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#
5
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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
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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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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
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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
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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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Infect Dis 67:127-131. 514
515
516
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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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