Phenotypic Characteristics Of Piglets Infected With JEV Strains Differentially Expressing NS1’ Protein

Japanese encephalitis virus(Japanese encephalitis virus,JEV)is a single-stranded positive-strand RNA arbovirus together with West Nile Virus(WNV)and Zika Virus(ZIKV),it belongs to the genus Flavivirus(Fructus genus)of the Flaviviridae family.The natural host range involves mosquitoes,pigs,waterfowl and bats.Pigs are the main augmentation host of JEV in nature.The JEV NS1’ protein is a product of ribosome translocation in NS2A resulting in early termination of translation during viral genome translation.It contains the complete sequence of NS1 protein and a C-terminal extension of 52 amino acids,which with a molecular weight of approximately 55 kDa.Encephalitis flaviviruses such as JEV,WNV and ZIKV can produce NS1’ protein.It has been reported that the NS1’ protein can increase the level of flavivirus RNA in avian cells and promote the production of virus.Recent studies have found that JEV NS1’ inhibits IFN-1 signaling in host cells.Expression of NS1’increases the neuroinvasiveness of WNV in mice.The role of NS1’ in JEV-infected pigs has not been reportedIn this study,the JEV SA14-14-2 point mutant strain rJEV SA14-14-2 A66G was successfully constructed in the early stage of the experiment,and the resuscitation was performed.JEV SA14-14-2 and rJEV SA14-14-2 A66G were infected with 90-day-old JEV antibody-negative piglets.From the viral infections of serum,nasal swabs,and various tissues and organsand,phenotypic differences in piglets infected with JEV strains that differentially express NS1’ were conpared.The role of NS1’ protein in JEV-infected piglets was initially explored.Histological observation of brain tissue and soft palate tonsils in JEV-infected piglets,preliminary investigation of cell preference in JEV-infected pig tissues.The specific research contents are as follows:1.Infection characteristics of piglets vaccinated with JEV SA14-14-2 strain differentially expressing NS1’ proteinIn the early stage of the laboratory,the rJEV SA14-14-2 strain was obtained by constructing the infectious clone of the JEV attenuated vaccine strain SA14-14-2.The construction of infectious clones combined with point mutation technology,the mutant strain rJEV SA 14-14-2 A66G was obtained,and the expression of JEV NS1’ was successfully obtained.Amplifying the E gene of rJEV SA14-14-2 A66G strain preserved in the laboratory by PCR,and the PCR product was sequenced and identified,it had 100%homology with rJEV SA14-14-2 E gene,the strain was identified as attenuated strain.The expression of NS1’ of rJEV SA14-14-2 A66G strain was successfully identified by western blot.The expression of NS1’ protein in BHK-21 cells,A549 cells and PK-15 cells infected with rJEV SA14-14-2 A66G strain was analyzed by Western blot.It was found that NS1’was stably expressed in three kinds of cells,and in three kinds of cells.NS1’ can be secreted outside the infected cells.In order to compare the differences between rJEV SA14-14-2 strain and rJEV SA14-14-2 A66G strain in infected piglets,the phenotypic differences in piglets infected with JEV strains differentially expressing NS1’ protein were investigated,the 90-day-old JEV antibody-negative piglets in the group were tested for inoculation,the group A was inoculated with rJEV SA14-14-2,the group B was inoculated with rJEV SA14-14-2 A66G,and the group C was inoculated with wild-type control JEV NJ2008,the inoculation dose of the three groups of piglets was 107TCID50,the inoculation site is subcutaneous in the neck and vein,and each part is injected with 1mL of virus solution,and the total volume is 2mL.Group D was a negative control group,and PBS was injected.The inoculation site and the injection dose were the same as those in the challenge group.Measuring the rectal temperature of the experimental piglets separately,observing the clinical symptoms,the pig serum was collected,the nasal swab was used to detect the infection of JEV,and the tissue organs were collected for the detection of JEV infection.Compared with the rJEV SA14-14-2 strain inoculation group,the rJEV SA14-14-2 A66G strain inoculation group showed viremia earlier in infected piglets,and JEV infection was detected earlier in nasal swabs.Organs showed a higher positive rate of tissue JEV infection.It was preliminarily proved that the differential expression of NS1’had a certain influence on the phenotypic differences of JEV-infected piglets.2.Study on tissue pathology and cell tropism of piglets infected with JEV strains differentially expressing NS1’ proteinTo further explore the phenotypic differences in piglets infected with JEV strains that differentially express NS1’ protein,tissue samples from each group of piglets brain tissue were prepared and HE stained for pathological changes.No obvious lesions were found in HE staining of brain tissue of group A.According to the experimental results of pig tissues in groups B and C,it was found that JEV infection caused non-suppurative encephalitis lesions in the cerebral cortex and thalamus,including necrosis of nerve cells,formation of vascular sleeves,satellite phenomenon and neurotropic phenomenon.Immunohistochemical detection of JEV-infected pig brain tissue and soft palate tonsils was performed to observe the distribution of JEV-infected tissues,and to explore the partiality of JEV-infected cells.The positive results of immunohistochemistry were found in group B and C.According to the observation results,basket cells and purkinje cells were positive for JEV infection;Immunohistochemical examination of the cerebral cortex and thalamus showed that the pyramidal cells were positive for JEV infection.The immunohistochemical results of soft palate tonsils showed that macrophages were the main metaphilic cells of JEV infection,and the infected positive macrophages were mainly distributed in the lymphoid nodules of the parenchyma of the soft palate,and some squamous epithelial cells were also infected with JEV.