Isabelle Schuffenecker

Human parechovirus infections, Lyon, France, 2008-10: evidence for severe cases.

BACKGROUND Although data documenting the frequency and severity of human parechovirus type 3 (HPeV-3) infection in infants have been published in Canada, the USA, the UK and the Netherlands, no data from France are available. OBJECTIVES To determine the detection frequency of HPeV in cerebrospinal fluid (CSF) samples collected from children aged <5 years hospitalized between 2008 and 2010 in the University Hospital of Lyon and to describe the clinical, virological and biological characteristics associated with HPeV infection. STUDY DESIGN A total of 1128 CSF samples were retrospectively tested using the Parechovirus-Rgene™ real-time RT-PCR assay. Positive samples were typed by sequencing using the CDC method. Retrospective analysis of the medical charts was performed. RESULTS Over a 3-year period, 33/1128 (2.9%) CSF samples were found to be HPeV-positive. In 2010, 9.3% of the children aged <3 months (32% in June) were detected HPeV-positive. The median age at diagnosis was 26 days (8-131 days). Most patients (86%) presented with fever or a sepsis-like syndrome. Three patients (2 with septic shock syndrome, 1 with severe respiratory distress) required hospitalization in an intensive care unit. An HPeV-3 acute infection was identified in an 11-day-old girl who died from sudden infant death syndrome. Of 29 patients genotyped, 28 were infected with HPeV-3 and one with HPeV-4. CONCLUSIONS HPeV is a significant cause of sepsis and severe sepsis in children <3 months. Routine screening for HPeV in CSF and blood should thus be performed more extensively and could improve clinical management.

European surveillance for enterovirus D68 during the emerging North-American outbreak in 2014

BACKGROUND In August and September 2014, unexpected clusters of enterovirus-D68 (EV-D68) infections associated with severe respiratory disease emerged from North-America. In September, the European Centre for Disease Prevention and Control (ECDC) asked European countries to strengthen respiratory sample screening for enterovirus detection and typing in cases with severe respiratory presentations. OBJECTIVES To provide a detailed picture of EV-D68 epidemiology in Europe by conducting a retrospective and prospective laboratory analysis of clinical specimens. STUDY DESIGN An initiative supported by the European Society for Clinical Virology (ESCV) and ECDC was launched to screen for EV-D68 in respiratory specimens between July 1st and December 1st 2014 in Europe and to sequence the VP1 region of detected viruses for phylogenetic analytic purposes. RESULTS Forty-two institutes, representing 51 laboratories from 17 European countries, analyzed 17,248 specimens yielding 389 EV-D68 positive samples (2.26%) in 14 countries. The proportion of positive samples ranged between 0 and 25% per country. These infections resulted primarily in mild respiratory disease, mainly detected in young children presenting with wheezing and in immuno-compromised adults. The viruses detected in Europe are genetically very similar to those of the North-American epidemic and the majority (83%) could be assigned to clade B. Except for 3 acute flaccid paralysis (AFP) cases, one death and limited ICU admissions, no severe cases were reported. CONCLUSIONS The European study showed that EV-D68 circulated in Europe during summer and fall of 2014 with a moderate disease burden and different pathogenic profile compared to the North-American epidemic.

Epidemiology of human enterovirus 71 infections in France, 2000-2009

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.

Blocking human enterovirus 71 replication by targeting viral 2A protease

OBJECTIVES Human enterovirus 71 (EV-71), a member of the Enterovirus genus, constitutes a major public health issue in the Asia-Pacific region, where it is associated with several severe neurological complications. There is currently no effective vaccine or antiviral against EV-71. The aim of this study was to determine whether the six amino acid peptide LVLQTM, which was previously shown to inhibit human rhinovirus (HRV) 2A protease (2A(pro)) activity in vitro and HRV replication in vivo in mice, could be of more general use against enteroviruses and more particularly against EV-71. METHODS To investigate whether the LVLQTM peptide was a pseudosubstrate of EV-71 2A(pro), a recombinant luciferase containing the LVLQTM sequence was designed so that recognition of this sequence by 2A(pro) led to luciferase activation. Direct interaction between EV-71 2A(pro) and the LVLQTM peptide was further confirmed by isothermal titration calorimetry. We then tested the effects of the peptide on EV-71 2A(pro) cleavage activity and EV-71 replication in HeLa cells. RESULTS We showed that the LVLQTM peptide behaved as an effective substrate analogue of EV-71 2A(pro), which binds into the active site of the protease with a dissociation rate constant of 9.6 μM. Moreover, LVLQTM significantly inhibited eIF4G cleavage activity of 2A(pro) as well as EV-71 replication in HeLa cells. CONCLUSIONS This study demonstrates that the LVLQTM peptide that has previously been shown to inhibit HRV replication is also an effective inhibitor of EV-71 2A(pro) and therefore of EV-71 replication, opening new doors in the development of new antivirals against EV-71.

Phylogenetic patterns of human coxsackievirus B5 arise from population dynamics between two genogroups and reveal evolutionary factors of molecular adaptation and transmission

ABSTRACT 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.

Epidemics of aseptic meningitis due to enteroviruses following national immunization days in Bahrain

We report two successive epidemics of aseptic meningitis due to enteroviruses (EV) observed after national immunization days against polio. Meningitis due to echovirus 30 occurred from July 1995 to the end of January 1996, mostly among children aged 0-12 years (95.1% of cases), and meningitis due to echovirus 4 occurred from May 1996 to the end of September 1996 in the same age group. There were 286 and 169 cases, respectively. Specimens from several representative cases were sent to the WHO Collaborating Center for Virus Reference and Research Laboratory for serological testing and virus detection, including polymerase chain reaction (PCR) studies. Using those tests, evidence of echovirus 30 infection was found in 44% of the children who were sampled during the first epidemic and 45.5% during the second. During echovirus 30 and echovirus 4 epidemics, a similar decline in the age-specific attack rate from 19.1/10,000 and 10.1/10,000 population aged 12 years to 2.4/10,000 and 3.6/10,000 population aged 13 years was observed, respectively.

Disseminated Rhinovirus C8 Infection with Infectious Virus in Blood and Fatal Outcome in a Child with Repeated Episodes of Bronchiolitis

ABSTRACT 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.

Ex Vivo and In Vivo Inhibition of Human Rhinovirus Replication by a New Pseudosubstrate of Viral 2A Protease

ABSTRACT Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model.

A new real-time RT-PCR targeting VP4-VP2 to detect and quantify enterovirus D68 in respiratory samples

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.