The Japanese Encephalitis Virus Of Genome CDNA Clone Of Newly Isolated From China

The Japanese encephalitis virus belongs to the Flaviviridae, Flavivirus. In accordance with the virus genome or part of the genome sequence of the Japanese encephalitis virus, it can be divided into five genotypes (Ⅰ,Ⅱ,Ⅲ,Ⅳ, and Ⅴ).The JEV GZ56strain was first to be isolated from cerebrospinal fluid of encephalitis patients in China in2006. The JEV XZ0934strain was isolated from Culex tritaeniorhynchus, collected from Tibet in2009. JEV XZ0934strain and JEV Muar strain are evolutionarily related, both belonging to the JEV genotypes Ⅴ. JEV GZ56and XZ0934were obtained from two deaths of neonatal rat brain, respectively, which were infected of JEV GZ56and XZ0934. To obtain purified JEV, we carried out plaque purification, and then, the whole genome sequences were obtained using the16fragments of these two purified viruses. Their pathogenic molecular basis was measured and analysed using the genomic sequence features of these two viruses with modern molecular biology theory and techniques. In this study, the JEV XZ0934strain genomic full-length was segmented amplification to four large fragments of ride for two to two stacked. And then, the segmentations were clone to the transformation vector pACYC-MCS which has a multiple restriction enzyme sites. The reverse genetics technology was used to elucidate the pathogenicity of the virus molecular mechanism, the development of recombinant virus vaccine and the development of genetically engineered vector.The full-length genome of the JEV GZ56is10965nt in length and encodes3432amino acids. The molecular evolution analysis of virus genome showed that JEV GZ56and M-28were in the same evolutionary branch. The homology of genome ranged from96.2%to98.6%in nucleotide and from98.2%to99.7%in amino acid respectively when it was compared with selected genotype Ⅰ of JEV strains in Genbank. There were11amino acid divergences in E protein when compared with the JE inactivated P3strain. However, there were14amino acid divergences in E protein when compared with the JE live attenuated vaccine SA14-14-2strain.The full-length genome of the JEV XZ0934is10983nt, coding3432amino acids. The molecular evolution analysis of virus genome showed that JEV XZ0934and Muar were in the same evolutionary branch. The homology of genome ranged from78.8%to 90.7%in nucleotide and from90.0%to98.3%in amino acid respectively when compared with selected23JEV strains in Genbank. Compared with the XZ0934and Muar homology of nucleotide and amino acid were90.7%and98.3%, the XZ0934and GZ56were79.4%and91.2%, respectively. There were47amino acid divergences in E protein when compared with the JE inactivated P3strain. However, there were52amino acid divergences in E protein when compared with the JE live attenuated vaccine SA14-14-2strain.Vector pACYC-MCS,2467bp in length, was constructed successfully, and it has ampicillin resistance. In addition, it has23restriction endonucleases at the multiple cloning sites. The JEV XZ0934genomic full-length was segmented amplification to four large fragment by RT-PCR, and these segmentswere2418bp,3002bp,2607bp,2983bp. And sequencing results showed that the total length of the viral genome has been connected to the transformation vector pACYC-MCS, and the pACYC-MCS-XZ0934genome length is13450bp.