Discovery and optimization of 2-((1H-indol-3-yl)thio)-N-benzyl-acetamides as novel SARS-CoV-2 RdRp inhibitors.

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic coronavirus disease (COVID-19), but no specific antiviral drug has been proven effective for controlling this pandemic to date. In this study, several 2-((indol-3-yl)thio)-N-benzyl-acetamides were identified as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors. After a two-round optimization, a new series of 2-((indol-3-yl)thio)-N-benzyl-acetamides was designed, synthesized, and evaluated for SARS-CoV-2 RdRp inhibitory effect. Compounds 6b2, 6b5, 6c9, 6d2, and 6d5 were identified as potent inhibitors with IC50 values of 3.35 ± 0.21 µM, 4.55 ± 0.2 µM, 1.65 ± 0.05 µM, 3.76 ± 0.79 µM, and 1.11 ± 0.05 µM, respectively; the IC50 of remdesivir (control) was measured as 1.19 ± 0.36 µM. All of the compounds inhibited RNA synthesis by SARS-CoV-2 RdRp. The most potent compound 6d5, which showed a stronger inhibitory activity against the human coronavirus HCoV-OC43 than remdesivir, is a promising candidate for further investigation.

Recent advances of enterovirus 71 [Formula: see text] targeting Inhibitors.

With CA16, enterovirus-71 is the causative agent of hand foot and mouth disease (HFMD) which occurs mostly in children under 5 years-old and responsible of several outbreaks since a decade. Most of the time, HFMD is a mild disease but can progress to severe complications such as meningitis, brain stem encephalitis, acute flaccid paralysis (AFP) and even death; EV71 has been identified in all severe cases. Therefore, it is actually one of the most public health issues that threatens children's life. [Formula: see text] is a protease which plays important functions in EV71 infection. To date, a lot of [Formula: see text] inhibitors have been tested but none of them has been approved yet. Therefore, a drug screening is still an utmost importance in order to treat and/or prevent EV71 infections. This work highlights the EV71 life cycle, [Formula: see text] functions and [Formula: see text] inhibitors recently screened. It permits to well understand all mechanisms about [Formula: see text] and consequently allow further development of drugs targeting [Formula: see text]. Thus, this review is helpful for screening of more new [Formula: see text] inhibitors or for designing analogues of well known [Formula: see text] inhibitors in order to improve its antiviral activity.

New opportunities for designing effective small interfering RNAs.

Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.

Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus.

NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.

Antiviral effects and mechanisms of Yinhuapinggan granule against H1N1 influenza virus infection in RAW264.7 cells.

Yinhuapinggan granule (YHPG), a modified prescription based on Ma-Huang-Tang (MHT), is used in traditional Chinese medicine (TCM) to treat influenza, cough, and viral pneumonia. In this study, we investigated the antiviral effects of YHPG by means of pre-, post-, and co-treatment, and its underlying mechanisms on regulating the levels of inflammatory-related cytokines, modulating the mRNA expressions of interferon-stimulated genes in influenza virus-infected murine macrophage cells (RAW264.7), and evaluating the protein expressions of key effectors in the Type I IFN and pattern recognition receptor (PRRs) signaling pathways. The results showed that YHPG markedly inhibited influenza virus (IFV) replication in pre-, post- and co-treatment assay, especially in post-treatment assay. Antiviral mechanisms studies revealed that YHPG (500 and 250 µg/mL) significantly up-regulated levels of IFN-ß, IFN-stimulated genes (Mx-1, ISG-15 and ISG-56) compared with the IFV control group, while the levels of IL-6 and TNF-α were significantly down-regulated. Furthermore, western blot analysis results revealed that the protein expressions of the phosphorylated forms of TBK1, IRF3, ERK1/2, P38 MAPK and NF-κB p65 were significantly down-regulated in RAW264.7 cells with the YHPG (500 and 250 µg/mL) treatment, while the expression of the phosphorylated form of STAT1 was significantly enhanced. Based on these results, YHPG had antiviral effects in IFV-infected RAW264.7 cells, which might be associated with regulation of the inflammatory cytokines production, evaluation of the levels of IFN-stimulated genes, and modulation of the protein expressions of key effectors in the Type I IFN and PRRs signaling pathways.

Protein disulfide isomerases as potential therapeutic targets for influenza A and B viruses.

Seasonal flu as well as potential pandemic flu outbreaks continuously underscores the importance of the preventive and therapeutic measures against influenza viruses. During screening of natural and synthetic small molecules against influenza A and B virus, we identified juniferdin as a highly effective inhibitor against both viruses in cells. Since juniferdin is known to inhibit protein disulfide isomerases (PDIs), multiple PDI inhibitors were tested against these viruses. Among PDI inhibitors, 16F16, PACMA31, isoquercetin, epigallocatechin-3-gallate or nitazoxanide significantly reduced the replication of influenza A and B viruses in MDCK and A549 cells. Furthermore, siRNAs specific to three PDI family members (PDI1, PDIA3 or PDIA4) also significantly reduced the replication of influenza A and B viruses in cells. These results suggest that PDIs may serve as excellent targets for the development of new anti-influenza drugs.

Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment.

BACKGROUND: Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)-Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy. RESULTS: At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. 'Jinshui no. 2') shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5'-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C. CONCLUSIONS: Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein.

The hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors.

A Lead-In with Silibinin Prior to Triple-Therapy Translates into Favorable Treatment Outcomes in Difficult-To-Treat HIV/Hepatitis C Coinfected Patients.

BACKGROUND: The efficacy of first-generation protease inhibitor based triple-therapy against hepatitis C virus (HCV) infection is limited in HIV/HCV-coinfected patients with advanced liver fibrosis and non-response to previous peginterferon-ribavirin. These patients have a low chance of achieving a sustained virologic response (SVR) using first generation triple-therapy, with a success rate of only 20%. We investigated the efficacy and safety of lead-in therapy with intravenous silibinin followed by triple-therapy in this difficult-to-treat patient group. METHODOLOGY: Inclusion criteria were HIV/HCV coinfection with advanced liver fibrosis and documented previous treatment failure on peginterferon-ribavirin. The intervention was a lead-in therapy with intravenous silibinin 20 mg/kg/day for 14 days, followed by triple-therapy (peginterferon-ribavirin and telaprevir) for 12 weeks, and peginterferon-ribavirin alone for 36 weeks. Outcome measurements were HCV-RNA after silibinin lead-in and during triple-therapy, SVR data at week 12, and safety and tolerability of silibinin. RESULTS: We examined sixteen HIV/HCV-coinfected patients with previous peginterferon-ribavirin failure, of whom 14 had a fibrosis grade METAVIR ≥F3. All were on successful antiretroviral therapy. Median (IQR) HCV-RNA decline after silibinin therapy was 2.65 (2.1-2.8) log10 copies/mL. Fifteen of sixteen patients (94%) had undetectable HCV RNA at weeks 4 and 12, eleven patients (69%) showed end-of-treatment response (i.e., undetectable HCV-RNA at week 48), and ten patients (63%) reached SVR at week 12 (SVR 12). Six of the sixteen patients (37%) did not reach SVR 12: One patient had rapid virologic response (RVR) (i.e., undetectable HCV-RNA at week 4) but stopped treatment at week 8 due to major depression. Five patients had RVR, but experienced viral breakthroughs at week 21, 22, 25, or 32, or a relapse at week 52. The HIV RNA remained below the limit of detection in all patients during the complete treatment period. No serious adverse events and no significant drug-drug interactions were associated with silibinin. CONCLUSION: A lead-in with silibinin before triple-therapy was safe and highly effective in difficult-to-treat HIV/HCV coinfected patients, with a pronounced HCV-RNA decline during the lead-in phase, which translates into 63% SVR. An add-on of intravenous silibinin to standard of care HCV treatment is worth further exploration in selected difficult-to-treat patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01816490.

Targeting RNA-protein interactions within the human immunodeficiency virus type 1 lifecycle.

RNA-protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA-protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5'-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by ¹H NMR spectroscopy.

Green tea phenolic epicatechins inhibit hepatitis C virus replication via cycloxygenase-2 and attenuate virus-induced inflammation.

Chronic hepatitis C virus (HCV) infection is the leading risk factor for hepatocellular carcinoma (HCC) and chronic liver disease worldwide. Green tea, in addition to being consumed as a healthy beverage, contains phenolic catechins that have been used as medicinal substances. In the present study, we illustrated that the epicatechin isomers (+)-epicatechin and (-)-epicatechin concentration-dependently inhibited HCV replication at nontoxic concentrations by using in vitro cell-based HCV replicon and JFH-1 infectious systems. In addition to significantly suppressing virus-induced cyclooxygenase-2 (COX-2) expression, our results revealed that the anti-HCV activity of the epicatechin isomers occurred through the down-regulation of COX-2. Furthermore, both the epicatechin isomers additively inhibited HCV replication in combination with either interferon-α or viral enzyme inhibitors [2'-C-methylcytidine (NM-107) or telaprevir]. They also had prominent anti-inflammatory effects by inhibiting the gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and inducible nitrite oxide synthase as well as the COX-2 in viral protein-expressing hepatoma Huh-7 cells. Collectively, (+)-epicatechin and (-)-epicatechin may serve as therapeutic supplements for treating HCV-related diseases.

Antiviral activity of four types of bioflavonoid against dengue virus type-2.

BACKGROUND: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC50) to inhibitory concentration 50 (IC50) for each compound. RESULTS: The half maximal inhibitory concentration (IC50) of quercetin against dengue virus was 35.7 µg mL-1 when it was used after virus adsorption to the cells. The IC50 decreased to 28.9 µg mL-1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 µg mL-1, respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC50 = 168.2 µg mL-1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC50 = 142.6 µg mL-1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 µg mL-1) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin. CONCLUSION: Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.

Inhibition of dengue virus polymerase by blocking of the RNA tunnel.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

Anti-enterovirus 71 activity screening of chinese herbs with anti-infection and inflammation activities.

Antipyretic and toxin-eliminating traditional Chinese herbs are believed to possess antiviral activity. In this study, we screened extracts of 22 herbs for activity against enterovirus 71 (EV71). We found that only extracts of Houttuynia cordata Thunb. could neutralize EV71-induced cytopathic effects in Vero cells. The 50% inhibitory concentration of H. cordata extract for EV71 was 125.92 +/- 27.84 mug/ml. Antiviral screening of herb extracts was also conducted on 3 genotypes of EV71, coxsackievirus A16 and echovirus 9. H. cordata extract had the highest activity against genotype A of EV71. A plaque reduction assay showed that H. cordata extract significantly reduced plaque formation. Viral protein expression, viral RNA synthesis and virus-induced caspase 3 activation were inhibited in the presence of H. cordata extract, suggesting that it affected apoptotic processes in EV71-infected Vero cells by inhibiting viral replication. The antiviral activity of H. cordata extract was greater in cells pretreated with extract than those treated after infection. We conclude that H. cordata extract has antiviral activity, and it offers a potential to develop a new anti-EV71 agent.

Seco-pregnane steroids target the subgenomic RNA of alphavirus-like RNA viruses.

Plants have evolved multiple mechanisms to selectively suppress pathogens by production of secondary metabolites with antimicrobial activities. Therefore, direct selections for antiviral compounds from plants can be used to identify new agents with potent antiviral activity but not toxic to hosts. Here, we provide evidence that a class of compounds, seco-pregnane steroid glaucogenin C and its monosugar-glycoside cynatratoside A of Strobilanthes cusia and three new pantasugar-glycosides of glaucogenin C of Cynanchum paniculatum, are effective and selective inhibitors to alphavirus-like positive-strand RNA viruses including plant-infecting tobacco mosaic virus (TMV) and animal-infecting Sindbis virus (SINV), eastern equine encephalitis virus, and Getah virus, but not to other RNA or DNA viruses, yet they were not toxic to host cells. In vivo administration of the compounds protected BALB/c mice from lethal SINV infection without adverse effects on the mice. Using TMV and SINV as models, studies on the action mechanism revealed that the compounds predominantly suppress the expression of viral subgenomic RNA(s) without affecting the accumulation of viral genomic RNA. Our work suggested that the viral subgenomic RNA could be a new target for the discovery of antiviral drugs, and that seco-pregnane steroid and its four glycosides found in the two medicinal herbs have the potential for further development as antiviral agents against alphavirus-like positive-strand RNA viruses.

Zinc is a negative regulator of hepatitis C virus RNA replication.

BACKGROUND/AIMS: Hepatitis C virus (HCV) infection is a significant global public health problem. In clinical studies, zinc has been closely related to the pathogenesis of chronic hepatitis C. However, the role of zinc in both viral replication and the expression of viral proteins remains unclear. We aimed to clarify the effect of zinc on the replication of HCV in vitro. METHODS: We incubated subgenomic HCV replicon cells (sO) and genome-length HCV RNA-replicating cells (O) treated with several chemicals including trace elements. Total RNAs were collected and subjected to real-time reverse-transcriptase polymerase chain reaction in order to examine the level of HCV RNA replication, and Western blotting was performed to confirm the expression of viral proteins. RESULTS: Iron salts and interferon-alpha suppressed HCV RNA replication and protein expression in both sO and O cells. Zinc salts effectively reduced the viral replication in the genome-length HCV RNA replication system but not in the subgenomic HCV replicon system. CONCLUSIONS: We demonstrated that zinc may play an important role as a negative regulator of HCV replication in genome-length HCV RNA-replicating cells. Zinc supplementation thus appears to offer a novel approach to the development of future strategies for the treatment of intractable chronic hepatitis C.

Inhibition of hepatitis viral replication by siRNA.

Small interfering RNA (siRNA)-mediated sequence-specific gene silencing is a powerful tool to inhibit endogenous and exogenous gene expression, and it holds great potential to prevent and eradicate viral infection, for which existing therapy is inadequate, such as HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV). A number of studies have documented the effectiveness of siRNA against HBV or HCV at various regions of the viral genome in infected human hepatoma cell lines. Selected siRNA may reduce the production of viral replicons, as well as structural or non-structural proteins by > 90%. Only a few in vivo studies that demonstrated the efficacy of siRNA in the suppression of HBV replication in mice are available. Thus, reliable models of HBV and HCV infection in small animals or non-human primates are needed to evaluate the delivery and efficacy of siRNA as a therapeutic modality for viral hepatitis.

Inhibitory potential of neem (Azadirachta indica Juss) leaves on dengue virus type-2 replication.

In the present study we report in vitro and in vivo inhibitory potential of crude aqueous extract of neem leaves and pure neem compound (Azadirachtin) on the replication of Dengue virus type-2. In vitro antiviral activity of aqueous neem leaves extract assessed in C(6/36) (cloned cells of larvae of Aedes albopictus) cells employing virus inhibition assay showed inhibition in dose dependent manner. The aqueous extract of neem leaves at its maximum non-toxic concentration of 1.897 mg/ml completely inhibited 100-10,000 TCID(50) of virus as indicated by the absence of cytopathic effects. The in vivo protection studies with neem leaves extract at its maximum non-toxic concentrations 120-30 mg/ml resulted in inhibition of the virus replication as confirmed by the absence of Dengue related clinical symptoms in suckling mice and absence of virus specific 511 bp amplicon in RT-PCR. The pure neem i.e. Azadirachtin did not reveal any inhibition on Dengue virus type-2 replication in both in vitro and in vivo systems.

The application of ribozymes to HIV infection.

During the past decade major advances have been made in combating HIV infection and reducing the incidence of AIDS in the western world. Despite optimism about such progress, there is accumulating evidence to suggest that new forms of therapy may be necessary to combat viral resistance to current drugs as well as to provide alternatives to life-long drug use. Genetic forms of therapy are considered to be an important alternative to current drug therapy. One therapeutic agent that can be tailored to inhibit viral infection is catalytic RNA or ribozymes. These RNAs can be engineered to site-specifically cleave targeted RNAs, thereby minimizing cellular toxicity associated with conventional drugs. A potential advantage of ribozymes over other forms of genetic therapy aside from target specificity is their potential for interfering with different stages of the viral life cycle. Ribozymes can be designed and expressed to interfere with viral entry, messenger RNA function and viral packaging. For the two simplest ribozyme motifs, the hammerhead and hairpin, there are hundreds of potential sites along the viral genome. Combinatorial use of ribozymes allows multiple HIV-1 sequences to be attacked simultaneously, thereby circumventing viral resistance through mutation. Ribozymes can also be designed to inhibit expression of cellular targets, which are required for HIV-1 infection. The successful applications of ribozymes against HIV-1 in preclinical settings has now set the stage for their testing in patient trials and several first phase clinical trials are currently underway.