Isolation And Identification Of Porcine Original Japanese Encephalitis Virus Strains

Japanese encephalitis virus(JEV)is a flavivirus transmitted by mosquitoes.Infections in human may lead to Japanese encephalitis(JE),which is a zoonotic disease.Infections in both boars and sows can lead to reproductive disorders,while piglets infection can lead to significant neurological symptoms.The widespread of JEV has caused enormous economic losses in the pig industry.It was proved that JEV infection in pigs was geographically associated with human infection.Therefore,the comprehensive understanding of JEV biological characteristics,prevention,and control measures is essential for public health.In this experiment,two virus strains,HN202101 and HN202102 were isolated from lung and brain tissues of a JEV suspected infected pig.The proliferation patterns of HN202101 and HN202102 were investigated in vivo and in vitro.The main contents of this experiment were as follows:1.Isolation and identification of JEVBrain and lung tissues from a JEV suspected infected pig were collected.Preliminary validation was performed by RT-PCR.The results demonstrated that the pig was infected by JEV.The hamster kidney cells(BHK-21)were inoculated with lung and brain tissue grinds.Typical CPE was observed.It demonstrated that the virus strains were successfully isolated.The isolated strains from lung and brain tissues were respectively named HN202101 and HN202102.Using indirect immunofluorescence assay(IFA),the specific green fluorescence was observed in two isolates,which was same as positive control.Using plaque assay,the virus titers of HN202101 and HN202102 were 1.67×10~7 PFU/m L and 3.53×10~7 PFU/m L,respectively.The phylogenetic analysis of the virus E gene showed that two isolates were both clustered in genotype III.The amino acid sequences of HN202101 and HN202102 were compared with SA14-14-2.HN202101 had 20mutations in contrast to SA14-14-2,while HN202102 had 19 mutations.And there were no mutations in the eight key virulence-determined amino acids.By comparing the isolates with the eight amino acids affecting the virulence of JEV,the results showed that both HN202101 and HN202102 isolates had the typical characteristics of a high virulent strain.The full genome length of two isolates was both 10 977 bp,while the 5′UTR was95 bp,3′UTR was 586 bp,and ORF was 10 296 bp.There were 15 nucleotide mutations and 8 amino acid mutations between the two isolates.2.Growth curves of two isolates on different cell linesP3,HN202101,and HN202102 were inoculated into swine testis cells(ST),BHK-21,human lung cancer cells(A549),and human glioma cells(U251).The supernatant samples of cell cultures were collected at four time points to determine the virus titers by plaque assay.No significant difference was found in the virus titers of the three strains on ST cells.On A549 cells,the virus titers of HN202101 were higher than those of HN202102 at 24,36,and 48 hours,indicating that the lung-derived strain HN202101 had higher proliferation ability on lung-derived A549 cells.While on U251 cells,the virus titers of HN202102 were higher than those of HN202101 at 12,24,36,and 48 hours,indicating that the brain-derived strain HN202102 had higher proliferation ability on brain-derived U251 cells.These results showed that P3,HN202101,and HN202102 had various proliferation abilities on different cell lines.This experiment laid the foundation for the preparation of high titer antigen and the development of JEV vaccine.3.Distribution of two isolates in different mice tissuesThe isolated strains,HN202101 and HN202102 were both intraperitoneal injected into mice with 300μL suspension(the virus titer was both 10~7 PFU/m L).Typically clinical symptoms were both observed in HN202101 infected mice and HN202102infected mice.Histopathological studies revealed that myocardial degeneration,epicardial fracture,and granular degeneration were observed in heart tissue.No significant lesion was observed in liver tissue.Increasing macrophage infiltration was observed in spleen tissue.The epithelium of the lung alveolar had degeneration,swelling,and necrosis.The alveolar wall was widened and had mild congestion.The bronchial mucosa cells were necrotic and detached.Glomerular capillaries and glomeruli were constricted.The epithelial cells were necrotic and detached.Inflammatory responses of blood vessels were observed in brain tissue.The distribution of HN202101 and HN202102 in the heart,liver,spleen,lung,kidney,and brain tissues of mice was detected by RT-q PCR.At day 2,JEV levels in heart,liver,spleen and kidney tissues were significantly different,while JEV levels in lung and brain tissues were not significantly different,and JEV levels in all tissues of mice in the brain isolate infection group were higher than those of the lung isolate.At day 5,JEV levels in heart,lung and kidney tissues of mice infected with HN202101 and HN202102 strains were not significantly different,and JEV levels in liver,spleen and brain tissues were significantly different,and JEV levels in all tissues of mice in the brain isolate infected group were higher than those in the lung isolate infected group,except for lung tissues.The results showed that the levels of JEV in various tissues of mice were different between the two isolates,which is important for the study of the extent of damage to peripheral and brain tissues and clinical diagnostic studies after JEV infection in animals.On day 1,3,and 5,the blood of infected mice was collected to detect the JEV RNA levels by RT-q PCR.On day 1,3,and 5,JEV was both detected in the blood of HN202101infected mice and HN202102 infected mice.It indicated the mice had viraemia.