Construction Of Mutant Library Of Pseudorabies Virus By Tn5 Transposition And Function Study Thereof

Pseudorabies (PR) caused by Pseudorabies virus(PRV)is an acute infectious disease that causes fever,itching(except for pigs)in pigs,cattle,sheep and other domestic and wild animals.Pig is the main host and the natural reservoir of this disease.PRV belongs to the Herpesviridae family,alpha-herpesvirinae.The virus genome is about 143 kb in length and encodes 70 genes.Although some genes have been studied,the functions of many genes in the huge viral genome are still unclear.In order to systematically analyze the function of the PRV genome,the Tn5 transposon was transposed into p BAC-JS2012 in vitro and then the recombinant plasmids which contain Tn5 transposon were electrotransformed into bacteriato establish a random mutation library of PRV JS-2012 genome.The recombinant plasmids were extracted and identified by sequencing the transposition sites.The mutant plasmids were transfecte into Vero cells to rescue the mutant viruses and the growth characteristics of the mutant viruses were analyzed.In this study,4000 mutant clones of JS-2012 genome were obtained through in vitro transposition technology combined with resistance screening.312 plasmids of mutant clones were randomly extracted and their transposon insertion sites were analyzed.The insertion sites of transposons in 249 recombinant plasmids had been identified.There were also double-peak sequencing results from 40 recombinant plasmids,which may be inserted by two or more Tn5 transposons.The results of 249 plasmids with single transposon insertion showed that the transposon was inserted into the viral genome in a random and uniform manner,including 59 genes with different functions and non-coding sequence regions.After transfecting the recombinant plasmids with single transposon insertion into Vero cells,169 cloned viruses with different mutation sites were obtained in total,including 46 genes.According to the results of the rescued virus,48 viral genes were divided into three categories: 20 essential genes,16 non-essential genes,and 12 non-essential genes that play an important role in virus replication.In addition,UL14,UL13,UL42,UL41,UL9 and other genetic mutation viruses played important role in of which the proliferation rate is significantly reduced on Vero cells.US6(g D)gene mutant strains appeared cytopathic effect after transfection of cells,but the mutant virus cannot continue to be passaged.Among the rescued mutant viruses,we found that the UL9 gene mutant virus can be stably passaged on Vero cells and showed a large area of syncytial lesions,which was different from the previous report that the UL9 gene was an essential gene in HSV-1.Therefore,the biological characteristics of the UL9 mutant virus were analyzed from two aspects: in vitro growth characteristics and virulence to mice.Compared with the parental strain JS-2012,the proliferation rate of UL9 mutant in vitro was significantly decreased.All mice survived form the UL9 mutant virus infection.The results of ELISA show that antibodies against PRV g B were developed in all survived mice.In summary,a mutant library of PRV genome was constructed with Tn5 transposition and the roles of 48 viral genes in virus replication were determined.The UL9 gene showed an important effect on viral propagation.These results may contribute to studying PRV gene function.It is of great significance for constructing a mutant library of large-segment DNA viruses,promoting the study of viral functional genomics,screening virus virulence and regulating host immune-related genes.