In vitro inhibition of human influenza A virus replication by chloroquine

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.     

Evaluation of the anti-hepatitis C virus effects of cyclophilin inhibitors, cyclosporin A, and NIM811

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. We recently discovered that the immunosuppressant cyclosporin A (CsA) and its analogue lacking immunosuppressive function, NIM811, strongly suppress the replication of HCV in cell culture. Inhibition of a cellular replication cofactor, cyclophilin (CyP) B, is critical for its anti-HCV effects. Here, we explored the potential use of CyP inhibitors for HCV treatment by analyzing the HCV replicon system. Treatment with CsA and NIM811 for 7 days reduced HCV RNA levels by 2-3 logs, and treatment for 3 weeks reduced HCV RNA to undetectable levels. NIM811 exerted higher anti-HCV activity than CsA at lower concentrations. Both CyP inhibitors rapidly reduced HCV RNA levels even further in combination with IFNalpha without modifying the IFNalpha signal transduction pathway. In conclusion, CyP inhibitors may provide a novel strategy for anti-HCV treatment.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

Frequent Detection of Hepatitis C Virus Subtype 3a (HCV-3a) Isolates in Thailand by PCR Using Subtype-Specific Primers

By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or ?2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and ?1b) and 2 (HCV-2a and ?2b).     

Prediction of HCV load using genotype,liver biomarkers,and clinical symptoms by a mathematical model in patients with HCV infection

Hepatitis C virus (HCV) infection is a major public health problem with about 1.75 million new HCV cases and 71 million chronic HCV infections worldwide. The study aimed to evaluate clinical, serological, molecular, and liver markers to develop a mathematical predictive model for the quantification of the HCV viral load in chronic HCV infected patients. In this cross‐sectional study, blood samples were taken from 249 recently diagnosed HCV‐infected subjects and were tested for liver condition, viral genotype, and HCV RNA load. Receiver operating characteristics (ROC) curves and multiple linear regression analysis were used to predict the HCV‐RNA load. Genotype 3 followed by genotype 1 were the most prevalent genotypes in Mashhad, Northeastern Iran. The maximum levels of viral load were detected in the mixed genotype group, and the lowest levels in the undetectable genotype group. The log of the HCV viral load was significantly associated with thrombocytopenia and higher serum levels of alanine transaminase (ALT). In addition, the log HCV RNA was significantly higher in patients with arthralgia, fatigue, fever, vomiting, or dizziness. Moreover, genotype 3 was significantly associated with icterus. A ROC curve analysis revealed that the best cut‐off points for serum levels of aspartate aminotransferase (AST), ALT, and alkaline phosphatase (ALP) were >31, >34, and ≤246 IU/L, respectively. Sensitivity, specificity, and positive predictive values for AST were 87.7%, 84.36%, and 44.6%, for ALT they were 83.51%, 81.11%, and 36%, and for ALP were 72.06%, 42.81%, and 8.3%, respectively. A mathematical regression model was developed that could estimate the HCV‐RNA load. Regression model: log viral load = 7.69 ? 1.01 × G3 ? 0.7 × G1 + 0.002 × ALT ? 0.86 × fatigue.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus.

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

Inhibition of the replication of hepatitis B virus in vitro by a novel 2,6-diaminopurine analog, beta-LPA

Antiviral therapy of chronic hepatitis B remains a major clinical problem worldwide. Like lamivudine, nucleoside analogs have become the focus of investigation of anti-hepatitis B virus (anti-HBV) drugs. Here, β-LPA is a novel 2,6-diaminopurine analog found to possess potent anti-HBV activity. In HepG2.2.15 cell line, β-LPA had a 50% effective concentration (EC₅₀) of 0.01 μM against HBV, as determined by analysis of secreted and intracellular episomal HBV DNA. Levels of HBV surface antigen (HBsAg) and e antigen (HBeAg) in drug-treated cultures revealed that β-LPA had no significant inhibitory effects on HBsAg and HBeAg. β-LPA didn’t show any cytotoxicity up to 0.4 μM with a 50% cytotoxic concentration (CC₅₀) of 50 μM. Furthermore, treatment with β-LPA resulted in no apparent inhibitory effects on mitochondrial DNA content. Considering the potent inhibition of HBV DNA synthesis and no obvious toxicity of β-LPA, this compound should be further explored for development as an anti-HBV drug.     

In vitro inhibition of herpes simplex virus type 1 replication by Mentha suaveolens essential oil and its main component piperitenone oxide

Several essential oils exert in vitro activity against bacteria and viruses and, among these latter, herpes simplex virus type 1 (HSV-1) is known to develop resistance to commonly used antiviral agents. Thus, the effects of the essential oil derived from Mentha suaveolens (EOMS) and its active principle piperitenone oxide (PEO) were tested in in vitro experimental model of infection with HSV-1. The 50% inhibitory concentration (IC₅₀) was determined at 5.1 μg/ml and 1.4 μg/ml for EOMS and PEO, respectively. Australian tea tree oil (TTO) was used as control, revealing an IC₅₀ of 13.2 μg/ml. Moreover, a synergistic action against HSV-1 was observed when each oil was added in combination with acyclovir. In order to find out the mechanism of action, EOMS, PEO and TTO were added to the cells at different times during the virus life-cycle. Results obtained by yield reduction assay indicated that the antiviral activity of both compounds was principally due to an effect after viral adsorption. Indeed, no reduction of virus yield was observed when cells were treated during viral adsorption or pre-treated before viral infection. In particular, PEO exerted a strong inhibitory effect by interfering with a late step of HSV-1 life-cycle. HSV-1 infection is known to induce a pro-oxidative state with depletion of the main intracellular antioxidant glutathione and this redox change in the cell is important for viral replication. Interestingly, the treatment with PEO corrected this deficit, thus suggesting that the compound could interfere with some redox-sensitive cellular pathways exploited for viral replication. Overall our data suggest that both EOMS and PEO could be considered good candidates for novel anti-HSV-1 strategies, and need further exploration to better characterize the targets underlying their inhibition.     

Effect of cell growth on hepatitis C virus (HCV) replication and a mechanism of cell confluence-based inhibition of HCV RNA and protein expression

An intimate relationship between hepatitis C virus (HCV) replication and the physiological state of the host liver cells has been reported. In particular, a highly reproducible and reversible inhibitory effect of high cell density on HCV replication was observed: high levels of HCV RNA and protein can be detected in actively growing cells but decline sharply when the replicon cells reach confluence. Arrested cell growth of confluent cells has been proposed to be responsible for the inhibitory effect. Indeed, other means of arresting cell growth have also been shown to inhibit HCV replication. Here, we report a detailed study of the effect of cell growth and confluence on HCV replication using a flow cytometry-based assay that is not biased against cytostasis and reduced cell number. Although we readily reproduced the inhibitory effect of cell confluence on HCV replication, we found no evidence of inhibition by serum starvation, which arrested cell growth as expected. In addition, we observed no inhibitory effect by agents that perturb the cell cycle. Instead, our results suggest that the reduced intracellular pools of nucleosides account for the suppression of HCV expression in confluent cells, possibly through the shutoff of the de novo nucleoside biosynthetic pathway when cells become confluent. Adding exogenous uridine and cytidine to the culture medium restored HCV replication and expression in confluent cells. These results suggest that cell growth arrest is not sufficient for HCV replicon inhibition and reveal a mechanism for HCV RNA inhibition by cell confluence.     

The inhibition of alcohol dehydrogenase in vitro and in isolated hepatocytes by 4-substituted pyrazoles

As a means of comparing the functional properties of an enzyme in dilute solution in vitro with those for the same enzyme acting in its normal cellular environment, a study was conducted with 4-substituted pyrazoles as inhibitors of rat liver alcohol dehydrogenase in vitro and ethanol oxidation in isolated rat hepatocytes. Inhibitor constants (Ki's) for the same set of pyrazole derivatives were also determined for human liver alcohol dehydrogenase. The best-fitting equations were derived to relate the Ki's to the chemical nature of substituents. These quantitative structure-activity relationships show that pyrazoles with stronger electron-withdrawing substituents are weaker inhibitors both for the enzyme in vitro and, to an equal extent, for ethanol oxidation by intact cells. Inhibitor effectiveness is also dependent on substituent hydrophobicity, but, while increasing hydrophobicity makes stronger inhibitors of the enzyme in vitro, it can diminish the effectiveness in vivo by decreasing permeability through the cell membrane. A structure-activity analysis of published Ki's for pyrazoles acting against human pi-ADH indicates that its active site differs from those in other alcohol dehydrogenases.     

Design and synthesis of spirocyclic compounds as HCV replication inhibitors by targeting viral NS4B protein

Two novel series of spirocyclic piperidine analogs appended to a pyrazolo[1,5-a]pyridine core were designed, synthesized and evaluated for their anti-HCV activity. A series of piperidine ketals afforded dispiro 6p which showed excellent in vitro anti-HCV activities (EC₅₀ of 1.5 nM and 1.2 nM against genotype 1a and 1b replicons, respectively). A series of piperidine oxazolidinones afforded 27c which showed EC₅₀’s of 10.9 nM and 6.1 nM against 1a and 1b replicons, respectively. Both compounds 6p and 27c bound directly to non-structural NS4B protein in vitro (IC₅₀’s = 10.2 and 30.4 nM, respectively) and exhibited reduced potency in replicons containing resistance mutations encoding changes in the NS4B protein.     

In vitro inhibition of HIV-1 replication in autologous CD4~+ T cells indicates viral containment by multifactorial mechanisms

HIV-1-specific cytotoxic T lymphocytes(CTLs) and neutralizing antibodies(NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells,primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro,which was mediated by natural killer cells(NKs) and dependent on an Fc-Fc receptor interaction.Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4~+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.     

Synergistic inhibition of pseudorabies virus replication by porcine alpha/beta interferon and gamma interferon in vitro

Interferon (IFN) is crucial for initiating the innate immune response and for the generation of the adaptive response. IFN, in most species, comprises IFN-alpha (IFN-alpha), IFN-beta (IFN-beta) and IFN-gamma (IFN-gamma). In this study, we compared the capacity of porcine IFN-alpha, -beta and -gamma, or a combination of them, to protect IBRS-2 cells (porcine kidney cells) from infection with pseudorabies virus (PRV). The results demonstrated that porcine IFN-beta (PoIFN-beta) was the most efficient of the three IFNs in conferring resistance PRV infection; 100 U/mL PoIFN-beta inhibited PRV plaque formation 5.3-fold. Compared with PoIFN-beta, porcine IFN-gamma (PoIFN-gamma) was less capable of inhibiting PRV plaque formation (3.3-fold inhibition). Porcine IFN-alpha (PoIFN-alpha) had the least capability of the three PoIFNs, and inhibited PRV plaque formation only 1.26-fold. The inhibitory capacity increased to only 2.3-fold with a treatment of 12,800 U/mL PoIFN-alpha. A combination of PoIFN-gamma and PoIFN-alpha or PoIFN-beta inhibited PRV plaque formation 12.8-fold or 100-fold, respectively. Treatment of IBRS-2 cells with PoIFN-alpha/beta and PoIFN-gamma inhibited PRV replication 29- or 146-fold. Additionally, real-time PCR analyses of the PRV immediate early (IE) gene revealed that IE mRNA expression was profoundly decreased in cells stimulated with PoIFN-alpha/beta and PoIFN-gamma (23.8-133.0-fold) compared with vehicle-treated cells. All the findings indicate that PoIFN-gamma acts synergistically with other PoIFNs (PoIFN-alpha and -beta) to potently inhibit PRV replication in vitro.     

Synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and molecular docking studies

Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and ¹H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM) while other four compounds exhibited good to moderate inhibition with IC₅₀ values between 29.45 ± 1.1 μM and 69.53 ± 0.9 μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds.