Background: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA). Objectives: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays. Study design: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens. Results: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log10 viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thawcycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity. Conclusion: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

We demonstrate the potent antiviral activity of a novel viral neuraminidase (sialidase) inhibitor, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GG167), administered by the intranasal route in comparison with those of amantadine and ribavirin in experimental respiratory tract infections induced with influenza A and B viruses. In an extended study in which mice were infected (day 0) with influenza A/Singapore/1/57 virus, with treatments given prophylactically plus twice daily over days 0 to 3 and with mice observed to day 10, we show that intranasally administered GG167 at 0.4 and 0.01 mg/kg of body weight per dose reduced mortality, lung consolidation, and virus titers in the lung, with no virus growing back following the cessation of treatment. In other studies with influenza B/Victoria/102/85 virus in which infected mice were culled after the cessation of treatment, the calculated intranasal dose required to reduce virus titers in the lungs of treated animals to 10% of that seen in untreated controls (EDAUC,o [where AUC is area under the virus titer days curve]) was 0.085 mg/kg per dose. GG167 was inactive against influenza viruses A and B when given by the intraperitoneal or oral route (EDAUC,0, >100 mg/kg per dose). GG167 was metabolically stable, with an elimination half-life of 10 min following intravenous administration. While readily bioavailable by systemic routes, it was poorly bioavailable by the oral route. Its potent efficacy by the intranasal route but lack of efficacy by other routes, relative to those of amantadine and ribavirin, was explicable in terms of its in vitro activity, bioavailability, and pharmacokinetic properties and with the extracellular activity of viral sialidase.

Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log 10 50% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n ‫؍‬ 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is a novel viral neuraminidase (sialidase) inhibitor which, following intranasal administration in ferrets, is at least 100 to 1,000 times more effective than ribavirin and amantadine against influenza A and B viruses. It retains its activity even when treatments are delayed until 24 h postinfection and has no effect on the serum antibody response to infection.Neuraminidase is primarily responsible for promoting the release of progeny virus from infected cells, and the selective neuraminidase inhibitor GG167 has been shown to inhibit the growth of a wide range of influenza A and B viruses in vitro (4, 5). When given intranasally to mice infected with influenza A and B viruses, GG167 was 100 to 1,000 times more active than amantadine or ribavirin and retained efficacy when treatments were delayed until the postinfection period (2). We now report on the efficacy of GG167 by the intranasal route against strains of influenza A and B viruses in a ferret model and the effects on efficacy of delaying treatments until the postinfection period.All reagents, medium components, MDCK cells, non-ferretadapted virus strains, and antiviral agents were obtained from sources given previously (2, 5). Ferrets (female, 0.6 to 1.2 kg) were obtained from Glaxo Wellcome Research and Development, Bury Green, Hertfordshire, United Kingdom, and were used in groups of three to six animals. Methods for inducing infection, for assessing and quantifying infection in terms of the area under the curve values (AUC) over days 1 to 9 of the nasal wash virus titers (log 10 50% tissue culture infective doses or PFU per milliliter), and for the telemetric monitoring of body temperature were as described previously (2, 4). Significant pyrexia is defined here as the elevation of body temperature to 1ЊC (or more) above the preinfection (baseline) temperature for at least 12 h within the 30-to 96-h-postinfection period. Unless stated otherwise, the standard treatment regimen used in efficacy tests involved 13 individual doses, starting with a single dose at Ϫ26 h (day Ϫ1) and continuing with doses at Ϫ2 and 5 h on the day of infection (day 0), at 22 and 30 h (day 1), and twice daily 8 h apart on days 2 to 5 inclusive. Where possible, ribavirin and amantadine were included at relevant dose levels and tested in parallel with at least one dose level of GG167 for each of the three viruses. Statistical analyses on nasal wash virus titer AUCs to compare treatment groups within experiments were conducted with analysis of variance; where only two groups were involved, comparisons were made with Duncan's multiple range test and the t test. Reduction in the incidence of significant pyrexia was analyzed with Fisher's exact test.The profiles of nasal wash virus titers and temperature responses over days 1 to 9 in 31 vehicle-dosed infected ferrets used in these studies were as described previously (4) and were similar for the three viruses at the challenge levels used. A total of 29 of the 31 ferret...

A randomized, double-blind, placebo-controlled, parallel-group trial performed at 5 residential units of the Finnish Defence Forces was conducted to assess the antiviral activity, efficacy and safety of inhaled zanamivir for the treatment of naturally acquired influenza. Conscripts were recruited within 2 d of onset of typical influenza symptoms and received inhaled zanamivir 10 mg via a Diskhaler twice daily for 5 d or matching placebo. Time to alleviation of clinically significant symptoms of influenza was the primary endpoint. Viral load measurements were made using quantitative real-time polymerase chain reaction assays. 435/588 patients (74%) had laboratory-confirmed influenza infection. The mean area under the curve for viral load during the first 48 h of treatment was 8.48 [95% confidence interval (95% CI) 2.85 to 14.11] log10 vRNA copies/ml x h lower in the zanamivir group compared with placebo (p = 0.003). Zanamivir reduced the time to alleviation of symptoms versus placebo in the influenza-positive group (medians 2.0 vs 2.33 d; 95% CI-0.17 to 1.0 d, p = 0.08). Zanamivir rapidly reduced viral load following the start of therapy compared with placebo and was well tolerated.