Genetic and phylogenetic analyses of influenza A H1N1pdm virus in Buenos Aires, Argentina

Abstract

An influenza pandemic caused by swine-origin influenza virus A/H1N1 (H1N1pdm) spread worldwide in 2009, with 12,080 confirmed cases and 626 deaths occurring in Argentina. A total of 330 H1N1pdm viruses were detected from May to August 2009, and phylogenetic and genetic analyses of 21 complete genome sequences from both mild and fatal cases were achieved with reference to concatenated whole genomes. In addition, the analysis of another 16 hemagglutinin (HA), neuraminidase (NA), and matrix (M) gene sequences of Argentinean isolates was performed. The microevolution timeline was assessed and resistance monitoring of an NA fragment from 228 samples throughout the 2009 pandemic peak was performed by sequencing and pyrosequencing. We also assessed the viral growth kinetics for samples with replacements at the genomic level or special clinical features. In this study, we found by Bayesian inference that the Argentinean complete genome sequences clustered with globally distributed clade 7 sequences. The HA sequences were related to samples from the northern hemisphere autumn-winter from September to December 2009. The NA of Argentinean sequences belonged to the New York group. The N-4 fragment as well as the hierarchical clustering of samples showed that a consensus sequence prevailed in time but also that different variants, including five H275Y oseltamivir-resistant strains, arose from May to August 2009. Fatal and oseltamivir-resistant isolates had impaired growth and a small plaque phenotype compared to oseltamivir-sensitive and consensus strains. Although these strains might not be fit enough to spread in the entire population, molecular surveillance proved to be essential to monitor resistance and viral dynamics in our country.

FIG. 1.

Summary of findings. (a) Total number of NPAs and viral etiology; (b) sex and age distributions of H1N1pdm cases.

FIG. 2.

Hierarchical clustering of samples. Distances were calculated for Argentinean samples; and replacement changes were plotted for the HA, NA, and M protein sequences (a) and whole-genome sequences (b).

FIG. 3.

Phylogenetic tree of concatenated complete genomes of Argentinean samples. Phylogeny was inferred with representative sequences retrieved from GenBank using Mr Bayes software (version 3.1; settings, 20,000,000 ngen, 5,000 samplefreq, 4 nchains, 5,000 burnin). Clades are indicated as 1 to 7, according to Nelson et al. (22). Argentinean strains are clustered in clade 7. The tree was rooted with H1N1pdm reference strain California/04 (clade 1).

FIG. 4.

Timeline distribution of nucleotide variants of N-4 fragment. The 27 different nucleotide variants that circulated from 27 May 2009 to 27 August 2009 were represented by the oldest sequence in the group. The number of sequences in each group is indicated in parentheses. Oseltamivir-resistant strains are indicated with an asterisk.

FIG. 5.

In vitro viral replication kinetics. Viral titrations in the time series were plotted for wild-type (HNRG15) and H275Y resistant (HNRG23) Argentinean strains (a) and isolates from mild cases (HNRG14) and fatal cases (HNRG5, HNRG45, and HNRG102) (b) in MDCK cells. Corresponding plaque characteristics are shown below.