CN113736749A

CN113736749A - Avian influenza virus strain and application thereof

Info

Publication number: CN113736749A
Application number: CN202110981666.XA
Authority: CN
Inventors: 傅芬芳; 苗玉和; 崔丽瑾
Original assignee: Fujian Shengwei Biotechnology Co ltd
Current assignee: Fujian Shengwei Biotechnology Co ltd
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-12-03
Anticipated expiration: 2041-08-25
Also published as: CN113736749B

Abstract

The invention provides an H9N2 subtype avian influenza virus strain and application thereof. The avian influenza subtype strain is named as SW001, and the preservation number is CGMCC No. 19387. The virus strain provided by the invention is a low-pathogenicity virus strain, has good specificity and immunogenicity, and the immunized chick can generate a specific HI antibody aiming at the H9 subtype avian influenza virus and can generate a good protection effect on the attack of the H9N2 subtype avian influenza virus. The SW001 strain of the H9N2 subtype avian influenza virus can be used as a vaccine strain to prepare a vaccine for preventing H9 subtype avian influenza of poultry, and has better economic benefit and social benefit.

Description

Avian influenza virus strain and application thereof

Technical Field

The invention relates to the field of animal virology, in particular to an avian influenza virus strain and application thereof.

Background

Avian Influenza (AI) is a syndrome caused by infection of poultry with Avian influenza virus A (AIV) ranging from no clinical symptoms to a decrease in respiratory disease and egg production to severe systemic disease with mortality rates approaching 100%. Avian influenza virus belongs to the family of orthomyxoviridae, the genus of influenza virus, Hemagglutinin (HA) and Neuraminidase (NA) are two main protective antigens of the virus, and influenza a virus can be divided into different subtypes according to the antigenic difference between HA and NA. Until now, it HAs been known that there are 16 subtypes of HA (H1-H16) and 10 subtypes of NA (N1-N10), and different combinations among them give influenza A virus multiple subtypes with no mutual immunity among the subtypes. According to pathogenicity, the avian influenza virus can be divided into high-pathogenicity AIV, mainly two serotypes of H5 and H7, the rest is low-pathogenicity avian influenza, and the clinical epidemic is mainly H9 subtype.

The H9N2 subtype avian influenza virus is widely distributed and exists, is one of the main subtypes of the current avian influenza epidemics, and a plurality of strains of avian influenza viruses with the same subtype are separated from different regions in China since the H9N2 subtype avian influenza virus is separated for the first time in 1994 in mainland. After the avian influenza virus H9N2 is infected, diseases such as secondary or concurrent escherichia coli and the like are frequently caused, so that a large number of chickens die, and serious economic loss is brought to the poultry industry in China. Although the H9 subtype avian influenza inactivated vaccine is widely used and is effectively controlled, in recent years, H9 subtype avian influenza still has a disease after an immune chicken flock, the problem is solved, the H9N2 avian influenza vaccine strain with good protection is selected by screening and separating the local epidemic strains, and the prevention and control of the avian influenza are more targeted and have practical effectiveness.

Disclosure of Invention

The invention aims to provide a novel avian influenza virus strain which is an H9N2 subtype avian influenza virus SW001 strain, wherein the HA protein of the strain HAs an amino acid sequence shown in SEQ ID No.1, and the NA protein of the strain HAs an amino acid sequence shown in SEQ ID No. 2. The H9N2 subtype avian influenza virus SW001 strain is preserved in China general microbiological culture Collection center (CGMCC No.9495 for short) of the institute of microbiology of China academy of sciences, No.2 of West Lu 1 of the North Cheng, Naja, Beijing, 26 days 03 and 26 days 2020, and is classified and named as H9N2 subtype avian influenza virus with the preservation registration number of CGMCC No. 19387.

The invention aims at providing an application of an avian influenza virus strain in preparing biological products, wherein the avian influenza virus strain is an H9N2 subtype avian influenza virus SW001 strain with the preservation registration number of CGMCC No. 19387.

According to the invention, the use of the avian influenza virus strain for the preparation of a biological product, preferably an avian influenza vaccine or a combined vaccine.

According to the invention, the avian influenza virus strain is used for preparing biological products, and the avian influenza vaccine is preferably an inactivated vaccine, wherein the antigen is the SW001 strain of the H9N2 subtype avian influenza virus which is inactivated.

The invention also aims to provide a biological product prepared by using the SW001 strain of the H9N2 subtype avian influenza virus.

Preferably, the biological product is any one of a product for detecting avian influenza and/or an avian influenza vaccine.

The avian influenza H9N2 subtype virus strain SW001 provided by the invention has good specificity and immunogenicity, and experiments show that the avian influenza H9N2 subtype virus strain SW001 can not be inhibited by anti-Newcastle Disease Virus (NDV) positive serum, anti-egg drop syndrome virus 76 strain (EDS76) positive serum, anti-infectious bronchitis virus (M41) positive serum, anti-H5 subtype avian influenza positive serum and anti-H7 subtype avian influenza positive serum, and is only inhibited by H9 subtype avian influenza positive serum. Biological property tests, serum neutralization tests and exogenous virus detection prove that the virus has good purity. The infection amount of SW001 strain to chick embryo is EID50 ═ 10^-8.520.1ml, the vaccine prepared by the SW001 strain can be used for immunizing chicks to generate specific antibodies aiming at H9 subtype, the average value reaches 6.5log2, and the vaccine has good protection effect on the attack of H9N2 subtype avian influenza virus.

The H9N2 subtype avian influenza virus strain provided by the invention can be used as a vaccine strain to prevent H9 subtype avian influenza of birds, and particularly has an avian influenza prevention effect on an epidemic focus separation source area superior to that of the existing avian influenza vaccine, and has important value on prevention and treatment of avian influenza virus.

Drawings

Fig. 1 shows the results of the HA gene homology analysis of the SW001 strain of H9N2 subtype avian influenza virus of the present invention.

Fig. 2 shows the results of the HA gene genetic evolution analysis of the SW001 strain of H9N2 subtype avian influenza virus of the present invention.

FIG. 3 shows the results of NA gene homology analysis of SW001 strain, a subtype H9N2 avian influenza virus strain according to the present invention.

Fig. 4 shows the results of NA gene genetic evolution analysis of the SW001 strain of H9N2 subtype avian influenza virus of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention.

The SPF chick embryos used in the examples of the present invention were purchased from Rihua agricultural science and technology Limited, Zhejiang, and positive sera against subtype H5, H7 and H9 avian influenza viruses were purchased from Haerbividae Biotechnology Limited, and positive sera against Newcastle Disease Virus (NDV), egg drop syndrome virus (EDS76) and infectious bronchitis virus M41 were purchased from China veterinary medicine inspection institute.

Unless otherwise indicated, the chemical reagents used in the examples were all routine laboratory analytical reagents, and the techniques used in the examples were all routine procedures well known to those skilled in the art.

The H9N2 subtype avian influenza virus SW001 strain provided by the invention is obtained by separating in the body of a diseased chicken in a chicken farm in Fujian in 2018 and 12 months. The chickens in the chicken farm begin to develop diseases at 21 days of age, and the clinical symptoms are mainly shown as follows: drinking water and ingestion are reduced, mouth breathing is performed, sudden death is occasionally caused, the death rate is increased after 3 days, and the death rate can reach 2%. The dissection shows that a few throats have hemorrhage, severe air sac inflammation, bronchial embolism, white cheese-like substances and lung hemorrhage. An H9N2 subtype avian influenza virus is obtained by separating and identifying in vivo from the sick chickens and is named as A/Chicken/Fujian/SW001/2018(H9N2) strain (SW 001 strain for short). The virus strain is preserved in China general microbiological culture Collection center (CGMCC No.9495 for short, the address: No.2, West Lu 1 of the university of Beijing, Chaoyang, and the institute of microbiology, Chinese academy of sciences, zip code 100101) at 26.03.26.2020, and is classified and named as H9N2 subtype avian influenza virus with the preservation number of CGMCC No. 19387.

The HA protein of the avian influenza H9N2 subtype virus strain SW001 provided by the invention HAs an amino acid sequence shown as SEQ ID No.1, and the NA protein HAs an amino acid sequence shown as SEQ ID No. 2.

The chick embryo allantoic fluid containing the isolate is used for agglutinating 1% of chick red blood cells, and Hemagglutination Inhibition (HI) experiments prove that the agglutination is not inhibited by single factor positive serum such as Newcastle disease, avian influenza H5, H7 subtype, egg drop syndrome and the like. Through the intracerebral inoculation index (ICPI) determination of 1 day old chicks, the avian influenza virus is proved to be a low-virulence strain.

The active ingredient of the avian influenza vaccine prepared in the invention is the inactivated SW001 strain virus, the prepared vaccine is a vaccine aiming at avian H9N2 subtype avian influenza virus, and the effect evaluation is carried out on the vaccine by using a serology method and an immune challenge virus protection test method. The result shows that the inactivated avian influenza vaccine prepared by the invention has good protection to birds.

Further, the biological product prepared by using the avian influenza H9N2 subtype avian influenza virus strain SW001 or the recombinant strain thereof also belongs to the protection scope of the invention.

The invention provides application of an avian influenza H9N2 subtype virus strain SW001 in preparation of an avian influenza vaccine.

Example 1: separation and identification of SW001 strain of H9N2 subtype avian influenza virus strain

1. Virus isolation

The applicant of the invention separates H9N2 subtype avian influenza virus SW001 strain from the bodies of diseased chickens in a certain broiler farm in Fujian province.

The chickens in the chicken farm begin to attack at 21 days of age, and the clinical symptoms are mainly shown as follows: drinking water and ingestion are reduced, mouth breathing is opened, sudden death is occasionally caused, and the death rate is increased after 3 days and can reach 2 percent. The dissection shows that a few throats have hemorrhage, severe air sac inflammation, bronchial embolism, white cheese-like substances and lung hemorrhage.

Collecting liver, spleen, lung and pancreas of sick chicken under aseptic condition, cutting, grinding, adding sterile PBS at weight ratio of 1:5, centrifuging at 4 deg.C and 8000r/min for 10min, collecting supernatant, filtering with 0.22 μm filter, and transferring into another aseptic 1.5ml EP tube. Inoculating 6 SPF (specific pathogen free) chick embryos of 10 days old according to the dose of 0.2 ml/embryo, discarding the chick embryos dying within 24 hours, observing once every 12 hours, collecting the dead chick embryos, storing at 4 ℃, taking all chick embryo allantoic fluid out after 96 hours, performing sample retention detection on each chick embryo, subpackaging the rest part according to the dose of 1 ml/tube, and storing at-80 ℃.

Hemagglutinating virus separated from sick chicken material, hemagglutinating test shows that the isolate can agglutinate chicken erythrocyte, and hemagglutinating inhibition test (HI) shows that the hemagglutinating property of the isolate can not be inhibited by Newcastle Disease Virus (NDV) serum, anti-AIV H5 subtype serum and anti-AIV H7 subtype serum, but can be inhibited by AIV H9 subtype positive serum.

2. Virus subculture

The virus was passaged further in 11-day-old SPF chick embryos to harvest allantoic fluid.

The SW001 strain isolate identified by a serological method is AIV H9N2 subtype and is named as A/Chicken/Fujian/SW001/2018(H9N2) strain (SW 001 strain for short). The isolate has been preserved in China general microbiological culture Collection center (CGMCC No.9495 for short, address: No.2 of West Lu 1 of the university of Beijing, Chaoyang, and Microbiol research institute of Chinese academy of sciences, zip code 100101) at 26.03.26.2020, and is classified and named as H9N2 subtype avian influenza virus with the preservation number of CGMCC No. 19387.

Example 2: determination of virulence of SW001 strain of H9N2 subtype avian influenza virus。

1. Determination of intracerebral pathogenicity index (ICPI) of 1 day old chicks

10 SPF chicks of 1 day old are taken and injected intracerebrally according to the ratio of 10^-1Dilution 0.05ml of diluted allantoic fluid containing virus. Another 2 of the virus-diluted solutions were injected in the same manner with 0.05ml of physiological saline. And (5) isolated breeding, observing for 8d, and recording test results.

After inoculation, the chicks were observed daily at a time corresponding to the time of inoculation and were scored as normal (manifested as agility, no ataxia in locomotion), sick (including those presenting as paralyzed, lying down, but not including those presenting as dull) and dead. The observation was continued for 8 days, the total of normal, diseased and dead chickens at the end of the observation was calculated, the scores were accumulated according to different weights (normal 0, diseased 1 and dead 2), and the results of the observation and recording are shown in table 1.

Based on the results in table 1, the total score was 60 and the total sum was 80, and the ICPI of the avian influenza virus SW001 strain was calculated to be 0.75 using the formula ICPI ═ total score/total sum. According to the international universal standard, when the ICPI is within the range of 1.6-2.5, the virus strain can be judged to be a strong pathogenic virus strain, and the ICPI of the avian influenza virus SW001 strain is lower than the lower limit value of the numerical range by 1.6, so that the separated virus strain is judged to be a low pathogenic avian influenza virus.

TABLE 1

2. Viral titer determination

Diluting the virus strain with sterile PBS 10 times, and taking 10 times^-7～10^-9Three dilutions were used to inoculate 10 day-old SPF chick embryos via allantoic cavity at a dose of 0.1 ml/embryo, 5 chick embryos were inoculated per dilution. Culturing chicken embryo at 37 deg.C for 120 hr after inoculation, detecting HA titer of chicken embryo one by one, and calculating virus EID by Reed-muench method₅₀The results are shown in Table 2.

TABLE 2

According to the results in the table 2, the infection amount of the SW001 strain of the H9N2 subtype avian influenza virus to the chick embryo is calculated to be EID₅₀Is 10^-8.170.1ml, i.e. the viral suspension is subjected to 10^-8.17After dilution, the diluted solution was inoculated into chicken embryos in an amount of 0.1ml, and 50% of the chicken embryos were infected.

Next, the H9N2 subtype avian influenza virus SW001 strain was assayed for virulence against chicken embryos.

Diluting the 3 rd generation virus of SW001 strain of H9N2 subtype avian influenza virus with sterile PBS 10⁴And (4) inoculating the strain to 10-day-old SPF chick embryos, and inoculating 0.2ml of the strain to each embryo for 15. After inoculation, the chick embryos were incubated at 37 ℃ for up to 120 hours, observed twice a day, and the dead embryos were recorded, the results are shown in Table 3.

TABLE 3

Next, the H9N2 subtype avian influenza virus SW001 strain was further tested for virulence against chicks.

SPF chickens 4 weeks old were inoculated with a total of 10 according to OIE criteria. After inoculation, the chickens were observed for survival and morbidity for 10 consecutive days, and the results are shown in Table 4. From the results in table 4, it can be seen that all the chickens survived and showed no obvious clinical onset symptoms and pathological changes, indicating that the SW001 strain of H9N2 subtype avian influenza virus of the present invention has low pathogenic virulence.

TABLE 4

Example 3: immunogenicity evaluation of avian influenza H9N2 subtype Virus Strain SW001 Strain

The SW001 strain virus liquid of H9N2 subtype avian influenza virus is inactivated by a diethylene imine (BEI) solution with the final concentration of 1mmol/L for 36 hours at 37 ℃, and is continuously stirred. After inactivation, 1mol/L filtration-sterilized sodium thiosulfate was added to a final concentration of 10mmol/L, and the mixture was stirred well and reacted at 37 ℃ for L hours to terminate inactivation. Mixing the solution containing the inactivated H9N2 subtype avian influenza virus SW001 strain and tween-80 according to the volume ratio of 96:4, and fully shaking until the tween-80 is completely dissolved to obtain a water phase. 94 parts of white oil for injection and 2 parts of aluminum stearate are added, the mixture is uniformly mixed and heated and stirred until the mixture is completely transparent, span-806 parts are added, and the mixture is fully mixed and then is sterilized under high pressure to obtain an oil phase. Mixing the water phase and the oil phase according to the volume ratio of 1:2, emulsifying and stirring to prepare the oil emulsion inactivated vaccine.

15 of 21-day-old SPF chickens were inoculated subcutaneously into the neck of 10 of the SPF chickens, 0.2ml of the above-prepared oil-emulsion inactivated vaccine was inoculated into each of the SPF chickens, and 5 of the SPF chickens were injected subcutaneously into the neck of the SPF chickens to prepare a control group. 15 chickens were separately housed, blood was taken 28 days later to isolate serum, Hemagglutination Inhibition (HI) test was performed with an avian influenza virus H9 subtype standard antigen and the HI average titer against the H9 subtype standard antigen was determined. At 28 days after immunization, the SW001 strain of the H9N2 subtype avian influenza virus is used for virus challenge, and the virus challenge dose is 10^8.0EID₅₀0.1 ml. 5 days after challenge, cotton swabs were collected, 10-day-old SPF chick embryos were inoculated and the strain isolated therefrom.

The results are shown in Table 5.

TABLE 5

The number of vaccinated animals is the number of diseased or dead animals.

As can be seen from the results in table 5, no clinical symptoms of suspected avian influenza appeared in both the immune group and the control group, and the pathogen separation was performed on the cotton swab, no virus was separated from any of the 10 chickens in the immune group, while viruses were separated from any of the 5 chickens in the control group, and the cotton swab results showed that the inactivated vaccine prepared by using the SW001 strain of H9N2 subtype avian influenza virus of the present invention can protect the chickens from the attack by the strain, indicating that the SW001 strain of H9N2 subtype avian influenza virus has good immunogenicity.

Example 4: evaluation of stability of SW001 Strain of H9N2 subtype avian influenza Virus

The SW001 strain of H9N2 subtype avian influenza virus strain was continuously passaged for 10 generations using 10-day-old SPF chick embryos, and allantoic fluid hemagglutination titer (HA) was examined for each generation of the virus, and the results are shown in Table 6.

Randomly selecting one passage of virus from the passage viruses, storing at-80 ℃, detecting HA titer of the virus in the stored 1 st, 3 rd, 6 th, 9 th and 12 th months, and taking the virus of 7 th-9 th generation as an experimental control. The results are shown in Table 7.

TABLE 6

TABLE 7

The results in tables 6 and 7 show that the SW001 strain of H9N2 subtype avian influenza virus of the present invention can be stably passaged and can maintain excellent titer stability under low-temperature storage conditions.

Example 5: analysis of HA gene and NA gene of SW001 strain of H9N2 subtype avian influenza virus

The kit is used for extracting RNA of P1 virus, HA gene and NA gene of the virus are amplified by PCR, PCR products are recovered and connected to pMD18-T vector, the vector is transformed to DH5 alpha sensing generation cells, and plasmids which are identified as positive are sent to the company Limited in the engineering bioengineering (Shanghai) to be subjected to sequencing analysis.

Sequencing results show that the full-length sequence of the HA gene of 1686bp is obtained by amplification, the specific sequence is shown as SEQ ID No.3, and the protein coded by the gene HAs the amino acid sequence shown as SEQ ID No. 1. The full-length sequence of the HA gene and the existing H9N2 strain in GenBank listed in Table 8 are subjected to genetic evolution analysis, and the result is shown in figure 2, the SW001 strain of the H9N2 subtype avian influenza virus is in the h9.4.2.5 branch which is popular in recent years, and the homology between the HA gene sequence and the h9.4.2.5 branch separated strain is more than 98.8% (see figure 1).

TABLE 8

Sequencing results show that 1401bp of the full-length fragment of the NA gene is obtained through amplification, the sequence is shown as SEQ ID No.4, and the amino acid sequence coded by the NA gene is shown as SEQ ID No. 2. The full-length sequence of the NA gene and the epidemic strains after 2018 in GenBank listed in Table 9 are subjected to homology sequence analysis, and the results are shown in figures 3 and 4, and the NA gene sequence of the SW001 strain of the H9N2 subtype avian influenza virus has the homology of more than 98 percent with the recent H9 subtype isolate strain, and has high homology and close relationship.

TABLE 9

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto without departing from the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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FUJIAN SHENGWEI BIOTECHNOLOGY Co.,Ltd.

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caagaggaat caaaattaga aagacagaga atagaggggg tcaagctgga atctgaagga 1560

acttacaaaa tcctcaccat ttattcgact gtcgcctcat ctcttgtgat tgcaatgggg 1620

tttgctgcct tcttgttctg ggccatgtct aatgggtctt gcagatgcaa catttgtata 1680

taa 1683

<210> 4

<211> 1401

<212> DNA

<213> Avian Influenza Virus (Avian Influenza virus)

<400> 4

atgatcccaa atcagaagat aatagcaatt ggctctgttt ctctaatcat tgcgataata 60

tgtctcctca tgcaaattgc aatcttaaca acgactatga cattacattt cgggcagaaa 120

gaatgcagca aaccatcgaa taatcaagtg atgccatgtg aaccgatcat aatagaaagg 180

aatacagtgc atttgaatag tactaccata gagagggaaa tttgtcccaa agtggcggag 240

tataaaaatt ggtcaaaacc acagtgtcta attacagggt tcgctccttt ctcaaaggac 300

aactcaatta ggctttctgc aggtggggat atctgggtaa caagagaacc ttatgtctca 360

tgcagtcccg acaaatgtta tcaatttgca cttgggcagg gaaccaccct gaaaaacgag 420

cactcaaatg gcactacaca cgatagaacc cctcacagaa ctcttttaat gaatgaatta 480

ggtgtcccat ttcatctggg aaccaaacaa gtgtgtatag catggtctag ttcaagctgc 540

tatgatggaa aagcatggtt acatatttgt gttactgggg acgataaaaa tgctactgct 600

agtatcatct atgatgggat gcttgttgac agtattggat catggtccaa aaacatcctc 660

agaactcagg agtcagaatg cgtttgcatc aatggaactt gtgcagtggt aatgactgat 720

ggcagtgcat caggagtggc cgacactaga gtattattca taagggaagg gaaaattata 780

aatattagac cattgtcagg aagcgctcag ttcgttgagg aatgctcctg ttatccccgg 840

taccctgaaa ttagatgtgt ttgcagagac aattggaagg gctccaatag gcccattata 900

tatataaata tggctgatta tagcattgaa tccagctata tgtgctcagg acttgttggc 960

gacacaccaa gaaatgatga tagctccagc agcagcaact gcagagaccc taacaacgaa 1020

agaggggccc caggagtgaa agggtgggcc tttgacgacg ggaatgatgt ttggatggga 1080

cggacaatca aaaatggttc acgctcaggt tatgagactt ttagggtcat aaatggttgg 1140

accatggcta attcaaagtc acagataaat aggcaagtca tagtcgacag tgacaactgg 1200

tctgggtatt ccggcatctt ctctgttgaa ggcaaagaat gcatcaacag gtgtttttat 1260

gtagagttga taagagggag accacaggaa cccagagtgt ggtggacatc aaatagcatc 1320

attgtattct gtggaacctc aggtacatat ggaacaggct catggcctga tggagcgaat 1380

atcaacttca tgcctatata a 1401

Claims

1. An avian influenza virus strain is an H9N2 subtype avian influenza virus SW001 strain with the preservation registration number of CGMCC No. 19387.

2. The avian influenza virus strain of claim 1, wherein the HA protein of the avian influenza virus strain HAs an amino acid sequence shown as SEQ ID No. 1.

3. The avian influenza virus strain according to claim 1, wherein the NA protein of the avian influenza virus strain has an amino acid sequence shown as SEQ ID No. 2.

4. The application of the avian influenza virus strain in the preparation of biological products is characterized in that: the avian influenza virus strain is an H9N2 subtype avian influenza virus SW001 strain, and the preservation registration number is CGMCC No. 19387.

5. The use according to claim 4, wherein the biological product is an avian influenza vaccine or a combination vaccine.

6. The use according to claim 5, wherein the avian influenza vaccine is an inactivated vaccine, wherein the antigen is the inactivated SW001 strain of avian influenza virus subtype H9N 2.

7. The use according to claim 4, wherein the biological product is an antigenic agent for the diagnosis of avian influenza virus.

8. The use according to claim 4, wherein the biological product is a positive serum preparation for diagnosing avian influenza virus.

9. A biological product prepared using the avian influenza virus strain of claim 1 or a recombinant virus strain thereof.