# Co-circulation of multiple enterovirus D68 subclades, including a novel B3 cluster, across Europe in a season of expected low prevalence, 2019/20

Sofie Elisabeth Midgley¹, Kimberley Benschop², Robert Dyrdak³,⁴, Audrey Mirand⁵,⁶, Jean-Luc Bailly⁶, Sibylle Bierbaum⁷, Stefan Buderus⁸, Sindy Böttcher⁹, Anna-Maria Eis-Hübinger¹⁰, Mario Hönemann¹¹, Veronika Vorobieva Jensen¹, Ulla Birgitte Hartling¹², Cécile Henquell⁵, Marcus Panning⁷, Marianne Kragh Thomsen¹³, Emma B Hodcroft¹⁴,¹⁵, Adam Meijer²

1. Department for Virus and Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark

2. Centre for Infectious Disease Research, Diagnostics and Laboratory Surveillance, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands

3. Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden

4. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

5. CHU Clermont-Ferrand, Centre National de Référence des entérovirus et parechovirus - Laboratoire Associé, Laboratoire de Virologie, Clermont-Ferrand, France

6. Université Clermont Auvergne, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France

7. Institute of Virology, University of Freiburg, Freiburg, Germany

8. Department of General Pediatrics, St.-Marien-Hospital, Bonn, Germany

9. National Reference Center for Poliomyelitis and Enteroviruses, Robert Koch-Institute, Berlin, Germany

10. Institute of Virology, University of Bonn Medical Center, Bonn, Germany

11. Institute of Virology, University of Leipzig, Leipzig, Germany

12. Department of Paediatrics, Aarhus University Hospital, Aarhus, Denmark

13. Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark

14. Biozentrum, University of Basel, Basel, Switzerland

15. Swiss Institute of Bioinformatics, Basel, Switzerland

Correspondence: Sofie Elisabeth Midgley (soi@ssi.dk)

**Citation style for this article:**

Midgley Sofie Elisabeth, Benschop Kimberley, Dyrdak Robert, Mirand Audrey, Bailly Jean-Luc, Bierbaum Sibylle, Buderus Stefan, Böttcher Sindy, Eis-Hübinger Anna-Maria, Hönemann Mario, Jensen Veronika Vorobieva, Hartling Ulla Birgitte, Henquell Cécile, Panning Marcus, Thomsen Marianne Kragh, Hodcroft Emma B, Meijer Adam. Co-circulation of multiple enterovirus D68 subclades, including a novel B3 cluster, across Europe in a season of expected low prevalence, 2019/20. Euro Surveill. 2020;25(2):pil=1900749. https://doi.org/10.2807/1560-7917.ES.2020.25.2.1900749

Article submitted on 07 Jan 2020 / accepted on 16 Jan 2020 / published on 16 Jan 2020

Enterovirus D68 (EV-D68) was detected in 93 patients from five European countries between 1 January 2019 and 15 January 2020, a season with expected low circulation. Patients were primarily children (n=67, median age: 4 years), 59 patients required hospitalisation and five had severe neurologic manifestations. Phylogenetic analysis revealed two clusters in the B3 subclade and subclade A2/D. This circulation of EV-D68 associated with neurological manifestations stresses the importance of surveillance and diagnostics beyond expected peak years.

Enterovirus D68 (EV-D68) is primarily a respiratory virus. Previously published data have suggested circulation with a biennial epidemic cycle in Europe and North America [1-3], but surveillance is not consistent. The Danish enterovirus surveillance detected two cases of EV-D68 infection in August 2019 and a further case in early September. Colleagues in other European countries were contacted, and France, Germany, the Netherlands and Sweden responded that they had also seen cases. Here we report on the start of seasonal circulation of EV-D68 in five European countries, a circulation which is still ongoing with further cases detected since the initial submission of this report.

## Epidemiological trend and description of cases

A total of 93 EV-D68 infections were reported between 1 January 2019 and 15 January 2020 (Denmark n=21, France n=6, Germany n=36, the Netherlands n=19, Sweden n=11). Epidemiological and clinical information was collected for cases where possible (Table 1). As surveillance samples and data are collected retrospectively, data for December 2019 and January 2020 are not complete.

Cases were identified through enterovirus surveillance (n=31), rhinovirus surveillance (n=3), influenza and respiratory infection community surveillance (n=12) or hospital-based diagnostics of respiratory infections (n=47). Following diagnostic testing of respiratory samples for enterovirus and/or rhinovirus using commercial or in-house assays, EV-D68 cases were identified by either EV-D68-specific real-time PCR or partial sequencing of the VP1 and/or VP4/VP2 region of the genome [9-11]. Most of the cases were identified during the usual enterovirus season starting in late summer (August n=5, September n=14, October n=17, November n=36 and December n=15; Figure 2).

<figure><img src="image_1.png" /></figure>
**FIGURE 1**

Yearly variation in detection of enterovirus D68 in five
European countries, 1 January 2007–15 January 2020
(n = 1,191)

<figure><img src="image_2.png" /></figure>

For Sweden, cases in 2014 include only those confirmed by sequencing, and only a limited number of samples were screened (n=30). Data from Germany represent five sites for the period 2013 to 2018 (Bonn, Düsseldorf, Freiburg, Leipzig and Würzburg), but only three for 2019 and 2020 (Bonn, Freiburg and Leipzig).

Of the Dutch cases, two became ill in Turkey and were
diagnosed with EV-D68 after being hospitalised for res-
piratory support upon their return to the Netherlands.
One Swedish patient had a recent travel history to East
Asia and one French patient had travelled to Portugal.
Most of the cases were children (n=67), with a median
age of 4 years (range: 16 days–89 years). Forty-six
patients were female.

Clinical manifestations of cases

Five patients, from Denmark, France and Germany, pre-
sented with severe neurological symptoms. Of these,
one was a 1 year-old, previously healthy boy who pre-
sented with acute flaccid myelitis (AFM) following a
febrile respiratory infection. The paralysis was asym-
metric, included all four limbs and the torso, with severe
paraesthesia in affected limbs. The second patient was
a 15-month-old boy who presented with AFM following
a digestive prodromal illness. The third patient was a
29-year-old woman who presented with cranial nerve
palsy. She underwent a caesarean section in week 40
of pregnancy because of the acute neurological symp-
toms. The fourth patient was a 6-year-old girl who
presented with paralysis of both legs and the bladder.
The fifth patient was a 16-year-old girl who presented
with loss of balance and coordination, double vision
and loss of sensation. In addition to the severe neuro-
logical cases, one further patient, a 27-year-old man,
presented with a discrete unilateral paresis of the right
leg. Two patients suffered seizures, most likely related
to underlying conditions.

The clinical manifestations for the remaining patients
ranged from mild cold-like symptoms of the upper res-
piratory tract to severe pneumonia requiring continuous
positive airway pressure and respirator support (Table
1). Fifty-nine patients (among the 73 for whom this
information was available) required hospitalisation,

either because of severity of symptoms or underlying
medical conditions. Twenty-one patients had underly-
ing medical conditions, including asthma, cancer, non-
HIV-related immunodeficiency, epilepsy and trisomy 14
mosaicism with growth retardation, cognitive impair-
ment and multiple malformations. For one Dutch case,
EV-D68 was identified in bronchoalveolar lavage and
this patient also had a pneumococci-positive antigen
test in urine. The patient died after 11 days of hospitali-
sation from bilateral pneumonia.

Phylogenetic analysis of enterovirus D68
strains

Full- or nearly full-length genome sequencing was suc-
cessfully carried out for seven strains (Denmark and
the Netherlands: in-house protocols, Sweden [12]).
Sequences which were available up to and including 6
December 2019 are available on GenBank (Table 2).

Phylogenetic analysis of VP1 sequence data was car-
ried out using the Nextstrain augur pipeline [13]. We
included samples from this study with ≥300 bp in
VP1 (Table 2), alongside all available VP1 sequences
in GenBank of ≥700 bp, randomly down-sampled to
20 samples per country per month to avoid sampling
bias and overrepresentation of some countries (par-
ticularly during the 2014 and 2016 epidemics; no 2019
samples were down-sampled). The code is available
at https://github.com/enterovirus-phylo/evd68-2019;
the analysis can be viewed at https://nextstrain.org/
community/enterovirus-phylo/evd68-2019/vp1-300.
This analysis will be updated with new sequence data
as this becomes available. EV-D68 has been charac-
terised into the major clades A, B, and C, with A and
B divided into the subclades A1–A2, and B1–B3. Some
studies designate A2 as D and subdivide it into D1 and
D2 (Figure 3).

The dominant subclades have varied between years
of upsurge: the 2014 outbreak was dominated by B1
strains, whereas the 2016 and 2018 outbreaks con-
sisted only of the B3 and A2/D subclades. Some clades
and subclades may no longer be circulating, as evi-
denced by the lack of clade C strains since 2010 and
the absence of A1, B1 and B2 strains since 2014/15. In
the current study, there was a close genetic relation-
ship between 15 strains from all five countries (Figure
4A). These formed a distinct cluster within clade B3
(EU19 in Figures 2 and 3), which did not include pre-
viously detected B3 strains from these countries and
was not well represented in the 2016 or 2018 epi-
demic (see Nextstrain analysis, https://nextstrain.org/
community/enterovirus-phylo/evd68-2019/vp1-300).
Fourteen of the sequences clustered closely, with
an estimated most recent common ancestor (MRCA)
in September 2018, suggesting that this cluster may
have circulated in Europe during the 2018 season. Four
German strains clustered within this group in the VP4/
VP2 region (https://nextstrain.org/community/enter-
ovirus-phylo/evd68-2019/vp4vp2). NL-03 (infection
presumably acquired in Turkey) was more distant, with

<figure><img src="image_3.png" /></figure>
TABLE 1A

<table><caption>Cases of enterovirus D68 in five European countries, 1 January 2019–15 January 2020 (n = 93)</caption><thead><tr><th>Case number</th><th>Sample date (month-year)</th><th>Age (years)</th><th>Sex</th><th>Hospitalised</th><th>Pre-existing disease</th><th>Travel abroad &lt; 2 weeks before sampling</th><th>Fever</th><th>Enteric symptoms</th><th>Respiratory symptoms</th><th>Neurological symptoms</th><th>Dermatological symptoms</th><th>Co-infections</th></tr></thead><tbody><tr><td>DK-01</td><td>Aug-19</td><td>15</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>None</td><td>Bronchiolitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-02</td><td>Aug-19</td><td>2</td><td>F</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>None</td><td>Common cold</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-03</td><td>Sep-19</td><td>3</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>None</td><td>Acute bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-04</td><td>Sep-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>None</td><td>ILI</td><td>AFM</td><td>NA</td><td>Adenovirus, parechovirus</td></tr><tr><td>DK-05</td><td>Sep-19</td><td>2</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>None</td><td>Obstructive bronchiolitis</td><td>N</td><td>Rash</td><td>Adenovirus</td></tr><tr><td>DK-06</td><td>Sep-19</td><td>29</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>None</td><td>Common cold</td><td>Cranial nerve palsies, dysphagia, dysaemia</td><td>N</td><td>N</td></tr><tr><td>DK-07</td><td>Sep-19</td><td>0</td><td>F</td><td>N</td><td>N</td><td>NA</td><td>Y</td><td>None</td><td>ILI</td><td>N</td><td>Rash</td><td>N</td></tr><tr><td>DK-08</td><td>Sep-19</td><td>0</td><td>F</td><td>Y</td><td>N</td><td>NA</td><td>Y</td><td>Diarrhoea, rumbling stomach</td><td>Common cold</td><td>N</td><td>N</td><td>Bordetella pertussis</td></tr><tr><td>DK-09</td><td>Oct-19</td><td>0</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>None</td><td>Pneumonia</td><td>N</td><td>N</td><td>Moraxella catarrhalis</td></tr><tr><td>DK-10</td><td>Oct-19</td><td>27</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>Diarrhoea</td><td>Pneumonia</td><td>Reduced strength in right leg, attenuated patellar and plantar reflexes</td><td>N</td><td>Suspected bacterial infection</td></tr><tr><td>DK-11</td><td>Oct-19</td><td>50</td><td>F</td><td>N</td><td>NA</td><td>NA</td><td>N</td><td>None</td><td>Common cold</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-12</td><td>Oct-19</td><td>30</td><td>M</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>None</td><td>ILI</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-13</td><td>Oct-19</td><td>0</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>None</td><td>Pneumonia</td><td>N</td><td>N</td><td>Haemophilus influenzae, Moraxella catarrhalis</td></tr><tr><td>DK-14</td><td>Nov-19</td><td>2</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>None</td><td>Pneumonia</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-15</td><td>Nov-19</td><td>2</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Obstructive bronchiolitis</td><td>N</td><td>N</td><td>Parechovirus, Haemophilus influenzae</td></tr><tr><td>DK-16</td><td>Nov-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>NA</td><td>N</td><td>Diarrhoea</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-17</td><td>Nov-19</td><td>16</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Nausea, vomiting</td><td>Y</td><td>Ataxic cerebral palsy, diplopia, hypoaesthesia</td><td>N</td><td>N</td></tr><tr><td>DK-18</td><td>Nov-19</td><td>2</td><td>M</td><td>Y</td><td>Y</td><td>NA</td><td>Y</td><td>None</td><td>Common cold</td><td>N</td><td>N</td><td>Haemophilus influenzae, Pseudomonas aeruginosa</td></tr><tr><td>DK-19</td><td>Nov-19</td><td>2</td><td>M</td><td>Y</td><td>Y</td><td>NA</td><td>N</td><td>NA</td><td>Respiratory distress</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DK-20</td><td>Dec-19</td><td>2</td><td>F</td><td>Y</td><td>Y</td><td>NA</td><td>NA</td><td>NA</td><td>Bilateral pneumonia</td><td>N</td><td>N</td><td>Pneumococcus</td></tr><tr><td>DK-21</td><td>Dec-19</td><td>1</td><td>M</td><td>N</td><td>Y</td><td>NA</td><td>NA</td><td>NA</td><td>Obstructive bronchiolitis</td><td>N</td><td>NA</td><td>Rhinovirus C22</td></tr><tr><td>NL-01</td><td>Jan-19</td><td>61</td><td>M</td><td>NA</td><td>NA</td><td>Y</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Haemophilus influenzae</td></tr><tr><td>NL-02</td><td>Jan-19</td><td>65</td><td>F</td><td>N</td><td>Y</td><td>N</td><td>NA</td><td>Yes</td><td>NA</td><td>NA</td><td>N</td><td>NA</td></tr><tr><td>NL-03</td><td>Apr-19</td><td>1</td><td>F</td><td>Y</td><td>NA</td><td>Y</td><td>NA</td><td>NA</td><td>Respiratory insufficiency</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-04</td><td>Aug-19</td><td>5</td><td>F</td><td>Y</td><td>NA</td><td>Y</td><td>NA</td><td>NA</td><td>Respiratory insufficiency</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-05</td><td>Aug-19</td><td>75</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Pneumonia</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-06</td><td>Sep-19</td><td>46</td><td>M</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>Diarrhoea</td><td>ILI</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-07</td><td>Oct-19</td><td>61</td><td>F</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>ARI, dyspnoea</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-08</td><td>Nov-19</td><td>1</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Common cold, dyspnoea</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-09</td><td>Nov-19</td><td>1</td><td>M</td><td>NA</td><td>NA</td><td>N</td><td>Y</td><td>NA</td><td>ARI, dyspnoea</td><td>NA</td><td>NA</td><td>Respiratory syncytial virus type A</td></tr></tbody></table>

AFM: acute flaccid myelitis; ARI: acute respiratory infection; DK: Denmark; FR: France; DE: Germany; ILI: influenza-like illness; N: no; NA: not available; NL: the Netherlands; SE: Sweden; URTI: upper respiratory infection; Y: yes.

<figure><img src="image_4.png" /></figure>
<caption>TABLE 1B</caption>

Cases of enterovirus D68 in five European countries, 1 January 2019–15 January 2020 (n = 93)

<table><thead><tr><th>Case number</th><th>Sample date (month-year)</th><th>Age (years)</th><th>Sex</th><th>Hospitalised</th><th>Pre-existing disease</th><th>Travel abroad &lt; 2 weeks before sampling</th><th>Fever</th><th>Enteric symptoms</th><th>Respiratory symptoms</th><th>Neurological symptoms</th><th>Dermatological symptoms</th><th>Co-infections</th></tr></thead><tbody><tr><td>NL-10</td><td>Nov-19</td><td>89</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-11</td><td>Nov-19</td><td>4</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-12</td><td>Nov-19</td><td>2</td><td>F</td><td>NA</td><td>NA</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-13</td><td>Nov-19</td><td>0</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-14</td><td>Nov-19</td><td>2</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-15</td><td>Dec-19</td><td>4</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-16</td><td>Dec-19</td><td>53</td><td>M</td><td>NA</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-17</td><td>Dec-19</td><td>7</td><td>F</td><td>NA</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-18</td><td>Dec-19</td><td>22</td><td>M</td><td>NA</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>NL-19</td><td>Mar-19</td><td>60</td><td>M</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>NA</td><td>Influenza virus A(H1N1)pdm09</td></tr><tr><td>SE-01</td><td>Aug-19</td><td>70</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Haemophilus influenzae</td></tr><tr><td>SE-02</td><td>Sep-19</td><td>2</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Rhinovirus</td></tr><tr><td>SE-03</td><td>Sep-19</td><td>0</td><td>F</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>NA</td><td>N</td></tr><tr><td>SE-04</td><td>Sep-19</td><td>5</td><td>F</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>SE-05</td><td>Oct-19</td><td>61</td><td>M</td><td>N</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>NA</td><td>Streptococcus pneumoniae</td></tr><tr><td>SE-06</td><td>Oct-19</td><td>2</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>SE-07</td><td>Oct-19</td><td>4</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>NA</td><td>N</td></tr><tr><td>SE-08</td><td>Oct-19</td><td>2</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>NA</td><td>Adenovirus<br>Rhinovirus Haemophilus<br>influenzae</td></tr><tr><td>SE-09</td><td>Nov-19</td><td>2</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>SE-10</td><td>Nov-19</td><td>5</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>SE-11</td><td>Jan-20</td><td>69</td><td>M</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>FR-01</td><td>Oct-19</td><td>0</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>FR-02</td><td>Oct-19</td><td>33</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Streptococcus pneumoniae</td></tr><tr><td>FR-03</td><td>Nov-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>Diarrhoea</td><td>N</td><td>AFM</td><td>N</td><td>N</td></tr><tr><td>FR-04</td><td>Nov-19</td><td>0</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>Diarrhoea</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>FR-05</td><td>Dec-19</td><td>51</td><td>F</td><td>Y</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>FR-06</td><td>Dec-19</td><td>66</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-01</td><td>Jul-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-02</td><td>Sep-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>Rhinovirus C17</td></tr><tr><td>DE-03</td><td>Sep-19</td><td>20</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>Multiple bacterial and viral infections</td></tr><tr><td>DE-04</td><td>Sep-19</td><td>9</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-05</td><td>Sep-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Rhinovirus A49</td></tr></tbody></table>

AFM: acute flaccid myelitis; ARI: acute respiratory infection; DK: Denmark; FR: France; DE: Germany; ILI: influenza-like illness; N: no; NA: not available; NL: the Netherlands; SE: Sweden; URTI: upper respiratory infection; Y: yes.

<figure><img src="image_5.png" /></figure>
TABLE 1C

<table><caption>Cases of enterovirus D68 in five European countries, 1 January 2019–15 January 2020 (n = 93)</caption><thead><tr><th>Case number</th><th>Sample date (month-year)</th><th>Age (years)</th><th>Sex</th><th>Hospitalised</th><th>Pre-existing disease</th><th>Travel abroad &lt; 2 weeks before sampling</th><th>Fever</th><th>Enteric symptoms</th><th>Respiratory symptoms</th><th>Neurological symptoms</th><th>Dermatological symptoms</th><th>Co-infections</th></tr></thead><tbody><tr><td>DE-06</td><td>Oct-19</td><td>0</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-07</td><td>Oct-19</td><td>0</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-08</td><td>Oct-19</td><td>6</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>Bilateral lower limb paralysis</td><td>N</td><td>N</td></tr><tr><td>DE-09</td><td>Oct-19</td><td>12</td><td>M</td><td>Y</td><td>N</td><td>NA</td><td>Y</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>DE-10</td><td>Nov-19</td><td>2</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-11</td><td>Nov-19</td><td>7</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Asthma exacerbation</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-12</td><td>Nov-19</td><td>2</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-13</td><td>Nov-19</td><td>5</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Acute bronchitis</td><td>Seizures</td><td>N</td><td>Clostridioides difficile</td></tr><tr><td>DE-14</td><td>Nov-19</td><td>22</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-15</td><td>Nov-19</td><td>1</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Acute bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-16</td><td>Nov-19</td><td>8</td><td>F</td><td>Y</td><td>Y</td><td>NA</td><td>NA</td><td>N</td><td>Y</td><td>NA</td><td>NA</td><td>Adenovirus</td></tr><tr><td>DE-17</td><td>Nov-19</td><td>78</td><td>F</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-18</td><td>Nov-19</td><td>8</td><td>M</td><td>Y</td><td>N</td><td>NA</td><td>NA</td><td>N</td><td>Y</td><td>NA</td><td>N</td><td>N</td></tr><tr><td>DE-19</td><td>Nov-19</td><td>0</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-20</td><td>Nov-19</td><td>5</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-21</td><td>Nov-19</td><td>4</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-22</td><td>Nov-19</td><td>2</td><td>M</td><td>Y</td><td>NA</td><td>NA</td><td>Y</td><td>NA</td><td>Y</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>DE-23</td><td>Nov-19</td><td>29</td><td>M</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-24</td><td>Nov-19</td><td>7</td><td>M</td><td>Y</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td><td>Y</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>DE-25</td><td>Oct-19</td><td>74</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>NA</td><td>N</td></tr><tr><td>DE-26</td><td>Nov-19</td><td>71</td><td>M</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-27</td><td>Nov-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-28</td><td>Nov-19</td><td>3</td><td>M</td><td>Y</td><td>N</td><td>NA</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-29</td><td>Nov-19</td><td>40</td><td>F</td><td>N</td><td>N</td><td>NA</td><td>Y</td><td>N</td><td>Y</td><td>NA</td><td>NA</td><td>N</td></tr><tr><td>DE-30</td><td>Dec-19</td><td>2</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-31</td><td>Dec-19</td><td>1</td><td>F</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Obstructive bronchitis</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-32</td><td>Dec-19</td><td>6</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Pneumonia</td><td>N</td><td>N</td><td>N</td></tr><tr><td>DE-33</td><td>Dec-19</td><td>2</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td><td>Bronchitis and pneumonia</td><td>N</td><td>N</td><td>Rhinovirus C55</td></tr><tr><td>DE-34</td><td>Dec-19</td><td>1</td><td>M</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td><td>Pneumonia</td><td>N</td><td>N</td><td>Bocavirus 1</td></tr><tr><td>DE-35</td><td>Dec-19</td><td>5</td><td>F</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Obstructive bronchitis</td><td>Seizures</td><td>N</td><td>Respiratory syncytial virus</td></tr><tr><td>DE-36</td><td>Dec-19</td><td>2</td><td>M</td><td>Y</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>URTI</td><td>N</td><td>N</td><td>N</td></tr></tbody></table>

AFM: acute flaccid myelitis; AR: acute respiratory infection; DK: Denmark; FR: France; DE: Germany; ILI: influenza-like illness; N: no; NA: not available; NL: the Netherlands; SE: Sweden; URTI: upper respiratory infection; Y: yes.

<figure><img src="image_6.png" /></figure>
Epicurve of enterovirus D68 cases by phylogenetic cluster, five European countries, 1 January 2019–15 January 2020 (n = 93)

<figure><img src="image_7.png" /></figure>

DK: Denmark; EU18: 2019 sequences with most recent common ancestor in large B3 cluster of European sequences from 2018; EU19: novel B3 cluster identified in this study; FR: France; DE: Germany; NA: sequence data for clade allocation not available or phylogenetic analysis not done; NL: the Netherlands; SE: Sweden; US18: 2019 sequences with most recent common ancestor in large B3 cluster predominantly containing sequences from the United States in 2018.

For clade allocation of cases see section *Phylogenetic analysis of enterovirus D68 strains*.

an estimated MRCA with the larger 2019 European B3 subclade of January 2016.

Seventeen other strains (US18 in Figures 2 and 3) formed two further clusters within clade B3, one cluster with five strains and an MRCA of May 2019, the other with 12 strains and an MRCA of September 2018 (Figure 4B). These two clusters had a common MRCA in August 2018. This common ancestor fell within a large American cluster circulating in 2018, which previously contained only three non-American sequences. Strains NL-09, DK-09 and four German strains also fell within this American cluster; NL-09 separately from the other 2019 VP1 samples and the other five in the

VP4/VP2 region (Figure 2, sequence data not shown). Samples DE-01 and DE-12 formed a pair and were descended from a cluster of B3 strains that circulated in Europe in 2018, including one of the Dutch cases detected in January (NL-02; cluster EU18 in Figures 2 and 3). The other Dutch case from January (NL-07; cluster A2/D in Figures 2 and 3), along with five other strains, belonged to clade A2/D (Figure 4C) and rooted, although separately, in a cluster circulating in Europe in autumn 2018. Five and 21 sequences, respectively, were long enough to be included in the Nextstrain full-length (≥6,000 bp) and VP1 (≥700 bp) builds; they can be viewed at https://nextstrain.org/enterovirus/d68/

<figure><img src="image_8.png" /></figure>
TABLE 2A

GenBank accession numbers and available sequence for phylogenetic analysis, enterovirus D68 strains from five European countries, 1 January–6 December 2019 (n=67)

<table><thead><tr><td>Case number</td><td>GenBank accession number</td><td>Sequence</td></tr></thead><tbody><tr><td>DK-01</td><td>MN896974</td><td>Partial genome</td></tr><tr><td>DK-02</td><td>MN896975</td><td>Partial genome</td></tr><tr><td>DK-03</td><td>NA</td><td>NA</td></tr><tr><td>DK-04</td><td>MN896976</td><td>Partial VP1</td></tr><tr><td>DK-05</td><td>NA</td><td>NA</td></tr><tr><td>DK-06</td><td>MN896977</td><td>Partial VP1</td></tr><tr><td>DK-07</td><td>MN896978</td><td>Full genome</td></tr><tr><td>DK-08</td><td>NA</td><td>NA</td></tr><tr><td>DK-09</td><td>MN896979</td><td>Partial VP4/VP2</td></tr><tr><td>DK-10</td><td>MN896980</td><td>Partial VP1</td></tr><tr><td>DK-11</td><td>NA</td><td>NA</td></tr><tr><td>DK-12</td><td>MN896981</td><td>Partial VP1</td></tr><tr><td>DK-13</td><td>MN896982</td><td>Partial VP1</td></tr><tr><td>DK-14</td><td>MN896983</td><td>Partial VP1</td></tr><tr><td>DK-15</td><td>MN896985</td><td>Partial VP1</td></tr><tr><td>DK-16</td><td>MN896984</td><td>Partial VP1</td></tr><tr><td>DK-17</td><td>NA</td><td>NA</td></tr><tr><td>DK-18</td><td>MN896986</td><td>Partial VP1</td></tr><tr><td>NL-01</td><td>MN764886</td><td>Partial VP1</td></tr><tr><td>NL-02</td><td>MN764887</td><td>Partial VP1</td></tr><tr><td>NL-03</td><td>MN764888</td><td>Partial VP1</td></tr><tr><td>NL-04</td><td>MN764889</td><td>Partial VP1</td></tr><tr><td>NL-05</td><td>MN726800</td><td>Full genome</td></tr><tr><td>NL-06</td><td>MN726801</td><td>Full genome</td></tr><tr><td>NL-07</td><td>MN726798</td><td>Full genome</td></tr><tr><td>NL-08</td><td>MN726799</td><td>Partial VP1</td></tr><tr><td>NL-09</td><td>MN809623</td><td>Complete VP1</td></tr><tr><td>NL-10</td><td>MN809624</td><td>Partial VP1</td></tr><tr><td>NL-11</td><td>MN809625</td><td>Partial VP1</td></tr><tr><td>NL-12</td><td>MN809626</td><td>Partial VP1</td></tr><tr><td>SE-01</td><td>MN935869</td><td>Full genome</td></tr><tr><td>SE-02</td><td>NA</td><td>NA</td></tr><tr><td>SE-03</td><td>NA</td><td>NA</td></tr><tr><td>SE-04</td><td>MN935870</td><td>Full genome</td></tr><tr><td>SE-05</td><td>NA</td><td>NA</td></tr><tr><td>SE-06</td><td>NA</td><td>NA</td></tr><tr><td>SE-07</td><td>NA</td><td>NA</td></tr><tr><td>SE-08</td><td>NA</td><td>NA</td></tr><tr><td>SE-09</td><td>NA</td><td>NA</td></tr><tr><td>FR-01</td><td>LR743438</td><td>Complete VP1</td></tr><tr><td>FR-02</td><td>LR743439</td><td>Complete VP1</td></tr><tr><td>FR-03</td><td>LR743440</td><td>Complete VP1</td></tr><tr><td>FR-04</td><td>LR743441</td><td>Complete VP1</td></tr><tr><td>DE-01</td><td>MN814240</td><td>Partial VP1</td></tr><tr><td>DE-02</td><td>MN814241</td><td>Partial VP1</td></tr><tr><td>DE-03</td><td>MN814242</td><td>Partial VP1</td></tr><tr><td>DE-04</td><td>MN814243</td><td>Partial VP1</td></tr><tr><td>DE-05</td><td>NA</td><td>NA</td></tr><tr><td>DE-06</td><td>MN814244</td><td>Partial VP1</td></tr></tbody></table>

NA: not available.

<figure><img src="image_9.png" /></figure>
TABLE 2B

GenBank accession numbers and available sequence for phylogenetic analysis, enterovirus D68 strains from five European countries, 1 January–6 December 2019 (n = 67)

<table><thead><tr><td>Case number</td><td>GenBank accession number</td><td>Sequence</td></tr></thead><tbody><tr><td>DE-07</td><td>MN832475</td><td>Partial VP4/VP2</td></tr><tr><td>DE-08</td><td>MN812202</td><td>Partial VP1</td></tr><tr><td>DE-09</td><td>MN832476</td><td>Partial VP4/VP2</td></tr><tr><td>DE-10</td><td>MN814245</td><td>Partial VP1</td></tr><tr><td>DE-11</td><td>MN814246</td><td>Partial VP1</td></tr><tr><td>DE-12</td><td>MN814247</td><td>Partial VP1</td></tr><tr><td>DE-13</td><td>MN814248</td><td>Partial VP1</td></tr><tr><td>DE-14</td><td>MN814249</td><td>Partial VP1</td></tr><tr><td>DE-15</td><td>MN814250</td><td>Partial VP1</td></tr><tr><td>DE-16</td><td>MN832477</td><td>Partial VP4/VP2</td></tr><tr><td>DE-17</td><td>MN832478</td><td>Partial VP4/VP2</td></tr><tr><td>DE-18</td><td>MN832479</td><td>Partial VP4/VP2</td></tr><tr><td>DE-19</td><td>MN832480</td><td>Partial VP4/VP2</td></tr><tr><td>DE-20</td><td>MN814251</td><td>Partial VP1</td></tr><tr><td>DE-21</td><td>MN832481</td><td>Partial VP4/VP2</td></tr><tr><td>DE-22</td><td>MN832482</td><td>Partial VP4/VP2</td></tr><tr><td>DE-23</td><td>MN832483</td><td>Partial VP4/VP2</td></tr><tr><td>DE-24</td><td>MN832484</td><td>Partial VP4/VP2</td></tr></tbody></table>

NA: not available.

genome and https://nextstrain.org/enterovirus/d68/vp1.

Discussion

Here we report the detection of EV-D68 infections associated with both respiratory and neurological manifestations in five European countries in the autumn of 2019. Following its first identification in 1962, only 699 cases of EV-D68 were reported in scientific literature before 2014 [14]. From 2014 onwards, the epidemiology appears to have changed, and EV-D68 has increasingly been associated with outbreaks of respiratory infections and a concurrent upsurge of AFM [15,16] with poor long-term prognosis [17,18]. We identified 15 sequences, collected from five countries, which formed a distinct cluster within subclade B3 not previously described in Europe, illustrating the rapid evolution and spread of EV-D68. The sequences most closely related to this novel B3 cluster were sampled in India in 2017 and from sewage in the United Kingdom in 2018. This would suggest either a very low circulation of strains of this cluster or a lack of sustained global surveillance for EV-D68 and/or submission of sequences in GenBank, or both. The estimated MRCA in mid-2018 suggests that these viruses were already circulating and diversifying during and after the 2018 EV-D68 epidemic, but were not sampled until August 2019. In previous European seasons of EV-D68, Denmark has only reported respiratory infections caused by EV-D68, whereas Germany, France, the Netherlands and Sweden have experienced paralytic cases [11,18,19].

The EV-D68 cases were detected through routine surveillance networks in each country, with alerts to physicians posted in Denmark and the Netherlands. The number of samples processed in each country was within the expected range for the enterovirus season, but the detection rate of EV-D68 was higher than expected in Denmark, Germany and Sweden, where the number of detections for the 2018 season had been unexpectedly low. Detection rates in the other countries were similar to those seen in previous years with low circulation. The rate of hospitalisation among cases was high, however, current surveillance systems primarily allow the detection of severe infections. In this study, 12 of 93 cases were detected through community-based surveillance and a further three cases through sequencing of rhinovirus-positive samples, highlighting the importance of including such surveillance when monitoring infections such as EV-D68. The detection of EV-D68 associated disease indicates a continuous, and most likely underestimated, global circulation of this enterovirus type that was recognised after the large outbreak in the United States in 2014 had sparked increased testing. This justifies the need for reinforced enterovirus surveillance based on a more systematic screening of respiratory samples, collected in hospitalised patients or through the surveillance of respiratory infections (such as influenza community surveillance). Our report also highlights the heterogeneity of the surveillance of EV-D68 in Europe, which may hamper both the comparison of the epidemiological pattern between countries and, more importantly, the early detection of an outbreak. Continuous surveillance will add to our understanding of how EV-D68

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>Phylogenetic analysis of enterovirus D68 with Nextstrain using partial VP1 sequences (n = 1,693)</figcaption></figure>

EU18: 2019 sequences with most recent common ancestor in large B3 cluster of European sequences from 2018; EU19: novel B3 cluster identified in this study; US18: 2019 sequences with most recent common ancestor in large B3 cluster predominantly containing sequences from the United States in 2018.

For sequences with a sample date up to and including 2018, VP1 fragments of ≥ 700 bp in length were included, for sequences from 2019, fragments of ≥ 300 bp were included.

Divergence between major subclade divisions is shown, sequences are colour-coded according to date of sampling. Brackets identify where sequences reported in this study cluster with previous samples.

<figure><img src="image_12.png" /></figure>
Phylogenetic analysis of three clusters of partial VP1 sequences of enterovirus D68 strains using Nextstrain, five European countries, 1 January–6 December 2019 (n = 36)

A. Cluster B3 EU19

<figure><img src="image_13.png" /></figure>

B. B3 US18 (partial)

<figure><img src="image_14.png" /></figure>

C. A2/D (partial)

<figure><img src="image_15.png" /></figure>

AFM: acute flaccid myelitis.

Zoomed in view of three phylogenetic clusters containing 2019 EV-D68 sequences. Nodes are coloured by country; the colour legend is the same for all three panels. Date estimate confidence intervals are shown as light grey bars for nodes leading to 2019 strains. Years are marked with dashed vertical lines. Red dots: sequences from this study; diamond shapes: neurological symptoms for 2019 cases.

Panel A shows a novel 2019 B3 cluster which is poorly represented in previous years. Panel B shows two 2019 clusters which descend from a large, United States-dominated, 2018 B3 cluster. The partial VP1 sequences of the upper seven strains with nodes having wide date estimate confidence intervals are identical. Panel C shows 2019 A2/D strains clustering with strains that circulated in France and Senegal in 2018.

<figure><img src="image_16.png" /></figure>
evolves outside of outbreak periods and whether low-
prevalence years vary in geographic or demographic
distribution. The detection of EV-D68 in a presumed
year of low activity and the presence of patients with
severe neurologic symptoms, stresses the importance
of continuous and systematic surveillance and avail-
ability of diagnostics for workup of clinical cases.

Acknowledgements

The authors wish to thank the European non-polio Enterovirus Network (ENPEN) for facilitating contact between the countries.

The authors are grateful to all the scientists and researchers
who have made their EV-D68 samples and metadata avail-
able publicly, often in a very timely manner, which enables
the analysis of recent sequences in a global context.

The Danish authors wish to thank the clinical microbiology
departments for sending entero- and rhinovirus-positive
samples to SSI for further characterisation. We also wish to
thank the clinicians in the hospitals for ensuring appropriate
sampling and diagnostic testing and providing us with clini-
cal information about enterovirus-positive cases. Finally, we
wish to thank Shukriya Barzinci and Mille Weismann Poulsen
for carrying out the laboratory analyses at SSI.

The Dutch authors wish to thank the Dutch medical mi-
crobiology laboratories for their contribution on the EV
surveillance programme and VIRO-TypeNed, in particular
thanks to the clinicians, molecular medical microbiologist
and technicians in the Dutch hospitals and laboratories for
providing us with sequence and clinical information about
the EV-D68 cases (Richard Molenkamp (Erasmus Medical
Center, Rotterdam), Judith Hoogenboom-Beuving (Reinier
Haga, Medical Diagnostic Center, Delft), Hubert GM Niesters,
Coretta Van Leer-Buter, Marjolein Knoester (University
Medical Center Groningen, Groningen), and Lieuwe Roorda
(Maasstad Hospital, Rotterdam)). We would also like to
thank Gé A. Donker coordinator of the Nivel Primary Care
database – sentinel Practices, Utrecht, The Netherlands and
technicians (Bas van der Veer, Anne-Marie van den Brandt,
Sharon van den Brink, Lisa Wijsman, Elsa Poorter, Barbara
Favier, Jeroen Cremer and Pieter Overduin) running molecu-
lar diagnostics and sequencing at RIVM. We also thank Harry
Vennema for bioinformatic assistance with analysis of NGS
data.

The Swedish authors wish to acknowledge the input of Jan Albert at the Department of Clinical Microbiology, Karolinska University Hospital, and Katherina Zakikhany at the Public Health Agency of Sweden. We also want to thank Lina Thebo at Karolinska Institutet for expert technical assistance, Tanja Normark at SciLifeLab for bioinformatics, and Anders Jonsson, Guillermo Martinez Gonzalez, and Rolf Ingemarsson running diagnostics at the Karolinska University Hospital.

The French authors thank Dr Gisèle Lagathu and Pr Vincent Thibault (University hospital of Rennes) and Dr Quentin Lepiller (University hospital of Besançon) for taking part in the enterovirus surveillance. We also thank Adeline Duard and Nathalie Rodde for technical assistance.

The German authors wish to thank all clinicians for submit-
ting patient samples for testing. We acknowledge Ortwin
Adams (University hospital of Düsseldorf), Christiane Prifert,
and Benedikt Weissbrich (University hospital of Würzburg)
who substantially contribute to the German EV-D68 surveil-
lance in the context of the RespVir network, as well as Sabine

Diedrich (Robert Koch Institute, Rolf Kaiser (University hos-
pital of Cologne), Souhaib Aldabbagh (Institute of Virology,
University of Bonn Medical Center) and all technicians in the
labs.

Analyses were run using sciCORE (http://scicore.unibas.ch/) scientific computing core facility at the University of Basel.

**Conflict of interest**

None declared.

Authors’ contributions

SEM: Initiated the study, wrote the first draft, edited subsequent drafts and prepared the final manuscript.

KB: Provided data, critically reviewed and edited all drafts of the manuscript.

RD: Provided data, critically reviewed and edited all drafts of the manuscript.

AMir: Provided data, critically reviewed and edited all drafts of the manuscript.

JLB: Critically reviewed and edited the manuscript.

SibB: Provided data, critically reviewed and edited the manuscript.

SteB: Provided data, critically reviewed and edited the manuscript.

SinB: Provided data, critically reviewed and edited the manuscript.

AMEH: Provided data, critically reviewed and edited the manuscript.

MH: Provided data, critically reviewed and edited the manuscript.

VVJ: Provided data, critically reviewed and edited the manuscript.

UBH: Provided data, critically reviewed and edited the manuscript.

CH: Critically reviewed and edited the manuscript.

MP: Provided data, critically reviewed and edited the manuscript.

MKT: Provided data, critically reviewed and edited the manuscript.

EBH: Carried out the phylogenetic analysis, critically re-
viewed and edited all drafts of the manuscript.

AMeij: Provided data, critically reviewed and edited all drafts of the manuscript.

**References**

1. Kramer R, Sabatier M, Wirth T, Pichon M, Lina B, Schuffenecker I, et al. Molecular diversity and biennial circulation of enterovirus D68: a systematic screening study in Lyon, France, 2010 to 2016. Euro Surveill. 2018;23(37):1700711. https:// doi.org/10.2807/1560-7917.ES.2018.23.37.1700711 PMID: 30229724

2. Messacar K, Pretty K, Reno S, Dominguez SR. Continued biennial circulation of enterovirus D68 in Colorado. J Clin Virol. 2019;113:24-6. https://doi.org/10.1016/j.jcv.2019.01.008 PMID: 30825833

3. Uprety P, Curtis D, Elkan M, Fink J, Rajagopalan R, Zhao C, et al. Association of enterovirus D68 with acute flaccid myelitis,

<figure><img src="image_17.png" /></figure>
Philadelphia, Pennsylvania, USA, 2009-2018. Emerg Infect Dis.
2019;25(9):1676-82. https://doi.org/10.3201/eid2509.190468
PMID: 31407660

4. Cottrell S, Moore C, Perry M, Hilvers E, Williams C, Shankar AG. Prospective enterovirus D68 (EV-D68) surveillance from September 2015 to November 2018 indicates a current wave of activity in Wales. Euro Surveill. 2018;23(46):1800578. https://doi.org/10.2807/1560-7917.ES.2018.23.46.1800578 PMID: 30458915

5. Bal A, Sabatier M, Wirth T, Coste-Burel M, Lazrek M, Stefic K, et al. Emergence of enterovirus D68 clade D1, France, August to November 2018. Euro Surveill. 2019;24(3):1800699. https://doi.org/10.2807/1560-7917.ES.2019.24.3.1800699 PMID: 30670143

6. Pellegrinelli L, Giardina F, Lunghi G, Uceda Renteria SC, Greco L, Fratini A, et al. Emergence of divergent enterovirus (EV) D68 sub-clade D1 strains, northern Italy, September to October 2018. Euro Surveill. 2019;24(7):1900090. https://doi.org/10.2807/1560-7917.ES.2018.24.7.1900090 PMID: 30782269

7. The United Kingdom Acute Flaccid Paralysis Afp Task Force. An increase in reports of acute flaccid paralysis (AFP) in the United Kingdom, 1 January 2018-21 January 2019: early findings. Euro Surveill. 2019;24(6):1900093. PMID: 30755296

8. González-Sanz R, Taravillo I, Reina J, Navascués A, Moreno-Docón A, Aranzamendi M, et al. Enterovirus D68-associated respiratory and neurological illness in Spain, 2014-2018. Emerg Microbes Infect. 2019;8(1):1438-44. https://doi.org/10.1080/22221751.2019.1668243 PMID: 31571527

9. Midgley SE, Christiansen CB, Poulsen MW, Hansen CH, Fischer TK. Emergence of enterovirus D68 in Denmark, June 2014 to February 2015. Euro Surveill. 2015;20(17):21105. https://doi.org/10.2807/1560-7917.ES2015.20.17.21105 PMID: 25955773

10. Meijer A, van der Sanden S, Snijders BE, Jaramillo-Gutierrez G, Bont L, van der Ent CK, et al. Emergence and epidemic occurrence of enterovirus 68 respiratory infections in The Netherlands in 2010. Virology. 2012;423(1):49-57. https://doi.org/10.1016/j.virol.2011.11.021 PMID: 22177700

11. Dyrdak R, Grabbe M, Hammas B, Ekwall J, Hansson KE, Luthander J, et al. Outbreak of enterovirus D68 of the new B3 lineage in Stockholm, Sweden, August to September 2016. Euro Surveill. 2016;21(46):30403. https://doi.org/10.2807/1560-7917.ES.2016.21.46.30403 PMID: 27918255

12. Dyrdak R, Mastafa M, Hodcroft EB, Neher RA, Albert J. Intra- and interpatient evolution of enterovirus D68 analyzed by whole-genome deep sequencing. Virus Evol. 2019;5(1):vez007. https://doi.org/10.1093/ve/vez007 PMID: 31037220

13. Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, et al. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018;34(23):4121-3. https://doi.org/10.1093/bioinformatics/bty407 PMID: 29790939

14. Holm-Hansen CC, Midgley SE, Fischer TK. Global emergence of enterovirus D68: a systematic review. Lancet Infect Dis. 2016;16(5):e64-75. https://doi.org/10.1016/S1473-3099(15)00543-5 PMID: 26929196

15. Dyda A, Stelzer-Braid S, Adam D, Chughtai AA, MacIntyre CR. The association between acute flaccid myelitis (AFM) and Enterovirus D68 (EV-D68) - what is the evidence for causation? Euro Surveill. 2018;23(3):e. https://doi.org/10.2807/1560-7917.ES.2018.23.3.17-00310 PMID: 29386095

16. Messacar K, Asturias EJ, Hixon AM, Van Leer-Buter C, Niesters HGM, Tyler KL, et al. Enterovirus D68 and acute flaccid myelitis-evaluating the evidence for causality. Lancet Infect Dis. 2018;18(8):e239-47. https://doi.org/10.1016/S1473-3099(18)0094-X PMID: 29482893

17. Yea C, Bitnun A, Robinson J, Mineyko A, Barton M, Mah JK, et al. Longitudinal outcomes in the 2014 acute flaccid paralysis cluster in Canada. J Child Neurol. 2017;32(3):301-7. https://doi.org/10.1177/0883073816680770 PMID: 28193112

18. Knoester M, Helfferich J, Poelman R, Van Leer-Buter C, Brouwer OF, Niesters HGM, et al. Twenty-nine cases of enterovirus-D68-associated acute flaccid myelitis in Europe 2016: a case series and epidemiologic overview. Pediatr Infect Dis J. 2019;38(1):16-21. https://doi.org/10.1097/INF.00000000000002188 PMID: 30234793

19. Antona D, Kossorotoff M, Schuffenecker I, Mirand A, Leruez-Ville M, Bassi C, et al. Severe paediatric conditions linked with EV-A71 and EV-D68, France, May to October 2016. Euro Surveill. 2016;21(46):30402. https://doi.org/10.2807/1560-7917.ES.2016.21.46.30402 PMID: 27918268

This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.

Any supplementary material referenced in the article can be
found in the online version.

This article is copyright of the authors or their affiliated institutions, 2020.

<figure><img src="image_18.png" /></figure>