| H9N2 subtype avian influenza virus in many countries and regions,including China,has brought great economic losses to the aquaculture industry,which seriously restricts the healthy development of aquaculture industry.Our country's environment and breeding model promoted the spread of the disease,at the same time,a large number of vaccine immunization also provides a favorable condition for the reorganization and mutation of H9N2 virus.This study was conducted to isolate and identify the virus samples from the live poultry market in Jiangsu from March 2016 to March 2017.We analysed the sequence and genetic evolution of 10 strains of H9N2 isolated.And the immune protective efficacy of 3 candidate strains A/CK/JS/04/2016,A/CK/JS/05/2016 and A/CK/JS/08/2016 vaccine was evaluated.The aim of this study was to provide experimental basis for the prevention and control of avian influenza.1.2016-2017 Jiangsu live poultry market H9N2 avian influenza epidemiological survey2016-2017,we collected swabs and serum of chickens,ducks and geese in Jiangsu live poultry market.Within a year we collected cloacal swabs and oral swabs for virus isolation of about 4000 copies,of which,about 2400 samples of chicken,duck samples of about 760,about 840 samples of geese.The results showed that the positive rate of serum H5 in AIV subtype 55.71%?92.69%subtype AIV,the positive rate of serum H7 in 2.29%?29.28%,the positive rate of serum H9 in AIV subtype 64.78%?93.15%,Newcastle disease virus serum positive rate was 52.96%?92.86%.In a word,the positive rate of virus antibody was higher in serum.2.Isolation,identification and genetic evolution analysis of H9N2 subtype avian influenza virusThe clinical samples were collected from some chicken farms in Jiangsu Province,and the virus was isolated by inoculating SPF chicken embryos.HA,NA,M,NS,NP,PB1,PB2,PA 8 gene fragments were amplified by PCR,sequenced and analysed genetic evolution.The results showed that 10 strains of H9N2 subtype viruses were isolated,and the external and internal genes of the virus were changed greatly.the HA cleavage sites of all 10 isolates were RSSR-GLF,which was consistent with the molecular characteristics of low pathogenic avian influenza virus.In the 198th receptor binding site,eight of ten strains were threonine(T),and two of ten strains were alanine(A).In the ten strains,the 234th receptor binding site were all leucine(L),and one of ten was histidine(H)in 191st site.The homology of nucleotide and amino acid of HA gene among 10 strains were 94.3%-99%and 93.8-99.6%,belonging to A/Duck/Hong subgroup of Kong/Y280/97 in Eurasian clade.Deletion of the NA gene of 10 strains in the 62-64 site resulted in the deletion of the 61 glycosylation sites.The NWS of the 402 position of NA gene showed the variation of asparagine(N)to aspartic acid(D).Eight strains of the virus had only six glycosylation sites in the ten strains.The homology of nucleotide and amino acid of NA gene is 94.8%?99.9%,95.2%?99%,which belongs to the A/Duck/Hong subgroup of Kong/Y280/97 in Eurasian clade.Most of the internal genes belong to SH/F/98-like sub branch or Y439-like sub branch.3.Cross immune protection of H9N2 subtype avian influenzaBased on the analysis of genetic evolution of H9N2 avian influenza virus,the H9N2 vaccine candidate strains were A/CK/JS/04/2016,A/CK/JS/05/2016,A/CK/JS/08/2016.Average HI antibody titer of candidate strains was 71og2,EID50 were between 6.5 and 7.5.The results showed that the immune A/CK/JS/04/2016 and A/CK/JS/08/2016 vaccine group had a good immune effect to 3 strains.The immune protection rate was well,and the protective rate of A/CK/JS/05/2016 vaccine against A/CK/JS/08/2016 strain was not very good.In conclusion,A/CK/JS/04/2016 and A/CK/JS/08/2016 vaccine group were good candidate vaccine strains. |
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