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.

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.

The Herpes Simplex Virus Type 1 Multiple Function Protein ICP27

The herpes simplex virus type 1 （HSV-1） infected-cell protein 27 （ICP27） is an essential, highly conserved protein involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection. It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts. Recently, many novel functions performed by the HSV- 1 ICP27 protein were shown, including leptomycin B resistance, inhibition of the type I interferon signaling, regulation of the viral mRNA translation and determining the composition of HSV-1 virions

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction with the UL13 kinase,mediates the phosphorylation of RNA polymerase Ⅱ.Both ICP22 and UL13 are required for the activation of cdc2,the degradation of cyclins A and B and the acquisition of a new cdc2 partner,the UL42 DNA polymerase processivity factor.The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase Ⅱα in an ICP22-dependent manner to promote L gene expression.In addition,ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase Ⅱ.

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.

Analysis of the Cellular Localization of Herpes Simplex Virus 1 Immediate-early Protein ICP22

Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 （ICP22）, in the nucleus by observing the localization of ICP22-EGFP fusion protein Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and is finally distributed to daughter cells. We subsequently constructed a mammalian cell expression system, which had tetracycline- dependent transcriptional regulators. Consequently, the location of ICP22-EGFP in the nucleus changed with distinct induction conditions. This suggests that the cellular location of ICP22 is also influenced by promoter regulation, in addition to its own structure. Our findings provide new clues for the investigation of transcriptional regulation of viral genes. In addition, the non-protease reporter system we constructed could be utilized to evaluate the role of intemal ribosome entry sites （IRES） on transcriptional regulation.

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

A Multiple Functional Protein:the Herpes Simplex Virus Type 1 Tegument Protein VP22

The herpes simplex virus type 1 （HSV-1） VP22, is one of the most abundant HSV-I tegument proteins with an average stoichiometry of 2 400 copies per virion and conserved among alphaherpesvirinae. Many functions are attributed to VP22, including nuclear localization, chromatin binding, microtubule binding, induction ofmicrotubule reorganization, intercellular transport, interaction with cellular proteins, such as template activating VP16, pU factor I （TAF-I） and nonmuscle myosin II A （NMIIA）, and viral proteins including pUS9 and pUL46, glycoprotein E （gE） and gD. Recently, many novel functions perform tegument protein ed by the HSV-1 VP22 protein have been shown, including promotion of protein synthesis at late times in infection, accumulation of a subset of viral mRNAs at early times in infection and possible transcriptional regulation function .

Herpes Simplex Virus Type 1 ICP27 Protein:Its Expression, Purification and Specific Antiserum Production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni 2+ -NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.

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.

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.

Human Herpes Virus Type 2 ( HSV2 ), Human Cytomegalovirus (HCMV) in the Male Genital Tract and Fertilization

The possibility of infection of the human male genital tract by human herpes virus type 2 (HSV2) or human cytomegalovirus (HCMV) is well established and their sexual transmission has been the object of many studies. Moreover, medically assisted procreation, which helps in numerous fertility problems, raises the question of new viral risks linked to the application of these new technologies. In this review, we shall consider current knowledge in terms of the presence of HSV 2 and HCMV in the different parts of the genital tract of immunocompetent or immunodepressed men. We shall also consider the possibility of viral transmission by the sexual act or by the various techniques used in medically assisted procreation. We shall describe studies in human beings and in animals.

Effect of Gardenia extract ZG on the adsorption quantity of herpes simplex virus type 1 (HSV-1)

To observe the effect of Gardenia extract ZG on the adsorption quantity of herpes simplex virus type 1 （HSV-1） so as to explore the mechanism of its antiviral activity, fluorescein isothiocyanate （FITC） was used as the fluorescent probe to label viruses and heparin sodium was used as control. Meanwhile, the effect of Gardenia extract ZG on the adsorption quantity on the surface of Hep-2 cells was determined by flow cytometry. It was demonstrated that adsorption of HSV-1 on the surface of Hep-2 cells exhibited the character of saturation and specificity and heparin sodium could prevent attachment of viruses on these ceils. These results are in accord with those reported previously. It was also proved that the manner of drug-use prior to adsorption or simultaneous use of drug and adsorption was better than adsorption prior to drug-use, and the inhibition rates of the former and latter manner were 84.76% and 82.92% respectively. Three manners of drug-use with Gardenia extract ZG were all effective to reduce the adsorption quantity of viruses, especially the manner of simultaneous use of drug and adsorption with an adsorption inhibition rate of 68.46%. From the above observation, it is apparent that the mechanism of anti-viral activity of Gardenia extract ZG may be via several steps involved in the HSV-1 adsorption.

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。

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.

Current Status of Natural Products from Plants as Anti-herpes Simplex Virus 1 Agents

Nucleoside analogues have been the mainstay of clinical treatment of herpes simplex virus 1 (HSV-1) infections since their development. However, the emergence of drug resistant strains has underlined the urgency of the discovery of novel anti-HSV-1 drugs. Natural products, which provided many novel drug leads, are known to be an important source of anti-HSV-1 agents. Herein, we present an overview of natural products with anti-HSV-1 activities isolated from a variety of plants reported in recent years. Several different compounds, mainly belonging to the three groups of polysaccharides, polyphenols and terpenes, showed antiviral effects against HSV-1, indicating their potential to be promising anti-HSV-1 agents.

PNKP Knockdown by RNA Interference Inhibits Herpes Simplex Virus-1 Replication in Astrocytes

Herpes simplex virus-1 (HSV-1) is a major pathogen that causes various central nervous system (CNS) diseases,including herpes simplex encephalitis and meningitis.According to recent studies,PNKP significantly affects the proliferation of HSV-1 in astrocytes.Here,we used viral proliferation curves to confirm the significant inhibitory effects of PNKP on HSV-1 proliferation.PNKP downregulation was also confirmed by analyzing the transcription of viral genes.We found that PNKP downregulation affects the viral DNA copy number.This study preliminarily confirms that PNKP affects viral proliferation by affecting HSV-1 genome cyclization.These results also suggest that astrocytes play a specific role in preventing HSV-1 infection.

Herpes simplex virus type 1 in peptic ulcer disease: An inverse association with Helicobacter pylori

AIM： To assess the frequency of herpes simplex virus type I in upper gastrointestinal tract ulcers and normal mucosa with the modern and better assays and also with a larger number of well characterized patients and controls and its relationship to Helicobacter pylori（H pylori）. METHODS： Biopsy specimens from 90 patients （34 with gastric ulcer of the prepyloric area and 56 with duodenal ulcer） were evaluated. Biopsies from 50 patients with endoscopically healthy mucosa were considered as the control group. The method used to identify herpes simplex virus-1 （HSV-1） was polymerase chain reaction. Hpylori was detected by the CLO-test and by histological method. RESULTS： Herpes simplex virus-1 was detected in 28 of 90 patients with peptic ulcer （31%） Ⅲ of 34 patients with gastric ulcer （32.4%） and 17 of 56 with duodenal ulcer （30.4%）1 exclusively close to the ulcerous lesion. All control group samples were negative for HSV-1. The likelihood of Hpylori negativity among peptic ulcer patients was significantly higher in HSV-1 positive cases than in HSV-1 negative cases （P = 0.009）. Gastric ulcer patients with HSV-1 positivity were strongly associated with an increased possibility of Helicobacter pylori negativity compared to duodenal ulcer patients （P = 0.010）. CONCLUSION： HSV-1 is frequent in upper gastrointestinal tract ulcers but not in normal gastric andduodenal mucosa. There is an inverse association between HSV-1 and Hpylori infection.

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.