| Pseudorabies virus(PRV)infection could cause high fever,respiratory distress,nervous symptoms in piglets and miscarriage,stillbirth,mummified fetuses in pregnant sows.PRV has the property of latency,once upon infection,the virus could persist in the host and excret for life time,which resulted in great economic loss world-wide.PRV infection has always been a problem in China,since the first case of PRV infection reported in 1947.The virus has been transmitted to swine,canine,rabbit,mink and many other economical animals.The epidemic high-virulent PRV since 2011 lead to massive PR infection in many pig farms in China,and had brought great loss for Chinese swine industry.In this study,the full-length genome sequence of PRV traditional Chinese strain LA was deteriminted and compared with those of high-virulent strain ZJ01.And two gI and gE gene-exchanging chimeric variants were generated by construction of BAC cloning system with ZJ01 and LA strains.In vivo virulence test of the two chimeric variants proved that the mutations in gI and gE genes partially contributed to the enhanced virulence of the epidemic PRV strains.We also constructed a recombinant gl/gE/TK gene-deleted PRV variant ZJ11?gI/gE/TK,based on a gI/gE gene-deleted PRV ZJ01 variant(ZJO1?gI/gE)previously made in our lab.Animal experiment showed the triple-gene-deleted variant offered same immunogenicity with ZJO1 ?gI/gE,but was more safer to pigs than ZJ01 ?gI/gE,which established foundations for development of new vaccine varieties.The contents of this study contain four parts as following:1 Full-genome sequence comparition of 2 PRV strains with different virulence.PRV ZJ01 strain,isolated by our lab in a vaccinated pig farm in the year 2012,had been proved to have much higher virulence than an earlier Chinese pathogenic strain LA,especially on piglets.The sequence of ZJ01 has been obtained by our lab.In order to investigate the molecular basis for virulence enhancement of ZJ01 strain,the full-genome sequencing to LA strains was determinded by using Ion Torrent PGM system,and compared with those of ZJ01 strain.Results showed that LA genome is 141428bp in length,with a GC content of 73.66%,and contains 69 open reading frames(ORFs),which resembles all other announced PRV genomes.Phylogenetic analysis results revealed that all known Chinese strains formed a totally different cluster from the European/American strains.And LA strain was on a different sub cluster from the epidemic high-virulent strains(e.g.ZJ01,JS2012).The LA strain shared 97.07%homology with ZJ01,and the homology rate of LA and ZJ01 with tthe European/American strains were 95.06%and 91.18%,respectively.Comparing with LA,there are many mutations in the coding regions of ZJ01 strain,which existing in glycoproteins,tegument proteins,enzymes,envelop proteins and non-structural proteins genes.2 Construction and virulence comparison of the gIgE gene-exchanging variants ofZJ01 and LA in pigs.The bacterial artificial chromosome(BAC)clones of PRV high-virulent strain ZJ01 and traditional Chinese strain LA were obtained by replacing gI and gE gene with a BAC vector.The BAC clones were used as backbone for contructing the gIgE exchanging chimeric variants of ZJ01 and LA,rZJ-LA/EI and rLA-ZJ/EI,in which the gI and gE genes of ZJ01 and LA were replaced by corresponding genes in the other strain,respectively.The ZJ01 and LA revertants,rZJ01 and rLA,were also constructed.80-day-old pigs(free of PRV)challeng experiment results showed that pigs challenged with rZJ01 strain all died within 6 days.The pigs in rZJ-LA/EI group all died as well,however,the survival time was prolonged to 10 days.All pigs in rLA infected group survived through the experiment,while 1 out of 5 pigs died in the rLA-ZJ/EI group.The virus loads in the lungs of pigs from rZJ01 and rZJ-LA/EI challenge groups were higher than those in rLA and rLA-ZJ/EI groups.These results revealed that the virulence of chimeric rZJ-LA/EI variant was reduced by the replacement of gIgE genes from LA,and virulence of chimeric LA variant was enhanced with the replacement of gIgE genes from ZJ01.It indicated that the mutations in gI and gE genes played important roles in the enhanced virulence of ZJ01 strain.3 Construction of gI/gE/TK triple-gene-deleted PRV ZJ01 variant.A gl/gE/TK triple-gene-deleted PRV variant ZJ01?gI/gE/TK was constructed based on a ZJ01AgI/gE variant previously made in our lab.Briefly,the homologous fragments H1 and H2,flanking both sides of ZJ01 UL23(TK)gene,were amplified and cloned into pUC19 vector,creating intermediate vector pUC19-H1H2.The intermediate vector was then cloned into the pHA2 vector,which contains the bacterial artificial chromosome(BAC)vector and gfp cassette,creating transfer vector pHA2-pUC19-H1H2.By co-transfection of pHA2-pUC19-H1H2 and ZJ01?gI/gE genomic DNA,the recombinant virus ZJ01-GFP?gI/gE/TK,with the UL23 gene replaced by BAC vector and GFP cassette,was obtained.Co-transfection of pUC19-H1H2 with ZJO1-GFP?gI/gE/TK genomic DNA could result in the deletion of BAC and GFP genes from ZJ01-GFP?gI/gE/TK,and the recombinant virus was named as ZJO1?gI/gE/TK.The pathological morphology of ZJO1?gI/gE/TK on cells were similar with its parent strain ZJ01.The virus titer remained stable during a series of twenty times passage,with no reverts in the region of deletion.These results established foundations for the research of new genetic engineering vaccines from ZJ01 strain.4 Comparative pathogenicity and immunogenicity of triple and double gene-deletion pseudorabies virus vaccine candidates.The pathogenicity and immunogenicity of triple-gene-deleted variant ZJ01?gI/gE/TK were compared with that of ZJ01 ?gI/gE by experiment with 5-week-old piglets free of PRV.The results showed that ZJ01?gI/gE/TK caused milder clinical symptoms in piglets than the ZJ01 ?gI/gE strain.The gB-specific antibody and virus neutralizing antibody level were similar in the two group.Once challenged with lethal dose of ZJ01,ZJ01?gI/gE/TK vaccinated group show no other symptoms but slightly higher body temperature,and all piglet in this group were fully resistant to ZJ01 infection.In sum,ZJ01?gI/gE/TK had low pathogenicity and high immunogenicity to pigs,and should be considered as a vaccine candidate for controlling of PRV infection.In summary,in this study,we established the sequence divergence between PRV ZJ01 and LA strain.And we firstly found the role of gIgE mutations in the enhanced virulence of Chinese epidemic PRV strains.We also constructed and evaluated a triple-gene-deleted PRV vaccine candidate against high-virulent strains.All these above contributed to the prevention and control of PRV in the future. |
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