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.

Antiviral activity of five Asian medicinal pant crude extracts against highly pathogenic H5N1 avian influenza virus

Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcuma longa(C. longa),Gynostemma pentaphyllum, Kaempferia parviflora(K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.Results: The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants,C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-a and IFN-b m RNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.Conclusions: To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.

Synthesis and in vitro Anti-hepatitis B Virus Activity of Some Ethyl 5-Hydroxy-4-substituted Aminomethyl-2-sulfinylmethyl-1H-indole-3-carboxylates

A novel series of ethyl 5-hydroxy-4-substituted aminomethyl-2-sulfinylmethyl-lH-indole-3-carboxylates 8a--8j and 11e--11f was synthesized and evaluated in HepG2.2.15 cells for their anti-hepatitits B virus（HBV） activity and cytotoxicity. Among them, six compounds showed more potent inhibitory activity than lamivudine. Compound 8e exhibited the most significant anti-HBV activity with an IC50 value of 1.62 μmol/L, which was 33-times more potent than the reference drug lamivudine（IC50=54.78μmol/L）.

Expression,Purification and Activity Detection of Structural Protein VP1 of Foot-and-Mouth Disease Virus Serotype A

The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.

Synthesis and in vitro-Anti-hepatitis B Virus Activities of Several Ethyl 5-Hydroxy-1H-indole-3-carboxylates

A series of ethyl 5-hydroxyindole-3-earboxylates 6a-10r was designed and synthesized. The structures of all the compounds were confirmed by IR, ^1H NMR, and MS and their anti-hepatitis B virus （HBV） activities were evaluated in 2.2.15 cells. Among them, compound 7g { ethyl 5-hydroxy-2- [ （ 3-methoxyphenylsulfinyl ） methyl ] -1-methyl-4- [ （4-methylpiperazin-1-yl） methyl ]-1H-indole-3-carboxylate} displays a significant anti-HBV activity, which is more potent than the positive control lamivudine.

Hepatitis C Virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-α through dephosphorylation

AIM： To study the effect of Hepatitis C virus nonstructural 5A （HCV NSSA） on IFNα induced signal transducer and activator of transcription-1 （STAT1） phosphorylation and nuclear translocation.METHODS： Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NSSA-expressed and non-HCV NSSA-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells.RESULTS： Expression of HCV NS5A was found in the cytoplasm of pCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells, STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction, STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to pRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot.CONCLUSION： HCV NSSA expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.

The anti-HIV-1 activity of polyphenols from Phyllanthus urinaria and the pharmacokinetics and tissue distribution of its marker compound, gallic acid

Objective:To investigate the in vitro anti-HIV-1 activities and its associated mechanism of action of an extract isolated from Phyllanthus urinaria (P.urinaria) and to develop an HPLC test method for detecting gallic acid (GA) in plasma and tissues to study its pharmacokinetics and tissue distribution in rats.Methods:An extract of P.urinaria was isolated and purified by phytochemistry and chromatography techniques.The anti-HIV-1 activities and toxicities of the extract and its component GA were determined in human T lymph cells (MT-4) by theMTTr method.The mechanism of its anti-HIV-1 action was studied to examine the in vitro binding of its components with HIV-1 target proteins by Biacore technique.The pharmacokinetics and tissue distribution of GA were investigated after oral administration of polyphenol extract (PE) and pure GA in rats.The concentrations of GA in plasma and tissues were determined by HPLC.Results:The PE and GA isolated from P.urinaria had anti-HIV-1 activities with IC50s of 0.61 μg/mL and 0.76 μg/mL,respectively.The Biacore study indicated that PE and GA interacted with HIV-1 RT,gp120,and P24.The pharmacokinetic parameters Tmax,Cm ax,AUC0-t,and T1/2 for GA were (60.0 ± 3.0) minutes,(2.87 ± 0.50) μg·mL-1,(343.5 ± 11.2) mg·min·L-1,and (113.3 ± 9.3) minutes while the parameters for GA in the PE were (10.0 ± 1.3) minutes,(3.89 ± 0.90) μg·mL-1,(394.7 ± 14.0) mg· min· L-1,and (81.7 ± 4.1) minutes,respectively.GA was detected in rat lungs,liver,kidneys,heart and spleen.Conclusion:APE isolated from P.urinaria containing GA has anti-HIV-1 activities.GA is quickly absorbed and slowly eliminated in rats after oral administration.The pharmacokinetics of GA administered as a PE is desirable,and it is widely distributed in the main tissues of lung and liver.Both its properties and anti-HIV-1 activities make it of interest for further studies.

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.

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.

Cost and safety of assisted reproductive technologies for human immunodeficiency virus-1 discordant couples

Due to significant advances in the treatment of human immunodeficiency virus type-1(HIV-1), HIV-1 infection gradually has become a treatable chronic disease. Successfully treated HIV-positive individuals can have a normal life expectancy. Hence, more and more HIV-1 discordant couples in Taiwan and the rest of the world are seeking fertility assistance. Pre-treatment of highly active antiretroviral therapy(HAART) combined with sperm washing and RT-polymerase chain reaction examination for HIV-1 viral load has become the standard procedure to assist them to conceive. However,in order to reduce the transmission risk to the lowest level for the couple and to diminish the cost of health care for the insurance institutes or government, in vitro fertilization(IVF)-intracytoplasmic sperm injection(ICSI) therapy provides the ideal solution for HIV-1 discordant couples with infected men. Intrauterine insemination(IUI) theoretically introduces more than 107 times of sperm counts or semen volume to uninfected women vs IVF-ICSI. However, since some regimens of HAART may significantly decrease the sperm motility, compared to IVF-ICSI, IUI only produces 1/5 to 1/2 pregnancy rates per cycle. Given the risk of seroconversion of HIV infection which actually happens after successful treatment, IVF-ICSI for these HIV-1 seropositive men is more cost-effective and should be the first line treatment for these cases.

Preparation and Characterization of Monoclonal Antibodies against VP1 Protein of Foot-and-mouth Disease Virus O/China99

Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

Synthesis of 6-sulfamoyl-4-oxoquinoline-3-carboxylic acid derivatives as integrase antagonists with anti-HIV activity

A series of novel 6-sulfamoyl-4-oxoquinoline-3-carboxylic acids derivatives have been synthesized and screened for HIV integrase inhibition activity. Their structures were confirmed by ESI-MS, ^1H NMR and ^13C NMR. 2009 Li Ming Hu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

In Vitro Anti-HIV Activity of a Chinese Fungus Extract

Objective To investigate the inhibitory effect of the mycelium extract from a Chinese fungus （MI） on HIV-I and its mode of action. Methods Several in vitro methods including time of action, time of addition and PCR were used to test the mode of action of M 1. Results M 1 inhibited acute HIV infection in vitro and was effective when it was added 12 h after infection. PCR analysis of infected cells demonstrated that MI delayed the appearance of late product of reverse transcription and HIV was blocked before its RNA expression. Conclusion The target of M 1 is post-integration of proviral DNA.

Autoimmune hepatitis in a patient infected by HIV-1 and under highly active antiretroviral treatment:Case report and literature review

Liver disease has recently been described as an im-portant cause of morbidity and mortality in patientsinfected with human immunodeficiency virus （HIV）.Liver test changes are useful surrogates of the burdenof liver disease. Previous studies have shown that trans-aminase elevations are frequent among these patients.The cause of those changes is harder to establish inHIV-patients. We present a 61-year-old caucasian male,diagnosed with HIV type 1 infection since 1998, underhighly active antiretroviral treatment （HAART）, withvirological suppression and immunological recovery.He presented in a follow-up laboratory workup highvalues of transaminases, arthralgia at the hip joints and hepatomegaly. Liver function tests were normal. Theantibodies to hepatitis viruses were negative. However,autoimmune study and liver biopsy were compatible with autoimmune hepatitis （AIH）. The AIH is a rare di-agnosis in HIV-infected patients perhaps because the elevation of transaminases and changes in liver function tests are often associated to HAART or to other possible liver diseases, namely viral hepatitis and non-alcoholic steatohepatitis. The diagnosis may be underestimated. There are no specifc recommendations available for the treatment of HIV-associated AIH although the immuno-supression with slower tapering seems the most reason-able approach.

水芹总酚酸体外抗HIV-1的作用

目的探讨水芹总酚酸体外抑制人类免疫缺陷病毒1型的作用。方法用不同稀释度的水芹总酚酸与TZM-bl细胞共同培养,使用MTT法检测活细胞的数量,观察水芹总酚酸对TZM-bl细胞的毒性作用;用人类免疫缺陷病毒1型实验室适应株SF33、BAL分别感染TZM-bl细胞,基于TZM-bl细胞的荧光素酶检测体系,采用荧光素酶活性检测方法检测水芹总酚酸抗人类免疫缺陷病毒1型的活性。结果水芹总酚酸对TZM-bl细胞表现出较低的细胞毒性,半数细胞毒浓度>1600μg·ml^(-1)。水芹总酚酸对TZM-bl细胞HIV-1 SF33、HIV-1 BAL病毒的半抑制浓度平均值分别为38.93和24.25μg·ml^(-1),治疗指数分别为>41和>66。结论水芹总酚酸在体外具有抑制人类免疫缺陷病毒1型复制活性的作用。

EB病毒LMP-1上调鼻咽癌细胞系AP-1的活性

为了探讨 EB病毒潜伏膜蛋白 1 ( LMP- 1 )通过信号传导途径介导的致瘤分子机制 ,由此首先构建了 AP- 1报告基因 ,用佛波酯诱导确定了其报道 AP- 1的功能 ;通过建立荧光素酶双报道系统 ,研究了 LMP- 1表达对 AP- 1和 NFκB活性的影响 .研究发现 :在 LMP- 1阴性鼻咽癌 ( NPC)细胞系 ,导入 LMP- 1表达质粒后 ,AP- 1和 NFκB的活性均升高 4～ 5倍 ;而在 LMP- 1阳性 NPC细胞系中 ,当导入 LMP- 1反义表达质粒 ,AP- 1和 NFκB的活性则受抑制 ,活性下调 3～ 4倍 .结果表明 ,LMP- 1能上调 NPC细胞系 AP- 1的活性 ,同时再次证实了 LMP- 1能活化 NFκB.

人参皂苷Rg3体外抗HSV-1活性与免疫调节效应

目的：研究人参皂苷Rg3体外对单纯疱疹病毒1型（HSV-1）的抑制作用与免疫调节效应。方法：采用结晶紫染色法观察Rg3对HSV-1致细胞病变效应的抑制作用。用MTT比色法检测Rg3对Vero细胞和小鼠脾细胞增殖的影响。以Rg3诱导小鼠脾细胞，用鼠脾T淋巴母细胞增殖分析法测定上清中IL-2活性，细胞病变抑制法测定IFN-γ活性。结果：浓度在1．56～25mg·L^-1的Rg3对HSV-1致细胞病变效应抑制作用显著高于病毒对照组（P〈0．05）；浓度小于25mg·L^-1的Rg3对传代Vero细胞及小鼠脾细胞的增殖作用与细胞对照组比较差异无显著性（P〉0．05）；3．13～6．25mg·L^-1的Rg3体外诱生IL-2和IFN-γ分泌水平显著高于对照组（P〈0．05）。结论：一定浓度的Rg3具有体外抗HSV-1活性，对传代Vero细胞及小鼠脾细胞无毒性，可显著促进IL-2和IFN-γ分泌，参与免疫调节效应。

EB病毒潜伏膜蛋白1通过TRAF/TRADD激活JNK信号途径

为了探讨在鼻咽癌细胞中EB病毒编码的潜伏膜蛋白 1(LMP1)激活c Jun氨基端激酶 (JNK)信号途径的分子机制 ,利用可调控表达LMP1的鼻咽癌细胞系L7,蛋白质印迹检测 ,发现LMP1能够促进JNK的活化 ;利用稳定表达LMP1的鼻咽癌细胞系HNE2 LMP1及其三种突变体HNE2 LMP1ΔCTAR 1、HNE2 LMP1ΔCTAR2、HNE2 LMP1ΔCTAR 1,2及LMP1阴性的HNE2 为材料 ,采用蛋白质印迹和报告基因法分析JNK和活化蛋白 1(AP1)活化情况 ,结果显示HNE2 LMP1和HNE2 LMP1ΔCTAR1中磷酸化JNK蛋白表达量和AP1活性都无显著差异 ,而与HNE2 LMP1ΔCTAR2、HNE2 LMP1ΔCTAR1,2、阴性对照HNE2及空白载体转染细胞的JNK蛋白表达和AP1活性具有显著差异 ;进一步比较转染TRAF、TRADD显性负性突变体鼻咽癌细胞系HNE2 LMP1中磷酸化的JNK量和AP1活性 ,结果显示 :TRAF DN和TRADD DN的导入使活化的JNK蛋白和AP 1活性显著降低 ,二者间无显著差异 ,提示TRAF和TRADD可能参与了LMP1对JNK和AP 1的活化 .以上结果提示在鼻咽癌细胞系中LMP1功能结构域CTAR2通过结合TRAF/TRADD激活JNK从而活化重要的转录因子AP1.

栀子提取物ZG对副流感病毒1型感染后宿主细胞膜的影响

为了探讨栀子提取物ZG抗病毒作用的生物学机制,观察了栀子提取物ZG对副流感病毒1型(PIV-1)感染后宿主细胞膜电位、膜Na+-K+-ATP酶活性和膜流动性的影响。以氯化乙酰胆碱为阳性对照,采用荧光探针Di-BAC4(3)标记Hep-2细胞膜电位,借助流式细胞仪检测膜电位;定磷法,分光光度计检测Na+-K+-ATP酶活性;荧光探针NBD-C6-HPC标记细胞膜磷脂,以荧光漂白恢复法和激光扫描共聚焦显微镜检测膜流动性。结果显示:PIV-1感染后宿主细胞膜电位下降,处于超极化状态;膜Na+-K+-ATP酶活性显著增加,膜流动性显著降低。栀子提取物ZG作用后,对宿主细胞膜的超极化状态没有明显影响;对膜Na+-K+-ATP酶活性没有明显影响;而对膜流动性则有明显的恢复作用。阳性对照药乙酰胆碱能明显改善病毒感染后膜电位的超极化状态。PIV-1感染后膜电位、Na+-K+-ATP酶活性和膜流动性等细胞膜能态和功能的改变,可能为病毒感染的生物学机制之一;栀子提取物ZG可能是通过改善细胞膜流动性,维持细胞膜的正常功能来发挥抗病毒感染的作用,而与膜电位和膜Na+-K+-ATP酶活性等能态来源的环节可能无关。

新型抗HIV-1蛋白GRFT的构建、表达及活性研究

根据已知的新型抗HIV-1蛋白Griffithsin(GRFT)基因氨基酸序列,推测其DNA编码序列,对密码子优化及修饰后进行全基因化学合成,连接到原核表达载体pET28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达,获得目的蛋白.SDS-PAGE和Western Blot分析结果表明,目的蛋白得到良好表达并具有抗原活性.对表达条件进行了优化,在最佳表达条件下,目的蛋白的表达量可占菌体总蛋白的55.84%.利用Ni2+-NTA柱亲和层析法进行目的蛋白的复性和纯化,凝胶成像系统扫描分析表明,其纯度可达94.06%.运用Dot-ELISA法进行复性蛋白的抗原结合活性检测发现,目的蛋白可与HIV-1膜蛋白抗原特异性结合,初步证明所表达的重组蛋白具有良好的体外结合活性.基于制备的能够表达HIV-1 gp120基因的靶细胞模型,运用IFA法开展目的蛋白的细胞结合活性研究,结果发现,目的蛋白能够与靶细胞发生特异性反应,表明成功制备了具有生物活性的新型抗HIV-1蛋白GRFT,为进一步研制新型抗HIV-1基因工程药物及其靶向治疗制剂奠定了基础.