RNA Interferrence Research On R And S Gene Of Porcine Hemagglutinating Encephalomyelitis Virus

PHEV causes encephalomyelitis, vomiting or wasting disease in suckling piglets. When piglets younger than three weeks old are infected with PHEV, their mortality rate ranges from 20-100%. Recently, infection rates of PHEV have increased in some countries; thus, pigs without antibodies to PHEV, such as SPF pigs, were threatened by a high risk of infection by PHEV. The study of PHEV replication and of the control or inhibition of this infection is of great significance because, currently, no effective preventative measures or cures exist for this disease.Firstly PHEV is a member of the coronaviruses.S possesses antigenic determinants that trigger the immune response for the production of antibodies that neutralize virus infectivity and inhibit HA activity. S is a large, multifunctional protein that forms large petal-shaped spikes on the surface of the virions, and plays a central role in the biology and pathogenesis of coronaviruses. The number and size of R genes differ between coronavirus species, and they are important for RNA replication and transcription. RNA interference (RNAi) pathways have emerged as important regulators of virus–host cell interactions. We designed specific siRNAs to target the S and R genes, in order to test whether RNAi could selectively target PHEV viral RNAs.Secondly The features of PHEV,which grown on porcine kidney PK-15 cell lines,were analyzed by using indirect immunofluorescent assay(IFA)and real-time PCR assay and virus titration assay to detect PHEV protein expression and virus genome RNA replication and assembly of matured virus particles,respectively.The results showed that the cells appear primary focus at 12 h post-infection,which were fully infected at 32 h.The virus genome RNA synthesized rapidly.,which reach its peak at 48 h .,while the TCID50 can be detected at 18 h.,which indicated the eclipse period of PHEV,and peaked at 48 h .At last in this study the specific effect of RNAi on the replication of PHEV was explored. Four species of small interfering RNA (siRNA), targeting different regions of the PHEV S and R genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHEV, examination of cytopathic effects (CPE) demonstrated that the four siRNAs were capable of protecting cells against PHEV invasion with very high specificity and efficiency. In addition, viral proliferation within cells was examined by indirect immunofluorescence microscopy. At 48 hours post-infection, only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Transfection with siRNAs also suppressed the production of infectious virus by up to 18-32-fold as assessed by a hemagglutination (HA) test and 93-494-fold as assessed by TCID50 assay. Furthermore, treatment with siRNAs caused a 53%-91% reduction in viral genome copy number as assessed by real time RT-PCR. These results suggested that the four species of siRNAs can efficiently inhibit PHE-CoV genome replication and infectious virus production, but the effects were transient (data not shown). To circumvent this problem, stably transfected lines were created. two siRNA expression plasmids (shR2 and shS2) were generated to target two different coding regions of PHEV. The shRNAs were transiently transfected into a porcine kidney cell line, PK-15, to determine whether these constructs inhibited PHEV production. Infected cells were evaluated for antiviral activity against the PHEV strain by indirect immunofluorescence microscopy (IFA), viral titration and real-time RT-PCR at 48h post-infection. Our results revealed that both shRNAs were highly capable of inhibiting viral RNA genome replication, especially shR2. Next, stable transfection of shr2 was used to produce two siRNA stably-expressing PK-15 cell clones (shR2-1 and shR2-2), and these two lines were infected with PHEV. The analysis of cytopathic effects (CPE) demonstrated that shR2-1 and shR2-2 were capable of protecting cells against PHEV invasion with very high specificity and efficiency. There was a 66-fold reduction of PHEV growth in the shR2-2 cells as assessed by a TCID50 assay at 72 h post-infection, and effective inhibition of viral replication persisted for up to 120 h.In short these results indicated that RNAi targeting of R and S gene could facilitate studies of the specific function of viral genes associated with PHEV replication and may have potential therapeutic applications.