Inhibition Of Marek's Disease Virus Replication By RNA Interference Targeted To GI Gene And GE Gene

Marek's disease is an immune suppression of tumor diseases caused by Marek 's disease virus. In recent years the emergence of certain MDV variants and their increasingly virulence, the Immuno- protection efficacy of current vaccine decreases gradually, leading to the enormous economic lost to the poultry industry. RNA interference technology has been widely used for anti-viral research. Previous reports has shown that MDV gI and gE are important glycoprotein gene, which are closely related to mediate effcient cell-to-cell spread,involved in virulence, function as a receptor for the Fc domain of immunoglobulin. So, on the level of studying the specific siRNA targeted to gI and gE on the effect of the virus replication may has played an important reference value to investigate of MDV propagation in vitro and dissemination mechanism.In the study, the whole sequence of chicken U6 promoter was amplified from chicken embryo fibroblast (CEF) cells DNA, then the shRNA expression cassettes were construct by overlapping PCR method with the sense primer and ten anti-sense primers designed according to siRNA interference sites of Marek's disease virus (MDV) gI and gE gene. These cassettes together with pEGFP-gI or pEGFP-gE plasmids were cotransfection into CEF, respectively. The results from fluorescence microscopy and flow cytometry showed that the expression of the two fusion proteins were inhibited between 8.5% and 79.1%. The optimized cassette was inserted into pGEM-T vector to construct recombinant plasmids pcU6-shgI735 and pcU6-shgE936, Meanwhile,the same method was used to construct the pmU6-shgI735 and pmU6-shgE936 recombinant plasmids with mouse derived U6 promoter.These recombinant plasmids were all co-transfected into DF-1, MDCK and Vero cell lines with plasmid pEGFP-gI and pEGFP-gE reporter plasmid respectively to measure gI and gE gene knock down efficiency in different cell lines and compare the efficency of shRNA expression and RNAi activity of the chicken U6 and mouse U6 promoter.This paper revealed that the inhibitoty ratio of pcU6-shgI735 plasmids to gI gene expression was 87.39%,76.19% and 60.09% in DF-1, MDCK and Vero cell lines ; while that of pcU6-shgE735 plasmids to gE gene expression was 85.04%,73.60% and 58.57% , respectively,compared with controls .The recombinant plasmid with the shRNA under the control of cU6-3 promoter was 1.21 to 1.45 times more efficient in DF1 than that in mammalian cells (i.e. Vero or MDCK cells)(p<0.05),avian U6 promoter confer more efficient shRNA expression than that of mouse promoter in DF1 cells, therefore the avian U6 promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in avian cellls; while the mouse U6 promoter transcription activity in mammalian cells was 1.24 to 1.54 times more higher than that of avian U6(p<0.05). This finding may reflect inherent differences in the divergence of RNA polⅢpromoter activities between mammalian and non-mammalian vertebrates.To investigate the feasibility of inhibiting MDV propagation by RNA interference in vitro,the previously shRNA expression recombinant plasmids were employed to construct stable shRNA eukaryotic expression vector pEcU6-shgI735, pEcU6-shgE936 and multi-target tandem expression vector pEcU6-shgE936-shgI735. CEF cells were transfected with seris of shRNA expression vector and infected with MDV(L-ZY strain)after 8h post transfection, 24 hours later RT-PCR assay was performed using specific primers for shRNA expression cassette to confirm siRNA expression. Then using semi-quantitative RT-PCR, viral plaque formation (PFU)assay and MTT protection assay to measure the efficiency of inhibition of the virus replication. In the seris of shRNA expression plasmids–transfected group, the results showed that gI /gE protein mRNA levels were reduced by 77.33%/59.93%, 75.94%/61.46% and 83.15%/80.97%; the reduction rates of PFU were 78.57%,77.88%and 83.64%, compared to negative control group, respectively. MTT assay results also showed that the specific siRNAs provide favorable protection results against viral infection in avian cells.The above results demonstrated that the screened gI and gE gene specificity siRNAs were capable to significantly inhibit MDV gI and gE gene expression, plaque forming of MDV and its propagation in CEFs.It helps us to understand the replication mechanism of MDV and provids a new approach to prevention and therapy of this virus.