The Study Of SiRNA Target Praeparatum On Highly Pathogenic Avian Influenza Virus Of H5N1 Subtype

Backgroud Highly pathogenic avian influenza (HPAI) is one of fatal infectious diseases caused by influenza A virus of subtypes H5 or H7 in avian. The pandemic caused by highly pathogenic avian influenza virus (HPATV) already resulted in approximately a hundred million poultry death and led to tremendously economic expenses. HPAI is a worldwide infectious disease with great damaging power, and its outbreak usually is destructive. So, it is a list A diseases by Office International des Epizooties (OIE). Firstly in 1997, highly pathogenic avian influenza virus of H5N1 subtype broke through the hindrance of species and genus to infect human beings in Hongkong. Since 1997, the prevalence of H5N1 HPAI has been on a rise, and the cases of directly H5N1-infected human beings continuously have appeared, which shocked the whole world. At present, H5N1 HPAIV transmitted from birds to human in 58 countries and regions and case-fatality rate exceeded 60%. The serious situation of H5N1 HPAI prevalence implied that at present, the vaccine and anti-ATV medicines (For example, TamifluTM) could not effectively control and cure HPAI of H5N1 subtype in human beings. H5N1 HPAI has become a more and more seriously infectious disease that threatens health of human beings. Concerning the epidemic of HPAI, this paper launched the preparation and research of target praeparatum using siRNA against H5N1 HPAI.Methods In this study, utilizing the polyethylenimine (PEI) characteristics of binding nucleinic acids and proteins with negative charge, we used a recombinant of HA Single-chain antibody fragment of V-region with a negative amino acid Tail (ScFv-Tail, Tail is D4SX5) to closely bind PEI as a target vector to deliver siRNA. This deliver method is a novel target system to deliver siRNA:ScFv-Tail can recognize HA antigen on the surface of virus-infected host cells, and after occurrence of this recognization between antibody and antigen, the deliver system may accurately locate to infected cells. And as a new positive polycation, PEI closely binds ScFv-Tail and siRNA with negative charge. So, by target effectiveness of ScFv-Tail, this system can deliver siRNA into infected cells. After siRNA entry, the replication of virus will be inhibited by RNA interference. We observed transfection effect of target praeparatum in MDCK cells by flow cytometry and detected the inhibition effect of H5N1 HPAIV replication before using target praeparatum and after using target praeparatum. In addition, we detected the relative mRNA levels of NP, PA and PB1 by real-time PCR, and observed expression of NP protein by western blot and IFA. Furthermore, we detected inhibition capacity of target praeparatum in lung virus titers and protection effect in BABL/c mice.Results Flow cytometry result showed transfection levels of target praeparatum group is higher than those of DNA/PEI group in infectious cells (P< 0.05). The result of virus titer determination (TCID50) showed when using this target system before virus challenge, virus titers slightly decreased (P> 0.05, comparing with PEI/siRNA group), while used after virus challenge, virus titers significantly decreased and showed a significant difference (P<0.05). The result of real-time PCR showed that the levels of NP mRNA, PA mRNA using ScFv-Tail/PEI/siRNA (STPP group) were lower (P<0.05), but the level of PBl mRNA showed insignificant difference (P>0.05, comparing with PP group). The result of Western blot showed that the level of NP protein in STPP group was lowest; IFA demonstrated that intensity of red fluorescence in STPP group was lowest. These results showed that ScFv-Tail/PEI/siRNA could greatly inhibit expression of NP protein and effectively suppress the replication of H5N1 HPAIV.In vivo, after target deliver sysytem (ScFv-Tail/PEI/siRNA) was intravenously injected into BABL/c mice, lung virus titers were lower (P<0.05, comparing with PP group). This result demonstrated that target deliver system could significantly decrease lung virus titers in BABL/c mice infected with H5N1 HPAIV. After H5N1 virus challenged BABL/c mice, survival rate of STPP group was 60%, survival rate of PP group is 40%, and control group mice all died, which showed a significant difference (P <0.05; log-rank test). The target deliver system could protect the mice challenged by H5N1 HPAIV partially.Conclution The results of cells and mice experiments showed this target praeparatum could effectively inhibit AIV replication in MDCK cells, and decrease lung virus titers in challenged mice, furthermore, the target deliver system could protect the mice challenged by H5N1 HPAIV partially. These results suggested this target praeparatum may be used to control H5N1 HPAI. Objective In this study, three siRNAs targeting the polymerase A (PA) gene of highly pathogenic avian influenza virus (HPAIV) H5N1 were designed and evaluated for their abilities to inhibit HPAIV replication in vitro and in vivo.Methods According to siRNA design principles, we designed three siRNAs targeting PA gene of H5N1 HPAIV, and constructed expression plasmids (ps-PA496, ps-PA1116, ps-PA1473). To evaluate their abilities for antiviral effect in MDCK cells, virus titer assay, real-time PCR and indirect immunofluorescence assay were carried out. In addition, according to the results of cell experiment, we choosed most effective siRNA (ps-PA496) to observe the inhibition abilities of AIV replication in lungs of challenged mice and protection effect against lethal challenge with H5N1 HPAIV.Results Among three siRNAs, ps-PA496 could significantly inhibit H5N1 HPAIV replication in MDCK cells, and the viral replication in the ps-PA496-treated cells was 78-fold lower than that of the control. The results of real-time PCR and indirect immunofluorescence assay showed three siRNAs could inhibit transcription and expression of PA gene, especially ps-PA496. In vivo, PA-specific siRNA (ps-PA496) significantly decreased lung virus titers in BABL/c mice infected with AIV (P< 0.05), moreover, ps-PA496 partially protected mice against lethal challenge with H5N1 HPAIV (Survival rate is 37.5%).Conclusions In this study, targeting conservative region of PAN (E154-R170), designed ps-PA496 can significantly inhibit H5N1 HPAIV replication, and the findings might have significant implications for the application of siRNA for prophylaxis and therapy of influenza virus infection. In addition, we certified the conservative region of PAN (E154-R170) might act as a target in design of anti-AIV drugs.