Study On Tissular Tropism And Tonsils Cellular Tropism In Japanese Encephalitis Virus Infected Piglets

Japanese encephalitis is a zoonotic disease caused by Japanese encephalitis virus(JEV)infection.It is mainly prevalent in East and South Asia,China is a high incidence area for the disease.JEV is a member Flavivirus transmitted by mosquito vector,especially by Culex mosquito.Pigs and wild waterfowl are the main natural hosts for JEV.In addition,as the measurable viremia in pigs increase mosquito vector virus-positive rate,pigs act as major amplifying hosts of virus in the nature and will promote human infection and transmission.Differ from the fatal encephalitis symptoms of humans and horses,commercial pigs are generally asymptomatic after infected.Pregnant sows’ manifest disease such as stillbirth and abortion while reproductive disorders such as orchitis occurred in boarsNS 1 is a glycoprotein which plays an important role in virus infection and transmission.NS1’ is a 52 extra amino acid C-terminal extension of NS1 reported to be a product by a-1 ribosomal frameshift and has a molecular weight of approximately 55 kDa.Some researches demonstrated that NS1’ share the same cellular distribution with NS1,and NS1’ is closely related to virus virulence.Abolishing the production of NS1’ in JEV will reduce neurovirulence and neuroinvasiveness in mice.In mice,with the recovery of NS1’expression,the live attenuated JEV vaccine strain JEV SA14-14-2,which does not express NS1’,showed higher virulence.Pig tonsils are a group of lymphoepithelial tissue that can participate in local and systemic cell-mediated immunity and humoral immunity.Tonsil is one of the important replication sites of JEV.The long-term presence of the virus in the tonsil is one of the reasons for the persistent infection pigs.It has been speculated that the persistent infection of tonsil may be related to the oral and nasal transmission of JEV piglets.Tonsil is an important peripheral organ in JEV-infected pigs,but the cellular tropism of tonsil in JEV-infected pigs is unknown.Immunofluorescence histochemistry were performed on the soft palate tonsils of piglets with positive JEV antigen in immunohistochemistry to determine the cellular tropism of JEV to macrophages and dendritic cells in piglets’ tonsils.The effect of the recovery expression to the virulence of JEV and the systemic distribution of the virus.The main research contents and results are as follows:1.Cellular tropism of Japanese Encephalitis Virus in the tonsils of pigletsThe results of immunohistochemical assays revealed that JEV-infected cells in swine soft palate tonsils are mainly distributed in the periphery of lymphatic follicles and in diffuse lymphoid tissues,most of JEV antigen positive cells in the soft palate tonsils were irregular in shape,large in nucleus and light in color,with the characteristics of macrophages and dendritic cells.In order to further explore the cellular tropism of tonsils in JEV infected piglets,soft palate tonsils with JEV antigen positive in immunohistochemistry were selected for immunofluorescence histochemical analysis.Cellular tropism of Japanese Encephalitis Virus in the tonsils of piglets.Through the co localization of CD11b,CD 163 and MHCⅡcell surface markers and virus antigens,JEV positive cells were identified as macrophages and dendritic cells.The results showed that cellular tropism of JEV in porcine soft palate tonsils are antigen presenting cells such as dendritic cells and macrophages.Apart from this,the number of antigen-presenting cells expressing MHCⅡ molecules in soft palate tonsil follicles after JEV infection in piglets was significantly reduced,and the distribution was restricted around the lymph follicles.Through the co localization of iNOS,TNF-α and CD 172a cell surface markers and virus antigens,It was further identified that the dendritic cells favored by JEV in the soft palate tonsil tissue of piglets were mainly cDC2.Given all that,these results laid the foundation for studying the mechanism of persistent infection of JEV in tonsil.2.Differences in tissue and cellular tropism of JEV strains differentially expressing NS1’ protein in pigletsIn this study,In order to investigate the differences in tissue tropism of piglets inoculated with the rJEV SA14-14-2 strain that does not express NS1’ protein,the mutant strain rJEV SA14-14-2 A66G that recovers NS1’ protein expressionand the wild-type strain JEV NJ2008 that naturally expresses NS1’ protein,immunohistochemical experiments were carried out on tissue samples(cerebellum,thalamus,brain,spinal cord,soft palate tonsil,lung,kidney,and ileum)of piglets saved and fixation in laboratory after inoculated.The rJEV SA14-14-2 strain couldn’t infect soft palate tonsils.The results demonstrated that rJEV SA14-14-2 A66G strain and JEV NJ2008 strain had same cellular tropism in soft palate tonsil of piglets.The cellular tropism of rJEV SA14-14-2 A66G strain in piglets soft palate tonsils are also antigen presenting cells such as dendritic cells and macrophages.The rJEV SA14-14-2 strain only infected large neurons in the spinal cord of piglets.JEV antigens were immunohistochemically detected in the pyramidal cells of cerebral cortex and thalamus while in the spinal cord JEV-positive large neurons were found.JEV antigens were found in lung airway pseudostratified ciliated columnar epitheliums,goblet cells and lymph nodes near the bronchi.In the ileum,columnar cells and goblet cells in the small intestine gland are presumed to be infected.The mutant strain rJEV SA 14-14-2 A66G and the JEV NJ2008 strain had similar tissue and cell tropism.No significant JEV infection was found in the kidneys.Compared with the distribution of the rJEV SA 14-14-2 A66G strain and the rJEV SA14-142 strain in piglets,initially proved that NS1’ can promote the distribution of JEV in piglets.