Molecular epidemiology and antigenic analyses of influenza A viruses
H3N2 in Taiwan
J.-H. Lin1,2, S.-C. Chiu1,3, J.-C. Cheng4, H.-W. Chang1, K.-L. Hsiao2, Y.-C. Lin2, H.-S. Wu1and H.-F. Liu2,5,6
1) Center for Research and Diagnostics, Centers for Disease Control, Taipei, Taiwan, 2) Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelong, Taiwan, 3) Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, 4) Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan, 5) Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan and 6) Institute of Public Health, National Yang-Ming University, Taipei, Taiwan
Abstract
The severity of an influenza epidemic season may be influenced not only by variability in the surface glycoproteins, but also by differ-ences in the internal proteins of circulating influenza viruses. To better understand viral antigenic evolution, all eight gene segments from 44 human H3N2 epidemic strains isolated during 2004–2008 in Taiwan were analyzed to provide a profile of protein variability. Comparison of the evolutionary profiles of the HA, NA and PB2 genes of influenza A (H3N2) viruses indicated that they were derived from a group of H3N2 isolates first seen in 2004. However, the PA, M and PB1 genes were derived from a different group of H3N2 isolates from 2004. Tree topology revealed the NP and NS genes could each be segregated into two groups similar to those for the polymerase genes. In addition, new genetic variants occurred during the non-epidemic period and become the dominant strain after one or two seasons. Comparison of evolutionary patterns in consecutive years is necessary to correlate viral genetic changes with anti-genic changes as multiple lineages co-circulate.
Keywords: Antigenicity, epidemiology, influenza virus, phylogenetic evolution, reassortment, Taiwan Original Submission: 6 December 2009; Revised Submission: 5 March 2010; Accepted: 20 March 2010 Article published online: 29 March 2010
Clin Microbiol Infect 2011; 17: 214–222 10.1111/j.1469-0691.2010.03228.x
Corresponding author: Dr H.-F. Liu, Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelong, Taiwan; Department of Medical Research, Mackay Memorial Hospital, Taiwan and Institute of Public Health, National Yang-Ming University, Taipei, Taiwan
E-mail: [email protected]
Introduction
Influenza viruses cause yearly epidemics worldwide in humans, and are some of the most active pathogens in Taiwan leading to significant morbidity and mortality [1,2]. Conventionally, analyses of influenza evolution have focused on individual viral genes, most often HA, without exploring the interactions among them. However, the evolutionary behaviour of the virus often involves cooperative changes within and between genes. Thus, there is important information about influenza evolutionary behaviour that may be deduced from correlated changes between nucleotide positions both within and between genes.
With the cocirculation of different influenza A subtypes, genetic reassortment can play an important role in antigenic variation [3,4]. Reassortment of the eight segments of influ-enza A viruses can lead to complicated phylogenetic pat-terns on the genomic scale [5–7]. Previous studies have shown that the virulence and growth characteristics of influenza viruses are influenced by changes in the internal proteins [8,9]. The M1 protein is a multifunctional protein that contributes to the control of virulence, growth [10– 13] and host specificity [14,15]. Changes in the NP, PB2 and M1 proteins were shown to play a role in host restric-tion in monkeys [14]. Considering these observarestric-tions, the severity of an influenza epidemic season may be influenced not only by variability in the surface glycoproteins, but also by differences in the internal proteins of circulating influ-enza viruses. In the present study, we examined the evolu-tionary dynamics of influenza A (H3N2) virus at genomic and epidemiological scales, as isolated during 2004–2008 in Taiwan, to provide a complete profile of protein variability as well as the viral antigenic evolutionary patterns of all gene segments of these viruses.
Materials and Methods
Isolates
Influenza strains circulating in 2004–2008 seasons were iso-lated from combined nose and/or throat swabs from selected patients with influenza-like illness presenting to phy-sicians in sentinel practices (http://www.cdc.gov.tw). The specimen were inoculated into Madin-Darby canine kidney cells and were confirmed by the indirect immunofluores-cence assay (IFA, Chemicon, Inc., Temecula, CA, USA) and RT-PCR. RT-PCR reactions were performed as described previously [16]. Supernatants from positive cultures were
harvested and stored at)80C for subsequent antigenic and
genetic analyses.
Genetic and antigenic analysis
Phylogeny construction and evaluation were performed with the maximum likelihood and Neighbour-joining (NJ) methods
in the PHYLIP software [17]. Empirical transition/transversion
ratio was estimated by the TREE-PUZZEL software [18] to
calculate the evolutionary distances. To determine the over-all selection pressures of each gene, we estimated the mean
numbers of nonsynonymous substitutions (dN) and
synony-mous substitutions (dS) per site (ratio dN/dS) using the SLAC
method within theHYPHYpackage [19] through the
Datamon-key web-based interface (http://www.datamonDatamon-key.org). The
dN/dS estimates were based on NJ trees under the GTR
substitution model. The full-genome sequences of 44 influ-enza A (H3N2) viruses obtained in the present study have been submitted to GenBank and assigned accession numbers: FJ805464–FJ805743.
Viruses were characterized by haemagglutination inhibition (HI) assays, as described previously [20,21], using post-infec-tion ferret antisera obtained from CDC, Atlanta, Georgia.
Results
Epidemic activities and antigenic analysis
Although the relative prevalence of influenza virus varies from season to season, influenza A (H3N2) viruses were the major epidemic strains isolated during the study period with the exception of the 2005–2006 season. H3N2 viruses fre-quently cocirculated with influenza B viruses as seen during the 2004–2005 and 2006–2007 seasons in Taiwan (Fig. 1).
A total of 2080 samples that tested positive for influenza viruses in the 2004–2005 season were confirmed by RT-PCR. Of these, 726 (34.9%) were influenza A (H3N2) viruses, 305 of which were selected for characterization in HI tests. Of those HI analyzed, 172 (56.4%) were A/Wyoming/3/03-like, with the remaining 133 (43.6%) isolates showing reduced
titres with antisera produced against A/Wyoming/3/03
(Fig. 2).
In the 2005–2006 season, a total of 858 samples tested positive for influenza and 228 (26.6%) of these were H3N2. Ninety-five isolates of the H3N2 positive samples were selected for further characterization by HI testing. Of those HI analyzed, 68 (71.6%) of them were A/California/7/04-like, with the other 27 (28.4%) showing reduced titres with anti-sera produced against A/California/7/04. In the subsequent 2006–2007 season, a total of 2175 samples tested positive for influenza. Of these, 770 (35.4%) were H3N2, 213 of which were selected for characterization in HI tests.
Ninety-Isolates B A (untyped) A (H3N2) A (H1N1) Positive rate 180 200 160 140 120 100 80 60 40 20 2 8 14 20 26 32 2004 2005 2006 2007 2008 37 43 49 3 9 15 21 27 33 39 45 51 4 10 16 22 28 34 40 46 52 6 12 18 24 30 36 42 48 2 8 14 20 26 32 38 40 500% 5% 10% 15% 20% 25% 30% 35% week
FIG. 1.Weekly distribution of Taiwanese influenza A (H3N2) isolates based on cell culture from 2004 to 2008, together with the positive rate
four (44.1%) of them were A/Wisconsin/67/05-like, with the remaining 119 (55.9%) having reduced titres with antisera produced against A/Wisconsin/67/05.
Analysis of individual gene segments
HA and NA genes. Comparison of the amino acid sequences encoded by the HA1 genes with that of the A/Wyoming/3/ 03 vaccine strain revealed six conserved amino acid changes (Table 1). The phylogenetic tree of these HA genes could be divided into three subgroups according to different influenza seasons: I, II and III (Fig. 3a). All isolates except those in sub-group Ia had an additional S227P change. Two additional amino acid changes at S193F and D225N were observed in both groups II and III. Isolates within subgroup IIIb had four additional changes at R142G, N144D, K173E and Y195H. Isolates in group IIIa lacked those four amino acids changes and could be further distinguished by an amino acid change at position G50E.
Similar to the results seen for HA, NA genes could be divided into three phylogenetic groups (Fig. 3b). This
observation suggested that the NA genes of recent H3N2 viruses, similar to their HA counterparts, have evolved as two distinct phylogenetic branches. A total of 24 amino acid substitution were observed compared to the NA gene of A/ Wyoming/3/03, and amino acid changes E199K, K221E and Q432E became fixed after 2004 (Table 1). The amino acids composition of group II was similar to group I with the exception of N93D, but differed from group III, which could be distinguished by the signature change H150R. N93D, V194I, Y310H L370S, S372L and N387K were signature changes for NA group IIIa, whereas group IIIb viruses all had changes at N86K, K296R, I307M and S335G. None of the established genetic markers for neuraminidase drug resis-tance at positions 119, 152, 274, 292 and 294 were found in our NA dataset [22].
Polymerase PB2, PB1 and PA genes. In the phylogenetic tree of the polymerase genes, the PB2 genes were divided into three groups, Using the classification of the HA genes, the PB2 genes of group Ia could be distinguished by amino acid
TABLE 1.Amino acid variation observed in HA1 and NA genes of human H3N2 viruses isolated from 2004 to 2008 in Taiwan
Protein HA1 NA 50 128 142 144 145 159 173 186 189 193 195 219 225 227 86 93 150 194 199 221 296 307 310 335 370 372 387 432 Wyoming/03 G A R N K Y K V S S Y Y D S N N H V E K K I Y S L S N Q I Ia T N F G N S D K E E Ib T N F G N S P D K E E II T N F G N F S N P K E E III IIIa E T N F G N F S N P D R I K E H S L K E IIIb T G D N F E G N F H S N P K R K E R M G E (a) (b)
FIG. 2.(a) Antigenic characterization of influenza viruses isolated from 2004 to 2007 in Taiwan. (b) Haemagglutination inhibition reactions of
influenza A (H3N2) virus using post-infection ferret antisera. Reference strains for 2004–2007 seasons, A/Wyoming/3/03, A/California/7/04, A/ Wisconsin/67/05, respectively.
(a) (b) (e) (f) 2007–2008 T aiwan/4/08 T a iwan/214/07 T a iwan/216/07 T a iwan/790/06 T a iwan/5/07 T a iwan/132/07 T a iwan/7/07 T a iwan/18/07 T a iwan/33/07 T a iwan/431/05 T a iwan/259/05 Taiwan/384/05 T a iwan/343/05 T a iwan/239/05 T a iwan/156/05 T a iwan/367/08 T a iwan/427/08 T a iwan/201/08 T a iwan/422/08 T a iwan/3/08 T a iwan/6/08 T a iwan/4/08 T a iwan/21/08 T a iwan/1/08 T a iwan/383/05 T a
iwan/350/04 Taiwan/249/04 Taiwan/292/04
T a iwan/20/04 T a iwan/129/05 T a iwan/351/04 T a iwan/300/04 T a iwan/296/04 T a iwan/330/04 T a iwan/413/04 T a iwan/33/07 T a iwan/18/07 T a iwan/5/07 Ta iwan/790/06 Ta iwan/216/07 T a iwan/214/07 T a iwan/7/07 T a iwan/132/07 T a iwan/431/05 T a iwan/17/06 T a iwan/239/05 T a
iwan/384/05 Taiwan/156/05 Taiwan/343/05
T a iwan/260/05 T a iwan/259/05 T a iwan/413/04 T a iwan/330/04 T a iwan/296/04 T a iwan/300/04 T a iwan/3/08 T a iwan/422/08 T a iwan/367/08 T a iwan/427/08 T a iwan/201/08 T a iwan/1/08 T a iwan/21/08 T a iwan/6/08 T a iwan/4/08 T a iwan/249/04 T a iwan/292/04 T a iwan/20/04 T a iwan/350/04 T a iwan/383/05 T a iwan/129/05 T a iwan/351/04 T a iwan/295/04 T a iwan/449/07 T a iwan/758/06 Ta iwan/754/06 T a iwan/448/07 T a iwan/799/06 T a iwan/133/06 T a iwan/211/06 T a iwan/295/04 T a iwan/448/07 T a iwan/449/07 T a iwan/799/06 T a iwan/260/05 T a iwan/17/06 T a iwan/754/06 T a iwan/758/06 T a iwan/133/06 T a iwan/211/06 T aiwan/3/08 Taiwan/422/08 T aiwan/367/08 T aiwan/427/08 T aiwan/201/08 IIIa IIIb III II II I II I II I I Ib Ia IIIa III IIIb II I T aiwan/6/08 T a iwan/21/08 T aiwan/1/08 T aiwan/448/07 T aiwan/758/06 Taiwan/754/06 T aiwan/799/06 T a iwan/21/06 T aiwan/133/06 T aiwan/216/07 T aiwan/214/07 Taiwan/790/06 T a iwan/18/07 Taiwan/33/07 T aiwan/7/07 Taiwan/5/07 T a iwan/132/07 T a iwan/156/05 Taiwan/431/05 T aiwan/259/05 T aiwan/17/06 T
aiwan/206/05 Taiwan/384/05 Taiwan/343/05
T aiwan/239/05 T aiwan/249/04 T aiwan/129/05 T aiwan/383/05 T aiwan/20/04 T aiwan/292/04 Taiwan/350/04 T aiwan/351/04 T aiwan/296/04 T
aiwan/300/04 Taiwan/330/04 Taiwan/413/04 T
aiwan/295/04 T aiwan/449/07 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W ellington/1/04 Calif ornia/7/04 W y oming/03/03 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W e llington/1/04 Calif ornia/7/04 W y oming/03/03 T
aiwan/3/08 Taiwan/427/08 Taiwan/448/07
T aiwan/201/08 T aiwan/1/08 T aiwan/367/08 T aiwan/4/08 Taiwan/6/08 T aiwan/21/08 T a iwan/422/08 T aiwan/449/07 T a iwan/754/06 T aiwan/758/06 T aiwan/799/06 T aiwan/5/07 Taiwan/18/07 T aiwan/90/06 T
aiwan/7/07 Taiwan/216/07 Taiwan/214/07Taiwan/312/07 Taiwan/33/07 T
a iwan/383/05 T aiwan/211/06 T aiwan/133/06 T aiwan/259/05 T aiwan/260/05 T aiwan/17/06 T aiwan/384/05 T aiwan/343/05 T aiwan/431/05 T aiwan/156/05 T aiwan/239/05 T aiwan/413/04 Taiwan/350/04 T aiwan/292/04 T aiwan/249/04 T aiwan/20/04 T aiwan/129/05 T aiwan/351/04 T aiwan/330/04 T aiwan/300/04 T aiwan/296/04 T aiwan/295/04 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W ellington/1/04 Calif ornia/7/04 W y oming/3/03 P anama/2007/99 P anama/2007/99 P anama/2007/99 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W e llington/1/04 Calif ornia/7/04 W y oming/3/03 P anama/2007/99 2006–2007 2006–2007 2005–2006 2004–2005 2004–2005 HA PB2 NP 0.002 0.001 0.002 0.001 NA FIG . 3 . Ph ylogenetic analysis of gene tic evoluti on of full lengt h, co mplete genome sequenc es of 44 human influen za A (H3N 2) viruses isola ted fro m 2004 to 2008 in Taiwan . T h e trees were in ferred from HA (a), NA (b) , PB2 (c), PB1 (d), PA (e) , N P (f), MP (g) and NS (h) gene data by th e Neighbo ur-joini ng method and using boo tstrap analysis (n = 1000 ) to determ ine the best-fittin tree . The color ed conne ctors in dicate the major isol ates that resul t from reasso rtment. Th e re ference strains are re presen ted in italic boldfac e. V accin e strains for 2004 –200 8 seaso ns, A/Cali for-ni a/7/04, A/Wi sconsin/ 67/05, A/Brisb ane/ 10/07, re spectivel y.
(c) (d) (g) (h) T aiwan/1/08 T aiwan/422/08 T aiwan/427/08 T aiwan/201/08 T a iwan/367/08 T aiwan/3/08 T aiwan/6/08 Taiwan/4/08 T aiwan/21/08 T aiwan/431/05 T aiwan/343/05 T aiwan/384/05 T aiwan/17/06 T aiwan/260/05 T aiwan/239/05 T aiwan/156/05 T aiwan/296/04 T aiwan/300/04 Taiwan/330/04 T aiwan/413/04 T aiwan/295/04 T a iwan/296/04 T aiwan/413/04 T aiwan/300/04 T a iwan/330/04 T aiwan/295/04 T aiwan/351/04 T a iwan/129/05 T aiwan/156/05 T aiwan/260/05 Taiwan/239/05 T aiwan/431/05 T aiwan/17/06 T a iwan/343/05 T aiwan/259/05 T a iwan/384/05 T a iwan/4/08 T aiwan/6/08 T aiwan/21/08 T aiwan/1/08 T aiwan/427/08 T aiwan/201/08 Taiwan/3/08 T aiwan/367/08 T aiwan/422/08 T aiwan/5/07 T aiwan/132/07 T a iwan/216/07 T aiwan/214/07 T aiwan/790/06 T a iwan/7/07 T aiwan/33/07 T aiwan/18/07 T aiwan/133/06 T aiwan/211/06 T aiwan/449/07 T aiwan/754/06 T aiwan/758/06 T aiwan/799/06 T aiwan/383/05 T a iwan/20/04 T
aiwan/350/04 Taiwan/292/04 Taiwan/249/04
T aiwan/448/07 T aiwan/351/04 T aiwan/249/04 T aiwan/20/04 T aiwan/292/04 T a iwan/350/04 T aiwan/383/05 T aiwan/129/05 T aiwan/259/05 T aiwan/799/06 T aiwan/449/07 T aiwan/133/06 T
aiwan/211/06 Taiwan/758/06 Taiwan/754/06
T aiwan/448/07 T aiwan/5/07 T aiwan/33/07 T aiwan/7/07 T a iwan/132/07 T aiwan/216/07 T aiwan/214/07 T aiwan/18/07 T a iwan/790/06 II I II I I II II I T aiwan/6/08 T aiwan/21/08 T aiwan/4/08 T a iwan/1/08 T aiwan/449/07 T aiwan/201/08 T a iwan/427/08 T aiwan/3/08 T aiwan/367/08 T aiwan/422/08 T
aiwan/448/07 Taiwan/754/06 Taiwan/758/06
T aiwan/431/05 Taiwan/211/06 T aiwan/133/06 T aiwan/260/05 Taiwan/343/05 T aiwan/156/05 T aiwan/17/06 Taiwan/384/05 T aiwan/18/07 T aiwan/214/07 T aiwan/216/07 T aiwan/790/06 T aiwan/5/07 T aiwan/33/07 T aiwan/132/07T aiwan/7/07 T aiwan/239/05 T aiwan/259/05 T aiwan/413/04 T aiwan/300/04 T aiwan/330/04 Taiwan/296/04 T aiwan/20/04 T aiwan/292/04 T aiwan/249/04 T aiwan/350/04 T aiwan/383/05 T aiwan/799/06 T aiwan/129/05 T aiwan/351/04 T aiwan/295/04 T aiwan/427/08 T aiwan/3/08 Taiwan/20/08 T aiwan/367/08 T aiwan/422/08 T aiwan/448/07 T aiwan/449/07 Taiwan/4/08 Taiwan/1/08 T aiwan/6/08 T aiwan/21/08 T aiwan/754/06 Taiwan/758/06 T aiwan/384/05 T aiwan/156/05 Taiwan/260/05 T aiwan/17/06 T
aiwan/431/05 Taiwan/211/06 Taiwan/133/06
T aiwan/343/05 T aiwan/239/05 T aiwan/214/07 Taiwan/216/07 T aiwan/790/06 T aiwan/18/07 T aiwan/132/07 T aiwan/7/07 T aiwan/5/07 T aiwan/33/07 T aiwan/413/04 T aiwan/296/04 Taiwan/300/04 Taiwan/330/04 T aiwan/799/06 T aiwan/129/05 T aiwan/351/04 T aiwan/383/04 T aiwan/20/04 Taiwan/292/04 T aiwan/350/04 T aiwan/249/04 T aiwan/295/04 T a iwan/259/05 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W ellington/1/04 Calif ornia/7/04 W y oming/3/03 P anama/2007/99 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W ellington/1/04 Calif ornia/7/04 W y oming/3/03 P anama/2007/99 P anama/2007/99 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W ellington/1/04 Calif ornia/7/04 W y oming/03/03 P anama/2007/99 Brisbane/10/07 Wisconsin/67/05 Hir oshima/52/05 W ellington/1/04 Calif ornia/7/04 W y oming/03/03 MP NS 0.001 0.001 0.002 0.002 PA PB1 FIG . 3 . (Conti nued)
changes at T64I and T683A. An amino acid substitution V461I was observed in group Ib, and the E249G amino acid substitution distinguished group IIIb. The grouping of the PA genes was similar to that seen for the PB1 gene (Fig. 3c,d,e). When examining the deduced amino acid sequences of the PB1 genes, all isolates had an N375S amino acid substitution, and all except group Ib contained a V113A amino acid change compared to A/Wyoming/3/03(Table 2). In addition, all of the PA genes had the amino acid changes E101G, I421V and Y437H, except for group Ib. An addition amino acid substitution at S208T was seen in both groups II and III. The subgroup Ib had unique changes at V62M, Q256K, E382D and V602I (Table 2).
NP, M and NS genes. Phylogenetic analysis of the NP, M and NS genes showed that these three genes of these viruses could each be segregated into two groups similar to those for the polymerase genes (Fig. 3f,g,h). Using the classification used for the HA genes (above), three amino acid substitu-tions (Y52H, V280A and V312I) were observed in the NP genes of both group Ib and group IIIa. The amino acid substi-tution S377G was present in all isolates except those in group Ib and additional amino acid changes at K31R and E495K were observed in group IIIb.
Signature change associated with adamantanes resistance S31N in M2 gene became fixed after 2004 in all isolates except those in group Ib. Group IIIb could be distinguished by the amino acid change T239N in the M1 gene and P10T and G58S changes in the M2 gene. The NS1 genes of Tai-wanese isolates all have the V124M amino acid difference compared to A/Wyoming/3/03, with group IIIa having an additional amino acid change at K221E (Table 2). There were no changes in NS2 compared to the A/Wyoming/3/03 vac-cine strain.
Measurement of selection pressures
The selection pressures acting on human H3N2 viruses were
higher than for previous reports [23,24]. The highest dN/dS
ratios were observed in the HA and NA (mean dN/dS0.41
and 0.37, respectively), most likely reflecting immune selec-tion pressure at a small number of amino acid sites [25], and
also NP, NS and M (mean dN/dS0.08, 0.32, and 0.08,
respec-tively), which are essential for transcription and replication.
Discussion
To date, molecular and phylogenetic analyses of influenza A H3N2 viruses spanning successive seasons have not been reported in Taiwan. The changing patterns of genetic diver-sity in viral isolates provide insight into the seasonal dynam-ics of influenza A and reveal the evolutionary interaction between subtypes; for example, recent seasons dominated by H1N1 were observed following H3N2 epidemics (Fig. 1). The amino acids in the H3 HA1 from 2004 to 2008 had a total of 17 amino acid substitutions, all of which were located at well known antibody antigenic sites or receptor binding sites.
Changes in the 140–146 region of antigenic site A are characteristic for antigenically distinct viruses of epidemic sig-nificance [26]. The amino acid substitution K145N has been fixed subsequent to the 2004–2005 season and we found two additional amino acid changes, R142G and N144D, in the antigenic site A of viruses isolated in 2007. Moreover, one region in HA at amino acids 225–227, which affects anti-genic site D, changed dramatically during the study period as a result of D225N and S227P mutations. The 225 and 227 positions are also located in the receptor binding pocket and thus could be expected to have effects beyond antigenic vari-ation. Two amino acid substitutions at positions 128 (A to T; position 126–128 are N,W and T) and 144 (N to D; position 144–146 are D,N and S) were observed that could affect N-linked glycosylation, which could conceivably allow escape from human immune pressure.
The NA genes of the A/Hiroshima/52/05 reference strain apparently evolved along a separate evolutionary pathway
Protein PB2 PB1 PA NP M1 M2 NS1 64 249 461 683 113 375 586 621 642 62 101 208 256 382 421 437 602 31 52 280 312 377 495 174 219 239 10 31 58 124 221 Wyoming/03 T E V T V N R R N V E S Q E I Y V K Y V V S E R I T P S G V K I Ia I A A S S G V H G N M Ib I S Q M K D I H A I K V M II A S G T V H G N M III IIIa A S K G T V H H A I G N M E IIIb G A S G T V H R G K N T N S M
and cocirculated with A/California/7/04-like and A/Wiscon-sin/67/05-like viruses between 2004 and 2007, thus providing an opportunity for mixed infections that might lead to the emergence of reassortants. These reassortants would not be expected to have any increased epidemic potential compared to their parents and, at this stage, there is no definitive evi-dence that the influenza A H3N2 reassortant viruses were more virulent than the nonreassortant ones in terms of clini-cal outcomes. A previous study suggested that new antigenic variants may arise from changes that affect N2 antigenic site B [27]. H3N2 viruses isolated from Taiwan possessed E199K and K221E changes in the NA gene, both of which are found in antigenic site B. We also detected amino acid substitutions in the neuraminidase gene that were not associated with cat-alytic or framework sites, and none of the neuraminidase substitutions conferring resistance to neuraminidase inhibi-tors have been observed in our study. Similarly, we did not observe amino acid changes in the N2 protein that would be expected to lead to a decrease in virus replication in eggs [28]; the amino acids known to allow efficient replication in eggs (Q119, K136 and Y347) were all present in the viruses examined.
Comparison of the evolutionary profiles of the HA, NA, and PB2 genes of influenza A (H3N2) viruses revealed that these genes of 2005–2008 isolates were derived from iso-lates having the group Ib HA gene, and PA, M and PB1 genes were derived from group Ia isolates of the 2004–2005 sea-son. The phylogenetic trees of HA and NA showed seasonal clusters but also co-circulating lineages within each season. It should be noted that, in the 2006–2007 season, the HA, NA, PA, PB1 and M genes of three viruses (A/Taiwan/799/2006, A/Taiwan/448/2007 and A/Taiwan/449/2007) were in group IIIa but the PB2 and NP genes were in group Ib. These new genetic variants were detected during the 2006 and 2007 non-epidemic periods and the reassortants isolated in 2006 were antigenically similar to A/California/7/04; however, the reassortants isolated in 2007 demonstrated a four-fold HI difference compared to A/Hiroshima/52/2005, both of which were vaccine strains recommended for the 2005–06 and 2006–07 season, respectively. Indeed, these reassortant viruses continued to be isolated and become the dominant strains during year 2008 (Fig. 3).
Reassortment among co-circulating H3N2 viruses similar to that observed in present study has been described previ-ously, including reassortment of the NA gene segment [29] and the internal segments [30–33]. However, a different phylogenetic pattern from previous report is observed dur-ing the 2007–08 season, and such a major difference in the phylogenetic signal strongly suggests that H3N2 viruses evolve rapidly, as a result not only of genetic drift, but also
natural selection. The complete genome analysis of recently sampled Taiwan A (H3N2) influenza viruses in the present study has revealed the co-circulation of multiple distinct groups and frequent intra-subtype reassortment events among them, and such evolution plays an important role in antigenic drift.
In addition, the data presented here reveal that the M2 proteins in all groups except group Ib possessed the substi-tution S31N. It is interesting to note that all isolates with this S31N amino acid change also have amino acid substitu-tions in PA and PB1 genes, including E101G, S208T, K256Q, D382D, I421I and Y437H in PA, and the V113A amino acid change in PB1. The role of PA is not well established, although it is known to be involved in RNA replication [34,35]. Recent studies indicate that PA is likely to be involved in transcription as well as replication [36,37]. It has also been reported that the N-terminal region of PA plays a critical role in multiple functions, such as cap-binding, endo-nuclease activity, etc. [38].
Genetic analyses of influenza H3N2 viruses provide infor-mation that is important for understanding the mechanisms involved in the emergence of pandemic influenza viruses. The present study covered the phylogenetic relationships of the external as well as internal genes and clearly demonstrates the value of analyzing the complete genetic composition of influenza viruses in order to understand fully the evolution-ary mechanisms and epidemiology of influenza. Furthermore, the present study explores in detail a reassortment event in internal genes leading to an epidemiologically significant out-come, namely the emergence of the ‘2008–2009 epidemic strain’ in the 2006–2007 season. Importantly, the data pre-sented here provide evidence suggestign that influenza A H3N2 viruses undergo internal gene reassortment and that important evolutionary processes may occur during non-epi-demic periods at least one or two season ahead. Although it is not yet clear how variant clades manage to persist along-side dominant strains, the fact that they do suggests the influenza virus has multiple adaptive abilities. The overall selection pressure also reveals that the former have not yet reached a global fitness peak characterized by little amino acid fixation.
Analyses of the genetic and antigentic data, together with epidemiological data for the sequenced isolates, have provided insights into the pattern of virus spread, the genetic diversity during seasonal epidemics, and the dynamics of subtype evolution. Continuous monitoring of viral genetic changes throughout consecutive years is necessary to formu-late hypotheses as to why antigenic evolution allows cer-tain variants to become dominant when multiple lineages co-circulate.
Acknowledgements
We are grateful to Dr Michael Shaw at the Influenza Divi-sion, Centers for Disease Control and Prevention, for critical reading and comments on the manuscript.
Transparency Declaration
This study was supported by the Centers for Diseases Control, Taiwan (grant DOH98-DC2007), and was funded in part by the National Science Council (grant NSC 95-2314-B-195-008) and Mackay Memorial Hospital (grant MMH 9633).
The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency.
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