Black Garlic as an Antiviral for Herpes Simplex Virus-2 in Lung Cells

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-GloTM H2O2 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.

The Chinese herbal prescription JieZe-1 inhibits caspase-1-dependent pyroptosis induced by herpes simplex virus-2 infection in vitro

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.

Role of viruses in periodontitis:An extensive review of herpesviruses,human immunodeficiency virus,coronavirus-19,papillomavirus and hepatitis viruses

Periodontitis is the inflammation of the supporting structures around the dentition.Several microbial agents,mostly bacteria,have been identified as causative factors for periodontal disease.On the other hand,oral cavity is a rich reservoir for viruses since it contains a wide variety of cell types that can be targeted by viruses.Traditionally,the focus of research about the oral flora has been on bacteria because the most widespread oral diseases,like periodontitis and dental caries,are outcomes of bacterial infection.However,recently and especially after the emergence of coronavirus disease 2019,there is a growing tendency toward including viruses also into the scope of oral microbiome investigations.The global high prevalence of periodontitis and viral infections may point out to a concomitant or synergistic effect between the two.Although the exact nature of the mechanism still is not clearly understood,this could be speculated through the manipulation of the immune system by viruses;hence facilitating the furthermore colonization of the oral tissues by bacteria.This review provides an extensive and detailed update on the role of the most common viruses including herpes family(herpes simplex,varicella-zoster,Epstein-Barr,cytomegalovirus),Human papillomaviruses,Human immunodeficiency virus and severe acute respiratory syndrome coronavirus 2 in the initiation,progression and prognosis of periodontitis.

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

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.

Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

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.

Value of avidity index of IgG and polymerase chain reaction in diagnosis of herpes simplex 2 virus in pregnancy

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.

SEARCH FOR HERPES SIMPLEX VIRUS TYPE2（HSV－2）AND HUMAN PAPILLOMAVIRUS（HPV）IN THE NORMAL AND ABNORMAL CERVICAL SAMPLES

The specimens of 111 cervical carcinomas. 68 chronic cervicitis and 43 normal cervical exfoliated epithelial cells were examined for the presence of HSV2 DNA sequences with DNA hybridization using HSV2 BgL Ⅱ N fragment probe labelled by 32PdCTP. The result showed that the infection rates of HSV2 in the samples of cervical cancer.chronic cervicitis and normal epithelial cells were 1 4. 41 %(16/111). 27.94%( 19/68) and 25.58% ( 11/43),respectively. It was implied that early stages carcinogenesis of cervical epithelial cells might be correlated with the HSV2 infection.Sixteen HSV 2 positive samples of cervical carcinomas were also examined for the presence of the sequences homologous to human papillomavirus (HPV) type 6B/11. 16 and 18 DNA using dot blot hybridization (Tm17℃). The result indicated that 13 out of 16 were HPV 16 DNA hybridization positive accounting for 81. 2% of all HSV-2 positive samples and none of them were positive for HPV type 6B/11 and 18. The result indicated that double infection of HSV 2 and HPV16 in the same cervical carcinoma tissues may provide a strong evidence of the viral synergistic interaction in the induction of female cervical

FAILURE OF HERPES SIMPLEX VIRUS TYPE 2 TO SUBSTITUTE FOR DIMETHYL-BENZANTHRACENE IN TWO-STAGE SKIN CARCINOGENESIS

There are some Indications thtt herpes aimplex virus (HSV) may be mutagenic. Specific chromosomal changes have also been demonstrated In cultured cells infected with HSV. To further Investigate the mutagenic activity of HSV type 2 (HSV-2) we used mouse skin as a model system for cardnogenetls. Inoculation of the back skin of 4-week-old Sencar mke with live virus twice per week for one week or with Inactivated virus twice per week for two weeks was used to Initiate the mouse akin. After Initiation with HSV-2, 12-O-tetradecanoylphorbol-13-acetale(TPA) was applied twice weekly for 50 weeks as a promoter. During a period of 52 weeks, no skin carcinoma was found In the experimental groups, whereas 55% of control mice treated with 9, 10-dlmethy1-1, 2- benzanthracene (DMBA ) and then with TPA-developed skin carcinoma. The results demonstrate that HSV-2 could not substitute for DMBA in this animal model of two-stage skin carcinogenesis.

Anti-herpes simplex virus activities of monogalactosyl diglyceride and digalactosyl diglyceride from Clinacanthus nutans, a traditional Thai herbal medicine

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.

ENHANCEMENT OF MORPHOLOGICAL AND ONCOGENIC TRANSFORMATION OF MOUSE CELLS WITH 12-O-TETRADECANOYL PHORBOL- 13-ACETATE FOLLOWING HERPES SINPLEX VIRUS TYPE 2 INFECTION

Sequential exposure of mouse embryo cells to HSV-2 and TPA gave rise to a synergistic enhancement of the transformation frequency. The transformants were selected for their ability to form dense foci of cells in medium containing 10% or 1%(low) fetal bovine serum. The average number of foci induced with HSV -2 followed by TPA was about 3 or 5(in low serum) fold greater than that induced with HSV- 2 alone. HSV- 2 antigen could be detected in about 10% of transformed cells before 27th passage with immunofluorescence technique. Of two cell lines established from single focus , one designated BL which was preferable to form foci in subcultures was tumorigenic after 21th passage. All of the tumors were sarcomas with interlacing bundles of pleomorphic fibroblasts. The other,designated NP was nontumorigenic until 50th passage. The BL cell line was composed of two distict cell types, i. e.,pigmented and unpigmented. No viral DNA sequences weredetected in the cells of tumors derived from BL cell line.

Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

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.

Herpes simplex virus 2-induced aseptic meningitis presenting with sudden deafness:A case report

BACKGROUND Aseptic meningitis is defined as meningeal inflammation caused by various etio-logies with negative cerebrospinal fluid(CSF)bacterial culture.The most common etiologies are viruses[enteroviruses,arboviruses,and herpes simplex virus type 2(HSV-2)].Aseptic meningitis can have various presentations,including sensori-neural deafness.While sensorineural deafness from mumps meningoencephalitis has been reported,cases of HSV-2-induced hearing loss are rare.Herein,we re-port a case of HSV-2-induced meningitis that presented with sudden deafness.CASE SUMMARY A 68-year-old man experienced a profound sudden onset of left-sided hearing loss for one day.Pure-tone audiograms demonstrated sudden left-sided sensorineural hearing loss(thresholds 80-90 dB).After treatment with high-dose steroids for 1 week,he experienced an acute consciousness change with left hemiparesis.The laboratory data showed no significant abnormalities.Brain computed tomography without contrast and magnetic resonance imaging revealed no intracranial hemo-rrhage or obvious brain lesion.The CSF analysis and the Multiplex PCR panels showed HSV-2 positivity.Hence,under the diagnosis of herpes meningoenceph-alitis,acyclovir was prescribed and his symptoms gradually resolved.CONCLUSION This case report further demonstrates that a viral infection could be a cause of sudden sensorineural hearing loss.

单纯疱疹病毒2型ICP27_(377-513)核酸疫苗联合IL-15核酸疫苗免疫效果的观察

目的构建表达单纯疱疹病毒2型感染细胞蛋白27(infected cells protein 27,ICP27)的重组质粒pcDNA3.1-ICP27377-513并观察白细胞介素(IL)-15核酸疫苗联合HSV-2-ICP27377-513核酸疫苗的免疫效果。方法采用分子克隆技术构建pcDNA3.1-ICP27377-513重组质粒。将BALB/c雌性小鼠随机分为pcDNA3.1-ICP27377-513联合pcDNA3.1-IL-15(pIL-15)组、pcDNA3.1-ICP27377-513组、pcDNA3.1组和pIL-15组,共免疫3次,每次间隔2周。末次免疫后28 d取血,用微量中和实验法检测血清中特异性中和抗体,ELISA法检测血清中IL-4、IL-2和干扰素-γ(IFN-γ)水平。阴道给予致死量HSV-2攻击小鼠,分别于接种后3 d、7 d和14 d收集阴道冲洗液,用荧光定量PCR法检测生殖道病毒载量。结果pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组中和抗体效价分别为40.00±8.16、28.67±4.47,但两组间差异无统计学意义(P>0.05)。pcDNA3.1-ICP27377-513联合pIL-15组IFN-γ、IL-4水平明显高于pcDNA3.1-ICP27377-513组(P<0.05),但IL-2水平两组间差异无统计学意义(P>0.05)。阴道给予致死量HSV-2攻击后,pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组小鼠阴道冲洗液中病毒载量随着时间延长逐渐减低,pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组相比差异有统计学意义(P<0.05)。结论IL-15核酸疫苗联合HSV-2-ICP27377-513核酸疫苗对小鼠有较好的免疫保护作用。

Regulation of Wnt/β-catenin signaling by herpesviruses

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.

Eosinophilic Meningitis in a Patient With Early HIV Infection,Latent Syphilis and Herpes Simplex Virus 2 Meningitis

Eosinophilic meningitis is an unusual condition,particularly in patients with HIV infection or syphilis.The detection of herpes simplex virus(HSV)-2 and HIV-1 in cerebrospinal fluid from a patient with acute eosinophilic meningitis has not been previously reported.Herein,we report a case of a 26-year-old cisgender man newly diagnosed with HIV-1 infection and latent syphilis,who presented with headache and low-grade fever.He was diagnosed with both acute eosinophilic meningitis and HSV-2 meningitis,and his condition improved after treatment with acyclovir and penicillin.The compelling case indicates that HSV-2 meningitis may manifest concurrently with acute eosinophilia meningitis in patients coinfected with HIV and Treponema pallidum.Metagenomics next-generation sequencing plays an important role in identifying and further confirming pathogens responsible for central nervous system infections.

川牛膝多糖硫酸酯的体外抗单纯疱疹病毒2型活性

从传统的川产道地中药材川牛膝中提取多糖RCP ,用氯磺酸 -吡啶法制备了它的硫酸酯衍生物S RCP .产物经红外鉴定 ,证实RCP硫酸酯化后羟基部分被硫酸基取代 ,在 12 5 0cm-1和 810cm-1处显示了硫酸基的特征吸收峰值 .w(S) =8.2 7% ,取代度Ds=0 .5 6 9.体外实验研究了RCP和S RCP对非洲绿猴肾细胞 (Verocellline)的细胞毒性和抗单纯疱疹病毒 2型 (HSV 2 )的活性 .细胞病变效应法 (CPE)和MTT法结果表明RCP在 6 .0mg/mL以下 ,S RCP在 4 .0mg/mL以下均无细胞毒性 ,RCP本身无抗病毒活性 ,经硫酸酯衍生后的S RCP获得抗病毒活性 ,0 .2 5～ 4 .0mg/mL的S RCP能很好地抑制HSV 2引起的细胞病变 .以 0 .5mg/mL的无环鸟苷 (ACV)作为阳性对照 ,结果显示当S RCP浓度为2 .0mg/mL和 4 .0mg/mL时有显著的抗病毒作用 ,效果与阳性ACV相当 .图 1表 8参 2

地塞米松联合阿昔洛韦对单纯疱疹病毒性脑炎小鼠预后及IL-2表达的影响

目的探讨地塞米松联合阿昔洛韦治疗单纯疱疹病毒性脑炎小鼠的预后,及其对IL-2表达的影响。方法颅内接种单纯疱疹病毒1型建立单纯疱疹病毒性脑炎小鼠模型,并设正常对照组、病毒感染组、阿昔洛韦组和地塞米松联合阿昔洛韦组。通过神经症状评分观察单纯疱疹病毒性脑炎小鼠的发病情况,并对其神经损伤症状进行评估;详细记录小鼠死亡的时间及数量,待病情不再发展时对各治疗组小鼠行生存率分析;应用免疫组化法测定小鼠脑组织中IL-2的表达并比较;采用Spearman相关分析描述神经症状评分与IL-2表达的相关性。结果病毒感染组小鼠神经症状评分最高、生存率最低,而地塞米松联合阿昔洛韦组神经症状评分最低、生存率最高,阿昔洛韦组则介于两者之间,四组间差异有统计学意义(P<0.05)。病毒感染组小鼠脑组织中IL-2的表达较正常对照组明显上调,阿昔洛韦组有所下降,地塞米松联合阿昔洛韦组明显下降,但仍高于对照组,四组间差异有统计学意义(P<0.05)。小鼠感染病毒第6天的神经症状评分与IL-2表达呈显著正相关(r=0.639,P=0.000)。结论阿昔洛韦联合糖皮质激素治疗小鼠单纯疱疹病毒性脑炎,较单纯抗病毒治疗生存率显著提高,临床症状减轻;单纯疱疹病毒性脑炎小鼠神经损伤程度与脑组织IL-2表达呈正相关。

白介素2基因佐剂协同单纯疱疹病毒1型糖蛋白D核酸疫苗免疫诱导的特异性免疫应答

目的研究白介素2(IL-2)cDNA协同单纯疱疹病毒1型(HSV-1)gD核酸疫苗免疫对机体体液免疫和细胞免疫应答的影响,以及在HSV-1病毒角膜攻击时的保护效果。方法利用pcDNA3.1载体分别构建HSV-1糖蛋白D和IL-2的真核表达质粒pgD和pIL-2,体外鉴定其表达。动物实验分为pcDNA3.1空载体对照组、pgD单独免疫组和pgD+pIL-2联合免疫组3组,具体为通过肌肉免疫接种BALB/c小鼠3次,间隔2周,每次接种质粒100μg,第3次免疫后2周,用酶联免疫吸附试验(ELISA)检测抗体滴度并做亚型分析,利用3H-TdR掺入法进行Th细胞增殖实验,ELISA试剂盒检测Th细胞分泌的IL-2、IFN-γ和IL-10水平,耳廓肿胀实验检测迟发型超敏反应(DTH)反应;HSV-1病毒角膜攻击后,裂隙灯显微镜观察角膜上皮病变。结果与pgD单独免疫组相比,pIL-2的协同免疫可以显著提高IgG2a抗体滴度、Th细胞增殖反应和DTH反应,Th细胞分泌IL-2和IFN-γ水平也显著提高,IL-10水平明显下降。在动物保护实验中,pIL-2的协同免疫明显增强了pgD疫苗在病毒攻击时对角膜的保护效果。结论IL-2cDNA的协同免疫可以显著提高pgD核酸疫苗诱导的体液免疫和细胞免疫应答水平,增加核酸疫苗的免疫保护效果。

小檗碱衍生物HB-13抗单纯疱疹病毒-2作用机制的研究

目的探讨小檗碱衍生物HB-13抗单纯疱疹病毒-2(HSV-2)的作用机制。方法采用细胞病变法检测HB-13是否对HSV-2有直接作用;空斑法检测HB-13是否抑制HSV-2的吸附和释放;荧光定量PCR检测HB-13是否影响HSV-2 DNA复制以及蛋白免疫印迹方法检测HB-13是否影响HSV-2蛋白质合成。结果HB-13在细胞外无直接杀灭HSV-2作用;对HSV-2的吸附和释放亦无影响;不抑制HSV-2 DNA复制;但HB-13浓度依赖性地抑制HSV-2蛋白质合成。结论HB-13可抑制HSV-2蛋白质合成,其抗HSV-2作用机制与ACV等核苷类药物不同。

敲减单纯疱疹病毒Ⅱ型(HSV-2)UL30蛋白表达可抑制HSV-2复制

按照shRNA(small hairpin RNA)设计要求,选择编码单纯疱疹病毒Ⅱ型DNA多聚酶催化亚单位的UL30(unique long 30,UL30)基因序列保守区域,设计、合成并构建表达UL30序列特异性siRNA(short interfering RNA)的质粒载体pUL30.通过磷酸钙转染法将其转染入HEK(human embryonic kidney)293细胞中,用蛋白印迹法检测对HSV-2 UL30蛋白表达的影响,观察受染细胞病变效应(cytopathic effect,CPE),终点滴定法测定细胞上清液中病毒感染滴度(50%tissue culture infective dose,TCID50).结果表明,针对UL30基因的siRNA能有效抑制UL30蛋白表达,同时显著抑制受染细胞的CPE,降低上清液中病毒感染滴度.提示本研究建立的针对UL30基因特异性siRNA能有效阻断HSV-2在HEK293细胞内的复制,UL30基因是一个潜在的抗HSV-2复制的药物靶标.