单纯疱疹病毒2型疫苗的研究进展

单纯疱疹病毒2型(herpes simplex virus type 2,HSV-2)是人类疱疹病毒家族中α家族成员,可引起多年龄段的人群生殖器疱疹及其他疱疹疾病。由于该病毒具有复杂的基因结构及感染病理,其感染至今尚无有效的治疗方法。该病毒可抑制机体免疫系统,在神经细胞潜伏感染,具有重激活特性,因此,其疫苗的研制亦需要复杂的技术,且面临重重的挑战。     

溶瘤I型单纯疱疹病毒的研究进展

溶瘤病毒(Oncolytic virus,OV)是可以靶向感染并杀伤肿瘤细胞的一类病毒,其中溶瘤I型单纯疱疹病毒(Oncolytic herpes simplex virus type 1,OHSV-1)是目前研究最多的溶瘤病毒之一,可通过多种策略进行构建,已有多种OHSV-1进入临床试验,大量结果显示其具有较好的安全性和有效性。本文主要介绍OHSV-1的分子生物学特性与优势、主要的开发及靶向性策略、各类OHSV-1的研究进展以及目前存在的问题等。     

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.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.     

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.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

us3基因的缺失对单纯疱疹病毒1型毒力和抗凋亡功能的影响

利用CRISPR/Cas9系统使单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1) ul7、ul41、LAT 基因缺失构建M3减毒株(M3株),在M3株基础上通过缺失 us3 得到M4突变株(M4株)。本研究旨在分析野毒株(McKrae株)、M3株与M4株在毒力和抗细胞凋亡方面的差异。结果表明,McKrae组出现明显的临床症状,且100%死亡(P<0.001),而M3、M4组未出现临床症状。M4组小鼠组织中病毒载量明显低于McKrae组和M3组;病理学检测表明,McKrae组出现蛛网膜出血、胶质小结等现象,而M3、M4组未见病理损伤,M4组炎性因子表达与McKrae、M3组相比也显著下降(P<0.01);免疫后M4组较M3组出现高水平的中和抗体、γ干扰素(interferon γ,IFN-γ)和白细胞介素4(interleukin 4,IL-4)抗原特异性T细胞;McKrae株再次感染时,M4组小鼠组织中病毒载量明显低于对照组和M3组;在人急性T细胞淋巴瘤细胞中,M4株相比McKrae株和M3株可明显诱导细胞凋亡。     

Pathogenesis of herpes simplex virus type 2 experimental genital infection in pregnant mice

The progression of herpes simplex-2 genital infection in pregnant mice was studied by detection of viral antigens using immunoperoxidase in tissue sections, electron microscopy and virus isolation. The majority of mice (66.66%) died at 8-9 days post-inoculation. Abortions were observed in 69.23% of the infected mice along with impairment of labor and delivery. Herpes antigens were detected in most of the autonomic nerves of the uterus, including those surrounding small arterioles in the myometrium and the Auerbach and Meissner plexa of the large bowel, but not in the abortions or placentas. The infection of uterine autonomic fibers and myometrial cells could explain the delivery impairment and could have provoked a decrease in blood flow leading to abortions.     

Chemical structure and antiviral activity of carrageenans from Meristiella gelidium against herpes simplex and dengue virus

Chemical and spectroscopic methods showed that the major KCl-precipitated galactans from Meristiella gelidium (Solieriaceae) are iota/kappa/nu-hybrid carrageenans with the former one in higher proportion. These carrageenans showed, by HPSEC-MALLS analysis, unimodal symmetrical peaks with MW of 425.6–956.7 kDa. The effectiveness of the crude extracts from M. gelidium against HSV-2 was higher than the corresponding extract from G. griffithsiae, previously determined. However, when considering the homogeneous carrageenans, the fractions obtained from both seaweeds showed the same level of activity. The extracts and carrageenan derived from M. gelidium were more effective inhibitors of DENV-2 if compared with G. griffithsiae samples and reference polysaccharides. The most active fraction obtained from M. gelidium showed a selectivity index against HSV-2 of 25,000, a value high enough to consider this carrageenan as a promising agent to be evaluated for the treatment of genital HSV-2 infections.     

T Cell Receptor Signaling Pathways： New Targets for Herpes Simplex Virus

Herpes simplex viruses (HSV-1 and HSV-2) cause global morbidity and synergistically correlate with HIV infection.HSV exists life-long in a latent form in sensory neurons with intermittent reactivation,in despite of host immune surveillance.While abundant evidence for HSV interfering with innate immune responses so as to favor the replication and propagation of the virus,several lines of evidence declare that HSV attenuates adaptive immunity by various mechanisms,including but not limited to the ablation of antigen presentation,induction of apoptosis,and interruption of cellular signaling.In this review,we will focus on the perturbative role of HSV in Tcells signaling.     

Intercellular trafficking of herpes simplex virus type 2 UL14 deletion mutant proteins

The UL14 gene product of herpes simplex virus is a 32kDa protein expressed late in infection and is a minor component of the virion tegument. We recently showed that the wild-type UL14 protein has heat shock protein (HSP)-like and/or molecular chaperone-like functions. In this study, the intracellular localization of UL14 wild-type and deletion mutant proteins was examined in transfected cells by immunofluorescence. We found that N-terminus deleted but not wild-type/C-terminus deleted mutant proteins showed a significant number of cytoplasmic, multi-cellular stains in transfected Vero cells. The effect was greatly intensified by subjecting cells to heat shock at 43 degrees C, whereas it was obstructed by treatment with the microfilament-disrupting drug cytochalasin D. The staining patterns of UL14 antigen-positive cells after heat shock suggested a cell-to-cell spread of the protein. Although the mechanism is unclear, the phenomenon seems to be an unprecedented type of intercellular trafficking.     

单纯疱疹病毒2型潜伏相关转录体ORF1的表达及其抗凋亡作用

旨在研究单纯疱疹病毒2型潜伏相关转录体 (LAT) 开放读码框1 (ORF1) 对放线菌素D诱导的凋亡作用的影响。以HSV-2 333基因组为模板PCR扩增ORF1片段,构建重组质粒pEGFP-ORF1,转染Vero细胞,RT-PCR鉴定ORF1的表达。放线菌素D诱导Vero细胞凋亡,通过荧光显微镜观察凋亡小体,Hochest33258荧光染色观察细胞形态变化,MTT检测细胞活性,流式细胞术检测细胞凋亡率。双酶切和测序确认pEGFP-ORF1构建成功,RT-PCR表明该真核表达载体能在Vero细胞中高效表达。转染了pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后,Hochest33258染色显示细胞形态正常。MTT结果表明转染了重组质粒pEGFP-ORF1的Vero细胞经放线菌素D凋亡诱导后Vero细胞活性与未经任何处理的正常对照组相比,无显著差异 (P＞0.05),但高于放线菌素D诱导凋亡的Vero细胞组及与转染空质粒pEGFP-C2且放线菌素D诱导凋亡的Vero细胞组,差异具有统计学意义 (P＜0.05)。流式结果表明,转染重组质粒pEGFP-ORF1且经放线菌素D诱导凋亡组与正常对照组凋亡率差异不显著 (P＞0.05),而显著低于放线菌素D诱导凋亡组和转染空质粒pEGFP-C2且经放线菌素D诱导凋亡组 (P＜0.05)。HSV-2 LAT ORF1具有抗放线菌素D诱导的Vero细胞的凋亡作用。     

Antiviral activity of recombinant cyanovirin-N against HSV-1

In this study, a standard strain of HSV-1 (strain SM₄₄) was used to investigate the antiviral activity of the recombinant Cyanovirin-N (CV-N) against Herpes simplex virus type 1 (HSV-1) in vitro and in vivo. Cytopathic effect (CPE) and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells. The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The results showed that CV-N had a low cytotoxicity on Vero cells with a CC₅₀ of 359.03±0.56 μg/mL, and that it could not directly inactivate HSV-1 infectivity. CV-N not only reduced the CPE of HSV-1 when added before or after viral infection, with a 50% inhibitory concentration (IC₅₀) with 2.26 and 30.16μg/mL respectively, but it also decreased the copies of HSV-1 DNA in infected host cells. The encephalitis model for HSV-1 infection was conducted in Kunming mice, and treated with three dosages of CV-N (0.5, 5 &; 10 mg/kg) which was administered intraperitoneally at 2h, 3d, 5d, 7d post infection. The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination (HE staining). Compared with the untreated control group, in the 5mg/kg CV-N and 10mg/kg CV-N treated groups, the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively. HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups, the brain cells did not show visible changes, except for a slight inflammation. Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo, and it would be a promising new candidate for anti-HSV therapeutics.     

Antiviral property and mechanisms of a sulphated polysaccharide from the brown alga Sargassum patens against Herpes simplex virus type 1

A sulphated polysaccharide (SP-2a) from the brown alga Sargassum patens (Kütz.) Agardh (Sargassaceae) was found to significantly inhibit the in vitro replication of both the acyclovir (ACV)-sensitive and -resistant strains of Herpes simplex virus type 1 (HSV-1), in dose-dependent manners, with 50% inhibitions occurring with 1.5–5.3 μg/ml of the polysaccharide. SP-2a exhibited extracellular virucidal activity only against the ACV-sensitive strains, but not the resistant strain, at the concentration of 100 μg/ml. The strongest antiviral activities against the different strains of HSV-1 were observed when this polysaccharide was present during and after adsorption of the virus to host cells. The inhibitory effect of SP-2a on virus adsorption occurred dose-dependently in all the HSV-1 strains tested, and the adsorption of the ACV-resistant DM2.1 strain was reduced by 81.9% (relative to control) with 4 μg/ml of the polysaccharide. This study clearly demonstrated that the antiviral mode of action of SP-2a is mediated mainly by inhibiting virus attachment to host cells, and this sulphated polysaccharide might have different modes of action against the ACV-sensitive and -resistant strains of HSV-1.     

单纯疱疹病毒II型糖蛋白G主要抗原表位的表达和抗原性检测*

利用PCR拼接技术,合成含单纯疱疹病毒II型糖蛋白G(gG2)抗原表位（氨基酸序列第561～578位）的片段,并进一步利用基因工程技术获得该表位的双拷贝片段,克隆入pET-KDO表达载体进行原核表达。经IPTG诱导后,高效表达出分子量大小约为39,000D的融合蛋白,经Western-blot检测具有良好的抗原性。表达的融合蛋白经凝血酶切割和亲和层析纯化,得到双拷贝gG2（561～578aa）目的蛋白,经ELISA检测具有良好的灵敏度和特异性。该重组抗原的构建和表达可用于HSV-2特异性血清学诊断的研究。     

1,2,4-Triazoloazine derivatives as a new type of herpes simplex virus inhibitors

A new class of inhibitors of herpes simplex virus replication was found. The compounds under study are derived from condensed 1,2,4-triazolo[5,1-c][1,2,4]triazines and 1,2,4-triazolo[1,5-a]pyrimidines, structural analogues of natural nucleic bases. Antiherpetic activity and cytotoxicity of the compounds were studied. The corresponding triphosphates of several active compounds were prepared and tested as inhibitors of DNA synthesis catalyzed by herpes simplex virus polymerase. The potential mechanism of their action is blocking of DNA dependent DNA polymerase, a key enzyme of viral replication.     

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.     

Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis

A new NADH oxidase, useful for the regeneration of NAD⁺, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10–15 U mg^–1. After purification by four chromatographic steps, an activity of 116 U mg^–1 was obtained with 14% yield. Highest activity was from pH 5.5–7 and at 40°C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The K _m value for NADH was 24 M.