Construction Of Porcine Parvovirus-like Particles Harboring Epitopes Of Japanese Encephalitis Virus And Generation Of Japanese Encephalitis Virus Infectious Clone

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, belongs to flaviviruses ofFlaviviridae family. It is a positive strand RNA virus, whose genome is roughly11kb. The genome ofJEV just has one ORF, which encodes three structural proteins, and seven non-structural proteins. JEVinfection will cause Japanese encephalitis (JE) which is a mosquito-borne zoonosis. Porcine parvovirus(PPV) is one of the main pathogenies of porcine reproductive disorder. PPV can cause abortion,infertility, stillbirth, deformities, mummy foetus of sows, which widely exists in pigs and results inseverely lose to pig industry. Up to now, there are no effective drugs to cure JE and PPV disease,therefore it is meaningful to research the virus pathogenic mechanism and develop the safety, efficacyvaccines.This research has two parts. In the first one, development of Virus-like particles of PPV:VLP (JEV)vaccine. The gene fragments of JEV encoding B-cell epitopes (aa150-156, aa386-399, aa307-316) andT-cell epitopes (aa436-445, aa60-68) were fused with the VP2gene of porcine parvovirus (PPV) andinserted into plasmid pCold-Ⅰto construct recombinant plasmid pMEP-VP2. The resulted plasmid wastransformed to E.coli BL21to express the recombinant protein. Virus-like particles of PPV:VLP(JEV)were able to self-assemble from the purified protein in vitro. The mice were immunized with theseparticles to study immunogenicity of this particles vaccine.In the second part, the construction of JEV infectious clone. The fragmented cDNA weresynthesized from JEV RNA genome by reverse transcription-PCR, and subsequently cloned intoplasmids respectively. A full-length JEV cDNA were generated by overlapping PCR, then JEV RNAwas synthesized in vitro and tranfected into BHK-21cells to gain the recovered virus. At last, therecovered virus was confirmed through the infective assay, RNA genome and viral proteins analysis.The contents of the paper are as follows:1. Porcine parvovirus-like particles harboring B-cell and T-cell epitopes of JEV. According toliteratures and the construct strategies of multi-epitopes vaccine, JEV encoding B-cell epitopes(aa150-156, aa386-399, aa307-316) and T-cell epitopes (aa436-445, aa60-68) were picked. In addition,these epitopes were separated by glycine and proline to facilitate the submission of antigen. To improveimmunogenicity of JEV epitope-vaccine, the gene fragments of JEV were fused with the VP2gene ofPPV and inserted into plasmid pCold-Ⅰ to construct recombinant plasmid pMEP-VP2. The resultedplasmid was transformed into E.coli BL21to express the recombinant protein, which was detected bywestern blot. The result showed that the recombinant protein can be distinguished by anti-PPVpolyclonal antibody and anti-JEV polyclonal antibody. Besides, virus-like particles of PPV:VLP (JEV)was able to self-assemble in vitro from the protein, which could be observed by electron microscopy.2. Immunogenicity of the porcine parvovirus-like particles harboring B-cell and T-cell epitopes ofJapanese encephalitis virus in mice model.4-6weeks old female BALB/c mice were randomlyseparated into6groups,10mice each group, mice were intraperitoneally (ip) immunized with JEV-MEP,PPV-VP2protein, PPV:VLP(JEV), JEV inactivated vaccine and PPV inactivated vaccine three times at2weeks intervals, and with PBS control. Level of antibody and CTL were detected to evaluate humoral immune and cellular immune responses of PPV:VLP(JEV). The results showed immunization withPPV:VLP(JEV) was able to induce stronger JEV humoral, cellular immune responses and PPV cellularimmune responses.3. The amplification of JEV complete cDNA and the generation of the recombinant JEV. As animportant viral zoonosis pathogen, JEV reverse genetics, particularly infectious clone, is a valuable toolwith applications to many areas of viral research including the generation of vaccine candidates andstudy on the virus pathogenic mechanism. However, this technology is sometimes insufficient for theconstruction cDNA clones as the genome sequences and/or encoding proteins of some viral agents maybe toxic to the host cells used for cloning. To circumvent this problem, we developed a polymerasechain reaction (PCR)-based protocol for generating a complete Japanese encephalitis virus (JEV) cDNA.The fragmented cDNA were synthesized from JEV RNA by reverse transcription-PCR, andsubsequently cloned into plasmids for use as templates for JEV cDNA synthesis. The fragmented cDNAwere amplified by PCR to generate a full-length JEV cDNA. Using this cDNA as a template, JEV RNAwas synthesized in vitro and transfected into BHK-21cells, and the cloned virus was rescuedsuccessfully, which can produce apparent CPE in BHK-21cells infected by second passage JEVrecovered virus. The infectivity of the recovered virus was verified by observation of CPE, indirectimmunofluorescence assay and RT-PCR. The recovered virus and the parental virus have the similarvirus-induced plaque morphology and the recovered virus grows fast compared to the parental virus.In conclusion, porcine parvovirus-like particles harboring B-cell and T-cell epitopes of JEV caninduce humoral and cellular immune responses in mice. So the research may lead to a new approach forthe development of JE-PPV vaccine. The method to gain the JE recovered virus based on a polymerasechain reaction (PCR)-based protocol for generating a complete JEV cDNA is a valuable tool to researchviral replication, transcription, viral assembling, the interaction between the virus and cells and themarker vaccine.