Isolation And Identification Of Japanese Encephalitis Virus From Swine And Construction Of Pseudotyped Virus Based On E Gene

Japanese encephalitis (JE) is caused by Japanese encephalitis virus (JEV). JEV belongs to a mosquito-borne Flavivirus within the family Flaviviridae and is mainly prevalent in the tropical and subtropical regions. China is a high incidence of JE, nearly 80% of globally reported cases occur in China. When the pigs were infected with JEV, the breeding pigs appear reproductive failure and the piglets appear neurological symptom. Swine is an important reservoir and overwintering host of JEV and plays a critical role in the human epidemic encephalitis. So the monitoring and prevention of pig's JE is helpful to prevent and control the human JE. In this study, we isolated two new JEV strains from piglets appeared the encephalitis symptom in Yunchen, Shanxi. Tts molecular evolution relations were analyzed and the map of distribution on JE was constructed in our country different area. These results will be useful for us to further prevent and control pig and human JE. In the meantime, pseudotyped viruses based on JEV E gene were constructed to investigate the functional domain of the E protein which interacted with cell receptor. It provided basic informations for future researching on the invasion mechanism and development of antiviral drugs to JEV.1,Isolation and identification of Japanese encephalitis virus from swineThe piglet brain tissues were collected from a pig farm in Yunchen, Shanxi which was suspected to be infected with JEV. Seven samples were detected by RT-PCR and immunohistochemistry. JEV specific C/prM 240 nucletise segment could be amplified from all seven samples and beglon to the genotype I JEV through sequencing and phylogenetic analyses (GenBank accession numbers:HQ893545～HQ893551).Two new JEV strains were isolated from the seven samples by BHK-21 or C6/36 cells and named as SX09S-01 and SX09S-02. Then the viruses were multiplied in suckling mouse brain and the virulent value was determined. There were approximate 40～60nm virus-like particles in the cytoplasm of Vero cells infected with SX09S-01 or SX09S-02 by electron microscope.In order to futher analyze its molecular characterization, the full-length genome of SX09S-01 were cloned and sequenced. Resluts indicated that it contains 10965 nucleotides in length and encoded a 10299-nucleotide with single ORF (3432 amino acid residues) flanked by a 96-nucleotide 5'-UTR and a 570-nucleotide 3'-UTR. There is an additional adenine (A) nucleotide insertion comparing with other genotype strains. A 13-nucleotide deletion immediately downstream of the translation stop codon was determined among the 3'-UTR of the SX09S-01 strains. The complete genomic sequence of SX09S-01 strain was compared with other 21 JEV strains available in GenBank. The SX09S-01 strain shares the highest nucleotide and amino acid sequence homology with XJ69 strains. Eight amino acid residues of E protein play a critical role on the viral neurovirulence. The E proteins of these two viruses had some typical characteristics of strong virulent JEV. To evaluate the neurovirulence and neuroinvasiveness,4-week-old female Balb/c mice were inoculated intracerebrally and intraperitoneally, and the neurovirulence and neuroinvasiveness of the SX09S-01 and SX09S-02 strians were 0.62,5.87 and 0.77,5.77, resepectively. Compared with the P3 strain, their neurovirulence and neuroinvasiveness were little lower. In addition, we used the sera of the genotypeⅢto neutralize the newly isolated virus belonged to genotype I in order to evaluate the efficiency of the current JEV vaccine. The result indicated that the genotypeⅢsera can neutralize the genotype I JEV and there were no significant differences.To analyze the epidemiology of JEV in China, we investigate the evolvment of genotypes of JEV and distribution in different regions. The genotype I JEV appeared after the 70's. Especially 2005 later, the genotype I JEV assumed the trend of escalation, and the frequency of occurences was higher than the genotypeⅢJEV. JEV had occurred in all provinces except the Qinghai province in China. There are genotype I and genotypeⅢJEV strains in the same province, such as Shanxi, Sichuan, Liaoning, Yunnan, Guangxi, Hubei, Henan, Shaanxi, Guizhou, Fujian and Shanghai. Genotype I only populars in Beijing, Xinjiang, Heilongjiang, Guangdong and Taiwan only popular genotypeⅢ, Gansu, Shandong, Zhejiang and Jiangxi..2,Construction of pseudotyped virus based on JEV E geneUp to date, the cellular receptors and the invasion mechanism of JEV are still unclear and E protein is a key protein in interaction with cell receptor. To study the functional domain of the E protein which interacted with cell receptor, host tropism, and antiviral drugs, pseudotyped virus based on JEV E gene were constructed with human immunodeficiency virus type 1 (HIV-1) vector system. E gene and its different fragments (E1437 (deletion one transmenbrance domain), E1299 (deletion both transmenbrance domains), E12 (domainⅠandⅡof E gene) and E3 (domainⅢ), were amplified by PCR. All the E gene fragments were ligated into the envelope expression plasmid vector, then harvesting the recombinant lentivirus by cotransfected 293FT cells with transfer vectors and helper plasmids.48 hours after cotransfection, the green fluorescence could be observed in the 293 FT cells, and all the E genes were expressive by the detection of western blotting in the 293FT cells.Mammalian cells 293 FT and BHK-21 were transduced with the recombinant lentiviruses. Lentiviral titers were determinated by flow cytometry. Fluorescence-positive signal was only detected on 293 FT cells,but all of the recombinant lentivirus had very low fluorescence, in which E12 and E3 genes recombinant lentivirus had slightly higher fluorescence, were 1.65±0.37×103and 1.32±0.95×103, respectively. But on BHK-21 cells all the samples were not fluorescence-positive signal.