The Gene Region Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus Live Vaccine (HuN4-F112strain) Of ORF1a For Inducing Neutralizing Antibodies

Porcine reproductive and respiratory syndrome(PRRS)was caused by PRRS virus,which is characterized by reproductive failure in pregnant sows and respiratory problems in all ages of pigs.PRRSV is an enveloped,positive polarity,single-stranded RNA virus,belonging to the Arteriviridae family.Currently,it is unclear about the mechanism of neutralizing antibodies production of PRRSV.It has been showed that there exist differences in inducing the production of neutralizing antibodies and cross-protection between the live vaccine HuN4-F112 strain of HP-PRRSV and the live vaccine CH-1R strain of classical PRRSV.The study of our laboratory showed that the ORF1 a of live vaccine HuN4-F112 strain was involved in virus neutralization in vitro and played an important role in the production of neutralizing antibodies.ORF1 a occupies about 50% of the genome and encode the nonstructural proteins(NSP)of PRRSV of NSP1-8.In order to explore which gene region play a role in inducing the production of neutralizing antibodies,we made the following exploration.In this study,we successful constructed the chimeric PRRSVs of rHuN4-F112-TNSP1,rHuN4-F112-TNSP2,rHu N4-F112-TNSP2-8 and rHuN4-F112-TNSP3-8 based on the skeleton of the full length infectious cDNA clone of rHu N4-F112.The NSP1,NSP2,NSP2-8 and NSP3-8 of rHuN4-F112 were replaced by the corresponding fragments of CH-1R,respectively.The growth kinetics showed that the growth characteristic of chimeric PRRSVs were basically identical with their parental virus rHuN4-F112.The chimeric PRRSVs were passed on Marc-145 cells to sequenced,and the results indicated rHuN4-F112-TNSP1 was found to be mutated and the other three chimeric PRRSVs were stable in vitro.Based on the neutralization test,the neutralizing antibody titers of sera collected from pigs inoculated with HuN4-F112 against rHu N4-F112-TNSP2 were significantly lower than rHu N4-F112 and the neutralizing antibody titers of sera collected from pigs inoculated with HuN4-F112 against the other three chimeric viruses were not significantly different from that of rHuN4-F112.PRRSV-free pigs were inoculated with rHuN4-F112-TNSP1,rHuN4-F112-TNSP2-8,rHuN4-F112-TNSP3-8 and rHu N4-F112.There were 3 piglets for each group and 1 group for control.Blood samples were collected once a week for the purpose of virus isolation,ELISA test and neutralization test.The viremia was not detected from the sera collected from pigs inoculated with HuN4-F112-TNSP2-8,but the viremia was detected from the sera collected from pigs inoculated with rHuN4-F112-TNSP1 and rHuN4-F112-TNSP3-8.There was a reverse mutation in the sequence of rHuN4-F112-TNSP1 but not in the sequences of rHu N4-F112-TNSP3-8.There was a reverse mutation in the sequence of rHuN4-F112-TNSP1 but not in the sequences of rHuN4-F112-TNSP3-8.The neutralizing tests results revealed that rHuN4-F112 can induce high levels of neutralizing antibody but the rHuN4-F112-TNSP2-8 induced low level of neutralizing antibody during the study period.The neutralizing antibody titers induced by the rHu N4-F112-TNSP3-8 were similar to that induced by the rHuN4-F112.The results suggest that the rHuN4-F112-TNSP1 could not be presented stably in vitro and in vivo.The neutralizing antibody titers of sera collected from pigs inoculated with HuN4-F112 against rHuN4-F112-TNSP2 were significantly lower than that of HuN4-F112.The neutralizing antibody titers of sera collected from pigs inoculated with Hu N4-F112 against the rHuN4-F112-TNSP1 and rHuN4-F112-TNSP3-8 were not significantly different from that of rHuN4-F112.The rHuN4-F112-TNSP2-8 could induce low level of neutralizing antibody.This study suggest the NSP2 gene region may play an important role in the production of neutralizing antibody of HuN4-F112 strain,and NSP1 and NSP3-8 gene region have no effect on the production of neutralizing antibody of HuN4-F112 strain.