Antiviral Drug-Resistant Influenza B Viruses Carrying H134N Substitution in Neuraminidase, Laos, February 2016

Abstract

In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused by viruses harboring this mutation.

Keywords: H134N substitution; Laos; antimicrobial resistance; antiviral drug–resistant; influenza; influenza B viruses; mutation; neuraminidase; viruses.

Figure 1

Neuraminidase gene segment (nts 399–497) of influenza B/Laos/0080/2016 virus carrying NA-H134 (A) and B/Laos/0654/2016, NA-N134 (B). RNA extracted from respiratory specimens was used for reverse transcription PCR (RT-PCR) amplification. Two primers, NA-B-242F (5′-CATACCCGCGTTTATCTTGC-3′, forward primer) and NA-B-426Rb (biotin-5′-CTGTCTCCTCTTGTTCCATTGTAG-3′, reverse biotinylated primer) were used in RT-PCR, essentially as described previously (10); primer NA-B-378Fs (5′-TGCAAACACTTTGCTTTAAC-3′) was used for pyrosequencing. Underlining indicates nucleotide triplet encoding amino acid residue 134.Shading indicates the nucleotides used to determine the proportion of H134 and N134 neuraminidase variants. Pyrosequencing dispensation order: E-Enzyme mixture; S-substrate mixture; G, C, A and T – nucleotides dGTP, dCTP; dATPαS and dTTP, correspondingly.

Figure 2

Characterization of influenza B viruses detected in Laos, February 2016. A) Thermostability of neuraminidase (NA) determined after viruses were incubated for 15 min at 4°C or at 30°C–57°C. NA enzyme activity was determined by a fluorescence-based assay (4). B) Replication kinetics of influenza B viruses in fully differentiated human primary NHBE cells that were inoculated with the designated viruses (multiplicity of infection 0.001). Apical washes were taken at indicated times after inoculation, and virus titers were determined on MDCK cells. The area under the virus titer curve from 2 to 72 h after inoculation (AUC_2–72) was determined and compared with that of the control virus by repeated-measures analysis of variance with the Dunnett posttest, using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Dashed line represents the limit of detection of the assay (1.75 log₁₀ 50% tissue culture infectious dose [TCID₅₀/mL]). Values shown are means and SDs from 2 independent experiments performed in duplicates (n = 4). Error bars represent SDs. NS, not significant.