Construction Of JE/DV2Chimeric Virus With JEV Vaccine Strain SA₁₄-14-2

The dengue virus (DV), a member of the family Flaviviridae and genus Flavivirus, includes four antigenically distinct serotypes (DV-1to DV-4). Dengue virus is the most important arboviral pathogen in tropical and subtropical regions throughout the world and responsible for dengue fever, dengue hemorrhagic fever and dengue shock syndrome. WHO estimates that2.5billion people at the risk of dengue and25000deaths worldwide every year. So far, no licensed vaccine is currently available for dengue virus.The development of infectious clone technology opened new avenues for flavivirus vaccine research. Within the family Flaviviridae, Japanese encephalitis virus (JE virus) and Dengue virus are both enveloped virus with a single-stranded positive-sense RNA genome of approximately11kb. The genome has only one open reading frame encoding for a polyprotein which produces three structural proteins and seven nonstructural proteins(5’NCR-C-PrM/M-E-NS1-NS2B-NS3-NS4A-NS4B-NS5-NCR3’). PrM/E,The membrane proteins, M and E, are involved in inducing neutralizing antibodies. The non-structural proteins and NCR are involved in virus replication and protein translation. The safety of Japanese encephalitis virus live-attenuated vaccine, strain SA14-14-2, are favorable. In this study, the full length cDNA clone of SA14-14-2was used as a genetic background for construction of a chimeric virus containing the structural proteins prM and E of Dengue virus, serotype2.Based on the JEV infectious clone pBRF\, two replicons of JEV were constructed. One’s prM/E gene was deleted completely (named as pFull△prM/E), the other’s prM/E gene was deleted partially (213bp of C terminal of E gene which may be the signal peptide sequence of NS1protein was reserved; named as pPartial△prM/E), and the deleted parts were replaced with the multiclone sites. NS2B-63、NS3-105、NS4A-225was known as the possible locus which located in NCR for attenuation of SA14-14-2. Results of sequence analysis showed that all of the three locus didn’t mutated in those two replicons vector.Replicons RNA were transfected into BHK-21cell. After24h,48h,72h,96h, Real-time PCR was used to identify replication ability. The results showed that after transfected intoBHK-21cell, as time went by, the quantity of RNA increased. Yellow Fluorescent Protein(YFP) gene was inserted into the multiclone sites of those two replicon vectors. Was After transfectedinto BHK-21cells, the expression of YFP was detected by the fluorescence microscopy and flow cytometer. Increasing trend of fluorescent signal and the rate of YFP positive cell was observed andtested. The results indicate that Full△prM/E Replicon andPartial△prM/E Replicon have the ability to duplicate itself and express the foreign protein.The prM/E gene of DV2was cloned into pFull△prM/E and pPartial△prM/E respectively. In the latter,213bp of C terminal of E gene of DV2was deleted. The chimeric plasmid was named as pJED2and pJED2-1770. The constructed chimeric plasmids were linearized and then were transcripted in vitro. RNA transcript was transferred into BHK-21cells via electroporation. Five to seven days later, Cytopathic effect (CPE) could be observed. Chimeric viruses rescued were named as JED2V and JED21770V. Supernatant of the first passage chimeric virus was passaged to BHK-21cells and C6/36cells respectively. CPE could be observed after C6/36cells were infected with chimeric virus for about4days. To determine the nucleotide sequence of chimeric virus, cDNA fragments were amplified by RT-PCR from viral RNAs extracted from virus-infected supernatant. The results of DNA sequencing show that the prM/E gene come from DV2and the NS1gene come from JEV. Indirect immunofluorescence analysis and western-blotting results demonstrate that the C6/36cells infected with the chimeric virus can react with DEN2-EⅢ-specific immune serum. However, the chimeric virus could not passage in BHK-21cell. In order to identify the immunogenicity of chimeric virus, Balb/C mice were inoculated intraperitoneally with chimeric viruses. Four weeks after been inoculating, DV2E protein specific IgG antibody was detected in mice serum, and titer of neutralizing antibody reached nearly1:10. Furthermore, in order to know the safety about chimeric viruses, chimeric viruses, DV2and JEV were diluted, from105to101TCID50/200ul. Sucking mice were injected intracerebrally with both viruses respectively at each dose level, and then were observed for14days. Survival curve were compared between different group by Log-rank test. The results showed that survival rate of the sucking mice in two chimeric virus groups were lower than those of JEV group(p<0.05). Compared with DV2group, survival rate of the sucking mice in two chimeric virus groups were lower than those of DV2group(p<0.05) in some levels of dose. However, in some levels of dose there were no statistical significance between the chimeric virus group and DV2group. Results of sucking mice experiment about neurotoxicity indicated that the rescued chimeric virus were not attenuated.In sum, two JEV replicons vector were constructed successfully, which lays the groundwork for the further use of the JEV vector. The chimeric viruses rescued successfully make us acquire some rewarding experience to develop the vaccine of DV in future work.