Title Epidemiological and Evolutionary Dynamics of Influenza BViruses in Malaysia , 2012-2014

Epidemiological and evolutionary dynamics of influenza B Victoria and Yamagata lineages remained poorly understood in the tropical Southeast Asia region, despite causing seasonal outbreaks worldwide. From 2012–2014, nasopharyngeal swab samples collected from outpatients experiencing acute upper respiratory tract infection symptoms in Kuala Lumpur, Malaysia, were screened for influenza viruses using a multiplex RT-PCR assay. Among 2,010/3,935 (51.1%) patients infected with at least one respiratory virus, 287 (14.3%) and 183 (9.1%) samples were tested positive for influenza A and B viruses, respectively. Influenza-positive cases correlate significantly with meteorological factors—total amount of rainfall, relative humidity, number of rain days, ground temperature and particulate matter (PM10). Phylogenetic reconstruction of haemagglutinin (HA) gene from 168 influenza B viruses grouped them into Yamagata Clade 3 (65, 38.7%), Yamagata Clade 2 (48, 28.6%) and Victoria Clade 1 (55, 32.7%). With neuraminidase (NA) phylogeny, 30 intra-clade (29 within Yamagata Clade 3, 1 within Victoria Clade 1) and 1 inter-clade (Yamagata Clade 2HA/Yamagata Clade 3-NA) reassortants were identified. Study of virus temporal dynamics revealed a lineage shift from Victoria to Yamagata (2012–2013), and a clade shift from Yamagata Clade 2 to Clade 3 (2013–2014). Yamagata Clade 3 predominating in 2014 consisted of intra-clade reassortants that were closely related to a recent WHO vaccine candidate strain (B/Phuket/3073/2013), with the reassortment event occurred approximately 2 years ago based on Bayesian molecular clock estimation. Malaysian Victoria Clade 1 viruses carried H274Y substitution in the active site of neuraminidase, which confers resistance to oseltamivir. Statistical analyses on clinical and demographic data showed Yamagata-infected patients were older and more likely to experience headache while Victoriainfected patients were more likely to experience nasal congestion and sore throat. This study describes the evolution of influenza B viruses in Malaysia and highlights the importance of continuous surveillance for better vaccination policy in this region. PLOS ONE | DOI:10.1371/journal.pone. 0136254 August 27, 2015 1 / 21 OPEN ACCESS Citation: Oong XY, Ng KT, Lam TT-Y, Pang YK, Chan KG, Hanafi NS, et al. (2015) Epidemiological and Evolutionary Dynamics of Influenza B Viruses in Malaysia, 2012-2014. PLoS ONE 10(8): e0136254. doi:10.1371/journal.pone. 0136254 Editor: Florian Krammer, Icahn School of Medicine at Mount Sinai, UNITED STATES Received: June 22, 2015 Accepted: August 1, 2015 Published: August 27, 2015 Copyright: © 2015 Oong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the Ministry of Education, Malaysia: High Impact Research H-500001-00-A000012-000001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.