Studies On RNAi For Anti-CSFV

Classical swine fever (CSF) is a highly contagious disease of pigs caused by CSFV, with high fever, bleeding, lymphocytes decreased and immune suppression as the main symptoms. It is a notifiable disease of the World Organization for Animal Health (OIE). Hog cholera lapinized virus (HCLV) made a significant contribution to prevention and control CSF in China. In recent years, CSF with lower morbility and mortality and mild, chronic, atypical symptoms was often observed, even in immune pigs herd. So the prevention of CSF has posed a severe challenge. Exploring new antiviral approaches has important practical significance to prevention CSF epidemic effectively. RNA interference (RNAi) is a natural post-transcriptional gene silencing mechanism. Since its discovery, RNAi has been regarded by virologists as a promising method for inhibition viral replicaton. It has been applied successfully to inhibit the replication of human and other animal viruses. With the development of transgenic technology, it can help to improve animal traits to animals by adding foreign genes to the gemome without the restriction of traditional breeding. Combination of these two methods, it can be bred transgenic animals carrying viral resistant genes and reduced the loss to the economy by virus deseases. In this study, we explored a new way of anti-CSFV in vitro and in vivo by RNAi combined with the technology of transgenic animal. This concept might help to reduce viral transmission and to prevent outbreak of infectious diseases.In this study, we designed and chemically synthesis 6 siRNA target sites targeting Npro, NS3 and NS5B genes, and then the sites were screened for efficient inhibition of CSFV replication. We obtained 3 target sites targeting NS3 gene (siNS3-1, siNS3-2 and siNS3-3), corresponding to reductions of up to 85.5% ,95.1% and 84.7% , and siNS3-2 was the most efficient, will be used for futher study.Based on siNS3-2 target site and the sites we previously screening (siN1, siN2 and siNS5B), we constructed CSFV-specific siRNA expression cassettes by using H1 promoters. The cassettes were inserted into the vectors PGKneotpAlox2 and pLox-siN1, and we obtained single anti-CSFV siRNA expression genes (pLox-siN2, pLox-siNS3-2 and pLox-siNS5B) and double anti-CSFV siRNA expression genes (pLox-siN1N2, pLox-siN1NS5B, pLox-siN1NS3-2). The quadruple anti-CSFV siRNA expression gene (pLox-siRNA4) were constructed by four CSFV-specific siRNA expression cassettes inserted into the vectors PGKneotpAlox2, using four different promers (hU6, hH1, mU6 and h7SK). The multiple anti-CSFV siRNA expression genes were confirmed by reporters and each siRNA was still functional when two or four different siRNAs were expressed in a single vector. Then, PK-15 cells were transfencted with the anti-CSFV genes and infected with CSFV. The results showed the anti-CSFV genes (pLox-siN1, pLox-siN2, pLox-siNS3-2, pLox-siNS5B, pLox-siN1N2, pLox-siN1NS5B, pLox-siN1NS3-2 and pLox-siRNA4) were significantly inhibited CSFV replication (P<0.01), corresponding to reductions of up to 84.8% ,87.9% ,87.0% ,83.2% ,90.1% ,94.0% ,91.8% and 96.8% . The multiple anti-CSFV siRNA expression genes inhibited CSFV replication much more effectively than single anti-CSFV siRNA expression genes, and quadruple anti-CSFV siRNA expression gene significantly inhibited CSFV replication than single anti-CSFV siRNA expression genes (P<0.05). The efficiency of inhibition of CSFV replication is additive and maintained by multiple anti-CSFV siRNA expression genes.In order to study on the inhibition efficiency of the anti-CSFV siRNA expression genes in vivo and explore breeding for CSFV resistance transgenic pigs, the anti-CSFV genes were digested with restriction enzyme and construction transgenic pigs by cooperation unit, leaving only the DNA sequence of G-418 expression cassette flanked by loxP sequences and the siRNA expression cassettes as the transgenic construct. Transgenic pigs No.0101, No.0103 and No.0107 were constructed by double anti-CSFV siRNA expression gene siN1N2, and transgenic pig No. 2019 was constructed by quadruple anti-CSFV siRNA expression gene siRNA4. Detection of exogenous gene integration was conducted by PCR, The results indicated that the transfected gene had integrated into genome. We isolated primary cells (umbilical vein endothelial cells, pigtial fibroblasts cells, white blood cells and kidney cells) of the 4 transgenic pigs for assessment of antivirus ability. The results shows Transgenic pigs No.0101 primary cells can inhibit CSFV infection and replication from different tissues; Transgenic pigs No.2019 umbilical vein endothelial cells can inhibit CSFV infection and replication; Transgenic pigs No.0103 and No. 0107 part of primary cells can inhibit CSFV infection and replication.To investigate whether CSFV could develop resistance to RNAi, growth studies were carried out in stable siRNA-expressing PK-15 cells. Ten lines of cells were infected with CSFV and passaged 10 times. Viral genomes from each line at the 10th passage were sequenced in the siRNA target region and compared with the corresponding region of the parental virus genome. No mutations were detected.In the present study, we studied on RNAi for anti-CSFV in vitro and in vivo, and explored new methods for prevention and controlling CSF. Further more we analysis of antivirus ability of transgenic-clone pig, which accumulated essential experimental data of construction anti-CSFV pigs. Our results also provide a reference for the establishment of other anti-virus breeding of transgenic animals.