The effect of the MDCK cell selected neuraminidase D151G mutation on the drug susceptibility assessment of influenza A(H3N2) viruses.

Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ∼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.

Cell culture-selected substitutions in influenza A(H3N2) neuraminidase affect drug susceptibility assessment.

Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.

Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.

BACKGROUND: Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS: Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS: With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS: This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Assessment of pandemic and seasonal influenza A (H1N1) virus susceptibility to neuraminidase inhibitors in three enzyme activity inhibition assays.

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC(50)s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC(50)s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC(50)s between the wild type and the variants, whereas the IC(50)s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.