Molecular Evolution Of Avian Influenza Virus And Serological Study Of Occupational Population In Anhui Province

Objective:In recent years,various subtypes of avian influenza viruses have been prevalent in birds,and even some subtypes of avian influenza viruses have broken through the species barrier and can infect mammals and spread widely,which seriously threatens public health and safety.There are three migratory routes for birds in China.The migratory activities of birds in winter and spring each year,the special breeding mode and the prosperous live poultry market also provide convenient conditions for the spread of avian influenza virus.However,people occupationally exposed to poultry have a higher risk of infection if they are exposed to the virus for a long time than the general population,which requires special attention.Methods:This study was divided into two parts.One part was to conduct surveillance and sampling in migratory birds and habitat environmental detection points in Anhui Province from 2018 to 2021,avian influenza pathogen monitoring points in the external environment,and sampling of human infection with avian influenza virus in hospitals in various cities,and conduct nucleic acid detection by real-time fluorescence quantitative q PCR.The positive samples were further subtyped to determine the subtypes.The sequences of low pathogenic virus samples were obtained by chicken embryo method and cell culture,and the sequences of high pathogenic virus samples were obtained by direct sequencing after nucleic acid extraction.Bioinformatics analysis of viral sequences was performed.TCID50 and receptor binding assays were used to assess the risk of infection in mammals by influenza viruses of different origins.The second part Is to collect the serum samples of the occupationally exposed population in the avian influenza pathogen surveillance points,and conduct HI test on the serum samples to detect the inobvious infection of the occupationally exposed population,and to prepare pseudovirus for neutralization test to detect whether the antibodies in the occupationally exposed population have immune protection against the main prevalent viruses.Results:1.A total of 759 samples were collected from the external environment,and 383 samples were positive for influenza A nucleic acid,383 samples were positive for H9 nucleic acid,17 samples were positive for H5 nucleic acid,and 10 samples were positive for H5/ H9 mixed nucleic acid.A total of 1137 samples were collected from migratory birds and habitats.Fourteen samples were positive for influenza A virus nucleic acid and 12 samples were positive for H9 virus nucleic acid.A dead sample was collected from the migratory bird monitoring station in Feidong County,which was positive for H5 subtype nucleic acid by typing.Thirty-six strains of H9N2 virus were isolated and cultured by egg and cell culture methods.All the viruses belonged to the G57 genotype,which was considered as low pathogenic avian influenza virus.Analysis of the receptor binding sites of 36 H9N2 viruses showed that some viruses had T155 N,158 N,A190 T/V mutations,and all viruses had H183 N,Q226L mutations.Analysis of other protein sites revealed that all viruses had a deletion in positions 62 to 64 of the NA protein.Some viruses had A588 V and E627 V mutations in PB2 protein,but no mutation was found in 701 site.The 702 site of 35 viruses was K,and the 702 site of 1 virus was R.The PB1 protein of 35 strains had I368 V mutation.Most of the viruses had A100 V and K356 R mutations in PA protein.Except for one strain,the 409 site of the virus was S,and the other 35 viruses were N.S31 N mutation was found in M2 protein of 36 strains.2.Phylogenetic analysis of the H5N8 virus strains showed that FD1203 belonged to the Eurasian lineage,and had high homology with the strains found in many countries along the migratory routes of birds,such as East-Australia,Central Asia and West Asia.The cleavage site of the HA protein of the virus indicated that the virus was a highly pathogenic avian influenza virus.Analysis of the receptor binding sites of HA protein of FD1203 showed that 133 site was L,160 site was A,no mutation was found,187 site was N,193 site was N,196 site was K.The 226-228 site was QRG.FD1203 has mutations in several important receptor-binding sites.Other protein molecular characterization results showed that several important NA protein sites of FD1203 were not mutated for oseltamivir resistance.No S31 N amino acid mutation was found in the amantane resistance site of M2 protein.K389 R and V589 T amino acid mutations were found in the PB2 protein of FD1203,and no E627 K amino acid mutation was found.3.Ten strains of H9N2 viruses with high similarity were selected for TCID50 assay and receptor binding assay.The TCID50 values of 10 strains of H9N2 viruses were between 102.875 and 106.335.The titer of environmental H9N2 viruses was slightly lower than that of human H9N2 viruses.However,there are differences in replication ability between the two.Receptor binding assay showed that human H9N2 virus and environmental H9N2 virus could bind to avian and human receptors,and human H9N2 virus had a stronger affinity with human receptors,while environmental H9N2 virus had a stronger affinity with avian receptors.4.A total of 418 serum samples were collected from workers occupationally exposed to poultry.The results showed that 30 samples were found to be seropositive,and the seropositive rate was low(7.17%).There was significant difference in the overall seropositive rate between male and female(P<0.05),including 8 H5N8 positive sera,7 H5N6 positive sera,5 H7N9 positive sera and 12 H9N2 positive sera.The positive rates of antibodies against H5,H7 and H9 subtypes were 3.59%,1.20% and 2.87%,respectively.Among them,one serum sample was tested positive for H5N6 and H7N9 antibodies,and one serum sample was tested positive for H5N8 and H7N9 antibodies.5.The results showed that the neutralization efficiency of 3 H5N8 positive serum samples and 2 H5N6 positive serum samples were more than 97% and 73%,respectively.All nine negative serum samples were less than 65% effective in neutralizing the pseudovirus when diluted 40 times.Conclusions:H9N2 subtype avian influenza virus was the main subtype in Anhui province from2018 to 2021,followed by H5 subtype avian influenza virus.H9N2 subtype avian influenza virus can be detected in migratory birds,external environment and human population.The virus can bind to human receptors and replicate in mammalian cells,and the virus gradually adapts to the mammalian host.The HA and NA genes of H5N8 virus from migratory birds belonged to Eurasian lineage.The H5N8 virus was still the avian receptor,but the virulence tended to increase.The inapparent infection of H9N2 was the most serious,followed by H5N8,H5N6,H7N9.However,most of the serum antibodies of people exposed to poultry were very low in neutralizing the H5N8 virus from migratory birds.It is necessary to be vigilant against the spread of H5N8 subtype avian influenza virus.