| West Nile virus disease(WND)is zoonosis with the main clinical symptoms of West Nile fever and West Nile encephalitis,which is caused by West Nile virus(WNV)infection via mosquito bites.Animals and human infections have occurred every year since the outbreak in 1999 New York,USA.According to statistics from the American Animal and Plant Health Inspection Service(APHIS)in 2002,12,527cases of horse infections were diagnosed in the United States,which was the highest in history.Vaccine prevention is one of the most effective measures for the infectious diseases.There are currently four strains of equine West Nile virus vaccines licenced.Three of them are inactivated vaccines,and another one is a live canarypox virus vaccine containing the West Nile virus structural gene(the live vaccine cannot be replicated in the body).Although there are good immune protection effects about the four vaccines,horses are needed to boost once a year for sustained immune protection after the first immunization,and the cost of vaccines and labor will increase accordingly.Therefore,there is an urgent need for a vaccine that can provide long protection from disease via a single or twice immunization.The Japanese encephalitis virus SA14-14-2 vaccine strain(JEV SA14-14-2)with the West Nile virus belongs to the flavivirus genus of the Flaviviridae family.It is an excellent vaccine vector for establishing a live West Nile virus chimeric strain.First of all,JEV SA14-14-2 is highly safe.Since the launch of JEV SA14-14-2 in 1989,this vaccine effectively controlled Japanese encephalitis and showed no obvious adverse reactions in China after more than 30 years of immunization.Second,the genetic structures of the two kinds of viruses are similar.It was relatively easier to construct chimeric West Nile virus vaccine candidate.The mortality of West Nile fever caused by WNV NY99 strain is high,which posed a potential threat to the safety of animals and humans.Although a WNV strain has been isolated from Culex pipens in China in 2011,there has been no reports about animal infection.Therefore,WNV NY99 strain with great potential threat were considered for vaccine study.Objective:To construct the chimeric virus ChiVax-WN01 containing the West Nile virus pr M?E gene,based on the established JEV SA14-14-2 reverse genetic system,and detected the immunogenicity of ChiVax-WN01,and lay the foundation for the development of a cost-effective West Nile virus vaccine candidate for horses.Methods:(1)Genome sequencing for JEV SA14-14-2.In this study,the plaque purification method by BHK-21 cells was used to obtain JEV SA14-14-2 monoclonal plaques after three rounds of purification,and BHK-21 cells were used to propagate the virus.Viral RNA was extracted and reverse transcribed into c DNA,and the c DNA was amplified by PCR into 11 segments,which were then connected to p EASY-Blunt cloning vectors for sequencing.The full-length genome sequence of the JEV SA14-14-2 was acquired after DNAStar and DNAMAN software alignment and splicing of the sequences.(2)Establishment of JEV SA14-14-2 reverse genetic system.The genomic sequence of JEV SA14-14-2 was analyzed by DNAStar and divided into 5 segments(F1?F5)by 4 enzyme digestion sites.The T7 promoter sequence,HDVr sequence,T7terminator sequence and intron sequences were introduced into 5 fragments by primers or synthesis.5 fragments were serially engineered to the p ACYC177 vector to construct an infectious clone,named p FLJEV.BSR T7/5 cells were transfected with p FLJEV by Lipofectamine 3000 to rescue the virus.Finally,the biological characteristics of the rescued and parent strains are identified and compared by plaque morphology,growth curve and viral nucleotides replication.(3)Construction and identification of ChiVax-WN01 vaccine candidate for West Nile virus.Based on the JEV SA14-14-2 reverse genetic system,the pr M?E gene of the JEV SA14-14-2 was replaced with the corresponding gene of WNV NY99 strain(DQ211652)to construct the chimeric infectious clone p ChiVax-WN01.The lipofectamine 3000 was employed to transfect p ChiVax-WN01 into BSR T7/5 cells to rescue the virus.The biological characteristics of the chimeric strain ChiVax-WN01were identified and compared with the parent strain.(4)Safety and immunization study of chimeric West Nile virus ChiVax-WN01.BALB/c mice were as animal model to be injected intraperitoneally with 10~5-10~1 PFU of chimeric ChiVax-WN01 and JEV SA14-14-2 respectively.The survival rate of the mice was observed for 15 days and analyzed the virus neuroinvasiveness.BALB/c mice model were injected intracranially with 100 PFU chimeric ChiVax-WN01 and100 PFU JEV SA14-14-2,and the survival rate of the mice was calculated for 15 days and analyzed the neurovirulence of the virus.In order to identified the immunogenicity of ChiVax-WN01,BALB/c mice were intraperitoneally inoculated with 100 PFU JEV SA14-14-2,100 PFU ChiVax-WN01 and PBS respectively.The plasma,brains,livers,spleens,lungs and kidneys of mice at 1 d,3 d,5 d,7 d and 9 d post inoculation were collected and detected the viral proliferation and distribution in mice by plaque assay.The neutralizing antibody titer of the mice were detected by the microneutralization assay.The number of IFN-?,IL-2 and IL-4 secreting cells in splenocytes was detected by ELISpot.Results:(1)The full-length of JEV SA14-14-2 c DNA was obtained and the Gen Bank accession number is MK585066.(2)The JEV SA14-14-2 infectious clone p FLJEV was sucessfully contructed,transfected and rescued the progeny viurs with no obvious difference in virus titer,growth kinetics and plaque morphology from the parent strain.Intact virions were observed via ultrathin sections of electron microscope.(3)The infectious clone of p ChiVax-WN01 has been successfully constructed,and the progeny virus ChiVax-WN01 has been successfully rescued.Intact virions have been observed under electron microscopy.(4)The neuroinvasiveness experiment showed that the survival rate of mice in the ChiVax-WN01 group was 100%,with no difference from that of JEV SA14-14-2 group and PBS group.The neurovirulence experiment showed that the survival rate of mice in the ChiVax-WN01 group was50%,which presented neurovirulence.The survival rate of mice in JEV SA14-14-2group and PBS group was 100%.It showed that there was no viral titer was detected in the viscera at various time points after ChiVax-WN01 inoculation.The chimeric ChiVax-WN01 can induce neutralizing antibodies production with a titer of 1:90,and can significantly increase the ratio of IFN-?and IL-2 secreting cells in splenocytes.The results above indicated that WNV chimeric vaccine candidate ChiVax-WN01 has higher safety and good immunogenicity by intraperitoneal immunization.Conclusion:The JEV SA14-14-2 reverse genetic system has been successfully established,and a vaccine candidate ChiVax-WN01 with higher safety and good immunogenicity for mice has been constructed based on the JEV SA14-14-2backbone. |
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