| Highly pathogenic avian influenza virus, which is originally from wild birds naturally without any symptom, can cause highly pathogenic death in poultry. Since 1997, The report that cases of people deaths caused by H5N1 influenza virus from birds in Hong Kong in China, prompted these virus can transmitted from infected birds to humans, and impacted a major worldwide concern. Luckily, these can not be capable of infected between humans at present. As of 19 March 2009, 254 deaths of 407 infected humans had been reported from WHO (mortality rate >60%). The situation, including more and more affected areas, more and more death cases, is very urgent; as a result, the outbreak of H5N1 influenza virus pandemic all over the world will happen in future.There are no effective medicine and ways to highly pathogenic avian influenza. Though vaccines are thought to be an effective way to anti-influenza virus, anti-H5N1 influenza vaccines for humans are in research because of hysteresis and variability. Historically, M2 ion channel protein inhibitors, including rimantidine and amantadine, first were for treating highly pathogenic avian influenza; but American CDC suggested to stop using them because of CNC side effect and the rapid occurrence of emergence of drug-resistant viral strains. Neuraminidase (NA) inhibitors are other anti-influenza drugs, including oseltamivir and zanamivir. It was reported that there were also drug-resistant viral strains in clinic. Therefore, it is of great importance to find a new target for anti-influenza virus of H5N1 subtype by a new technique. Hemagglutinin (HA), is necessary in the process of Entry which is crucial first step in virus life cycle, may be a useful target for research new anti-influenza drugs. HA is constituent of two subunits HA1 and HA2, which are relevant to recognizing and binding the receptor in the surface of host cells and mediate the process of membranes fusion respectively. Our experiment wants to build a new pharmacy model for anti-H5N1 highly pathogenic influenza virus in order to research and develop new drugs.Objective:The research is to establish a cell-based pharmacy model targeting for HA in the surface of different subunits influenza virus specifically and to screen more than 2000 compounds selected from marine and land by the model and to research the mechanism of compounds anti-influenza virus activity.Methods:(1) HA plasmids and NA plasmid were co-transfected with env-deficient HIV vector (pNL4-3.luc.R-E-) by using modified Ca3(PO4)2 transfection method to set up HA(Viet)/HIV-luc, HA(QH)/HIV-luc, HA(H5N2)/HIV-luc, VSVG/ HIV-luc , VSVG/MLV-GFP; (2) Detected the expression and activity of HA protein in the producer cells by western blot and antibody binding assay ; Diluted pseudotyped virions in different proportion (1:2, 1:4, 1:8, 1:16, 1:32 and 1:64) and determined Relative Luciferase activity units (RLUs) by using FB15 fluorescence detector, and drew Virus titer-RLUs curve;(3) More than 2000 compounds were screened by HA (Viet)/ HIV-luc, then used HA (Viet)/MLV-GFP, VSVG/ HIV-luc, VSVG/MLV-GFP to confirm the target for HA-SA by Susceptibility assay, Fluorescence and flow cytometry;(4) To detected the process three kinds of HA/HIV Pseudotyped virions infected host cells were inhibited by activity compounds, drew quantity-activity curve and calculated IC50 value;(5) Cytotoxic of activity compounds to 293ET and A549 cell lines can be detected by MTT assay;(6) Effect of HA (Viet)/HIV-luc and VSVG/HIV-luc on the receptor on the surface of host cells, which were incubated for 15min and 24 hours with compounds, were detected;(7) The processes of HA in influenza virus infected host cells, including binding and mediating fusion, were definite by HA-mediated cell cell fusion assay.Results:(1) We set up cell-based pseudotyped virions model with luciferase report genes by modified Ca3 (PO4)2 transfection method successfully. Infected abilities can be detected through intension of Relative Luciferase activity units (RLUs). The RLUs of 293ET cells which infected by HA (Viet)/HIV virus (diluted by 1:1) was 106-107, which is ten times than HA (QH)/HIV-luc and HA (H5N2)/HIV-luc (diluted by 1:1).(2) Western blot result revealed HA protein can express in host cells, but cell alone and HIV vector alone groups did not showed the bind; Anti-HA polyclonal antibody can inhibit HA (Viet)/HIV-luc viruses from infecting host cells. The results that the same antibody incubated with VSVG/HIV viruses or HA (Viet)/HIV-luc viruses with serum from rabbit were adverse. HA (Viet) /HIV-luc can infect many kinds of cell lines from human organs and had highly infected abilities to 293ET and A549 cell lines; The Virus titer-RLUs curves showed clearly dose-dependent effect between the level of the viral replication and the expression level of the luciferase report gene. So these pseudotyped virus models could be used to screening the drugs which aimed at the target for HA and the reproduction of HIV-1. Therefore, the models can be used for screening compounds.(3) Among more than 2000 compounds, three compounds, YCUA-6, YC-72 and GC-29, can inhibit HA (Viet)/HIV-luc and HA (Viet)/MLV-GFP virus, but not VSVG/HIV-luc and VSVG/MLV-GFP virus.(4) Activity compounds can inhibit HA (Viet)/HIV infecting 293ET and A549 cell lines with inhibition rate of 50%-70% at the concentration of 10μM.(5) MTT assay results showed that activity compounds were no toxic to host cells, 293ET and A549 cell lines, at the concentration of 10μM for 48h, 72h and 96h.(6) Activity compounds incubated with 293ET and A549 cells for 24h, compared with 15min, can not change HA (Viet)/HIV-luc and VSVG/HIV-luc virus infectivity degree.(7) Activity compounds can inhibit three subtype viruses, HA (Viet)/HIV-luc, HA (QH)/HIV-luc and HA (H5N2)/HIV-luc from infecting host cells effectively with the IC50 value ranges from one to tenμM.(8) HA-mediated cell-cell fusion assay showed that activity compounds can affect the infectivity process with inhibition rate 40%-60%, especially, YCUA-6 was the most obvious effect in the three compounds.Conclusions:(1) Three subtype HA-mediated cell-based pseudotyped virions pharmacy models, which target for HA in the surface of influenza viruses, were set up and optimized successfully.(2) Approximately 2000 compounds were screened using HA(Viet)/HIV-luc virus model and we get three activity compounds, which can also dose-dependent inhibit HA(QH)/HIV-luc and HA(H5N2)/HIV-luc, through compared with different models specifically. (3) The mechanism of YCUA-6, YC-72, GC-29 from marine anti-H5N1 influenza virus activity may they affect the congenerous protein sequence of HA from different influenza viruses or conservative protein sequence of HA, but not the regulation of HA receptor on host cells surface.
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