The Studies Of The Biological Properties Of Japanese Encephalitis Vaccine Mutant Andthe Genome Characterization Of A Prevalent Strain Of Duck Tembusu Virus

Japanese encephalitis virus(JEV)is the medically important human pathogen of viral encephalitis.JEV can cause the serious infectious disease with the central nervous system damage and higher mortality.And JEV is also the major horse pathogen of fatal encephalitis,in swine herds,JEV also cause reproductive disorder in pregnant sow,such as abortion,stillbirths,mummified fetuses and orchitis in breeding pigs.Viral encephalitis and the high death rate are observed in piglets when infected with JEV.JEV is transmitted in a zoonotic cycle particularly between Culex species and pigs and most of vertebrates are the susceptible host of JEV.The enzootic cycle involves mosquitoes as vectors,pigs as the major reservoir/amplifying hosts,Birds as carrying host may contribute to the long-distance dissemination of JEV into new geographic locations because of the life habit of them.JEV is a small enveloped virus with a positive-sense single-stranded RNA genome and is the prototype virus of the JE serogroup of the genus Flavivirus.The single ORF is translated into one large putative polyprotein,which is co-and post-translationally cleaved by host and viral proteases to yield three structural proteins and seven nonstructural proteins.However,in the JEV serogroup,the conserved slippery heptanucleotide(YCCUUUU)and the downstream 39-adjacent potential pseudoknot in the 5’ end of the NS2A-coding gene result in-1 ribosomal frameshift during translation,this leads to the synthesis of NS1’,an extended NS1 protein product,including the N-terminal 9 amino acids of NS2A and 43 amino acids after the frameshift,and ends up with a stop codon.In this study,we have examined the nt 66 in the viral NS2A gene was one of critical site for the GC-rich stable pseudoknot and resulted in the synthesis of NS1’ through the reverse genetics system.Animal experiments demonstrated the NS1’ protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain.But the secreted NS1’ may server as early surrogate biomarker for viraemia to distinguish the virulent virus infection from the vaccine inoculation.Furthermore,we also demonstrated NS1’ protein can inhibit the IFN-βsignal pathway transduction.The thesis contains four parts as the following:1.The functional studies of Japanese encephalitis virus E279 on viral plaque morphology and neurovirulenceJapanese encephalitis(JE),widely distributed in the Asia region,is the most prevalent viral encephalitis caused by Japanese encephalitis virus(JEV).There are still no specific drugs available to treat JE;active immunization is the most effective method to against JEV infection for human and susceptible host.Here,we have investigated the qualities and molecular characters of the commercial live attenuated vaccines SA14-14-2 both for swine and human.The result showed that all the commercial vaccines meet the requirement;both the viral RNA copies and virus titers of the commercial vaccines could reach 105 in every sample.Unexpectedly,we found the plaque morphology of JEV strain SA14-14-2 from two swine live-attenuated JE vaccines were significantly smaller than others during detecting the virus titers of commercial JE vaccines by plaque forming assay.Then the complete genome of JEV strain SA14-14-2 from four commercial vaccines were sequenced and the alignment results showed several nucleotide mutations occurred in JEV from the two swine vaccines,particularly within 5’ nontranslated region(NTR)and E gene.However,the 1813th nucleotide of complete genome was the only identical mutation of JEV from the two swine vaccines in comparison with that from other commercial JE vaccines.In some nucleotide sites,there were two nucleotide fluorescent signals detected at the same nucleotide site,this indicated the substituted nucleotides were not completely replaced that of the attenuated JEV from commercial vaccines.Further analysis of amino acid sequence,to our surprised,one crucial amino acid was substituted in the hinge region of the E protein(at position of 279)of the JEV strain SA 14-14-2 from vaccine P1 and vaccine P2 in comparison with others or the published vaccine strains SA14-14-2.The plaque purification and the complete genome analysis showed that the E279 may results in the small-plaque morphology of JEV on BHK-21 cells when it was substituted from Met to Lys.Furthermore,this mutation in E279 may also increase the virus titer of progeny virions in cultural supernatant.The mice neurovirulence assays showed The M279K mutation in envelope protein might remarkably enhance the neurovirulence of the virus to suckling mice.In this study,we found one crucial site of the genome of the JEV strain SA14-14-2 was substituted during the production process and provided useful information for helping the manufacturer to deeply carry for the surveillance and evaluations of the commercial vaccines.2.The production of molecular mechanism of NS1’ protein in attenuated JEV vaccine strain and the functional studies of NS1’ protein in JEV infectionIn this study,we have developed a reliable reverse genetics system for the JEV SA14-14-2 strain by construction of the full-length infectious cDNAs clone.Then,we have examined the mechanism for the synthesis of NS1’ protein in JEV serogroup and have investigated the role of NS1’ protein during virus infection through the reverse genetics system.NS1’ is an additional form of NS1 protein with 52 amino acids carboxy-terminal extension and is expressed conditionally by the members of the JEV serogroup due to the translation frameshift.And JEV live attenuated vaccines SA14-14-2 ablates NS1’ formation due to the nucleotide mutation.A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1’.The NS1’protein had no significant efficiency in the virus replication properties in BHK-21 cells.Animal experiments demonstrated the NS1’ protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain.But the NS1’-expressing viruses(rA66G)could induce a higher immune response than the NS1’-non-expressing viruses(rSA14-14-2).Furthermore,NS1’ protein can be detected in the serum of infected animal and in the cultural media of infected mammalian cells.Interesting,only the dimer of NS1’ can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1’ was in agreement with that of the secreted virion.Although NS1’ have the restricted role on viral neurovirulence,in compare with the Japanese encephalitis-live vaccine virus ablates formation of NS1’,most of the virulent JEV strains produce the NS1’ protein.And the secreted NS1’ may server as early surrogate biomarker for viraemia to distinguish the virulent virus infection from the vaccine inoculation.In total,we have examined the nt 66 in the viral NS2A gene was one of critical site for the-1 programmed ribosomal frameshift to produce the NS1’ protein and demonstrated the NS1’ may be used for diagnostic biomarker during JEV infection.3.Simulation of the ribosomal frameshift in vitro and the characterization of cellular localization of NS1’ proteinNS1’ is an additional form of NS1 protein with 52 amino acids carboxy-terminal extension and is expressed conditionally by the members of the JEV serogroup due to the translation frameshift.In this study,we have simulated the-1 ribosomal frameshift of JEV in vitro for eukaryotic expression of NS1’.We have detected the NS1’ protein in transfected cells as early as 12h after transfection.The expression of NS1’ was the time dependent at the early stage of transfection.Furthermore,The NS1’ secreted protein in dimerization was also found in cultural media of the NS1’ expressed-plasmid transfected BHK-21 cells.NS1’ and NS1 localized to the same cellular compartments when expressed from plasmid DNAs and had the same cellular localization.4.The affect of JEV NS1’ protein in the IFN-P signal pathway transductionIn this study,we used the dual-luciferase reporter assay system to analyze the affect of NS1’ protein in the IFN-βsignal pathway transduction.First,we have constructed five dual-luciferase reporter plasmids with different IFN-βpromoter positive regulatory domains.The result showed that the positive regulatory domainsⅠ/ⅢandⅡwere critical for IFN-βpromoter activity,the absence of positive regulatory domainsⅠ/ⅢandⅡresulted in no activity of IFN-βpromoter in PK-15 cells with or without POLY(I:C)treatment.The other reporter plasmids had an activity in PK-15 cells with or without POLY(I:C)treatment.NS1’ protein could inhibit the promoter activity of IFN-βin PK-15 cells with or without the Poly(I:C)stimulating.And IRF7 could not reduce the inhibition of the promoter activity of IFN-βby NS1’ protein in PK-15 cells.Furthermore,NS1’ protein could inhibit the nuclear translocation of NF-κB after the Poly(I:C)stimulating in PK-15 cells.Virus infection demonstrated NS1’protein could facilitate production of JEV mRNA in A549 cells and could inhibit the mRNA of IFN-βat the early stage of infection.We have detected that NS1’ protein might interact with Heat shock proteins,Vimentin and TRIM21 in PK-15 cells or A549 cells by co-immunoprecipitation and mass chromatographic analysis.And NS1’ protein may inhibit the IFN-βsignal pathway transduction through interacting with these proteins.5.The genome characterization of a prevalent strain of duck Tembusu virusDuck Tembusu virus(DTMUV)is a mosquito-borne flavivirus to cause severe drops in egg production,declines in feed uptake,and even mortality of the affected ducks.During investigations into the outbreak of DTMUV infection in 2011 in China,a DTMUV strain(DTMUV-AH2011)was isolated from the affected ducks.The length of the genome of the DTMUV-AH2011 strain was found to be 11,064 nucleotides and to possess 10,278 nucleotides of one open reading frame(ORF),flanked by 94 nucleotides of the 5’non-translated region(NTR)and 692 nucleotides of the 3’ non-translated region(NTR).In comparison with 5 fully sequenced TMUV genomes,the genome of DTMUV-AH2011 had 74 nucleotides insertion in the 3’ NTR.Comparison of the DTMUV-AH2011 fully deduced amino acid sequences with those of other Tembusu virus strains reported recently in China showed they had a highly conserved polyprotein precursor,sharing 98.9%amino acid identities,at least.The overall divergences of amino acid substitutions were randomly distributed among viral proteins except for the protein NS4B,the protein NS4B was unchanged.Knowledge of the biological characters of DTMUV and the potential role of the insertion in the 3’NTR in RNA replication will be useful for further studies of the mechanisms of virus replication and pathogenesis.