Virology 492 (2016) 21–24

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Brief Communication

Local persistence and global dissemination play a significant role in thecirculation of influenza B viruses in Leyte Island, Philippines

Yuki Furuse a,1, Takashi Odagiri a,1,2, Raita Tamaki a, Taro Kamigaki a, Hirono Otomaru a,Jamie Opinion b, Arlene Santo c, Donna Dolina-Lacaba d, Edgard Daya d, Michiko Okamoto a,Mariko Saito-Obata e, Marianette Inobaya f, Alvin Tan f, Veronica Tallo f, Socorro Lupisan f,Akira Suzuki g,3, Hitoshi Oshitani a,n

a Department of Virology, Tohoku University Graduate School of Medicine, Sendai, Japanb Tacloban City Health Office, Tacloban City, Philippinesc Tanauan Rural Health Unit, Tanauan, Philippinesd Leyte Provincial Health Office, Palo, Philippinese Tohoku-RITM Research Center for Emerging and Reemerging Infectious Diseases, Muntinlupa City, Philippinesf Research Institute for Tropical Medicine, Muntinlupa City, Philippinesg Virus Research Center, Clinical Research Division, Sendai Medical Center, Sendai, Japan

a r t i c l e i n f o

Article history:Received 6 January 2016Returned to author for revisions31 January 2016Accepted 2 February 2016Available online 18 February 2016

Keywords:Influenza B virusTransmissionMolecular epidemiologyPhilippines

x.doi.org/10.1016/j.virol.2016.02.00122/& 2016 The Authors. Published by Elsevier

esponding author.ail address: oshitanih@med.tohoku.ac.jp (H. Oese authors contributed equally to this studyesent address: Division of International Healthf Medical and Dental Sciences, Niigata, Japanesent address: Department of Pediatrics, Tf Medicine, Sendai, Japan.

a b s t r a c t

The local and global transmission dynamics of influenza B virus is not completely understood mainlybecause of limited epidemiological and sequence data for influenza B virus. Here we report epidemio-logical and molecular characteristics of influenza B viruses from 2010 to 2013 in Leyte Island, Philippines.Phylogenetic analyses showed global dissemination of the virus among both neighboring and distantareas. The analyses also suggest that southeast Asia is not a distributor of influenza B virus and canintroduce the virus from other areas. Furthermore, we found evidence on the local persistence of thevirus over years in the Philippines. Taken together, both local persistence and global dissemination play asignificant role in the circulation of influenza B virus.& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Influenza B virus causes acute febrile disease with respiratorysymptoms similar to influenza A virus. Although influenza B virusused to be considered as less virulent and consequently less of apublic health threat than influenza A virus (Glezen, 1982; Nolan etal., 1980), it has recently been shown that influenza B virus alsohas a significant global disease burden, particularly among chil-dren (Peltola et al., 2003; Poehling et al., 2006). There are twomajor viral surface glycoproteins in the influenza B virus:hemagglutinin (HA) and neuraminidase (NA). Binding of the virusto its cellular receptors is mediated by HA (Wang et al., 2007).Infected individuals produce antibodies against HA, which can

Inc. This is an open access article u

shitani).., Niigata University Graduate.ohoku University Graduate

prevent subsequent infections (Berton and Webster, 1985). How-ever, naturally induced mutations in the HA gene during replica-tion can alter antigenicity of the virus (antigenic drift), enablingthe virus to escape from the host's immunity developed by pre-vious infections. With this mechanism, influenza B virus succes-sively evolves and causes epidemics almost every year (Chen andHolmes, 2008; Webster and Berton, 1981). Unlike influenza Avirus, influenza B virus does not have subtypes, which are deter-mined based on the characteristics of HA and NA. However, cur-rently circulating influenza B viruses are divided into two phylo-genetically and antigenically distinct lineages: B/Victoria/2/87-like[Victoria lineage] and B/Yamagata/16/88-like [Yamagata lineage](Rota et al., 1990).

In the temperate regions, influenza B virus causes epidemicsfrom winter to early spring with a clear peak, whereas year-roundcirculation and minor peaks are observed in the tropical regions(Chew et al., 1998; Finkelman et al., 2007). However, factors con-tributing to the different seasonality between temperate and tro-pical regions remain elusive (Chew et al., 1998; Tamerius et al.,2011). Although there is a year-round circulation of the virus in

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Y. Furuse et al. / Virology 492 (2016) 21–2422

southern China, the subtropical region, new antigenic variants arealso introduced there (Tan et al., 2013). It has been proposed thatany new antigenic variant of the influenza A virus first emerges inSoutheast Asia and then spreads to other parts of the world(Bedford et al., 2010; Rambaut et al., 2008; Russell et al., 2008),although this hypothesis remains controversial (Bahl et al., 2011).Phylogeographic analyses of influenza B viruses have revealed thatviruses circulate outside Southeast Asia for several years withoutevidence of seeding from East and Southeast Asia (Bedford et al.,2015). Similarly, viruses within Southeast Asia can exclusivelycirculate in Southeast Asia for 41 year (Bedford et al., 2015).However, local and global transmission dynamics of the influenzaB virus is not completely understood mainly because of limitedepidemiological and sequence data for this virus, which is far lessthan those for influenza A virus. In particular, data from the tro-pical region, including Southeast Asia are limited. In this study, wereported the epidemiological and molecular characteristics ofinfluenza B viruses in the Philippines from 2010 to 2013 andcompared their HA gene sequence with strains circulating in otherparts of the world for the better understanding of the transmissiondynamics of the virus.

Results and discussion

From January 2010 to December 2013, a total of 2185 naso-pharyngeal swab specimens were collected from patients withinfluenza-like illness (ILI). Of these, 128 specimens (5.9%) werepositive for isolation and typing of influenza B virus. The medianage of patients positive for influenza B virus was 4.9 (range, 0.3–18.3) years, whereas the median age of all ILI patients was 2.3(range, 0.1–56.0) years. Demographical and clinical information ofpatients positive for influenza B virus has been discussed in detailin our previous study (Otomaru et al., 2015). We detected threeepidemics of influenza B virus infection; they occurred in 2010(Phi-2010), between 2011 and 2012 (Phi-2011–12), and in 2013(Phi-2013) (Fig. 1). Influenza A virus strains A(H1N1)pdm09 and A(H3N2) and influenza C virus were also detected.

We then sequenced the HA gene and constructed a phylogenetictree (Supplemental Fig. 1). All strains from the Phi-2010 and Phi-2013 epidemics were classified as the Victoria lineage. Those virusesisolated in the Phi-2011–12 epidemic included strains of both theVictoria lineage and Yamagata lineages. We also obtained allsequence data of influenza B viruses collected from all over theworld between 2009 and 2014 from the Influenza Virus Resource(http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html; accessed onNovember 12, 2015). We constructed phylogenetic trees with ourstrains and sequences obtained from the database (Fig. 2A). Moststrains in the Victoria lineage and Yamagata lineage can be dividedinto four (Vic1–4) and two (Yam1 and 2) clades, respectively. The

Fig. 1. Epidemic curve of cases positive for influenza virus. Monthly distribution oftotal ILI cases and cases positive for influenza A, B, and C viruses. Three epidemicsof influenza B virus infection (Phi-2010, Phi-2011–12, and Phi-2013) are indicatedby square brackets.

phylogenetic trees showed that strains isolated in the Philippineswere genetically close to or even identical to strains collected fromother countries, including China, Malaysia, Vietnam, Singapore,Japan, the United States, and Australia. This suggests that there was aglobal dissemination of the virus in both neighboring and distantareas. During the study period, the Phi-2010 and Phi 2013 epidemicscaused by Victoria lineage lasted for 48 months, whereas the Phi-2011–12 epidemic caused by the Yamagata lineage lasted for o3months (Figs. 1 and 2A). The Phi-2010 epidemic was caused bystrains from two clades: Vic1 and Vic2. Furthermore, the Phi-2010strains in the Vic1 clade clustered into two groups and strains in theVic2 clade clustered into three groups (Fig. 2A). Similarly, Phi-2013strains clustered into three groups within the Vic4 clade. These datasuggest that there were multiple introductions of virus in thiscountry, which may be a reason for the relatively long epidemicscaused by the Victoria lineage. In contrast, the Phi-2011–12 strains inthe Yamagata lineage clustered into a single group. The short epi-demic duration was possibly due to the single introduction of theYamagata lineage (Fig. 2A). It should also be noted that the Phi-2011–12 strains in the Victoria lineage were similar to some of thePhi-2010 strains (indicated by an arrow in Supplemental Figs. 1 and2A), suggesting local persistence of the virus.

Fig. 2B shows the temporal distribution of the phylogeneticclades in different countries where robust genetic sequence datafor influenza B virus are available; these include the Philippines,Vietnam, Thailand, Malaysia, China, Japan, the United States,Australia, and New Zealand. As for strains of the Yamagata lineage,we identified two different co-circulating clades (Yam1 and Yam2)in some countries (Thailand, Malaysia, and the United States) andexclusive circulation of one clade at a given time in other countries(Philippines, Vietnam, and China). It is difficult to draw any firmregarding the timing or direction of transmission of the Yamagatalineage among these countries. As for the Victoria lineage, Vic1strains circulated in Southeast Asia in 2010, although these strainswere scarcely detected in other areas at this time (Fig. 2B). Further,strains in the Vic1 clade had been detected in Southeast Asia since2004 (data not shown). In contrast, Vic4 strains have been circu-lating in several countries, including Japan and the United States,since 2010. The Vic4 strains in the Philippines and Vietnam werenot identified until the early 2013. It is likely that the Vic4 strainsin the Philippines were introduced from outside the country.These findings suggest relatively persistent and local circulationwith sporadic global transmission of the virus. Furthermore, thesealso suggest that Southeast Asia is not a distributor of the influ-enza B virus but is susceptible to virus introduction from otherareas (Bedford et al., 2015).

In the present study, we reported the molecular epidemiologyof influenza B virus in the Philippines from 2010 to 2013 andanalyzed local and global transmission dynamics of the virus. Ourfindings suggest that both local persistence and global dis-semination play a significant role in circulation of influenza B virusin the Philippines.

Materials and methods

We conducted a prospective study on the epidemiology andetiology of ILI in Leyte Island, Philippines. This study was conductedin the outpatient department of three health facilities: Leyte Pro-vincial Hospital, Tacloban City Health Center, and Tanauan RuralHealth Unit. Nasopharyngeal swab specimens were collected frompatients who attended three facilities with ILI. ILI was defined asfever Z38 °C or feverish and either cough or nasal discharge beingpresent. Details of the study design have been described previously(Otomaru et al., 2015). Virus isolation from specimens and typingusing the hemagglutination inhibition test for influenza B virus were

Fig. 2. Phylogenetic tree for the HA gene and trend of strains in different countries. (A) Phylogenetic trees with strains from other areas for Victoria lineage (1410 strains, 699nucleotides) and Yamagata lineage (888 strains, 696 nucleotides). Each tree was rooted using the sequence of B/Beijing/1/87 for Victoria lineage and B/Yamagata/16/88 forYamagata lineage. Identical sequences from the same region were excluded. Clustering of strains (Vic1–4, and Yam1 and 2) is indicated by square brackets. In this study,strains isolated from each epidemic are also described along with clade names. Similar strains in Phi-2010 and Phi-2011–12 are indicated by an arrow. (B) Time-course ofproportion of strains in each clade (as described in panel A) is shown for the countries indicated. Data from the Philippines consist of our sequences in this study (n¼128)and other sequences publicly available (n¼11). Data from Australia and New Zealand are combined. Lines are smoothed using Kernel average smoother (3 months window).

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performed to detect influenza B virus among ILI cases. The HA geneof the influenza B viruses isolated in the study was sequenced (dataavailable at Genbank; accession number LC107150-LC107277) andphylogenetic trees were constructed by Maximum Likelihoodmethod using MEGA 6 (Tamura et al., 2013).

This study was conducted as a component of ILI national sur-veillance under the Philippine Integrated Disease Surveillance andResponse (PIDSR) program. For children aged o12 years, writteninformed consent for participation in the study was obtained fromtheir parents or guardians. For children aged 12–18 years, writteninformed consent/assent was required prior to the collection ofepidemiological and clinical information in addition to the parentor guardian's consent. In case of patients aged 418 years, writtenconsent was obtained from the participants themselves. The studyprotocol was approved by the ethical review board of TohokuUniversity Graduate School of Medicine.

Acknowledgments

We thank the staff of Leyte Provincial Hospital, Tacloban CityHealth Center, Tanauan Rural Health Unit, and Tohoku-RITM Col-laborative Research Team. This work was supported by Grants-in-Aid from the Japan Initiative for Global Research Network onInfectious Diseases (J-GRID) (Grant no. 15fm0108013h0001) fromJapan Agency for Medical Research and Development (AMED), andScience and Technology Research Partnership for SustainableDevelopment (SATREPS) (Grant no. 15jm0110001h0006) fromAMED and Japan International Cooperation Agency (JICA). Thefunders were not involved in the study design; data collection,analysis, or interpretation; the writing of the article; or the deci-sion to submit for publication.

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.virol.2016.02.001.

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