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.

Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSVMT) in golden syrian hamster

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSVMT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSVMT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSVΔGMT-SΔ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding SΔ21 of Delta-variant can induce more potent immune responses compared with those encoding SΔ21 of Omicron- or WA1-strain. VSVMT is a promising platform to develop a mucosal vaccine for countering COVID-19.

PI3K inhibitor ZSTK474 enhancing the antitumor of vesicular stomatitis virus A51 against osteosarcoma / 解剖学报

Objective To explore whether PI3K inhibitor combined with oncolytic virus can play an effective oncolytic effect on osteosarcoma. Methods The cytotoxicity to tumor cells was detected by MTT method, and the mechanism of enhancing the anti-tumor activity was explored by observation of the swelling of endoplasmic reticulum using electron microscope and the expression of apoptosis-related proteins using Western blotting. The tumor clearance ability of the combination of the PI3k inhibitor ZSTK474 and vesicular stomatitis virus A51 (VSVA51) was verified by anti-tumor experiment in vivo. The apoptosis of tumor cells was verified by immunohistochemistry. Results PI3K inhibitor could be used as sensitizers of oncolytic VSVA51, and confirmed that the)' promoted the strong apoptosis of osteosarcoma cells by aggravating the stress of endoplasmic reticulum in tumor cells (P < 0 . 01). In vivo experiments also showed that PI3K inhibitors combined with VSVA51 could significantly promote the oncolytic effect of osteosarcoma (P<0.001), and this combination therapy enhanced the infiltration of immune cells in the tumor (P<0.001). Conclusion PI3K inhibitors combined with oncolytic virus is a potential therapy for osteosarcoma.

Observation on effect of Livin on VSV-induced apoptosis of A549 cells / 肿瘤研究与临床

Objective To investigate the effect of Livin expression on VSV-induced apoptsis of A549 cells.Methods The expression of Livin of A549 cells was inhibited by RNA interference.VSV-induced apoptosis of A549 cells was observed by Tunel assay.Protein Level of livin was detected by Western blot.Caspase-3 activity was detected by the fluorescence-based quantitative method.Results Livin downregulation VSV-induced apoptosis of A549 cells.Inhibited the expression of Livin of A549 cells had increased Caspase-3 activity.Conclusion The effect of Livin on VSV-induced apoptotic of A549 cells could be increased by RNA interference.

Reviewing host proteins of Rhabdoviridae: Possible leads for lesser studied viruses.

Rhabdoviridae, characterized by bullet-shaped viruses, is known for its diverse host range, which includes plants, arthropods, fishes and humans. Understanding the viral–host interactions of this family can prove beneficial in developing effective therapeutic strategies. The host proteins interacting with animal rhabdoviruses have been reviewed in this report. Several important host proteins commonly interacting with animal rhabdoviruses are being reported, some of which, interestingly, have molecular features, which can serve as potential antiviral targets. This review not only provides the generalized importance of the functions of animal rhabdovirus-associated host proteins for the first time but also compares them among the two most studied viruses, i.e. Rabies virus (RV) and Vesicular Stomatitis virus (VSV). The comparative data can be used for studying emerging viruses such as Chandipura virus (CHPV) and the lesser studied viruses such as Piry virus (PIRYV) and Isfahan virus (ISFV) of the Rhabdoviridae family.

Retroviral Expression System and Its Application in Efficient Foreign Gene Expression / 中国生物工程杂志

Retroviral expression system which consists of retroviral vector,envelop protein vector and packaging cell line is an efficient expression system for recombinant protein.It has great potential in gene therapy and biopharmacy.Transcriptional active genome regions are the preferred targets for retrovirus integration.Furthermore,VSV-G protein enables this system a broader host range and makes virus integration more efficient.After infection of high-titer virus,high production clones can be selected through simple screening.So far,the research on retrovirus expression system has developed into application in bio-pharmacy industry.Here the composition of this system and the mechanism of virus transduction and summarize the application and prospect of retroviral expression system are introduced.

Interferon consensus sequence binding protein: Not essential for interferon alpha-mediated antiviral response to vesicular-stomatitis virus infection in HL-60 cells

BACKGROUND: The role of the interferon consensus sequence binding protein (ICSBP), a member of interferon regulatory factor family, in protecting against a vesicular stomatitis virus (VSV) infection has not been firmly elucidated. Thus, it was investigated utilizing the human promyelocytic leukemia HL-60 cells which do not express ICSBP. METHODS: HL-60 cells were stably transfected with plasmid containing cDNA for either ICSBP or DNA binding domain (DBD) and tested for their VSV-susceptibilities. The susceptibility of each transfectant group to a VSV infection was determined by a plaque assay at 1 h, 24 h, and 48 h post-infection in the presence (500 IU/ml) or absence of interferon alpha(IFN alpha). RESULTS: In the absence of IFN alpha, the three groups showed similar sensitivities to a VSV infection. However, when pre-treated with IFN, the viral titers in both the ICSBP and control clones steadily decreased over 48 h of incubation, indicating the existence of IFN alpha-mediated protection against VSV infection. The IFN alpha-treated ICSBP clones appeared to be more resistant to infection compared with the control clones, although the difference was not great . On the contrary, the viral titers in the IFN alpha-treated DBD clones increased at 24 h then decreased by 48 h. CONCLUSION: The expression of truncated ICSBP (DBD) does not appear to underlie the impaired protection against a VSV infection in the DBD clones, since even the control clones lacking ICSBP were protected from a VSV infection. This suggests that ICSBP does not play a critical role in the IFN alpha-mediated anti-VSV response of HL-60 cells, although it appears to confer some resistance to a VSV infection.

Effect of proteolytic digestion on the function of vesicular stomatitis virus ribonucleoprotein complex.

The nucleocapsid protein (49 Kd) of vesicular stomatitis virus is tightly bound to the genome rendering the latter transcriptionally competent. Controlled digestion with chymotrypsin removed a 12 Kd peptide from the complex. The resulting complex failed to serve as template for genome transcription in vitro when the polymerase components L and NS proteins were added. A template-associated protein kinase activity was also lost upon chymotrypsin treatment. However, the cleaved nucleocapsid protein (37 Kd) was still capable of binding tightly with the genome template and retained the epitope recognized by a monoclonal antibody. These results suggest that the nucleocapsid protein possesses separate domains that mediate binding to polymerase complex and maintain the structural integrity of the template.