Studies On The Inhibition Of Replication And Multiplication Of Some Silkworm And Swine Viruses By Targeting ShRNAs

Over the past decade RNA interference (RNAi) plays an important role in biology, especially for silencing gene expression. RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked down with high specificity and selectivity. Methods of mediating the RNAi effect involve small interfering RNA (siRNA) and short hairpin RNA (shRNA). In various applications, RNAi has been used to create model systems, to identify novel molecular targets, to study gene function in a genome-wide fashion, and to create new avenues for clinical therapeutics. In this study, we developed a system of RNAi for the inhibition of replication and multiplication of some silkworm and swine viruses by targeting shRNAs. Our results suggest that RNAi technology might serve as an experimental basis and technical insight into the researches on virus disease therapeutics.1Porcine circovirus type2(PCV2) is the primary causative agent of an emerging swine disease, it caused postweaning multisystemic wasting syndrome (PMWS) in pig farms in many swine-producing areas in the world in recent years, moreover, no antiviral treatment is available. To exploit the possibility of using RNA interference as a therapeutic approach against the PCV2, the291recombinant plasmid short hairpin RNAs (shRNAs) were constructed to target the Rep, Cap, ORF3and ORF4of PCV2genome. Transfection of these shRNAs into cultured PK15cells caused a different level of reduction in viral DNA replication. These results indicated that shRNAs are capable of inhibiting PCV2infection in vitro and thus may constitute an effective therapeutic strategy for PCV2infection.2Porcine reproductive and respiratory syndrome (PRRS) is now considered one of the most important diseases in countries with intensive swine industries. However, at present there is no effective method to prevent and control the disease. Therefore it is needed to develop the new antiviral strategies. In this study, the recombinant plasmid expressing shRNAs, specifically targeting the GP5/M gene RNAs of PRRSV, were generated and used to inhibit the production of GP5/M gene RNAs. It was found that shRNAs could down-regulate effectively specific gene expression and inhibit viral replication in Marc-145cells when compared to the controls. The highest activity displayed in shRNAs of the ORF6e sequences, which the inhibition rate reached to99.09%. The results suggest that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore the transgenic Mac-145cell line of piggyBac transpo son-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus was established, which is necessary for research works on transformed pigs studies.3The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to silk industry. In this study, we developed a system of RNAi for preventing the BmNPV infection using the piggyBac transpo son-derived targeting shRNA interference. The shRNAs targeting the genes of ie-1, lef1, lef2and lef3of.BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in BmN cells. The highest activity was presented in the shRNA targeting the iel-c of BmNPV, which the inhibition rate reached94.5%. Further a stable BmN cell line of piggyBac transpo son-derived targeting shRNA interference against BmNPV was established, which presented highly efficacious suppression of virus proliferation. This efficient method of stable gene transformation opens a way for promising research and biotechnology application on silkworm lethal diseases.