Epidemiologic Study of Human Influenza Infection in Korea ...jcm.asm.org/content/early/2010/04/14/JCM.00209-10.full.pdf60 of amino acid variation in HA is clustered in variable antigenic

Jan 25, 2010 1

Mar 26, 2010 (revised) 2

3

Epidemiologic Study of Human Influenza Infection in 4

Korea from 1999 to 2007: Origin and Evolution of 5

A/Fujian/411/2002-like strains 6

Seokha Kang,1* In Seok Yang,2* Joo-Yeon Lee,2 Yiho Park,1 Hee-Bok Oh,2 Chun 7

Kang,2** and Kyung Hyun Kim1** 8

Department of Biotechnology & Bioinformatics, College of Science & Technology, 9

Korea University, Chungnam, Korea,1 Center for Infectious Diseases, Korea Centers 10

for Disease Control and Prevention, Seoul, Korea,2 11

12

*S.K. and I.S.Y. contributed equally to this work 13

14

**Corresponding authors: C. Kang. Center for Infectious Diseases, Korea Centers for 15

Disease Control and Prevention, Korea. [email protected]; K. H. Kim. Department of 16

Biotechnology & Bioinformatics, Korea University, Korea. [email protected] 17

18

19

Running Title: Influenza epidemiology in Korea from 1999 to 200720

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00209-10 JCM Accepts, published online ahead of print on 14 April 2010

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ABSTRACT 21

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Influenza epidemics arise through the accumulation of viral genetic changes, 23

culminating in a novel antigenic type that is able to escape host immunity. Following 24

an outbreak of the A/Fujian/411/2002-like strains in Asia including China, Japan and 25

Korea in 2002, Australia and New Zealand experienced substantial outbreaks of the 26

same strains in 2003 and subsequently worldwide in the 2003-2004 season. The 27

emergence of A/Fujian/411/2002-like strains coincided with a higher level of 28

influenza-like illness in Korea than what is seen at the peak of a normal season, and 29

there was at least a year’s difference between Korea and the U.S.A. Genetic 30

evolution of human influenza A/H3N2 viruses was monitored by sequence analysis 31

of hemagglutinin (HA) genes collected in Asia including 269 (164 new) HA genes 32

isolated in Korea from 1999 to 2007. The Fujian-like influenza strains were 33

disseminated with rapid sequence variation across the antigenic sites of the HA1 34

domain, which sharply distinguished between the A/Moscow/10/1999-like and 35

A/Fujian/411/2002-like strains. This fast variation, equivalent to approximately 10 36

amino acid changes within a year, had occurred in Asia, and would be the main 37

cause of the disappearance of the reassortants, although the reassortant and non-38

reassortant Fujian-like strains simultaneously circulated in Asia. 39

40

Keywords: influenza virus, H3N2, genetic variation, hemagglutinin 41


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INTRODUCTION 42

Influenza is an important respiratory infectious disease causing seasonal epidemics or 43

occasional pandemics across the world with considerable morbidity and mortality. The 44

influenza outbreaks are associated with antigenic variation of influenza viruses. Annual 45

influenza epidemics typically occur during the winter season in temperate regions, whereas 46

tropical regions may function as permanent mixing pools for viruses, providing a ready 47

source of extended viral transmission (7, 26). Antigenic and genetic analysis revealed that 48

there was a continuous circulation of human influenza A/H3N2 viruses in East and Southeast 49

Asia via a regional network from which epidemics in the temperate regions were seeded (22). 50

In particular, southern China was considered a potential epicenter for emergence of novel 51

influenza virus strains (23). 52

The enveloped influenza virus contains eight segments of negative-sense single-stranded 53

RNA, each of which codes for particular viral protein(s). The gene segment coding for a 54

surface glycoprotein hemagglutinin (HA) is of major importance because HA is the primary 55

target of immune response and the primary component of influenza vaccine. HA is a 56

homotrimeric protein synthesized as a single polypeptide HA0 that is cleaved into two 57

subunits, HA1 and HA2, for receptor binding and cell entry (18). Antibodies against HA are 58

elicited during virus infection to inhibit binding with receptor effectively (27). Accumulation 59

of amino acid variation in HA is clustered in variable antigenic sites around the receptor 60

binding site, which leads to gradual antigenic drift in the influenza viruses. It was previously 61

proposed that an antigenic drift variant of epidemiological importance usually requires 62

changes of at least four amino acids across two or more antigenic sites but a single amino 63

acid substitution at one antigenic site can cause sufficient antigenic change (9, 10). The 64

influenza virus can also acquire a new subtype by reassortment of one or more gene 65


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segments, which, combined with antigenic drift, provides the basis for the remarkable 66

antigenic variability in viral populations (12). 67

A/Moscow/10/1999-like or antigenically equivalent A/Panama/2007/1999-like strains of 68

H3N2 had been circulating worldwide since 1999. The emergence of A/Fujian/411/2002-like 69

strains caused an epidemic in China, Japan and Korea in 2002 (3, 13, 21). It was shown that 70

two amino acid substitution was attributed to be critical for antigenicity distinct from 71

A/Panama/2007/1999 (14). Interestingly, a descendant of the Fujian strain reassorted, which 72

caused an unusually severe influenza season in Australia and New Zealand in 2003 and in 73

North America, Europe and worldwide in the 2003-2004 season (2, 4, 13). This reassortment 74

caused a minor clade to provide a HA gene that later became part of the dominant strain at 75

the same season (4, 13), reaching South America after an additional 6 to 9 months (22). The 76

appearance of the Fujian strains thus prompted a change in the selection of vaccine 77

components in 2004. However, non-reassortant strains were the dominant strains in Asia in 78

the 2002-2003 season and thereafter. 79

The elucidation of how and when a new influenza virus emerges as an epidemic strain 80

requires a deeper understanding of the mechanisms that underlie viral evolution. We have 81

determined the nucleotide sequence of the HA gene segments of influenza viruses in nasal 82

swabs collected from infected patients aged 6 months and older during the 1999-2007 83

influenza seasons in Korea. At the same time, influenza-like illness was monitored during 84

each season and the phylogeny of HA sequences available worldwide was analysed to 85

investigate the origin and evolution of the H3N2 Fujian strains. 86


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MATERIALS & METHODS 87

Virus collection and isolation. Nasopharyngeal swabs were obtained from outpatients 88

with symptoms of ILI, residing in Seoul and other cities in Korea from 1999 and 2007. The 89

samples in viral transport medium were transported to the Influenza Virus Disease Team at 90

the Korea National Institute of Health (KNIH) in Seoul on the day of collection. One 91

hundred microliter aliquots of the supernatants of the nasopharyngeal swabs were inoculated 92

onto Madin-Darby Canine Kidney (MDCK) cells in 48-well multiple plates which were 93

prepared at 37oC with 5% CO2. Virus growth was monitored at 34

oC with reference to 94

cytopathic effects. The viruses were passaged three times to obtain sufficient virus titers for 95

virus identification. All isolates were typed and subtyped by the hemagglutination inhibition 96

assay (15). 97

98

RNA extraction and PCR. One hundred-microliter aliquots of the supernatant after the 99

third culture passage were used for viral RNA extraction with an Extragen II kit (Kainos, 100

Tokyo, Japan), according to the manufacturer’s instructions. RNA was transcribed to cDNA 101

with the influenza A virus universal primer and the HA gene of H3N2 viruses was amplified 102

with segment-specific primers (15). 103

104

Nucleotide sequencing and sequence analysis. The PCR products were purified with a 105

MicroSpin S-300 HR column (GE Healthcare, Uppsala, Sweden), labeled by using a BigDye 106

Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster, CA), according to the 107

manufacturer’s instructions, and analyzed on an ABI 3100 automatic DNA sequencer. 108

Sequence alignment of HA1 domain was performed using MUSCLE program (8). The 109

maximum parsimony tree of Korean strains was inferred using MEGA 4.0 program with 110


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Close-Neighbor-Interchange algorithm (25). In this analysis, 116 amino acid sequences of 111

Korean strains, representatives of 269 strains after excluding 153 strains that have identical 112

amino acid sequences, were included for the reconstruction of a phylogenetic tree. 113

A/Beijing/32/1992 was used for rooting the tree and 7 other vaccine strains were included as 114

reference strains for each year’s epidemics. In addition, 6 strains isolated in 2009 were 115

combined to the dataset to determine the ancestral lineage of recent H3N2 strains. HA and 116

neuraminidase (NA) maximum likelihood (ML) trees were determined to identify Fujian-117

reassortants in Asia during two consecutive seasons 2002-2004. A total of 30 New York 118

strains including 11 reassortant strains in figure 1 of reference (19) were incorporated to 119

assess the phylogenetic positions of reassortants. The node support was calculated by 120

approximate likelihood ratio test (aLRT) using PhyML (1, 11). To examine the evolution of 121

A/Fujian/411/2002-like strains, 521 HA1 nucleotide sequences from the National Center for 122

Biotechnology Informatics (NCBI) GenBank and newly sequenced Korean isolates were 123

subjected to ML analysis. The dataset for ML phylogenetic analysis contained HA1 of H3N2 124

viruses collected globally from 2001 to 2002 except for the sequences less than 950bp or 125

ambiguous sequences. Two Korean strains diverged prior to A/Sydney/5/1997, i.e. 126

A/Gyeongbuk/2/2002 and A/Gyeongbuk/304/2002, were excluded. After 127

A/Moscow/10/1999 was used as the phylogenetic root and 2 vaccine strains in 2004 as 128

representatives of the following season’s dominant strains were combined, we re-analysed 129

53 strains from the ML tree to pinpoint the evolutionary pathway of the Fujian/411 origin. 130

All ML analyses were performed using PhyML software with GTR+I+Γ4 model (11). 131

132

Nucleotide sequence accession number. A total of 164 nucleotide sequences for H3N2 133

subtype from KNIH were deposited in the NCBI GenBank, (accession nos. CY054107-134


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CY054270), and 105 H3N2 nucleotide sequences of Korean isolates were retrieved from the 135

NCBI Influenza Virus Resource, which are listed in Table S1. 136

137

138

RESULTS 139

Prevalence of influenza in Korea from 1999 to 2007. The relative prevalence of 140

influenza virus subtypes varied from season to season. Human influenza A/H3N2 viruses 141

were the dominant circulating strain in Korea during the seasons from 1999 to 2007 (Fig. 142

1A). In addition, the 2001-2002 and 2005-2006 seasons were marked by the spread of 143

influenza A/H1N1 viruses in peak winter weeks and B viruses in the following spring. Our 144

data showed that H3N2 has become widespread and co-circulated with H1N1 and B viruses 145

during the 2000-2007 seasons. 146

A/Moscow/10/1999-like strains were dominant in the seasons from 1999 to 2001 (Fig 147

2A). The change of dominant strains in H3N2 from A/Moscow/10/1999 to 148

A/Fujian/411/2002 caused a big epidemic worldwide in 2003-2004 (3, 4, 17, 21, 22). 149

However, the influenza activity of the A/Fujian/411/2002-like viruses was already observed 150

in China and Korea in 2002 (6). Our epidemiologic study showed that the emergence of 151

A/Fujian/411/2002 coincided with higher level of influenza-like illness in Korea than what is 152

typically seen at the peak of a normal season. It was particularly notable that monitoring of 153

ILI during the 2002-2004 seasons demonstrated a one-year difference between the ILI 154

patient data of Korea with those of United States (Fig 1B). Further, the composition of 155

influenza subtypes circulated in United States essentially resembled that in Korea in each 156

previous year (Fig 1C). 157

158


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A/H3N2 influenza viruses. HA genes of 269 human influenza A/H3N2 viruses that had 159

been collected and sequenced in Korea from 1999 to 2007 were used in this study. The 160

maximum parsimony tree comprising 228 amino acid changes revealed that there are 8 161

subgroups from 2000 to 2007 in Korea (Fig. 2A). Three isolates of 1999 and two of 2002 162

seasons were nested between A/Beijing/32/1992 and A/Sydney/5/1997. The other three 163

isolates of 1999 formed a separate clade from A/Moscow/10/1999. In 2002, 12 of 16 isolates 164

were Fujian/411-like, excluding A/South Korea/C5-4/2002 and A/Kwangju/219/2002 (Fig. 165

2A). Five isolates of the 02-03 groups were found to possess essentially identical amino acid 166

sequences with A/Fujian/411/2002, and additional three isolates could be regarded identical 167

if the missing or ambiguous sequences were supposed to be the same with the Fujian 168

sequence. This suggests that the Fujian strains were introduced and widespread prior to the 169

2002-2003 season in Korea. 170

A reassortant strain emerged in New Zealand and Australia in the summer of 2003 and 171

was later spread to United States and Europe during the 2003-04 season (2, 4, 13). The 172

epidemiological and genetic data of the Fujian-like influenza viruses including 30 New York 173

strains (19) indicated that both the reassortants and non-reassortants circulated in Asia during 174

the 2002-2004 seasons (Fig. 3A & 3C). All of 11 reassortant New York strains were 175

separated from the non-reassortant New York strains and formed a clade (gray-lined 176

branches in a circle) with some Asian strains in both of HA and NA trees (Fig. 3B & 3D). 177

The reassortment event was confirmed by the incongruence of the two trees, i.e. the deep 178

branching between 11 reassortants and A/New York/406/2002 (marked as a gray dot on the 179

trees) which had appeared prior to the reassortants in the HA tree and close relationship 180

among them in NA. The reliability of the reassortant clade was supported not only by the 181

moderate aLRT values (0.785 and 0.983 for the HA and NA trees, respectively), but also by 182


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the fact that no incongruence was found between both trees (i.e. no strain was located in the 183

reassortant clade in the HA tree but was simultaneously positioned in the non-reassortant 184

group in the NA tree, or vice versa.). 185

Unlike the Fujian-reassortants, all of Korean strains were separated from the Fujian-186

reassortant clade with the exception of two strains A/Korea/124/2003 and A/Jeju/218/2004 187

(Fig. 2A). The phylogenetic tree of HA demonstrated that the non-reassortant strains rapidly 188

predominated in Korea as well as in Asia during the 2002-2004 seasons (Fig. 3A). While the 189

reassortment event was known to be the source of increased fitness of the virus in Australia, 190

United States and Europe, it is evident that the non-reassortants became the predominant 191

strains in the following seasons. The Fujian-reassortants were indeed significantly reduced in 192

the 2004-2005 season and disappeared (data not shown). In this context, it is not surprising 193

to observe that the epidemic strains to date were descended from the non-reassortant lineage. 194

Our results thus strongly suggest that the antigenic drift of HA alone gave sufficient fitness 195

increase to the virus in and outside of Asia (Fig. 3A & 4A). The 06-07A and 06-07B clades 196

of the non-reassortants were predominant during the 2006-2007 season, and the recent 197

strains were originated from the minor clade which has the G50E mutation in the 2005-2006 198

season. 199

200

Amino acid variation. A total of 105 sites were found to be subjected to amino acid 201

substitutions which were marked retrospectively on the maximum parsimony tree from 202

A/Sydney/5/1997 to 2007 strains, excluding uninformative amino acid changes (Fig. 2). It is 203

clearly evident that a rapid evolution which had occurred between 2001 and 2002 resulted in 204

the emergence of the Fujian/411 strain. These include antigenic sites A(Ile144), B(His155, 205

Ser186) and E(Glu83), receptor binding site (Gly225) and the positive selection sites 206


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(Gln156, Ser186) which also contribute to receptor-binding adaptation (5, 18, 27). Multiple 207

changes were also found in amino acid residues 50, 142, 144 and 225. The R50G mutation 208

took place in the 00-01 group with additional changes in the clade containing 209

A/Brisbane/10/2007 and 2009 strains. The amino acid residue 144, located at antigenic site 210

A, was altered from Ile to Asn between 1999 and 2001. Additional mutations simultaneously 211

occurred in some of the 03-04 and 06-07A strains from Asn to Asp. Amino acid residue 225 212

was mutated twice at the emergences of the 02-03 and 05-06 strains. At the 06-07 season, 213

Arg142 was mutated far back to its original glycine residue which could be found at 214

A/Sidney/5/1997. 215

216

Origin of A/Fujian/411/2002. We reconstructed a phylogenetic tree to estimate the 217

position of A/Fujian/411/2002 among 521 strains isolated from around the globe in 2001 and 218

2002 (Fig. 4A) and chose 53 strains located near the emergence of A/Fujian/411/2002. 219

Sequential amino acid changes at key antigenic sites along the evolutionary pathway of the 220

Fujian strains are shown in the maximum likelihood tree (Fig. 4B). The S186G and A131T 221

mutations occurred in 2001, which was followed by mutations of both L25I and H75Q. 222

Since any intermediate strain having one mutation of either L25I or H75Q was not observed, 223

the order of the two mutations could not be determined. Phylogenetic position of 224

A/India/C3-45/2002 and A/Taiwan/8/2002, which have mutations L25I and H75Q but not 225

H155T, suggested that the H155T mutation occurred after the population spread of the 226

simultaneous mutations of L25I and H75Q. The phylogenetic tree also indicated that 227

A/Fujian/411/2002 already has additional DNA substitutions from the trunk strains 228

(A/Hunan/407/2002, A/Cheju/311/2002, A/Chungnam/447/2002, and 229

A/Kyongnam/347/2002), despite their identical amino acid sequences. 230


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Close comparative analyses of the sequences and amino acid changes revealed a couple 231

of prominent features. First, most of the intermediates and Fujian-like strains were isolated 232

from Asian countries, whereas 7 out of 53 strains were not Asian. In addition, none of the 233

earliest strains at each clade were isolated from non-Asian countries. Given the sampling 234

bias that only 33.7% (175 of 521) is isolated in Asia (Fig. 4A), our results strongly suggest 235

that these mutational events associated with the Fujian strains took place in Asia. Second, the 236

sequences of the isolates collected during the 2001-2002 season allowed us to estimate the 237

evolutionary history and inferred date of introduction to Asian population of the Fujian 238

strains. Closely dated phylogeny from Dec 26, 2001 through Aug 11, 2002 showed that the 239

antigenic evolution of the H3N2 Fujian strains had periods of rapid antigenic changes, 240

equivalent to 10 amino acid changes per year (Fig. 4C). Different subtypes evolve at 241

different rates such that H3N2 viruses change more rapidly than H1N1 viruses, with the 242

average rate of 3.6 amino acid substitutions per year (24). In this regard, the change in the 14 243

amino acids that had accumulated from the Moscow/10/1999 clade to the Fujian/411/2002 244

clade showed an exceptionally fast rate of evolution. However, genetic distance of vaccine 245

strains from a clade to a subsequent clade was merely two (A/Wellington/01/2004 to 246

A/California/7/2004, or A/California/7/2004 to A/Wisconsin/67/2005) or three amino acid 247

substitutions (A/Sydney/5/1997 to A/Moscow/10/1999, or A/Fujian/411/2002 to 248

A/Wellington/01/2004). Taken together, our results demonstrated that the antigenic 249

evolution of the Fujian strains was initiated by rapid antigenic change that had occurred in 250

Asia, which was then continued by relatively modest changes. 251

252

253


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DISCUSSION 254

Human influenza H3N2 viruses have been the dominant strain in most years since they 255

first emerged in 1968, and have been responsible for one of the most serious respiratory 256

infections until the novel swine-origin influenza H1N1 virus emerged in 2009 causing a new 257

pandemic (28). The phylogenetic tree of 269 HA sequences of human influenza H3N2 258

viruses collected in Korea showed that the viral genes formed seasonal phylogenetic clusters 259

which have evolved from A/Beijing/32/1992 strains to A/Brisbane/10/2007 strains via 260

A/California/7/2004 strains from 1999 to 2007. The results indicated that progressive 261

antigenic drift occurred at the HA antigen in these seasons. Notably, the strains of different 262

clusters often co-circulated within the same season, which was most apparent with the 263

identification of multiple subclades of H3N2 viruses in 1999 and 2002. Moreover, as shown 264

in Fig. 1A, seasonal H1N1, H3N2 and B viruses have circulated simultaneously during the 265

seasons. 266

A change of dominant strains in H3N2 from A/Moscow/10/1999 to A/Fujian/411/2002 267

caused a worldwide epidemic, since the H3N2 vaccine strain for the 2002-2003 season 268

(A/Moscow/10/1999) did not antigenically match the circulating A/Fujian/411/2002-like 269

viruses, reducing effectiveness against virus-caused illness (3, 14, 22). The emergence of 270

A/Fujian/411/2002-like strains thus coincided with higher level of influenza-related 271

morbidity, and the 2002-2003 season was clearly a turning point with regard to circulating 272

influenza H3N2 viruses in Asia. The Fujian-like strains had undergone a rapid change in 273

amino acid sequence of HA in the 2001-2002 season, and relatively slow and constant 274

antigenic changes were subsequently observed from 2003 to 2007. Interestingly, a 275

reassortant strain emerged early in the New Zealand winter, followed by the appearance of 276

similar viruses in Australia (2), which was later seen in United States, Europe and Brazil 277


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during the 2003-04 season. While the HA sequence of these viruses demonstrated only 278

minor differences from the Fujian H3N2 strain, NA and other internal genes (NS, NP, M) 279

were different from those of circulating non-reassortant H3N2 viruses (Fig 3A & 3C) (13). 280

Both the reassortant and non-reassortant viruses circulated not only in the United States and 281

Europe, but also in Asia possibly due to the re-introduction of the reassortant strains from the 282

Southern hemisphere. Nevertheless, the non-reassortant strains, not the reassortants, became 283

predominant in the following years. 284

It was not known what features of the A/Fujian/411/2002 strains were responsible for the 285

global spread and how the non-reassortant strains were again dominant against the 286

reassortant ones. The 2001-2002 season strains matched the A/Moscow/10/1999 vaccine 287

strain. Remarkably, a total of 14 amino acid changes (plus 4 amino acid changes from the 288

most common recent ancestor of A/Moscow/10/1999 and A/Fujian/411/2002 to the Moscow 289

strain) were found across the antigenic sites of the HA1 domain, which distinguished the 290

Fujian strain from the Moscow strain (Fig. 2A & 2B). It was previously reported that a total 291

of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino 292

acid changes had occurred in HA from 1968 to 2000 (20). By contrast, most mutations in the 293

Fujian strain appeared in a short period of time from Dec 2001 to Aug 2002 (Fig. 3C). 294

Among the 14 amino acid changes, 10 of them were located in one of the five antigenic sites: 295

H75Q and E83K (site E), A131T and I144N (site A), H155T, Q156H, S186G and T192I 296

(site B), and D172E and W222R (site D). It was shown that two residues 155 and 156 are 297

responsible for the major antigenic differences between the A/Moscow/10/1999 and 298

A/Fujian/411/2002-like strains (13). H155T and Q156H were indeed present in the isolates 299

from the 2002-2003 season, whereas some intermediate isolates with the replacement at site 300

155 but not at site 156 were also identified at the same season. 301


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In the 2003-2004 season, the Korean non-reassortant strains had at least three additional 302

amino acid substitutions in antigenic sites B and D, namely Y159F, S189N and S227P, from 303

the Fujian/411 strain, whereas two reassortants had only one or two mutations from the 304

Fujian strain. The reassortants were found to have eventually faded away, suggesting that the 305

non-reassortant viruses were more antigenically advanced than the reassortants. The 306

epidemic of the Fujian-like strains during the previous season might have hampered the 307

introduction of reassortant strains to Asia since the reassortant strains gained scant antigenic 308

difference from the original Fujian/411 HA. It was recently reported that newly dominant 309

A/California/7/2004-like strains, which featured two key amino acid changes in the 310

polymerase PA segment, grew to higher titers in MDCK cells (19). Influenza strains thus can 311

be selected through mutations in replicative fitness and virulence. Taken together, it is 312

strongly suggestive that the collapse of the reassortants is mainly caused by the precession of 313

antigenic change of non-reassortants bearing A/California/7/2004-like strains. 314

The isolates collected during successive seasons were further observed to undergo a 315

progressive antigenic drift from A/California/7/2004-like strains to A/Brisbane/10/2007-like 316

strains. Both A/Fujian/411/2002-like and A/California/7/2004-like viruses were prevalent in 317

the 2004-2005 season, as A/California/7/2004-like viruses circulated as a new strain. There 318

were 8 amino acid differences between A/Fujian/411/2002 and A/California/7/2004. The 5 319

mutations which were commonly found in 2004-2005 isolates (A/California/7/2004-like) 320

were placed in antigenic sites: K145N (site A), Y159F, S189N (site B), V226I, and S227P 321

(site D). A/California/7/2004-like viruses then became a new predominant strain in the 322

successive seasons. In the 2005-2006 season, S193F and D225N substitutions were 323

accumulated to the H3N2 strains, and the R142G mutants dominated during the 2006-2007 324


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season. However, it was notable that recent strains were descended from a minority group of 325

the 2005-2006 season which has G50E mutation. 326

A/Fujian/411/2002 was first collected in Aug 2002 in China, and a Fujian strain in Korea 327

was first detected in Busan on 20 November 2002, followed by simultaneous appearance 328

throughout Korea. Notably, the 2002 FIFA World Cup was held in Korea and Japan from 31 329

May to 30 June, 2002 . And the 14th

Asian games were held in Busan, Korea from 330

September 29 to October 14, 2002 with a total number of 18,000 athletes and officials from 331

44 countries . Asian countries have intensive contact through air travel, which could 332

contribute to viral transmission patterns. Recent analysis of air-traffic patterns showed a 333

strong correlation between the international travel and 2009 H1N1 transmission (16). It was 334

proposed that the variability of influenza H3N2 epidemics may form an E-SE Asian 335

circulation network that maintains influenza virus in the region by passing from epidemic to 336

epidemic (22). A network of monitoring efforts for international events can be employed in 337

preparation of a novel influenza outbreak. 338

339

ACKNOWLEDGEMENTS 340

We wish to acknowledge the technical support from Mr. C. H. Gong and Mr. Joon Seung 341

Lee at the department of Biotechnology & Bioinformatics, Korea University. This work was 342

supported by the Korea National Institute of Health (K.H.K) and a grant from the BioGreen 343

21 Program (K.H.K). 344

345

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Klimov, T. Kageyama, N. Komadina, A. S. Lapedes, Y. P. Lin, A. Mosterin, M. 406

Obuchi, T. Odagiri, A. D. Osterhaus, G. F. Rimmelzwaan, M. W. Shaw, E. 407 Skepner, K. Stohr, M. Tashiro, R. A. Fouchier, and D. J. Smith. 2008. The global 408

circulation of seasonal influenza A (H3N2) viruses. Science 320:340-6. 409

23. Shortridge, K. F. 1997. Is China an influenza epicentre? Chin Med J 110:637-41. 410

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25. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular 414

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424

425

Conflict of interest statement. None declared. 426


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Fig. 1. Weekly influenza epidemic time series. (A) Influenza virus isolates in Korea over 427

the period from 2000 to 2007. Different subtypes are shown in different gray shades. 428

(B) Epidemics in Korea and United States. The y axis shows influenza-like illness over 429

the period from 2000-2007. (C) Proportions of influenza viruses A/H1N1, A/H3N2, B of 430

Korea (KOR) and the United States in each season. Unsubtyped influenza A viruses of 431

the United States were assumed to have identical proportion of subtyped ones. 432

433

Fig. 2. Phylogenetic analysis and amino acid substitutions of HA of influenza 434

A/H3N2 viruses circulating in Korea from 1999 to 2007. (A) The maximum 435

parsimony tree. Reference vaccine strains are highlighted in gray background. Six 436

2009 strains from the US and Philippines are combined. Amino acid substitutions on 437

major clades are described under the internal branches, and the substitutions to 438

extant lineage are highlighted in bold. Only one representative strain was presented 439

in bold with the number of identical strains when there were more than two strains 440

having identical amino acid sequence. Numbers in parentheses under group names 441

indicate the number of isolates of that group. (B) Locations of the amino acid 442

substitutions that were found on major clades on HA structure model (PDB ID 443

1HA0). 444

445

Fig. 3. Maximum likelihood (ML) phylogenies reconstructed by PhyML program to 446

identify the Fujian-reassortants in Asia during 2002-2004. The clade A strains in 447

Figure 1 of reference (19) were used for the reference sequences of reassortants. 448

The closely related non-reassorted strain of the clade A, A/New York/406/2002, is 449


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19

marked as a gray dot on the ML trees. The node support values for reassortant 450

clades were estimated using approximate likelihood ratio test incorporated in PhyML 451

(1). (A) ML tree of HA1 nucleotide sequences of 637 Asian strains, 30 New York 452

strains and A/Moscow/10/1999 as a phylogenetic root. The Fujian/411 clade is 453

shown and branch marked by circles means a reassortant clade. (B) The 454

reassortant clade shown in (A). (C) ML tree of neuraminidase (NA) nucleotide 455

sequences of 161 Asian strains, 30 New York strains and two outgroup strains 456

(A/New York/313/1998 and A/New York/328/1998). (D) The reassortant clade 457

shown in (C). 458

459

Fig. 4. Maximum likelihood (ML) trees and timeline of the emergence of 460

A/Fujian/411/2002 strain. Both trees are rooted by A/Moscow/10/1999. (A) The ML 461

tree of HA1 domain nucleotide sequence of 521 human influenza A/H3N2 virus 462

isolated worldwide during 2001-2002. The circle indicates the locations of the strains 463

for further analysis shown in (B). (B) The ML tree of 53 strains around the 464

emergence of the Fujian-411 strain. Two vaccine strains isolated in 2004 and 465

A/Moscow/10/1999 as root were included. The earliest isolates of the clades are 466

highlighted in bold. The amino acid changes are described under the internal 467

branches. (C) Timeline of the appearance of A/Fujian/411/2002 in Asia. The earliest 468

day of each clade is described under the timeline. 469


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0

50

100

150

200

250

300

350

26 36 46 4 14 24 34 44 2 12 22 32 42 52 10 20 30 40 50 8 18 28 38 48 6 16 26 36 46 3 13 23 33 43 1 11 21

Fig. 1

A

B

2000-2001 2001-2002 2006-20072005-20062004-20052003-20042002-2003

2000-2001 2001-2002 2006-20072005-20062004-20052002-2003 2003-2004

2001-2002 2006-20072005-20062004-20052003-20042002-2003 2000

-2001

2007

-2008

US KOR US KOR US KOR US KOR US KOR US KOR USKOR

Pre

va

len

ce

%

C

0

1

2

3

4

5

6

7

8

9

0

2

4

6

8

10

12

26 36 46 4 14 24 34 44 2 12 22 32 42 52 10 20 30 40 50 8 18 28 38 48 6 16 26 36 46 4 14 24 34 44 2 12 22

Korea

United States

B

A/H3N2

A/H1N1

0

20

40

60

80

6

100

Nu

mb

er

of is

ola

tes

Virus isolation in Korea

Weeks

% IL

I (U

nite

d S

tate

s)

IL

I p

er

1,0

00

(K

ore

a)

Weekly surveillance of ILI

Weeks

B

A/H3N2

A/H1N1 (KOR)

A/H1N1 & H1N2 (US)

Weekly influenza epidemic time series. (A) Influenza virus isolates in Korea over the

period from 2000 to 2007. Different subtypes are shown in different gray shades. (B)

Epidemics in Korea and United States. The y axis shows influenza-like illness over

the period from 2000-2007. (C) Proportions of influenza viruses A/H1N1, A/H3N2,

B of Korea (KOR) and the United States in each season. Unsubtyped influenza A

viruses of the United States were assumed to have identical proportion of subtyped

ones.


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5 AA

25

5057

83

172

227226225222

145144142140

160156155202131

202189192193186

Fig. 2

A

B

Phylogenetic analysis and amino acid substitutions of HA of influenza A/H3N2 viruses circulating in Korea from 1999 to 2007. (A) The maximum parsimony tree. Reference vaccine strains are highlighted in gray background. Six 2009 strains from the US and Philippines are combined. Amino acid substitutions on major clades are described under the internal branches, and the substitutions to extant lineage are highlighted in bold. Only one representative strain was presented in bold with the number of identical strains when there were more than two strains having identical amino acid sequence. Numbers in parentheses under group names indicate the number of isolates of that group. (B) Locations of the amino acid substitutions that were found on major clades on HA structure model (PDB ID 1HA0).

00-01

(8)

02-03

(14)

03-04

(32)

04~05A

(31)

05-06

(50)

06-07A

(57)

06-07B

(41)

2009

I144ND172ET192I

R50G

L25IH75QE83KA131TH155TS186GV202I

W222RG225D

Q156H

Y159FS189NS227P

K145NV226I

G50E

K140I

P227SK326T

S193FD225N

N6IS45NL157SK173E

R142G

N144D

R57Q

04~05B

(22)

G142R

A/Incheon/422/2005

A/Jeju/266/2006

A/Fujian/411/2002

A/Daegu/1783/2007

A/Gyeongnam/717/2004

A/Wisconsin/67/2005

A/Jochiwon/04/2001

A/Gyeonggi/1995/2007

A/Philippines/16/2009

A/Korea/770/2002

A/South/Korea/C5-3/2002

A/Gyeonggi/743/2006

A/Korea/124/2003

A/Daegu/1755/2007

A/Busan/44/2001

A/Incheon/1356/2007

A/Daegu/24/2001

A/Daejeon/2419/2007

A/Busan/805/2004

A/Gangwon/741/2006

A/Jeju/187/2006

A/Seoul/1154/2004

A/Daegu/58/2001

A/Daejeon/284/2006_25

A/Jeju/218/2004

A/Jeonbuk/746/2006_5

A/Wellington/01/2004

A/NewYork/1685/2009

A/Kwangju/105/1999_2

A/Incheon/677/2006

A/Inchon/81/1999

A/Kyungnam/122/2005

A/Ulsan/198/2005

A/Incheon/57/2005_5

A/Gwangju/554/2004

A/Jeju/274/2002_2

A/Busan/808/2004_3

A/Incheon/1909/2004

A/Incheon/459/2005_11

A/Busan/3/2005

A/Kwangju/219/2002

A/Ulsan/509/2004

A/Kyunggi/205/2005A/Incheon/1723/2004_2

A/Iowa/03/2009

A/Jeonbuk/1165/2004

A/Jeounbuk/2217/2007

A/Kangwon/88/1999_2

A/Daegu/203/2005

A/Jeonbuk/243/2006


A/Incheon/380/2005_2

A/Jeonbuk/674/2005

A/Incheon/707/2004


A/Busan/11/2001

A/Daejeon/457/2006

A/Kangwon/11/1999

A/Incheon/241/2006

A/Gyeongbuk/989/2006_2

A/NewYork/3274/2009

A/Cheonbuk/1072/2005

A/Gangwon/259/2006

A/Daegu/77/2005

A/Ulsan/689/2006_4

A/NewYork/3104/2009

A/Daegu/56/2001

A/Jeju/958/2006

A/South/Korea/C5-4/2002


A/Gwangju/1032/2005

A/Incheon/2012/2007_2

A/Daegu/277/2004

A/Chungbuk/1822/2004

A/Jeonbuk/2189/2007_9

A/Gwangju/560/2004_5

A/Jeonbuk/1144/2004

A/Daegu/826/2004

A/Beijing/32/1992

A/Jeju/308/2006

A/Incheon/1608/2007_2

A/Kyugnam/170/2005_2

A/Incheon/1574/2007_35

A/Moscow/10/1999

A/CHU/2-524/2005


A/NewYork/1670/2009

A/Kangwon/12/1999

A/Jeonnam/336/2004

A/Daegu/234/2005_3

A/Ulsan/50/2005_3

A/Sydney/5/1997

A/Chungbuk/43/2005_2

A/Incheon/243/2005

A/Jeonbuk/1429/2007

A/Jeju/288/2004_7

A/Daejeon/514/2006

A/Chungnam/2288/2007

A/Gyeongnam/291/2005_2

A/Busan/06/2007_2


A/Jeounbuk/2195/2007

A/Kwangju/117/1999_2

A/Jeonbuk/1117/2004_2

A/Chungnam/271/2002

A/Kyongnam/347/2002_5


A/Chungbuk/5/2005

A/Daejeon/1228/2005_11

A/Gyeongnam/7/2005

A/Incheon/34/2005

A/Seoul/230/2004

A/Chungbuk/1471/2007

A/Daegu/637/2004

A/Jeju/1965/2007

A/Jeonbuk/1115/2004

A/Kyeongbuk/304/2002A/Gyeongbuk/2/2002

A/California/7/2004

A/Jeonbuk/2211/2007

A/Daejeon/700/2006_3

A/Pusan/504/2002_2

A/Pusan/71/2001

A/Korea/KO-37-06/2005

A/Incheon/928/2006

A/Daejeon/1720/2007_20

A/Busan/16/2001

A/Jeju/1984/2007

A/Brisbane/10/2007

A/Seoul/323/2004


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0.785

A/HongKong/CUHK22510/2004




A/NewYork/33/2004

A/NewYork/5/2004

A/NewYork/271/2003


A/NewYork/69/2004

A/NewYork/42/2003

A/HongKong/HKU2/2004







A/NewYork/39/2003

A/NewYork/17/2003

A/NewYork/26/2003

A/Taiwan/4183/2004

A/NewYork/18/2003

A/NewYork/6/2004

A/NewYork/15/2003

0.983

A/Malaysia/768/2003


A/Kobe/3/2004

A/NewYork/42/2003




A/Kitakyushu/2/2004

A/Sapporo/193/2003




A/NewYork/69/2004

A/NewYork/5/2004

A/Jeju/218/2004

A/NewYork/18/2003

A/Malaysia/1522/2004

A/Singapore/107/2003

A/Taiwan/1569/2004

A/NewYork/6/2004

A/NewYork/271/2003

A/NewYork/17/2003


A/NewYork/26/2003


A/Osaka/25/2003


A/NewYork/33/2004

A/NewYork/39/2003

A/Korea/124/2003




A/Taiwan/TW-1548/2004

A/NewYork/15/2003


Fig. 3

A B

C D

Fujian/411

clade

Maximum likelihood (ML) phylogenies reconstructed by PhyML program to identify the Fujian-reassortants

in Asia during 2002-2004. The clade A strains in Figure 1 of reference (21) were used for the reference

sequences of reassortants. The closely related non-reassorted strain of the clade A, A/New York/406/2002, is

marked as a gray dot on the ML trees. The node support values for reassortant clades were estimated using

approximate likelihood ratio test incorporated in PhyML (3). (A) ML tree of HA1 nucleotide sequences of

637 Asian strains, 30 New York strains and A/Moscow/10/1999 as a phylogenetic root. The Fujian/411 clade

is shown and branch marked by circles means a reassortant clade. (B) The reassortant clade shown in (A). (C)

ML tree of neuraminidase (NA) nucleotide sequences of 161 Asian strains, 30 New York strains and two

outgroup strains (A/New York/313/1998 and A/New York/328/1998). (D) The reassortant clade shown in (C).


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0.003 (nucleotide subst./site)

S186G

A131T

L25I

H75Q

H155T

Q156H

Asia

North

America

Oceania

World-

wide

Fujian/411

clade

A/Wellington/01/2004A/California/7/2004

A/Chungnam/447/02.12.02

A/Hawaii/HI-02-4092/02.12.28A/Kumamoto/102/02.12.25

A/Ishikawa/102/02.11.26A/Singapore/C5-7/2002

A/Bangladesh/C5-6/2002

A/Hong Kong/CUHK53327/02.12.27

A/Kyongnam/347/02.12.27

A/Stockholm/26/2002

A/Jeju/274/02.11.25A/Daejeon/390/02.11.27

A/Pusan/504/02.11.20

A/Jeonnam/432/02.11.26

A/Chungnam/271/02.11.22

A/Cheju/311/02.12.02

A/SouthKorea/C5-3/2002

A/Hunan/407/02.08.20


A/Bangkok/247/02.07.15

A/China/C1-5/2001

A/Guangzhou/603/02.06.15


A/Switzerland/7494/2002

A/Philippines/C3-15/2002

A/Wuhan/12/02.01.10



A/Anhui/550/02.12.01



A/China/C1-7/2002A/Zhejiang/8/02.01.08


A/Beijing/51/02.12.20

A/Taiwan/8/02.09.25


A/Philippines/471/02.07.18

A/Fujian/354/02.07.23

A/Fujian/411/02.08.11

A/Perth/201/02.01.29


A/Belgium/C2-11/2002

A/Wuhan/16/02.01.17

A/India/C3-45/2002

A/Townsville/4/02.07.17

A/Hebei/22/02.01.10

A/NewJersey/4/02.11.04


A/Beijing/178-NEW-AGN/02.11.21

A/China/C1-3/2001A/China/C1-2/2001


A/Hong Kong/C1-9/2002

Fujian/411

clade

A B

World-

wide

C

Fig. 4 Maximum likelihood (ML) trees and timeline of the emergence of A/Fujian/411/2002 strain. Both trees are

rooted by A/Moscow/10/1999. (A) The ML tree of HA1 domain nucleotide sequence of 521 human influenza

A/H3N2 virus isolated worldwide during 2001-2002. The circle indicates the locations of the strains for

further analysis shown in (B). (B) The ML tree of 53 strains around the emergence of the Fujian-411 strain.

Two vaccine strains isolated in 2004 and A/Moscow/10/1999 as root were included. The earliest isolates of the

clades are highlighted in bold. The amino acid changes are described under the internal branches. (C) Timeline

of the appearance of A/Fujian/411/2002 in Asia. The earliest day of each clade is described under the timeline.

S186G

26 DEC 2001

A/Hong Kong/CUHK51431/2001

A131T

2001 (date not available)

A/China/C1-2/2001

A/China/C1-3/2001

L25I, H75Q, H155T

1 JUN 2002

A/Hong Kong/CUHK24044/2002

Q156H

11 AUG 2002

A/Fujian/411/2002

First Fujian-like strain

detected in Korea

20 NOV 2002

A/Pusan/504/2002

20022001 2003


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Epidemiologic Study of Human Influenza Infection in Korea ...jcm.asm.org/content/early/2010/04/14/JCM.00209-10.full.pdf60 of amino acid variation in HA is clustered in variable antigenic