ABSTRACT
ObjectiveTo investigate the antiviral effect of Menispermi Rhizoma total alkaloids and its relationship with the type Ⅰ interferon （IFN-Ⅰ） signaling pathway. MethodThe effects of Menispermi Rhizoma total alkaloids on the intracellular replication of influenza A virus （H1N1）， vesicular stomatitis virus （VSV）， and cerebral myocarditis virus （EMCV） were detected by fluorescent inverted microscope， flow cytometry， Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot. A mouse model infected with H1N1 was constructed， and the mice were divided into a control group， H1N1 model group， Menispermi Rhizoma total alkaloids groups （10， 20， 30 mg·kg-1）， and oseltamivir group （40 mg·kg-1）， so as to study the effects on the weight and survival rate of infected mice. Real-time PCR was used to detect the activation effect of Menispermi Rhizoma total alkaloids on the IFN-Ⅰ pathway in cells， and the relationship between the antiviral effect of Menispermi Rhizoma total alkaloids in IFNAR1 knockout A549 cells （IFNAR1-/--A549） and IFN-Ⅰ pathway was detected. ResultCompared with the control group， the virus proliferated significantly in the model group （P<0.01）. Compared with the model group， Menispermi Rhizoma total alkaloids could significantly inhibit the replication of H1N1， VSV， and EMCV in vitro （P<0.01）， inhibit the weight loss of the mice infected with the H1N1 in vivo， and improve the survival rate of mice （P<0.05）. In addition， Menispermi Rhizoma total alkaloids activated the IFN-I pathway and relied on this pathway to exert the function of antiviral infection. ConclusionMenispermi Rhizoma total alkaloids exert antiviral effects in vivo and in vitro by activating the IFN-Ⅰ pathway.
ABSTRACT
@#Objective To construct recombinant chimeric vesicular stomatitis virus(VSV)expressing G protein of rabies virus(RV)using VSV as vector.MethodsTo rescue the recombinant virus,G gene of VSV antigenome was replaced with G gene of RV vaccine CTN-1 strain,and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and proteins N,P and L of VSV. The expression of RV G gene and G protein was detected by RT-PCR,immunofluorescence assay and Western blot. The recombinant virus was subcultured in Vero cells,the virus titer of different generations was detected and the virus growth curve was drawn.ResultsThe recombinant virus VSV-RVG was successfully rescued. RTPCR results demonstrated that the RV G gene was successfully inserted into the genome of the recombinant virus,and the expression of RVG protein was detected by immunofluorescence assay and Western blot. The recombinant virus was continuously passaged for 5 generations,and the virus titer was stable within 7. 5 ～ 8. 5 lgTCID50/mL.ConclusionThe recombinant chimeric VSV expressing RV G protein was successfully constructed with good genetic stability,which lays a foundation of the construction of reverse genetics technology platform based on VSV vector.