Allicin, an antioxidant, is known for providing garlic with its unique fragrance and taste, as well as for its antimicrobial properties. Black garlic, a fermented form of garlic, contains higher levels of antioxidants...Allicin, an antioxidant, is known for providing garlic with its unique fragrance and taste, as well as for its antimicrobial properties. Black garlic, a fermented form of garlic, contains higher levels of antioxidants than fresh garlic. Antioxidants play a vital role in alleviating cellular stress during viral infections. Viral infections result in oxidative stress through the production of reactive oxidative species (ROS). A prolonged state of oxidative stress can result in cell death, DNA damage, and disease progression. In this study, black garlic extract (BGE) is evaluated for its ability to mitigate cytopathic effects and oxidative stress caused by herpes simplex virus-2 (HSV-2) infections in vitro. Antiviral assays were performed to determine the percent of viral inhibition resulting from treatment with the BGE. ROS-Glo<sup>TM</sup> H<sub>2</sub>O<sub>2</sub> assays were then completed to measure the post-infection ROS levels of BGE-treated virus and cells. The results thus far suggest that BGE may inhibit viral infection and decrease levels of oxidative stress.展开更多
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...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 100μmol/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,coimmunoprecipitation,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 domaincontaining 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.展开更多
In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mu...In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.展开更多
AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of...AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.展开更多
Objective:The goal of the present study is to highlight the significance of the determination of IgG avidity to herpes simplex2virus(HSV2) compared to detection of viremia by polymerase chain reaction(PCR) in relation...Objective:The goal of the present study is to highlight the significance of the determination of IgG avidity to herpes simplex2virus(HSV2) compared to detection of viremia by polymerase chain reaction(PCR) in relation to recurrent abortions.Methods:Serum samples from pregnant patients with bad obstetric histories and control subjects were analyzed for quantitative determination of IgG specific for HSV2 by using ELISA with and without sample treatment with 8 M urea as protein-denaturing agents for measurement of avidity index(AI).PCR was performed for samples to determine HSV2 viremia.Results:Regarding avidity index 11.5%had avidity index = 100.0,11.5%has avidity index = 47%and 76.9%had avidity index】60%.Control group had AI】70%.There was statistically insignificant difference between OD of IgG in patients with viremia before urea treatment and after urea treatment compared to healthy control.Also there was statistically insignificant difference in OD before urea and after urea in patients with viremia.Conclusion:From this study we can conclude that viremia for HSV2 is common finding in patients with bad obstetric history.Low avidity immunoglobulin G was not common finding in those patients.This may be explain that even reactivation of latent HSV 2 infection is associated with fetal loss.展开更多
Objective: To evaluate the monogalactosyl diglyceride(MGDG) and digalactosyl diglyceride(DGDG) from Clinacanthus nutans(C. nutans) for their in vitro antiviral activities against herpes simplex virus type 1(HSV-1) and...Objective: To evaluate the monogalactosyl diglyceride(MGDG) and digalactosyl diglyceride(DGDG) from Clinacanthus nutans(C. nutans) for their in vitro antiviral activities against herpes simplex virus type 1(HSV-1) and type 2(HSV-2) by plaque reduction assay.Methods: MGDG and DGDG were extracted with chloroform from C. nutans leaves.MGDG and DGDG were separated from chloroform crude extract using column chromatography, characterized by thin layer chromatography and quantified by high performance liquid chromatography. The anti HSV-1 and 2 activity against pre-treatment and posttreatment of the compounds was evaluated using plaque reduction assay. The cytotoxicity of the extract and the compounds on Vero cells were performed by MTT assay.Results: MGDG and DGDG obtained by column chromatography showed identical profiles as standard MGDG and standard DGDG using thin layer chromatography and high performance liquid chromatography. MGDG and DGDG from C. nutans showed 100%inhibition of HSV-1 replication at the post step of infection at noncytotoxic concentration with IC50 values of 36.00 and 40.00 mg/m L, and HSV-2 at 41.00 and 43.20 mg/mL,respectively. Moreover, MGDG and DGDG from C. nutans were demonstrated to have antiherpes simplex activity at the same level as standard synthetic compounds. In contrast, pretreatment of Vero cells with MGDG and DGDG before HSV-1 and HSV-2 infection did not show inhibitory effect against these viruses. MGDG and DGDG exhibited antiviral activity against HSV-1 with selectivity index of 26.00 and 23.00 and HSV-2 of 23.30 and 21.30.Conclusions: MGDG and DGDG from C. nutans, a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.展开更多
Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell ...Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.展开更多
The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact ...The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/β-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/β-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/β-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/β-catenin pathway to promote a virus-centric agenda.展开更多
Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exte...Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exten-sively;however,further genetic engineering of HSV-1 is hindered by its complex genome structure.In the present study,we designed and constructed a synthetic platform for HSV-1 based on H129-G4.The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination(TAR)in yeast,and was named H129-Syn-G2.The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus.According to growth curve assay and electron microscopy results,the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus.This synthetic platform will facilitate further manipulation of the HSV-1 genome for the devel-opment of neuronal circuit tracers,oncolytic viruses,and vaccines.展开更多
目的了解上海市闵行区婚检人群单纯疱疹病毒2型(herpes simplex virus type 2,HSV-2)感染情况及其影响因素。方法对上海市闵行区2013—2014年的婚检人群进行艾滋病相关知识、性行为情况问卷调查。采集血样检测HIV、HSV-2及梅毒感染情况...目的了解上海市闵行区婚检人群单纯疱疹病毒2型(herpes simplex virus type 2,HSV-2)感染情况及其影响因素。方法对上海市闵行区2013—2014年的婚检人群进行艾滋病相关知识、性行为情况问卷调查。采集血样检测HIV、HSV-2及梅毒感染情况。结果共调查2 116名婚检对象,共有92人感染HSV-2,感染率为4.35%。其中男性感染率为3.69%,女性感染率为5.01%。多因素Logistic回归分析显示,对于男性婚检人群,文化程度为高中及以下(OR=2.47,95%CI:1.195~5.108),未婚妻为HSV-2阳性(OR=9.29,95%CI:4.279~20.164)者更容易感染HSV-2。对于女性婚检人群,年龄≥25岁(OR=9.29,95%CI:4.279~20.164),非上海市户籍(OR=2.19,95%CI:1.091~4.378),文化程度为高中及以下(OR=3.37,95%CI:1.721~6.596),从不使用安全套(OR=3.45,95%CI:1.265~9.392),未婚夫为HSV-2阳性(OR=8.46,95%CI:3.700~19.351)者更容易感染HSV-2。结论上海市闵行区婚检人群的HSV-2阳性率较低,然而安全套使用率低导致未婚夫妻中任何一方感染HSV-2就会增加另一方的感染风险,提示需进一步提高未婚夫妻安全套使用。展开更多
文摘Allicin, an antioxidant, is known for providing garlic with its unique fragrance and taste, as well as for its antimicrobial properties. Black garlic, a fermented form of garlic, contains higher levels of antioxidants than fresh garlic. Antioxidants play a vital role in alleviating cellular stress during viral infections. Viral infections result in oxidative stress through the production of reactive oxidative species (ROS). A prolonged state of oxidative stress can result in cell death, DNA damage, and disease progression. In this study, black garlic extract (BGE) is evaluated for its ability to mitigate cytopathic effects and oxidative stress caused by herpes simplex virus-2 (HSV-2) infections in vitro. Antiviral assays were performed to determine the percent of viral inhibition resulting from treatment with the BGE. ROS-Glo<sup>TM</sup> H<sub>2</sub>O<sub>2</sub> assays were then completed to measure the post-infection ROS levels of BGE-treated virus and cells. The results thus far suggest that BGE may inhibit viral infection and decrease levels of oxidative stress.
基金supported by grants from the National Natural Science Foundation of China (No. 81874483)
文摘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 100μmol/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,coimmunoprecipitation,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 domaincontaining 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.
文摘In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.
基金Supported by National Natural Science Foundation of China(No.81273212,81100651)Project of Science and Technology of Shandong Province(No.2014GSF118044)
文摘AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.
文摘Objective:The goal of the present study is to highlight the significance of the determination of IgG avidity to herpes simplex2virus(HSV2) compared to detection of viremia by polymerase chain reaction(PCR) in relation to recurrent abortions.Methods:Serum samples from pregnant patients with bad obstetric histories and control subjects were analyzed for quantitative determination of IgG specific for HSV2 by using ELISA with and without sample treatment with 8 M urea as protein-denaturing agents for measurement of avidity index(AI).PCR was performed for samples to determine HSV2 viremia.Results:Regarding avidity index 11.5%had avidity index = 100.0,11.5%has avidity index = 47%and 76.9%had avidity index】60%.Control group had AI】70%.There was statistically insignificant difference between OD of IgG in patients with viremia before urea treatment and after urea treatment compared to healthy control.Also there was statistically insignificant difference in OD before urea and after urea in patients with viremia.Conclusion:From this study we can conclude that viremia for HSV2 is common finding in patients with bad obstetric history.Low avidity immunoglobulin G was not common finding in those patients.This may be explain that even reactivation of latent HSV 2 infection is associated with fetal loss.
基金Supported by Department of Medical SciencesMinistry of Public Health,Thailand(Grant No.RMSc-3Nk-RD-27-2011)
文摘Objective: To evaluate the monogalactosyl diglyceride(MGDG) and digalactosyl diglyceride(DGDG) from Clinacanthus nutans(C. nutans) for their in vitro antiviral activities against herpes simplex virus type 1(HSV-1) and type 2(HSV-2) by plaque reduction assay.Methods: MGDG and DGDG were extracted with chloroform from C. nutans leaves.MGDG and DGDG were separated from chloroform crude extract using column chromatography, characterized by thin layer chromatography and quantified by high performance liquid chromatography. The anti HSV-1 and 2 activity against pre-treatment and posttreatment of the compounds was evaluated using plaque reduction assay. The cytotoxicity of the extract and the compounds on Vero cells were performed by MTT assay.Results: MGDG and DGDG obtained by column chromatography showed identical profiles as standard MGDG and standard DGDG using thin layer chromatography and high performance liquid chromatography. MGDG and DGDG from C. nutans showed 100%inhibition of HSV-1 replication at the post step of infection at noncytotoxic concentration with IC50 values of 36.00 and 40.00 mg/m L, and HSV-2 at 41.00 and 43.20 mg/mL,respectively. Moreover, MGDG and DGDG from C. nutans were demonstrated to have antiherpes simplex activity at the same level as standard synthetic compounds. In contrast, pretreatment of Vero cells with MGDG and DGDG before HSV-1 and HSV-2 infection did not show inhibitory effect against these viruses. MGDG and DGDG exhibited antiviral activity against HSV-1 with selectivity index of 26.00 and 23.00 and HSV-2 of 23.30 and 21.30.Conclusions: MGDG and DGDG from C. nutans, a traditional Thai herbal medicine illustrated inhibitory activity against HSV-1 and HSV-2, probably by inhibiting the late stage of multiplication, suggesting their promising use as anti-HSV agents.
文摘Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.
文摘The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/β-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/β-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/β-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/β-catenin pathway to promote a virus-centric agenda.
基金Wuhan Institute of Virology for financial support for the research(grant no.EISA020201).
文摘Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exten-sively;however,further genetic engineering of HSV-1 is hindered by its complex genome structure.In the present study,we designed and constructed a synthetic platform for HSV-1 based on H129-G4.The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination(TAR)in yeast,and was named H129-Syn-G2.The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus.According to growth curve assay and electron microscopy results,the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus.This synthetic platform will facilitate further manipulation of the HSV-1 genome for the devel-opment of neuronal circuit tracers,oncolytic viruses,and vaccines.
