RNA interference against influenza A (H1N1) virus
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Influenza virus is a RNA virus which causes human and animals to suffer influenza, leading to acute upper respiratory tract infection. The existing vaccines and drugs have limited role in the treatment of influenza virus subtype H1N1. We prepared the small interfering RNA targeting RNA polymerase (PA) gene of influenza A (H1N1) virus and studied its effect of inhibiting virus replication. We designed and synthesized three pairs of siRNA targeting PA gene of influenza A (H1N1) virus, as well as constructed expression plasmid pS-PA646, pS-PA841 and pS-PA1537, being transfected into MDCK cells and chicken embryos respectively and infected with influenza virus subtype H1N1, to detect effects of siRNA on inhibiting influenza virus replication. We conducted viral HA titer determination, real-time RT-PCR. The results show that in the designed 3 pairs of siRNA, pS-PA1537 can inhibit the replication of influenza A (H1N1) virus in MDCK cells and chicken embryos, laying the foundation for the development of therapeutic agents resistant H1N1.Cite
In March 2013, a patient infected with a novel avian influenza A H7N9 virus was reported in China. Since then, there have been 458 confirmed infection cases and 177 deaths. The virus contains several human-adapted markers, indicating that H7N9 has pandemic potential. The outbreak of this new influenza virus highlighted the need for the development of universal influenza vaccines. Previously, we demonstrated that a tetrameric peptide vaccine based on the matrix protein 2 ectodomain (M2e) of the H5N1 virus (H5N1-M2e) could protect mice from lethal infection with different clades of H5N1 and 2009 pandemic H1N1 influenza viruses. In this study, we investigated the cross-protection of H5N1-M2e against lethal infection with the new H7N9 virus. Although five amino acid differences existed at positions 13, 14, 18, 20, and 21 between M2e of H5N1 and H7N9, H5N1-M2e vaccination with either Freund's adjuvant or the Sigma adjuvant system (SAS) induced a high level of anti-M2e antibody, which cross-reacted with H7N9-M2e peptide. A mouse-adapted H7N9 strain, A/Anhui/01/2013m, was used for lethal challenge in animal experiments. H5N1-M2e vaccination provided potent cross-protection against lethal challenge of the H7N9 virus. Reduced viral replication and histopathological damage of mouse lungs were also observed in the vaccinated mice. Our results suggest that the tetrameric H5N1-M2e peptide vaccine could protect against different subtypes of influenza virus infections. Therefore, this vaccine may be an ideal candidate for developing a universal vaccine to prevent the reemergence of avian influenza A H7N9 virus and the emergence of potential novel reassortants of influenza virus.
Ectodomain
H5N1 genetic structure
Pandemic
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The behavior in mice of two thermosensitive (ts) mutants (denoted ts217 and ts700) of the recombinant influenza virus S/N (H2N1) was studied. The parental thermoresistant (tr) virus and both of the mutants were capable of inducing protection against pneumotropic A/Singapore (H2N2) and A/WS (H0N1) challenge viruses. Immunity against the Singapore virus, with which the S/N virus shared the hemagglutinin, developed earlier than against the WS virus, with which the S/N virus shared the neuraminidase. The tr and ts217 viruses were immunologically more active than the ts700 virus. The first two viruses grew markedly better in mouse lungs than did the latter. In the course of ts217 virus replication in vivo, revertants capable of growing at 39 degrees C appeared readily. On the other hand, the ts700 virus proved to be genetically stable. These data seem to provide evidence of a linkage between the stability of the ts phenotype, reproductive capacity in mouse lungs, and immunogenicity in the viruses examined.
Recombinant virus
H5N1 genetic structure
Reverse Genetics
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Aptamer
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Pathogenesis
H5N1 genetic structure
Reverse Genetics
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PB1-specific siRNAs of H5N1 highly pathogenic avian influenza virus(HPAIV) were designed and detected for their abilities to inhibit virus replication in vitro.Two siRNAs(ps-PB1-777 and ps-PB11282) were designed and constructed expression plasmids.Virus titer assay,Real-time PCR and indirect immuno-fluorescence experiment were used to observe their inhibition effect in MDCK cells challenged with H5N1 HPAIV.The results suggested virus titers of ps-PB1-777 were lower(P0.05,8 times comparing with the control group);after treatment of ps-PB1-777,the levels of PB-1 mRNA greatly decreased(P0.05);the levels of proteins of H5N1 HPAIV decreased.However,ps-PB1-1282 did not show the abilities to antiviral effect.As a result,ps-PB1-777 can effectively suppressed the replication of H5N1 HPAIV,and the findings might have significant applications for prophylaxis and therapy of influenza virus.
Highly pathogenic
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Active circulation of pandemic influenza and new variants of influenza H3N2 strains requires monitoring of antiviral efficacy of drugs permitted for influenza therapy in the Russian Federation.Assessment of antiviral efficacy of «Kagocel» substance against influenza viruses H1N1, H1N1pdm09 and H3N2 in vitro.Cytotoxic effect of «Kagocel» substance on MDCK cells had been determined by stained with MTS. Antiviral efficacy of «Kagocel» substance against influenza infection has been studied in vitro in the culture of MDCK cells infected with influenza virus strains: A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong/4801/2014 (H3N2). The antiviral activity of «Kagocel» substance was tested by its effect on the infectious titer of the influenza viruses and on its impact on the expression level of viral antigens in the enzyme immunoassay test system.«Kagocel» substance had low toxicity for MDCK cells. «Kagocel» inhibited the infection titer of influenza virus strains A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong /4801/2014 (H3N2) in the MDCK cell culture with equal efficacy. Study of the impact of «Kagocel» substance on the expression level of viral antigens by ELISA also revealed its antiviral efficacy for all tested strains. Dose dependence was observed from concentration of substance and from infective dose of virus.Effective suppression of the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in the different sublines of MDCK cells with «Kagocel» was shown by the different methods. These results give the possibility to suggest that along with the ability to induce interferons, «Kagocel» can impact on the reproduction of influenza virus, but the further research is needed.«Kagocel» substance effectively inhibits the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in vitro. At the same time, the selectivity index is quite high.
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Abstract Background Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state. Results In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice. Conclusions Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.
RIG-I
Antiviral drug
H5N1 genetic structure
Pandemic
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Early in 2017,the mutant H7N9 influenza virus appeared in China and it had showed the characteristic of highly pathogenic for poultry.Base on the hemagglutinin(HA) gene sequences of this highly pathogenic H7N9 influenza virus(HP-H7N9),specific primers and probe were designed,a real time one-step RT-PCR method was developed to detect HP-H7N9.It could specifically detect HP-H7N9,non-crossreaction with low pathogenice H7N9 influenza virus(LP-H7N9),H7N3 influenza virus(H7N3),H3N2 influenza virus(H3N2),H5N1 influenza virus(H5N1),H9N2 influenza virus(H9 N2),Newcastle disease virus(NDV),H1N1 influenza virus(H1N1),Infectious bursal disease virus(IBDV),avian infectious bronchitis virus(IBV).The minimum detection limit for positive plasmid was 35.45 copies which showed good sensitivity.It will play an important role in the diagnosis,surveillance,and control of highly pathogenic H7N9 influenza virus.
Newcastle Disease
H5N1 genetic structure
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