Reassortment and mutations associated with emergence and spread of oseltamivir-resistant seasonal influenza A/H1N1 viruses in 2005-2009

Abstract

A dramatic increase in the frequency of the H275Y mutation in the neuraminidase (NA), conferring resistance to oseltamivir, has been detected in human seasonal influenza A/H1N1 viruses since the influenza season of 2007-2008. The resistant viruses emerged in the ratio of 14.3% and quickly reached 100% in Taiwan from September to December 2008. To explore the mechanisms responsible for emergence and spread of the resistant viruses, we analyzed the complete genome sequences of 25 viruses collected during 2005-2009 in Taiwan, which were chosen from various clade viruses, 1, 2A, 2B-1, 2B-2, 2C-1 and 2C-2 by the classification of hemagglutinin (HA) sequences. Our data revealed that the dominant variant, clade 2B-1, in the 2007-2008 influenza emerged through an intra-subtype 4+4 reassortment between clade 1 and 2 viruses. The dominant variant acquired additional substitutions, including A206T in HA, H275Y and D354G in NA, L30R and H41P in PB1-F2, and V411I and P453S in basic polymerase 2 (PB2) proteins and subsequently caused the 2008-2009 influenza epidemic in Taiwan, accompanying the widespread oseltamivir-resistant viruses. We also characterized another 3+5 reassortant virus which became double resistant to oseltamivir and amantadine. Comparison of oseltamivir-resistant influenza A/H1N1 viruses belonging to various clades in our study highlighted that both reassortment and mutations were associated with emergence and spread of these viruses and the specific mutation, H275Y, conferring to antiviral resistance, was acquired in a hitch-hiking mechanism during the viral evolutionary processes.

Figure 1. The frequency of the H275Y substitution in NA genes and monthly distribution of influenza isolates confirmed in Taiwan from 2005 to 2009.

(A) The NA genes of seasonal influenza A/H1N1 viruses isolated in Taiwan from 2005 to 2009 were sequenced and analyzed for the mutation associated with oseltamivir resistance. The number of isolates tested and their H275Y ratio are illustrated by a black line and white bar, respectively. The H275Y positivity rate rose quickly from 4.2% (2/47) before August to 100% (37/37) in December in 2008. (B) The number of influenza isolates and their positivity rates. Seasonal influenza A/H1N1 viruses were the predominant circulating subtypes in the 2005–2006, 2007–2008 and 2008–2009 epidemics and the influenza B viruses were predominant in 2006–2007. The seasonal A/H3N2 viruses also co-circulated during these four influenza seasons. Since July 2009, the pandemic H1N1 viruses started to replace the seasonal influenza viruses and became dominant. (C) The clade-based epidemiological curves of the seasonal influenza A/H1N1 viruses. The 1585 isolates were classified into four distinct clades: 1, 2A, 2B (2B-1 and 2B-2) and 2C (2C-1 and 2C-2) according to the phylogenetic analyses and amino acid substitutions of HA sequences. The major viruses in 2005–2006, 2007–2008 and 2008–2009 seasons were clade 2A, 2B-1 and 2B-2, respectively. Minor species from other clades circulated during this period.

Figure 2. Phylogenetic relationships of the HA and PB2 segments of influenza A/H1N1 viruses in Taiwan.

The phylogenetic analyses were constructed using the neighbor-joining method with 1000 bootstrap replications. Branch values of more than 75 are indicated. All of the phylogenies were rooted with the A/New Caledonia/20/1999, which was the vaccine strain recommended by WHO during the 2001–2007 influenza seasons. The genome sequences of A/Solomon Islands/3/2006, A/Brisbane/59/2007 and the early isolates carrying the H275Y substitution, A/England/494/2006, A/England/594/2006 and A/Kansas/UR06-0104/2007 obtained from the NCBI database also were included. Different clades were shown by different colors.

Figure 3. Phylogenetic relationships of the PB1 and M segments of influenza A/H1N1 viruses in Taiwan.

The phylogenetic analyses were constructed using the neighbor-joining method with 1000 bootstrap replications. Branch values of more than 75 are indicated. All of the phylogenies were rooted with the A/New Caledonia/20/1999. Different clades were shown by different colors.

Figure 4. Amino acid changes in various proteins between clades 2B-1 and 2B-2 in the 92 global isolates.

Amino acids substitutions at positions 158, 200, 202, 206 in HA, 275, 354 in NA, 642 in PB1, 30, 41 in PB1-F2, and 411, 453 in PB2 are represent by various colors. Each column represents the amino acid position indicated. Amino acids (single-letter abbreviations are used) are indicated by different colors, as shown in the key. Each row represents a single isolate and the 92 isolates analyzed are displayed in this figure in the order of collection time. The 92 isolate names are listed in Table S1.