| The Japanese encephalitis virus(JEV), which belongs to the family Flaviviridae, mainly infects the central nervous system of humans and equines, and causes stillbirths in swine. Japanese encephalitis virus(JEV) was transmitted to humans by persistently-infected mosquitoes and maintained in infected vertebrate reservoirs, especially in viremia-amplifying hosts such as swine and avian. In humans, JEV mainly causes acute encephalitis with fatality rates ranging from20%to as high as50%and most of the survivors display persistent neurological or psychological sequelae. In domestic animals such as swine and horses, JEV infection causes stillbirth, encephalitis and even death. Swine is an important host, amplifier and infection sources of Japanese encephalitis virus (JEV) in the natural environment. On the other way, the Japanese encephalitis(JE) is one of the important diseases of Swine, which can cause abortion and stillbirth of Sows, orchid’s of Boar and nervous symptoms of piglets. In the view of economics, the pig industry suffered serious lossed from the viral infection as a result of the reproduce failure in sows and death in piglets.In recent years, the region of prevalence of JE is exaggerating and the aera of pathogenesy is raising in China. The WHO has internalized JE to the diseases which were controlled intensively. Until now, Many experiments demonstrated that there are many correlations between Japanese encephalitis of Swine and Japanese encephalitis of Human. The Prevention for Japanese encephalitis of Swine is a very important intervention for human’health. So, separating Japanese encephalitis virus in pig-farm and analyzing the characters of the isolates is very stringent. According to the features of prevalence and propagation of the Japanese encephalitis virus, the new methods of separating JEV were adopted in this Experiment. New isolates were analyzed in details included Molecular biological characteristic and features of prevalence. The contents of the paper contain three parts as following:1The isolate of Japanese encephalitis virusMosquitoes were collected and killed by freezing in liquid N2for transport and storage. Approximately100mosquitoes were homogenized in2mL of cell culture media without FBS. Homogenate were centrifuged at4℃,12,000rpm for20min. Supernatant fluid was filtered using0.22μm filtration membrane. Serum was separated from swine blood samples. Serum was diluted, filtered and inoculated on the monolayers of BHK-21cells. Swine sperm samples were performed three freeze/thaw cycles. Then, centrifuged at4℃,12, OOOrpm for20min. Supernatant fluid was filtered with0.22μm filtration membrane. The Filtrates were inoculated on the monolayers of BHK-21cells. Following adsorption for1.5h, filtrates were discarded and fresh medium was supplemented with2%fetal bovine serum and antibiotics.The cells were incubated in a5%CO2incubator at37℃. Incubating for2-3days, culture supernatant fluids were harvested by twice freeze/thaw cycles and were inoculated on new monolayers of BHK-21cells. Virus strains isolated were passaged5times consecutively, Culture were harvested and stored at-40℃for use.2The identification of Japanese encephalitis virus from isolatersIndirect Immunofluorescence Assay (IFA) was performed in a96-well plate. BHK-21cells monolayer grown on96-well plate was fixed with chilled acetone/alcohol (6:4) for10min at RT about20h after virus inoculation. After washing out excess unbound reagent with PBS, the fixed cells were incubated with JEV specific antibody at37℃for1h in humid chamber, Washing three times with PBS, and then stained with FITC conjugated anti-rabbit IgG. After washing in PBS, the cells were examined by fluorescence microscope.Identification for JEV by RT-PCR.Viral RNAs was extracted from Culture supernatants using the TRIZOL reagent according to the manufacturer’s instruction. The precipitated RNA was dissolved in DEPC-treated water and cDNA fragments were synthesized using M-MLV reverse transcriptase with specific oligonucleotides according to the manufacturer’s instruction. The partial M gene were amplified by the use of a rTaq DNA polymerase with primers3Molecular biological characteristic analyses of Japanese encephalitis virus isolatesTo characterize Japanese encephalitis virus (JEV) strains recently prevalent in China, JEV separation was performed in pig farm from2008to2009in the southeast of China. Fifteen new JEV isolates were obtained and compared with previous isolates from China and other Asian countries. All of the new isolates were classified into genotype3by nucleotide sequence analysis of the E gene. Nucleotide homology of the E gene sequences were similar by between99.0%and99.9%when15new isolates compared with each other; However, while new isolates compared with vaccine strain P(3)(1949), except for Mo/Zhenjiang/1/2009strain and Mo/Jintan/9/2008strain being higher Nucleotide homology99.7%and99.6%, the others were between97.3%and97.9%. These results suggested that JEV strains isolated from the same geographic area at close time points were very similar, but that emerge of genetic variation occurred among JEV strains from diverse regions or those isolated at different time points in the same region. The present study indicated that:(ⅰ) Japanese encephalitis virus (JEV) strains recently prevalent are genotype3in China;(ⅱ) The prevalence of JEV strains is very general and widespread the natural world; and (ⅲ) New JEV isolates are included in the same subcluster within genotype3(G3), but there are also a wide range of diversity when compared with JEV strains isolated previously from China;... |
PDF Full Text Request