Epidemiological and Clinical Insights into the Enterovirus D68 Upsurge in Europe 2021-2022 and Emergence of Novel B3-Derived Lineages, ENPEN Multicentre Study.

Enterovirus D68 (EV-D68) infections are associated with severe respiratory disease and acute flaccid myelitis (AFM). The European Non-Polio Enterovirus Network (ENPEN) aimed to investigate the epidemiological and genetic characteristics of EV-D68 infections and its clinical impact during the fall-winter season of 2021-2022. From 19 European countries, 58 institutes reported 10 481 (6.8%) EV-positive samples of which 1004 (9.6%) were identified as EV-D68 (including 852 respiratory samples). Clinical data were reported for 969 cases; 78.9% of infections were reported in children (0-5 years); and 37.9% of cases were hospitalized. Acute respiratory distress was commonly noted (93.1%) followed by fever (49.4%). Neurological problems were observed in 6.4% of cases including 6 diagnosed with AFM. Phylodynamic/Nextstrain and phylogenetic analyses based on 694 sequences showed the emergence of 2 novel B3-derived lineages, with no regional clustering. In conclusion, we describe a large-scale European EV-D68 upsurge with severe clinical impact and the emergence of B3-derived lineages.

Assessment of twelve echovirus virus-neutralisation assays in Europe: recommendations for harmonisation of non-polio enterovirus sero-surveillance studies.

Non-polio enteroviruses (NPEV) cause significant disease worldwide. Population-based sero-surveillance, by measuring antibodies against specific NPEV types, provides additional information on past circulation and the prediction for future upsurges. Virus neutralisation assays (VNA), the current method of choice for measuring NPEV type specific antibodies, are not entirely standardised. Via the European Non-Polio Enterovirus Network, we organised a VNA quality assessment in which twelve laboratories participated. We provided five echovirus (E) types (E1, E18, E30 G2, E30 G6 and E6) and intravenous immunoglobulins (IVIG) as a sample for the NPEV VNA quality assessment. Differences in VNA protocols and neutralising Ab (nAb) titres were found between the participating laboratories with geometric coefficients of variation ranging from 10.3-62.9 %. Mixed-effects regression analysis indicated a small but significant effect of type of cell line used. Harmonisation of cell line passage number, however, did not improve variation between laboratories. Calibration by making use of a reference sample, reduced variation between laboratories but differences in nAb titres remained higher than two log2 dilution steps. In conclusion, sero-surveillance data from different laboratories should be compared with caution and standardised protocols are needed.

Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

Molecular characterization of enterovirus detected in cerebrospinal fluid and wastewater samples in Monastir, Tunisia, 2014-2017.

BACKGROUND: Enteroviruses (EVs) are considered the main causative agents responsible for aseptic meningitis worldwide. This study was conducted in the Monastir region of Tunisia in order to know the prevalence of EV infections in children with meningitis symptoms. Detected EV types were compared to those identified in wastewater samples. METHODS: Two hundred CSF samples collected from hospitalized patients suspected of having aseptic meningitis for an EV infection between May 2014 and May 2017 and 80 wastewater samples collected in the same time-period were analyzed. EV detection and genotyping were performed using PCR methods followed by sequencing. Phylogenetic analyses in the 3'-VP1 region were also carried-out. RESULTS: EVs were detected in 12% (24/200) CSF and in 35% (28/80) wastewater samples. EV genotyping was reached in 50% (12/24) CSF-positive samples and in 64% (18/28) sewage. Most frequent types detected in CSF were CVB3, E-30 and E-9 (25% each). In wastewater samples, the same EVs were identified, but also other types non-detected in CSF samples, such as E-17,CVA9 and CVB1 from EV species B, and EV-A71 and CVA8 from EV-A, suggesting their likely lower pathogenicity. Phylogenetic analysis showed that within the same type, different strains circulate in Tunisia. For some of the EV types such as E-9, E-11 or CVB3, the same strains were detected in CSF and wastewater samples. CONCLUSIONS: Epidemiological studies are important for the surveillance of the EV infections and to better understand the emergence of certain types and variants.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

An imported case of vaccine-derived poliovirus type 2, Spain in the context of the ongoing polio Public Health Emergency of International Concern, September 2021.

The monthly retrospective search for unreported acute flaccid paralysis (AFP) cases conducted as a complementary component of the Spanish AFP surveillance system identified a case of AFP in a child admitted in Spain from Senegal during August 2021. Vaccine-derived poliovirus 2 was identified in the stool in September 2021. We present public health implications and response undertaken within the framework of the National Action Plan for Polio Eradication and the Public Health Emergency of International Concern.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016.

IntroductionEnterovirus A71 (EV-A71) is an emerging pathogen that causes a wide range of disorders including severe neurological manifestations. In the past 20 years, this virus has been associated with large outbreaks of hand, foot and mouth disease with neurological complications in the Asia-Pacific region, while in Europe mainly sporadic cases have been reported. In spring 2016, however, an EV-A71 outbreak associated with severe neurological cases was reported in Catalonia and spread further to other Spanish regions.AimOur objective was to investigate the epidemiology and clinical characteristics of the outbreak.MethodsWe carried out a retrospective study which included 233 EV-A71-positive samples collected during 2016 from hospitalised patients. We analysed the clinical manifestations associated with EV-A71 infections and performed phylogenetic analyses of the 3'-VP1 and 3Dpol regions from all Spanish strains and a set of EV-A71 from other countries.ResultsMost EV-A71 infections were reported in children (mean age: 2.6 years) and the highest incidence was between May and July 2016 (83%). Most isolates (218/233) were classified as subgenogroup C1 and 217 of them were grouped in one cluster phylogenetically related to a new recombinant variant strain associated with severe neurological diseases in Germany and France in 2015 and 2016. Moreover, we found a clear association of EV-A71-C1 infection with severe neurological disorders, brainstem encephalitis being the most commonly reported.ConclusionAn emerging recombinant variant of EV-A71-C1 was responsible for the large outbreak in 2016 in Spain that was associated with many severe neurological cases.

Acute flaccid paralysis (AFP) surveillance: challenges and opportunities from 18 years' experience, Spain, 1998 to 2015.

Acute flaccid paralysis (AFP) surveillance is key for global polio eradication. It allows detecting poliovirus (PV) reintroductions from endemic countries. This study describes AFP surveillance in Spain from 1998 to 2015. During this time, 678 AFP cases were reported to the Spanish National Surveillance Network. The mean notification rate was 0.58 AFP cases/100,000 population under 15 years old (range: 0.45/100,000-0.78/100,000). Two periods (P) are described: P1 (1998-2006) with the AFP notification rate ranging from 0.66/100,000 to 0.78/100,000, peaking in 2001 (0.84/100,000); and P2 (2007-2015) when the AFP rate ranged from 0.43/100,000 to 0.57/100,000, with the lowest rate in 2009 (0.31/100,000). No poliomyelitis cases were caused by wild PV infections, although two Sabin-like PVs and one imported vaccine-derived PV-2 were detected. Overall, 23 (3.4%) cases met the hot case definition. Most cases were clinically diagnosed with Guillain-Barré syndrome (76.9%; 504/655). The adequate stool collection rate ranged from 33.3% (7/21) to 72.5% (29/40). The annual proportion of AFP cases with non-polio enterovirus findings varied widely across the study period. AFP surveillance with laboratory testing for non-polio enteroviruses must be maintained and enhanced both to monitor polio eradication and to establish sensitive surveillance for prompt detection of other enteroviruses causing serious symptoms.

Molecular epidemiology of enterovirus and parechovirus infections according to patient age over a 4-year period in Spain.

The epidemiology and clinical association of enterovirus (EV) and parechovirus (HPeV) infections, as well as the type-distribution-according-to-age, were determined during a 4-year study period in Spain. During 2010-2013, a total of 21,832 clinical samples were screened for EV and the detection frequency was 6.5% (1,430). Of the total EV-negative samples, only 1,873 samples from 2011 to 2013 were available for HPeV testing. HPeV was detected in 42 (2%) of them. Positive samples were genotyped using PCR and sequencing. EV infections occurred in all age groups of patients: neonates (17%), children 28 days to 2 years (29%), children 2-14 years (40%), and adults (14%). Thirty-four different EV types were identified. HPeV infections were detected exclusively in infants <8 m (70% neonates, P < 0.05). All but one HPeV were HPeV-3. Differences in type frequency detection were found according to age and clinical manifestation. Coxsackievirus (CV)-B4 (61%), CV-B5 (83%), and HPeV-3 (64%) were more frequent in neonates than in older patients (P < 0.05). Echovirus (E)-3 (60%), E-18 (47%), E-25 (62%), CV-A6 (61%), CV-A16 (72%), and EV-71 (75%) were mainly detected in children 28 days to 2 years (P < 0.05), whereas, E-6 (79%), E-20 (88%), and E-30 (85%) were predominant in children >2 years and adults (P < 0.05). Clinically, meningitis was associated with EV (P < 0.01) whereas, encephalitis was more frequent in HPeV-infected patients. CV-B types were associated with myocarditis (90%; P < 0.05) and EV species A with hand-foot-mouth-disease/atypical exanthema (88%; P < 0.05). J. Med. Virol. 89:435-442, 2017. © 2016 Wiley Periodicals, Inc.

Molecular epidemiology and the evolution of human coxsackievirus A6.

Coxsackievirus A6 (CV-A6) is a major aetiologic agent for hand, foot and mouth disease (HFMD) in recent years. HFMD outbreaks associated with CV-A6 resulted from the evolutionary dynamics of CV-A6 and the appearance of novel recombinant forms (RFs). To examine this, 151 variants collected in 2013 and 2014 from Germany, Spain, Sweden, Denmark and Thailand were genotyped for the VP1 capsid and 3Dpol genes. Analysis of the VP1 gene showed an increasing correspondence between CV-A6 genome recombination and sequence divergence (estimated substitution rate of 8.1×10-3 substitutions site-1 year-1 and RF half-life of 3.1 years). Bayesian phylogenetic analysis showed that recent recombination groups (RF-E, -F, -H, -J and -K) shared a common ancestor (RF-A). Thirty-nine full-length genomes of different RFs revealed recombination breakpoints between the 2A-2C and the 5' UTRs. The emergence of new CV-A6 recombination groups has become widespread in Europe and Asia within the last 8 years.

Molecular epidemiology of coxsackievirus B3 infection in Spain, 2004-2014.

Epidemiological and clinical characteristics of coxsackievirus B3 infections in Spain were investigated. This enterovirus (EV) type was detected mainly in young children (<6 months) and was associated with neurological (78 %) and respiratory diseases (10 %) but also with myo/pericarditis (10 %). Two myocarditis cases were fatal. Phylogenetic analysis of the VP1 region showed that genotype III circulated in the country between 2004 and 2008 and was replaced by genotype V in 2010. Furthermore, phylogenetic analysis of the 3D region indicated that recombination events have occurred and contributed to the genetic evolution of this EV type.

Clinical characteristics and molecular epidemiology of Enterovirus infection in infants <3 months in a referral paediatric hospital of Barcelona.

UNLABELLED: Enterovirus (EV) infection is common in infants, but the information with regard to the molecular epidemiology and the associations between types and clinical variables is very scarce. This study includes 195 children <3 months old with fever, attended from March 2010 to December 2012 in an emergency department of a tertiary paediatric hospital in whom EV infection was confirmed by real-time PCR in blood and/or cerebrospinal fluid. Clinical and epidemiological data was prospectively collected. In 152 (77.9 %) patients, EVs could be typed. The most common type was Echovirus-5 (E5; 32, 21.1 %), followed by Echovirus-11 (E11; 18, 11.8 %), Echovirus-21 and Echovirus-25 (E21, E25; 11 each one, 7.2 %) and Coxsackievirus-B4 (CVB4; 6, 6.6 %). The majority of types appeared in spring, but E5 and E25 were found mainly during summer (p < 0.01). E21 was associated with high-grade fever (p < 0.01); E5 with exanthema (p = 0.03) and CVB4 tended to cause meningitis more often than the other types (p = 0.07). CONCLUSION: The most common EV types were Echovirus-5 and Echovirus-11. Some significant associations between types and epidemiologic and clinical findings were observed. What is Known-What is New • Enteroviruses cause a normally benign illness in young infants, except in some cases. • The molecular epidemiology of Enterovirus infection is not well known in European countries. • This study describes a large number of infants with Enterovirus infection and shows the seasonality of different types, and their associations with epidemiologic and clinical variables.

Comparison of epidemiology and clinical characteristics of infections by human parechovirus vs. those by enterovirus during the first month of life.

UNLABELLED: Human parechoviruses (HPeV) have been recently recognized as important viral agents in paediatric infections. The aims of this study were to investigate the HPeV infection prevalence in infants <1 month in Spain and, secondly, to analyse the clinical and epidemiological characteristics of the infected patients compared with those infected by enterovirus (EV). Infants <1 month with neurological or systemic symptoms were included in a multicentre prospective study. EV and HPeV detection by RT-PCR and genotyping were performed in cerebrospinal fluids (CSF), sera or throat swabs. Out of the total of 84 infants studied during 2013, 32 were EV positive (38 %) and 9 HPeV positive (11 %). HPeV-3 was identified in eight cases and HPeV-5 in one. Mean age of HPeV-positive patients was 18 days. Diagnoses were fever without source (FWS) (67 %), clinical sepsis (22 %) and encephalitis (11 %). Leukocytes in blood and CSF were normal. Pleocytosis (p = 0.03) and meningitis (p = 0.001) were significantly more frequent in patients with EV infections than with HPeV. CONCLUSIONS: Although HPeV-3 infections were detected less frequently than EV, they still account for approximately 10 % of the cases analysed in infants younger than 1 month. HPeV-3 was mainly associated with FWS and without leukocytosis and pleocytosis in CSF. In these cases, HPeV screening is desirable to identify the aetiologic agent and prevent unnecessary treatment and prolonged hospitalization.

Recombination and evolutionary dynamics of human echovirus 6.

Enterovirus (EV) infections are associated with a wide array of often severe disease presentations including aseptic meningitis, encephalitis, and acute flaccid paralysis. Surveillance for polioviruses and other EVs is therefore important as a public health measure both for patient management and epidemiological studies. From 1988 to 2008, echovirus (E) 30 was the predominant genotype in Spain (33.7% of the total typed EVs). E6 was also endemic throughout this period although isolated less frequently (12.5%). In 2009, however, a substantial increase in the incidence of E6 was detected (60%), displacing E30 type (2%). To investigate the evolution and recombination in the epidemiology and transmission of E6 in Spain, a genetic analysis in VP1 and 3Dpol regions of 67 Spanish strains collected during the period 2004-2010 was performed. All VP1 sequences clustered monophyletically in the assigned genogroup C, subgroup 9, currently the predominant circulating strains identified in Europe and elsewhere in the last 10 years. 3Dpol sequences were interspersed with other species B EVs resulting from several recombination events that generated at least 12 different recombinant forms (RFs) among study samples. These showed typically minimal divergence in VP1. The co-circulation of different lineages of E6 in the same geographical area associated with its mainly endemic pattern of transmission may have contributed to the extremely short estimated half-life of E6 RFs (0.87 years). This pattern contrasts markedly with other species B EVs and EV71 where VP1 lineage expansion and extinction occurred in step with defined recombination events and periodic changes in incidence.

Impact of the bacterial nasopharyngeal microbiota on the severity of genus enterovirus lower respiratory tract infection in children: A case-control study.

INTRODUCTION: Rhinoviruses (RV) and enteroviruses (EV) are among the main causative etiologies of lower respiratory tract infection (LRTI) in children. The clinical spectrum of RV/EV infection is wide, which could be explained by diverse environmental, pathogen-, and host-related factors. Little is known about the nasopharyngeal microbiota as a risk factor or disease modifier for RV/EV infection in pediatric patients. This study describes distinct nasopharyngeal microbiota profiles according to RV/EV LRTI status in children. METHODS: Cross-sectional case-control study, conducted at Hospital Sant de Déu (Barcelona, Spain) from 2017 to 2020. Three groups of children <5 years were included: healthy controls without viral detection (Group A), mild or asymptomatic controls with RV/EV infection (Group B), and cases with severe RV/EV infection admitted to the pediatric intensive care unit (PICU) (Group C). Nasopharyngeal samples were collected from participants for viral DNA/RNA detection by multiplex-polymerase chain reaction and bacterial microbiota characterization by 16S rRNA gene sequencing. RESULTS: A total of 104 subjects were recruited (A = 17, B = 34, C = 53). Children's nasopharyngeal microbiota composition varied according to their RV/EV infection status. Richness and diversity were decreased among children with severe infection. Nasopharyngeal microbiota profiles enriched in genus Dolosigranulum were related to respiratory health, while genus Haemophilus was specifically predominant in children with severe RV/EV LRTI. Children with mild or asymptomatic RV/EV infection showed an intermediate profile. CONCLUSIONS: These results suggest a close relationship between the nasopharyngeal microbiota and different clinical presentations of RV/EV infection.

Metagenomic sequencing, molecular characterization, and Bayesian phylogenetics of imported type 2 vaccine-derived poliovirus, Spain, 2021.

Introduction: In 2021, a type 2 vaccine-derived poliovirus (VDPV2) was isolated from the stool of a patient with acute flaccid paralysis (AFP) admitted to Spain from Senegal. A virological investigation was conducted to characterize and trace the origin of VDPV2. Methods: We used an unbiased metagenomic approach for the whole-genome sequencing of VDPV2 from the stool (pre-treated with chloroform) and from the poliovirus-positive supernatant. Phylogenetic analyses and molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology were used to determine the geographical origin and estimate the date of the initiating dose of the oral poliovirus vaccine for the imported VDPV2. Results: We obtained a high percentage of viral reads per total reads mapped to the poliovirus genome (69.5% for pre-treated stool and 75.8% for isolate) with a great depth of sequencing coverage (5,931 and 11,581, respectively) and complete genome coverage (100%). The two key attenuating mutations in the Sabin 2 strain had reverted (A481G in the 5'UTR and Ile143Thr in VP1). In addition, the genome had a recombinant structure between type-2 poliovirus and an unidentified non-polio enterovirus-C (NPEV-C) strain with a crossover point in the protease-2A genomic region. VP1 phylogenetic analysis revealed that this strain is closely related to VDPV2 strains circulating in Senegal in 2021. According to Bayesian phylogenetics, the most recent common ancestor of the imported VDPV2 could date back 2.6 years (95% HPD: 1.7-3.7) in Senegal. We suggest that all VDPV2s circulating in 2020-21 in Senegal, Guinea, Gambia, and Mauritania have an ancestral origin in Senegal estimated around 2015. All 50 stool samples from healthy case contacts collected in Spain (n = 25) and Senegal (n = 25) and four wastewater samples collected in Spain were poliovirus negative. Discussion: By using a whole-genome sequencing protocol with unbiased metagenomics from the clinical sample and viral isolate with high sequence coverage, efficiency, and throughput, we confirmed the classification of VDPV as a circulating type. The close genomic linkage with strains from Senegal was consistent with their classification as imported. Given the scarce number of complete genome sequences for NPEV-C in public databases, this protocol could help expand poliovirus and NPEV-C sequencing capacity worldwide.

Rare Recombinant GI.5[P4] Norovirus That Caused a Large Foodborne Outbreak of Gastroenteritis in a Hotel in Spain in 2021.

Noroviruses are among the most important causes of acute gastroenteritis (AGE). In summer 2021, a large outbreak of norovirus infections affecting 163 patients, including 15 norovirus-confirmed food handlers, occurred in a hotel in Murcia in southeast Spain. A rare GI.5[P4] norovirus strain was identified as the cause of the outbreak. The epidemiological investigation determined that norovirus transmission might have been initiated through an infected food handler. The food safety inspection found that some symptomatic food handlers continued working during illness. Molecular investigation with whole-genome and ORF1 sequencing provided enhanced genetic discrimination over ORF2 sequencing alone and enabled differentiation of the GI.5[P4] strains into separate subclusters, suggesting different chains of transmission. These recombinant viruses have been identified circulating globally over the last 5 years, warranting further global surveillance. IMPORTANCE Due to the large genetic diversity of noroviruses, it is important to enhance the discriminatory power of typing techniques to differentiate strains when investigating outbreaks and elucidating transmission chains. This study highlights the importance of (i) using whole-genome sequencing to ensure genetic differentiation of GI noroviruses to track chains of transmission during outbreak investigations and (ii) the adherence of symptomatic food handlers to work exclusion rules and strict hand hygiene practices. To our knowledge, this study provides the first full-length genome sequences of GI.5[P4] strains apart from the prototype strain.