Study Of Replication Capacity Of Duck H6N1 Subtype Avian Influenza Virus In Mice,in Vitro Swine Lung And Human Lung Tissue

Backgrund and Objectives Over the past ten years,highly pathogenic avian influenza viruses,such as H5N1 and H7N9,frequently cross interspecies barriers from avians to humans,which pose a great threat to human health and public health security.Worryingly,in May 2013,the world’s first human H6N1 influenza virus appeared in Taiwan,china.Although H6N1 is a low pathogenic virus,but prevalent in poultry.H6N1 could evolved into the new flu virus with pandemic potential.In addition to being beware of highly pathogenic avian influenza viruses such as H5N1 and H7N9,we can not ignore the potential threat of the low pathogenic avian influenza virus H6N1 subtype.On the basis of previous studies,this paper explores whether duck H6N1 avian influenza viruses could infect mammals and humans potentially,and further screens the dominant strains.It provides experimental basis for the study of gene characteristics in interspecies transmission.Methods1.The amplification of avian influenza virus and EID50 test:the 5 strains of avian influenza virus that from the H6N1 subtype and H9N2 that has been screened subtype were amplified by chicken embryo inoculation.The amplified embryos of the chick embryo allantoic fluid were centrifuged and stored at-80℃.Use the virus solution gradient dilution to do EID50(egg infections dose)experiment,the virus inlution was determined before the animal infection experiment.2.Experiment of BABL/c mice infected by avian influenza virus:with frozen dilution has been determined to dilute the number of five strains of avian influenza virus were simulated natural environment virus transmission pathways,the use of nasal drops,eye drops,pharyngeal drops inoculation of infection 6-8(BABL/c mice)were used to monitor the changes of body weight and release the virus within one week after infection.The HA was detected by HE staining in the spleen,lung,kidney,brain and turbinate tissues.The histopathological changes were observed and the virus NP antigen was detected by the method of immunohistochemistry.3.Avian influenza virus infection swine lung tissue in vitro:was performed in the free lung tissue of the swine lung tissue.The tissue was stained as a small tissue in a 6-well culture plate.The lungs were infected with gradient dilution.The titer of avian influenza virus HA in the liquid and tissue grinding fluid was observed by HE staining,and the virus NP antigen was detected by the method of immunohistochemistry.4.Avian influenza virus infection human lung tissue in vitro:infection in clinical trials to obtain fresh free human lung tissue,diluted with avian influenza virus in vitro infection,regular sampling to monitor the culture supernatant and tissue grinding fluid poultry Influenza virus HA titer,and embedded sections were stained to observe the histopathological changes after viral infection and immunohistochemistry to explore the avian influenza virus human lung tissue affinity infection ability.Results1.Duck H6N1 subtype avian influenza virus infection in mice:H6N1 subtype avian influenza virus could infect mice and amplify and release mature virus in their tissue cells.The mice were infected with poor activity,decreased eating,weight loss in 1 week.After the expression of avian influenza antibody,the symptoms improved.HE staining indicated that the lung tissue structure changed,the congestion was serious,and the necrotic inflammatory reaction occurred in the mice.The antigen of the virus could be detected by the method of immunohistochemistry.2.Duck H6N1 subtype avian influenza virus infection in swine lung tissue in vitro:The H6N1 subtype avian influenza virus can infect swine lung tissue in vitro.Tissue mill supernatant amplification and HA detection were positive.HE staining showed local necrosis in lung tissue after viral infection.Immunohistochemistry could detect the virus in the lung tissue of the swine.3.Duck H6N1 subtype avian influenza virus infection in human lung tissue in vitro:The H6N1 subtype avian influenza virus can infect human lung tissue in vitro.Tissue mill supernatant amplification and HA detection were positive.HE staining showed local necrosis in lung tissue after viral infection.Immunohistochemistry could detect the virus in the lung tissue of the human.Conclusions1.Duck H6N1 subtype avian influenza virus has the ability to directly infect mammals,BABL/c mice,swine and human lung from waterfowl,and enriches the theory of influenza virus evolution and transmission across species.2.The avian influenza virus strain of H6N1 subtype DK/GX/767/2010 was better than other four strains of avian influenza virus infection,and could be used as a dominant strain for the study of avian influenza.