Epidemiological and Evolutionary Dynamics of Influenza B Viruses 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 2-HA/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 Victoria-infected 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.


Introduction
Influenza A and B viruses are important pathogens to humans, contributing to a large proportion of morbidity and mortality in respiratory infections worldwide [1].Both viruses have a segmented negative-stranded genome with an enveloped structure [2].Due to the limited host range (with no natural animal hosts other than humans and seals) of influenza B viruses as compared to influenza A viruses [3], antigenic shift does not occur in influenza B viruses, and hence they have little pandemic potential [4].However, influenza B viruses evolve through antigenic drift, enabling them to escape host immunity and continue to adapt to new environments [5], causing significant disease burden to the global population [6].Two major genetically and antigenically distinct influenza B lineages, B/Victoria/2/87-like (Victoria lineage) and B/Yamagata/16/88-like (Yamagata lineage) have been detected since 1983 [7,8].However, in recent decades, the changing demographics and rapid movement of human populations between countries [9] have resulted in the co-circulation and recurring outbreaks of two influenza B lineages in many regions of the world [6,[10][11][12][13][14][15][16][17][18][19].
Studies have shown that the tropical East and Southeast Asia region played a role in reseeding new genetic variants of influenza A (H3N2) viruses in the temperate region, causing an annual H3N2 epidemic worldwide [20,21].In contrast, recent phylogeographic analyses from 2000 to 2012 revealed that influenza B Victoria and Yamagata lineage viruses within East and Southeast Asia region commonly circulate exclusively within this region for several years with limited spreading to other regions [22].Whether this region continues to play a role in global influenza B epidemics still requires further investigation as recent dominant strains isolated in several countries in both Northern and Southern hemispheres during the 2014/2015 season were closely related to strains with a Southeast Asia origin [23,24].
A recent report on the evolution and epidemiology of influenza B virus in Malaysia form 1995-2008, which generally involved the children population, described the incidence and evolutionary changes of predominant circulating Victoria and Yamagata lineages over the years [25].In view of this, we had conducted an influenza surveillance on more adult and elderly patients with respiratory infections and investigated the epidemiological and evolutionary dynamics of influenza B lineages circulating in Malaysia between 2012 and 2014 using phylogenetic methods.Clinical presentation and demographic profile of patients infected by both lineages were compared.Although studies in temperate regions have highlighted the role of humidity and temperature in shaping influenza seasonality [26][27][28], such studies were lacking in the tropical Southeast Asia region.Hence, we also aimed to identify possible meteorological predictors that drive the seasonal periodicity of influenza viruses by associating climatic variables to influenza activities.

Ethical Statement
This study was approved by the University of Malaya Medical Centre (UMMC) Medical Ethics Committee (MEC890.1).Standard, multilingual consent forms validated by the Medical Committee were used.Written consent was obtained from all study participants.

Clinical Specimen Collections
A total of 3,935 nasopharyngeal swab samples were collected from outpatients experiencing symptoms of acute upper respiratory tract infection at the Primary Care Clinic of UMMC in Kuala Lumpur, Malaysia between February 2012 and May 2014.The presence of symptoms associated with acute respiratory tract infection (sneezing, nasal discharge, nasal obstruction, headache, sore throat, hoarseness of voice, muscle ache and cough) was self-recorded by the patient on a questionnaire, which was designed based on previously reported criteria [29].The nasopharyngeal samples were then transported in universal transport medium to the laboratory and stored at -80°C before further processing.

Rapid Detection, Amplification and Sequencing of Influenza B Viruses
Total nucleic acids from nasopharyngeal samples were extracted using the NucliSENS easy-MAG automated nucleic acid extraction system (BioMérieux, France) [30,31].Influenza B viruses and other respiratory viruses (including influenza A viruses) were then detected using the xTAG Respiratory Virus Panel (RVP) FAST multiplex RT-PCR assay (Luminex Molecular Diagnostics, USA) [32,33].Both extraction and detection procedures were performed based on manufacturer's protocol.
A two-step reverse transcription PCR (RT-PCR) approach was adopted following protocols recommended by the World Health Organization (WHO) on influenza B-positive samples [34].The HA and NA genes were amplified as overlapping halves using WHO-recommended gene specific primers (primer sequences and cycling conditions are listed in S1 Table ).PCR products were purified and sequencing was performed in an ABI PRISM 3730XL Genetic Analyzer using the BigDye Terminator v3.1 cycle sequencing kit chemistry (Applied Biosystems, USA).The HA and NA sequence reads were assembled into a contig and aligned with the WHO vaccine and reference sequences.

Phylodynamic Analysis of the HA and NA Genes
Evolutionary analyses of HA and NA gene sequences were carried out on all Malaysian influenza B viruses and other related virus strains worldwide.Our sequence data set included those from the Global Initiative on Sharing all Influenza Data (GISAID) [35] and GenBank within the study period.WHO recommended vaccine and reference strains were also included [23,24].All gene sequences were edited and aligned in the BioEdit 7.2 [36].Phylogenetic trees of the HA and NA genes were reconstructed using maximum likelihood (ML) method heuristically inferred using subtree pruning and regrafting and nearest neighbor interchange algorithms with general time-reversible (GTR) nucleotide substitution model, a proportion of invariant sites (+I) and four categories of gamma rate heterogeneity (+Γ 4 ), implemented in PAUP version 4.0 [37].Robustness of the branching orders was evaluated by bootstrap analysis of 1,000 replicates.
Molecular clock dating analysis using the Bayesian Markov chain Monte Carlo (MCMC) method implemented in BEAST 1.7 [38] was used to estimate the timescale for the emergence of the B/Phuket/3073/2013-like viruses detected in the region, as previously described [14,[39][40][41].The uncorrelated lognormal relaxed clock model with GTR+I+Γ 4 nucleotide substitution model and Bayesian skyline plot (BSP) model was employed to estimate the posterior distribution of phylogenies, nucleotide substitution rates, and the time of most recent common ancestor (tMRCA) of B/Phuket/3073/2013-like viruses.Three independent MCMC runs of 50 million steps sampled for every 50,000 states were performed on HA and NA genes separately.The MCMC sampling was assessed for convergence (effective sampling size > 200) after 10% burn-in using Tracer 1.4 (http://tree.bio.ed.ac.uk).Bayesian maximum clade credibility (MCC) trees were annotated using TreeAnnotator in BEAST package and visualized in FigTree (http:// tree.bio.ed.ac.uk/software/figtree/) to determine the posterior probability of B/Phuket/3073/ 2013-like clade.

Statistical Analysis on Meteorological, Demographical and Clinical Data
To understand the seasonality of influenza viruses in Malaysia, meteorological data were obtained from the Malaysian Meteorological Department (Kuala Lumpur) (03°06'N, 101°39'E).Meteorological factors including daily rainfall amount, number of rainy days, relative humidity, ground temperature and particulate matter with size less than 10 micrometers in diameter (PM10) were collected between February 2012 and May 2014.The associations between these data and influenza-positive cases were determined through linear correlation (bivariate and partial) and regression analysis using the Statistical Package for Social Sciences version 22.0 (SPSS Inc., Chicago, USA).Demographic and clinical features between patients infected with influenza B Victoria lineage and Yamagata lineage were also compared using independent samples t-test.Association between symptoms and lineages was accessed using Pearson's chisquare or Fischer's exact test and binary logistic regression [42].

Nucleotide Sequence Accession Numbers
HA and NA nucleotide sequences of the Malaysian influenza B viruses generated in this study are available in GenBank under accession numbers KR073326-KR073659.

Seasonality of Influenza Viruses in Malaysia
During the study period from February 2012 to May 2014, a total of 287 (14.3%) and 183 (9.1%) samples tested positive for influenza A and B viruses, respectively among 2,010/3,935 (51.1%) patients infected with at least one respiratory virus.We found that the seasonality of influenza viruses showed similar patterns with meteorological factors such as the total amount of rainfall, relative humidity and number of rain days, such that peaks were observed between October to May, and hit their lowest marks around June to September in 2012 and 2013 (Fig 1A and 1C).However, both particulate matter (PM10) and ground temperature showed opposite trends as influenza activity was reduced during an increase in particular matter and ground temperature (Fig 1B and 1C).
The bivariate correlation between three meteorological factors (total amount of rainfall, relative humidity and number of rain days) and number of influenza cases were significantly positive (p < 0.05), while both particulate matter and ground temperature showed significant negative correlation (p < 0.05) with number of influenza cases (Table 1).Though, partial correlation showed that only ground temperature was significantly associated with the number of influenza cases (p < 0.05).A multiple linear regression analysis showed that the linear combination of all five meteorological factors (non-ordered predictors) was significantly related to the number of influenza cases (outcome) (R 2 = 0.458, adjusted R 2 = 0.329, F (5, 21) = 3.549, p < 0.05).Based on both linear correlational and regression analyses, ground temperature is perhaps the main predictor (standard regression coefficient, beta = -0.545,t (26) = -2.308,p < 0.05) which accounts for 40.5% (r: -0.636, R 2 = 0.405) of the variance of the number of influenza cases, while the other variables contributed only an additional 5.3% (45.8%-40.5% = 5.3%).However, such judgments about the relative importance of these predictors are challenging because they are also correlated among themselves (range of r: 0.407-0.931,p < 0.05, data not shown).
Notable characteristic waves of influenza viruses were also observed (Fig 1C): First, influenza A cases consistently peaked ahead of influenza B and fell between September and January, whereas influenza B cases peaked later between February and April when influenza A cases decreased.Second, the overall prevalence of influenza B infection was consistently lower than that of influenza A virus.

Phylogenetic Classification of Influenza B Viruses
A total of 168 full-length HA and 166 full-length NA gene sequences were obtained from 170 patients in the present study (S2 Table ).Additional 23 Malaysian influenza B viruses with fulllength HA and NA gene sequences and collection dates from January 2012 to June 2014 were also retrieved from GISAID and GenBank databases.These published sequences were originated from the National Influenza Centre at the Institute of Medical Research (IMR) Malaysia.Hence, a total of 193 Malaysian influenza B viruses were included for phylogenetic classification.
Phylogenetic analysis of the HA sequences (1,758bp) shows that 67.3% (113/168) of Malaysian influenza B viruses from this study belonged to Yamagata lineage, while 32.7% ( 55

S2 Fig). Both phylogenies indicate that all Malaysian
Victoria viruses detected had Victorialineage HA and Yamagata-lineage NA.This inter-lineage reassortment has long been seen in B/Brisbane/60/2008-like viruses in previous studies [13,18,43,44], which were derived from B/Brisbane/32/2002-like viruses.They have a HA gene segment that evolved from B/Shangdong/7/97-like viruses of Victoria lineage and NA gene segment that evolved from B/Sichuan/ 379/99-like viruses of Yamagata lineage [39].
We further assessed the phylogenetic positions of 2,005 global full-length HA and NA sequences retrieved from GISAID and GenBank databases with a collection year of 2012-2015, and we identified 446 sequences from other countries that fell into this new subclade which had HA and NA gene that derived from Wisconsin/01-like subclade and Stockholm/12-like subclade respectively (S3 and S4 Figs).Interestingly, a recent WHO recommended candidate vaccine strain for the Northern and Southern Hemisphere of the 2015-2016 influenza season -B/Phuket/3073/2013 strain from Thailand, fell into this new subclade as well [23,24].Thus, we conveniently denote this new subclade as Phuket/3073-like subclade.Though it is noteworthy that B/Malaysia/U2462/2013 was by far the earliest strain detected in this subclade (on 31st May 2013)-5 months before B/Phuket/3073/2013 was sampled (on 21st November 2013).The

Evolutionary Dynamics of Phuket/3073-like Subclade
The emergence time of Phuket/3073-like subclade was investigated, by including all 32 Malaysian viruses and 446 global influenza B viruses retrieved from GISAID that were phylogenetically grouped under this subclade (S3-S6 Figs).
Applying a relaxed molecular clock model in the Bayesian MCMC analysis obtained the estimates of the evolutionary rates for both HA and NA gene at 2.2 (95% HPD: 1.9-2.6)x 10 −3 and 3.0 (2.5-3.4)x 10 −3 substitutions/site/year, respectively (Table 2), similar to previously published data [39,41] 2).These estimates collectively suggested that the intra-clade reassortment event could have occurred in March 2013 or earlier, which is about 10 months before the B/Phuket/3073/2013 vaccine strain was first isolated in Thailand.The maximum clade credibility (MCC) tree reconstruction for HA (Fig 5A ) and NA (Fig 5B ) gene of Phuket/3073-like subclade showed that B/Malaysia/U2462/2013 virus consistently occupied the basal position of this subclade while other Malaysian viruses intermingled with global viruses.This suggested that Malaysia could possibly be the place where early Phuket/3073 subclade-like viruses have been circulating, from which the virus was disseminated to other places, and that re-introduction back to Malaysia have also occurred.

Influenza B HA and NA Protein Sequence Analysis
In general, all Malaysian Vic-1, Yam-2 and Yam-3 viruses shared more than 99.0% average nucleotide and amino acid similarity with representative candidate vaccine strains B/Brisbane/ 60/2008, B/Massachusetts/02/2012 and B/Wisconsin/01/2010, for both HA and NA gene (S3 Table ).These similarities generally decreased every year, reflecting that the Malaysian viruses were evolving away from respective candidate vaccine strains (S3 Table ).
A total of 99 amino acid substitutions on the HA protein were detected in all three clades when compared to respective representative candidate vaccine strains (S4-S6 Tables).Among these substitutions, only 15 substitutions occurred on positively selected sites of the four major epitopes of HA1 subunit and their surrounding regions as reported previously [46][47][48][49]  (Table 3).In particular, N116K substitution (shared by 64 Yam-3 viruses) and H122Y substitution (shared by 22 Vic-1 viruses) were found in the 120-loop of HA1 subunit.I146V substitution was shared by 67 Vic-1 viruses in the 150-loop.These substitutions occurred on regions that serve as antibody binding sites, and could contribute to the structural alteration of the HA protein, leading to changes in antigenicity [15,48,50] [47].This substitution (N-glycosylation) was detected in all Malaysian viruses, and was also found in the recent Thailand [18] and Beijing [15] strains.Substitution at this amino acid position has been known to alter the antigenicity of the virus [51] and may help to shield the 190-helix epitope from antibody recognition [47], though the role of D194N substitution in increasing viral fitness requires additional investigation.
On the NA protein, a total of 127 amino acid substitutions were found in all 3 clades when compared to respective representative candidate vaccine strains (S7-S9 Tables).Only 2/68 (2.94%) Malaysian viruses from Vic-1 displayed H274Y substitution in the NA active sites, which was a neuraminidase inhibitor (oseltamivir)-resistant substitution [52].No other substitutions were observed on the NA active sites that confer to neuraminidase inhibitor resistance, as reported elsewhere [53][54][55][56][57], suggesting that the virus would still be sensitive to neuraminidase inhibitors.Notably, three Malaysian viruses from Vic-1 and all viruses from Yam-3 including the Phuket/3073-like subclade had a novel D463N substitution on a non-active site, which introduced a potential glycosylation at that position, potentially shielding the nearby active site.Further functional study of this substitution is warranted.
Comparison of protein sequences with candidate vaccine strains (B/Brisbane/60/2008 for Vic-1, B/Massachusetts/02/2012 for Yam-2 and B/Wisconsin/01/2010 for Yam-3) allowed us to identify several signature amino acid substitutions shared by major clades and subclades.Interestingly, 21 out of 32 Malaysian viruses within Phuket/3073-like subclade had an additional L172Q substitution apart from sharing N116K, K298E and E312K substitutions on the HA protein with Wisconsin/01-like subclade (Table 4).However, in the NA protein, all viruses within Phuket/3073-like subclade had additional I45V, E320K and E340D substitutions besides sharing L73P and K343E substitutions on the NA protein with Stockholm/12-like subclade.

Comparison of Demographic and Clinical Characteristics between Influenza Lineages
A comparison of demographic and clinical characteristics between Yamagata and Victoria lineage-infected patients is shown in Table 5. Patients infected by the Yamagata lineage viruses were significantly older than patients infected by the Victoria lineage virus, with mean age (years±S.D) for Yamagata being 43.58±18.22versus Victoria being 32.11±16.18(p<0.001;Independent Samples t-Test).Similarly, a higher proportion of adults over 56 years old were infected by the Yamagata lineage than the Victoria lineage (51.8% versus 14.3%; p = 0.006; Pearson's chi-square test).In contrast, we found no significant difference in day(s) onset of  symptoms (number of days after the first appearance of symptoms) between the two different lineages.Besides, only two symptoms: nasal congestion and sore throat were found to have an association with patients infected by Victoria lineage viruses (p = 0.033, OR = 2.22 and p = 0.020, OR = 2.49 respectively; Pearson's chi-square test).
In addition, when binary logistic regression was used to predict the likelihood of patients infected with either Yamagata or Victoria lineage virus, four factors made a unique statistically significant contribution (p<0.05) to the regression model: patients 56 years old, nasal congestion, headache and sore throat.The strongest predictors of a Yamagata lineage-infection were headache and age 56 years old (aOR: 3.603, 95% CI: 1.316-9.896,p: 0.013 and aOR: 4.33, 95% CI: 1.184-15.838,p: 0.027, respectively), while the strongest predictors of a Victoria lineage-infection were nasal congestion and sore throat (aOR: 2.833 (1/0.353)95% CI: 1.038-   5).

Discussion
Environmental factors such as higher amount of rainfall, higher relative humidity and colder temperature were found to increase the risk of seasonal influenza transmission [58,59].These factors may affect virus survivability by lengthening the protective effect of droplets on viruses trapped on fomites or aerosols [26,[58][59][60].In particular, colder temperature has been found promoting influenza virus survival in aerosols [25], whereas higher humidity might increase the amount of virus particles that is deposited on the surface, hence encouraging contact transmission of the virus [57].Malaysia has a tropical equatorial climate accompanied by two monsoon seasons, the Southwest Monsoon (May to September) and Northeast Monsoon (November to March).The Northeast Monsoon brings in more rainfall compared to the Southwest Monsoon [61].In this study, as expected, we observed that the peak of the total amount of rainfall, number of rain days and relative humidity coincided with the Malaysian Northeast Monsoon season (Fig 1).The lowest ground temperature was also recorded during this period.Based on bivariate correlation, higher number of influenza cases was found to associate significantly with lower temperature and higher amount of rainfall, rain days and relative humidity (Table 1).Similar association for influenza A was reported in previous studies as well [26,28].However, when partial correlation and multiple linear regression was performed, only ground temperature shows significant negative association with no. of influenza cases and was the strongest predictor among all meteorological factors (Table 1).The actual causal relationship between ground temperature and influenza seasonality remains in question.Though, these findings suggest that the influenza A and B seasonal activity probably coincided with a combination of colder and rainier Malaysian Northeast Monsoon instead of the Southwest Monsoon.A significant negative correlation between particulate matter (PM10) and influenza activity was also found in this study (Table 1).Similar relationship was reported in Southern China [62,63], which postulated that lower PM10 would result in higher ultraviolet radiation (UVR) that may decrease immune function.However, the role of UVR in increasing host susceptibility to influenza remains controversial and requires further investigation.
There was a difference in the overall prevalence between influenza A and B viruses during the study period.The number of influenza B cases was consistently lower compared to influenza A cases every year, and there is a time lag between their peak activities (Fig 1C).On the basis of several reports indicating lower rates of nucleotide mutation and selection pressure in influenza B viruses compared to influenza A viruses [39,64], it is tempting to suggest that they may play a role in the lower prevalence of influenza B viruses.Though such description remains speculative as prevalence studies could be affected by sampling artifacts or the scale of surveillance.While both influenza and B viruses continue to co-circulate, we also observed a slight increase of influenza B incidence when influenza A incidence decreased) ( However, we hypothesize that the turnover of antigenically distinct lineages from Victoria lineage in 2012 to Yamagata lineage in 2013 could be a result of immune selection due to accumulated herd immunity in the human population [39].We suggest that the less dominant Yamagata lineage with distinct antigenicity may regain dominance when the predominating Victoria lineage has induced sufficient herd immunity in the hosts, either through recovery from infection Whether the signature amino acid substitutions may play a role in antigenic drift will require further in-depth molecular experiments to confirm, as antigenic characterization was not performed such that the detection of antigenic drift relied on prediction based on protein sequences.Though, overall, it is worth highlighting that a combination of meteorological factors, influenza population prevalence, genetic reassortment and antigenic drift and possibly other factors (vaccination uptake by the susceptible young or elderly population, socio-economic factors etc.) may shape the epidemiological and evolutionary dynamics of influenza B viruses in the lineage, clade and subclade levels.
Our analysis on the evolutionary dynamics of Phuket/3073-like subclade provides further evidence that Southeast Asia region with tropical and subtropical climate might be a regional and global hub for the emergence of novel influenza viruses [20,22].Notably, there is almost a two-year difference between the estimated time of intra-clade reassortment event (February-March 2013) (Table 2) and time when B/Phuket/3073/2013 vaccine strain was announced on September 2014 for the Southern Hemisphere and on February 2015 for the Northern Hemisphere [23].This suggests that influenza B surveillance in Malaysia and other Southeast Asian countries should be intensified for early detection of emerging strains with epidemic potential.
Our observation of notable age difference between influenza B lineages, with Yamagata viruses were more likely to infect the older adults (56 years old) in the population, has also been observed in previous studies (Table 5) [14,42].This could be the result of a difference in background population immunity, in which older adults with weaker immunity are more susceptible to the current Yamagata strains due to their recent evolution.However, lineage-specific transmissibility among older adults is still currently unclear.We also observed that Yamagata-infected older patients were more likely to experience headache while Victoriainfected patients were more likely to experience nasal congestion and sore throat.Since this is the first association found between these symptoms with specific lineages, it would require additional data and studies to provide a more conclusive evidence of this association.
In summary, this study highlights the importance of continuous surveillance of influenza B viruses in order to better understand the current epidemiology and evolutionary dynamics of these viruses in Malaysia.The main limitation of this study is that only HA and NA surface genes were sequenced and analyzed, which were unable to detect the reassortment involving other internal genes.Phylogenetic analysis of all gene segments will provide a better understanding on influenza B evolution.
/168) viruses belonged to Victoria lineage (Fig 2, S1 Fig).In contrast, evaluation of the NA sequences (1,401bp) shows that all 166 viruses from this study belonged to the Yamagata lineage (Fig 3,

Fig 2 .
Fig 2. Phylogenetic analysis of the HA gene of influenza B viruses in Malaysia from 2012 to 2014.HA sequences of Malaysian influenza B viruses were compared with WHO recommended candidate vaccine and reference strains.The phylogeny was reconstructed using maximum likelihood (ML) method.Bootstrap values (>60%) and amino acid substitutions are mapped to key branches.Intra-and inter-clade reassortants are indicated as boxes.Yamagata Clade 3 (Yam-3) (blue), Yamagata Clade 2 (Yam-2) (orange) and Victoria Clade 1 (Vic-1) (green) are indicated.Scale bar represents a genetic distance of 0.008 substitutions/site.Phylogenetic tree for HA gene with complete taxa identity is shown in S1 Fig. doi:10.1371/journal.pone.0136254.g002

Fig 3 .
Fig 3. Phylogenetic analysis of the NA gene of influenza B viruses in Malaysia from 2012 to 2014.NA sequences of Malaysian influenza B viruses were compared with WHO recommended candidate vaccine and reference strains.The phylogeny was reconstructed using maximum likelihood (ML) method.Bootstrap values (>60%) and amino acid substitutions are mapped to key branches.Intra-and inter-clade reassortants are indicated as boxes.Yamagata Clade 3 (Yam-3) (blue), Yamagata Clade 2 (Yam-2) (orange) and Victoria Clade 1 (Vic-1) (green) are indicated.Scale bar represents a genetic distance of 0.008 substitutions/site.Phylogenetic tree for NA gene with complete taxa identity is shown in S2 Fig. doi:10.1371/journal.pone.0136254.g003

Fig 5 .
Fig 5. Maximum clade credibility (MCC) tree reconstruction of Phuket/3073-like subclade.(A) HA gene and (B) NA gene.The 95% highest posterior density (HPD) for the ancestral node is indicated.Timescale is shown at the bottom of the tree.MCC tree with complete taxa identity is shown in S5 and S6 Figs.doi:10.1371/journal.pone.0136254.g005
or vaccination.It was expected that such alteration of dominance would continue and hence Victoria lineage would become dominant again in 2014.Surprisingly, the Yamagata lineage continued its domination in the influenza B viral population with a clade shift from Yam-2 in 2013 to Yam-3 in 2014 that consists mainly of B/Phuket/3073/2013-like viruses (Fig 4).Besides possessing an intra-clade reassortment property (HA from Wisconsin/01-like subclade and NA from Stockholm/12-like subclade) (Figs 2 and 3), Phuket/3073-like subclade viruses also shared several signature amino acid substitutions (Table 4).Haemagglutination inhibition (HI) tests by WHO have shown that the representative B/Phuket/3073/2013 strains have acquired significant antigenic drift (4 folds of titer reduction) from the B/Wisconsin/1/ 2010 strains and (2 folds of titer reduction) from the B/Stockholm/12/2011 strains, in which both are representatives of B/Phuket/3073/2013 HA and NA parental clades, respectively [65].

Table 1 .
Linear correlations and regression between meteorological factors and number of influenza positive cases (March 2012 -May 2014).

Table 2 .
Evolutionary rate and age of influenza B Phuket/3073-like subclade.

Table 3 .
Amino acid substitutions found on the HA protein of influenza B viruses.

Table 4 .
Major signature amino acid substitutions.These substitutions are summarized from S4-S9 Tables.

Table 5 .
Comparison on the demographic and clinical characteristics of patients infected by influenza B Victoria and Yamagata lineages.