| Porcine reproductive and respiratory syndrome (PRRS) is considered to be the most widespread infectious disease in swine, and has caused huge economic losses in the swine industry worldwide. Since 2006, a highly pathogenic PRRSV has emerged in China, causing huge economic losses in the swine industry. Guangxi province has large number of pig farms. PRRSV is one of the major pathogens affecting its pig industry. Prevention and controling of PRRSV is essential for development swine industry. To investigate the prevalence and genetic variation of PRRSV in Guangxi province,475 of clinical samples were collected based on the geographical distribution that covered almost places of Guangxi province from 2013 to 2014. RT-PCR results showed that PRRSV positive rate was 32.54%. PRRSV strains were isolated from MARC-145 cells inoculated with RT-PCR prositive samples and furthrt identified by RT-PCRand IFA.31 of 65 strans sequences of NSP2 hypervariable region and 58 ORF5 sequences were amplified and sequenced for further analysis. The results showed that the 31 sequences of NSP2 hypervariable region displayed 73.4?100% nucleotide identity and 83.1?100% amino acid homology with each other,and shared 41.4?99.5% nucleotide identity and 28.4?99.2% amino acid homology with reference sequence available in GenBank. The amino acid analysis of Nsp2 showed that all 30 strains contained a 29+1-amino acid deletion which is considered as hallmarker of HP-PRRSV. Moreover, PRRSV stain GXYL1403d has 124-amino acid deletion in NSP2. Phylogenetic analysis based on the NSP2 indicated that all PRRSVstrains obtained from this study belong to HP-PRRSV. The 58 sequences of ORF5 displayed 86.4-100% nucleotide identity and 84.5?100% amino acid homology with each other, and shared 62.9?100% nucleotide identity and 56.6?100% amino acid homology with reference sequence avalible in GenBanked. Phylogenetic analysis based on the ORF5 gene indicated that GXYL1403aand GXLB1403 shared a high identity with the VR-2332 and belong to VR-2332-like subgroup; GXGG1305a, GXGG1305b, GXGG1306 with the reference strains Em2007, HB-1/2(sh)-2002 belong to the intermediate subgroup; 53 strains belonged to JXA1-like subgroup and they shared a high identity with the JXA1 representative strain. Sequence and phylogenetic analysis of NSP2 and ORF5 demonstrated that HP-PRRSV has been becoming the dominant in the field of Guangxi, but classic and intermediate strainsare still circularingexist.To explore the biological characteristic of the isolated PRRSVs and genetic diversity in PRRSV isolates from China, the complete genome of the 4 PRRSV isolates, designated GXYL1403d, GXGG1305a, GXGG1305b and GXGG1306, were analyzed. The full-length sequence analysis showed that full-length sequence GXGG1305a, GXGG1305b and GXGG1306 contained 15,300nt and GXYL1403d was 15018nt in length.Genomic sequence analysis showed that the 4 PRRSVs shared 96.3?99.6% nucleotide identity with each other, and shared 60.7?98.1% nucleotide identity with reference sequence.Analysis of NSP2 revealed that GXYL 1403d has a 1 and 123-amino acid deletion at positions 481 and 511-635, unlike other HP-PRRSV isolates. Like HP-PRRSV, GXGG1305a, GXGG1305b and GXGG1306 have 1+29-amino acid deletionin NSP2. Genetic variability of GP5 and GP3 proteins was evident. Phylogenic analysis showed that GXYL 1403d belonged to JXAl-like subgroup and GXGG13O5a, GXGG1305b and GXGG1306 belong to the intermediate subgroup.To assess the susceptibility of PRRSVs srains to hyperimmune serum and determine the relationship between genetic variation of GP5 and the degree of cross neutralization among PRRS viruses,A panel of 30 PRRSV isolates with 1 vaccine strain and a hyperimmune monospecific sera were used in cross-neutralization assays. The results of this study indicated that PRRSV isolates differ in their sensitivity, they can be classified in 3 categories based on differences in their neutralization phenotype, susceptible(1:16? VN titers<1:64), moderate resistant (1:16< VN titers?1:8)and resistant(VN titers< 1:8). The susceptibility to neutralization by anti- JXM100 was serum evaluated in the context of amino acid differences in GP5 indicated that there is a certain relationship between the sensitivity of GP5 amino acid sequence homology and the level of neutralizing antibody, Further comparison of GP5 sequence showed that sensitive, moderate resistant strains and resistant strains of 32-34aa variation, GXLB1403?GXYL1403a had one amino acid mutation(Q58 to N58) in 57-59aa,they also had two amino acid mutation(S137 to A137 and R151 to T151). Analysis of neutralizing epitope of B was also found that GXLB1403, GXYL1403 had a mutation (I39 to L39). The changes of these sites, are likely to lead to different strains have difference sensitivity to neutralizing antibody. |
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