Developments towards antiviral therapies against enterovirus 71

Enterovirus 71 (EV71) has emerged as a clinically important neurotropic virus that can cause acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. Recurring outbreaks of EV71 have been reported in several countries. The current lack of approved anti-EV71 therapy has prompted intense research into antiviral development. Several strategies--ranging from target-based chemical design to compound library screenings--have been employed, while others revisited compound series generated from antiviral developments against poliovirus and human rhinoviruses. These efforts have given rise to a diversity of antiviral candidates that include small molecules and non-conventional nucleic-acid-based strategies. This review aims to highlight candidates with potential for further clinical development based on their putative modes of action.     

金银花-连翘药对抗肠道病毒71型的初步探究

目的采用体内体外的方法初步探讨金银花-连翘(以下简称"银翘")药对提取物对肠道病毒71型的抑制作用。方法采用MTT法检测"银翘"药对提取物对Hela细胞的毒性以及抗EV71作用;采用腹腔注射20μl EV71建立感染模型,观察小鼠的一般体征,并在光学显微镜下观察心脏组织病理学变化。结果 "银翘"药对提取物对Hela细胞的半数有毒浓度(TC_(50))为123.3 mg/ml,最大无毒浓度(TC_0)为15.6 mg/ml(按生药计)。无论同时加入药物和病毒,还是药物在病毒感染后加入均能够有效抑制病毒,并呈现出一定程度上的剂量依赖关系。体内抗病毒研究显示,中、高剂量组的"银翘"药对提取物均能显著改善小鼠的不良症状和心脏组织的病理变化,效果接近利巴韦林。结论 "银翘"药对提取物能有效地抑制EV71,为其临床应用提供理论依据。     

Antiviral activity of Rheum palmatum methanol extract and chrysophanol against Japanese encephalitis virus

Rheum palmatum, Chinese traditional herb, exhibits a great variety of anti-cancer and anti-viruses properties. This study rates antiviral activity of R. palmatum extracts and its components against Japanese encephalitis virus (JEV) in vitro. Methanol extract of R. palmatum contained higher levels of aloe emodin, chrysophanol, rhein, emodin and physcion than water extract. Methanol extract (IC₅₀ = 15.04 μg/ml) exhibited more potent inhibitory effects on JEV plaque reduction than water extract (IC₅₀ = 51.41 μg/ml). Meanwhile, IC₅₀ values determined by plaque reduction assay were 15.82 μg/ml for chrysophanol and 17.39 μg/ml for aloe-emodin, respectively. Virucidal activity of agents correlated with anti-JEV activity, while virucidal IC₅₀ values were 7.58 μg/ml for methanol extract, 17.36 μg/ml for water extract, 0.75 μg/ml for chrysophanol and 0.46 μg/ml for aloe-emodin, respectively. In addition, 10 μg/ml of extract, chrysophanol or aloe emodin caused 90 % inhibition of JEV yields in cells and significantly activated gamma activated sequence-driven promoters. Hence, methanol extract of R. palmatum and chrysophanol with high therapeutic index might be useful for development of antiviral agents against JEV.     

Spiramycin and azithromycin,safe for administration to children,exert antiviral activity against enterovirus A71 in vitro and in vivo

Hand-foot-mouth disease (HFMD) is a common viral disease in young children, mainly caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Specific antiviral agents are not commercially available yet. Here we report that the macrolide antibiotics spiramycin (SPM) and azithromycin (AZM) possess antiviral activities against EV-A71 and CV-A16. SPM significantly reduced EV-A71 RNA and protein levels, most likely through interfering with viral RNA replication. The SPM-resistant EV-A71 variants showed similar resistance to AZM, indicating a similar anti-EV-A71 mechanism by which these two drugs exert their functions. The mutations of these variants were reproducibly mapped to VP1 and 2A, which were confirmed to confer resistance to SPM. Animal experiments showed that AZM possesses stronger anti-infection efficacy than SPM, greatly alleviated the disease symptoms and increased the survival rate in a mouse model severely infected with EV-A71. In all, our work suggests that AZM is a potential treatment option for EV-A71-induced HFMD, whose proved safety for infants and children makes it even more promising.     

Development of a broad-spectrum antiviral with activity against Ebola virus

We report herein the identification of a small molecule therapeutic, FGI-106, which displays potent and broad-spectrum inhibition of lethal viral hemorrhagic fevers pathogens, including Ebola, Rift Valley and Dengue Fever viruses, in cell-based assays. Using mouse models of Ebola virus, we further demonstrate that FGI-106 can protect animals from an otherwise lethal infection when used either in a prophylactic or therapeutic setting. A single treatment, administered 1 day after infection, is sufficient to protect animals from lethal Ebola virus challenge. Cell-based assays also identified inhibitory activity against divergent virus families, which supports a hypothesis that FGI-106 interferes with a common pathway utilized by different viruses. These findings suggest FGI-106 may provide an opportunity for targeting viral diseases.     

The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell

Two homogeneous sulfated polysaccharides obtained from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata, the kappa/iota/nu carrageenan G3d and the dl-galactan hybrid C2S-3, were assayed for their antiviral properties against the four serotypes of dengue virus (DENV) in different host cell types. Both seaweed derivatives were selective inhibitors of DENV-2 multiplication in Vero cells with inhibitory concentration 50% (IC50) values around 1 microg/ml and selectivity indices > 1000. The compounds had a lower antiviral effect against DENV-3 (IC50 values in the range 13.9-14.2 microg/ml), an even lower effect against DENV-4 (IC50 values in the range 29.3 to > 50 microg/ml) and were totally inactive against DENV-1. With respect to the host cell, the polysulfates were inhibitors of DENV-2 and DENV-3 in the human hepatoma HepG2 and foreskin PH cells, with similar antiviral effectiveness as in Vero cells, but were totally inactive in mosquito C6/36 HT cells. Mechanistic studies demonstrated that G3d and C2S-3 were active DENV-2 inhibitors only when added together with the virus or early after infection, and both initial processes of virus adsorption and internalization are the main targets of these compounds. Therefore, the variations in antiviral activity of the polysaccharides depending on the viral serotype and the host cell may be ascribed to differences in the virus-cell interaction leading to virus entry.     

Natural antiviral activity of mouse macrophages against encephalomyocarditis virus

Resident mouse peritoneal cells (PC) express a significant antiviral activity against encephalomyocarditis virus (EMCV) in vitro, as judged by decreased virus yield from infected mouse embryo fibroblasts (MEF). This natural antiviral activity of PC was not due either to enhanced lysis of virus-infected cells, as these were protected from lysis rather than destructed by PC, or to interferon (IFN) production, as no direct correlation between IFN and anti-EMCV activity was found. Among PC, macrophages (M phi) appear to be responsible for the anti-EMCV activity, which was indeed attributable to a Thy 1.2-negative, adherent mononuclear cell. Moreover, M phi-defective C3H/HeJ mice showed a significant impairment of anti-EMCV activity, whereas M phi of mice defective for natural killer (NK) activity (bg/bg, SJL/J) or for mature T cells (nu/nu) possessed an intact antiviral capacity.     

Identification of fukinolic acid from Cimicifuga heracleifolia and its derivatives as novel antiviral compounds against enterovirus A71 infection

Human enterovirus 71 (EV-A71) infections cause a wide array of diseases ranging from diarrhoea and rashes to hand-foot-and-mouth disease and, in rare cases, severe neurological disorders. No specific antiviral drug therapy is currently available. Extracts from 75 Chinese medicinal plants selected for antiviral activity based on the Chinese pharmacopeia and advice from traditional Chinese medicine clinicians were tested for activity against EV-A71. The aqueous extract of the rhizome of Cimicifuga heracleifolia (Sheng Ma) and Arnebia euchroma (Zi Cao) showed potent antiviral activity. The active fractions were isolated by bioassay-guided purification, and identified by a combination of high-resolution mass spectrometry and nuclear magnetic resonance. Fukinolic acid and cimicifugic acid A and J, were identified as active anti-EV-A71 compounds for C. heracleifolia, whereas for A. euchroma, two caffeic acid derivatives were tentatively deduced. Commercially available fukinolic acid analogues such as L-chicoric acid and D-chicoric also showed in vitro micromolar activity against EV-A71 lab-strain and clinical isolates.     

EV71非结构蛋白的结构和功能及其为靶点的药物研究进展

近年肠道病毒71型(EV71)等引起的手足口病在中国大陆呈现上升的流行趋势,其正链的RNA基因组翻译成一个多聚蛋白,进一步自剪切为结构蛋白和非结构蛋白;随着研究的深入,非结构蛋白在病毒生命周期中的功能逐一被鉴定,本文就EV71非结构蛋白的结构功能及针对这些靶点的抗病毒药物进行综述。     

Protection of pigs against mosquito-borne Japanese encephalitis virus by immunization with a live attenuated vaccine

Pigs were vaccinated with an attenuated Japanese encephalitis virus (JEV) vaccine and challenged with virulent JEV, either by subcutaneous injection or by exposure to infected mosquitoes. The vaccinated pigs developed circulating antibodies to JEV. After challenge they did not develop viremia detectable by inoculation of their serum in suckling mice. They were also unable to transmit virus to mosquitoes fed on their skin. In contrast, unvaccinated pigs, whether challenged by injection or by mosquito bites, developed viremia and did transmit virus to mosquitoes which were allowed to bite them. Transmission seemed possible for only 3 days post-infection.     

In vitro antiviral activity of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose against hepatitis B virus

This study examined the antiviral activity of the root of Paeonia lactiflora PALL. Among the solvent fractions of the crude drug, the ethyl acetate fraction showed anti-hepatitis B virus (HBV) activity (IC50, 8.1 microg/ml) in an HBV-producing HepG2.2.15 cell culture system. The active anti-HBV principle was isolated and identified as 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) from the crude drug by activity-guided fractionation. PGG isolated from P. lactiflora was examined for the inhibition of HBV multiplication by measurement of HBV DNA and hepatitis B surface antigen (HBsAg) levels in the extracellular medium of HepG2.2.15 cells after 8-d treatment. PGG decreased the level of extracellular HBV (IC50, 1.0 microg/ml) in a dose-dependent manner. PGG also reduced the HBsAg level by 25% at a concentration of 4 microg/ml. The gallate structure of PGG may play a critical role in the inhibition of anti-HBV activity. These results suggest that PGG could be a candidate for developing an anti-HBV agent.     

Improved antiviral activity in vitro of ribavirin against measles virus after complexation with cyclodextrins

Despite vaccination, measles remains a burden in both developed and developing countries and complications may necessitate an efficient therapy. Measles virus (MEV) is susceptible to ribavirin (RBV), but the use of this drug is limited by its toxicity. Cyclodextrins (CDs) can form complexes with numerous molecules, improving their bioavailability and their biological properties. We have evaluated in vitro the antiviral effects of complexes of RBV with alpha-, beta- or gamma-CD against two clade A laboratory strains of MEV (Edmonston and CAM/RB) grown on Vero cells. Complexation of RBV with alpha-CD or beta-CD lead to a five-fold or a two-fold decrease in the 50% inhibitory concentration, respectively, against both MEV strains. In contrast, gamma-CD complexation showed no modification.     

Carbovir: the (-) enantiomer is a potent and selective antiviral agent against human immunodeficiency virus in vitro.

In this paper we describe the in vitro antiviral activity of the (-) enantiomer of carbocyclic 2',3'-deoxydidehydroguanosine, (-) carbovir, a nucleoside analogue that has selective and potent anti-HIV activity in a series of lymphocyte culture systems. The cellular cytotoxicity of this compound has also been evaluated in a number of systems and compared to the saturated dideoxynucleoside analogues AZT and ddC.     

Substituted benzaldehyde thiosemicarbazone with antiviral activity against poliovirus

A series of eight benzaldehyde thiosemicarbazone derivatives with a variable number of -OH substituents at different positions on the aromatic ring were prepared and evaluated for in vitro antiviral activity against poliovirus types I, II and III. Some of the compounds significantly inhibited the replication of poliovirus at a mean ED50 of 2, 5.7, 10.5 and 9.7 micrograms/ml. A comparison between chemical structure and biological activity suggests that the inhibitory effect depends on the relative position of the -OH and the thiosemicarbazone group. The compounds possess antiviral action only when the two groups are distant enough to prevent the formation of intramolecular hydrogen bonds.     

A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is emerging as a significant human pathogen in many regions of the world, including Africa, Asia, and Europe. In this report, we describe a simple screening method for discovering new antiviral compounds directed against CCHFV. Antiviral activity was determined by assaying infected SW-13 cells (human adrenal gland carcinoma) for protection from cytopathic effect (CPE). By using an in vitro neutral red uptake assay, we were able to quantitatively measure CPE induced by CCHFV. As a proof of concept, we used this method to evaluate the antiviral activity of ribavirin and a series of structural analogs (ribamidine, 6-azauridine, selenazofurin, and tiazofurin) against four geographically diverse strains of CCHFV. Ribavirin inhibited the replication of CCHFV as reported previously using plaque reduction assays. One drug, ribamidine, showed antiviral activity that was 4.5- to 8-fold less than that of ribavirin, and the other three drugs (6-azauridine, selenazofurin, and tiazofurin) did not show significant antiviral activity. There were no significant differences in drug sensitivities among the CCHFV strains. Development of this simple and reliable assay will potentially allow high-throughput screening for discovering additional antiviral drugs to combat this important public health threat.     

Synergistic antiviral activity of acyclovir and vidarabine against herpes simplex virus types 1 and 2 and varicella-zoster virus

Acyclovir and vidarabine both exhibit anti-herpetic activity. Because different mechanisms of action of vidarabine and acyclovir have been reported, we analyzed their combined anti-herpetic activity on plaque formation of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV) by isobolograms. The results indicate that acyclovir and vidarabine have a synergistic effect on wild type HSV-1, HSV-2, and VZV. The susceptibility of thymidine kinase-deficient HSV-1 to vidarabine was not affected by the presence of acyclovir, suggesting that phosphorylation of acyclovir is essential for synergism. The combined anti-HSV activity of acyclovir and vidarabine against phosphonoacetic acid-resistant HSV-1 with DNA polymerase mutation did not show synergism in contrast to that against wild-type herpesviruses. Alteration of the substrate specificity of viral DNA polymerase to acyclovir and vidarabine annihilated the synergism. Thus, the nature of their binding sites on DNA polymerase is important to the synergistic anti-herpesvirus activity of acyclovir and vidarabine.     

糖皮质激素联合抗病毒药物治疗单纯疱疹病毒性脑炎临床比较

目的 探讨糖皮质激素联合抗病毒药物治疗单纯疱疹病毒性脑炎临床效果。方法 80例单纯疱疹病毒性脑炎患者,按照随机数字表法分为观察组和对照组,每组40例。对照组患者接受抗病毒药物,观察组患者在对照组基础上加以糖皮质激素治疗。比较两组患者治疗第3、6、9天T淋巴细胞亚群值变化情况。结果 治疗第3、6天,两组患者的CD4^+、CD8^+和CD4^+/CD8^+水平比较差异无统计学意义(P>0.05);治疗第9天,观察组患者的CD4^+(31.44±5.16)%和CD4^+/CD8^+(0.96±0.33)均低于对照组的(36.40±3.91)%、(1.20±0.27), CD8^+(32.38±4.11)%高于对照组的(28.63±2.37)%,差异具有统计学意义(P<0.05)。结论 在单纯疱疹病毒性脑炎患者发病期给予糖皮质激素联合抗病毒药物治疗能够在短期内有效减轻患者急性炎性反应,改善免疫细胞损伤情况。     

Potent antiviral activity of brequinar against the emerging Cantagalo virus in cell culture

In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5 μM BQR reduced virus progeny production by >90%, revealing an EC₅₀ (drug concentration required to inhibit 50% of virus replication) of 0.017 μM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures.     

P1-substituted symmetry-based human immunodeficiency virus protease inhibitors with potent antiviral activity against drug-resistant viruses

Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.