| Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is one of the most economically significant viral pathogens for production worldwide. The etiological agent of PRRS is porcine reproductive and respiratory syndrome virus (PRRSV). Since HP-PRRSV outbreak in china in 2006, the evolving rate of PRRSV strains is alarming. The highly pathogenic PRRSV has brought great economic losses to the pig industry and its ever changing genome makes the prevention and treatment of the disease face severe challenges.The nsp2 gene has the highest genetic diversity in the genomes of PRRSV field isolates and also is used as an epidemiological genetic marker. To investigate the amino acid difference among PRRSV isolates, a pair of specific primers for amplification of nsp2 hypervairable region was designed. The hypervariable region of nsp2 gene of 34 PRRSV isolates from different areas of Guangxi during 2013 to 2014 was amplified sequenced and analysed by software Lasergene and MEGA5.1. The result showed the amplified nsp2 hypervariable region exhibited various sizes in length. Compared to strains VR-2332,1 of 34 nsp2 sequences were 1772 nt in length, same as those of VR-2332 strains. GXYL1310, GDHZ1401 and GXBH1404 were found to contain a discontinuous 120 aa deletion. Strains GXBS1401a, GXNN1396 and GXYL1403e contained a discontinuous 31 aa,49, and 123aa deletion, respectively. We also noted that one isolated strain (GX1407a) contained 30aa deletion and laa insertions.33 out of 34 nsp2 HV region sequences had the same length of 1600 nt, containing the same 30-aa deletion at aa 482 and aa 533-561 as JXA1 and other HP-PRRSV strains, compared with VR-2332.Homology analysis showed that the homology of nucleotide and amino acid sequences of the 34 strains of isolates of nsp2 gene sequence compared CH-la,JXAl, HuN4 strains isolated since 2006 were 77.3%～99.7% and 65.9%～99.2%; Compared to strains VR-2332,the nucleotide and amino acid homology were respectively between 77.4% to 99.7% and 65.9% and 99.2%. Especially the homology of nucleotide and amino acid sequences of the strains GXYL1403a compared strain VR-2332 were respectively 99.7% and 99.2%, compared MLV isolates were respectively 99.7% and 99.2%. A phylogenetic tree showed that the PRRSV isolates could be divided into three major branches. Strain GXYL1403a, belonging to subgroup I, was closely related to the MLV RespPRRS/Repro vaccine. The other 33 isolates, which were highly homologous to each other, formed a large cluster in subgroup I and were highly homologous to JXA1, which are representative HP-PRRSV isolates, indicating that the HP-PRRSV epidemic spread widely in Guangxi.To investigate the role of aa deletion in nsp2 on the replication in culture cells, a series of mutants in which contain different nucleotide numbers deletion was contructed based on the infectious cDNA clone pMJX0612-NM. The position of aa deletion in nsp2 was existed in some field PRRSV isolates. BHK-21 cells were transfected with mutants and pMJX0612-NM. The supernatants of the transfected BHK-21 cells was collected after 48 hours and was used to infect fresh MARC-145 cells. The results showed the the 87nt and 264nt deletion were not essential for virus viability but the other deletions were lethal. To confirm whether mutations introduced into the viral RNA were maintained in the replicating viruses, the nsp2 regions of the mutant viruses were amplified by RT-PCR and sequenced. The result showed the existence of the engineered mutation in all of the passaged viruses, and no other mutations were detected in the nsp2 coding region.To characterize the virological properties of the mutant viruses, MARC-145 cells were infected with the mutants and parental virus at an MOI of 0.01. The viral titers in supernatants harvested at regular intervals (6tol08 h.p.i.) were determined. The results showed that growth kinetics were indistinguishable among rMJX0612-NM-D87 and parental virus pMJX0612-NM, while rJX0612-NM-D264 showed somewhat a peak titer 1 log higher than those of the parental virus. The plaque morphologies of mutants and parental virus were assessed using MARC-145 cells. As shown in Fig 3-15, the plaques produced by rMJX0612-NM-D87 appeared similar to those of the parental virus, while rJX0612-NM-D264 showed somewhat bigger plaque size than the parental virus. These results indicated that aa deletion (D264) in nsp2 increase the production of infectious viruses.Finally, the expression levels of inflammatory cytokines in the primary PAM cells infected with the mutants were studied. The results show that the expression levels of inflammatory cytokines in the primary PAM cells represent varying degrees of up regulation. Compared with the parental virus, the mutant virus can lead to the expression levels of IL-8 and IL-10 increased more significantly. In 24 hours after infection, the two mutant virus can cause the expression of ISG54 increased significantly, but parent virus is opposite. The experimental results confirmed that the nsp2 deletion in different positions can indeed change the cellular immune response induced by parent virus. In this study, by studying the biological characteristics of the mutant virus and parental strain in vitro, we understand the influence of the deletion of nsp2 on PRRSV replication, cell apoptosis and the expression of immune related cytokines. Research data is great helpful for understanding the virus pathogenesis, virulence variation and the development of new vaccines. |
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