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Viruses are suspected to play a role in the multifactorial pathogenesis of sudden infant death. We described a sudden and unexpected death in a 5-month-old boy, with detection of both enterovirus and parechovirus RNA in the blood. This is the first report of a dual viraemia of enterovirus and parechovirus and its potential association with a sudden unexpected infant death. Extensive sampling and testing especially using molecular methods currently available is needed to better understanding the “hypothetical” link between viral infections and sudden infant death.

International audience; Acute flaccid myelitis (AFM) is an acute paralysis syndrome defined by a specific inflammation of the anterior horn cells of the spinal cord. From 2014, worrying waves of life-threatening AFM consecutive to enterovirus infection (EV-D68 and EV-A71) have been reported. We describe 10 children displaying an AFM with an EV infection, the treatments performed and the 1 to 3-years follow-up. Two groups of patients were distinguished: 6 children (“polio-like group”) had severe motor disability whereas 4 other children (“brainstem group”) displayed severe brainstem weakness requiring ventilation support. Electrodiagnostic studies (n = 8) support the presence of a motor neuronopathy associated to myelitis. The best prognosis factor seems to be the motor recovery after the first 4 weeks of the disease.

Infectious agents including viruses are thought to play a role in the pathogenesis of necrotizing enterocolitis, a well-known gastrointestinal emergency in newborns. Enteroviruses are common pathogens in neonates and have been associated with outbreaks in neonatal units. Enterovirus-associated necrotizing enterocolitis has been described in 3 preterms. Spatiotemporal and molecular analyses have provided evidence of nosocomial transmission.

Human enteroviruses (EV) are the most common cause of viral meningitis in children. Human parechoviruses (HPeV) are increasingly being recognized as a cause of central nervous system (CNS) infections and sepsis-like disease in children. Both viruses belong to Picornaviridae family. The clinical picture in EV and HPeV infections is usually nonspecific. Therefore, molecular detection of both viruses is needed for etiological diagnosis. In this case report, we describe and discuss clinical and laboratory findings of two consecutive episodes of viral meningitis caused by EV and HPeV, respectively, occurring in the first month of a newborn’s life.

International audience; We report a seasonal increase of enterovirus D68 (EV-D68) cases in France, with 54 cases detected between 19 August and 14 November 2018. Molecular typing revealed that 20 of 32 of the isolates belonged to clade D1, only sporadically detected before in France. Median age of D1-cases was 42 years, 10 developed severe respiratory signs and one had neurological complications. The 2018-D1 viruses showed a genetic divergence of 3.34 % with D1 viruses identified previously.

Picornaviruses (family Picornaviridae) are small, nonenveloped, positive-sense, single-stranded RNA viruses. The members of this family are currently classified into 47 genera and 110 species. Of picornaviruses, entero-and parechoviruses are associated with aseptic meningitis. They are transmitted via fecal-oral and respiratory routes, and occasionally, these viruses may cause a brief viremia and gain access to central nervous system (CNS). During the diagnostic screening of entero-and parechovirus types in Finland in year 2013-14, we detected a cluster of echovirus 4 (E4) infections in young adults and adolescents. As E4 is infrequently detected in Finland, we contacted several Northern and Central European laboratories that conduct routine surveillance for enteroviruses and, for those who have had E4 cases, we send a query for E4 sequences and data. Here we report CNS infections caused by E4 in Finland, Sweden, Norway, Denmark, Iceland and Germany in 2013 and 2014, and show that the E4 detected in these countries form a single lineage. In contrast, E4 strains circulating in these countries preceding the year 2013, and those circulating elsewhere in Europe during 2013-2014, formed several independent clusters.

International audience

Background Understanding enterovirus D68 (EV-D68) circulation patterns as well as risk factors for severe respiratory and neurological illness is important for developing preventive strategies. Methods: Between 2010 and 2016, 11,132 respiratory specimens from hospitalised patients in Lyon, France, were screened for EV-D68 by PCR. Phylogenetic relationships of the viral-protein-1 sequences were reconstructed using maximum-likelihood and Bayesian-Markov-Chain-Monte-Carlo approaches. Results: Overall, 171 infections with a biennial pattern were detected, including seven, one, 55, none, 42, one and 65 cases annually during 2010–16. Children (Conclusions: Reinforcement of diagnostic capacities and clinical-based surveillance of EV-D68 infections is needed in Europe to assess the EV-D68 burden.

BackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a notemplate-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6% to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

International audience; Diagnosis: Atypical hand, foot, and mouth disease caused by coxsackievirus A6 (CVA6).

Causing an international outbreak of respiratory disease, Enterovirus D68 quickly entered the closed circle of emerging viral pathogens of public health significance. As rapid and accurate detection of EV-D68 is essential for an efficient clinical management, we designed and validated a new highly efficient one-step quantitative rRT-PCR specific to EV-D68 VP4-VP2 region. With 100% specificity and 95.6% sensitivity to all EV-D68 strains, this new assay can be reliably used to detect and quantify EV-D68 in respiratory samples and represents an interesting additional tool for diagnosis as it targets an original region of the genome.

Background The human parechoviruses (HPeVs) were recently recognized as important viral pathogens involved in various illnesses in young children. However, routine detection is not performed in most clinical laboratories. Therefore, in this study, we aim to assess the relevance of HPeV detection in cerebrospinal fluid (CSF) of infants, according to clinical presentation. Methods A total of 120 CSF specimens collected during 2012 from infants aged less than 1 year and previously reported negative for Herpes simplex virus (HSV) and enterovirus were selected. HPeV detection was performed with a commercially available real-time RT-PCR and HPeV strains from positive samples were subsequently genotyped by sequencing. Results HPeV RNA was detected in nine (7.5%) CSF samples. The median age of infected children was 41 days (range: 19–122 days). HPeV genotyping could be performed on five samples and three HPeV-3, one HPeV-1, and one HPeV-4 were identified. Hyperthermia associated with mottled skin was the predominant clinical presentation. Most clinical presentations of HPeV-infected infants were mild with a final diagnosis of sepsis-like illness. The median hospital stay was 3.5 days and five children received antibiotics. Conclusion Routine detection of HPeV in CSF may allow differential diagnosis of enterovirus infection and improve etiologic identification of sepsis-like illness in children.

The aim of this study was to gain insights into the tempo and mode of the evolutionary processes that sustain genetic diversity in coxsackievirus B5 (CVB5) and into the interplay with virus transmission. We estimated phylodynamic patterns with a large sample of virus strains collected in Europe by Bayesian statistical methods, reconstructed the ancestral states of genealogical nodes, and tested for selection. The genealogies estimated with the structural one-dimensional gene encoding the VP1 protein and nonstructural 3CD locus allowed the precise description of lineages over time and cocirculating virus populations within the two CVB5 clades, genogroups A and B. Strong negative selection shaped the evolution of both loci, but compelling phylogenetic data suggested that immune selection pressure resulted in the emergence of the two genogroups with opposed evolutionary pathways. The genogroups also differed in the temporal occurrence of the amino acid changes. The virus strains of genogroup A were characterized by sequential acquisition of nonsynonymous changes in residues exposed at the virus 5-fold axis. The genogroup B viruses were marked by selection of three changes in a different domain (VP1 C terminus) during its early emergence. These external changes resulted in a selective sweep, which was followed by an evolutionary stasis that is still ongoing after 50 years. The inferred population history of CVB5 showed an alternation of the prevailing genogroup during meningitis epidemics across Europe and is interpreted to be a consequence of partial cross-immunity.

We report 59 cases of severe paediatric conditions linked with enterovirus (EV)-A71 and EV-D68 in France between May and October 2016. Fifty-two children had severe neurological symptoms. EV sequence-based typing for 42 cases revealed EV-A71 in 21 (18 subgenotype C1, detected for the first time in France) and EV-D68 in eight. Clinicians should be encouraged to obtain stool and respiratory specimens from patients presenting with severe neurological disorders for EV detection and characterisation.

From January to April 2015, Réunion experienced a major outbreak of acute haemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24, which heavily impacted the healthcare system. According to the general practitioners’ (GP) sentinel network, the number of medical consultations due to conjunctivitis during this period was estimated at ca 100,000. This report describes the characteristics of the outbreak, which were obtained through several different yet complementary surveillance systems on the island. These included the network of hospital emergency departments (OSCOUR network), the GPs’ sentinel network, an Internet-based population cohort (‘Koman i lé’) participating in a survey on distinct symptoms including ‘red eyes’ and the monitoring of eye drop sales. Overall the results of the different surveillance approaches were in good agreement regarding the outbreak dynamic. A peak of patients with conjunctivitis was detected in the first 15 days of March (week 10 and 11), coinciding with increased eye drop sales on the island. Strains recovered from outbreak cases belonged to genotype IV and were most closely related to strains identified in AHC outbreaks in China, Egypt and Japan since 2010. Continued surveillance of AHC in Réunion remains important not only locally, but also because frequent exchanges between the island and mainland France may lead to introduction of this virus in Europe.

Background Human enterovirus 71 (EV-71) emerged as a significant pathogen able to cause large outbreaks involving severe neurological cases and children fatalities in Asia. Objectives To describe epidemiology of EV-71 infections in France. Study design Fifty-nine patients admitted in 12 different hospitals from 1994 to 2009 were included. The entire VP1 coding gene of 58 EV-71 strains was sequenced and phylogenetic analyses were performed to assign strains to genogroups/subgenogroups and to compare French isolates to European and worldwide isolates. Results The median age of the patients was 1.04 years (9 days to 7 years). Among 46 documented EV-71 infections, 39 were self-limited. Seven children developed severe sepsis-like, respiratory or neurological complications. Among them, 2 children died from acute respiratory distress syndrome. All the EV-71 strains belonged to genogroup C: 31 isolates belonged to subgenogroup C1, 26 to subgenogroup C2 and 1 to subgenogroup C4. All the strains were genetically related to other European strains isolated at the same period of time. Although C1 isolates were predominant between 1994 and 2005, C2 strains have been predominant since 2007. No association was found between any genotype and the age or the clinical symptoms. Conclusions The C4 subgenogroup, which was associated with large outbreaks in China, did not spread in France. It is important to monitor more carefully the EV-71 strains circulating in France to detect the introduction of new genetic variants that could be associated with major outbreaks.

Human enterovirus 71 (EV-71) is a cause of seasonal epidemics of hand, foot and mouth disease, and of less common but severe neurological manifestations. Uncertainty persists regarding the circulation of virus populations in several geographical areas and the timescale of their dissemination. We determined EV-71 sequences at loci 1D (VP1 capsid protein) and 3CD (non-structural proteins) in 86 strains recovered in Austria, France and Germany and performed an evolutionary genetic study of extant virus populations. Phylogenetic analyses positioned 78 of the 86 sequences within two clades among subgenogroups C1 and C2. A minor sequence cluster was assigned to subgenogroup C4. Analyses incorporating the available sequences estimated the substitution rate in genogroup C at 3.66 x 10(-3) and 4.46 x 10(-3) substitutions per site year(-1) for loci 1D and 3CD, respectively, assuming a relaxed molecular-clock model for sequence evolution. Most of the 'European' strains belonged to clades C1b and C2b, which originated in 1994 [95 % confidence interval (CI), 1992.7-1995.8] and 2002 (95 % CI, 2001.6-2003.8), respectively. Estimates of divergence times for locus 3CD were consistent with those measured for locus 1D. Intertwining between clades representing EV-71 subgenogroups and clades corresponding to other enterovirus types (notably early coxsackievirus A prototype strains) in the 3CD phylogeny is highly indicative of ancestral recombination events. Incongruent phylogenetic patterns estimated for loci 1D and 3CD show that a single tree cannot model the epidemic history of circulating EV-71 populations. The evolutionary timescale of genogroup C estimated for both loci was measured only in decades, indicating recent dissemination.

International audience; Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

External quality assurance for serological detection of chikungunya virus infection was performed to assess the diagnostic quality of expert laboratories. Of 30 participants, only six correctly analysed all reference samples with their respective tests. Thirteen laboratories gave at least 85% correct results, and 11 laboratories 75% or less. IgM antibodies were detected less frequently than IgG antibodies (p

The chikungunya virus (CHIKV), an arbovirus of the genus Alphavirus, family Togaviridae, is mainly transmitted by Aedes mosquitoes. It causes an acute infection, characterized by high fever, polyarthralgia and rash and was responsible for a major outbreak which started in 2005 and spread over many islands of the south western Indian Ocean before it hit the Indian subcontinent. As nucleic acid amplification can be used only during the viremic period, serological tests are most widely used for the diagnosis of CHIKV infections. CHIKV IgM and IgG antibodies can be detected as soon as 3-6 days after clinical onset, respectively. Presently only in-house ELISA and immunofluorescence tests exist for analysing the CHIKV specific immune response. The first commercial indirect immunofluorescence test (IIFT) (EUROIMMUN AG, Lüebeck, Germany) was evaluated using two sera panels of patients from La Reunion and travellers returning with CHIKV infections from the Indian Ocean region. The IgM IIFT shows a specificity of 98.3% and a sensitivity of 96.9%. The specificity and sensitivity for the IgG IIFT are 100.0% and 95.4%, respectively. This commercial IIFT is a valuable tool for the diagnosis of CHIKV infections and antibody seroprevalence studies.

International audience; An equine West Nile virus (WNV) outbreak occurred in 2004 in the Camargue, a wetland area in the south of France where the virus was first reported in 1962 and re-emerged in 2000. WNV neutralizing antibodies were detected in resident birds and two isolates from a House Sparrow (Passer domesticus) and a Common Magpie (Pica pica) were completely sequenced. Phylogenetic analyses revealed that these isolates are closely related to strains previously found in horses in southern Europe and North Africa. More extensive investigation is required to determine whether WNV has been re-introduced or has become endemic in the Camargue.

Background The monitoring and genotyping of Enterovirus (EV) infections can help to associate particular or severe clinical manifestations with specific EV types and to identify the aetiology of infectious outbreaks. Objectives To describe the epidemiological features of EV infections diagnosed during the year 2013 in the Greater Paris area (Ile de France). Study design During 2013, 2497 samples taken from 470 patients in 33 hospitals of Ile-de France were tested for EV genome by RT-PCR. EV genotyping was performed by the National Reference Centre (NRC) laboratories. EV infections were retrospectively reviewed by retrieving clinical and genotyping data from the NRC database. Results Of the 2497 samples, 490 (19.6%) was positive for EV genome detection. These EV infections represented 88.7% and 24.1%, respectively, of all reported regional and national infections. Twenty-seven different genotypes were identified. Echovirus 30 (E-30) accounted for 54.1% of all characterized strains and caused a large outbreak. Four severe neonatal infections were reported, of which two were caused by EV-A71. Respiratory infections involving EV-D68 were observed in two adults. One fatal case of Coxsackievirus A2-associated myocarditis was reported. Conclusion Monitoring EV infections in combination with EV genotyping via the French EV network characterized the epidemiology of EV infections in the Ile de France region in 2013 and documented severe EV infections associated with EV-A71 or CV-A2.

Introduction: During the summer op 2014 an outbreak of enterovirus-D68 (EV-D68) was observed in North-America. While clinical cases were rapidly accumulating, beginning in the USA no similar signal was detected in Europe. The question was raised if there was indeed no circulation of this virus in Europe, or whether the near absence of reports were due to insufficient testing or detection. Methods: The European society for Clinical Virology launched the European EV-D68 initiative, in collaboration with the ECDC, to investigate the circulation of EV-D68 both retrospectively and prospectively. Laboratories that contributed to the initiative, tested respiratory materials for EV-D68, and collected clinical information. In addition, the VP1 frangment of isolated EV-D68 was sequenced by these laboratories, to investigate if the stains found in Europe were related to the ones found in North America. Results: Overall, 42 laboratories from 17 countries participated. A total of 17,248 specimens were analysed, yielding 389 EV-D68 positives (2.26%) in 14 countries. The prevalence ranged between 0 to 25%. These infections were mainly detected in young children presenting with wheezing and immunocompromised adults. Te viruses were epidemiologically linked to the USA epidemic. Conclusions: EV-D68 has been circulating all over Europe during summer and fall 2014, at an unprecedented frequency but with a very mild disease burden. Through detection and virus typing adapted tools, the European laboratories rapidly provided a clear picture of the circulation of this emerging virus.. This study emphasizes the need of a better epidemiological surveillance of respiratory pathogens.

We report a fatal case of acute lower respiratory tract disease with human rhinovirus C (HRV-C) as the unique cause in a 19-month-old girl with a history of repeated episodes of bronchiolitis. HRV-C type 8 nucleic acids were observed in respiratory, stool, and cerebrospinal fluid samples, and infectious virions were isolated from patient serum after inoculation onto reconstituted airway epithelia.

International audience; 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 southeast 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 southeast 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.

Journal de Gynecologie Obstetrique et Biologie de la Reproduction - Vol. 35 - N° 6 - p. 578-583

Resume Introduction Une transmission materno-fœtale du virus chikungunya a ete observee au decours de l’epidemie survenue dans le sud de l’ile de la Reunion (Ocean Indien) des mars 2005 puis dans le nord de l’ile a la fin de l’annee 2005. Les premiers cas de femmes enceintes atteintes de cette arbovirose ont ete detectes des juin 2005. Observations De juin 2005 au 31 janvier 2006, 84 femmes enceintes ont souffert du chikungunya pendant leur grossesse. Dans 88% des cas (n = 74), les nouveau-nes ont ete asymptomatiques a la naissance ; toutes ces femmes avaient fait leur episode pathologique a distance de l’accouchement. Dix nouveau-nes ont eu des atteintes severes (4 encephalites, 3 coagulations intravasculaires disseminees) necessitant un sejour prolonge en neonatologie (dont 6 enfants en reanimation neonatale avec necessite de ventilation assistee). Il n’y a pas eu de deces neonatal dans cette serie, mais une complication grave d’hemorragie cerebrale par thrombopenie severe chez un des nouveaux nes. Tous ces cas pathologiques ont ete documentes par serologie a IgM et/ou PCR specifiques aussi bien chez les meres que chez les nouveau-nes. A noter que dans tous les cas pathologiques, les meres ont fait leur viremie (et hyperthermie) en per-partum.

To the Editor: Enterovirus 71 (EV-71) causes primarily asymptomatic or benign infections in children

Hand, foot and mouth disease (HFMD) is classically defined as a childhood fever accompanied by a rash with vesicles or erosions of the oral mucosa, hands, feet and sometimes the buttocks. Severe neurological complications are associated with enterovirus 71 outbreaks in Asia. Recently, it has been suggested that HFMD is related to coxsackie virus A6 (CV-A6) when there is an atypical rash. The objective of the study is to determine the dermatological pattern of HFMD and to identify the virus serotypes associated with a specific dermatological pattern.A prospective, cross-sectional study was conducted in 7 pediatric dermatology units in France from March 2010 to February 2012. All children with clinically suspected diagnosis of HFMD were included. Clinical data were collected and swabs from the nasopharynx and vesicles were taken for reverse transcription polymerase chain reaction and genotyping. Only children with confirmed HFMD--defined by clinical diagnosis of HFMD and positive enterovirus polymerase chain reaction results--were included for analysis.One hundred and four children consulted for suspected HFMD, including 89 (mean age: 25.7 months; sex ratio M/F 1.54) with confirmed HFMD. Seventy-eight (87.6%) had skin lesions on sites other than hand, feet and mouth. Thirty-seven (41.5%) had 5 or more anatomical sites involved (hand, feet and mouth, buttocks, legs, arms and trunk) considered as widespread exanthema. Widespread vesicular exanthema was observed with both CV-A6 and CV-A16. Peri-oral rash was associated with CV-A6 (P0.001).HFMD has a clinical spectrum ranging from classical to generalized vesicular exanthema. Generalized and atypical exanthema were observed with both CV-A6 and CV-A16 infections. CV-A6 is associated with peri-oral rash.

Chikungunya virus (CHIKV) infection has been reported in West Africa since 1966, with the last outbreaks from Senegal in 1996 and 1997. We report a cluster of CHIKV infection among travelers returning from Senegal in 2006. Eight imported cases of dengue-like syndrome with fever, joint pain, and skin manifestations were investigated.