| H9N2 subtype Avian influenza(AI)has been widely used in domestic poultry.With the increasing virulence of Avian influenza virus(AIV),the gradual weakening or disappearance of the species barrier,and the high mortality caused by secondary infections,the poultry industry has brought huge economic losses and poses a serious threat to human public health security.Therefore,the establishment of a comprehensive H9N2 subtype avian influenza surveillance program,screening strains with broad antigenic spectrum and excellent immunogenicity,and developing a vaccine with high protection rate against the current popular H9N2 avian influenza is very important for poultry production and public health.1.Genetic evolution analysis of H9N2 AIVTimely monitoring of changes in the H9N2 subtype avian influenza virus and effective measures against changes are an effective way to prevent outbreaks,reduce economic losses in livestock production,and remove threats to public health.In this study,18 strains of H9N2 AIV with 6 log2 or more isolated from Jiangsu Province in 2015-2017 were subjected to whole-genome sequencing and genetic analysis software such as MEGA and DNA Star were used for genetic evolution analysis.The results showed that the variation of HA and NA of the 18 strains of surface proteins was relatively large,and the changes of internal genes M,NP,NS,PA,PB1 and PB2 were relatively small,among which HA,NA,NP,NS and PB1 belonged to CK/SH/F/98-like,M,PB2 belong to Qa/HK/G1/97-like,PA mostly belongs to CK/SH/F/98-like,and a small part belongs to DK/HK/Y280/94-like.There is no continuous basic amino acid in the HA cleavage site of all isolates,which has the standard characteristics of low pathogenic avian influenza virus.And the 226 sites of the 18 strains of HA are all leucine(L),indicating that H9N2 AIV has the ability to bind to human influenza virus receptors,thereby increasing the possibility of causing human disease.The loss of the neck of the 18 strains of virus NA showed that the virus virulence was enhanced compared with the intact NA protein intact H9N2 AIV.The 27,30,and 31 sites of the M gene are mutated and have resistance to adamantane drugs.Other genes are relatively conserved,and amino acid site variation is relatively small.2.Immunoprotection study of H9N2 AIVThe avian influenza virus is rapidly mutating and the virulence is gradually increasing,and the phenomenon of immune failure sometimes occurs.Vaccination is one of the most effective ways to control H9N2 avian influenza in addition to biosafety in China.It is imperative to develop a new vaccine to prevent and control the current H9N2 subtype avian influenza induction.Based on the genetic evolution characteristics of 18 H9N2 AIVs isolated in 2015-2017,combined with the relevant biological characteristics,five representative strains were selected,namely CZ1603,CZ1704,CZ1705,WX1512 and JR1609.Cross-HI and cross-immunoprotective studies were performed on 5 representative strains.The results showed that the average HI antibody titer of CZ1603,CZ1704,CZ1705,WX1512,JR1609 was 8.16 log2,the cross-HI difference was below 2.4 log2,and the antigen-related value was 0.77.Between?1;the virus content of the 5 representative strains were 108.17 EID50,108.17 EID50,107.5 EID50,108.22 EID50 and 109.25 EID50,respectively.The inactivated vaccine was used to immunize SPF chickens with 5 representative strain antigens.When the HI antibody reached 6 log2 or more,the blood was separated and serum was separated.Each vaccine immunized chicken was challenged with each strain of virus,and the unimmunized chicken was used as the control group.Five viruses were attacked by each virus.The challenged chicken throat swabs and cloaca swabs were collected 5 days after challenge.The detoxification of SPF chicken embryos was determined.The results showed that the virus isolation ratios of the control chickens after challenge were:4/5,4/5,4/5,5/5,5/5,except for the CZ1705 immunization group attacking the CZ1704 strain and the WX1512 immunization group attacking the CZ1705 strain,the virus isolation ratio was 1/5,and the other attack groups were The virus was not isolated,and the CZ1603,CZ1704,and JR1609 immunization groups could produce 100%immunoprotective effects against the 5 strains of viruses.The virus content,antigen spectrum and cross-immunoprotection test results of 5 representative strains were combined to determine JR1609.The strain virus is a vaccine candidate strain,which lays a foundation for the development of commercial vaccines. |
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