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.