The Anti-PRRSV Activity And Mechanism Of Swine Ribonuclease L

Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ears disease, is a highly contagious disease which is caused by porcine reproductive and respiratory syndrome virus (PRRSV). The disease is an acute infectious disease characterized by sow abortion, preterm birth, estrus, reproductive disorders, mummified, weak or dead piglets and the respiratory symptoms of pigs of different ages. The typical characteristics of pigs infected with PRRSV is the destruction of the immune system and immune suppression, resulting in disorders of innate immune response, slown acquired immunity, more easily leading to co-infection and secondary infection by bacteria or other virus. The disease is one of the most economically significant viral diseases of swine, frustrating challenge to the global swine industry.Viruses can induce a variety of cytokines (CK) when invading the body and CK plays a very important role in antiviral immunity. Interferon (IFN) is a protein with multiple functions in response to the stimulation of the virus in human and animal cells. RNase L is an interferon induced antiviral protein (AVP) involved in specific and nonspecific immunity which plays an important role in antiviral immune process. Activated RNase L has an inhibition effect on a variety of RNA viruses and DNA viruses. We found that the swine RNase L (sRNase L) also has a certain inhibition effect on the proliferation of PRRSV in host and activated sRNase L significantly reduces the titer of PRRSV. Several experiments were conducted to study the anti-PRRSV activity and mechanism of sRNase L:1. Construction and identification of sRNase L eukaryotic expression vectorBased on the Sus scrofa RNase L complete gene sequence from GenBank, a pair of primers was designed with an enhanced expression Kozak sequence in N-terminal and HA tag sequence in C-terminal. The full-length of sRNase L gene from PK-15 cells (porcine kidney epithelial cells) was amplified by RT-PCR after stimulated with poly (I:C) and cloned into pXJ41 eukaryotic expression vector to obtain pXJ41-sRNase L by double enzyme digestion. Recombinant expression vector pXJ41-sRNase L was confirmed to be successfully constructed by double enzyme digestion and sequencing analysis. Results of western blotting showed that RNase L was successfully expressed.2. Establishment of a absolute and relative quantative real-time PCR for detection of PRRSV viral load and IFN-β mRNA levelThree pairs of specific primers were designed according to the PRRSV N protein gene and IFN-β, GADPH gene sequences. The standard plasmid containing the sequence of PRRSV N protein gene was constructed and used as a positive standard substance. The standard curve for the quantitative detection of the PRRSV N protein gene copies and SYBR Green I real-time PCR detection system for relative quantitative detection of IFN-β were established. These detection systems showed good linear relationship, high sensitivity and specificity. The real-time PCR products showed a single melting peak, respectively. The established real-time PCR method could be used to detect PRRSV viral load and IFN-p mRNA level.3. The anti-PRRSV activity and mechanism of sRNase L RNase L plays an important role in antiviral immunity process, howerer, anti-PRRSV activity of sRNase L has not been determined yet. The effects of 2-5A/sRNase L on the proliferation of PRRSV under different conditions were explored in this study. We found that sRNase L could inhibit the proliferation of PRRSV, the titer of PRRSV was significantly reduced by sRNase L when activated by 2-5A. PRRSV viral load was significantly decreased as detected by real-time PCR and PRRSV N protein expression was also significantly reduced as shown by IFA. The antiviral mechanism was further explored through the detection of IFN-β mRNA level and ISRE promoter activity. IFN-β mRNA level and ISRE promoter activity were significantly increased when MARC-145 cells were transfected with pXJ41-sRNase L as detected by real-time PCR and luciferase reporter gene assay. These data suggested that sRNase L could enhance the expression of IFN-P and ISRE activity simultaneously to strengthen the anti-PRRSV activity. This study is important to clarify the mechanism of anti-PRRSV activity of sRNase L and design new antiviral agents.