Isolation, Characterization And Phylogenetic Analysis Of The Field Japanese Encephalitis Virus Isolates From Boars Farm In Henan Province

Japanese encephalitis (JE) is a viral zoonosis caused by Japanese encephalitis virus (JEV). JEV infection causes hominine nervous system disorder and lead serious consequences, which also cause porcine reproductive failure, such as abortion, stillbirth and orchitis. Therefore, it is one of the most important reproductive disorder diseases. JEV is a mosquito-borne flavivirus and pigs are the most important amplifying and maintenance hosts. In this study, a total of 19 boar seminal fluid (BSF) of clinical diseased boars from the large-scale pig farms distributed in Henan province was collected for virus isolation and characterization. Phylogenetic analysis was simultaneously performed for studying the current molecular epidemiology of JEV in Henan province. This study provides a scientific basis for further control of JE in large-scale pig farming.According to JEV M and E gene sequences previously reported in GenBank database, two pairs of specific primers were designed using Primer Premier 5.0 based on the conserved homologous sequences. Firstly, samples of boar BSF containing JEV virus were identified by the method of RT-PCR. Among all the detected BSF samples, two of them were identified positive. Animal experiments also showed that two of the supernatant of seminal fluids caused typical clinical nervous symptoms or death of suckling mouse at about 10 days post intracellular inoculation and the JEV M and E genes can be similarly amplified from the brains of diseased suckling mouse.The virus infections of the 2 new isolates on BHK-21 cells were detected by indirect immunofluorescence assay (IFA) using JEV-specific monoclonal antibody at 60 h post-infection. This indicated that these two isolates are JEV virulent strains, namely as BSF.ZZ-1 or BSF.ZZ-3, respectively. Both of the two JEV isolates were propagated on BHK-21 cells for three passages. Virus titration showed that the virus titers of the first passage of cell cultures were low, which are 103.2±0.2 TCID50/mL and 101.6±0.1 TCID50/mL for the isolates BSF.ZZ-1 and BSF.ZZ-3, respectively. After three passages on BHK-21 cell line, virus titers of BSF.ZZ-1 and BSF.ZZ-3 obviously increased to 104.5±0.1 TCID50/mL and 104.2±0.2 TCID50/mL, respectively.Using bio-software MEGA 4.0, two phylogenetic trees based on the M or E gene sequences of the new isolates together with other 17 reference JEV strains belonging to four distinct subtypes were constructed. The results showed that all the 19 JEV isolates were classified into four clusters and the new isolates, BSF.ZZ-1 and BSF.ZZ-3, are adjacent to each other and both belong to the subtypeⅢ, together with the isolates of SA14, SH-3, Ha-3 and the vaccine strain SA14-14-2 which were previously isolated in China.