| Porcine reproductive and respiratory syndrome (PRRS) is characterized by severe reproductive failure and a high rate of late abortion and early farrowing in sows, and respiratory disease and mortality in young pigs. It is caused by a small, enveloped positive-strand RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), The disease suddenly emerged in the late1980s in the US and Europe, and has since spread worldwide, causing huge economic losses in the swine industry. Since2006, atypical PRRS(so-called porcine high fever syndrome(PHFS) in china was pandemic in china. Numerous reports have indicated the emergence of potentially highly pathogenic variants of PRRSV as the cause for large-scale outbreaks with high mortality in China. Highly pathogenic variants PRRSV (HP-PRRSV) belonging to North America genotype was confirmed as the causative agent of this outbreak. HP-PRRSV have a unique non-contiguous30-amino acid deletion in Nsp2. Highly pathogenic PRRSV TJ strain was isolated by our laboratory. Its attenuated descendent on Marc-145cell, TJM strain, had been shown to be avirulent in pigs with no clinical signs when inoculated. In order to study the mechanism of replication and intensive virulence of PRRSV, Nsp2genes of series of passages of PRRSV TJ strain were cloned and sequenced. Using the technology of eukaryotic expression and RNA interference, we investigated the effect of Nsp2on viral replication. Reverse genetics technology platform was established. And the cDNA clone of HP-PRRSV TJ and its attenuated TJM were successfully constructed.One pair primers were designed according to the full length genomic sequenee of HP-PRRSV TJ, based on the comparing result of several Nsp2gene of PRRSV from Asia region. The hypervariable region of HP-PRRSV TJ Nsp2of14different passages before and post plaque-cloned and Nsp2complete gene of TJ-F3and TJM-F92were amplicated, PCR products were cloned to pMD18-T, and sequenced. Amino acid sequences were analysed and predicted the second structure. The results showed120amino acids deletion in Nsp2of PRRSV TJ strain was appeared in passage18and the deletion stably existed through passage122. The second structure prediction of the deleted120amino acids and full Nsp2showed the deletion lead to the obvious second structure change. It is speculated the deletion may be related to the attenuation of PRRSV TJM. The result comparing six pairs of virulent parental/attenuated vaccine strains of PRRSV indicated the sequence difference of TJ/TJM pairs was distinct. It is not easy to recover for TJM.To investigate the effect of the Nsp2protein on porcine reproductive and respiratory syndrome virus replication, the Nsp2gene of highly pathogenic PRRSV TJ and attenuated TJM were amplified with RT-PCR and cloned into the plasmid pEGFP-N1, which containing enhanced green fluorescent protein expression box. The constructed plasmid pEGFP-TJ Nsp2and pEGFP-TJM Nsp2were transfected into Marc-145cells and screened by G418. Anti-G418Marc-145-TJ(Nsp2) and Marc-145-TJM(Nsp2) cells were obtained and the expression of Nsp2protein in anti-G418Marc-145-TJ(Nsp2) and Marc-145-TJM(Nsp2) cells was proved by PCR, RT-PCR. The Marc-145-TJ(Nsp2) and Marc-145-TJM(Nsp2) cells were infected by PRRSV, and determined TCID50. Results showed the cells expressing Nsp2gene of highly pathogenic PRRSV TJ and attenuated TJM were stable. PRRSV replication was fast in early stage on these cells. Nsp2protein play a positive role in early phase of PRRSV proliferation, and the effect of Nsp2protein of highly pathogenic PRRSV TJ was more obvious. K-factor of PRRSV on Marc-145-TJM(Nsp2) cells was less than that on Marc-145-TJ(Nsp2) cells. It was thought the120aa deletion might affect the role of Nsp2in viral replication.To study the effect of Nsp2gene siRNA on porcine reproductive and respiratory syndrome virus replication, three shRNA recombinant expression vectors specific to non-structural protein2(Nsp2) gene were constructded. The vectors identified by sequencing were transfected into Marc-145cells. Then, we infected the Marc-145cells with PRRSV TJ strain after6h post infection. CPE was observed every day. Sampling at series times was measured by RT-PCR aimed Nsp2mRNA and TCID50assay was used to titer. The results showed three siRNAs of Nsp2all inhibited the replication of PRRSV on Marc-145cells successfully, among which the effect of S-1and S-2were visible. The two siRNAs decreased the virus titre not less than10.A significant difference between S-1, S-2and the null vector (t Test, P<0.001).A total of seven fragments, covering the complete PRRSV TJ strain genome and TJM strain genome, were PCR amplified, respectively. All PCR-amplified fragments were gel purified and cloned into the pCR-Blunt shuttle vector. Two ribozyme sequences (HAM and HDV) and two enzymes(SnaB I andNot I) were engineered into5’end and3’end downstream of the poly(A) tail of both TJ and TJM genome for the further linearization of the full-length cDNA. The C at position15143and the C at position15148were mutated to A and T by PCR, respectively, to create a restriction enzyme site Mlu I as the genetic marker. Seven fragments confirmed by enzyme digestion and sequence were subcloned into eukaryotic expression vector pcDNA3.1. full-length cDNA clone of TJ and TJM were eliminated endotoxin and transfected into Marc-145cells. The freezing-thawing supernatant from transfected monolayers were serially passaged on Marc-145cells.The virus rescued from this newly assembled cDNA clone of TJ and TJM were identified by IFA and the detection of the genetic marker. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. The availability of full-length cDNA clone of PRRSV TJ strain and its attenuated TJM play a important role for further investigation of PRRSV molecular biology property and pathogenic mechanism and development of effective marked vaccine. |
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