Characterization of substitutions in the neuraminidase of A(H7N9) influenza viruses selected following serial passage in the presence of different neuraminidase inhibitors.

Avian A(H7N9) infections in humans have been reported in China since 2013 and are of public health concern due to their severity and pandemic potential. Oseltamivir and peramivir are neuraminidase inhibitors (NAIs) routinely used for the treatment of A(H7N9) infections, but variants with reduced sensitivity to these drugs can emerge in patients during treatment. Zanamivir and laninamivir are NAIs that are used less frequently. Herein, we performed in vitro serial passaging experiments with recombinant viruses, containing the neuraminidase (NA) from influenza A/Anhui/1/13 (H7N9) virus, in the presence of each NAI, to determine whether variants with reduced sensitivity would emerge. NA substitutions were characterized for their effect on the NA enzymatic activity and surface expression of the A/Anhui/1/13 (Anhui/1) NA, as well as NAs originating from contemporary A(H7N9) viruses of the Yangtze River Delta and Pearl River Delta lineages. In vitro passage in the presence of oseltamivir, peramivir and laninamivir selected for substitutions associated with reduced sensitivity (E119D, R292K and R152K), whereas passage in the presence of zanamivir did not select for any viruses with reduced sensitivity. All the NA substitutions significantly reduced activity, but not the expression of the Anhui/1 NA. In contemporary N9 NAs, all substitutions tested significantly reduced NA enzyme function in the Yangtze River lineage background, but not in the Pearl River Delta lineage background. Overall, these findings suggest that zanamivir may be less likely than the other NAIs to select for resistance in A(H7N9) viruses and that the impact of substitutions that reduce NAI susceptibility or enzyme function may be less in A(H7N9) viruses from the Pearl River lineage.

Characterization of Influenza B Virus Variants with Reduced Neuraminidase Inhibitor Susceptibility.

Treatment options for influenza B virus infections are limited to neuraminidase inhibitors (NAIs), which block the neuraminidase (NA) glycoprotein on the virion surface. The development of NAI resistance would therefore result in a loss of antiviral treatment options for influenza B virus infections. This study characterized two contemporary influenza B viruses with known resistance-conferring NA amino acid substitutions, D197N and H273Y, detected during routine surveillance. The D197N and H273Y variants were characterized in vitro by assessing NA enzyme activity and affinity, as well as replication in cell culture compared to those of NAI-sensitive wild-type viruses. In vivo studies were also performed in ferrets to assess the replication and transmissibility of each variant. Mathematical models were used to analyze within-host and between-host fitness of variants relative to wild-type viruses. The data revealed that the H273Y variant had NA enzyme function similar to that of its wild type but had slightly reduced replication and transmission efficiency in vivo The D197N variant had impaired NA enzyme function, but there was no evidence of reduction in replication or transmission efficiency in ferrets. Our data suggest that the influenza B virus variant with the H273Y NA substitution had a more notable reduction in fitness compared to wild-type viruses than the influenza B variant with the D197N NA substitution. Although a D197N variant is yet to become widespread, it is the most commonly detected NAI-resistant influenza B virus in surveillance studies. Our results highlight the need to carefully monitor circulating viruses for the spread of influenza B viruses with the D197N NA substitution.