Rapid communications Case of seasonal reassortant A(H1N2) influenza virus infection, the , March 2018

Adam Meijer1,6, Corien M Swaan¹, Martin Voerknecht², Edin Jusic¹, Sharon van den Brink¹, Lisa A Wijsman¹, Bettie CG Voordouw1,6, Gé A Donker³, Jacqueline Sleven⁴, Wendelien W Dorigo-Zetsma⁵, Sanela Svraka⁵, Michiel van Boven¹, Manon R Haverkate¹, Aura Timen¹, Jaap T van Dissel¹, Marion PG Koopmans6, Theo M Bestebroer⁶, Ron AM Fouchier⁶ 1. Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, the Netherlands 2. General practitioner participating in the Primary Care Database sentinel surveillance coordinated by NIVEL Netherlands institute for health services research, , the Netherlands 3. Coordinator NIVEL Primary Care Database sentinel surveillance, NIVEL Netherlands institute for health services research, Utrecht, the Netherlands 4. Municipal Health Services ‘ en Vechtstreek’, Bussum, the Netherlands 5. Central Bacteriology and Serology Laboratory, Tergooi Hospitals, Hilversum, the Netherlands 6. Department Viroscience, Erasmus University Medical Center, Rotterdam, the Netherlands Correspondence: Adam Meijer ([email protected])

Citation style for this article: Meijer Adam, Swaan Corien M, Voerknecht Martin, Jusic Edin, van den Brink Sharon, Wijsman Lisa A, Voordouw Bettie CG, Donker Gé A, Sleven Jacqueline, Dorigo- Zetsma Wendelien W, Svraka Sanela, van Boven Michiel, Haverkate Manon R, Timen Aura, van Dissel Jaap T, Koopmans Marion PG, Bestebroer Theo M, Fouchier Ron AM. Case of seasonal reassortant A(H1N2) influenza virus infection, the Netherlands, March 2018. Euro Surveill. 2018;23(15):pii=18-00160. https://doi. org/10.2807/1560-7917.ES.2018.23.15.18-00160

Article submitted on 29 Mar 2018 / accepted on 12 Apr 2018 / published on 12 Apr 2018

A seasonal reassortant A(H1N2) influenza virus har- NIC from a systematic selection of patients presenting bouring genome segments from seasonal influenza with ILI or another acute respiratory infection (ARI). viruses A(H1N1)pdm09 (HA and NS) and A(H3N2) (PB2, Diagnostic GP and hospital laboratories forward influ- PB1, PA, NP, NA and M) was identified in March 2018 enza virus-positive specimens for further characterisa- in a 19-months-old patient with influenza-like illness tion to the NIC. All influenza viruses are subtyped by (ILI) who presented to a general practitioner partici- real-time RT-PCR and a subset is further characterised pating in the routine sentinel surveillance of ILI in by sequencing, virus isolation and haemagglutination the Netherlands. The patient recovered fully. Further inhibition and neuraminidase inhibition assays. epidemiological and virological investigation did not reveal additional cases. Case detection In the Netherlands, the 2017/18 influenza epidemic has In the routine sentinel general practitioner (GP) surveil- been dominated by influenza viruses of the B/Yamagata lance of influenza in the Netherlands, a new seasonal lineage (Figure 1). Since the beginning of March, both reassortant A(H1N2) influenza virus was identified in A(H3N2) and A(H1N1)pdm09 influenza viruses have natural human infection in March 2018. Here we report been increasingly detected. By week 11/2018 the inci- the case detection, molecular characterisation of the dence of ILI had been above the epidemic threshold for virus and the results of follow-up epidemiological and 14 weeks. In week 10 on 5 March, a sentinel GP was virological investigation. consulted for a 19 months-old patient with acute onset of ILI on 2 March characterised by fever, malaise, sore Influenza surveillance in the Netherlands throat, coughing, shortness of breath, rhinorrhoea and In the Netherlands, influenza surveillance is a collabo- diarrhoea. rative effort of the National Influenza Centre (NIC) (con- sisting of the National Institute for Public Health and Virus characterisation the Environment (RIVM) in Bilthoven and the Erasmus MinION nanopore whole genome sequencing confirmed University Medical Centre (Erasmus MC) in Rotterdam) on 18 March that the patient was infected by a seasonal and the NIVEL Primary Care Database sentinel sur- reassortant A(H1N2) influenza virus, with the HA and veillance in Utrecht [1,2]. The GPs participating in the non-structural (NS) segments derived from seasonal sentinel surveillance report weekly the incidence of A(H1N1)pdm09 influenza virus and all other segments influenza-like illness (ILI) according to the case defi- (polymerase components PB2, PB1 and PA, nucleopro- nition by Pel (sudden onset of symptoms, fever of at tein (NP), NA and matrix (M)) from seasonal influenza least 38 °C and at least one of the following: cough, A(H3N2) virus. The genome sequence of influenza virus rhinorrhoea, sore throat, frontal headache, retroster- A/Netherlands/10407/2018 H1N2 is available from the nal pain, myalgia) [3]. They take nose and throat swabs GISAID EpiFlu database (EPI_ISL_304183). for influenza virus detection by real-time RT-PCR at the

www.eurosurveillance.org 1 Figure 1 ILI incidence and number of specimens from patients with ILI collected by sentinel general practitioners in which influenza virus was detected, the Netherlands, 21 March 2018 (n = 395)

60 180 Influenza B/Yam Influenza B/Vic 160

50 ILI incidence/100,000 Influenza A(H1N1)pdm09 140 Influenza A(H3N2) 40 Influenza A(H1N2) 120 ILI incidence 100 30 Epidemic threshold 80 inhabitants Detected viruses 20 60

40 10 20

0 0 40 41 42 43 44 45 46 47 48 49 50 51 52 1 2 3 4 5 6 7 8 9 10 11

2017 2018 Year and week of specimen collection

ILI: influenza-like illness.

Left axis: influenza virus-positive specimens from patients with ILI; right axis: ILI incidence per 100,000 inhabitants. Data are presented by week of specimen collection (ISO week numbering).

The HA and NA segments were highly similar to those of influenza A(H3N2) viruses isolated during the 2017/18 the 2018 A(H1N1)pdm09 and A(H3N2) influenza viruses season in the sentinel surveillance in the Netherlands in the Netherlands detected through the sentinel sur- that had an NA protein sequence similar to that of the veillance (Figure 2 and Figure 3). influenza A(H1N2) virus were tested for susceptibil- ity to oseltamivir and zanamivir in the neuraminidase Because no full genomes of other 2018 Dutch influ- inhibition assay as described [4]. All eight were inhib- enza viruses were available, the other segments were ited normally by oseltamivir (median IC50: 0.20; range: compared with those from 100 A(H1N1)pdm09 and 200 0.17–0.28 nM) and zanamivir (median IC50: 0.31; range: A(H3N2) influenza viruses collected in 2018 from coun- 0.21–0.38 nM). Together, these findings indicate that tries in the World Health Organization European Region. this influenza A(H1N2) virus was highly likely to be The NS segment was closely related to NS segments sensitive to oseltamivir and zanamivir. As the HA pro- of 2018 A(H1N1)pdm09 viruses (Figure 4) and the PB2, tein sequence was similar to that of recent influenza PB1, PA, NP and M segments were closely related to A(H1N1)pdm09 viruses and interim 2017/18 season the corresponding segments of 2018 A(H3N2) viruses estimates of vaccine effectiveness for Europe showed (Supplement 1). 55–68% effectiveness in preventing laboratory-con- firmed influenza A(H1N1)pdm09 virus infection [5], we We observed a number of nucleotide substitutions consider that the currently used vaccine for the north- in the segments of this influenza A(H1N2) virus com- ern hemisphere offers protection against the influenza pared with the consensus sequences of other 2018 A(H1N2) virus. viruses. However, the majority of them were silent; unique amino acid substitutions were only identified in Unfortunately, attempts to isolate the A(H1N2) virus the M2 protein (V68I and D88N) and in the NA protein in cell culture and embryonated chicken eggs were (I30L). M2 harboured S31N, similar to all recent influ- unsuccessful, probably because of the low viral enza A(H3N2) viruses, causing resistance against the load in the clinical specimen at the time of sampling adamantane class of influenza antiviral drugs. In NA, as shown by a Ct value of 29.2 in the RT-PCR target- no amino acid substitutions were detected that have ing M. Experimental reassortant influenza virus with been associated previously with reduced sensitivity HA from A(H1N1)pdm09 influenza virus and the other to NA inhibitors, again similar to the vast majority of segments from influenza A(H3N2) virus showed only recent influenza A(H3N2) viruses. Furthermore, eight slightly attenuated growth characteristics in human

2 www.eurosurveillance.org Figure 2 Phylogenetic analysis of the influenza A(H1N2) virus haemagglutinin genome segment in the context of a reference influenza virus data seta and recent influenza viruses from the Netherlands

52 A/Netherlands/10178/2018 71 86 A/Netherlands/10218/2018 A/Netherlands/10285/2018 V250A 99 A/Netherlands/10142/2018 A/Netherlands/10407/2018 28 A/Netherlands/10172/2018 A/Paris/1289/2017 A/Netherlands/10081/2018 35 A/Netherlands/10238/2018 98 A/Netherlands/10093/2018 A/Netherlands/10371/2018 A/Netherlands/3546/2017 47 A/Netherlands/10378/2018 35 A/Netherlands/10303/2018 90 S183P 99 A/Netherlands/10456/2018 100 A/Netherlands/10273/2018 P137S, N124T A/Netherlands/10278/2018 53 A/Netherlands/10066/2018 55 A/Netherlands/10417/2018 T120A A/Netherlands/10146/2018 49 92 S164T 97 A/Netherlands/10145/2018 A/Netherlands/10039/2018 98 A/Netherlands/10424/2018 6B.1 S183P, E235D, N260D, V193A 40 A/Netherlands/10442/2018 84 A/Netherlands/3523/2017 99 A/Netherlands/10010/2018 64 A/Netherlands/10360/2018 94 A/Netherlands/10374/2018 A/Netherlands/10208/2018 S74R, I295V 95 A/Hong Kong/2214/2017 33 A/Hong Kong/2245/2017 A/Norway/2146/2017 I166V, S183P, R205K, G155E A/Torino/93/2017 98 A/Netherlands/3792/2016 99 90 A/Netherlands/502/2017 A/Netherlands/050/2016 51 A/Bayern/81/2017 48 39 A/Slovenia/2903/2015 42 A/Dakar/09/2017 A/Thuringen/130/2017 S84N, S162N (+CHO), I216T 99 I324V95 93 A/Hong Kong/2111/2017 A/Michigan/45/2015 VACCINE VIRUS V152T, V173I, 30 A/OSAKA/170/2017 E164G, D174E A215G 97 A/Hong Kong/2199/2017 100 A/Jilin-Ningjiang/SWL1297/2017 K163Q, A256T 66 A/Israel/Q-504/2015 A/Ghana/FS-16-0249/2016 K283E, E172K 97 91 100 A/Netherlands/1037/2015 D97N, S185T 44 A/South Africa/3626/2013 A/Dakar/04/2014 27 A/Hong Kong/5659/2012 S124N 29 45 A/St. Petersburg/27/2011 28 A/Czech Republic/32/2011 56 A/St. Petersburg/100/2011 52 A/Norway/120/2013 E47K 35 A/Dakar/20/2012 A/Astrakhan/1/2011 S203T 93 A/Christchurch/16/2010 I295V A/Hong Kong/3934/2011 48 A/Hong Kong/2212/2010 A/Bayern/69/2009 A/California/7/2009 VACCINE VIRUS

0.005

HA: haemagglutinin. a Reference dataset provided by the World Health Organization Collaborating Centre London through the European Centre for Disease prevention and Control for seasonal phylogenetic analysis. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences retrieved from GISAID’s Epiflu database that were used in this analysis (Supplement 2).

The evolutionary history was inferred using the neighbour-joining method [12]. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches [13]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method [14] and are in the units of the number of base substitutions per site. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA7 [15]. Common amino acid substitutions (black: HA1, orange: HA2) compared with the root virus are shown at branches that characterise the cluster of viruses after that branch. HA of the influenza A(H1N2) virus (red dot) clustered with HA of recent 2018 Dutch clade 6B.1 A(H1N1)pdm09 influenza viruses characterised by common amino acid substitution V250A.

www.eurosurveillance.org 3 Figure 3 Phylogenetic analysis of the influenza A(H1N2) virus neuraminidase genome segment in the context of a reference influenza virus data seta and recent influenza viruses from the Netherlands

I392M 66 A/Netherlands/10393/2018 A/Netherlands/10436/2018 A/Netherlands/10415/2018 48 A/Netherlands/10407/2018 [I30L] A/Netherlands/10358/2018 46 A/Netherlands/10461/2018 A/Netherlands/3535/2017 47 A/Netherlands/10279/2018 A/Netherlands/10379/2018 A/Netherlands/10013/2018 33 A/Netherlands/10159/2018 HA clade 3C.2a 23 A/Netherlands/10193/2018 A/Netherlands/10419/2018 41 A/Netherlands/10391/2018 S334R A/Netherlands/10160/2018 34 A/Netherlands/10174/2018 A/Netherlands/3521/2017 P386S 42 64 A/Netherlands/10018/2018 22 A/Netherlands/10214/2018 I176M 31 A/Netherlands/10077/2018 A/Qatar/10-VI-17-0001178/2017 A/Hong Kong/3090/2017 N329S 34 13 A/Greece/4/2017 99 A/Netherlands/3768/2016 A/Netherlands/2009/2017 A/Norway/3806/2016 57 A/Hong Kong/3163/2017 71 A/Netherlands/10377/2018 K220N, 87 A/Netherlands/10260/2018 26 80 A/Netherlands/10438/2018 V303I P126L 63 A/Netherlands/10042/2018 A/Netherlands/10297/2018 L338F 23 A/Hong Kong/2286/2017 13 A/Netherlands/10406/2018 10 A/Netherlands/10373/2018 25 S315R, E344K, G346D 98 A/Netherlands/10402/2018 A/Bolzano/7/2016 A/Saudi Arabia/252773/2017 A/Oman/2585/2016 A/Singapore/Infimh-16-0019/2016 VACCINE VIRUS D339N 37 31 A/Hong Kong/3042/2017 20 A/Antananarivo/2398/2017 A/Netherlands/2653/2017 L140I 57 43 34 A/Netherlands/10029/2018 A/Hong Kong/3160/2017 A/Hong Kong/3115/2017 68 I380V 83 79 A/Lithuania/6165/2017 A/Hong Kong/3156/2017 I176M 99 A/Victoria/10/2017 S245N, S247T, G93D, D339N, A/Cote D Ivoire/544/2016 T267K, P468H 99 A/Hong Kong/3105/2017 H468L 99 A/Hong Kong/3125/2017 N161S 58 A/Norway/2603/2017 69 A/Antsirabe/2961/2017 94 45 V149A, G346V, D356E 77 A/Analavory/2821/2017 A/Netherlands/3782/2016 A/Hong Kong/4801/2014 VACCINE VIRUS E221D 94 A/Florida/48/2017 I392T 95 A/Switzerland/9715293/2013 VACCINE VIRUS 75 60 A/Stockholm/6/2014 I26V, M51V, S331R, A/Iceland/69/2017 L338S, D463N 99 A/Netherlands/2329/2017 A/Samara/73/2013 A/Texas/50/2012 VACCINE VIRUS

0.002

NA: neuraminidase; HA: haemagglutinin. a Reference dataset provided by the World Health Organization Collaborating Centre London through the European Centre for Disease prevention and Control for seasonal phylogenetic analysis. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences retrieved from GISAID’s Epiflu database that were used in this analysis (Supplement 2).

Phylogenetic analysis was carried out as described in the Figure 2 legend. Common amino acid substitutions compared with the root virus are shown at branches that characterise the cluster of viruses after that branch. The NA of the influenza A(H1N2) virus (red dot) clustered with NA of recent 2018 Dutch influenza A(H3N2) viruses characterised by common amino acid substitutions I176M and P386S. The NA of influenza A(H1N2) virus is derived from recent HA clade 3C.2a A(H3N2) influenza virus and has in addition the I30L amino acid substitution.

4 www.eurosurveillance.org Figure 4 tots ca 2.5–4-years-old) were reported ill from mid- Phylogenetic analysis of the influenza A(H1N2) virus NS January through February. We do not know how many genome segment in the context of recent vaccine strains and selected seasonal influenza viruses representative of of these children had ILI or laboratory-confirmed those circulating in the Netherlands in 2016 and 2017 and influenza. However, given the fact that the influenza in other European countries in January and February epidemic was at its peak at that time, it is likely that 2018a some of these children had influenza. From the end of February, children were reported ill only sporadically. A/Centre/043/2018 A/Netherlands/10407/2018 In the sentinel surveillance and in the GP and hospital A/England/130/2018 66 A/Champagne Ardenne/876/2018 laboratory surveillance of influenza virus detections, A/England/80420871/2018 A/Paris/290/2018 no other influenza A(H1N2) cases were detected up to A/Champagne Ardenne/895/2018 A/Saint-Petersburg/RII-7/2018 20 March. The main diagnostic laboratory in the region A/Denmark/2/2018 29 65 A/Paris/322/2018 was contacted to submit all type A influenza virus-pos- A/Denmark/3/2018 A/England/80760594/2018 itive specimens collected in March for subtyping; no 36 A/England/81/2018 A/Sweden/10/2018 reassortant viruses were identified among specimens A/Sweden/9/2018 35 A/Netherlands/3523/2017 from 24 patients; one of them was 5-weeks-old, while NS1: D2E, E55K, L90I, I123V, 39 A/Moscow/3/2018 E125D, K131E, N205S A/Denmark/17/2018 the rest had a median age of 73 years (range: 31–91 NS2: D2E, N29S, T48A, M83I 78 A/England/80/2018 A/Michigan/45/2015 VACCINE VIRUS years). A/Netherlands/3792/2016 NS1: K64E; NS2:G154R 98 A/Netherlands/502/2017 A/California/07/2009 VACCINE VIRUS Discussion 0.005 Human seasonal reassortant A(H1N2) influenza viruses harbouring the H1 from human seasonal A(H1N1) and NS: non-structural protein. seven segments from human seasonal influenza A(H3N2) virus have been detected before during an out- a Sequences were selected from ca 100 influenza A(H1N1)pdm09 break in China in 1988/89 and from 2000 to 2003 start- viruses after phylogenetic analysis. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences ing in Asia and spreading worldwide [7-10]. However, retrieved from GISAID’s Epiflu database that were used in this the latter failed to co-circulate in the long term or to analysis (Supplement 2). replace the A(H1N1) or A(H3N2) influenza viruses that Phylogenetic analysis was carried out as described in the Figure 2 were in circulation at the time. A sporadic case of sea- legend. Red dot: influenza A(H1N2) virus described in this study. sonal reassortant A(H1N2) influenza virus harbouring the H1 from human influenza A(H1N1)pdm09 virus and the other seven segments from human seasonal influ- enza A(H3N2) virus was detected in India during the influenza pandemic in 2009 [11]. In a mixture of reas- tracheobronchial epithelial (HTBE) cells with reduced sortants generated by in vitro coinfection with sea- amount of virus produced early in infection compared sonal A(H1N1)pdm09 and A(H3N2) influenza viruses, to parental A(H3N2) virus [6], suggesting that highly sporadic virus with the current 2:6 constellation of likely the current reassortant influenza A(H1N2) virus genome segments was detected [6]. Furthermore, in is capable of substantial growth. However, combined a transmission experiment with guinea pigs infected with the NS segment of influenza A(H1N1)pdm09 virus with this mixture of reassortant viruses, the influenza this effect might have an impact on the success of A(H1N2) virus with the NS segment from influenza isolation of the current reassortant influenza A(H1N2) A(H1N1)pdm09 virus was not detected in contact ani- virus, especially when combined with low viral load mals [6]. in the clinical specimen. Further phenotypic charac- terisation of the virus awaits rescue of the virus using In conclusion, this is the first time a seasonal reassor- reverse genetics techniques. tant A(H1N2) influenza virus with HA and NS from influ- enza A(H1N1)pdm09 virus and the other six segments Epidemiological and virological follow-up of influenza A(H3N2) virus is reported in a human natu- The patient consulted the GP again on 8 March with ral infection. The patient recovered fully and there is otitis media for which antibiotic treatment (amoxicil- no evidence of extended spreading of the reassortant lin 40 mg/kg 3 daily doses for 7 days) was prescribed. virus in humans. Published evidence indicates lim- Within a few days, the patient made a full recovery. ited capacity for transmission of seasonal reassortant Contact investigation revealed that the parents had A(H1N2) influenza viruses [6,11]. However, laboratories, been ill in the last two weeks of February with symp- especially national influenza centres, should stay vigi- toms of gastro-intestinal infection before onset of dis- lant for reassortant human seasonal influenza viruses ease in the index case. No laboratory diagnostics was with mixed genome segments from A(H1N1)pdm09 and performed. None of the parents had close contacts with A(H3N2) influenza viruses. animals before the disease episode. The patient visited day care a couple of days per week; after onset of dis- ease, the patient remained at home. In the day care centre, more than 20 children from different groups (babies 0–1-year-old, toddlers 1–ca 2.5-years-old and

www.eurosurveillance.org 5 Acknowledgements 8. Guo YJ, Xu XY, Cox NJ. Human influenza A (H1N2) viruses isolated from China. J Gen Virol. 1992;73(Pt 2):383-7. https:// We gratefully acknowledge the authors, originating and doi.org/10.1099/0022-1317-73-2-383 PMID: 1538194 submitting laboratories of the sequences retrieved from 9. Gregory V, Bennett M, Orkhan MH, Al Hajjar S, Varsano N, GISAID’s Epiflu database that were used in this analysis Mendelson E, et al. Emergence of influenza A H1N2 reassortant viruses in the human population during 2001. Virology. (Supplement 2). 2002;300(1):1-7. https://doi.org/10.1006/viro.2002.1513 PMID: 12202200 RF, TB and MK received funding through European Union’s 10. Chen MJ, La T, Zhao P, Tam JS, Rappaport R, Cheng SM. Genetic Horizon 2020 grant COMPARE (643476). AM received funding and phylogenetic analysis of multi-continent human influenza through European Union’s Horizon 2020 grant I-MOVE-plus A(H1N2) reassortant viruses isolated in 2001 through 2003. Virus Res. 2006;122(1-2):200-5. https://doi.org/10.1016/j. (634446) and ECDC Specific Contracts no 7 – ECD.6594, no virusres.2006.07.010 PMID: 16971014 8 – ECD.6646 and no 9 – ECD.7946 implementing activities 11. Mukherjee TR, Agrawal AS, Chakrabarti S, Chawla-Sarkar to Framework Contract no ECDC/2014/026 I-MOVE. M. Full genomic analysis of an influenza A (H1N2) virus identified during 2009 pandemic in Eastern India: evidence of We thank Pieter Overduin and Rachel Scheuer for virus se- reassortment event between co-circulating A(H1N1)pdm09 and quencing and Thierry Janssens for technical assistance A/Brisbane/10/2007-like H3N2 strains. Virol J. 2012;9(1):233. https://doi.org/10.1186/1743-422X-9-233 PMID: 23057530 in the phylogenetic analysis and amino acid substitution 12. Saitou N, Nei M. The neighbor-joining method: a new annotation. method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406-25. PMID: 3447015 The parents of the case provided informed consent for fur- 13. Felsenstein J. Confidence limits on phylogenies: an approach ther investigation and to publish the case. using the bootstrap. Evolution. 1985;39(4):783-91. https://doi. org/10.1111/j.1558-5646.1985.tb00420.x PMID: 28561359 14. Jukes TH, Cantor CR. Evolution of protein molecules. In Munro HN, editor, Mammalian Protein Metabolism, pp. 21-132, 1969, Conflict of interest Academic Press, New York. 15. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary None declared. Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870-4. https://doi.org/10.1093/molbev/ msw054 PMID: 27004904 Authors’ contributions

AM, GD and RF designed the study. AM carried out the analy- License and copyright sis of the data and wrote the manuscript. MV performed primary clinical diagnostics and contributed to the follow- This is an open-access article distributed under the terms of up. EJ, SvdB, LW and TB performed diagnostic tests and the Creative Commons Attribution (CC BY 4.0) Licence. You sequencing of the virus. JS, CS, BV and MH carried out the may share and adapt the material, but must give appropriate follow-up and together with AM, RF, MvB, AT, JvD and MK credit to the source, provide a link to the licence, and indi- formed the response team. WD-Z and SS provided regional cate if changes were made. diagnostic data and viruses. All authors critically reviewed the manuscript and provided valuable comments. This article is copyright of the authors, 2018.

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