Pathogenicity Of Avian Influenza Virus H6N6 Subtype Of Chicken In Mice And Replication In Swine And Human Respiratory Tissues

Background and purpose:Avian influenza virus?AIV?H6N6 subtype is widely popular in Eurasia wild birds and poultry,and its host range gradually expanded to swines.Some strains even acquire the ability to recognize and bind to human-type receptor,and evolve into direct infecting humans potential.Therefore,H6N6 virus poses a significant threat to human health and public health potential.However,it is not clear how H6N6 subtype avian influenza virus can cross the interspecific barrier from avian to mammals and humans.In our study,mice infected in vivo and respiratory tissue of swines and humans in vitro by avian influenza virus H6N6 subtype from chicken were performed.Then the pathogenicity of the H6N6 virus in mice,the replication of virus in pig and human respiratory tissue were observed.At the same time,we used gene sequencing method to compare the variation of H6N6 virus gene fragments,and screen the genetic characteristics of H6N6 virus infected mice,and replicated in swines and humans.These results might provide useful basis for revealing the potential of H6N1 viruses crossing interspecies barriers from avians to human and key molecular markers for virus replication in humans.Methods:1.Proliferation of influenza virus and test of EID₅₀ and TCID₅₀:Three strains of H6N6 subtype AIV from l terrestrial bird A/CK/JX/20490/2014?JX20490?,A/CK/ZZ/1923/2015?ZZ1923?,A/CK/ZZ/346/2014?ZZ346?were inoculated in Chicken embryo.Detection of EID50 and TCID50 values to determine viral dilution in BALB/c mice infection in vivo,and swine and human tissues in vitro.2.BALB/c mice were infected with H6N6 virus:BALB/c mice were inoculated through intranasal and eye routes.Symptoms and weight were recorded every day after inoculation.Pharyngeal swabs were collected on 1,3,5and 7 days.Three mice were euthanized on 3,5 and 7 days,then lung and trachea tissues were collected.Half of the tissues were used for grinding and testing viral titers.The other half were fixed with 10%formalin for HE staining and immunohistochemistry.Blood samples were collected 14 days after inoculation for detecting serum antibodies.3.Replication of H6N6 virus in swine respiratory tract tissue in vitro:Fresh swine respiratory tissue were collected.The virus solution was added to the swine respiratory tissue,and culture supernatant and tissue explants were harvested at regular intervals for the isolation and identification of viruses.In addition,tissue explants were fixed with 10%formalin and used for HE staining and immunohistochemistry for virus antigen detection.4.Replication of H6N6 virus in human respiratory tract tissue in vitro:Fresh human respiratory tissue were collected.The virus solution was added to the human respiratory tissue,and culture supernatant and tissue explants were harvested at regular intervals for the isolation and identification of viruses.In addition,tissue explants were fixed with 10%formalin and used for HE staining and immunohistochemistry for virus antigen detection.5.Isolation of influenza virus:The samples collected for isolation and culturing in the previous test were divided into two parts,one was inoculated in chicken embryo,the other was inoculated in MDCK cells.Then HA test was performed to detect the virus titer.6.Detection of serum antibodies in infected animals by HI test:On 14 day after infection,blood sample was collected and the upper serum was collected after centrifugation,and serum antibodies were detected by HI test.7.HE staining was used to observe the pathological changes of virus infection in animals and tissues:Respiratory tissues of the anaesthetized mice,swine and human respiratory tract tissues infected in vitro were fixed and sliced,stained with HE,and then pathological changes were observed under optical microscope.8.Detection of virus antigen by immunohistochemistry:Respiratory tissues of the anaesthetized mice,swine and human respiratory tract tissues infected in vitro were fixed and sliced,and immunohistochemical staining was performed to detect virus antigens.9.Gene sequencing and selection key molecular markers:Viral RNA was extracted,amplified by PCR and purified.Then,the virus genome was sequenced on the Solexa system of Illumina,and the relevant virus data were prepared by using the global initiative on sharing all influenza data,GISAID).Sequence comparison and homology analysis of gene segments were carried out by using Bioedit,and the genetic characteristics of pathogenicity in BALB/c mice and replication in swine and human respiratory tract were screened.Results:1.ZZ346 strain can replicate effectively in mice after inoculation with virus.The respiratory tract tissue abrasive solution was inoculated in chicken embryo and MDCK cells,and virus isolation were positive.Epithelial cells in trachea necrosis and inflammatory cell infiltration in lung tissue,positive of NP antigen in the cells from trachea to alveolus of mice after inoculation with ZZ346 viruses strain were observed.Epithelial cells necrosis and positive of NP antigen of trachea after inoculation with JX20490 virus strain,but virus isolation were negative,pathological changes in lung tissues were not observed,and the NP antigen of lung tissues were negative.However,all were negative after inoculation of ZZ1923 virus strain in mice.After 14 days of inoculation,all the serum samples of the three virus strains in mice were positive by HI test.2.Three chicken H6N6 subtype AIV can replicate effectively in swine respiratory tract tissue in vitro.Positive of virus isolation,local cell necrosis and positive of NP antigen in alveolar cells were observed.3.Three chicken H6N6 subtype AIV in human respiratory tract tissue in vitro can be isolated after inoculation in chicken embryo.Local cell necrosis and positive of NP antigen in lung tissue of human after inoculation with ZZ346viruses strain were observed.But pathological changes and virus NP antigen of human lung tissue after inoculation with JX20490 and ZZ1923 virus strains were not observed.4.Gene sequencing analysis showed that the HA cleavage site pattern of H6N6 subtype AIV was all PQIETG/GL,which was the characteristics of a single basic amino acid cleavage site and belonged to a low-pathogenic virus.All three viruses HA showed P186T mutation,but only HA158 of Z346 virus had amino acid deletion and 11 amino acid?59-69?deletion in the NA neck region.Conclusions:1.Chicken H6N6 subtype influenza virus can effectively replicate in the respiratory tract of mice and cause respiratory inflammatory,indicating that the chicken H6N6 subtype influenza virus can across the inter-species barrier to directly infect mice.2.Chicken H6N6 subtype influenza virus can effectively replicate in swine respiratory tissues in vitro.Among the 3 virus strains,ZZ346 virus strains can effectively replicate in swine and human lung tissues,suggesting that this virus strain has the potential to infect swine and human and the prefer to bind to human-type SA–?2,6-gal receptors.3.The HA cleavage site pattern of H6N6 subtype AIV belongs to the low-pathogenic virus.P186T mutation,amino acid deletion site 158 in HA and11 amino acid?59-69?deletion in NA neck region of ZZ346 virus strain were observed.Combined with the results that ZZ346 strains can replicate effectively in human alveolar tissues,it is suggested that these gene mutation contribute to the effectively replication of H6N6 subtype AIV in human alveolar tissues.