Isolation And Identification Of Low Pathogenic Avian Influenza Virus From Wild Birds In Ebinhu Lake

Objective: A number of studies have shown that wild birds carry a variety of pathogenic microorganisms,and in the case of avian influenza,migration of wild birds and the trade of live poultry in the market promote the recombination of influenza viruses in different regions,different bird species and different subtypes.The detection of wild bird virus in Aibi Lake Wetland Nature Reserve plays an early warning role in animal husbandry and epidemic prevention and control in Xinjiang.Methods: In this study,avian influenza virus was detected by using avian influenza specific primer M229 in the supernatant of cotton swabs of feces collected from wild birds in Ebinhu Nature Reserve of Xinjiang,and PCR amplification,sequencing and analysis were performed on positive avian influenza samples using HA universal primer.After the sequence analysis,9-day-old common chicken embryos were used to isolate the avian influenza virus,and the allantoic fluid of the isolated chicken embryos was used to identify the genotype of the virus with the common primers of avian influenza virus.The bioinformatics of hemagglutinin and neuraminidase were predicted and the whole genome sequence of the two strains was analyzed.After the virus was obtained,the whole genome sequence of the isolated strains was amplified,and then transformed into DH5? competent cells after connecting with the T vector.After coating the plate,a single colony was selected for shaking and sequencing.Finally,female balb/c mice and 4-week-old chickens were infected respectively to observe and determine the virulence of the isolates.Results: A total of 80 swabs were collected in this study,and the positive rate of avian influenza was18.75%.Two representative strains of low-pathogenicity avian influenza viruses were isolated.They have been named A/Wide Bird/Ebinur Lake/40/2017(H1N2)and A/Wide Bird/Ebinur Lake/47/2017(H3N8).The hemagglutinin cleavage sites of the isolates did not contain multiple consecutive basic amino acids,which was consistent with the sequence characteristics of low pathogenic avian influenza amino acids.Both hemagglutinin and neuraminidase of the isolates were hydrophilic.The splicing site of A/Wide Bird/Ebinur Lake/40/2017(H1N2)hemagglutinin signal peptide is 0.8117,which belongs to secretory protein.There are 12 B cell epitopes with more than 10 consecutive amino acids,with 25 antigenic determinants and 59 peptides.A/Wide Bird/Ebinur Lake/40/2017(H1N2)neuraminidase,9 B cell epitopes with more than 10 consecutive amino acids,15 antigenic determinants and 55 peptides.The splicing site of A/Wide Bird/Ebinur Lake/47/2017(H3N8)hemagglutinin signal peptide is AFS-QD,(0.4754),which belongs to secretory protein.There are 12 B cell epitopes with more than 10 consecutive amino acids,with17 antigenic determinants and 51 peptides.There are 9 B cell epitopes of A/Wide Bird/Ebinur Lake/47/2017(H3N8)with more than 10 consecutive amino acids,with 16 antigenic determinants and 46 peptides.The two isolates could replicate in female balb/c mice and 4-week-old unimmunized chickens,and had no lethal effect on female balb/c mice and 4-week-old unimmunized chickens.The intravenous vaccination index of A/Wide Bird/Ebinur Lake/40/2017(H1N2)was 0.11,the intravenous inoculation index of wide Bird/Ebinur Lake/47/2017(H3N8)was 0.08,both were less than 1.2,which further confirmed that it was a low pathogenic avian influenza virus.Hemagglutination inhibition test was carried out on the isolated serum of each chicken on the 14 th day of infection,and the positive rates of H1N2,H1N2,H3N8 and H3N8 were 70%,40%,40% and 20%,respectively,but the hemagglutination inhibition titers of each group were very low.Conclusion: In this study,two strains of avian influenza virus were successfully isolated from the fecal swabs of Ebinur Lake wild birds,and the positive rate of avian influenza in fecal swabs was 18.75%,which confirmed the existence of avian influenza virus in Ebinur Lake wild birds.Through the whole genome sequence analysis,the pathogenicity of female balb/c mice and four-week-old unimmunized chickens and the bioinformatics analysis of the isolated strains,it was confirmed that the two strains were low pathogenic avian influenza viruses,and the isolates had multiple subtypes of virus recombination,which could infect female balb/c mice and replicate and spread in four-week-old unimmunized chickens.