Concept: Viral neuraminidase
Without baseline human immunity to the emergent avian influenza A(H7N9) virus, neuraminidase inhibitors are vital for controlling viral replication in severe infections. An amino acid change in the viral neuraminidase associated with drug resistance, NA-R292K (N2 numbering), has been found in some H7N9 clinical isolates. Here we assess the impact of the NA-R292K substitution on antiviral sensitivity and viral replication, pathogenicity and transmissibility of H7N9 viruses. Our data indicate that an H7N9 isolate encoding the NA-R292K substitution is highly resistant to oseltamivir and peramivir and partially resistant to zanamivir. Furthermore, H7N9 reassortants with and without the resistance mutation demonstrate comparable viral replication in primary human respiratory cells, virulence in mice and transmissibility in guinea pigs. Thus, in stark contrast to oseltamivir-resistant seasonal influenza A(H3N2) viruses, H7N9 virus replication and pathogenicity in these models are not substantially altered by the acquisition of high-level oseltamivir resistance due to the NA-R292K mutation.
Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here a new class of specific, mechanism-based anti-influenza drugs that function via the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and confirm this mode of action via structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.
The wizard of OS (resistance): The binding difference of neuraminidase inhibitors (zanamivir versus oseltamivir (OS)) was used to establish an assay to identify the influenza subtypes that are resistant to OS but still sensitive to zanamivir. This assay used a zanamivir-biotin conjugate to determine the OS susceptibility of a wide range of influenza viruses and over 200 clinical isolates.
Oseltamivir, an anti-influenza virus drug, has strong antipyretic effects in mice (Ono et al., 2008) and influenza patients. In addition, hypothermia has been reported as an adverse event. The prodrug oseltamivir is converted to oseltamivir carboxylate (OC), an active metabolite of influenza virus neuraminidase. In this study, core body temperature was measured in mice, and oseltamivir and OC were administered intracerebroventricularly (i.c.v.) or intraperitoneally (i.p). Low i.c.v. doses of oseltamivir and OC dose-dependently produced hypothermia. Zanamivir (i.c.v.), another neuraminidase inhibitor, did not produce hypothermia. These results suggested that the hypothermic effects of oseltamivir (i.p. and i.c.v.) and OC (i.c.v.) are not due to neuraminidase inhibition. OC (i.p.) did not lower body temperature. Although mecamylamine (i.c.v.) blocked the hypothermic effect of nicotine administered i.c.v., the hypothermic effects of oseltamivir and OC (i.c.v.) were not blocked by mecamylamine (i.c.v.). The effect of oseltamivir (i.p.) was markedly increased by s.c.-preadministered mecamylamine and also hexamethonium, a peripherally acting ganglionic blocker, suggesting their potentiating interaction at peripheral sites. The hypothermic effect of nicotine (i.c.v.) was decreased by lower doses of oseltamivir (i.c.v.), suggesting the anti-nicotinic action of oseltamivir. These results suggest that oseltamivir (i.p.) causes hypothermia through depression of sympathetic temperature regulatory mechanisms via inhibition of nicotinic receptor function and through unknown central mechanisms.
The first structure determination of a neuraminidase inhibitor, oseltamivir phosphate, the active component of the anti-influenza agent Tamiflu, was achieved by single crystal X-ray diffraction with synchrotron radiation. The structure reproduces to a great extent the binding of the inhibitor in the neuraminidase active site and provides more accurate structural parameters as well as detailed insight into the binding preferences of the inhibitor.
Zanamivir, laninamivir, and CS-8958 are three neuraminidase inhibitors that have been clinically used to combat influenza. We report herein a novel organocatalytic route for preparing these agents. Only 13 steps are needed for the assembly of zanamivir and laninamivir from inexpensive D-araboascorbic acid by this synthetic route, which relies heavily on a thiourea-catalyzed enantioselective Michael addition of acetone to tert-butyl (2-nitrovinyl)carbamate and an anti-selective Henry reaction of the resulting Michael adduct with an aldehyde prepared from D-araboascorbic acid. The synthetic procedures are scalable, as evident from the preparation of more than 3.5 g of zanamivir.
Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.
ABSTRACT We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NA(K292) virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NA(R292) and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NA(K292) virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NA(R292) virus, the A/Shanghai/1/2013-NA(K292) virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.
Background. The impact of neuraminidase inhibitors (NAI) treatment on clinical outcomes of public health importance during the 2009-10 pandemic has not been firmly established.Methods. We conducted a systematic review and meta-analysis, searching 11 databases (2009 through April 2012) for relevant studies. We used standard methodology conforming to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Pooled odds ratios (OR) and 95% confidence intervals (CI) were estimated using random effects models.Results. Regarding mortality we observed a non-significant reduction associated with NAI treatment (at any time) vs none (OR, 0.72 [95% CI, 0.51 - 1.01]). However we observed significant reductions for early treatment (≤48h after symptom onset) vs late (OR, 0.38 [95% CI, 0.27 - 0.53]); and for early treatment vs none (OR, 0.35 [95% CI, 0.18 - 0.71]). NAI treatment (at any time) vs none was associated with an elevated risk of severe outcome (OR, 1.76 [95% CI, 1.22 - 2.54]); but early treatment vs. late reduced the likelihood (OR, 0.41 [95% CI, 0.30 - 0.56]).Conclusions. During the 2009-10 influenza A(H1N1) pandemic, early initiation of NAI treatment reduced the likelihood of severe outcomes compared with late or no treatment.Prospero Registration. CRD42011001273.
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