Establishment Of Porcine Tonsil Epithelial Cell Line And Analysis Of Virus Susceptibility

Japanese encephalitis is an important zoonotic disease caused by the Japanese encephalitis virus,which leads to serious encephalitis disease all over the world.About 60000 to 80000 cases of JEV infection were reported,and about 15000 to 20000 deaths every year.Pig is the pivotal intermediate and amplify hosts,which causes the circulating transmission between pigs,mosquitoes and pigs.JEV infection lead to sow abortion,mummified fetus,The infection of boar lead to orchitis.JEV persist in the tonsils for a long time.As the amplify host of JEV,pig is the basis of viral transmission and epidemics,can produce viremia for a long time.However,the reason why pigs are the amplified hosts is still unclear.Tonsil is a special organ that load JEV in a high level and persist JEV infection for a long time,it may be related to the persistent viremia of pigs.At the same time,pig tonsils are also the organs that carry many pathogens,a variety of important pathogens persist infect in the tonsils for a long time.The mechanism of JEV infection in tonsil is not clear,the main reason is the lack of corresponding tonsil cell lines to support research.In this experiment,a porcine tonsil cell?PT?was established.The susceptibility of PT cell to major swine pathogens was analyzed.It was found that PT cell was susceptible to JEV and classical swine fever virus?CSFV?.Next,we analyzed the infection process of JEV on PT cells,and found some characteristics of PT cells on the expression of viral receptors and surface antiviral molecules manbe impact on PT persistent toxicity.Furthermore,the differences of gene expression of pig-derived cell lines:porcine tonsil cells?PT?,porcine testis cells?ST?and porcine hip artery endothelial cells?PIEC?after JEV infection were detected and compared by RNA-Seq deep sequencing.The mechanism of persistent infection of JEV was further analyzed.Firstly,the palatal tonsil of SPF pig was isolated and primary cells of palatal tonsil epithelium were taken.PCK immunofluorescence staining was performed to identify the positive rate of isolated cells.HIV-1,HBV,HCV,mycoplasma,bacteria,yeast and fungi were measured to ensure no infectious pathogens was contaminated in cells.Then the lentiviral infection method was used to transfect the SV40plasmid,followed by subculture for 10 generations and then puromycin selection.The tonsil epithelial cell line that was screened by purine pressure and identified as positive was expanded and cultured to construct an immortalized cell line,named PT.The established cell lines were cultured,biologically and morphologically identified,and the results showed that the subcultured cell lines had been successfully established and showed basic experimental functions.Secondly,the susceptibility of important infectious pathogens?PRRSV?CSFV?PCV2?PEDV?PRV?JEV?of pigs to PT cells was tested.PT cells were inoculated with 5 MOI purified virus,and the inoculated cells were detected by real-time quantitative PCR and TCID₅₀.The results showed that PRRSV?PCV2?PEDV?and PRV could not infect PT cells,which viral nucleic acids and viral particles were not detected in the cells for 48 hours infection.CSFV and JEV showed infectious ability on PT cells,which viral nucleic acids and virus particles were detected in PT cells.Among them,PT is more susceptible to JEV,which can produce high-titer JEV particles at 48 hours post inoculation.These results indicated that PT cells can be used for JEV and CSFV study in future.Thirdly,the process and characteristics of PT cells infected with JEV were analyzed.By comparing the process of JEV infection in different pig-derived cell lines,it was found that entry of JEV was more quickly in PT cells than other cells.Analysis of the potential receptor of JEV proved that LMAN2L may be the main viral receptor of JEV to invade PT cells.In addition,IFITM3,a molecule that affects virus invasion and infection on the cell surface,was also studied.The results showed that the level of IFITM3on the surface of PT cells was relative low,and IFITM3 in PT cells after JEV infection showed a low change level.The study revealed the process and characteristics of JEV infection in tonsils,and basicly explained the reason of JEV persistent infection.Fourthly,the gene expression of pig-derived cell lines?PT?ST?and PIEC?after JEV infection was analyzed by RNA-Seq deep sequencing.It was found that there were differences in antiviral response and immune response between PT cells and ST?PIEC cells after infection.JEV infection induce the up-regulation of PT cell innate immune response?interferon-stimulated gene?,but the role of innate immune response gene is limited,the antiviral ability is not strong,and JEV in cells cannot be completely eliminated.In this study,the pig tonsil cell line was established for the first time,the biological characteristics of the cells were analyzed,and exploration was made on the persistent infection of swine pathogens.It laid the foundation for the persistent infection and host transmission of swine pathogens,especially JEV,and also expanded the research model of swine disease and swine biology.