To investigate the mechanism of the major capsid protein VP5 (encoded by the UL19 gene) of oncolytic herpes simplex virus type Ⅱ (oHSV2) in regulating the antitumor function of immune cells, we constructed a mouse breast cancer cell line 4T1-iRFP-VP5-GFP stably expressing VP5 protein, near-infrared fluorescent protein (iRFP), and green fluorescent protein (GFP) by using the PiggyBac transposon system. Flow cytometry and Western blotting were employed to screen the monoclonal cell lines expressing both GFP and VP5 and examine the expression stability of UL19 in the constructed cell line. The results of SYBR Green I real-time PCR and Western blotting showed that the copies of UL19 and the expression level of VP5 protein in the 15th passage of 4T1-iRFP-VP5-GFP cells were significantly higher than those in the 4T1 cells transiently transfected with UL19, demonstrating the stable insertion of UL19 into the 4T1 cell genome. The real-time cell analysis (RTCA) was employed to monitor the proliferation of 4T1-iRFP-VP5-GFP cells, which showed similar proliferation activity to their parental 4T1 cells. Further studies confirmed that NK92 cells exhibited stronger cytotoxicity against 4T1-iRFP-VP5-GFP cells than against 4T1 cells. This study layed a foundation for elucidating the role of VP5 protein in regulating immune cells, including T cells and NK cells, via HLA-E in 4T1 cells to exert the anti-tumor function.
Animals ; Mice ; DNA Transposable Elements/genetics* ; Cell Line, Tumor ; Capsid Proteins/biosynthesis* ; Transfection ; Green Fluorescent Proteins/metabolism* ; Oncolytic Viruses/genetics* ; Female ; Simplexvirus/genetics*

OBJECTIVE: JieZe-1 (JZ-1), a Chinese herbal prescription, has an obvious effect on genital herpes, which is mainly caused by herpes simplex virus type 2 (HSV-2). Our study aimed to address whether HSV-2 induces pyroptosis of VK2/E6E7 cells and to investigate the anti-HSV-2 activity of JZ-1 and the effect of JZ-1 on caspase-1-dependent pyroptosis. METHODS: HSV-2-infected VK2/E6E7 cells and culture supernate were harvested at different time points after the infection. Cells were co-treated with HSV-2 and penciclovir (0.078125 mg/mL) or caspase-1 inhibitor VX-765 (24 h pretreatment with 100 μmol/L) or JZ-1 (0.078125-50 mg/mL). Cell counting kit-8 assay and viral load analysis were used to evaluate the antiviral activity of JZ-1. Inflammasome activation and pyroptosis of VK2/E6E7 cells were analyzed using microscopy, Hoechst 33342/propidium iodide staining, lactate dehydrogenase release assay, gene and protein expression, co-immunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay. RESULTS: HSV-2 induced pyroptosis of VK2/E6E7 cells, with the most significant increase observed 24 h after the infection. JZ-1 effectively inhibited HSV-2 (the 50% inhibitory concentration = 1.709 mg/mL), with the 6.25 mg/mL dose showing the highest efficacy (95.76%). JZ-1 (6.25 mg/mL) suppressed pyroptosis of VK2/E6E7 cells. It downregulated the inflammasome activation and pyroptosis via inhibiting the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (P < 0.001) and interferon-γ-inducible protein 16 (P < 0.001), and their interactions with apoptosis-associated speck-like protein containing a caspase recruitment domain, and reducing cleaved caspase-1 p20 (P < 0.01), gasdermin D-N (P < 0.01), interleukin (IL)-1β (P < 0.001), and IL-18 levels (P < 0.001). CONCLUSION JZ-1 exerts an excellent anti-HSV-2 effect in VK2/E6E7 cells, and it inhibits caspase-1-dependent pyroptosis induced by HSV-2 infection. These data enrich our understanding of the pathologic basis of HSV-2 infection and provide experimental evidence for the anti-HSV-2 activity of JZ-1. Please cite this article as: Liu T, Shao QQ, Wang WJ, Liu TL, Jin XM, Xu LJ, Huang GY, Chen Z. The Chinese herbal prescription JieZe-1 inhibits caspase-1-dependent pyroptosis induced by herpes simplex virus-2 infection in vitro. J Integr Med. 2023; 21(3): 277-288.
Caspase 1/metabolism* ; Inflammasomes/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Simplexvirus/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Herpes Simplex/drug therapy* ; Humans

BACKGROUND: The dramatic increase in use of the IgG test for toxoplasma, rubella, cytomegalovirus (CMV), and herpes simplex virus (HSV) [TORCH] has led to the requirement for a high-efficiency method that can be used in the clinical laboratory. This study aimed to compare the results of BGI-Array ELISA TORCH IgG (BGI-GBI, China) screening method to those of Virion/Serion TORCH IgG ELISA (Virion/Serion, Germany). METHODS: Serum specimens (n=400) submitted for routine IgG testing by Virion/Serion ELISA were also tested using the BGI-Array ELISA method. The agreements of these two kinds of method were analyzed by kappa-coefficients calculation. RESULTS: Following repeat testing, the BGI-Array ELISA TORCH IgG assays demonstrated agreements of 99.5% (398/400 specimens), 98% (392/400 specimens), 99% (396/400 specimens), and 99.5% (398/400 specimens), respectively. The BGI-Array ELISA IgG assays provided results comparable to Virion/Serion ELISA results, with kappa-coefficients showing near-perfect agreement for the HSV (kappa=0.87), rubella (kappa=0.92) and CMV (kappa=0.93) and substantial agreement for the toxoplasma (kappa=0.80) IgG assays. The use of the BGI-Array ELISA TORCH IgG assays could reduce the turnaround time (1.5 hr vs. 5 hr by Virion/Serion ELISA for 100 specimens) and were easy to use. CONCLUSIONS: BGI-Array ELISA TORCH IgG shows a good agreement with Virion/Serion ELISA methods and is suitable for clinical application.
Antibodies, Viral/blood ; Cytomegalovirus/immunology/*metabolism ; *Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/*analysis/blood ; Protozoan Infections/diagnosis ; Reagent Kits, Diagnostic ; Rubella virus/immunology/*metabolism ; Sensitivity and Specificity ; Simplexvirus/immunology/*metabolism ; Toxoplasma/immunology/*metabolism ; Virion/*immunology/metabolism ; Virus Diseases/diagnosis

OBJECTIVETo develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)).

METHODSTumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination.

RESULTSHSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol.

CONCLUSIONRecombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

Animals ; Cell Line, Tumor ; Cercopithecus aethiops ; Green Fluorescent Proteins ; metabolism ; Humans ; Neoplastic Cells, Circulating ; metabolism ; pathology ; Recombinant Proteins ; metabolism ; Sensitivity and Specificity ; Simplexvirus ; metabolism ; Vero Cells

OBJECTIVE: To determine the effect and molecular mechanism of DNA damage caused by suicide gene therapy system HSV-TK/GCV under Tet-On regulation in human breast cancer cell line MCF-7 infected by recombinant adeno-associated virus (rAAV). METHODS: We used comet assay to detect the effect of HSV-TK/GCV suicide gene regulation system on MCF-7 DNA damage, and analyzed the expression change of relative DNA damage response active genes and proteins with RT-PCR and Western blot. RESULTS: Compared with other control groups, the comet assay showed that MCF-7 cells with HSV-TK/GCV treatment had obvious comet tails, and the expression level of DNA damage response active genes and proteins changed obviously in the HSV-TK/GCV treatment group,such as ATM, p53 and p27,but CyclinE and CDK2 did not change. CONCLUSION DNA damage on MCF-7 cells is resulted from HSV-TK/GCV in suicide gene therapy system through a p53-dependent signal pathway, causing cell cycle arrest and cell death.
Breast Neoplasms ; genetics ; pathology ; therapy ; DNA Damage ; Dependovirus ; genetics ; Female ; Ganciclovir ; metabolism ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Humans ; MCF-7 Cells ; Recombinant Fusion Proteins ; genetics ; metabolism ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics

OBJECTIVE: To investigate the mechanism and effect of diallyl disulfide (DADS) on the bystander effect of herpes simplex virus kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system which was mediated by recombinant adeno-associated virus (rAAV) in lens epithelial cells. METHODS: Immunohistochemistry method was used to determine the distribution of connexin 43 (Cx43) protein in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene. Cx43 protein was measured and analyzed in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene that was induced by various DADS. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: DADS increased the Cx43 protein expression in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene, and especially in 50 μmol/L DADS. After combining with DADS, the bystander effect was significantly improved (P<0.05). CONCLUSION DADS can elevate the expression of Cx43 protein and enhance the bystander effect of HSV-tk/GCV suicide gene therapy system.
Adenoviridae ; genetics ; metabolism ; Allyl Compounds ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Bystander Effect ; drug effects ; Cells, Cultured ; Connexin 43 ; metabolism ; Disulfides ; pharmacology ; Epithelial Cells ; metabolism ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Lens, Crystalline ; cytology ; drug effects ; metabolism ; Plant Oils ; Rabbits ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism

OBJECTIVETo observe the effect of ultrasound microbubble carrying herpes simplex virus thymidine kinase hepatocellular carcinoma in mice.

METHODSKunming mice were inoculated subcutaneously with H22 tumor cells. 40 male mice bearing subcutaneous hepatoma were randomized into 4 groups: PBS (group A), HSV1-TK (group B), HSV1-TK (group C), and microbubble carrying HSV1-TK (group D) were injected into the tail vein every 3 days. Mice in group C and D were exposed to ultrasound. The expression of TK protein was detected by western blot. Ganciclovir (GCV) was intraperitoneally injected at a dose of 100 mg x kg (-1) x d(-1) in group B, group C and group D. The tumor size was measured every 2 days.

RESULTSTK gene could be injected precisely into hepatocellular carcinoma with ultrasound monitor, and the expression of TK protein was found in all 4 groups. Expression in group D was higher than others (P < 0.05). The rate of tumor growth inhibition were 0 in group A, 3.90%+/-1.80% in group B, 22.70%+/-2.86% in group C, 41.25%+/-3.20% in group D (group B vs group C, P < 0.05; group D vs group C, P < 0.05; group D vs group B, P < 0.05).

CONCLUSIONUltrasound microbubble not only improve target gene therapy, but also enhance transfection efficiency.

Animals ; Carcinoma, Hepatocellular ; therapy ; Cell Line, Tumor ; Genes, Transgenic, Suicide ; Genetic Therapy ; Liver Neoplasms ; therapy ; Male ; Mice ; Mice, Inbred Strains ; Microbubbles ; Simplexvirus ; genetics ; metabolism ; Thymidine Kinase ; genetics ; Treatment Outcome ; Ultrasonics

Animals ; Apoptosis ; Herpes Simplex ; physiopathology ; virology ; Humans ; MicroRNAs ; genetics ; metabolism ; Simplexvirus ; genetics ; physiology ; Virus Latency

OBJECTIVETo study the effect of cotransfection of genes of insulin-like growth factor I (IGF-I) and herpes simplex virus thymidine kinase (HSV-tk) on wound healing.

METHODSThirty male Wistar rats were inflicted with 30% TBSA full-thickness scald. They were then divided into A group (4.6 microg pcDNA3.1/IGF-I+Lipofectamine 2000+saline), B group (3.6 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), C1 group and C2 group (2.3 microg pcDNA3.1/IGF-I+1.8 microg pcDNA3.1/HSV-tk+Lipofectamine 2000+saline), and D group (3.0 microg pcDNA3.1+Lipofectamine 2000+saline) according to the random number table, with 6 rats in each group. The above-mentioned mixtures were subcutaneously injected into left back of each rat the moment after injury and on post scald day (PSD) 7, 14, 21, and 28. Gancyclovir (2.5 mg/100 g) was hypodermically injected into rats in C2 group on PSD 29, 30, 31, 32. Changes in body weight of rats were measured. Wound healing rates were calculated. On PSD 35, the expressions of IGF-I gene in local wound and liver tissue were determined with immunohistochemical staining. The serum expression of IGF-I was determined with radioimmunoassay. Expression of HSV-tk gene in local wound was determined with RT-PCR. Apoptosis of fibroblast in C1 and C2 groups was observed under transmission electron microscope. Data were processed with one-way analysis of variance and Turkey method.

RESULTSBody weight of rats in A, C1, and C2 groups increased from PSD 7 through 35, and the difference between former three groups and B, D groups was statistically significant (with F value respectively 2.764, 4.519, 5.009, 13.449, 5.877, P values all below 0.05). Wound healing rates of rats in A, C1, and C2 groups were higher than those in B, D groups (with F value respectively 5.286, 100.880, 152.380, 127.850, 147.750, P values all below 0.05). IGF-I gene was positively expressed in wound fibroblast in A, C1 and C2 groups, but negatively in liver tissues of all the rats. There was no significant statistical difference among groups in serum content of IGF-I [from (1185+/-170) to (1270+/-130) ng/mL, F=0.355, P=0.838]. HSV-tk gene was positively expressed in rat skin tissue in B, C1 and C2 groups. Fibroblast apoptosis was observed under transmission electron microscope in C2 group, but it was not observed in C1 group.

CONCLUSIONSCotransfection of pcDNA3.1/IGF-I and pcDNA3.1/HSV-tk mediated by liposome can promote wound healing, and inhibit the scar proliferation to some extent.

Animals ; Burns ; genetics ; metabolism ; therapy ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Wound Healing

Capsid Proteins ; genetics ; metabolism ; Simplexvirus ; genetics ; metabolism