| Porcine Reproductive and Respiratory Syndrome(PRRS)caused by PRRS virus(PRRSV) is characterized by respiratory problems in piglets and reproductive failure in sows. In 2006 an outbreak of highly pathogenic PRRS(HP-PRRS) caused by HP-PRRSV occurred in China and resulted in disastrous economic loss to swine industry. Compared to the classical PRRSV, HP-PRRSV could transmit rapidly and showed strong pathogenicity, serious inflammatory response and immune suppression. As an important immuno-regulatory cytokine, IL-10 plays an significant role in virus mediated immune suppression. Previous studies reported that PRRSV infection enhances systemic interleukin-10 production in infected pigs, which indicated that IL-10 may involve in the impairment of the immune system during PRRSV infection. The mechanism of the up-regulation of IL-10 level by HP-PRRSV needs to be clarified and our study will provide valuable data to determine the mechanism of immune evasion of PRRSV infection and contributed to the elimination and control PRRS.Our previous study suggested that the IL-10 expression level existed a big difference during PRRSV HuN4 and HuN4-F112 infection, high level of IL-10 was found when piglets infected with HuN4, while the level of IL-10 in piglets infected with HuN4-F112 was very low. To explain this, we first investigated the level of IL-10 expression when PAMs infected with HuN4 or HuN4-F112. The results revealed that high level of IL-10 was found when PAMs infected with HuN4, the level of IL-10 in PAMs infected with HuN4-F112 was very low. To further analysing the difference of IL-10 expression during PAMs infected with HuN4 or HuN4-F112, we measured key cytokines involved in IL-10 pathway. The result showed that the expression of TLR2 and TLR4 in PAMs infected with HuN4 were higher than that infected with HuN4-F112, while the expression of TLR7 and TLR9 were lower in the same case. There was no difference in TLR3 expression during HuN4 or HuN4-F112 infection. Then we investigated the expression of TRIF and MyD88 during HuN4 and HuN4-F112 infection, the results showed that higher TRIF expression was observed during HuN4 infection than that of HuN4-F112, but there was no significant difference of MyD88 expression among the HuN4 and HuN4-F112. The phosphorylation of p38 and ERK were further tested, and higher phosphorylation of p38 was founded when PAMs infected with HuN4 than that of HuN4-F112. No difference of ERK phosphorylation was found during each infection. All these results indicted the difference IL-10 expression between HuN4 and HuN4-F112 infection in PAMs; HuN4 could induced higher level of IL-10 expression than HuN4-F112, which could modulated MAPK pathway through p38 phosphorylation which was beneficial for IL-10 expression.To determine the relevant region in virus genome during IL-10 expression, we infected PAMs with the chimeric viruses rHuN4-F5-ORF1 a, rHuN4-F5-ORF1 b, rHuN4-F5-ORF2-7, rHuN4-F112-ORF1 a, rHuN4-F112-ORF1 b, rHuN4-F112-ORF2-7, which rescued by our lab before. The result indicted that ORF1 a and ORF2-7 contribute to the IL-10 expression during virus infection in PAMs. Then nsps(including nsp1, nsp2, nsp9-10 and nsp9-11) were exchanged between HuN4-F5 and HuN4-F112 using reverse genetics system, and the rescued viruses were named rHuN4-F5-nsp1, rHuN4-F5-nsp2, rHuN4-F5-nsp9-10, rHuN4-F5-nsp9-11(genetic backbone of HuN4-F5) and rHuN4-F112-nsp1, rHuN4-F112-nsp2, rHuN4-F112-nsp9-10, rHuN4-F112-nsp9-11(genetic backbone of HuN4-F112), respectively. The result showed that when PAMs infected with rHuN4-F5-nsp2, the level of IL-10 expression was significantly lower than PAMs infected with HuN4-F5, and when PAMs infected with rHuN4-F112-nsp2, the level of IL-10 expression was significantly higher than PAMs infected with HuN4-F112. Our results showed that PRRSV nsp2 region can modified IL-10 expression during PRRSV infected PAMs in vitro. |
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