Passage And Complete Genome Sequence Analysis Of Japanese Encephalitis Virus Isolated From Pig

Japanese encephalitis virus (JEV) is a member of Flavivirus genus of theFlaviviridae family. The virus circulates in humans, mosquitoes, and animals to causeserious viral encephalitis in humans named as Japanese encephalitis (JE) with a highdeath rate and most patients leaving neurological sequelae. There are more than onebillion of people living in the endemic regions of the JEV, which are expandingconstantly in recent years. JE has become a public health problem of global concernand Asia is the main endemic region. There are no effective drugs for the therapy ofJEV infection and the most important controlment is to use vaccine immunization toprevent clinical cases.A lot of genetic information of JEV can be obtained by sequencing the viralgenomes. The complete genome of JEV is about11kb long, which belongs to thepositive-strand RNA viruses. There is a type I cap structure in5’-end of the viralgenome, a CU-OH structure in the3’-end while without poly A tail. JEV genomeencodes a single open reading frame (from sites96to10394) encoding a precursorpolyprotein (composed of3432amino acids) and can translates three structuralproteins (C, M&E) and seven non-structural proteins (NS1-NS5). The structuralprotein E is the most important structural protein with a plurality of epitopes and playsan important role in invading the host. Changing the gene sequences of protein E maylead to amino acid changes and further, among which the E176, E138and other sites(such as E177, E279and NS2b-63) may directly or indirectly relates to the virulenceof the virus. To date, genome sequencing of a total of130JEV Chinese isolates hasbeen completed, of which63genomes have been published in the GenBank data base.However, only five of them are isolated from swine, and the rest are most frommosquito and human. Thus, it is meaningful for understanding the JEV epidemiologyand the genome characteristics more directly through the genome-wide sequencing ofJEV isolates from pigs.In the present study, the genetic stability of E gene of JEV isolates BSF.ZZ-1andBSF.ZZ-3passaged in BHK-21cells was first studied. The results showed that the E gene was becoming stable after60passages in BHK-21cells. After serialsucultivation, stable amino acid mutation occured at the sites E21(Fâ†'L), E200(Tâ†'A), E244(Gâ†'E), E279(Nâ†'S) and E426(Gâ†'D) in isolate BSF.ZZ-1and sitesE244(Gâ†'E), E255(Fâ†'L), E285(Mâ†'L), E368(Lâ†'S) and E97(Nâ†'S) in isolateBSF.ZZ-3. No mutations were observed at part of the virulence correlated aa sites,such as E107, E138, E176, E177and E315, similar to those of the vaccine strainSA14-14-2. However, mutations at the virulence correlated amino acid sites of E279（Mâ†'Kâ†'M）of BSF.ZZ-3happened repeatedly. Whether these mutations werecorrelated with the virulence and adaptability of JEV to host cells needed to be furtherstudied. Sequencing of the viral genomes showed that the length of both of thecomplete genomes of BSF.ZZ-1-p60and BSF.ZZ-3-p60were10977nt. Compared tothe vaccine strain SA14-14-2, the homologies of nucleotide sequences ofBSF.ZZ-1-p60and BSF.ZZ-3-p60were99.6%and95.9%, and the homologies ofamino acid sequences were96.8%and85.0%, respectively. The phylogenetic analysiswith other30reference isolates that covered all the five JEV genotypes, indicated thatBSF.ZZ-1-p60and BSF.ZZ-3-p60located in a same evolutionary branch andbelonged to the genotype III.