Virus Research 330 (2023) 199089 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Detection of reassortant influenza B strains from 2004 to 2015 seasons in Barcelona (Catalonia, Spain) by whole genome sequencing Cristina Andrés a, b, Margarita del Cuerpo c, d, Núria Rabella c, d, Maria Piñana a, b, Manuel Jesús Iglesias-Cabezas a, b, Alejandra González-Sánchez a, b, Juliana Esperalba a, b, Ariadna Rando a, b, Maria Carmen Martín a, Francisco Fuentes a, Susana Rubio a, Narcís Saubi a, b, Tomàs Pumarola a, b, *, Andrés Antón a, b a Respiratory Viruses Unit, Virology Section, Microbiology Department, Vall d’Hebron Hospital Universitari, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035, Barcelona, Spain b Centro de Investigación Biomédica en red de Enfermedades Infecciosas CIBERINFEC, Instituto Carlos III, Madrid, Spain c Microbiology Department, Hospital de la Santa Creu i Sant Pau. Universitat Autónoma de Barcelona (UAB). Sant Pau Biomedical Research Institute (IIB Sant Pau), Barcelona, Spain d Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: Background: Influenza B viruses (FLUBV) have segmented genomes which enables the virus to evolve by segment Influenza B viruses reassortment. Since the divergence of both FLUBV lineages, B/Victoria/2/87 (FLUBV/VIC) and B/Yamagata/ Reassortment event 16/88 (FLUBV/YAM), PB2, PB1 and HA have kept the same ancestor, while some reassortment events in the Lineage other segments have been reported worldwide. The aim of the present study was to find out reassortment epi­ Molecular epidemiology sodes in FLUBV strains detected in cases attended at Hospital Universitari Vall d’Hebron and Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) from 2004 to 2015 seasons. Methods: From October 2004 to May 2015, respiratory specimens were received from patients with respiratory tract infection suspicion. Influenza detection was carried out by either cell culture isolation, immunofluorescence or PCR-based assays. A RT-PCR was performed to distinguish both lineages by agarose gel electrophoresis. Whole genome amplification was performed using the universal primer set by Zhou et al. in 2012, and subsequently sequenced using Roche 454 GS Junior platform. Bioinformatic analysis was performed to characterise the se­ quences with B/Malaysia/2506/2007 and B/Florida/4/2006 corresponding sequences as reference of (B/VIC) and (B/YAM), respectively. Results: A total of 118 FLUBV (75 FLUBV/VIC and 43 FLUBV/YAM), from 2004 to 2006, 2008–2011 and 2012–2015 seasons, were studied. The whole genome of 58 FLUBV/VIC and 42 FLUBV/YAM viruses was suc­ cessfully amplified. Based on HA sequences, most FLUBV/VIC viruses (37; 64%) belonged to clade 1A (B/Bris­ bane/60/2008) except to 11 (19%), which fell within clade 1B (B/HongKong/514/2009) and 10 (17%) to B/ Malaysia/2506/2004. Nine (20%) FLUBV/YAM viruses belonged to clade 2 (B/Massachusetts/02/2012), 18 (42%) to clade 3 (B/Phuket/3073/2013) and 15 (38%) fell within Florida/4/2006. Numerous intra-lineage reassortments in PB2, PB1, NA and NS were found in 2 2010–2011 viruses. An important inter-lineage reas­ sortment event from 2008 to 2009 (11), 2010–2011 (26) and 2012–2013 (3) FLUBV/VIC (clade 1) strains to FLUBV/YAM (clade 3) was found, in addition to 1 reassortant NS in 2010–2011 B/VIC virus. Conclusions: Intra- and inter-lineage reassortment episodes were revealed by WGS. While PB2-PB1-HA remained in complex, NP and NS reassortant viruses were found in both lineages. Despite reassorment events are not often, the characterisation only by HA and NA sequences might be underestimating their detection. * Corresponding author at: Respiratory Viruses Unit, Virology Section, Microbiology Department, Vall d’Hebron Hospital Universitari, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, Passeig Vall d’Hebron 119-129, 08035, Barcelona, Spain. E-mail address: virusrespiratoris@vallhebron.cat (T. Pumarola). https://doi.org/10.1016/j.virusres.2023.199089 Received 8 February 2023; Received in revised form 7 March 2023; Accepted 9 March 2023 Available online 5 April 2023 0168-1702/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). C. Andrés et al. Virus Research 330 (2023) 199089 1. Background 2.2. Characterisation of FLUBV Influenza viruses (FLUV), as causative agents of respiratory in­ A lineage reverse transcription RT-PCR (one-step RT-PCR system; Qia­ fections, contribute to morbidity and mortality in the community, gen, Hilden, Germany) from an adapted protocol of the Centre for Control mainly in patients at high-risk infections such as elderly, those with Disease and Prevention (CDC) was performed to initially distinguish both underlying comorbidities or pregnant women (Tewawong et al., 2017; FLUBV lineages based on the HA sequence by agarose gel electrophoresis Medina and García-Sastre 2011). (WHO Chinese NIC, n.d.) (Fig. 1). The primer sequences were BVf FLUV are single-stranded, negative-sense, segmented RNA viruses (5′ -ACATACCCTCGGCAAGAGTTTC-3′ ), BVr (5′ -TGCTGTTTTGTTGTTGT belonging to the Orthomyxoviridae family (Schrauwen et al., 2014; CGTTTT-3′ ), BYf (5′ -ACACCTTCTGCGAAAGCTTCA-3′ ) and BYr (5′ -CAT­ Bouvier and Palese 2008). Three FLUV types (A, B and C), which infect AGAGGTTCTTCATTTGGGTTT-3′ ), for FLUBV/VIC and FLUBV/YAM line­ humans, can be described based on genetic and antigenic features and, ages, respectively. amongst influenza B viruses (FLUBV), two lineages genetically and antigenically different can be distinguished, B/Victoria/2/87-like 2.3. Whole-genome amplification (WGA) and 454-pyrosequencing (FLUBV/VIC) and B/Yamagata/16/88-like (FLUBV/YAM) (Tewawong et al., 2015). The WGA was carried out using universal primer set and PCR pro­ Both influenza A viruses (FLUAV) and FLUBV evolve through point tocol by Zhou and Wentworth (2012) with SuperScript III one-step mutations mainly in the surface glycoproteins (antigenic drift), hae­ RT-PCR kit (Invitrogen, USA) and subsequently sequenced with Roche magglutinin (HA) and neuraminidase (NA), to escape the existing 454 GS Junior platform. Briefly, full genome PCR amplicons were human immunity and persist in the population, leading to the recurrent quantified using Quant-iT™ PicoGreen® Kit (Molecular Probes, Thermo seasonal epidemics (Steinhauer 1999; Bedford et al., 2015). In addition, Scientific™, USA) in an Infinite 200 PRO Tecan fluorimeter. A library for these viruses also evolve through the exchange of segments (antigenic each patient was constructed by nebulisation and prepared using the GS shift) between clades, subtypes (FLUAV) or lineages (FLUBV) due to its DNA Rapid Library Preparation Kit (GS Junior Titanium Series, 454 segmented genome. These reassortment events can cause pandemics and Roche Life Sciences) according to manufacturer’s recommendations. FLUAV are considered the major threat over FLUBV owing to their large Each purified DNA was ligated to a multiplex identifier (MID). The number of reservoirs compare to FLUBV (Steinhauer and Skehel 2002). resulting fragments were size-selected using Agencourt AMPure XP bead Since its divergence in 1983, both FLUBV lineages have co-circulated sizing (Beckman Coulter, USA), quantified again to make a dilution with in the human population and reassortment episodes, despite being few, the molecules needed to proceed with the emulsion PCR (emPCR) and have been reported later than 1992 because of lineage mixing (Oong then, ten samples per amplification were pooled. After emPCR, the et al., 2017; Dudas et al., 2015). While most of FLUBV segments have emulsion was disrupted and the beads containing clonally amplified suffered some reassortment events as the NA or the non-structural pro­ DNA were enriched. The beads were separated and deposited into in­ tein (NS), another ones like the polymerase subunits 1 (PB1) and 2 (PB2) dividual picotiter-plate wells and combined with sequencing enzymes. or the HA have maintained separate FLUBV/VIC or FLUBV/YAM line­ Pyrosequencing was carried out on the picotiter-plate by successive flow ages (Dudas et al., 2015). of dNTPs (Deyde and Gubareva 2009; Margulies et al., 2005; Raca et al., Next generation sequencing (NGS) should help to recognise reas­ 2010). sortant FLUBV strains through the study of the whole viral genome. The aim of this study was to find out reassortment events in FLUBV strains 2.4. Bioinformatics analysis of the data detected in cases attended at two hospitals in Barcelona (Catalonia, Spain) from 2004 to 2015 by whole-genome sequencing (WGS). Sequencing data was obtained as standard flow gram format (SFF) files from the GS Run Processor after quality control filtering and read 2. Materials and methods trimming using the manufacturer’s default parameters (see 454 Sequencing System software manual version 2.5). The reads were From October 2004 (week 40/2004) to May 2015 (week 20/2015), trimmed using the MIDs for each sample, and mapped using the GS upper (nasopharyngeal aspirates or swabs) and lower (bronchoalveolar Reference Mapper with the reference sequences B/Malaysia/2506/2004 lavages, bronchoaspirates and tracheal aspirates) respiratory tract for FLUBV/VIC and B/Florida/4/2006 for FLUBV/YAM viruses. specimens were collected for influenza laboratory-confirmation from patients with suspicion of acute respiratory tract infection who were attended at the Emergency Department or admitted to Hospital de la 2.5. Phylogenetic analysis and molecular characterisation Santa Creu i de Sant Pau (HSCSP) and Hospital Universitari Vall d’He­ bron (HUVH). Respiratory samples were processed within the first 24 h, Phylogenetic analyses of the complete eight-segment large contigs being kept at 2–4 oC in several aliquots until its use. Based on medical were performed using the reference sequences by FLUBV lineage as records, demographic features (age and sex) and clinical data were recommended by season in the Northern Hemisphere by the European retrospectively collected from FLUBV laboratory-confirmed cases. Centre for Disease Prevention and Control (ECDC). Nucleotide se­ quences were aligned using the MUSCLE algorithm, and the molecular 2.1. Detection of FLUV from respiratory specimens evolutionary model of nucleotide substitutions were fitted to the mul­ tiple sequence alignments, both conducted in MEGA v11 (Tamura et al., The detection of FLUV was carried out by either immunofluorescence 2011). The phylogenetic trees were constructed using a neighbor-joining (D3 Ultra 8TM DFA Respiratory Virus Screening & Identification Kit distance method as implemented in MEGA v11 with the evolutionary Diagnostic HYBRIDS, USA), PCR-based assays (Anyplex II RV16 Detec­ tion Kit Seegene, Korea and GeneXpert Flu, Cepheid, USA) or cell culture isolation by Madin-Darby canine kidney (MDCK) SIAT 1 cells (WHO, 2011). Prior to PCR-based assays, total nucleic acids were extracted using NucliSense easyMAG (bioMérieux, Marcy l’Etoile, France) ac­ cording to the manufacturer’s instructions, and kept frozen (− 20 oC) until use. Fig. 1. Agarose gel electrophoresis of lineage RT-PCR. 2 C. Andrés et al. Virus Research 330 (2023) 199089 model with the lowest Bayesian information criterion score (Tamura et al., 2011). The topological accuracy of the internal branch was evaluated by the bootstrap method (1000 replicates). In order to detect amino acid substitutions in the different FLUBV segments, the translated amino acid sequences from aligned nucleotide sequences were related to reference sequences recommended by ECDC for influenza virus characterisation (B/Yamagata: B/Florida/04/2006, GISAID EPI134356; and B/Victoria: B/Malaysia/2506/2004, GISAID EPI117513). 2.6. Statistical analysis Statistical analysis was performed using SPSS v22 (SPSS Inc., Chi­ cago, IL, USA). Categorical variables were described through fre­ quencies and proportions. Chi square test was calculated to assess associations between categorical variables. P values <0.05 were Fig. 3. Summary of the reassortment events observed in the present study at considered to be statistically significant. the lineage and clade level. 3. Results (11V1231), which showed genetic similarities to clade 1A in HA, but did not show any in NA (Supplementary Fig. 1). Regarding the polymerase A total of 118 FLUBV (75 FLUBV/VIC and 43 FLUBV/YAM) strains subunits, the same genetic groups were maintained as observed in HA from the 2005–2006, the 2008–2011 and the 2012–2015 seasons were and NA, but three 2010–2011 FLUBV/VIC viruses (11V500, 11V1231 studied. During the 2006–2008 and the 2011–2012 seasons, FLUBV and 11V2568) shifted between clades. While 11V1231 did not show strains could not be detected due to the low FLUBV circulation (Insituto genetic similarities with clade 1 neither in PB1 nor in PB2, it fell within Carlos III, n.d.). Whole genome was successfully amplified and clade 1A in polymerase subunit A (PA). On the other hand, 11V500 fell sequenced from 58 (77%) FLUBV/VIC and 42 (98%) FLUBV/YAM vi­ within clade 1A in PB1, and shifted to clade 1B in the other subunits as it ruses. Most of FLUBV laboratory-confirmed studied cases (98) were also happened to 11V2568, which fell within clade 1B in PB1 and children under 5 years of age (40; 41%) and children between 5 and 14 changed to clade 1A in the other segments (Supplementary Fig. 2). years (35; 36%) with a 58:40 proportion of male and female (p = 0.375). Phylogenetic analysis of the nucleoprotein (NP) revealed an important The phylogenetic analysis based on HA sequences revealed that most inter-lineage reassortment event from 2008 to 2009 (11), 2010–2011 FLUBV/VIC viruses (37; 64%) belonged to clade 1A represented by B/ (26) and 2012–2013 (3) FLUBV/VIC (clade 1) strains to FLUBV/YAM Brisbane/60/2008 and the remaining sequences to clade 1B (11; 19%) (clade 3) as shown in Figs. 3 and 4. The molecular characterisation of represented by B/HongKong/514/2008 and B/Malaysia/2506/2004 these strains revealed the acquisition of characteristic FLUBV/YAM (10; 17%). Most FLUBV/YAM strains belonged to clade 3 (18; 42%) amino acid changes like A17T, A60D or V171I. Amongst the matrix represented by B/Phuket/3073/2013 and B/Florida/4/2006 (15; 38%) protein (MP), the same genetic groups were kept as seen in HA, but in and the leftover sequences to clade 2 (9; 20%) represented by B/Mas­ the NS, 11V500 shifted to clade 1B and 11V1231 reassorted to FLUBV/ sachusetts/02/2012. The comparison of HA and NA phylogenetic trees YAM gaining the typical FLUBV/YAM amino acid changes as N4D or showed at first congruent classification between the different genetic E138D (Supplementary Fig. 3) and other mutations (I98V, S109P, groups as shown in Fig. 2. In addition, molecular characterisation of NA T115S, N152S and V263M). sequences revealed no mutations related to reduce sensibility to neur­ Regarding the clinical data, hospitalisation, ICU-admission and aminidase inhibitors (NAIs) (Francisco Pozo, 2013; Nguyen et al., 2012). mortality rates are summarised in Table 1. The main clinical symptoms Several intra- and inter-lineage reassortment events were found by of patients carrying reassortant strains (40/98; 41%) were fever (21; phylogenetic analysis of each segment as summarised in Fig. 3. Firstly, 52%) and respiratory disease (18; 45%), such as upper (laryngitis or corresponding classification was observed amongst HA and NA glyco­ pharyngitis) or lower (bronchitis) respiratory tract infections. No proteins except to one reassortant 2010–2011 FLUBV/VIC strain Fig. 2. Phylogenetic trees based on the HA and NA coding-regions sequences of FLUBV strains using the Tamura 3-parameter method with gamma distribution 0.4. Sequences from the present study and the reference ones are labelled according to the legend on the right. The different coloured tree branches refer to the genetic groups of each lineage. Amino acid substitutions, relative to the reference sequences of every lineage, that define these genetic groups are labelled in blue. 3 C. Andrés et al. Virus Research 330 (2023) 199089 of both FLUBV/VIC and FLUBV/YAM in 1983, several reassortment events have been reported between clades and lineages (Dudas et al., 2015; Oong et al., 2015, 2017), so these episodes might play a role in determining the evolutionary changes in both lineages. While PB1, PB2 and HA segments have kept the same ancestor (FLUBV/VIC or FLUB­ V/YAM) later than the lineage divergence, the other segments have suffered several reassortment episodes like PA, NP, NA and MP amongst FLUBV/YAM and NS amongst FLUBV/VIC viruses (Dudas et al., 2015). In order to detect these reassortment events, WGS is the ideal technique to study the whole viral genome such as its genetic features or the ex­ change of segments to have a better understanding of the genetic characteristics. In addition, the usage of universal primers avoids the selection of viral variants, and thus, the results are more reliable to further studies. Herein, the detection of different inter- and intra-lineage reassortment events in FLUBV strains from 2004 to 2015 seasons in Catalonia by WGS are described. FLUBV, like FLUAV, have a variable and unpredictable circulation, with the shift of one lineage to another or their co-circulation season after season. In this study, the different FLUBV clades from both lineages are represented, which improves the study of reassortment episodes amongst all these strains. Dudas et al. (2015) reported in 2014 that PB1, PB2 and HA were the only segments to maintain separate FLUBV/VIC or FLUBV/YAM lineages in an almost 20-year study. They observed that the PB1-PB2-HA complex remained as a pure lineage, in comparison with the other segments. This could be caused because of the lack of reassortment between these segments due to their linkage disequilib­ rium since having a pure lineage of this complex might be beneficial for the whole viral fitness (Dudas et al., 2015). Nevertheless, in the present study, reassortment episodes of PB1-PB2-HA complex were observed (Fig. 3), in particular, amongst PB1 and PB2 segments, though they were detected only intra-lineages rather than inter-lineages. Thereby, it is worth to highlight these events due to the possible emergence of reas­ sortant strains which might carry different lineage segments, as the appearance of A(H1)pdm09 amongst FLUAV (Garten et al., 2009; Fowlkes et al., 2011; Antón et al., 2012). Fig. 4. Phylogenetic tree based on the NP coding-region sequences of FLUBV FLUBV/VIC and FLUBV/YAM lineages have suffered numerous strains. Sequences from the present study and the reference ones are labelled reassortment events in the past as other authors reported (Kim et al., according to the legend The reassortment event is red squared. Amino acid 2016; Dudas et al., 2015; Tewawong et al., 2015, 2017). Differences in substitutions, relative to the reference sequence of B/YAM lineage, that define phylogenetic groups between HA and NA are well known and have been the genetic group B/Phuket/3073/2013, are labelled in blue. deeply studied compared to the remaining segments due to their importance in the worldwide influenza surveillance (Antón et al., 2016). Table 1 Thus, one of the reasons to carry out this work by WGS was to study the Hospitalisation, ICU-admission and mortality data from studied patients car­ whole viral genome and detect reassortment episodes in the other seg­ rying or not reassortant strains. ments, which just with the study of HA and NA would not be possible. Patients carrying reassortant strains Yes (%) No (%) The FLUBV/YAM NP has reassorted three times since 1995, in which the last one was in 2008 (Dudas et al., 2015). In this study, the NP segment Hospitalisation Total: 22 p-value from the 2008–2009, 2010–2011 and 2012–2013 FLUBV/VIC strains Yes 6 (15) 16 (28) 0,296 No 33 (82) 40 (69) reassorted to FLUBV/YAM lineage (Fig. 4) and it corresponded to what ICU-admission Total: 7 Dudas et al. reported in 2014. These strains belonged initially to clade 1A Yes 1 (3) 6 (10) 0,297 and 1B from FLUBV/VIC lineage and then, these viruses gained No 38 (95) 50 (86) distinctive amino acid changes related to clade 3 from FLUBV/YAM Mortality Total: 5 Yes 0 (0) 5 (9) 0,081 lineage. However, no relevant clinical differences could be distinguished No 40 (100) 53 (91) between the patients carrying reassortant strains with those who did not TOTAL 40 (41) 58 (59) 98 carry. Percentages for Hospitalisation, ICU-admision and Mortality were calculated As well as the reassortant NP segment, the NS segment from one vertically, depending on the total patients carrying or not reassortant strains. 2010–2011 FLUBV/VIC strain shifted to FLUBV/YAM lineage in this study, acquiring those amino acid changes characteristic from that lineage. In addition, it gained another amino acid substitution different differences amongst hospitalisation, ICU-admission or mortality rates from the other strains (I98V, S109P, T115S, N152S and V263M), but were found between reassortant and non-reassortant strains. these amino acids were from the same group, according to the side chain, with minor differences. Thus, these mutations might be 4. Discussion compensating the reassortment into a different lineage. Though, this is a singular case (11V1231), which, as observed in Fig. 3, has shifted either The limited number of FLUBV reservoirs causes that these viruses between clades amongst the polymerase subunits, HA, NA and MP evolve through punctual mutations and, to a lesser degree, by the ex­ segments or between lineages in the NP and NS segments. However, change of segments, compared to FLUAV. However, since the divergence regarding its clinical data, no proper differences could be found, and the 4 C. Andrés et al. Virus Research 330 (2023) 199089 patient was a four-year-old child, who did not have any comorbidities, Supplementary materials only fever as the unique symptom. Influenza surveillance is based on the molecular characterisation of Supplementary material associated with this article can be found, in only the HA and NA proteins, without taking into account the other the online version, at doi:10.1016/j.virusres.2023.199089. segments, which could be misleading the emergence of novel strains carrying reassortant segments. The active implementation of the WGS in References the influenza surveillance network would improve the detection of these strains and would give a wider view of what is really circulating (WHO), World Health Organisation, 2011. WHO global influenza surveillance network. Manual For the Laboratory Diagnosis and Virological Surveillance of Influenza. worldwide. Although no clinical differences could be found amongst the WHO Library Cataloguing-in-Publication Data. patients carrying reassortant strains, further studies concerning its Antón, A., Marcos, M.A., Torner, N., Isanta, R., Camps, M., Martínez, A., Domínguez, A., possible relationship are needed. Jané, M., Jiménez de Anta, M.T., Pumarola, T., 2016. Virological surveillance of influenza and other respiratory viruses during six consecutive seasons from 2006 to In summary, several intra- and inter-lineage reassortment episodes 2012 in Catalonia, Spain. Clin. Microbiol. Infect. 22 (6), 564. https://doi.org/ were herein revealed by WGS and phylogenetic analysis. PB1-PB2-HA 10.1016/j.cmi.2016.02.007 e1-9. https://www.ncbi.nlm.nih.gov/pubmed remained in complex within lineages as reported before, although /26939538. Antón, A., Pozo, F., Niubó, J., Casas, I., Pumarola, T., 2012. Influenza A(H1N1)pdm09 some inter-clade reassortment events were detected in three strains. In virus: viral characteristics and genetic evolution. Enferm. Infecc. Microbiol. Clin. 30 addition, an important reassortment in the NP and another in the NS (Suppl 4), 10–17. https://doi.org/10.1016/S0213-005X(12)70099-X. http://www. segments were found in both lineages. Therefore, surveillance must be ncbi.nlm.nih.gov/pubmed/23116787. Bedford, T., Riley, S., Barr, I.G., Broor, S., Chadha, M., Cox, N.J., Daniels, R.S., continued due to the continue emergence of reassortant FLUBV, Gunasekaran, C.P., Hurt, A.C., Kelso, A., Klimov, A., Lewis, N.S., Li, X., McCauley, J. although they are not often, but with the implementation of WGS. W., Odagiri, T., Potdar, V., Rambaut, A., Shu, Y., Skepner, E., Smith, D.J., Suchard, M.A., Tashiro, M., Wang, D., Xu, X., Lemey, P., Russell, C.A., 2015. Global Ethical approval circulation patterns of seasonal influenza viruses vary with antigenic drift. Nature 523 (7559), 217–220. https://doi.org/10.1038/nature14460. http://www.ncbi.nlm. nih.gov/pubmed/26053121. Institutional Review Board approval (PR(AG)329/2014) was ob­ Bouvier, N.M., Palese, P., 2008. The biology of influenza viruses. Vaccine 26 (Suppl 4), tained from the Vall d’Hebron University Hospital (HUVH) Clinical D49–D53. http://www.ncbi.nlm.nih.gov/pubmed/19230160. Deyde, V.M., Gubareva, L.V., 2009. Influenza genome analysis using pyrosequencing Research Ethics Committee. method: current applications for a moving target. Expert Rev. Mol. Diagn. 9 (5), 493–509. https://doi.org/10.1586/erm.09.21. http://www.ncbi.nlm.nih.gov/pub CRediT authorship contribution statement med/19580433. Dudas, G., Bedford, T., Lycett, S., Rambaut, A., 2015. Reassortment between influenza B lineages and the emergence of a coadapted PB1-PB2-HA gene complex. Mol. Biol. Cristina Andrés: Conceptualization, Visualization, Formal analysis, Evol. 32 (1), 162–172. https://doi.org/10.1093/molbev/msu287. https://www.ncb Methodology, Writing – original draft, Writing – review & editing. i.nlm.nih.gov/pubmed/25323575. Fowlkes, A.L., Arguin, P., Biggerstaff, M.S., Gindler, J., Blau, D., Jain, S., Dhara, R., Margarita del Cuerpo: Conceptualization, Visualization, Formal anal­ McLaughlin, J., Turnipseed, E., Meyer, J.J., Louie, J.K., Siniscalchi, A., Hamilton, J. ysis, Methodology, Writing – original draft, Writing – review & editing. J., Reeves, A., Park, S.Y., Richter, D., Ritchey, M.D., Cocoros, N.M., Blythe, D., Núria Rabella: Writing – review & editing, Conceptualization, Visual­ Peters, S., Lynfield, R., Peterson, L., Anderson, J., Moore, Z., Williams, R., McHugh, L., Cruz, C., Waters, C.L., Page, S.L., McDonald, C.K., Vandermeer, M., ization, Writing – review & editing. Maria Piñana: Writing – review & Waller, K., Bandy, U., Jones, T.F., Bullion, L., Vernon, V., Lofy, K.H., Haupt, T., editing. Manuel Jesús Iglesias-Cabezas: Writing – review & editing. Finelli, L., 2011. Epidemiology of 2009 pandemic influenza A (H1N1) deaths in the Alejandra González-Sánchez: Writing – review & editing. Juliana United States, April-July 2009. Clin. Infect. Dis. 52 (Suppl 1), S60–S68. https://doi. Esperalba: Writing – review & editing. Ariadna Rando: Writing – re­ org/10.1093/cid/ciq022. https://www.ncbi.nlm.nih.gov/pubmed/21342901. Pozo, F., Lina, B., Rebelo de Andrade, H., Enouf, V., Kossyvakis, A., Broberg, E., view & editing. Maria Carmen Martín: Methodology. Francisco Daniels, R., Lackenby, A., Meijer, A., 2013. Guidance for clinical and public healt Fuentes: Methodology. Susana Rubio: Methodology. Narcís Saubi: laboratories testing influenza virus antiviral drug susceptibility in Europe. J. Clin. Writing – review & editing. Tomàs Pumarola: Conceptualization, Virol. 7. Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., Sessions, W.M., Visualization, Writing – review & editing. Andrés Antón: Conceptual­ Xu, X., Skepner, E., Deyde, V., Okomo-Adhiambo, M., Gubareva, L., Barnes, J., ization, Visualization, Writing – review & editing. Smith, C.B., Emery, S.L., Hillman, M.J., Rivailler, P., Smagala, J., de Graaf, M., Burke, D.F., Fouchier, R.A., Pappas, C., Alpuche-Aranda, C.M., López-Gatell, H., Olivera, H., López, I., Myers, C.A., Faix, D., Blair, P.J., Yu, C., Keene, K.M., Dotson, P. Declaration of Competing Interest D., Boxrud, D., Sambol, A.R., Abid, S.H., St George, K., Bannerman, T., Moore, A.L., Stringer, D.J., Blevins, P., Demmler-Harrison, G.J., Ginsberg, M., Kriner, P., The authors declare no conflicts of interest. Waterman, S., Smole, S., Guevara, H.F., Belongia, E.A., Clark, P.A., Beatrice, S.T., Donis, R., Katz, J., Finelli, L., Bridges, C.B., Shaw, M., Jernigan, D.B., Uyeki, T.M., Smith, D.J., Klimov, A.I., Cox, N.J., 2009. Antigenic and genetic characteristics of Data availability swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325 (5937), 197–201. https://doi.org/10.1126/science.1176225. http://www.ncbi.nlm. nih.gov/pubmed/19465683. Data will be made available on request. Kim, J.I., Lee, I., Park, S., Bae, J.Y., Yoo, K., Lemey, P., Park, M.S., Song, J.W., Kee, S.H., Song, K.J., 2016. Reassortment compatibility between PB1, PB2, and HA genes of the two influenza B virus lineages in mammalian cells. Sci. Rep. 6, 27480. https://doi. Acknowledgements org/10.1038/srep27480. https://www.ncbi.nlm.nih.gov/pubmed/27270757. Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Braverman, M.S., Chen, Y.J., Chen, Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., This study was supported by the Spanish Ministry of Economy and Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L., Competitiveness, Fondo de Investigación Sanitaria (Grants FIS PI08/ Jarvie, T.P., Jirage, K.B., Kim, J.B., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz, S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., 0118, FIS PI11/01864, FIS PI14/01838) del Instituto de Salud Carlos III, McKenna, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., co-financed by the European Development Regional Fund (ERDF) "A Ronan, M.T., Roth, G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., way to achieve Europe", Spanish Network for Research in Infectious Tartaro, K.R., Tomasz, A., Vogt, K.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P., Begley, R.F., Rothberg, J.M., 2005. Genome sequencing in Diseases (REIPI RD16/0016/0003), and by CIBER -Consorcio Centro de microfabricated high-density picolitre reactors. Nature 437 (7057), 376–380. Investigación Biomédica en Red- (CB 2021) del Instituto de Salud Carlos https://doi.org/10.1038/nature03959. http://www.ncbi.nlm.nih.gov/pubmed III, Ministerio de Ciencia e Innovación and Unión Europea – /16056220. Medina, R.A., García-Sastre, A., 2011. Influenza A viruses: new research developments. NextGenerationEU. Nat. Rev. Microbiol. 9 (8), 590–603. https://doi.org/10.1038/nrmicro2613. http ://www.ncbi.nlm.nih.gov/pubmed/21747392. Nguyen, H.T., Fry, A.M., Gubareva, L.V., 2012. Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods. Antivir. Ther. 17 (1 Pt B), 5 C. Andrés et al. Virus Research 330 (2023) 199089 159–173. https://doi.org/10.3851/IMP2067. http://www.ncbi.nlm.nih.gov/pub Steinhauer, D.A., 1999. Role of hemagglutinin cleavage for the pathogenicity of med/22311680. influenza virus. Virology 258 (1), 1–20. https://doi.org/10.1006/viro.1999.9716. Oong, X.Y., Ng, K.T., Lam, T.T., Pang, Y.K., Chan, K.G., Hanafi, N.S., Kamarulzaman, A., http://www.ncbi.nlm.nih.gov/pubmed/10329563. Tee, K.K., 2015. Epidemiological and evolutionary dynamics of influenza B viruses in Steinhauer, D.A., Skehel, J.J., 2002. Genetics of influenza viruses. Annu. Rev. Genet. 36, Malaysia, 2012-2014. PLoS One 10 (8), e0136254. https://doi.org/10.1371/journal. 305–332. https://doi.org/10.1146/annurev.genet.36.052402.152757. http://www. pone.0136254. https://www.ncbi.nlm.nih.gov/pubmed/26313754. ncbi.nlm.nih.gov/pubmed/12429695. Oong, X.Y., Ng, K.T., Tan, J.L., Chan, K.G., Kamarulzaman, A., Chan, Y.F., Sam, I.C., Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: Tee, K.K., 2017. Whole-genome phylogenetic analysis of Influenza B/Phuket/3073/ molecular evolutionary genetics analysis using maximum likelihood, evolutionary 2013-like viruses and unique reassortants detected in Malaysia between 2012 and distance, and maximum parsimony methods. Mol. Biol. Evol. 28 (10), 2731–2739. 2014. PLoS One 12 (1), e0170610. https://doi.org/10.1371/journal.pone.0170610. https://doi.org/10.1093/molbev/msr121. http://www.ncbi.nlm.nih.gov/pubmed https://www.ncbi.nlm.nih.gov/pubmed/28129386. /21546353. WHO Collaborating Centre for Reference and Research on Influenza Chinese National Tewawong, N., Suntronwong, N., Korkong, S., Theamboonlers, A., Vongpunsawad, S., Influenza Centre. National Institute for Viral Disease Control and. 155 Changbai Poovorawan, Y., 2017. Evidence for influenza B virus lineage shifts and reassortants Roadm changping district. 102206 Beijing China: Chinese centre for disease control circulating in Thailand in 2014-2016. Infect. Genet. Evol. 47, 35–40. https://doi. and prevention. org/10.1016/j.meegid.2016.11.010. https://www.ncbi.nlm.nih.gov/pubmed Instituto Carlos III, "Sistema de Vigilancia de la Gripe en España." Available at: http:// /27845268. vgripe.isciii.es/gripe/inicio.do. Tewawong, N., Suwannakarn, K., Prachayangprecha, S., Korkong, S., Vichiwattana, P., Raca, G., Jackson, C., Warman, B., Bair, T., Schimmenti, L.A., 2010. Next generation Vongpunsawad, S., Poovorawan, Y., 2015. Molecular epidemiology and sequencing in research and diagnostics of ocular birth defects. Mol. Genet. Metab. phylogenetic analyses of influenza B virus in Thailand during 2010 to 2014. PLoS 100 (2), 184–192. https://doi.org/10.1016/j.ymgme.2010.03.004. http://www.ncb One 10 (1), e0116302. https://doi.org/10.1371/journal.pone.0116302. https i.nlm.nih.gov/pubmed/20359920. ://www.ncbi.nlm.nih.gov/pubmed/25602617. Schrauwen, E.J., de Graaf, M., Herfst, S., Rimmelzwaan, G.F., Osterhaus, A.D., Zhou, B., Wentworth, D.E., 2012. Influenza A virus molecular virology techniques. Fouchier, R.A., 2014. Determinants of virulence of influenza A virus. Eur. J. Clin. Methods Mol. Biol. 865, 175–192. https://doi.org/10.1007/978-1-61779-621-0_11. Microbiol. Infect. Dis. 33 (4), 479–490. https://doi.org/10.1007/s10096-013-1984- http://www.ncbi.nlm.nih.gov/pubmed/22528160. 8. http://www.ncbi.nlm.nih.gov/pubmed/24078062. 6