Biological Characteristics Of Pseudorabies Virus Isolate From Immunized Hogpen In Henan Province And Construction Of GE And TK-deletion Recombinant Strain

Pseudorabies?PR?is an acute infectious disease caused by Pseudorabies Virus?PRV?that can cause a variety of livestock and wildlife.Pigs are the natural host of the disease,which can cause stillbirth and premature birth of sows.It has caused huge losses to the world pig industry for a long time.Some developed countries such as the United States,Japan,and Denmark have already purified the disease in their own farms and successfully eradicated PR.However,China has not yet launched such projects.Depending on immunization of the traditional vaccines such as Bartha-K61,Ea,and HB-98 are the main methods of preventing pigs in China.In the past 30 years,the control of the disease has been relatively stable.However,some research in recent years have shown that new variant strains have appeared in hogpens in many areas,which has made new difficulties in preventing on farms.In 2015,PR broke out in a pig farm in Xinyang,Henan Province.In this study,the Pathological material was collected from dead piglets and cultured in VERO cells.After three rounds of proliferation and purification,the isolate showed typical CPE on VERO cells,including rounding,swelling,and formation of large syncytia,a strain of PRV was successfully isolated and named HNXY.Several important antigens and virulence-related genes?gB,gC,gD,gE,TK,RR1,RR2?were sequenced,and nucleotides were detected in early European and American strains represented by Bartha,Kaplan and Becker.After sequence alignment of nucleotide and amino acid,2001the homology of them is slightly higher than that of the TK gene,which is above 99%,and the other genes have a slightly lower homology,which is basically less than 99%.Compared with the SC,LA,Ea,and Fa of Chinese strains in the early 1990s,the homology of gB,gC,gD,and gE genes is almost below 99.5%,and the homology of TK,RR1,and RR2 genes is above 99.5%.Compared with the Chinese popular strains after 2012,the homologous ratio is the highest,and all genes are basically above 99.5%.Compared with the classic European and American strains Bartha,kaplan,Becker,the isolate HNXY,the Chinese strains in the early years and the Chinese popular strains after 2012,except for the RR2 gene,the sequence alignment of amino acids showed regular changes in amino acids?including insertions,deletions,or mutations?;The isolate HNXY has the same evolutionary relationship with all the Chinese epidemic strains after 2012.In addition to the RR2 gene,the other three genes of the classic European and American strains Bartha,kaplan and Becker in one separate branch,but were far away from the isolate HNXY;the earlier isolates of Chinese strains LA,Ea,and Fa were in a large branch,and the TK,RR1,and RR2 genes were all on one branch,gB,gC,gD and the gE gene form a small branch,and the gB,gD,gE and RR1 genes of the SC strain are closely related to the isolate,but the gC,TK and RR2 genes are the same as the three classical European and American strains.The one-step growth curve was determined by F5 generation.The results showed that the isolated strain showed a standard proliferation rule.0-36h was the logarithmic growth phase,and reached the highest value of about 10^6.5TCID₅₀ at 36h,after which the virus titer gradually decreased.The pathogenicity test of mice showed that there was no abnormal behavior within 24 hours after infection of the isolate.At 30 hours,the10^5.0TCID₅₀0 infection group began to die,reached a peak at 48 hours,and the10^4.0TCID₅₀ infection group began to die.At 72h,all the two groups died.At this time,10^3.0TCID₅₀ and 10^2.0TCID₅₀ also began to die.Between 72h and 96h,10^3.0-10^1.0TCID₅₀ infected group died successively,and the above infected mice Symptoms such as itching,hair removal,biting injection site,and injection site ulceration were observed.The LD₅₀ of HNXY strain was calculated by Reed-Muench method,which was 10^2.3TCID₅₀.The neutralization test showed that the anti-Bartha-K61 strain had poor neutralizing activity against the isolated strain,and the neutralization titer is only 1:12.7,while the neutralizing activity against Bartha-K61 strain itself was stronger and the neutralization titer is 1:128.On the basis of the isolate,the gE and TK genes of the virus were deleted in two steps by homologous recombination.First,each of the 1200 bp genes upstream and downstream of the gE and TK genes were amplified as homologous arms.The vectors pUC-gE-UD and pUC-TK-UD were constructed separately,and then the pCDNA3.1-EGFP vector was used as a template to amplify the CMV-EGFP-BGH ployA expression frame by primers,and the LoxP site was introduced at both ends,which used to remove the EGFP marker after recombination,and the vectors pUC-gE-UD-EGFP and pUC-TK-UD-EGFP were constructed by using the cleavage site based on the vectors pUC-gE-UD and pUC-TK-UD,and then amplified the homologous arm and the EGFP expression framework.And then recovered and purified the amplicon,and then to run the recombination by transfecting the PRV genome DNA and the amplicon into HEK293 cells,and the recombinant virus clones were purified by plaque dilution method which runned in VERO cells,each subcloning of the recombinant was performed in three rounds,and found that the efficiency of screening positive clones was 100%.The first step was to first delete the gE gene,and then use the Cre enzyme to remove the EGFP marker.Then delete the TK gene and the EGFP marker by the same method.The deletion strain has the same proliferation rule as the parent strain on the cell,and the virus titer is similar,but the cytopathic effect is changed,the cell infected by the deletion strain cannot form a syncytium,and the plaque diameter becomes smaller.The results of mouse pathogenicity test showed that the mice infected with the parent strain and the deletion strain showed normality within 24 hours,but the groups infected by parent strain began to die from 36h,reached the peak at 48h,and all died at 72h.The former mice showed symptoms such as itching,hair removal,bite injection site,and ulceration at the injection site.However,there was no death in the infected strain and the DMEM control group.The virulence of the virus was significantly weakened after deletion of gE and TK genes.Newborn piglets security experimental results show that the infection in 10⁵ TCID₅₀ dose of nasal mucosa and muscle injection newborn piglets,observing 25 d piglets without any clinical symptoms and adverse reactions,and also to maintain normal body temperature,by ELISA tests revealed piglets were produced after inoculation gB antibody,proved that the strain can stimulate the body's immune response,all infected piglets gE antibodies are negative,proved that the strain horizontal transmission ability in pigs is limited,the possibility of shedding.In conclusion,this study isolated and identified a new variant strain from the pig farm where immunized the traditional Bartha-K61 vaccine but PR still broken out,and this result once again verified the existence of new variant strains.Attenuated vaccines lacking TK and gE double virulence genes were prepared by genetic engineering,which laid a foundation for preventing infection caused by such strains.