Study On Artificial Attenuation And N-Linked Glycan Function Of Japanese Encephalitis Virus NJ2008 Strain

Japanese encephalitis virus (JEV) belongs to the genus Flavivirus, and the genus Flavivirus include many clinically important pathogens, such as Dengue virus (DENV), West Nile virus (WNV), et al. Japanese encephalitis virus (JEV) mostly causes infection of the central nervous system in humans and equines and stillbirths in swine. The virus is zoonotic, cycling between birds and mosquitoes, and is transmitted to humans by infected mosquitoes. Since swine serve as a reservoir and amplifier of the virus, Thus mass vaccination of swine can prevent the disease in swine and help to prevent JE epidemics in humans. As this stage, no treatment effects for JEV, and it is particularly important to prevent the disase. Vaccines are the best way to controlling the disease, and seeking a stable and secure JE vaccine of swine has become the main research directions for JEV infection in recent years.Therefore, the development of a swine vaccine against JEV is a high priority, as it could help prevent epidemics in humans.The advent of reverse genetics (infectious clone) has revolutionized the study of RNA viruses. It makes it possible to manipulate the RNA viruses through its complementary DNA copy, imitating the real viral life cycle, to explore the viral genome structure and function and virus-host interaction, and in-depth study the nature of RNA viruses and develop new products. Since application of this technology to JEV, great progresses in pathogenesis, molecular expression and regulation mechanism, cell growth characteristics and virulence determine gene of JEV had been made. In this study, two full-length mutant cDNA clone of JEV strain NJ2008 was constructed (E138 and N154 mutant cDNA clones). Potency of E138 and N154 mutant cDNA clones as useful tool for studying the attenuated live vaccine of the Japanese encephalitis virus for swine was evaluated. Meanwhile, mutation of the putative N-glycosylation site N154 in the E protein of JEV should be further investigated as a potential DNA vaccine against JEV. All studies were listed as below: 1 Genomic Sequence Analysis of Japanese Encephalitis Virus Strain NJ2008 Isolated from SwineThe swine Japanese encephalitis virus (JEV), strain NJ2008, was isolated from brain tissues of aborted fetuses of sow in Nanjing, China in 2008. In the present study, we determined the complete nucleotide sequence of the NJ2008 strain using RT-PCR. The10,961bp genome of strain NJ2008 has a 16 nucleotide deletion in the 3’ non-translated variable region. Phylogenetic trees based on the E and complete gene shows that the NJ2008 strain is most closely related to JEV genotype III viruses. Nucleotide sequence comparison of the NJ2008 strain genome with other 32 complete JEV genomes revealed nucleotide homology ranging from 99.4%(JaGAr01 strain) and 88%(KV1899 strain) and with other 45 JEV E genomes revealed homology ranging from 99.2%(JaGAr01 strain) and 87.7%(KV1899 strain). Amino acids sequence comparison of the NJ2008 strain genome with other 32 complete JEV sequence revealed nucleotide homology ranging from 99%(JaGAr01 strain) and 89.1%(KV1899 strain) and with other 45 JEV E sequence revealed homology ranging from 99.5%(JaGAr01 strain) and 89.7%(KV1899 strain). In comparison with other 16 additional swine isolates, we found that the substitution of NJ2008 strain at E-309 (Tyr-Ger) was unique, and within amino acid residues 307-309 that constitute a linear B-cell epitope in the E protein.2 Construction and Identification of Infectious Full-length cDNA Clones of Japanese Encephalitis Virus Strain NJ2008A full-length cDNA clone of JEV strain NJ2008 was constructed. Four cDNA fragments (J1-J4) were synthesized from genomic RNA through RT-PCR to cover the complete JEV genome. SUP was obtained by overlap PCR of Jl and J2, and HYPO was obtained by overlap PCR of J1 and J2. Then SUP and HYPO were cloned together into plasmids to yields 5’and 3’-halves of genome of NJ2008 strain, respectively. The 5’and 3’-halves of genome were assembled into plasmid pBlueseriptⅡSK to form the full-length cDNA clone of JEV (pSK-SUP, pSK-HYPO). The complete JEV cDNA is positioned under the control of T7 promoter elements for invitro transcription. In order to observe the infectivity of RNA from the full-length cDNA clones, capped RNA transcript synthesized from the linearized full-length cDNA Plasmid by using T7 RNA Polymerase was electroporated into BHK-21 cells and reeovered. The results of IFA used to detect viral protein expression in BHK-21 cells transfected with JEV RNA. No qualitative differences in CPE were observed between parental and recovered viruses, and one-step growth curves were similar for both recombinant and parental viruses on BHK-21 cell.3 Construction and Virus Rescue of an Infectious Clone of Japanese Encephalitis Virus E-138 MutationJapanese encephalitis virus (JEV) mostly causes infection of the central nervous system in humans and equines and stillbirths in swine. Previous studies have demonstrated clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV. A mutant Japanese encephalitis virus strain NJ2008 infectious clone was constructed by a single Glu-to-Lys mutation at aa 138 of the envelope protein, and the resultant E138 mutant full-length cDNA clone of JEV was transfected into BHK-21 cells. After three consecutive passages, the transfected cells detected by indirect immunofluorescent、real-time RT-PCR and plaque assay. Analysis of virial copies showed that the relative amount of viral RNA produced by the E138 mutant was lower than that of the WT virus, and the E138 mutant produced plaques smaller than those formed in the cells transfected with WT-derived RNA. The immunofluorescent analysis showed that the number of immunofluorescent of E138 mutant lower than that of the WT virus. Mutant of JEV E138 resulted in a marked reduction in pathogenicity compared to that of the WT when the virus was administered to mice via a peripheral route. Therefore, E138 mutant infectious clone was a useful tool for studying the attenuated live vaccine of the Japanese encephalitis virus for swine.4 N-Linked Glycosylation Site in the Japanese Encephalitis Virus Envelepe Protein is Associated with Virus Particle Release, Subcellular Localization and Pathogenicity in MiceJEV strain NJ2008 envelope protein (E) contains a single N-linked glycosylation site, at Asn-154. N-linked oligosaccharide side chains in flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here we report the effects of mutagenizing the N-linked glycan site on JEV E protein on infectious virion particle release, cytoplasmic localization and pathogenicity in mice. Elimination of JEV E protein N-glycosylation resulted in a 2-fold reduction in infectious virion particle release at 12 and 24 hours post-transfection. However, loss of N-glycosylation did not affect the intracellular levels of viral RNA, and the wild type infectious virus titers were not significantly higher than the N154A mutant at 2 days post-transfection. By indirect immuno fluorescence assay, we can conclude that the protein of N154 A virus accumulated in the cytoplasm. The substitution of Ala for Asn at N-linked glycosylation site had an effect on subcellular localization of the virus or E protein. Mutant of JEV N154 resulted in a marked reduction in pathogenicity compared to that of the WT when the virus was administered to mice via a peripheral route. Our findings indicate that this highly conserved N-glycosylation motif in JEV envelope is crucial for virus release, subcellular localization and pathogenicity in mice. Overall, N154 mutant infectious clone was a useful tool for studying the pathogenicity mechanism of attenuated live vaccine of the Japanese encephalitis virus for swine.5 Construction and Eukaryotic Expression for Mutant of Putative N-Linked Glycosylation Sites in Japanese Encephalitis Virus Premembrane and Envelope ProteinsSwine are an important host of Japanese encephalitis virus (JEV). The two membrane glycoproteins of JEV, prM and E, each contain a potential N-linked glycosylation site, at positions N15 and N154, respectively. We constructed plasmids that contain the genes encoding wild-type prME (contain the signal of the prM, the prM, and the E coding regions) and three mutant prME proteins, in which the putative N-linked glycosylation sites are mutated individually or in combination by site-directed mutagenesis. Vero cells were then transfected with this recombinant plasmid. Recombinant protein was showed to transcript and express effectively by indirect immunofluorescence. To determine whether the mobility shift observed for the JEV prME mutant proteins actually reflected the loss of glycans at the potential N-linked glycosylation sites, we treated or mock-treated immunoprecipitated lysates with PNGase F, which cleaves all types of N-linked glycans, and migration pattern were different between those of wild-type prME and three mutant prME proteins, Western blot showed the protein can interact with JEV monoclonal anibody, proved its biological activity and antigenicity.6 Mutation of Putative N-Linked Glycosylation Sites in Japanese Encephalitis Virus Premembrane and Envelope Proteins Enhances Humoral Immunity in BALB/C Mice after DNA VaccinationIn order to investigate the immunogenic efficiency of recombinant plasmids, fifty of JEV negative mice (4-week-old, female) were randomly divided into five groups with ten of them each group. Mice were inoculated intramuscularly pVAXI-prME、pVAXI-prME-M1、pVAXI-prME-M2 and pVAXI-prME-M3 at a dose of 100μg, and the blank eukaryotic expression plasmid pVAXI was used as controls. Our results indicate that immunizing mice with DNA vaccines that contain the N154A mutation results in elevated levels of interleukin-4 secretion, induces the IgGl antibody isotype, generates greater titers of anti-JEV antibodies, and shows complete protection against JEV challenge. We conclude that mutation of the putative N-glycosylation site N154 in the E protein of JEV significantly enhances the induced humoral immune response and suggest that this mutant should be further investigated as a potential DNA vaccine against JEV.