Interaction Of Sars-cov, Ev71, And The Interferon System

SectionⅠ: Some exploration on the interaction of SARS-Cronavirus with the interferon systemThe first aim of this study is to compare the antiviral activity of different interferons and explore the anti-SARS-CoV effector pathways of interferons. The second is to study the inhibition effects of some accessory proteins and nonstructural proteins of SARS-CoV on the IFN-β's promoter and ISG's promoter.In this report three type I IFNs (IFN-α, -βand -ω) showed different antiviral effects. IFN-βwas a more potent inhibitor of SARS-CoV than IFN-αand -ω. In PCR array analysis, there was no great difference in genes expression profiles among SARS-CoV-infected cells treated with different IFNs (IFN-α, -βand -ω). Some genes related to IFNs expressed differentially in SARS-CoV-infected cells with IFNs treatment compared to without IFNs treatment. OAS1 mRNA increased approximately 20 fold and was further evaluated about its antiviral effect against SARS-CoV in transient transfection assay. Expressions of several interferon-inducible genes, G1P3, IFI27, IFI44 and IFI44L, were increased dramatically, which maybe were related with interferon antiviral effect against SARS-CoV.The anti-SARS-CoV activity of IFN-βhad been verified. So the IFN effector proteins were studied to elucidate their anti- SARS-CoV effect.There are three effect pathways which have been verified, including PKR, 2'-5'OAS and MxA pathway. So the recombinant plsmids containing the three interested genes fragments respectively were constructed and correspond plasmids were transfected instantly into the Vero E6 cells. The anti- SARS-CoV activity was measured by Real-Time PCR. As we expected that the viral copies of cells expressing OAS protein was obviously reduced compared with that of the other two effector proteins. So we can figure out that OAS-1 is one of effetor pathways with which Type I IFNs are against SARS-CoV.The interaction of some SARS-CoV accessory and non-structural proteins with IFN-βpromoter was explored, including NSP6, NSP7, NSP8, ORF3b, ORF7b, ORF 6, UK6, ORF8a, ORF8b, ORF9b and NSP1. The results showed that ORF3a and ORF7a could activate the IFN-βpromoter, but NSP6 and ORF8a could significantly inhibit the expression of luciferase promoted by IFN-βpromoter or by ISG54 promoter. SectionⅡ: Some exploration on the interaction of EV71 with the interferon systemEnterovirus 71 (EV71) belongs to enterovirus. Most people infected with EV71 don't show any symptoms, only very few will come on, especially the children under 5 years of age. IFNs are used in the anti-virus therapy and can function effectively. So the interaction of EV71 with IFNs is under investigation.The complete genome of EV71 was sequenced and the genotype was identified. The complete genome size is 7324 nt besides the 3'UTR and translated from the 743 site of the 5'UTR, terminated at the 7324 nt site. This isolates have the 97 percent in common with several strains prevalent in Fuyang of Anhui province.VP1-based comparison with EV71 strains showed this strains have the 99% homologous with the 518-01F/SD /CHN / 07 strain prevailed in 2007 and this isolate belong to genogroup C4 .The anti-EV71 activity of IFN-βand IFN-ωwas evaluated in Vero E6 cells by use of CPE, crystal staining, Q-PCR, TCID50 and cell morphological observation. IFN could inhibit EV71's replication when either added 24h before infection or added 6h after infection. The eralier IFN is added after EV71's infection, the better it works.EV71 was found not to avtivate IFN-βand ISG54 promoters. IRF3, as an essential transcriptional molecular in IFN-β's expression, was analyzed. EV71 could not induce IRF-3 activation such as hyperphosphorylation, homodimer formation and nuclear translocation compared with the IFN-inducing control virus NDV. Our data suggest that EV71 appear to block IRF-3 activation in order to escape activation of the IFN system.