isolation and characterization of influenza c viruses in...

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Isolation and characterization of influenza C viruses in the Philippines and Japan. 1

2

Takashi Odagiri1, Yoko Matsuzaki

2, Michiko Okamoto

1, Akira Suzuki

1, Mariko Saito

1,3, 3

Raita Tamaki1,3

, Socorro P Lupisan4, Lydia T Sombrero

4, Seiji Hongo

2, Hitoshi 4

Oshitani1#

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1Department of Virology, Tohoku University Graduate School of Medicine, 2-1 7

Seiryou-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan 8

2Department of Infectious Diseases, Yamagata University Faculty of Medicine, 9

Iida-Nishi, Yamagata 990-9585, Japan 10

3RITM-Tohoku Research Collaborating Center for Emerging and Reemerging diseases, 11

Muntinlupa City, the Philippines 12

4Resarch Institute for Tropical Medicine, Muntinlupa City, the Philippines 13

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Running title: Isolation of influenza C in the Philippines and Japan 15

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#Corresponding author: Hitoshi Oshitani 17

Department of Virology, Tohoku University Graduate School of Medicine 18

JCM Accepts, published online ahead of print on 31 December 2014J. Clin. Microbiol. doi:10.1128/JCM.02628-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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2-1 Seiryou-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan 19

Tel: +81-22-717-8213 20

Fax: +81-22-717-8212 21

Email: [email protected] 22

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Abstract 24

From November 2009 to December 2013, 15 influenza C viruses were isolated, using 25

MDCK cells, from specimens obtained from children with severe pneumonia and 26

influenza-like illness (ILI), in the Philippines. This is the first report of influenza C virus 27

isolation in the Philippines. In addition, from January 2008 to December 2013, 7 28

influenza C viruses were isolated from specimens that were obtained from children with 29

acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with 30

monoclonal antibodies to the hemaggultinin–esterase (HE) glycoprotein showed that 19 31

strains (12 from Philippines and 7 from Japan) were similar to the reference strain 32

C/Sao Paulo/378/82(SP82). Phylogenetic analysis of the HE gene showed that strains 33

from the Philippines and Japan formed distinct clusters within an SP82-related lineage. 34

The clusters including the Philippine and Japanese strains were shown to have diverged 35

from a common ancestor around 1993. In addition, phylogenetic analysis of internal 36

genes showed that all strains isolated in the Philippines and Japan had emerged through 37

reassortment events. The composition of the internal genes of the Philippine strains was 38

different from that of the Japanese strains, although all strains were classified into an 39

SP82-related lineage by HE gene sequence analysis. These observations suggest that the 40

influenza C viruses analyzed here had emerged through different reassortment events; 41


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however, the time and place at which the reassortment events occurred could not be 42

determined. 43

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Introduction 45

Influenza C virus usually causes a mild upper respiratory illness, but it can also cause 46

lower respiratory infections, such as bronchitis and pneumonia (1). Seroepidemiological 47

studies have revealed that influenza C virus is widely distributed throughout the world 48

(2-6) and that recurrent infection with this virus occurs frequently in children, as well as 49

in adults (7). However, the virus has been isolated only occasionally by cell culture, and 50

long-term monitoring of influenza C viruses is rarely conducted. Monitoring of 51

influenza C among children in Yamagata and Miyagi Prefectures in Japan since 1988 52

has revealed that outbreaks of influenza C virus occur in winter or early summer at 1- or 53

2-year intervals (8-10). Influenza C virus infections, detected using molecular detection 54

methods, have recently been reported from several countries including Spain, France, 55

Cuba, Canada, Italy, India, and Finland (7, 11-16). A serological study conducted in the 56

Philippines in 1984 indicated the existence of influenza C viruses , but the viruses 57

themselves were not detected. 58

The genome of influenza C virus consists of seven RNA segments that encode three 59

polymerase proteins (PB2, PB1, and P3), the hemagglutinin–esterase glycoprotein (HE), 60

the nucleoprotein (NP), the matrix protein (M), the CM2 protein, and two nonstructural 61

proteins (NS1 and NS2) . Antigenic variation exists among influenza C virus isolates, as 62


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demonstrated by antigenic analysis with anti-HE monoclonal antibodies (MAbs) 63

(17-19). However, analysis with polyclonal immune sera showed a high degree of 64

cross-reactivity among all the isolates examined so far (17, 19-21), indicating that 65

influenza C virus is antigenically more homogenous than human influenza A and B 66

viruses. Early studies analyzing the molecular characteristics of isolates have suggested 67

that influenza C virus epidemiology might be characterized by the presence of multiple 68

lineages (22, 23). Antigenic and sequence analyses of the HE gene revealed the 69

existence of six lineages, which are represented by C/Taylor/1233/47(Taylor/47), 70

C/Kanagawa/1/76(KA176), C/Mississippi/80(MS80), C/Aichi/1/81(AI181), 71

C/Yamagata/26/81(YA2681), and C/Sao Paulo/378/82(SP82) (19), and that influenza C 72

viruses belonging to different lineages can be co-circulating in a single community (10, 73

17). Thus, mixed infections with influenza C viruses belonging to different lineages 74

may be occurring in a single host, resulting in the emergence of reassortant viruses, 75

characterized by exchange of genomic segments between two different strains (19, 24). 76

Long-term surveillance studies carried out in Yamagata and Miyagi Prefectures in Japan 77

also revealed that reassortment between viruses of different lineages had occurred 78

frequently, and that newly emerged reassortant viruses had replaced previously 79

circulating viruses (10). The virus antigenically similar to KA176, which reemerged in 80


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Miyagi Prefecture in 1996 for the first time in 20 years and subsequently spread 81

throughout Japan, acquired its internal genes from the previous epidemic virus, which 82

belongs to the YA2681 lineage, through a reassortment event (10). These observations 83

indicate that the genome composition of influenza C viruses may affect their ability to 84

spread among humans, and that reassortment events can be means of evolution for 85

influenza C viruses. 86

From 2011 to 2013, we isolated influenza C viruses from cases with severe pneumonia 87

and influenza-like illness (ILI) in the Philippines, for the first time. We also isolated 88

influenza C viruses through acute respiratory illness (ARI) surveillance conducted in 89

Sendai city, Miyagi, Japan from 2008 to 2013. In this study, we analyzed the influenza 90

C strains collected in the Philippines and Japan to characterize circulating influenza C 91

viruses in these two countries. This characterization included sequence analysis of all 92

seven RNA segments. 93

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Materials and methods 95

96

Virus isolation 97

We conducted two prospective studies of respiratory viruses in the Philippines. One of 98


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these studies is a pediatric pneumonia study conducted at Eastern Visayas Regional 99

Medical Center (EVRMC) on Leyte Island, since January 2010. Beginning in August 100

2012, the pediatric pneumonia study was expanded to include Biliran Provincial 101

Hospital (BPH) in Naval in Biliran Island and Ospital ng Palawan (ONP) in Puerto 102

Princesa City on Palawan Island (Fig. S1). The other is an ILI study conducted in the 103

outpatient clinics of three medical facilities on Leyte Island: Leyte Provincial Hospital 104

(LPH), Tacloban City Health Catchment Center (TCHCC), and Tanauan Rural Health 105

Unit (TRHU), since November 2009. Nasopharyngeal swab specimens were collected 106

following the Integrated Management of Childhood Illness (IMCI) guideline, from 107

hospitalized children with the clinical diagnosis of severe pneumonia, and from the 108

patients who visited the outpatient clinic of LPH, TCHCC or TRHU with ILI; ILI was 109

defined as fever ≥38 ºC or, feverish and either cough or nasal discharge. Details of the 110

study design have already been described (25). 111

From November 2009 to December 2013, a total of 5,343 nasopharyngeal swab 112

specimens were collected from children with severe pneumonia or ILI (age range, 113

0.0–14.9 years; mean age, 1.2 years), and the specimens were transferred to the 114

Research Institute of Tropical Medicine (RITM), Metro Manila, the Philippines, for 115

virus isolation. All specimens were inoculated into Madin–Darby canine kidney 116


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(MDCK), HEp-2, VeroE6, and human fetal lung fibroblast cells by using the microplate 117

method for isolation of viruses (26). These cell lines were used to detect common 118

respiratory viruses, such as influenza A, B, and C viruses, enteroviruses, human 119

metapneumovirus (hMPV), respiratory syncytial virus (RSV), and human adenoviruses 120

(HAdV). When cytopathic effects (CPE) were observed in MDCK cells, the culture 121

supernatant was tested with the hemagglutination (HA) test using chicken or turkey 122

erythrocytes. Influenza C virus causes agglutination in the chicken or turkey 123

erythrocytes, but does not cause agglutination of the guinea pig cells (27). If HA tests 124

were positive with chicken or turkey erythrocytes, but negative with guinea pig 125

erythrocytes, influenza C virus isolation was suspected. Then, the presence of influenza 126

C in these isolates was confirmed by reverse transcription-polymerase chain reaction 127

(RT-PCR) method using Influenza C virus-specific primer (28). 128

We also conducted an ARI study at the outpatient pediatric clinic from January 2008 to 129

December 2013 in Sendai city, Japan (Fig. S2). A total 1,845 nasopharyngeal and throat 130

swab specimens were collected from children with ARI (age range 0.1–14.9 years, mean 131

age 5.3), and the samples were transferred to Tohoku University Graduate School of 132

Medicine (Sendai, Japan) for virus isolation. The isolation of influenza C virus was 133

performed using same method that was used in the studies in the Philippine. 134


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135

Hemagglutination inhibition (HI) test for antigenic analysis 136

All HI tests were performed using isolates that were propagated in embryonated hen’s 137

eggs, because previous research has shown that viruses with high HA titers can be 138

obtained from embryonated eggs without an alteration in antigenicity (29). Therefore, 139

clinical specimens testing positive for influenza C virus were re-inoculated into the 140

amniotic cavity of 9-day-old embryonated hen’s eggs, and amniotic fluid was used in 141

the following analysis. Four previously characterized anti-HE MAbs (J14, Q5, U4, and 142

MS2) (30, 31) were used for antigenic analysis during HI testing. Briefly, 50 μl of virus 143

suspensions (8 HA units/50 μl) were added to each well of a microtiter plate containing 144

50 μl of two-fold-diluted MAbs. After incubation for 30 min at room temperature, 100 145

μl of 0.5% chicken erythrocytes were added to all wells, and the plates were stored at 146

4°C for 60 min. HI titer was expressed as the reciprocal of the highest antibody dilution 147

that completely inhibited hemagglutination. (9). 148

149

Nucleotide sequencing and phylogenetic analysis 150

Nucleotide sequencing and analyses were carried out as previously described (9, 10). 151

Briefly, viral RNA was extracted from 100 μl of the virus-containing amniotic fluid by 152


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using an RNeasy mini kit (Qiagen, Hilden, Germany). Viral RNA was then transcribed 153

into cDNA with a primer complementary to positions 1–12 at the 3'-end of the RNA 154

containing all of the influenza C virus RNA segments (18). Using this synthesized 155

cDNA as a template, the individual segments were amplified using gene-specific 156

primers (18). The coding region of the HE gene, corresponding to nucleotide positions 157

from 64 to 1989, was sequenced by using the BigDye Terminator v3.1 Kit (Applied 158

Biosystems, Foster City, CA,) and an ABI Prism 3130 sequencer (Applied Biosystems). 159

In addition to the HE gene, the partial nucleotide sequences of the PB2, PB1, P3, and 160

NP genes, as well as the complete coding region of the M and NS genes were 161

determined. The oligonucleotide primers used for sequencing were previously described 162

(10, 18, 32, 33). Sequence data were analyzed with MEGA software (version 5.1), and 163

phylogenetic trees of individual genes were constructed by the neighbor-joining method 164

(34), with p-distance as a substitution model, bootstrapped 1,000 replicates together 165

with previously reported sequences (9, 10, 19, 21, 24, 32, 35, 36) using the same 166

software. The divergence time for influenza C viruses detected in the Philippines and 167

Japan was estimated by using the Bayesian Markov chain Monte Carlo (MCMC) 168

approach implemented in the BEAST package v1.8.1. Lognormal relaxed clock 169

(uncorrected) was used with a tree prior of Bayesian Skyline, and the 170


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general-time-reversal (GTR) substitution model with gamma-distributed and invariant 171

sites as site heterogeneity model. The MCMC chains were run for two billion iterations, 172

with sampling at every 250,000 and the first 1,000 trees were discarded as burn-in. The 173

MCMC process was analyzed by using TRACER v1.5. A maximum clade credibility 174

tree was generated by using FigTree v1.3.1. The nucleotide sequences determined in 175

this study have been submitted to the DDBJ/GenBank databases and assigned accession 176

numbers AB978548 to AB978566. 177

178

Results 179

180

Isolation of influenza C viruses 181

A total of 15 (0.28%) strains of influenza C were isolated from 5,343 specimens 182

collected in the Philippines between 2009 and 2013, including 6 strains in 2011 (6 of 183

1324; 0.45%) and 9 strains in 2013 (9 of 1694; 0.53%) (Fig. 1A). Influenza C virus was 184

isolated as the single etiological agent in 14 patients. However, respiratory syncytial 185

virus was also isolated from one patient with ILI, usnig HEp-2 cells. In Japan, 7 186

(0.38%) strains of influenza C were isolated from children (age range, 0.9–5.7 years), 187

including 5 strains in 2008 (5/358, 1.40%) and 2 strains in 2012 (2/351, 0.57%) (Fig. 188


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1B). No other respiratory viruses, such as influenza A and B viruses, enteroviruses, 189

hMPV, RSV, and HAdV, were isolated from any of these seven patients. 190

Clinical manifestations of influenza C virus-positive patients are shown in Table 1. In 191

the Philippines, 7 (7 of 3118; 0.22%) strains of influenza C virus were isolated from 192

severe pneumonia patients (age range, 0.4–5.5 years), and 8 were isolated from ILI 193

patients (8 of 2225; 0.36%) (age range, 0.4–2.9 years) (Table 1). Among influenza C 194

virus-positive children with severe pneumonia, chest indrawing was seen in all cases, 195

while difficulty in breathing and fever over 38°C was seen in 6 cases. Regarding 196

outcome of these cases, six were discharged, and one refused admission. The primary 197

symptoms seen in influenza C virus-positive ILI cases in the Philippines and ARI cases 198

in Japan were cough, nasal discharge, and fever. 199

200

Antigenic analysis of influenza C viruses isolated in the Philippines and Japan 201

Among 15 positive specimens in the Philippines, only 12 specimens, including 5 202

specimens collected in 2011 and 7 in 2013, were available for re-inoculation into the 203

amniotic cavity of embryonated eggs. Influenza C viruses were isolated from 204

embryonated eggs inoculated with each of these 12 specimens. For Japanese specimens, 205

influenza C viruses were isolated from embryonated eggs inoculated with 7 specimens, 206


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including 5 from 2008 and 2 from 2012. The antigenicity of all 19 strains and the HI 207

titers of reference strains belonging to five antigenic groups (KA176, MS80, AI181, 208

YA2681 and SP82) are shown in Table 2. All the strains isolated in this study were 209

highly reactive with MAbs J14, Q5, and U4, but were unreactive or very weakly 210

reactive with MS2. The reactivity patterns of all isolates analyzed were similar to that of 211

SP82. 212

213

Phylogenetic analyses of individual RNA segments of influenza C virus strains 214

In order to confirm the results of the antigenic analyses, the sequence of the HE gene 215

from each of the 19 strains (nucleotide 64 to 1989) was determined. Previous studies 216

revealed that the HE genes of influenza C viruses could be classified into six discrete 217

lineages, represented by Taylor/47, KA176, YA2681, AI181, SP82, and MS80. As 218

shown in Fig. 2, all strains isolated in the Philippines and Japan were classified as 219

members of the SP82-related lineage. The nucleotide sequences of the HE genes of 220

strains isolated in the Philippines in 2011 and 2013 were highly homologous 221

(99.3–100% nucleotide identity). The seven strains isolated in 2008 and 2012 in Japan 222

were also highly homologous (99.3–100% nucleotide identity). The strains isolated in 223

the Philippines and Japan formed distinct clusters within the SP82-related lineage; 224


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although, the sequences within this branch, including those from the Philippines and 225

Japan, shared high sequence homology (98.2–98.8% nucleotide identity). 226

To determine the genomic compositions of the strains isolated in the Philippine and 227

Japan, and to determine the occurrence of reassortment event(s), the nucleotide 228

sequences of internal genes were also determined for the 10 strains from the Philippines 229

(C/Leyte/1/2011, C/Leyte/2/2011, C/Leyte/3/2011, C/Leyte/1/2013, C/Leyte/2/2013, 230

C/Leyte/3/2013, C/Biliran/1/2013, C/Biliran/2/2013, C/Biliran/3/2013, and 231

C/Palawan/1/2013) and the 6 strains from Japan (C/Sendai/TU1/2008, 232

C/Sendai/TU2/2008, C/Sendai/TU3/2008, C/Sendai/TU5/2008, C/Sendai/TU1/2012, 233

and C/Sendai/TU2/2012). As shown in Fig. 3, all of the 10 strains isolated in the 234

Philippines that had an HE gene of the SP82-related lineage, were classified into the 235

YA2681-related lineage based upon the phylogenetic trees constructed with PB1, M, 236

and NS, but classified into the MS80-related lineage in the phylogenetic trees 237

constructed with P3 and NP. Moreover, in the phylogenetic tree constructed using PB2, 238

all strains from the Philippines were classified into the 239

C/Pig/Beijing/115/81(PB11581)-related lineage. On the other hand, all six strains 240

isolated in Japan that had an HE gene of the SP82-related lineage were classified into 241

the YA2681-related lineage based upon the phylogenetic trees constructed with PB1, M, 242


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and NS. They were also classified into the MS80-related lineage using phylogenetic 243

trees constructed with the P3 gene; the same lineage as the Philippine strains. However, 244

they were classified into the PB11581-related lineage based upon phylogenetic trees 245

constructed with the NP gene, and into the KA176-related lineage using trees 246

constructed with the PB2 gene. One strain (C/Sendai/TU2/2012) was classified into the 247

PB11581-related lineage based upon a tree constructed with the PB2 gene, as were the 248

Philippine strains. Interestingly, in the phylogenetic trees constructed with the six 249

internal genes, all the Philippine strains were closely related to C/Miyagi/9/96, 250

C/Miyagi/2/2000, C/Saitama/3/2000, and C/Hiroshima/246/2000, which have HE genes 251

belonging to the KA176-related lineage, and were detected in Japan between 1996 and 252

2000 (10, 37). The genome compositions of the strains in this study, determined by 253

phylogenetic analyses, are summarized in Table 3. These results confirmed that all the 254

strains isolated in the Philippines and Japan are reassortant viruses and that the 255

composition of the internal genes of the Philippine strains is different from those of the 256

Japanese strains. 257

The phylogenetic analyses revealed that different reassortment events occurred in the 258

Philippine and Japanese strains. And on HE gene tree, the Philippine and Japanese 259

strains formed distinct clusters within the SP82-related lineage. A Bayesian evolutionary 260


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tree of the HE gene was generated to determine when these clusters diverged from their 261

last common ancestor. As shown in Fig. 4, the clusters that included the strains isolated 262

in the Philippines and Japan diverged from their last common ancestor at the estimated 263

node age of 1993. 264

265

Discussion 266

This study is the first report of influenza C virus isolation in the Philippines. During this 267

study period in the Philippines, 15 strains (0.28%) of influenza C virus were isolated 268

from children with severe pneumonia and ILI. In contrast, seven strains (0.38%) of 269

influenza C virus were isolated from children with ARI in Japan. The detection rates, 270

which were similar in the Philippines and Japan, are also in agreement with other 271

studies, which reported positive rates from 0.18 to 0.79% (1, 11, 13, 38). In the 272

Philippines, although the overall influenza C detection rate was low, the detection rate 273

in pediatric severe pneumonia cases (0.22%) was not significantly different than that in 274

ILI cases (0.36%). The symptoms of hospitalized pediatric patients included chest 275

indrawing and difficulty breathing, suggesting that influenza C virus may also cause 276

severe lower respiratory tract infections in children. 277

Long-term surveillance of influenza C virus infections in Japan has revealed that 278


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biennial influenza C virus epidemics have occurred during the even-numbered years 279

since 1996 (8-10). In the Philippines, influenza C virus was detected in 2011 and 2013, 280

which suggests that epidemics of the virus may be occurring in odd-numbered years. A 281

previous report revealed that seasonal peaks of influenza C are different from those of 282

influenza A, and that epidemics of influenza C usually occur after peaks of influenza A 283

in Japan (8). In the Philippines, influenza A virus is circulating almost throughout the 284

year without clear seasonality (39). However, influenza C viruses were isolated mainly 285

between January and July in the Philippines (Fig. 1). Therefore, there is a possibility 286

that there might be some seasonality for influenza C in the Philippines. Long-term 287

studies should be conducted to define the epidemic interval and seasonality of influenza 288

C virus infections in the Philippines. 289

Regarding antigenicity, all 19 strains detected in the Philippines and Japan belong to the 290

SP82 antigenic group (Table 2), suggesting that antigenically similar influenza C viruses 291

may be circulating throughout the region. Previous studies reported that the KA176 292

antigenic group and the SP82 antigenic group were dominant strains in Yamagata, Japan 293

between 2002 and 2004, and between 2006 and 2012, respectively (8, 40); and that, the 294

strains of the KA176 and SP82 antigenic groups were co-circulating in 2012. However, 295

no strains belonging to the KA176 antigenic group were isolated in the Philippines or 296


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Japan during the study period. 297

For influenza C virus, the phylogenetic classification based upon the HE gene 298

corresponds with the antigenic classification. Our results also confirmed that both 299

antigenic phylogenetic analyses identified all isolates from the Philippines and Japan as 300

SP82-like viruses. Influenza C viruses belonging to the KA176-related lineage were 301

identified in Singapore in 2006. In Caen, France, it was reported that influenza C 302

viruses detected from 2004 to 2007 were classified into two lineages: a YA2681-related 303

lineage and a SP82-related lineage (11). Additionally, in 2011, an influenza C virus with 304

an HE gene of the YA2681-related lineage was detected in Eastern India (16). 305

Co-circulation of strains classified into the KA176-related lineage and the SP82-realated 306

lineage have been reported in Catalonia, Spain (2009-2010 season) (13); Milan, Italy 307

(2008-2009 and 2009-2010 season) (15); Alberta, Canada (2010-2011 season) (41); and 308

Yamagata, Japan (2011-2012 season). In our study, all of the strains isolated in the 309

Philippines and Japan were classified into the SP82-related lineage. In 2012, strains of 310

the SP82-related lineage were also detected in Mie Prefecture, Japan (42). These studies 311

suggest that similar influenza C viruses are circulating worldwide, and that viruses 312

classified into the SP82-related lineage may be increasing. 313

One of the aims of our study was to clarify the evolutionary process of influenza C virus 314


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through the analysis of reassortment events. A previous report demonstrated that most of 315

the strains isolated in various regions of the world in the 1970s and 1980s had the same 316

genomic compositions as contemporary Japanese strains and suggested the possibility 317

that genetically similar influenza C viruses were circulating all over the world (37). 318

Although all strains in the Philippines and Japan were classified into the SP82-related 319

lineage based upon the phylogenetic tree constructed with the HE gene, the internal 320

gene compositions of the strains detected in the Philippines and Japan were different 321

(Table 3). This finding suggests that different reassortment events occurred for strains 322

detected in these countries. A Bayesian evolutionary tree of the HE gene revealed that 323

the strains in the Philippines and Japan diverged from a common ancestor around 1993 324

(Fig. 4), suggesting that the strains isolated in the Philippines and Japan evolved with 325

reassortment events independently after divergence from a common ancestor. 326

Interestingly, different strains identified in Catalonia in Span in 2009 were classified 327

into different clusters that included Philippine and Japanese strains in the phylogenetic 328

tree constructed with the HE gene, for example, C/Catalonia/1430/2009 in the 329

Philippine cluster, and C/Catalonia/1318/2009 and C/Catalonia/1284/2009 in the 330

Japanese cluster (Fig. 2). This suggests that viruses in these clusters might be circulating 331

not only in Asia, but also in the other parts of the world. 332


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The studies in the Philippines were carried out on three different islands. Although 333

Leyte and Biliran Islands are geographically close, Palawan Island is far from these 334

islands (Fig. S1). However, one strain isolated in Palawan Island in 2013 was highly 335

homologous with strains detected on Leyte and Biliran Island in all genes (Fig. 2 and 3). 336

This suggests that an almost identical strain was circulating throughout the Philippines. 337

In Japan, C/Sendai/TU2/2012 had a different internal genetic composition than the other 338

strains isolated in our study (Table 3). Although the bootstrap value of the internal gene 339

tree was low, C/Sendai/TU2/2012 was assigned to different clusters in the trees 340

constructed with the PB1 and P3 genes (Fig. 3B and 3C). Therefore, our observation 341

suggests that multiple reassortment events could have occurred in the same population 342

and generated viruses with different gene compositions. 343

The biological significance of these reassortment events remains unresolved. However, 344

interestingly, the internal genomic composition of all of the Philippine strains was same 345

as that of strains having HE genes of the KA176-related lineage isolated in Japan 346

between 1996 and 2000, such as C/Miyagi/9/96, C/Miyagi/2/2000, C/Saitama/3/2000, 347

and C/Hiroshima/246/2000 (Table 3). The KA176-related lineage reemerged in 1996 for 348

the first time in 20 years, by acquiring these internal genes through reassortment events. 349

It had been dominantly circulating in Japan until 2004. This fact suggests that there is a 350


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possibility that this internal gene composition may confer some advantage for virus 351

fitness. Reassortment might play a role in the acquisition of such advantageous internal 352

genes. 353

In conclusion, we isolated influenza C viruses in both in Japan and the Philippines. This 354

was the first report of influenza C virus isolation in the Philippines. Epidemiological 355

information for influenza C virus is still limited, especially in tropical countries. 356

Therefore, our data are a valuable contribution to our understanding of the 357

epidemiology of influenza C virus in the tropics. However, long-term monitoring of the 358

virus is necessary to define the etiological significance and epidemiology of influenza C 359

in the Philippines. We also revealed that strains in the Philippines and Japan have 360

emerged through different reassorment events. However, the role of reassorment events 361

in the evolution influenza C virus is still to be defined. 362

363

364


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Acknowledgements 365

This work was supported by a grant-in-aid from the Japan Initiative for Global Research 366

Network on Infectious Diseases (J-GRID) from the Ministry of Education, Culture, 367

Sports, Science and Technology (MEXT), Japan, Science and a Technology Research 368

Partnership for Sustainable Development (SATREPS) from the Japan Science and 369

Technology Agency (JST), Japan International Cooperation Agency (JICA) and JSPS 370

KAKENHI Grant Number 24249041. 371

We thank the staff of health facilities in the Philippines and pediatricians in Sendai city, 372

Miyagi, Japan for providing specimens for virus isolation. 373

374


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515

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Tables 517

Table 1 Clinical manifestations of influenza C virus-positive patients. 518

519

Table 2 Antigenic analysis of influenza C virus isolates, including representative strains, 520

using the hemagglutinin inhibition (HI) test performed with monoclonal antibodies 521

against the hemagglutinin-esterase (HE). 522

523

Table 3 Genome compositions of previous isolates and representative influenza C 524

viruses isolated in this study. 525

526

527


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Figure Legends 528

Fig. 1. Monthly distribution of numbers of specimens tested and influenza C viruses 529

isolated in the Philippines (A) and Sendai city, Japan (B). 530

531

Fig. 2. Phylogenetic tree of influenza C virus hemagglutinin-esterase (HE) genes. The 532

region between nucleotide positions 64 and 1989 was used for this analysis. The 533

numbers above the branches are the bootstrap probabilities (%) for the each branch, 534

determined using MEGA software (version 5.1). Each showed a value greater than 75%. 535

The Philippine strains are marked (■); strains isolated in 2011 are blue and strains 536

isolated in 2013 are purple. The Japanese strains are marked (●); strains isolated in 2008 537

are green and strains isolated in 2012 are red. 538

539

Fig. 3. Phylogenetic trees for the PB2 (A), PB1 (B), P3 (C), NP (D), M (E), NS (F) 540

genes of influenza C isolates. The nucleotide sequences of the following regions were 541

used for analysis: nucleotide positions from 52 to 520 for the PB2 gene, from 50 to 425 542

for the PB1 gene, from 49 to 420 for the P3 gene, from 71 to 670 for the NP gene, from 543

26 to 1147 for the M gene, and from 28 to 889 for the NS gene. Numbers below or 544

above the branches are the bootstrap probabilities (%) of each branch, determined using 545


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the MEGA software (version 5.1) and hide value lower than 75%. The Philippine strains 546

are marked (■); strains isolated in 2011 are blue and strains isolated in 2013 are purple. 547

The Japanese strains are marked (●), strains isolated in 2008 are green and strains 548

isolated in 2012 are red. 549

550

Fig. 4. Bayesian evolutionary tree of influenza C virus based on the nucleotide sequence 551

of the HE gene. The Philippine strains are marked (■); strains isolated in 2011 are blue 552

and strains isolated in 2013 are purple. The Japanese strains are marked (●); strains 553

isolated in 2008 are green and strains isolated in 2012 are red. This Bayesian tree was 554

generated using BEAST software. 555


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Table 1 Clinical manifestations of influenza C virus-positive patients. The Philippines Japan*

Pediatric pneumonia

(n = 7)

Influenza like illness (n = 8)

Acute respiratory infection (n = 5)

Age in years, median (range) 1.1 (0.4 – 5.5) 1.1 (0.4 – 2.9) 2.9 (0.9 – 7.9) Male gender 4 4 4 Sign and symptoms Cough 7 8 5 Nasal discharge 7 8 5 Difficulty breathing 6 0 0 Chest indrawing 7 0 0 Alar flaring 2 0 0 Grunting 0 0 0 Cyanosis 0 0 0 Inability to feed or drink 2 0 0 Convulsions 1 0 0

Fever 37.0-38.0°C 0 5 2 >38.0°C 6 1 3

*Clinical information was available for only 5 of 7 patients.


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Table 2 Antigenic analysis of influenza C virus isolates, including representative strains,

using the hemagglutinin inhibition (HI) test performed with monoclonal antibodies

against the hemagglutinin-esterase (HE).

HI titer of anti-HE MAbs

Antigenic group Virus strain J14 Q5 U4 MS2

KA176 C/Kanagawa/1/76 512000 2560 40 <

MS80 C/Mississippi/80 409600 < < 25600

AI181 C/Aichi/1/81 1024000 160 5120 <

YA2681 C/Yamagata/26/81 128000 12800 80 <

SP82 C/Sao Paulo/378/82 256000 128000 12800 <

C/Leyte/1/2011 64000 25600 3200 <

C/Leyte/2/2011 64000 25600 3200 <

C/Leyte/3/2011 128000 25600 3200 <

C/Leyte/4/2011 64000 25600 3200 <

C/Leyte/5/2011 128000 51200 6400 <

C/Leyte/1/2013 128000 25600 3200 <

C/Leyte/2/2013 64000 12800 3200 20

C/Leyte/3/2013 64000 51200 3200 <

C/Biliran/1/2013 64000 25600 3200 20

C/Biliran/2/2013 256000 51200 6400 <

C/Biliran/3/2013 64000 25600 3200 20

C/Palawan/1/2013 256000 25600 3200 20

C/Sendai/TU1/2008 128000 12800 1600 <

C/Sendai/TU2/2008 128000 12800 3200 <

C/Sendai/TU3/2008 128000 12800 3200 <

C/Sendai/TU4/2008 64000 25600 6400 <

C/Sendai/TU5/2008 64000 51200 6400 20

C/Sendai/TU1/2012 64000 25600 1600 <

C/Sendai/TU2/2012 64000 25600 3200 <

<, titer below 20

Strains isolated in this study are indicated in boldface.

Abbreviations: KA176, C/Kanagawa/1/76; MS80, C/Mississippi/80; AI181,

C/Aichi/1/81; YA2681, C/Yamagata/26/81; SP82, C/Sao Paulo/378/82.


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Table 3 Genome compositions of previous isolates and representative influenza C viruses isolated in this study. RNA segment

Lineage Virus strain PB2 PB1 P3 HE NP M NS

AI181-related lineage C/Aichi/1/81 A A A A A A A

MS80-related lineage C/Mississippi/80 M M M M M M M

YA2681-related lineage C/Yamagata/26/81 Y Y Y Y Y Y Y

KA176-related lineage C/Kanagawa/1/76 K K K K K Y Y C/Miyagi/9/96 P Y M K M Y Y C/Miyagi/2/2000 P Y M K M Y Y C/Saitama/3/2000 P Y M K M Y Y C/Hiroshima/246/2000 P Y M K M Y Y

SP82-related lineage C/Sao Paulo/378/82 S Y S S Y S S C/Leyte/1/2011 P Y M S M Y Y C/Leyte/2/2011 P Y M S M Y Y C/Leyte/3/2011 P Y M S M Y Y C/Leyte/1/2013 P Y M S M Y Y C/Leyte/2/2013 P Y M S M Y Y C/Leyte/3/2013 P Y M S M Y Y C/Biliran/1/2013 P Y M S M Y Y C/Biliran/2/2013 P Y M S M Y Y C/Biliran/3/2013 P Y M S M Y Y C/Palawan/1/2013 P Y M S M Y Y C/Sendai/TU1/2008 K Y M S P Y Y C/Sendai/TU2/2008 K Y M S P Y Y C/Sendai/TU3/2008 K Y M S P Y Y C/Sendai/TU5/2008 K Y M S P Y Y C/Sendai/TU1/2012 K Y M S P Y Y

C/Sendai/TU2/2012 P Y M S P Y Y Strains isolated in this study are indicated in boldface. Abbreviations: A, AI181-related lineage; S, SP82-related lineage; Y, YA2681-related lineage; K, KA176-related lineage; M, MS80-related lineage; P, PB11581-related lineage.


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