Sequence Analysis Of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 And Antigenic Epitopes Mapping Of Envelope Protein

Japanese encephalitis virus(JEV), being a member of the family Flaviviridae, is usually infected to the human body and animal through mosquitoes and then may cause acute viral encephalitic and neurologic disease. Therefore, it is commonly regarded as a serious public health problem, with JEV being the infectious agent in many domestic and wild animals, especially with swine(viral associated abortions). Pig is considered the main vertebrate host and represents an important amplifier and reservoir for JEV. Therefore prevention of JE in swine can benefit for swine production and health of human being. Hence, it is essential to prevent swine JE, which is useful for swine production as well as health of human being.Four cDNA fragments covering the complete genome of Japanese encephalitis virus(JEV) strain SA14-14-2-B20 were cloned and sequenced respectively, and its complete genomic sequence was assembled. The genome of SA14-14-2-B20 strain consisted of 10,977bp, and contained a large open reading frame encoding a polyprotein of 3,432 amino acids, which was flanked by untranslated regions(UTRs), 95 bases at the 5'-end and 583 bases at the 3'-end. Compared with the sequence of other six SA14 derival viruses, the homology of the nucleotide sequence and deduced amino acid sequence were both more than 99%, the mutation sites were located in different regions. And at some sites, SA14-14-2-B20, SA(A), and SA14 were same at nucleotide and amino acid level, but different from SA14-14-2. It was found that there was a G insertion near the middle of the 3'UTR at 10,701 site. In comparison with 3'UTR of 31 fully sequenced JEV genomes, results suggested this site might be vulnerable to variation. Phylogenetic analysis of SA14-14-2-B20 and other 31 fully sequenced JEV genomes, would be useful for studying the genetic relationships among JEV isolates. The sequence of this gene will be helpful to discover the pathogenic and attenuated mechanism, furthermore helpful in quality control of the vaccine manufacture, providing molecular basis on supervising of the revert mutation. In addition, the cDNA clones of JEV virus genome will be useful for the study of the infectious clone.The envelope glycoprotein, a major structural antigen, is associated with viral attachment, fusion, hemagglutination, cellular tropism, viral virulence, and induction of protective immune response. In this study, three truncated fragments, EF1(1～291aa), EF2(292～402 aa) and EF3(403～500 aa) of the envelope glycoprotein were expressed by fusion with GST in a pGEX-6p-1 vector respectively. ELISA and Western blot results both demonstrated that the 1~402aa fragment of the E protein was the antigenic dominant region. To map the antigenic epitope of this antigenic dominant region, a set of 52 partially overlapping fragments spanning the 1~402aa fragment were synthesized and fused with GST and expressed respectively. Eleven antigenic epitopes, E1(1～16 aa), E10(73～88 aa), E11(81～96 aa), E19 (145～160 aa), E33(257～272 aa), E39(305～320 aa), E45(353～368 aa), E47(369～384 aa), E48(377～392 aa), E49(385～402 aa) and E63(373～388 aa) were identified by ELISA and Western blot analysis, respectively. Furthermore the identified epitope were subdivided, nine antigenic epitopes, E1(1～16 aa), E10-4(77～84 aa), E11-2(83～94 aa), E19(145～160 aa), E33(257～272 aa), E39-2(309～316 aa), E45-3(359～362 aa), E48-1(377～384 aa) and E49(385～402 aa) were ascertained ultimately. Immunization of mice with each of the nine antigenic epitope-fused proteins revealed that all nine proteins could elicit short peptide specific antisera. E10(73～88 aa), E39(305～320 aa) were linearity neutralizing epitopes through neutralization test verification. E10 has early been reported, E39 has been affirmed recently. The identified epitopes were synthesized as polypeptides. Elisa plates were coated with mixing synthesized polypeptide to detect porcine sera. Identification of JEV envelope protein antigenic epitopes may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for JE, and further structural and function analysis of envelope protein.