Antiserum Preparation And Recombination Analysis Of NSP2 Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV) is the causative agent of PRRS, which can cause late-term reproductive failure in sows and sever pneumonia in neonatal pigs. Genomic sequence comparisons have shown that PRRSV consisted of two genotypes: European type and North American type. In 2006, the outbreak of highly pathogenic PRRS(HP-PRRS) in China has increased the threat to the swine industry worldwide. NSP2 is the largest and most diversity protein in PRRSV and has been considered an important region for monitoring the genetic and epidemiological evolution of PRRSV. More and more studies indicate that it has significant relationship with viral replication, host immunity, autophagy and apoptosis.In this study, HP-PRRSV HH12 strain isolated from Heilongjiang Province of China in 2012 was identified. NSP2 gene of three HP-PRRSV strains including HH12, Jilin TN1 and Jilin TN2 strain was amplified by RT-PCR and then cloned into p MD18-T Easy Vector. Recombinant clones were sequenced and the length of NSP2 was 2850 bp for each strain. Comparing with classical PRRSV strain, unique deletion of 30 amino acid of HP-PRRSV NSP2 was found in three isolates. Gene similarity of NSP2 among three isolates and HP-PRRSV JXA1 strain was 95.5%~98.7%. Phylogenetic analysis showed that three isolates positioned close to HB0701 strain and clustered to subgroup JXA1.NSP2 gene of Jilin TN1 strain after deleting partial hydrophobic region was amplified by RT-PCR and then cloned into p GEX 6p-1 prokaryotic vector. The positive recombinant plasmid p GEX 6p-NSP2 was transformed into E. coli Rosetta(DE3). The recombinant NSP2 protein(131.5 ku) was expressed and could be recognized by specific PRRSV antiserum in Western-blot. Then the purified and refolding protein was immunized into the New Zealand rabbit in order to prepare the antiserum. Indirect ELISA assays showed that the titer of the obtained antiserum was 1:215. Also Western-blot showed that it had highly specialty and reactivity. IFA test demonstrated that it could recognize Marc-145 cells infected with highly pathogenic as well as classic PRRSV strains. Besides, it could react with transient expressed NSP2 protein in BHK-21 cells. Both HP-PRRSV and classical type PRRSV infection could be inhibited by the obtained antibody and the inhibition rates were up to 76.0% and 58.6% respectively. These results indicated the antiserum had good biological activities which had potential use in further research of biofunction of PRRSV NSP2 protein.All publicly available NSP2 sequences of North American type in Gen Bank were examined for their recombination. Recombination events were detected with RDP4.2, genetic similarity was analyzed with SIMPLOT and phylogenetic analysis was carried out with MEGA5.2. Evidences of recombination were found in 37 out of 624 strains including 3 traditional type strains, 7 classical type strains and 27 variant type strains. However, recombinant signals were not observed in NSP2 of HH12, Jilin TN1 and Jilin TN2 strains. Furthermore, 7 inter-genotype recombinant events and 3 intra-genotype recombinant events were found. All the intra-genotype recombinant strains were composed of variant type and normal/classical type and showed distinctive topologies in phylogenic tree. These results indicated that recombination might exist in NSP2. The appearance of variant type speeded up the viral evolution and derived the variation of PRRSV more intricately. Furthermore, recombination may induce type changing so far as to produce new type of PRRSV, and that may give rise to novel virus outbreak. Therefore, corresponding prevention and control measures should be given more attention.