1 INTRODUCTION

Enteroviruses (EVs) are single positive-strand RNA viruses with a 7.2–8.5 kb genome containing a single open reading frame (ORF), flanked by untranslated regions (UTRs). This ORF is translated into a single polyprotein that autocleavages into three precursors: P1 encoding four structural capsid proteins (VP1–4), and P2 and P3 containing seven proteins for the virus replication.^{1, 2} EVs constitute a genus, in the Picornaviridae family, classified into 15 species (EV A-L, and Rhinovirus (RV) A-C).^{3, 4} Humans can be infected by more than 100 serotypes from EV A-D and RV A-C. These serotypes are mainly associated with mild infections. However, some of them are responsible for severe clinical presentations, such as polioviruses responsible for poliomyelitis, and acute flaccid paralysis (AFP),⁵ and other non-polio EVs, such as EV-A serotype 71 (EV-A71) and EV-D serotype 68 (EV-D68), that can cause pneumonia, meningitis, rhombencephalitis, and acute flaccid myelitis (AFM).^{6, 7}

EV-D68 was isolated for the first time in 1962, from patients with mild clinical pictures. Since then, few EV-D68 respiratory disease cases were reported, but after 2005, an increase in small EV-D68 respiratory outbreaks was documented around the world,⁷ and in 2014, a large EV-D68 severe respiratory infection outbreak was observed in the United States at the same time that there was an increase of AFM cases in children.⁸ AFM is a polio-like neurological disorder characterized by flaccid paralysis of the extremities, mostly affecting the upper limbs and lesion in the anterior horn of the gray matter along the spinal cord and sometimes in the brainstem, with significant associated morbidity, and sometimes irreversible effects in infected patients. Most of the patients also experience acute respiratory syndrome, up to 2 weeks before the onset of limb weakness.⁹ This type of paralysis may develop in children and adults with only viral-induced wheezing without any known underlying condition.⁹

Since 2014, several outbreaks of EV-D68 infection have been described worldwide, which correlate with a global increase in AFM cases.⁸ The EV-D68 circulation mainly follows a biennial pattern upsurge, with infection peaks in late summer-autumn of even-numbered years, starting in 2014.^{8, 10} In Europe, 343 EV-D68 positive samples and 4 patients with AFP were detected in 2014,¹¹ and 416 EV-D68 positive samples and 29 patients with AFM in 2016.¹² France, Italy, and Wales registered an increase of EV-D68 infected patients in 2018, with 61, 21, and 114 EV-D68 positive cases, respectively,^13-15 and the United Kingdom reported 65 EV-D68 infected patients, 40 of them with AFM.¹⁶ Furthermore, 93 patients mainly from Germany, Denmark, and the Netherlands were EV-D68 positives in 2019, a year in which a low detection rate was expected.¹⁰

A 2014–2018 study in Spain ¹⁷ reported no significant increase of EV-D68 positive cases in 2014, while around 140 and 45 cases were detected in 2016, and 2018, respectively. During this period, nine of the patients suffered AFP, and eight meningitis or meningoencephalitis, most of them in 2016 or 2018.

In 2012, the Valencia Hospital Surveillance Network for the Study of Influenza and Other Respiratory Viruses (VAHNSI) was established in the Valencia Region of Spain. This is an active surveillance network analyzing respiratory viruses, including EV-D68, in influenza-like illness (ILI) cases requiring hospital admission.¹⁸ Therefore, the objectives of this study were to describe the epidemiology of EV-D68 upsurges in the Valencia Region linked to VAHNSI ILI admissions between late 2014 and early 2020, and to examine the genetic variability and phylogenetic relationships of the isolated strains.

2 METHODS

3 RESULTS

Routine respiratory virus screening from a total of 19,639 respiratory samples identified 2210 (11.25%) EV/RV positives, during six consecutive surveillance periods, from late 2014 to early 2020 (Table 1). Of those EV/RV positive, 454 (20.54%) specimens corresponded to EV, with 68 isolates subtyped as EV-D68. The yearly distribution was: 2014 (13/235, 5.5%), 2015 (2/246, 0.8%), 2016 (1/249, 0.4%), and 2018 (52/605, 8.6%). No EV-D68 cases were detected in 2017, 2019, or 2020, although in 2020, sample collection was stopped on March 10th due to the SARS-CoV-2 pandemic (Figure 1 and Table 1).

In 2014, EV-D68 samples were isolated in autumn–winter (n = 11, n = 2; respectively); in 2015 (n = 2) and in 2016 (n = 1) in winter; whereas in 2018—expanded surveillance window—in summer–autumn (n = 16, n = 36; respectively) (Figure 1 and Table 1).

Table 2 shows the main characteristics of patients with ILI and EV-D68 infection (anonymized aggregated data on clinical and demographic characteristics of the patients are available under reasonable request). In 2014, 62% of the patients were <16 years old and 31% presented acute bronchitis or bronchiolitis (n = 4) —one of them was coinfected with bocavirus; whereas the remaining 38% were ≥16 years old and were diagnosed with different diseases—an 89-years-old patient who had an underlying heart disease was diagnosed with dyspnea and respiratory disturbances, resulting in a fatal outcome. In 2015, two patients were 2 and 73 years old and presented bronchitis and bronchiectasis, respectively, while in 2016, a 91-year-old patient suffered from pneumonia. In contrast, in 2018, only 4% of the EV-D68-positive patients (2/52) were <16 years old. Another two (4%) were 30- to 45-year-old —one of them with acute respiratory infection of the lower tract and asthma as underlying condition was admitted to the intensive care unit (ICU)—. In 2018, the remaining 48 out of 52 patients (91.2%) were >50 years old. Up to 12 of these patients were diagnosed with acute respiratory infection from the lower respiratory tract, resulting in two fatal outcomes >80-years-old patients (81 and 91) with no chronic conditions; 12 were diagnosed with pneumonia (one patient with Streptococcus pneumoniae and another with Haemophilus influenzae co-infection); 9 with chronic obstructive lung disease; and 14 with different diseases. Finally, 79% of the EV-D68 positive in-patients presented underlying diseases—mainly bronchitis, heart disease, and obesity—96% of them ≥16 years old and diagnosed at admission mainly with chronic obstructive lung disease (n = 9), acute respiratory infection of the lower respiratory tract (n = 13), and/or pneumonia (n = 11). We have no evidence of AFM symptoms in the patients at discharge.

3.2 Phylogenetic analyses and signature substitutions in the VP1 protein

Consistent with BLASTN analyses, phylogenetic reconstruction showed that the sequences from 2014 clustered mainly in B2 (6/12), and B1 (5/12) subclades, but also in A2/D1 (1/12), whereas sequences from 2015 (n = 1) and 2016 (n = 1) belonged to B3 and A2/D1 respectively. Besides, almost all sequences from 2018 (42/45) belonged to a divergent subgroup within the A2/D1 subclade, recently reclassified as divergent D1-like¹⁴ or D3 subclade,³¹ and only 3/45 belonged to the B3 subclade (Figure 2 and Table 3). While B1 and B2 viruses were mainly detected in children (80% and 67%, respectively) (median age 5 years in both subclades), B3 viruses were equally distributed between children and adults (median age 19 years), and A2/D1 and A2/D3 were observed only in adults (median age 84 and 78 years, respectively) (Figure 2 and Table 4). Furthermore, all viruses isolated from patients diagnosed with acute infection of the lower respiratory tract (n = 12), or chronic obstructive lung disease (n = 7) belonged to clade A2/D3, as well as 10 of the 11 isolates from patients with pneumonia (Table 4).

Table 3. Distribution of EV-D68 clades during the 2014–2020 influenza seasons, VAHNSI network, Valencia, Spain.

EV-D68 subclade	All (n = 68)	2014 (n = 13)	2015 (n = 2)	2016 (n = 1)	2018 (n = 52)
A1	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
A2	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
A2/D1	2 (2.94%)	1 (7.69%)	0 (0%)	1 (100%)	0 (0%)
A2/D3	42 (61.76%)	0 (0%)	0 (0%)	0 (0%)	42 (80.77%)
B1	5 (7.35%)	5 (38.46%)	0 (0%)	0 (0%)	0 (0%)
B2	6 (8.82%)	6 (46.15%)	0 (0%)	0 (0%)	0 (0%)
B3	4 (5.88%)	0 (0%)	1 (50%)	0 (0%)	3 (5.77%)
Clade C	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Not sequenced	9 (13.24%)	1 (7.69%)	1 (50%)	0 (0%)	7 (13.46%)

Table 4. Characteristics of EV-D68 positive children and adult patients and distribution of viral clades.

Characteristics	Children <16 years (n = 10)	Adults ≥16 years (n = 49)	p Value
Sex (male; n, %)	7 (70)	20 (41)	0.162
ICU admission (n, %)	0 (0)	1 (2)	>0.999
Current tobacco use (n, %)	0 (0)	10 (20)	0.186
Obesity (n, %)	0 (0)	16 (33)	0.049
Reasons for admission
ARI	3 (30)	20 (41)	0.725
Fever	5 (50)	9 (18)	0.032
Pneumonia	0 (0)	13 (26)	0.098
COPD	1 (10)	11 (22)	0.670
Asthma	0 (0)	5 (10)	0.577
Dyspnea/Tachypnea	5 (50)	31 (63)	0.433
Cough	3 (30)	21 (43)	0.506
Apnea	0 (0)	1 (2)	>0.999
ACS	0 (0)	1 (2)	>0.999
Heart failure	0 (0)	4 (8)	>0.999
Metabolic failure	0 (0)	1 (2)	>0.999
Multiorgan failure	0 (0)	1 (2)	>0.999
Convulsions/confusion	0 (0)	2 (4)	>0.999
Sepsis/SIRS	1 (10)	0 (0)	0.169
Comorbidities
Presence of comorbidities	2 (20)	45 (92)	<0.001
Heart disease	1 (10)	20 (41)	0.080
Cerebrovascular disease	0 (0)	2 (4)	>0.999
Peripheral arteriopathy	0 (0)	3 (6)	>0.999
Asthma	1(10)	10 (20)	0.670
Bronchitis	0 (0)	21 (43)	0.010
Diabetes	0 (0)	10 (20)	0.186
Endocrine disorder (other than diabetes)	0 (0)	7 (14)	0.590
Anemia	0 (0)	8 (16)	0.329
Chronic hepatic disease	0 (0)	1 (2)	>0.999
Chronic renal disease	0 (0)	5 (10)	0.577
Immunodeficiency	0 (0)	1 (2)	>0.999
Neuromuscular disorder	0 (0)	6 (12)	0.577
Neoplasia	0 (0)	5 (10)	0.577
Autoimmune disease	0 (0)	4 (8)	>0.999
Dementia	0 (0)	7 (14)	0.590
EV-D68 (Sub)clade
Subclade A1	0 (0)	0 (0)	NA
Subclade A2	0 (0)	0 (0)	NA
Subclade A2/D1	0 (0)	2 (4)	>0.999
Subclade A2/D3	0 (0)	42 (86)	<0.001
Subclade B1	4 (40)	1 (2)	0.002
Subclade B2	4 (40)	2 (4)	0.006
Subclade B3	2 (20)	2 (4)	0.130
Clade C	0 (0)	0 (0)	NA
Clade not determined	0 (0)	0 (0)	NA

• Abbreviations: ACS, acute coronary syndrome; ARI, acute respiratory infection; COPD; chronic obstructive pulmonary disease; ICU, intensive care unit; NA, not applicable; SIRS, systemic inflammatory response syndrome.

The analysis of the VP1 sequences showed several amino acid substitutions as compared to the Fermon prototype (Figures 2 and 3, Table SII). EV-D68 clades A1, A2/D, B, and C presented seven common amino acid substitutions. One of them in the VP1 antigenic region BC-loop, and three in the C-terminal region. Furthermore, all clades, except for some subgroups, also showed three more substitutions, including one in the DE-loop and another in C-terminus which is involved in a “KER…A” substitution pattern. In addition, each clade/subclade/subgroup also presented its own substitution patterns in the VP1 gene, most of them affecting antigenic loops and the C-terminal region (Figures 2 and 3, Table SIIA–G). Of the total of mutated positions, 53.6% were found in antigenic sites in clade A (40% of them in the DE-loop); 52.2% in subclade A2/D2 (41.7% in the C-terminus); 54.5% in subclade A2/D1 (41.7% in the BC-loop); 52% in subclade A2/D3 (46.2% in the BC-loop); 60% in clade C (26.7% in both the BC- and DE-loops); 62.5% in subclade B2 (33.3% in both the BC- and DE-loops); 60% in subclade B1 (40.7% in the BC-loop); and 68.2% in subclade B3 (40% in the BC-loop).

Forty-four sequences were retrieved in this study in 2014 (n = 1), 2016 (n = 1), and 2018 (n = 42), clustered in clade A (Figure 2 and Table 3). The two isolates from 2014 and from 2016 belonged to an A2/D1 subclade subgroup including viruses present in Canada and Europe in 2014 and related to sequences from 2013. They carried the substitutions that define the A2/D1 subclade and also T98I (BC-loop), V243I, and A277T (Figure 3, Table SIIB). In contrast, the 42 isolates we retrieved from 2018 belonged to the subclade A2/D3, with the most recent closely related strain isolated in France (2015) (Figure 2). One of these sequences is closely related to isolates detected in Senegal (2018) and Europe (2019), and showed A96E (BC-loop), T98I (BC-loop), and V243I substitutions, while S95E (BC-loop) was replaced by S95G. The remaining 41 A2/D3 sequences had close relationship with isolates detected in Europe (2018), with T98V (BC-loop). Twenty-two sequences showed the reversion I13V, and the other 19 the I17M substitution (Figure 3, Table SIIB).

Fifteen sequences were retrieved in this study in 2014 (n = 11), 2015 (n = 1), and 2018 (n = 3), clustered in clade B (Figure 2 and Table 3). Five sequences from 2014 belonged to two different subgroups of the B1 subclade. One of them is composed of samples isolated from Europe (2014), and the United States (2015), having as their basal taxon a US sample (2013) isolated from a patient with AFM. The other subgroup encompasses sequences from the Americas, Europe, and Japan (2014), which basal taxon is, again, a sample from the United States (2013). This latter subgroup presented M194I and the reversion S218N (GH-loop) (Figure 3, Table SIID). The remaining six sequences from 2014 belonged to subclade B2 (Figure 2, Table 3). Two of them clustered with a French sample (2014), and the other four sequences with a subgroup composed mainly of European sequences (2014), with T146S (DE-loop) and the reversion E283D (C-terminal) (Figure 3, Table SIIE). The most recent closely related isolate of these two subgroups was isolated in the United States (2012) (Figure 2). The only sequence isolated in this study in 2015, and three sequences from 2018, belonged to the B3 subclade (Figure 2, Table 3). The sample from 2015 clustered with a subgroup mainly composed of sequences from Asia (2013–2016), that also carried S1L and N218S (GH-loop) substitutions (Figure 3, Table SIIF). The three sequences isolated in 2018 were closely related to other strains obtained that same year. They carried N218T (GH-loop) and the reversion S143N (DE-loop) and whereas two of them belonged to a European subgroup where N2D was replaced by N2E and also presented S1I, the third sequence was closely related to two US strains (one of them from a patient with AFM) and showed a replacement from S95A (BC-loop) substitution to S95T, the reversion A98T (BC-loop) and the substitution T146A (DE-loop) (Figure 3, Table SIIF).

4 DISCUSSION

During the epidemiological hospital-based, active ILI surveillance periods carried out at the VAHNSI network in the Valencia Region of Spain from 2014 to 2020, 68 patients tested positive for EV-D68. EV-D68 upsurges took place in the expected even years 2014 (n = 13) and 2018 (n = 52), but not in 2016 (n = 1), in contrast to a wider study from Spain, where the upsurge was observed in 2016 but not in 2014.¹⁷ The differences may be explained because (i) before the SARS-CoV-2 pandemic, VAHNSI was mainly focused on influenza detection, therefore the collection of nasopharyngeal samples followed the influenza season, which missed the warm months in Valencia (May–November); and (ii) the national study in Spain is not based on active surveillance¹⁷ and detected the upsurge in 2016, but not the first 2014 perhaps because lack of awareness of clinicians. Although following the expected seasonal distribution,^{8, 10} the peak of infection was later in 2014 than in 2018, with most of the EV-D68 infections in late autumn, with a peak in December (n = 9) in 2014, and in late summer and early autumn reaching a peaking in September (n = 36) in 2018. In our study, most of the EV-D68 positive samples were isolated from adults (11 children and 57 adults), in contrast to the wider Spanish study that spanned the same time period, and obtained more EV-D68 positive samples from children (138 children and 34 adults).¹⁷ Different inclusion criteria (our study being focused on ILI and respiratory disease) and cohort structure may explain these differences. However, we observed in our study that the 2014-upsurge included fewer cases than the 2018-upsurge, with more than half of those EV-D68 positives being <16 years old, while most infected patients in 2018 were >50 years old mostly infected by new EV-D68 clade A2/D3 viruses. We have no evidence of AFM symptoms in the patients at discharge. However, some EV-D68 cases were associated with fatal or severe outcomes: Three >80-year-old patients died in 2014 (n = 1; with chronic conditions) and 2018 (n = 2; without chronic conditions), and a 43-year-old patient was admitted to the ICU in 2018 with previous underlying disease. Some age and gender-related differences were observed. Most of the children <16 years old were diagnosed with bronchitis or bronchiolitis, while adult females were mainly diagnosed with acute lower respiratory tract infection, bronchitis, and pneumonia, and adult males showed mainly chronic obstructive pulmonary disease and pneumonia.

Finding viral characteristics associated with periodic upsurges and/or disease outcomes will help to define EV-D68 epidemiological trends. In this study, 59 VP1 gene sequences were successfully sequenced. The phylogeny showed clustering by year of infection. Our local 2014 isolates clustered in subclades B1, B2, and A2/D1, with strains circulating in Europe and the United States in 2014,^{11, 32} whereas almost all isolates from 2018 belonged to the recently described A2/D3 subclade and some to B3, also detected in other regions from Spain¹⁷ and other European countries in 2018.^{13, 14} B3 subclade has been circulating in Asia since, at least, 2013³³ and in Europe since 2014 or earlier.³⁴ The introduction of subclade B3 in Spain was previously estimated in 2016,¹⁷ but the current study shows that this subclade was present in the Valencia Region since 2015. In 2016, this subclade B3 replaced the others in the European epidemics (including Spain) and was later widespread worldwide^{17, 35} mostly associated with neurological outcomes.³⁶ However, in our region, the B3 subclade had a very low prevalence in ILI, with only one positive detected in 2016 belonging to subclade A2/D1. On the other hand, A2/D3 subclade viruses were first detected in China in 2016 in co-circulation with B3,³¹ but this A2/D3 subclade was detected for the first time in the Valencia Region in 2018, becoming the most prevalent subclade that year, as in the rest of Spain, Italy, and France.^{13, 14, 17} In this study, A2/D1 and A2/D3 subclades were detected only in adult patients, a trend that had previously been observed in Spain, and other countries.^{13, 14, 17, 31} All but one of the patients were >50 years old and presented respiratory infections, resulting in two fatal outcomes and one (43 years old) ICU admission. Furthermore, all isolated viruses from adult patients diagnosed with acute lower respiratory tract infection, chronic obstructive lung disease, or pneumonia also belonged to A2/D1 and A2/D3 subclades. Interestingly, a neurotropic potential of the A2/D1 subclade was suggested,^{13, 17} but increased pathogenicity in older adults is not yet demonstrated. Alternatively, these new subclades could be antigenically distant from the ones previously infected older adults, resulting in a more vulnerability to reinfection and/or more severe infections,^{37, 38} whereas children infected by these subclades may not show significant disease to require admission.

Escape from antibody neutralization is probably important in EV upsurges and epidemics. Analysis of VP1 gene sequences revealed that EV-D68 strains circulating worldwide since, at least, the 1990s presented 10 common amino acid substitutions as compared to the Fermon prototype, two of them in the BC- and DE-loops, and four in the C-terminus, showing a “KER…A” substitution pattern that is present in the sequence of an EV-D68-specific peptide that was immunoreactive in AFM patients –no analysis was performed in EV-D68 positive patients who developed severe respiratory diseases without the development of AFM–.³⁹ The subclade with more mutations at antigenic sites in our isolates was B3 with 68.2% of VP1 substitutions located in the BC-, DE-, GH-loops and C-terminal regions—important for cell receptor viral attachment⁴⁰—, followed by subclade B2, clade C, subclade B1, subclade A2/D1, clade A1, subclade A2/D2, and finally the subclade A2/D3 (52%). Mutations were more frequent in the BC-loop for subclades B1, B3, A2/D1, and A2/D3; in the DE-loop for clade A; in BC- and DE-loops for clade C and subclade B2; and in the C-terminal region for the A2/D2 subclade. These results support the hypothesis that different EV-D68 clades/subclades/subgroups may have different antigenicity.⁴⁰

In summary, two EV-D68 epidemics linked to ILI hospitalized cases occurred in the Valencia Region during 2014–2020, one in 2014 and another in 2018. The first one affected more children and males and the second one, adults and females. We have no evidence of neurological pathologies among the patients, but most of them presented acute respiratory infections, with three fatal outcomes and one ICU admission. The EV-D68 strains circulating in our region in these epidemics were closed relatives to those concurrently circulating in Europe and worldwide. A limitation of our study is that ILI surveillance was mostly limited to the analysis of respiratory samples during influenza seasons (with the exception of the 2017/2018 and 2018/2019 seasons). Thus, we are missing a time window with significant EV circulation, and this may explain why we were not able to detect the 2016 upsurge while other regions confirmed it.¹⁶ Another limitation of our study is related to the diagnosis of AFM. As the main objective of the VAHNSI network is to select the diagnosis most closely related to influenza or its complications, if the AFM diagnosis is in diagnostic positions other than the primary and secondary positions, we do not have access to this information. Furthermore, the VAHNSI network does not follow up with patients after discharge, so we also do not know if some of them developed AFM a few weeks after their discharge for severe respiratory infection due to EV-D68.

Our results and other published studies support the need to reinforce the epidemiological surveillance of enteroviruses in ILI all year-round and carry out a clinical follow-up of infected patients after discharge. Identification of viral clusters linked to severe outcomes (either respiratory or neurological) will likely need a collaborative effort and multicentre studies,⁴¹ including more detailed genomic characterization (e.g., whole-genome sequencing using NGS) to detect recombination and mutational profiles potentially linked to EV-D68 virulence, and for a better understanding of EV-D68 circulation at local and global levels.

AUTHOR CONTRIBUTIONS

Beatriz Mengual-Chuliá: Conceptualization; data curation; formal analysis; investigation; visualization; methodology; writing. Rafael Tamayo-Trujillo: Investigation; visualization, writing. Ainara Mira-Iglesias: Data curation; formal analysis; methodology; writing. Laura Cano: Investigation. Sandra García-Esteban: Investigation. Maria Loreto Ferrús: Investigation. Joan Puig-Barberà: Methodology. Javier Díez-Domingo: Investigation; project administration; supervision. F. Xavier López-Labrador: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; supervision; writing.

VAHNSI Group author contributions: Mario Carballido-Fernández: Investigation; project administration. Juan Mollar-Maseres: Investigation; project administration. Miguel Tortajada-Girbes: Investigation; project administration. Germán Schwarz-Chavarri: Investigation; project administration. Vicente Gil-Guillén: Investigation; project administration. Ramón Limón-Ramírez: Investigation; project administration. Empar Carbonell-Franco: Investigation; project administration. Ángel Belenguer-Varea: Investigation; project administration. Concepción Carratalá-Munuera: Investigation; project administration. José Vicente Tuells-Hernández: Investigation; project administration.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

The Ethics Research Committee of the Dirección General de Salud Pública-Centro Superior de Investigación en Salud Pública (DGSP-CSISP) and of the Hospital Arnau de Vilanova approved the protocol of the study. All patients or caregivers signed written informed consent before inclusion in the study.