Replication Characteristics Of H6N2 Subtype Avian Influenza Virus In BALB/c Mice And Human Respiratory Tissues

Background and Object:In recent years,some studies have shown that the H6 subtype of avian influenza virus was widespread in poultry and spreaded to mammals,even occasionally infected humans.When H6 AIV circulate in poultry with H5 and H9,different subtypes of viruses may undergo gene reassortmentand change some genetic characteristics of the virus,it will promote the evolution of the virus to expand the host range and increase the chance of binding to the human-like receptor SA?-2,6Gal.However,the evolutionary evolution of the H6N2 subtype and its genetic characteristics have not been studied clearly until now.To explore whether the H6 AIVevolution and cross species transmission,BALB/c mice were infected by H6N2 subtype avian influenza virus isolated from ducks and its replicated in human respiratory tract in vitro,evaluate its pathogenicity to mice and its replication ability in human respiratory tract.Meanwhile,the related gene characteristics of H6N2 subtype avian influenza virus infecting mice and human were screened out.Methods:1.Selection and replication of viruses:Three avian influenza virus strains of H6N2 isolated from ducks:A/DK/GX/141/2005,A/DK/GX/3101/2018,and A/DK/GX/5220/2018.H9N2 virus A/DK/ST/3208/2010 was used as a positive control virus strain.Firstly,strainses were amplified in10 days chicken embryos for 48 hours.The next,collect the allantoic fluid was collected and HA was detected2.Determine the dose of viral infection by EID₅₀and TCID₅₀:The virus of infection experiment was inoculated in chicken embryo to determine EID₅₀(50%egg infection dose)and in MDCK(Madin-Darby canine kidney)to determine TCID₅₀(50%tissue infection dose).According to the results,the amount of virus inoculated in mice and human respiration tissue in vitro was calculated.3.BALB/c mice infected with H6N2 subtype avian influenza virus:BALB/c mice were randomly divided into infection group,positive control group and blank control group.BALB/c mice were inoculated with virus fluid through intranasal and eye.Then the symptoms and temperature,weight of mice were recorded in every day.The next,throat swab and blood were collected post inoculation 3,5,7 day after they were anesthetized with ether.When three mice of each group were euthanizedin,trachea and lung tissue were collected and divided into two parts.One of them was used for detection virus by inoculated into chicken embryos and MDCK cells.Another was fixed in 10%formalin for pathological examination.Post inoculation 14 day,blood of the last 3 mice were collected.4.H6N2 subtype of AIV replication in human respiratory tissue in vitro:The bronchus and lung tissues of human were collected from surgery of affiliated hospital of Guangxi Medical University.The bronchus and lung were removed and cleaned by medium and then cut into small pieces of tissue.The tissues were incubated with viruses for 1-2 h in 37?incubator.Post inoculation with virus 12,24,36,48 and 72 h,the tissue culture medium and tissue block were collected,respectively.5.Isolation of viruses and detection of serum antibody:Tissue grinding supernatant and culture medium were inoculated in chicken embryo and MDCK for 48 h,respectively.Then virus titer was detected by HA.Also,the serum antibody of mice was determined by HI.6.Pathology and detection of virus antigens in tissue:After tissue dehydrated,paraffin embedded and sliced,HE staining was used to observe the histopathological changes and immunohistochemistry was used to detect the virus antigen.7.Sequencing and analysis of virus genes:After the virus RNA was extracted for PCR,whole gene sequence of the virus was detected and then edited and processed by GCG 10.2.The sequence comparison and homology analysis were used by Mega 7.0 and Bioedit 7.0.9.Based on the results of mice infection and replication in human respiratory tract,the gene characteristics of H6N2 subtype influenza virus infecting BALB/c mice and replicating in human respiratory tract were analyzed.Results:1.The results of EID₅₀and TCID₅₀:EID₅₀values were measured in the inoculated chicken embryos,and the test results were GX141 was stronger infection capacity in embryo,while GX3101 and GX5220 strains were weaker slightly.The detection of TCID₅₀in MDCK cells were the GX141 strain could cause half of the MDCK cells infected at higher dilution degree,followed by GX5220,the lowest was GX3101.2.Pathogenicity of H6N2 strain in BALB/c mice:After mice were inoculated with three strains,positive virus isolation were detected.,The titers of GX3101,GX5220 and GX141were 1:1024,1:1024,and 1:512,respectively.However,in MDCK culture,only GX141 was positive,the titer only 1:16.About,tracheal tissue,only GX141 of tracheal tissue was positive virus isolation in chicken embryo and MDCK cells.The alveolar structure of the mice infected by the three virus strains were destroyed and inflammatory cell infiltrated,and the tracheal mucosa cells were exfoliated with inflammatory cell infiltration.IHC showed positive expression of viral NP protein in lung tissue cells of GX141 and GX3101,and negative expression of GX5220.The IHC results of tracheal tissue showed that only the GX141 group showed positive expression of viral NP protein in tracheal mucosal epithelial cells.3.Replication of H6N2 strain in human respiratory tract tissues:The 3 strains were inoculated in lung and bronchus tissues in vitro.The culture medium and tissue grinding were isolated in chicken embryo and MDCK cells,and there was no significant difference in the HA titer in chicken embryo and MDCK in each group.In the lung tissue of mice infected with GX141 and GX3101 strains,there were inflammatory reactions in varying degrees,such as destruction of alveolar structure and infiltration of inflammatory cells.In GX141 group,there were the bronchial mucosa cells exfoliation and inflammatory cells infiltration.IHC results showed that lung cells were positive expression of viral NP protein in group GX141 and GX3101,while those in group GX5220 was negative.However,bronchial tissue was negative of viral NP protein in all three strains.4.The results of virus gene sequencing:The HA cleavage site of three H6N2viruses was PQIETG/GL,which indicated that the cleavage site was a single base with low pathogenicity.T263K and T318Ldouble mutations were found in the HA protein of GX141,while only E190V amino acid substitution was found in GX3101 and GX5220.N66N amino acid substitution in PB1-F2 was found in GX141.Conclusions:1.The H6N2 virus could infect BABL/c mice,especially GX141,which could infect trachea and lung tissues.The result suggested that H6N2 subtype avian influenza virus can across the interspecies barrier directly to infect mice.2.The H6N2 virus could replicate in human lung tissue,but not in tracheal tissue.This indicating that the virus had the potential to infect humans,but it can notrecognize and bind to human-like SA?2,6Gal receptor.3.The HA amino acid sites of H6N2 avian influenza virus showed T263K and T318L double mutations and the N66S amino acid substitution of b1-F2 protein could promote the replication of the virus in mammals.However,the amino acid substitution at the HA protein E190V site of H6N2 AIV failed to across the species barrier to infect mammals and humans stability.Combined with the pathogenicity of H6N2 virus to mice and the replication characteristics in human lung tissue,T263K and T318L double mutations in the amino acid site of HA and N66S amino acid substitution in PB1-F2 of H6N2 avian influenza virus can promote the replication of the virus in mammals and humans,while only the amino acid substitution in E190v site of HA protein can not stably cause the replication of H6N2 subtype AIV in mammals and humans.