| Objective:In recent years,the infection rate and mortality rate of Lassa virus have been increasing,and there is still no effective vaccine or treatment.Natural products are an important source of new drug research and development,and tapping the important biological activities of traditional medicinal plants is of great significance to the research and development of new drugs.This study aimed to evaluate the crude extract antiviral activity of several medicinal plants by constructing a cell fusion model of Lassa virus and its pseudoviral infection model,so as to screen out medicinal plant extracts with anti-Lassa virus activity,and construct pseudoviral and cell-cell fusion models by using different lineage Lassa virus envelope proteins,further evaluate the antiviral activity of candidate medicinal plant extracts,and conduct a preliminary study on the antiviral mechanism of the extract.It provides a theoretical basis and experimental basis for the in-depth study of anti-Lassa virus drugs and the main components of antivirals.Method:1.The cell-cell fusion models of four lineages of Lassa virus were used to establish the corresponding pseudovirus infection models,and the antiviral activities of three types of medicinal plant extracts,including Yunnan pine tower,Sophora davidii,and purple elm,were preliminarily evaluated and screened.2.The cytotoxicity of medicinal plant extracts was evaluated by in vitro culture of 293 T and Huh-7,and the best antiviral activity was further determined by cell fusion model and pseudoviral infection model,and its antiviral activity in various lineages of LASV was evaluated.3.In order to explore the antiviral mechanism of Sophora davidii extract,Sophora davidii extract was added at different time points by the pseudovirus infection model of Lassa IV.(Josiah)to initially explore the period of action of Sophora davidii extract.The Lassa IV.(Josiah)pseudovirus infection model was used as a Time of addition experiment to further study which stage of viral infection the Sophora davidii extract acted on,the Sophora davidii extract was removed after pre-incubation with target cells,and then the target cells were infected with pseudovirus,and the Sophora davidii extract and pseudovirus were removed after incubation,and then the pseudo-virus infected cells was used to explore whether the Sophora davidii extract targeted to pseudoviruses or target cells.In addition,the Sophora davidii extract After co-incubation with pseudovirus,pseudovirus was collected with PEG6000 precipitation for semi-quantification of Western-Blot to further confirm whether the Sophora davidii extract had the effect of lysing pseudovirus.Result:1.Successfully constructed p AAV-IRES-GFP-Lassa I.(LP),p AAV-IRES-GFP-Lassa II.(803213),p AAV-IRES-GFP-Lassa III(GA391)and p AAV-IRES-GFP-Lassa IV.(Josiah)recombinant plasmids,and successfully expressed Lassa I.(LP),Lassa II.(803213),and Lassa I(LP),Lassa II.(803213)after transfection into 293 T cells,respectively Lassa III(GA391),Lassa IV(Josiah)Lassa virus envelope protein gene and green fluorescent protein gene.After transfection,293T/GFP/Lassa I.(LP)cells,293T/GFP/Lassa II.(803213)cells,293T/GFP/Lassa III.(GA391)cells,293T/GFP/Lassa IV.(Josiah)cells and Huh-7 cells were mixed and cultured for 12 h,and then started with fusion at low p H,compared to the negative control,after 30 min,Under the fluorescence microscope,it can be found that the arrangement of green fluorescent protein changes significantly,the volume of fusion cells increases to form cell fusions,the fluorescence intensity becomes weaker,and a large range of cell fusions with larger volume is formed after 2h,and the cell-cell fusion model is successfully constructed.The Lassa virus envelope protein genes of Lassa I(LP),Lassa II.(803213),Lassa III(GA391)and Lassa IV.(Josiah)were optimized by codons and inserted into the mammalian cell expression vector pc DNA3.1(+),respectively,to obtain recombinant plasmids pc DNA3.1-Lassa I.(LP),pc DNA3.1-Lassa II.(803213),pc DNA3.1-Lassa III(GA391)and pc DNA3.1-Lassa IV(Josiah).Each recombinant plasmid was co-transfected with HIV lentiviral backbone(p NL4-3.Luc.R-E-)plasmids into 293 T cells,and pseudoviral particles in the supernatant were obtained after 72 h,which detected a high level of enzyme activity after infection with Huh-7 cells,indicating that the pseudoviral infection model was successfully constructed.2.Using the Lassa IV.(Josiah)cell-cell fusion model,the fusion inhibitory activity of several medicinal plant extracts was detected,and their IC50(semi-inhibitory concentration)was obtained,among which,Sophora davidii extract:37.19±0.85μg/m L,pine tower extract: 83.07±8.95 μ g/m L,purple elm(ethyl acetate site)extract: 105.89 ±3.09 μ g/m L,purple elm(n-butanol site)extract: 86.53 ±10.02μ g/m L。 The antiviral activity of different medicinal plant extracts was detected by using the pseudovirus infection model of Lassa IV.(Josiah),and their IC50 were obtained,among which,Sophora davidii extract: 0.64±0.04μg/m L,pine tower extract: 98.71±15.95μg/m L,purple elm(ethyl acetate)extract: 21.78±2.01μg/m L,purple elm(n-butanol)extract: 19.49±2.94μg/m L.The cytotoxicity of each medicinal plant extract to the cell fusion model was different,among which the value of CC50(half toxic concentration)of Sophora davidii extract: 223.5± 6.2μg/m L,while the extracts of pine tower,purple diyu(n-butanol site)and purple elm(ethyl acetate site)did not have obvious toxic effects on cells even if the concentration of medicinal plant extract reached 500 μg/m L.The cytotoxicity of each medicinal plant extract to pseudoviral infection models was different,among which the CC50 value of Sophora davidii extract: 31.60±2.44μg/m L,the CC50 value of pine tower extract:562.37±10.89 μg/m L,the CC50 value of purple elm(ethyl acetate site):99.48±4.82μg/m L,and the CC50 value of purple elm(n-butanol site)extract:312.17±12.01μg/m L.Several medicinal plant extracts can exert antiviral effects in safe doses.3.The drug candidate alba extract also has inhibitory effect on different lineages of Lassa virus,among which the IC50 s of Sophora davidii extract for the fusion of various types of viruses were Lassa I.(LP): 63.16±7.13μg/m L,Lassa II.(803213):90.64±14.58 μg/m L,Lassa III.(GA391): 46.07±7.02μg/m L.The IC50 s for each type of pseudovirus were Lassa I.(LP): 2.66± 1.09 μg/m L,Lassa II.(803213): 1.29± 0.42μg/m L,Lassa III.(GA391): 1.58± 0.64 μg/m L.4.Using Lassa IV.(Josiah)as the antiviral mechanism of Sophora davidii extract,it was found that Sophora davidii extract exerts its antiviral effect in the early stage of pseudovirus infection.Time of addition results showed that the antiviral effect of Sophora davidii extract was exerted in the viral entry stage,mainly in the fusion stage,and that Sophora davidii extract targeted pseudoviruses rather than target cells.The pseudovirus was co-incubated with Sophora davidii extract and washed and recovered,and Western Blotting semi-quantification was performed,and the results showed that the pseudovirus did not decrease after incubation,and Sophora davidii extract did not have a lytic effect on Lassa IV.(Josiah)pseudoviral particles.Conclusion:1.Successfully constructed a cell-cell fusion model based on Lassa I(LP),Lassa II(803213),Lassa III(GA391),Lassa IV.(Josiah)and its pseudovirus infection model.All four medicinal plant extracts have anti-Lassa virus activity,and the antiviral effect of Sophora davidii extract is the most prominent.All four medicinal plant extracts exert their antiviral effects at safe doses.2.Sophora davidii extract in medicinal plant extracts has antiviral effects on different lineages of Lassa virus,and has a broad-spectrum anti-Lassa virus effect.3.Sophora davidii extract acts on the early stage of Lassa virus infection,mainly exerts antiviral effect in the fusion stage in the entry stage,and the antiviral effect of Sophora davidii extract targets pseudoviruses.Whitespine flower extract has no lysing effect on Lassa virus pseudoviral particles and may block the viral membrane fusion process by interacting with residues in the HR1 region. |
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