Research On Molecular Biology Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) In Guangxi

In recent, some pig farms in Guangxi occur frequently an infectious disease , which characters are abortion , death of embryo , mummified fetuses , and respiratory problems in piglet . Pathogen of the disease was considered as porcine reproductive and respiratory syndrome virus (PRRSV) . In this study , we identified that the sample collected from the farm in Nanning was positive by RT-PCR with a pair of specific N gene primers of PRRSV . Based on it, permissive cells , Marc-145 cells were inoculated with the sample . After six blind passages , typical cytopathogenic effect (CPE) was observed on cells as well as the positive control .And PRRSV was positive in the cells and culture medium by RT-PCR . The first PRRSV strain in Guangxi was isolated successfully, named GXA strain . And its infection titer was 105.33 TCID50/ml.According to VR-2332 and CH-1a strain , designed three pairs of primers E1/E2 , M1/M2 , N1/N2 ,which amplified the fragments of GXA strain's E , M , N gene , respectively . The amplified fragments were cloned and sequenced . The length of E , M and N gene fragment is 668 ,582 and 443 base pairs (bps), respectively . Processed the sequences by DNAstar software , obtained the length of E , M and N gene sequence is 603 ,524 and 372 nucleotides(nts), encoding 200 , 174 and 123 amino acids .respectively . Comparison of the homology among GXA , VR-2332 , MLV , BJ-4 , Lelystad virus (LV) and other 9 strains of PRRSV , the results shown that the homology of nucleotide sequences and deduced amino acid sequences of E gene among GXA and Norther American strains was 88.7-99.7% and 93.8-100% , respectively . And it was higher than the homology between GXA and typical European strain LV , which was only 64.7% and 66.3%, respectively . The homology of nucleotide sequences and deduced amino acid sequences of M gene among GXA and North American strains was 94.5-100% and 96.6-100% , respectively . It was higher than the homology between GXA and LV , which was only 69.2% and 78.9%, respectively. The homology of nucleotide sequences and deduced amino acid sequences of N gene among GXA and North American strains was 93.8-100% and 95.2-100% , respectively . It was higher than the homology between GXA and LV , which was only 66.3% and 63.9%, respectively. Furthermore , it was found that the homology was very high among GXA and VR-2332 , MLV , BJ-4 strains, but the homology was very low among GXA and LV .And the phylogenetic trees revealed that GXA strain perhaps has a close relation to VR-2332 , MLV and BJ-4 strains. These results suggested that GXA strain belongs to North American type , and probably comes from the vaccine strain.E , M and N gene of GXA strain were obtained from recombinant plasmids pMD-E , pMD-M and pMD-N , then subcloned to prokaryotic expression vector PET-32a . And constructed the prokaryotic expression recombinant plasmids PET- E, PET-M and PET-N .They were identified by being digested with restriction endonucleases , the results showed that these recombinant plasmids were succeessfully constructed.