Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic

Abstract

Pandemic influenza viruses can emerge through continuous evolution and the acquisition of specific mutations or through reassortment. This study assessed the pandemic potential of H5N1 viruses isolated from poultry outbreaks occurring from July 2006 to September 2008 in the Lao People's Democratic Republic (PDR). We analyzed 29 viruses isolated from chickens and ducks and two from fatal human cases in 2007. Prior to 2008, all H5N1 isolates in Lao PDR were from clade 2.3.4; however, clade 2.3.2 was introduced in September 2008. Of greatest concern was the circulation of three isolates that showed reduced sensitivity to the neuraminidase (NA) inhibitor oseltamivir in an enzyme inhibition assay, each with different NA mutations - V116A, I222L and K150N, and a previously unreported S246N mutation. In addition, six isolates had an S31N mutation in the M2 protein, which conferred resistance to amantadine not previously reported in clade 2.3.4 viruses. Two H5N1 reassortants were isolated whose polymerase genes, PB1 and PB2, were homologous to those of Eurasian viruses giving rise to a novel H5N1 genotype, genotype P. All H5N1 viruses retained avian-like receptor specificity, but four had altered affinities for alpha2,3-linked sialic acid. This study shows that, in a genetically similar population of H5N1 viruses in Lao PDR, mutants emerged with natural resistance to antivirals and altered affinities for alpha2,3-linked sialic acids, together with reassortants with polymerase genes homologous to Eurasian viruses. These changes may contribute to the emergence of a pandemic influenza strain and are critical in devising surveillance strategies.

Fig. 1.

Geographical location of H5N1 poultry outbreaks in Lao PDR and phylogenetic relationships of the H5 HA. Nucleotide sequences were analysed using the Bayesian approach. Clade credibilities (presented on the tree on the left) were calculated using the parameters described in Methods. The tree was rooted to the HA gene of A/swine/Henan/wy/04 (H5N1). The locations of sites of clade 2.3.4 H5N1 influenza virus isolation and outbreaks that occurred in early 2006 (□), mid-2006 and 2007 (•), 2008 (▵) and clade 2.3.2 H5N1 virus isolation in 2008 (◊) are shown on the right. bhg, Bar-headed goose; ck, chicken; cmag, common magpie; cph, Chinese pond heron; dk, duck; gf, guinea fowl; gs, goose; jwe, japanese white-eye; le, little egret; mdk, Muscovy duck; pf, peregrine falcon; ws, whooper swan. Bar, 0.1 nucleotide substitution per site.

Fig. 2.

Phylogenetic relationships of the genes NA and M. Lao H5N1 isolates are highlighted in blue and the H3N8 isolate in green. H5N1 viruses with mutations conferring reduced sensitivity to antivirals are in red. Nucleotide sequences were analysed using the Bayesian approach. Clade credibilities (presented on the trees) were calculated using the parameters described in Methods. The phylogenetic trees were rooted to the A/swine/Henan/wy/04 (H5N1) virus. bhg, Bar-headed goose; ck, chicken; dk, duck; gs, goose; sck, silky chicken; mdk, Muscovy duck; tky, turkey; ts, tree swallow. Bars, 0.1 nucleotide substitution per site.

Fig. 3.

Phylogenetic relationships of the genes of the polymerase complex (PB1, PB2 and PA) and NP. Lao H5N1 isolates are highlighted in blue and the H3N8 isolate in green. Nucleotide sequences were analysed using the Bayesian approach. Clade credibilities (presented on the trees) were calculated using the parameters described in Methods. The phylogenetic trees were rooted to the PB1 and PB2 genes of A/swine/Henan/wy/04 (H5N1). bhg, Bar-headed goose; ck, chicken; ct, common teal; dk, duck; gs, goose; sck, silky chicken; mdk, Muscovy duck; ms, mute swan; tky, turkey; ts, tree swallow. Bars, 0.1 nucleotide substitution per site.

Fig. 4.

Direct binding of viruses using sialyl glycopolymers. H5N1 viruses isolated in 2007 (a–c) or 2008 (d–f) were measured for their affinity to α2,3-linked (▪) or α2,6-linked (•) sialic acids. Data are means±sd of triplicate experiments.