Construction And Immunogenic Analysis Of Chimeric Viruses Between Attenuated Classical And Highly Pathogenic PRRSV Vaccine Strains

Porcine reproductive and respiratory syndrome(PRRS) is a serious swine viral disease which was caused by PRRS virus(PRRSV), it’s characterized by reproductive failure in sows and respiratory disease in pigs of all ages. The virus was first isolated in China in 1996. In early 2006, a highly pathogenic PRRSV(HP-PRRSV) was emerged in China and had brought severe losses to the Chinese pig production, which had become an economically important disease for the pig industry. Currently, vaccination is the most effective method to prevent and control the disease, but there are some different aspects between classic and highly pathogenic PRRSV vaccines, which include the production of neutralizing antibodies and the cross-protection in vivo. The molecular mechanisms for such differences are unclear, we choose the attenuated classical and high pathogenic PRRSV vaccine strains as the research objects, to reveal the reasons or genetic regions for such different immunological response by construction of chimeric viruses and provide new strategies to study the genomic structure and function of PRRSV.In this study, to construct the full-length infectious cDNA clone of CH-1R, the respective genes of HuN4-F112 were replaced by the counterparts of CH-1R using the reverse genetics technology, which was based on the skeleton of infectious cDNA clone of pHuN4-F112.The C in position 454 was mutated to T by site-directed mutagenesis PCR to create a unique restriction site SpeI as the genetic marker. Capped RNA was transcribed in vitro from a full-length cDNA clone of pCH-1R and transfected into BHK-21 cells for the virus packing and then passaged onto MARC-145 cells to rescue the virus. This newly rescued virus was identified by cell pathogenic effect(CPE) observation, indirect immunofluorescence assay(IFA) and detection of the genetic marker. In vitro studies demonstrated that the rescued virus maintained similar growth properties to its parental virus. Above all, these results show the infectious clone pCH-1R of PRRSV CH-1R was constructed successfully.In present study, based on the infectious clone of attenuated classical PRRSV of pCH-1R, three chimeric full-length cDNA plasmids were constructed, in which the ORF1 a, ORF1 b and ORF2-7 genes of pCH-1R were replaced by the corresponding genes from HP-PRRSV vaccine strain HuN4-F112, respectively. The full-length RNAs were prepared from the three infectious cDNA clones by in vitro transcriptions and transfected into BHK-21 cells for the virus packaging and then passaged in MARC-145 cells to rescue the chimeric virus, correspondingly designated rCH-1R-T1 a, rCH-1R-T1 b and rCH-1R-T27. Firstly, the growth kinetics revealed that three chimeric viruses showed similar growth patterns to their parental virus rCH-1R. Then, the animal experiment results showed that the seroconverted percentages of the antibodies against the PRRSV N protein from the inoculated piglets were 3/3 for rCH-1R-T1 a, 2/3 for rCH-1R-T1 b and 1/3 for rCH-1R-T27, but no anti-N protein antibody was detected in the rCH-1R inoculated piglets. Our results indicated that the ORF1 a and ORF1 b of PRRSV play an important role in the procedure of inducing the specific antibodies of N protein in vivo.