| Respiratory syncytial virus(RSV) is a common pathogen causing acute lower respiratory tract infection in infants and young children. RSV may also infect immune-compromised population, organ transplant patients and the elderly, causing high fatality. RSV was first isolated from chimpanzee rhinitis secretion in 1956 and the next year reports have shown that RSV may infect infants and young children causing bronchitis. Belonging to the paramyxovirus family, RSV is non-segmented single strand negative sense enveloped virus. The genome is 15.2 kb, encoded 11 proteins. Divided into RSV-A and RSV-B, the virus is prevalence by alternative each year. RSV affects about 30 million children each year, with developing countries being most severely influenced. There is currently no vaccine nor effective antiviral therapy. So it is urgent to develop effective antiviral drugs and vaccines.Surprisingly the development of formalin-inactivated vaccine couldn’t provide adequate protection of the virus, moreover it has caused immune enhancement disease leading to death of the recipient infants. Currently the only approved drug for treatment is ribavirin, but due to uncertain effectiveness and high toxicity, it is only used for high-risk children. Although a human monoclonal antibody palivizumab has been approved. It is difficult to be widely applied because of high cost. In order to identify novel anti-RSV compounds, we developed a cell based high-throughput screening assay by detecting cytopathic effect(CPE) caused by RSV infection. Our screening assay is based on cytopathic effect caused by syncytia of respiratory syncytial virus infection. Compounds were added shortly before infection, after co-culture with virus for 4 days. Results were detected through Cell titer glo luminescent reagent. This method may identify compounds that target at all replication steps. After optimization, we decide screening condition, with 5000 HEp-2 cells per 96 well plate, compounds were diluted to a final concentration of 2.5 μ g / ml, mix thoroughly and left 10 minutes to join the multiplicity of infection(MOI= 0.5) virus solution, after incubation for 96 h the results were measured by luminescence assay. Through primary screening, 7 natural compounds that show an inhibition of higher than 40% were conferred further validation. Among them CPA shows the best efficacy and is used for further mechanism revelation.CPA, a intracellular calcium ATPase inhibits RSV replication and progeny viral production at low toxicity concentrations. We verified the inhibitory effect of CPA on RSV with two kinds of invitro assays(Cytopathic effect assay and virus yield assay) CPA achieves EC50 4.2 μ m, CC50 33.9 SI 8.2. Moreover CPA inhibits replication of both RSV A, B and human parainfluenza virus type 3(PIV-3). However, it does not affect replication of enterovirus virus 71(EVA-71).Through time of addition experiment, CPA shows inhibition of viral replication or transcriptional process. While result in minigenome assay confirms the mechanism. In addition, CPA also inhibits syncytial formation. We then examine two other Calcium-ATPase inhibitors(Thapsigargin and BHQ) on viral replication, and find that these two intracellular calcium pump inhibitors inhibit viral replication. 10 n M Thapsigargin reduces viral titer by 124.5 times. We hypothesis that a calcium increase in intracellular caused by calcium pump inhibition may hamper virus replication process. Then we exame two kinds of calcium ion ionophere(A23187, Ionomycin) as well as a kind of Na+/K+-ATPase. All of them showed viral replication inhibitory effect. Down regulation of intracellular calcium concentration using calcium channel inhibitor on the membrane(Nifedipine, Nimodipine, and Tetrandrine) could not inhibit the replication of the virus. These results indicate an novel antiviral strategy that increasing intracellular calcium concentration may have an inhibitory effect on RSV replication. |
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