Antiviral Chemistry and Chemotherapy

The RNA interference (RNAi) phenomenon is a recently discovered process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of mRNA containing the same sequence. We designed mammalian expression vectors that direct the synthesis of small interfering RNA (siRNA)-like transcripts and examined them for their siRNA-mediated gene interference targeting the env gene (NL4–3:7490–7508, E7490). We constructed siRNA expression vectors for two different strands (sense and antisense; tandem promoter) and for siRNA expressed from the short hairpin RNA (shRNA). The inhibition efficacy on HIV–1 replication differed between these two vectors. Notably, the shRNA vector pU6-env-shRNA inhibited p24 production more effectively than the tandem promoter expression vector pU6-env-siRNA. Furthermore, we examined the ability of lentiviral vectors expressing shRNA to suppress HIV-1 expression in HIV-1-infected SupT1 cells. The env-shRNA (E 7490) almost completely suppressed HIV-1 expression in infected cells for up to 15 days.

We studied the combination effect of rimantadine hydrochloride and oseltamivir phosphate on mice infected with influenza A/Aichi/2/68 (H3N2) virus. Compounds were simultaneously administered in a 5-day-treatment course, starting 4 h before intranasal infection with 10 or 20 viral 50% mouse lethal doses. Initially, we tested combinations of oseltamivir (0.05, 0.1 and 0.2 mg/kg/day) and rimantadine (2.5, 5.0 and 7.5 mg/kg/day). Significant differences were recorded between combinationtreated groups, and groups with separately applied compounds and the placebo group, such as: protection index of oseltamivir with 5.0 or 7.5 mg/kg rimantadine varied between 34–41% and 43–87%, respectively, whereas the individual effects of oseltamivir, 5 mg/kg of rimantadine and 7.5 mg/kg of rimantadine were 0–10%, 0% and 18.7–29.6%, respectively; mean survival time in combination-treated groups was lengthened by 3.1–6.9 days, in oseltamivir groups by 0–1.9 days, and in rimantadine groups by 0.8–1.3 days at 5 mg/kg and 2.6–3.2 days at 7.5 mg/kg. The three-dimensional method of Prichard and Shipman characterized the combination effect as synergistic. Further, we studied the activity of 0.05 mg/kg/day of oseltamivir combined with 5 mg/kg of rimantadine. Lung virus titre in Madin Darby canine kidney cells, lung index and consolidation score proved the high effectiveness of the combination. When compared with the placebo group, a 2.8 log₁₀ lower titre of 50% cell culture infectious dose (CCID₅₀) was recorded in the combination-treated group at 48–60 h post-infection (the peak of lung virus growth). This is in contrast to the 0.1–1.0 log₁₀ and 1.1–1.4 log₁₀ reduction in CCID₅₀ titre observed in the oseltamivir and rimantadine groups, respectively. These data emphasize the high anti-influenza A potential of the combination.

Previous work has shown that the secretion of enveloped hepatitis B virus (HBV) DNA and the HBV middle envelope protein (MHBs) are sensitive to glucosidase inhibition. Here, it is shown that HBV DNA secretion remains depressed after the removal of the glucosidase inhibitor and long after glucosidase function returns to normal. For example, glycoprocessing and the secretion of α-1 anti-trypsin returned to normal within 3 h of the removal of the glucosidase inhibitor. In contrast, the secretion of HBV did not return to normal for more than 7 days after the removal of the inhibitor. Consistent with the inhibition of HBV virion secretion, the levels of HBV L and HBV M proteins were also reduced by treatment with the glucosidase inhibitor and remained reduced for 7 days after compound withdrawal. The implications of the prolonged antiviral effect against HBV and the use of glucosidase inhibitors as antiviral agents are discussed.

4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)amino]-N-(4,6-dimethyl-2-pyrimidinyl)-benzenesulphonamide (SPIII-5H) and related compounds were tested for antiviral activity against influenza A (H1N1, H3N2, and H5N1) and B viruses in Madin Darby canine kidney (MDCK) cell culture. Among the compounds tested, SPIII-5H and four derivatives (5-chloro [SPIII-5Cl], 5-bromo [SPIII-5Br], 5-methyl [SPIII-5Me] and N-acetyl [SPIII-NA]) showed similar antiviral potencies, with only the 5-fluoro (SPIII-5F) derivative being ineffective. Fifty percent effective concentration (EC₅₀) values were determined in cytopathic effect (CPE) inhibition assays quantified by neutral red dye uptake. By this method, the active compounds were inhibitory to the H1N1 strain of influenza A at 2.7–5.2 µg/ml, to the H3N2 strain of influenza A at 13.8–26.0 µg/ml, to the H5N1 strain of influenza A at 3.1–6.3 µg/ml and to influenza B at 7.7–11.5 µg/ml. Confirmatory virus yield reduction studies against influenza A (H1N1) virus demonstrated antiviral activity (90% inhibition) at concentrations of 2–10 µg/ml. No cytotoxic effects were evident in actively growing uninfected cells or stationary monolayers at 100 µg/ml. Potencies of the compounds were similar to those of ribavirin, but much less than those of oseltamivir carboxylate against the various viruses. Time-of-addition studies indicated the compounds inhibited an early step in the virus replication cycle, probably virus adsorption/penetration, and no virucidal activity was evident. The basic molecule is amenable to diverse chemical modifications, which may improve water solubility and antiviral potency.

Compounds approved for therapeutic use and in vitro inhibitors of severe acute respiratory syndrome coronavirus (SARS-CoV) were evaluated for inhibition in the mouse SARS-CoV replication model. A hybrid interferon, interferon alpha (IFN-α) B/D, and a mismatched double-stranded (ds) RNA interferon (IFN) inducer, Ampligen® (poly I:poly C₁₂₄), were the only compounds that potently inhibited virus titres in the lungs of infected mice as assessed by CPE titration assays. When mice were dosed intraperitoneally (i.p.) with IFN-α B/D once daily for 3 days beginning 4 h after virus exposure, SARS-CoV replication in the lungs of infected mice was reduced by 1 log₁₀ at 10,000 and 32,000 IU; at the highest dose of 100,000 IU, virus lung titres were below detectable limits. Ampligen® used i.p. at 10 mg/kg 4 h prior to virus exposure also reduced virus lung titres to below detectable limits. Nelfinavir, β-D-N⁴-hydroxycytidine, calpain inhibitor VI, 3-deazaneplanocin A and Alferon® (human leukocyte IFN-α-n3) did not significantly reduce lung virus titres in mice. Anti-inflammatory agents, chloroquine, amodiaquin and pentoxifylline, were also inactive in vivo, suggesting that although they may be useful in ameliorating the hyperinflammatory response induced by the virus infection, they will not significantly reduce the replication of the virus, the inducer of inflammatory response. Thus, anti-inflammatory agents may only be useful in treating virus lung infections if used in combination with agents that inhibit virus replication. In summary, the data suggest that induction of IFN by mismatched dsRNA or actual treatment with exogenous IFN-α can inhibit SARS-CoV replication in the lungs of mice.

The recent outbreak of severe acute respiratory syndrome (SARS), which is an acute respiratory illness, is caused by newly discovered SARS coronavirus (SARS-CoV). Herein we describe the antiviral activity of several classes of nucleoside analogues evaluated against SARS-CoV in Vero 76 cells, some of which exhibited moderate activity.