Enterovirus A71 crosses a human blood-brain barrier model through infected immune cells.

Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an in vitro blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.

Increase over time of antibody levels 3 months after a booster dose as an indication of better protection against Omicron infection.

The third, "booster", vaccination increases the overall immune response against SARS-CoV-2 variants. However, after the initial peak at around 3 weeks post-vaccination, anti-spike antibody levels decline. Post-booster kinetics of cellular response has been less investigated and there is no documented evidence of a true boosting effect. Furthermore, multiple studies underline the less effective immune responses against Omicron, the latest variant of concern, at both humoral and cellular levels. In this letter, we analyse humoral (anti-RBD IgG levels) and cellular (IFN-γ release assay) immune response in 205 health care workers 3 weeks and 3 months after administration of an mRNA-based booster dose, either mRNA-1273 or BNT162b2. Since all subjects were SARS-CoV-2 infection-naïve, we also looked at the incidence of Omicron infection between 3 and 6 months post-booster.At both timepoints, 3x mRNA-1273 vaccination had the highest overall antibody and IFN-γ levels, followed by 3x BNT162b2 vaccination and heterologous mRNA-based regimens. Heterologous ChAdOx1-mRNA-based regimen had the lowest antibody levels while cellular response equal to that of 3x BNT162b2 vaccination and heterologous mRNA-based regimens. Our results show that both humoral and cellular responses waned at 3 months for all vaccination regimens. However, we identified three trajectories of dosage variation. Interestingly, the subgroup of subjects with increasing anti-RBD IgG levels over time had a lower incidence of Omicron infection. Whether increasing humoral response at 3 months post-booster is more indicative of protection than a high initial peak remains to be confirmed in a larger cohort.

Evidence of co-infections during Delta and Omicron SARS-CoV-2 variants co-circulation through prospective screening and sequencing.

OBJECTIVES: To describe Delta/Omicron SARS-CoV-2 variants co-infection detection and confirmation during the fifth wave of COVID-19 pandemics in France in 7 immunocompetent and epidemiologically unrelated patients. METHODS: Since December 2021, the surveillance of Delta/Omicron SARS-CoV-2 variants of concern (VOC) circulation was performed through prospective screening of positive-samples using single nucleotide polymorphism (SNP) PCR assays targeting SARS-CoV-2 S-gene mutations K417N (Omicron specific) and L452R (Delta specific). Samples showing unexpected mutational profiles were further submitted to whole genome sequencing (WGS) using three different primer sets. RESULTS: Between weeks 49-2021 and 02-2022, SARS-CoV-2 genome was detected in 3831 respiratory samples, of which 3237 (84.5%) were screened for VOC specific SNPs. Unexpected mutation profiles suggesting a dual Delta/Omicron population were observed in 7 nasopharyngeal samples (0.2%). These co-infections were confirmed by WGS. For 2 patients, the sequence analyses of longitudinal samples collected 7 to 11 days apart showed that Delta or Omicron can outcompete the other variant during dual infection. Additionally, for one of these samples, a recombination event between Delta and Omicron was detected. CONCLUSIONS: This work demonstrates that SARS-CoV-2 Delta/Omicron co-infections are not rare in high virus co-circulation periods. Moreover, co-infections can further lead to genetic recombination which may generate new chimeric variants with unpredictable epidemic or pathogenic properties that could represent a serious health threat.

Coxsackievirus A6 Recombinant Subclades D3/A and D3/H Were Predominant in Hand-Foot-And-Mouth Disease Outbreaks in the Paediatric Population, France, 2010-2018.

Coxsackievirus A6 (CVA6) emerged as the most common enterovirus of seasonal outbreaks of hand-foot-and-mouth disease (HFMD). We investigated CVA6 genetic diversity among the clinical phenotypes reported in the paediatric population during sentinel surveillance in France between 2010 and 2018. CVA6 infection was confirmed in 981 children (mean age 1.52 years [IQR 1.17-2.72]) of whom 564 (58%) were males. Atypical HFMD was reported in 705 (72%) children, followed by typical HFMD in 214 (22%) and herpangina in 57 (6%) children. Throat specimens of 245 children were processed with a target-enrichment new-generation sequencing approach, which generated 213 complete CVA6 genomes. The genomes grouped within the D1 and D3 clades (phylogeny inferred with the P1 genomic region). In total, 201 genomes were classified among the recombinant forms (RFs) A, B, F, G, H, and N, and 12 genomes were assigned to 5 previously unreported RFs (R-V). The most frequent RFs were A (58%), H (19%), G (6.1%), and F (5.2%). The yearly number of RFs ranged between 1 (in 2012 and 2013) and 6 (2018). The worldwide CVA6 epidemic transmission began between 2005 and 2007, which coincided with the global spread of the recombinant subclade D3/RF-A.

Decline of Humoral and Cellular Immune Responses Against SARS-CoV-2 6 Months After Full BNT162b2 Vaccination in Hospital Healthcare Workers.

Clinical trials and real-world evidence on COVID-19 vaccines have shown their effectiveness against severe disease and death but the durability of protection remains unknown. We analysed the humoral and T-cell immune responses in 110 healthcare workers (HCWs) vaccinated according to the manufacturer's recommended schedule of dose 2 three weeks after dose 1 from a prospective on-going cohort in early 2021, 3 and 6 months after full vaccination with the BNT162b2 mRNA vaccine. Anti-RBD IgG titres were lower in HCWs over 60 years old 3 months after the second dose (p=0.03) and declined in all the subjects between 3 and 6 months with a median percentage change of -58.5%, irrespective of age and baseline comorbidities. Specific T-cell response measured by IGRA declined over time by at least 42% (median) in 91 HCWs and increased by 33% (median) in 17 others. Six HCWs had a negative T-cell response at 6 months. Ongoing follow-up should provide correlates of long-term protection according to the different immune response profiles observed. COVIDIM study was registered under the number NCT04896788 on clinicaltrials.gov.

A large-scale outbreak of hand, foot and mouth disease, France, as at 28 September 2021.

We report a large-scale outbreak of hand, foot and mouth disease (HFMD) in France. As at 28 September 2021, 3,403 cases have been reported (47% higher than in 2018-19). We prospectively analysed 210 clinical samples; 190 (90.5%) were enterovirus-positive. Most children presented with atypical HFMD. Coxsackievirus (CV)A6 (49.5%; 94/190) was predominant; no enterovirus A71 was detected. Dermatological and neurological complications of HFMD justify prospective syndromic and virological surveillance for early detection of HFMD outbreaks and identification of associated types.

Retrospective Study of the Upsurge of Enterovirus D68 Clade D1 among Adults (2014-2018).

Enterovirus D68 (EV-D68) has emerged as an agent of epidemic respiratory illness and acute flaccid myelitis in the paediatric population but data are lacking in adult patients. We performed a 4.5-year single-centre retrospective study of all patients who tested positive for EV-D68 and analysed full-length EV-D68 genomes of the predominant clades B3 and D1. Between 1 June 2014, and 31 December 2018, 73 of the 11,365 patients investigated for respiratory pathogens tested positive for EV-D68, of whom 20 (27%) were adults (median age 53.7 years [IQR 34.0-65.7]) and 53 (73%) were children (median age 1.9 years [IQR 0.2-4.0]). The proportion of adults increased from 12% in 2014 to 48% in 2018 (p = 0.01). All adults had an underlying comorbidity factor, including chronic lung disease in 12 (60%), diabetes mellitus in six (30%), and chronic heart disease in five (25%). Clade D1 infected a higher proportion of adults than clades B3 and B2 (p = 0.001). Clade D1 was more divergent than clade B3: 5 of 19 amino acid changes in the capsid proteins were located in putative antigenic sites. Adult patients with underlying conditions are more likely to present with severe complications associated with EV-D68, notably the emergent clade D1.

Monitoring of enterovirus diversity in wastewater by ultra-deep sequencing: An effective complementary tool for clinical enterovirus surveillance.

In a one-year (October 2014-October 2015) pilot study, we assessed wastewater monitoring with sustained sampling for analysis of global enterovirus (EV) infections in an urban community. Wastewater was analysed by ultra-deep sequencing (UDS) after PCR amplification of the partial VP1 capsid protein gene. The nucleotide sequence analysis showed an unprecedented diversity of 48 EV types within the community, which were assigned to the taxonomic species A (n = 13), B (n = 23), and C (n = 12). During the same period, 26 EV types, of which 22 were detected in wastewater, were identified in patients referred to the teaching hospital serving the same urban population. Wastewater surveillance detected a silent circulation of 26 EV types including viruses reported in clinically rare respiratory diseases. Wastewater monitoring as a supplementary procedure can complement clinical surveillance of severe diseases related to non-polio EVs and contribute to the final stages of poliomyelitis eradication.

Multirecombinant Enterovirus A71 Subgenogroup C1 Isolates Associated with Neurologic Disease, France, 2016-2017.

In 2016, an upsurge of neurologic disease associated with infection with multirecombinant enterovirus A71 subgenogroup C1 lineage viruses was reported in France. These viruses emerged in the 2000s; 1 recombinant is widespread. This virus lineage has the potential to be associated with a long-term risk for severe disease among children.

Assessment of blood enterovirus PCR testing in paediatric populations with fever without source, sepsis-like disease, or suspected meningitis: a prospective, multicentre, observational cohort study.

BACKGROUND: Enteroviruses are the most frequent cause of acute meningitis and are seen increasingly in sepsis-like disease and fever without source in the paediatric population. Detection of enterovirus in cerebrospinal fluid (CSF) specimens by PCR is the gold standard diagnostic test. Our aim was to assess a method of detecting enterovirus in blood specimens by PCR. METHODS: We did a prospective, multicentre, observational study at 35 French paediatric and emergency departments in 16 hospitals. We recruited newborn babies (aged ≤28 days) and infants (aged >28 days to ≤2 years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged >2 years to ≤16 years) with suspected meningitis, who were admitted to a participating hospital. We used a standardised form to obtain demographic, clinical, and laboratory data, which were anonymised. Enterovirus PCR testing was done in blood and CSF specimens. FINDINGS: Between June 1, 2015, and Oct 31, 2015, and between June 1, 2016, and Oct 31, 2016, we enrolled 822 patients, of whom 672 had enterovirus PCR testing done in blood and CSF specimens. Enterovirus was detected in 317 (47%) patients in either blood or CSF, or both (71 newborn babies, 83 infants, and 163 children). Detection of enterovirus was more frequent in blood samples than in CSF specimens of newborn babies (70 [99%] of 71 vs 62 [87%] of 71; p=0·011) and infants (76 [92%] of 83 vs 62 [75%] of 83; p=0·008), and was less frequent in blood samples than in CSF specimens of children (90 [55%] of 163 vs 148 [91%] of 163; p<0·0001). Detection of enterovirus was more frequent in blood samples than in CSF specimens of infants aged 2 years or younger with fever without source (55 [100%] of 55 vs 41 [75%] of 55; p=0·0002) or with sepsis-like disease (16 [100%] of 16 vs nine [56%] of 16; p=0·008). Detection of enterovirus was less frequent in blood than in CSF of patients with suspected meningitis (165 [67%] of 246 vs 222 [90%] of 246; p<0·0001). INTERPRETATION: Testing for enterovirus in blood by PCR should be an integral part of clinical practice guidelines for infants aged 2 years or younger. This testing could decrease the length of hospital stay and reduce exposure to antibiotics for low-risk patients admitted to the emergency department with febrile illness. FUNDING: University Hospital Clermont-Ferrand.

Monitoring human enteric viruses in wastewater and relevance to infections encountered in the clinical setting: a one-year experiment in central France, 2014 to 2015.

BackgroundHuman enteric viruses are resistant in the environment and transmitted via the faecal-oral route. Viral shedding in wastewater gives the opportunity to track emerging pathogens and study the epidemiology of enteric infectious diseases in the community. Aim: The aim of this study was to monitor the circulation of enteric viruses in the population of the Clermont-Ferrand area (France) by analysis of urban wastewaters. Methods: Raw and treated wastewaters were collected between October 2014 and October 2015 and concentrated by a two-step protocol using tangential flow ultrafiltration and polyethylene glycol precipitation. Processed samples were analysed for molecular detection of adenovirus, norovirus, rotavirus, parechovirus, enterovirus (EV), hepatitis A (HAV) and E (HEV) viruses. Results: All wastewater samples (n = 54) contained viruses. On average, six and four virus species were detected in, respectively, raw and treated wastewater samples. EV-positive samples were tested for EV-D68 to assess its circulation in the community. EV-D68 was detected in seven of 27 raw samples. We collected data from clinical cases of EV-D68 (n = 17), HAV (n = 4) and HEV infection (n = 16) and compared wastewater-derived sequences with clinical sequences. We showed the silent circulation of EV-D68 in September 2015, the wide circulation of HAV despite few notifications of acute disease and the presence in wastewater of the major HEV subtypes involved in clinical local cases. Conclusion: The environmental surveillance overcomes the sampling bias intrinsic to the study of infections associated with hospitalisation and allows the detection in real time of viral sequences genetically close to those reported in clinical specimens.

Phylogeography of Coxsackievirus A16 Reveals Global Transmission Pathways and Recent Emergence and Spread of a Recombinant Genogroup.

Coxsackievirus A16 (CV-A16; Picornaviridae) is an enterovirus (EV) type associated with hand, foot, and mouth disease (HFMD) in children. To investigate the spatial spread of CV-A16, we used viral sequence data sampled during a prospective sentinel surveillance of HFMD in France (2010 to 2014) and phylogenetic reconstruction. A data set of 168 VP1 sequences was assembled with 416 publicly available sequences of various geographic origins. The CV-A16 sequences reported were assigned to two clades, genogroup B and a previously uncharacterized clade D. The time origins of clades B and D were assessed in 1978 (1973 to 1981) and 2004 (2001 to 2007), respectively. The shape of the global CV-A16 phylogeny indicated worldwide cocirculation of genetically distinct virus lineages over time and across geographic regions. Phylogenetic tree topologies and Bayes factor analysis indicated virus migration. Virus transportation events in clade B within Europe and Asia and between countries of the two geographic regions were assessed. The sustained transmission of clade D viruses over 4 years was analyzed at the township level in France and traced back to Peru in South America. Comparative genomics provided evidence of recombination between CV-A16 clades B and D and suggested an intertype recombinant origin for clade D. Time-resolved phylogenies and HFMD surveillance data indicated that CV-A16 persistence is sustained by continuing virus migration at different geographic scales, from community transmission to virus transportation between distant countries. The results showed a significant impact of virus movements on the epidemiological dynamics of HFMD that could have implications for disease prevention.IMPORTANCE Coxsackievirus A16 is one of the most prevalent enterovirus types in hand, foot, and mouth disease outbreaks reported in Southeast Asia. This study is based on epidemiological and viral data on HFMD caused by CV-A16 in a European country. The phylogeographic data complemented the syndromic surveillance with virus migration patterns between geographic regions in France. The results show how viral evolutionary dynamics and global virus spread interact to shape the worldwide pattern of an EV disease. CV-A16 transmission is driven by movements of infected individuals at different geographic levels: within a country (local dynamics), between neighboring countries (regional dynamics), and between distant countries (transcontinental dynamics). The results are consistent with our earlier data on EV-A71 and confirm the epidemiological interconnection of Asia and Europe with regard to EV infections.

Ambulatory Pediatric Surveillance of Hand, Foot and Mouth Disease as Signal of an Outbreak of Coxsackievirus A6 Infections, France, 2014-2015.

The clinical impact of enteroviruses associated with hand, foot and mouth disease (HFMD) is unknown outside Asia, and the prevalence of enterovirus A71 (EV-A71) in particular might be underestimated. To investigate the prevalence of enterovirus serotypes and the clinical presentations associated with HFMD in France, we conducted prospective ambulatory clinic-based surveillance of children during April 2014-March 2015. Throat or buccal swabs were collected from children with HFMD and tested for the enterovirus genome. Physical examinations were recorded on a standardized form. An enterovirus infection was detected in 523 (79.3%) of 659 children tested. Two epidemic waves occurred, dominated by coxsackievirus (CV) A6, which was detected in 53.9% of enterovirus-infected children. CV-A6 was more frequently related to atypical HFMD manifestations (eruptions extended to limbs and face). Early awareness and documentation of HFMD outbreaks can be achieved by syndromic surveillance of HFMD by ambulatory pediatricians and rapid enterovirus testing and genotyping.

Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011-2013): identification of virus variants cocirculating in France.

Acute enterovirus (EV) meningitis is a frequent cause of hospitalisation, and over 100 EV serotypes may be involved. A total of 215 patients of all ages with meningitis signs were investigated in 2 Tunisian hospitals. Their cerebrospinal fluid (CSF) was analysed retrospectively for EVs with a TaqMan real-time RT-qPCR. The virus strains were typed, and their evolutionary relationships were determined by Bayesian phylogenetic methods. An EV genome was detected in 21/215 patients (9.8%). The CSF viral loads ranged from 3.27 to 5.63 log10 genome copies/mL. The strains were identified in 13/21 patients and assigned to EV-B types. Viruses identified in Tunisian patients were genetically related to variants detected in France. The viral loads were similar in Tunisian and French patients for most EV types. The phylogenetic data and viral loads determined in Tunisian and French patients suggest that close EV variants were involved in aseptic meningitis in the 2 countries over a same period.

Transmission patterns of human enterovirus 71 to, from and among European countries, 2003 to 2013.

Enterovirus 71 (EV-71) is involved in epidemics of hand, foot, and mouth disease (HFMD) and has been reported to occur with severe neurological complications in eastern and south-east Asia. In other geographical areas, the transmission of this virus is poorly understood. We used large sequence datasets (of the gene encoding the viral protein 1, VP1) and a Bayesian phylogenetic approach to compare the molecular epidemiology and geographical spread patterns of EV-71 subgenogroups B4, B5, C1, C2, and C4 in Europe relative to other parts of the world. For the study, European countries considered were European Union (EU) Member States and Iceland, Norway and Switzerland. Viruses of the B4, B5, and C4 subgenogroups circulate mainly in eastern and south-east Asia. In Europe sporadic introductions of these subgenogroups are observed, however C1 and C2 viruses predominate. The phylogenies showed evidence of multiple events of spread involving C1 and C2 viruses within Europe since the mid-1990s. Two waves of sporadic C2 infections also occurred in 2010 and 2013. The 2007 Dutch outbreak caused by C2 and the occurrence of B5 and C4 infections in the EU between 2004 and 2013 arose while the circulation of C1 viruses was low. A transmission chain involving a C4 virus was traced from Japan to the EU and then further to Canada between 2001 and 2006. Recent events whereby spread of viruses have occurred from, to, and within Europe appear to be involved in the long term survival of EV-71, highlighting the need for enhanced surveillance of this virus.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood-brain barrier.

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.

Evaluation of real-time RT-PCR Rhino&EV/Cc r-gene(®) (bioMérieux) kit versions 1 and 2 for rhinovirus detection.

BACKGROUND: Human rhinoviruses (HRVs) are frequent etiologic agents of tract infections, ranging from benign upper to potentially life-threatening lower respiratory tract infections. Diagnosis is based on molecular methods. 169 HRV types, belonging to species A, B and C, have been identified. This high genetic diversity makes it difficult to accurately detect circulating HRVs and to diagnose severe infection. OBJECTIVES: To comparatively assess the ability to detect HRV clinical isolates of the first version (V1) of the commercial real-time RT-PCR Rhino&EV/Cc r-gene(®) (bioMérieux) kit, of an in-house RT-PCR followed by genotyping, considered as the reference method, and of the second version of this commercial test (V2). STUDY DESIGN: From September 2011 to April 2013, HRVs were prospectively detected in 2525 respiratory specimens, using V1 in combination with the in-house reference RT-PCR. In November 2013, 85 specimens that had given initially false negative results with V1 were retested simultaneously with V1 and V2 and the in-house RT-PCR. In addition, 421 negative specimens with the in-house assay were prospectively tested with V2. RESULTS: Among the 2525 specimens, V1 detected 80.7% (502/622) of in-house RT-PCR positive isolates: 85.3% (220/258) of HRV-A, 84.4% (27/32) of HRV-B and 74.9% (176/235) of HRV-C. Among the 85 respiratory samples tested with V1, V2 and the in-house RT-PCR, V2 was more efficient than V1 in detecting 16 HRV isolates: 11/33 (33.3%) of HRV-A and 5/47 (10.6%) of HRV-C tested. The analytical sensitivity of V2 was greater for 8/18 HRV-A genotypes and 2/22 HRV-C genotypes. Relative to the in-house assay, the specificity of V2 was 100% (421/421). CONCLUSIONS: This study showed a slightly higher sensitivity of V2. However, diverse genotypes, especially HRV-C, were undetected.

Variations in cerebrospinal fluid viral loads among enterovirus genotypes in patients hospitalized with laboratory-confirmed meningitis due to enterovirus.

BACKGROUND: Acute enterovirus (EV) meningitis is a major cause of hospitalization among adults and children. It is caused by multiple EV genotypes assigned to 4 species (EV-A, EV-B, EV-C, and EV-D). METHODS: We determined viral loads in the cerebrospinal fluid (CSF) of 156 patients of all ages with EV meningitis during a 5-year observational prospective study. The virus strains were genotyped, and their time origin was determined with Bayesian phylogenetic methods. RESULTS: The CSF viral loads ranged between 3.4 and 7.5 log10 copies/mL (median, 4.9 log10 copies/mL). They were higher in neonates than in infants and children (P = .02) but were comparable in adults. Viral loads were associated with EV genotypes (P < .001). The EV strains were identified in 152 of 156 patients and assigned to 23 genotypes within the EV-A and EV-B species. The most frequent genotypes, echoviruses 6 and 30, were associated with different viral loads (P < .001). The highest viral loads were in meningitis cases caused by coxsackievirus A9, B4, and B5 genotypes. Most patients infected by a same genotype were infected by a major virus variant of recent emergence. CONCLUSIONS: The variations in CSF viral loads in patients at the onset of EV meningitis are related to genotypic differences in the virus strains involved.