgag tctggaaggc tcatagact taaggat gtaatggagt caatgaacaa 540 agaagaaatg gggatcacaa ctcattttca gagaaagaga cgggtgagag acaatatgac 600 taagaaaatg ataacacaga gaacaatggg taaaaagaag cagagattga acaaaaggag 660 ttatctaatt agagcattga ccctgaacac aatgaccaaa gatgctgaga gagggaagct 720 aaaacggaga gcaattgcaa ccccagggat gcaaataagg gggtttgtat actttgttga 780 gacactggca aggagtatat gtgagaaact tgaacaatca gggttgccag ttggaggcaa 840 tgagaagaaa gcaaagttgg caaatgttgt aaggaagatg atgaccaatt ctcaggacac 900 cgaactttct ttcaccatca ctggagataa caccaaatgg aacgaaaatc agaatcctcg 960 gatgtttttg gccatgatca catatatgac cagaaatcag cccgaatggt tcagaaatgt 1020 tctaagtatt gctccaataa tgttctcaaa caaaatggcg agactgggaa aagggtatat 1080 gtttgagagc aagagtatga aacttagaac tcaaatacct gcagaaatgc tagcaagcat 1140 cgatttgaaa tatttcaatg attcaacaag aaagaagatt gaaaaaatcc gatcgctctt 1200 aatagagggg actgcatcat tgagccctgg aatgatgatg ggcatgttca atatgttaag 1260 cactgtatta ggcgtctcca tcctgaatct tggacaaaag agatacacca agactactta 1320 ctggtgggat ggtcttcaat cctctgacga ttttgctctg attgtg aatg cacccaatca 1380 tgaagggatt caagccggag tcgacaggtt ttatcgaacc tgtaagctac ttggaatcaa 1440 tatgagcaag aaaaagtctt acataaacag aacaggtaca tttgaattca caagtttttt 1500 ctatcgttat gggtttgttg ccaatttcag catggagctt cccagttttg gggtgtctgg 1560 gatcaacgag tcagcggaca tgagtattgg agttactgtc atcaaaaaca atatgataaa 1620 caatgatctt ggtccagcaa cagctcaaat ggcccttcag ttgttcatca aagattacag 1680 gtacacgtac cgatgccata gaggtgacac acaaatacaa acccgaagat catttgaaat 1740 aaagaaactg tgggagcaaa cccgttccaa agctggactg ctggtctccg acggaggccc 1800 aaatttatac aacattagaa atctccacat tcctgaagtc tgcctaaaat gggaattgat 1860 ggatgaggat taccaggggc gtttatgcaa cccactgaac ccatttgtca gccataaaga 1920 aattgaatca atgaacaatg cagtgatgat gccagcacat ggtccagcca aaaacatgga 1980 gtatgatgct gttgcaacaa cacactcctg gatccccaaa agaaatcgat ccatcttgaa 2040 tacaagtcaa agaggagtac ttgaggatga acaaatgtac caaaggtgct gcaatttatt 2100 tgaaaaattc ttccccagca gttcatacag aagaccagtc gggatatcca gtatggtgga 2160 ggctatggtt tccagagccc gaattgatgc acggattgat ttcgaatctg g aaggataaa 2220 gaaagaagag ttcactgaga tcatgaagat ctgttccacc attgaagagc tcagacggca 2280 aaaatagtga atttagcttg tccttcatga aaa 2313 <210> 9 <211> 2206 <212> RNA <213> Influenza A virus <400> 9 gtactgatcc aaaatggaag attttgtgcg acaatgcttc aatccgatga ttgtcgagct 60 tgcggaaaaa acaatgaaag agtatgggga ggacctgaaa atcgaaacaa acaaatttgc 120 agcaatatgc actcacttgg aagtatgctt catgtattca gattttcact tcatcaatga 180 gcaaggcgag tcaataatcg tagaacttgg tgatccaaat gcacttttga agcacagatt 240 tgaaataatc gagggaagag atcgcacaat ggcctggaca gtagtaaaca gtatttgcaa 300 cactacaggg gctgagaaac caaagtttct accagatttg tatgattaca aggagaatag 360 attcatcgaa attggagtaa caaggagaga agttcacata tactatctgg aaaaggccaa 420 taaaattaaa tctgagaaaa cacacatcca cattttctcg ttcactgggg aagaaatggc 480 cacaaaggca gactacactc tcgatgaaga aagcagggct aggatcaaaa ccagactatt 540 caccataaga caagaaatgg ccagcagagg cctctgggat tcctttcgtc agtccgagag 600 aggagaagag acaattgaag aaaggtttga aatcacagga acaatgcgca agcttgccga 660 ccaaagtctc ccgccgaact tctccagcct tgaaaatgg t agagcctatg tggatggatt 720 cgaaccgaac ggctacattg agggcaagct gtctcaaatg tccaaagaag taaatgctag 780 aattgaacct tttttgaaaa caacaccacg accacttaga cttccgaatg ggcctccctg 840 ttctcagcgg tccaaattcc tgctgatgga tgccttaaaa ttaagcattg aggacccaag 900 tcatgaagga gagggaatac cgctatatga tgcaatcaaa tgcatgagaa cattctttgg 960 atggaaggaa cccaatgttg ttaaaccaca cgaaaaggga ataaatccaa attatcttct 1020 gtcatggaag caagtactgg cagaactgca ggacattgag aatgaggaga aaattccaaa 1080 gactaaaaat atgaagaaaa caagtcagct aaagtgggca cttggtgaga acatggcacc 1140 agaaaaggta gactttgacg actgtaaaga tgtaggtgat ttgaagcaat atgatagtga 1200 tgaaccagaa ttgaggtcgc ttgcaagttg gattcagaat gagtttaaca aggcatgcga 1260 actgacagat tcaagctgga tagagctcga tgagattgga gaagatgtgg ctccaattga 1320 acacattgca agcatgagaa ggaattattt cacatcagag gtgtctcact gcagagccac 1380 agaatacata atgaaggggg tgtacatcaa tactgccttg cttaatgcat cttgtgcagc 1440 aatggatgat ttccaattaa ttccaatgat aagcaagtgt agaactaagg agggaaggcg 1500 aaagaccaac ttgtatggtt tcatcataaa aggaagatcc cacttaagg a atgacaccga 1560 cgtggtaaac tttgtgagca tggagttttc tctcactgac ccaagacttg aaccacataa 1620 atgggagaag tactgtgttc ttgagatagg agatatgctt ataagaagtg ccataggcca 1680 ggtttcaagg cccatgttct tgtatgtgag aacaaatgga acctcaaaaa ttaaaatgaa 1740 atggggaatg gagatgaggc gttgcctcct ccagtcactt caacaaattg agagtatgat 1800 tgaagctgag tcctctgtca aagagaaaga catgaccaaa gagttctttg agaacaaatc 1860 agaaacatgg cccattggag agtcccccaa aggagtggag gaaagttcca ttgggaaggt 1920 ctgcaggact ttattagcaa agtcggtatt caacagcttg tatgcatctc cacaactaga 1980 aggattttca gctgaatcaa gaaaactgct tcttatcgtt caggctctta gggacaacct 2040 tgaacctggg acctttgatc ttggggggct atatgaagca attgaggagt gcctgattaa 2100 tgatccctgg gttttgctta atgcttcttg gttcaactcc ttccttacac atgcattgag 2160 ttagttgtgg cagtgctact atttgctatc catactgtcc aaaaaa 2206 <210> 10 <211> 1537 <212> RNA <213> Influenza A virus <400> 10 tagataatca ctcactgagt gacatcaaaa tcatggcgtc tcaaggcacc aaacgatctt 60 acgaacagat ggagactgat ggagaacgcc agaatgccac tgaaatcaga gcatccgtcg 120 gaaaaatgat tgg tggaatt ggacgattct acatccaaat gtgcaccgaa ctcaaactca 180 gtgattatga gggacggttg atccaaaaca gcttaacaat agagagaatg gtgctctctg 240 cttttgacga aaggagaaat aaataccttg aagaacatcc cagtgcgggg aaagatccta 300 agaaaactgg aggacctata tacaggagag taaacggaaa gtggatgaga gaactcatcc 360 tttatgacaa agaagaaata aggcgaatct ggcgccaagc taataatggt gacgatgcaa 420 cggctggtct gactcacatg atgatctggc attccaattt gaatgatgca acttatcaga 480 ggacaagagc tcttgttcgc accggaatgg atcccaggat gtgctctctg atgcaaggtt 540 caactctccc taggaggtct ggagccgcag gtgctgcagt caaaggagtt ggaacaatgg 600 tgatggaatt ggtcagaatg atcaaacgtg ggatcaatga tcggaacttc tggaggggtg 660 agaatggacg aaaaacaaga attgcttatg aaagaatgtg caacattctc aaagggaaat 720 ttcaaactgc tgcacaaaaa gcaatgatgg atcaagtgag agagagccgg aacccaggga 780 atgctgagtt cgaagatctc acttttctag cacggtctgc actcatattg agagggtcgg 840 ttgctcacaa gtcctgcctg cctgcctgtg tgtatggacc tgccgtagcc agtgggtacg 900 actttgaaag ggagggatac tctctagtcg gaatagaccc tttcagactg cttcaaaaca 960 gccaagtgta cagcctaatc agaccaaatg a gaatccagc acacaagagt caactggtgt 1020 ggatggcatg ccattctgcc gcatttgaag atctaagagt attaagcttc atcaaaggga 1080 cgaaggtgct cccaagaggg aagctttcca ctagaggagt tcaaattgct tccaatgaaa 1140 atatggagac tatggaatca agtacacttg aactgagaag caggtactgg gccataagga 1200 ccagaagtgg aggaaacacc aatcaacaga gggcatctgc gggccaaatc agcatacaac 1260 ctacgttctc agtacagaga aatctccctt ttgacagaac aaccattatg gcagcattca 1320 atgggaatac agaggggaga acatctgaca tgaggaccga aatcataagg atgatggaaa 1380 gtgcaagacc agaagatgtg tctttccagg ggcggggagt cttcgagctc tcggacgaaa 1440 aggcagcgag cccgatcgtg ccttcctttg acatgagtaa tgaaggatct tatttcttcg 1500 gagacaatgc agaggagtac gacaattaaa gaaaaat 1537 <210> 11 <211> 1000 <212> RNA <213> Influenza A virus <400> 11 gtagatattg aaagatgagt cttctaaccg aggtcgaaac gtacgtactc tctatcatcc 60 cgtcaggccc cctcaaagcc gagatcgcac agagacttga agatgtcttt gcagggaaga 120 acaccgatct tgaggttctc atggaatggc taaagacaag accaatcctg tcacctctga 180 ctaaggggat tttaggattt gtgttcacgc tcaccgtgcc cagtgagcga ggactgcagc 240 gtagacg ctt tgtccaaaat gcccttaatg ggaacgggga tccaaataac atggacaaag 300 cagttaaact gtataggaag ctcaagaggg agataacatt ccatggggcc aaagaaatct 360 cactcagtta ttctgctggt gcacttgcca gttgtatggg cctcatatac aacaggatgg 420 gggctgtgac cactgaagtg gcatttggcc tggtatgtgc aacctgtgaa cagattgctg 480 actcccagca tcggtctcat aggcaaatgg tgrcaacaac caatccacta atcagacatg 540 agaacagaat ggttttagcc agcactacag ctaaggctat ggagcaaatg gctggatcga 600 gtgagcaagc agcagaggcc atggaggttg ctattcgggc taggcaaatg gtgcaggcaa 660 tgagaaccat tgggactcat cctagctcca gtgctggtct gaaaaatgat cttcttgaaa 720 atttgcaggc ctatcagaaa cgaatggggg tgcagatgca acggttcaag tgatcctctc 780 attattgcct caartatcat tgggatcttg cacttgayat tgtggattct tgatcgtctt 840 tttttcaaat gcatttaccg tctctttaaa tacggtttga aaagagggcc ttctacggaa 900 ggagtgccaa agtctatgag ggaagaatat caaaaggaac agcagagtgc tgtggatgct 960 gacgatggtc attttgtcag catagagctg gagtaaaaaa 1000 <210> 12 <211> 861 <212> RNA <213> Influenza A virus <400> 12 tgacaaaaac ataatggatc caaacactgt gtcaagcttt caggtagatt g ctttctttg 60 gcatgtccgc aaacgagttg cagaccaaga actaggtgat gccccattcc ttgatcggct 120 tcgccgagat cagaaatccc taagaggaag gggcagtact ctcggtctgg acatcaagac 180 agccacacgt gctggaaagc agatagtgga gcggattctg aaagaagaat ccgatgaggc 240 acttaaaatg accatggcct ctgtacctgc gtcgcgttac ctaactgaca tgactcttga 300 ggaaatgtca agggactggt ccatgctcat acccaagcag aaagtggcag gccctctttg 360 tatcagaatg gaccaggcga tcatggataa gaacatcata ctgaaagcga acttcagtgt 420 gatttttgac cggctggaga ctctaatatt gctaagggct ttcaccgaag agggagcaat 480 tgttggcgaa atttcaccat tgccttctct tccaggacat actgctgagg atgtcaaaaa 540 tgcagttgga gtcctcatcg gaggacttga atggaatgat aacacagttc gagtctctga 600 aactctacag agattcgctt ggagaagcag taatgagaat gggagacctc cactcactcc 660 aaaacagaaa cgagaaatgg cgggaacaat taggtcagaa gtttgaagaa ataagatggt 720 tgattgaaga agtgagacac aaactgaaga taacagagaa tagttttgag caaataacat 780 ttatgcaagc cttacatcta ttgcttgaag tggagcaaga gataagaact ttctcgtttc 840 agcttattta gtactaaaaa a 861 <210> 13 <211> 28 <212> DNA <213> Artificial Sequ ence <220> <223> HA1F <400> 13 tccgaagttg ggggggatgg aggcagta 28 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Sub HA F <400> 14 tcaacggtca acaaggaaga attaattatt attg 34 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sub HA R <400> 15 taattcttcc ttgttgaccg ttgacaagag 30 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> HA R <400> 16 gggccgccgg gttattttat atacaaatgt tgc 33 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> N2-1F <400> 17 tccgaagttg ggggggagca aaagcaggag 30 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> N2-1413R <400> 18 gggccgccgg gttattagta gaaacaagga gt 32 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Phw-PB2-1F <400> 19 tccgaagttg ggggggagcg aaagcaggtc 30 <210> 20 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Phw-PB2 -2341R <400> 20 gggccgccgg gttattagta gaaacaaggt cg 32 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Phw-PB1-1F <40 0> 21 tccgaagttg ggggggagcg aaagcaggca 30 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Phw-PB1-2341R <400> 22 gggccgccgg gttattagta gaaacaaggc at 32 <210> 23 <211 > 30 <212> DNA <213> Artificial Sequence <220> <223> Phw-PA-1F <400> 23 tccgaagttg ggggggagcg aaagcaggta 30 <210> 24 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Phw-PA-2233R <400> 24 gggccgccgg gttattagta gaaacaaggt ac 32 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Phw-NP-1F <400> 25 tccgaagttg ggggggagca aaagcagggt 30 <210> 26 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Phw-NP-1565R <400> 26 gggccgccgg gttattagta gaaacaaggg ta 32 <210> 27 <211> 30 <212 > DNA <213> Artificial Sequence <220> <223> Phw-M-1F <400> 27 tccgaagttg ggggggagca aaagcaggta 30 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Phw -M-1027R <400> 28 gggccgccgg gttattagta gaaacaaggt ag 32 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Phw-NS-1F <400> 29 tcc gaagttg ggggggagca aaagcagggt 30 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Phw-NS-890R<400> 30 gggccgccgg gttattagta gaaacaaggg tg 32

Claims (15)

A method for producing an H9N2-type recombinant avian influenza A virus comprising the steps of:
(a) H9N2-type low pathogenic avian influenza A virus-derived HA (hemagglutinin) gene consisting of the nucleotide sequence of SEQ ID NO: 5 and H9N2-type low pathogenic avian influenza A virus-derived NA (neuraminidase) gene consisting of the nucleotide sequence of SEQ ID NO: 3 into vectors, respectively cloning to prepare a recombinant plasmid;
(b) Nucleotide sequences encoding PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) derived from low pathogenic avian influenza A virus cloning each into a vector to prepare a recombinant plasmid;
(c) transfecting the recombinant plasmid of steps (a) and (b) into a packaging cell; and
(d) obtaining H9N2-type recombinant avian influenza A virus from the culture supernatant of the packaging cells.
The method of claim 1, wherein the H9N2-type low pathogenic avian influenza A virus of step (a) is A/chicken/Korea/LBM314/2020(H9N2).
According to claim 1, wherein the amino acid Leu-Met-Gly located at positions 234 to 236 of HA consisting of the amino acid sequence of SEQ ID NO: 2 in the HA protein derived from H9N2 low pathogenic avian influenza A virus in step (a) is Gln -Gln-Gly substituted, H9N2-type genetically recombinant avian influenza A virus production method.
The method according to claim 1, wherein the HA gene from H9N2 low pathogenic avian influenza A virus in step (a) is nucleotides 700 to 705 of HA consisting of the nucleotide sequence of SEQ ID NO: 1 in which TTGATG is substituted with CAACAA A method for producing a human, H9N2-type recombinant avian influenza A virus.
The method of claim 1, wherein the low pathogenic avian influenza A virus in step (b) is A/PR/8/34 (H1N1).
According to claim 1, wherein the low pathogenic avian influenza A virus-derived from step (b) PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non -Structural protein) encoding the nucleotide sequence, each consisting of the nucleotide sequence of SEQ ID NOs: 7 to 12, H9N2-type recombinant avian influenza A virus production method.
According to claim 1, wherein the packaging cell of step (c) is one or more isolated cells selected from the group consisting of 293T, MDCK, Vero, DF1, PK15, and ST1 cells, H9N2-type recombinant avian influenza A A method for producing a virus.
H9N2-type recombinant avian influenza A virus comprising a negative-sense ssRNA of the following gene:
(i) HA (hemagglutinin) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 5;
(ii) NA (neuraminidase) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 3; and
(iii) PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) genes derived from low pathogenic influenza A virus.
The method according to claim 8, wherein the amino acid Leu-Met-Gly located at positions 234 to 236 of HA consisting of the amino acid sequence of SEQ ID NO: 2 is Gln-Gln-Gly in the HA protein derived from the H9N2-type low pathogenic avian influenza A virus. The substituted, H9N2-type recombinant avian influenza A virus.
The H9N2-type gene according to claim 8, wherein in the HA gene derived from the H9N2-type low pathogenic avian influenza A virus, nucleotides TTGATG located at 700 to 705 of HA consisting of the nucleotide sequence of SEQ ID NO: 1 are substituted with CAACAA. Recombinant avian influenza A virus.
According to claim 8, wherein the low pathogenic influenza A virus-derived PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) genes are H9N2-type recombinant avian influenza A virus, each consisting of the nucleotide sequence of SEQ ID NOs: 7 to 12.
Vaccine composition against H9 serotype avian influenza A virus, including H9N2 recombinant avian influenza A virus containing negative-sense ssRNA of the following gene:
(i) HA (hemagglutinin) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 5;
(ii) NA (neuraminidase) gene derived from H9N2-type low pathogenic avian influenza A virus consisting of the nucleotide sequence of SEQ ID NO: 3; and
(iii) PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) genes derived from low pathogenic influenza A virus.
The vaccine composition according to claim 12, wherein the H9 serotype avian influenza A virus comprises a Y280 family HA gene.
The method according to claim 12, wherein the low pathogenic influenza A virus-derived PB2 (polymerase B2), PB1 (polymerase B1), PA (polymerase A), NP (nucleocapsid), M (matrix protein) and NS (non-structural protein) genes are Each of the nucleotide sequences of SEQ ID NOs: 7 to 12, the vaccine composition.
The vaccine composition according to claim 12, wherein the recombinant H9N2-type avian influenza A virus in the vaccine composition is inactivated.

Images