Effect and Mechanism of Menispermi Rhizoma Total Alkaloids on Antiviral Infection / 中国实验方剂学杂志

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.

Construction and verification of recombinant chimeric vesicular stomatitis virus expressing rabies virus G protein / 中国生物制品学杂志

@#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.

Validation and preliminary application of VSV-G qPCR assay for detection replication competent lentivirus / 中华微生物学和免疫学杂志

Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.

Vesicular Stomatitis Virus G Glycoprotein and ATRA Enhanced Bystander Killing of Chemoresistant Leukemic Cells by Herpes Simplex Virus Thymidine Kinase/Ganciclovir

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

O polietilenoglicol aumenta a penetração do vírus da diarréia viral bovina, do vírus da estomatite vesicular e do vírus sincicial respiratório bovino em células de cultivo / Polyethylene glycol increases the penetration of bovine viral diarrhea virus, vesicular stomatitis virus and bovine respiratory syncytial virus in cultured cells

A baixa eficiência de penetração de alguns vírus em células de cultivo pode representar uma dificuldade para o isolamento e a multiplicação viral in vitro. No presente estudo investigou-se o efeito do polietilenoglicol (PEG) na replicação de sete vírus bovinos em células de linhagem de rim bovino (MDBK). A eficiência de penetração e replicação foi mensurada pela contagem do número de placas virais produzidas em tapetes celulares, após adsorção do inóculo viral (100 DICC50 mL-1) com ou sem a adição de PEG a 5 por cento (peso molecular 6.000). A adição de PEG ao inóculo resultou em aumentos significativos do número de placas para o vírus da diarréia viral bovina (BVDV) (aumento de 3,4 vezes), vírus da estomatite vesicular (VSV) (2,2 vezes) e vírus respiratório sincicial bovino (BRSV) (1,5 vezes). A adição de PEG não produziu aumento significativo no número de placas dos herpesvírus bovinos 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5). Por outro lado, o PEG produziu uma redução do número de placas (1,4 vezes) produzidas pelo vírus da parainfluenza bovina (bPI-3V). A adição de PEG a 5 por cento também aumentou a sensibilidade de detecção (entre 10 e 100 vezes) do BVDV no soro de três bezerros persistentemente infectados. Para o BRSV, a adição de PEG aumentou em duas vezes a sensibilidade do isolamento viral de secreções nasais de duas ovelhas infectadas experimentalmente. Esses resultados demonstram que o PEG aumenta a eficiência de infecção do BVDV, VSV e BRSV em células de cultivo, podendo ser utilizado para aumentar a sensibilidade de detecção desses vírus em amostras clínicas (isolamento viral) e/ou, para aumentar os títulos de vírus produzidos em cultivo celular.

The low efficiency of penetration of some viruses in cultured cells may represent an obstacle for viral isolation and/or viral multiplication in tissue culture. This study investigated the effect of polyethylene glycol (PEG) on the penetration and replication of seven bovine enveloped viruses in culture cells. Penetration efficiency was measured by counting the number of viral plaques produced in bovine kidney cells (MDBK). The addition of 5 percent PEG (molecular weight 6.000) to the viral inoculum containing 100 TCID50 mL-1 (tissue culture median infectious dosis) of each virus, during adsorption for 2h at 37ºC, resulted in a significant increase in the number of plaques for bovine viral diarrhea virus (BVDV) (increase of 3.4-fold), vesicular stomatitis virus (VSV) (2.2-fold) and bovine respiratory syncytial virus (BRSV) (1.5-fold). The addition of 5 percent PEG to the inoculum of bovine herpesviruses 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) did not increase the number of viral plaques. On the other hand, PEG produced a reduction in the number of plaques by bovine parainfluenza virus (bPI-3V) (1.4-fold). Furthermore, the addition of 5 percent PEG produced a 10- to 1000-fold increase in the sensitivity of BVDV detection in the serum of three persistently infected calves; and doubled the sensitivity of detection of BRSV in nasal secretions of two experimentally infected sheep. These results demonstrate that PEG enhances the efficiency of infection by BVDV, VSV and BRSV in cultured bovine cells and therefore may be used to increase the sensitivity of virus detection in clinical samples (viral isolation), and/or to increase virus titers in cell cultures.

Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus.

The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine- Dalgarno sequence and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed a correlation among the expression levels, accessibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides between the Shine-Dalgarno sequence and ATG for optimal gene expression.