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.

Phylogenetic analysis of Echovirus 30 isolated during the 2005 outbreak in France reveals existence of multiple lineages and suggests frequent recombination events.

BACKGROUND: Echovirus 30 (E-30) was responsible in France for a major aseptic meningitis outbreak during 2005 summer season. However, the virological mechanisms responsible for the periodic emergence of the epidemic strains remain to be investigated. OBJECTIVES: To assess the genetic diversity of two genome regions, VP1 and 3Dpol, of echovirus 30 strains isolated during the 2005 aseptic meningitis outbreak in Champagne Ardenne (CA) area (France). STUDY DESIGN: Partial VP1 genomic region of 23 E-30 strains isolated in CA was sequenced and compared with 73 E-30 strains originating from different French areas to estimate the number and the diversity of E-30 lineages. Partial sequences for 3D polymerase (3Dpol) were analyzed to detect potential recombination events within the non-structural (NS) region of the genome of EV neurotropic strains. RESULTS: Phylogenetic analysis of the VP1 evidenced the co-circulation of 6 distinct E-30 lineages responsible for the 2005 aseptic meningitis outbreak in France of which three had co-circulated in CA. Partial sequencing of the 3Dpol coding region showed that all of the E-30 strains exhibited different phylogenetic links between VP1 and 3Dpol genomic regions, suggesting multiple intra- or inter-serotypic recombination events within the NS part of the genome. CONCLUSIONS: Our findings revealed existence of multiple lineages and suggested frequent recombination events among E-30 strains having co-circulated in a restricted area during a short time outbreak period. Moreover, our data demonstrated that study of single VP1 genome region analysis could not accurately describe the phylogenetic origin of E-30 isolates.

Echovirus 6 strains derived from a clinical isolate show differences in haemagglutination ability and cell entry pathway.

Two echovirus 6 (EV6) strains were isolated from a clinical sample after successive sub-cultures in PLC (human hepatocellular carcinoma) and HeLa (human cervical adenocarcinoma) cells. The first strain retained its haemagglutinating capacity (HAEV6) while the second became non-haemagglutinating (NHAEV6). Virus binding assay showed that HAEV6 was capable of binding to DAF-expressing cells but not NHAEV6 confirming the role of DAF in EV6 haemagglutination. The lack of competition between the two viral strains during coinfections suggested that each strain used a different cell entry pathway. We provide evidence showing that HAEV6 used preferentially the lipid raft-dependent caveolae pathway, whereas NHAEV6 followed the clathrin-mediated pathway. Comparison of the sequences of HAEV6 and NHAEV6 revealed five amino acid changes in the VP1, VP2 and VP3 capsid proteins distributed in domains which are known to be highly immunogenic or suggested to be involved in receptor binding, virion stability and pathogenicity.

Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR.

Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.

New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo.

Surveillance of acute flaccid paralysis often identifies enteroviruses not typeable by virus neutralization in cell culture. During 2000 and 2001, 186 isolates from 138 children with acute flaccid paralysis in the Democratic Republic of the Congo were sent for typing to the National Reference Centre for Enteroviruses in Lyon, France. The 5' UTR of the viral genome could be amplified by PCR for 158 isolates from 114 patients. Isolates from 89 patients were neutralizable, and contained non-polio enterovirus types. Seventeen children were infected with more than one entero- or adenovirus; another three were co-infected with both these viruses. Serological typing failed with 19 isolates from 13 (9%) patients. The VP1 region of these strains could be amplified by PCR and sequenced, which revealed that five children were infected with CV-A17, EV-70, EV-76, EV-77, or CV-A13. Two patients were doubly infected, one with CV-A24 and E-9, and another with E-27 and EV-81. Isolates from six children contained strains with divergent VP1 region. The amino acid sequences of these complete VP1 regions diverged >or=28% from published types indicating that they represented two new enterovirus types, tentatively designated EV-93 belonging to HEV-B and EV-94 within HEV-D. The latter enterovirus has in parallel been isolated from sewage in Egypt. In conclusion, there was a high frequency of "untypable" enterovirus isolates from cases with acute flaccid paralysis in the Democratic Republic of the Congo. Six of these were shown to represent two enteroviruses not previously described.

Outbreak of acute hemorrhagic conjunctivitis in French Guiana and West Indies caused by coxsackievirus A24 variant: phylogenetic analysis reveals Asian import.

An outbreak of acute hemorrhagic conjunctivitis occurred in French Guiana between April and July 2003, with approximately 6,000 cases in the two major cities Kourou and Cayenne. Since acute hemorrhagic conjunctivitis is not a notifiable disease in France, there was no registration of the number of cases. Therefore, these were estimated by comparing the consumption of antibiotic eye drops and ophthalmic ointments during 2002 and 2003. The outbreak rapidly spread into the Caribbean Islands, causing an outbreak in Guadeloupe in October. Viral isolates from conjunctival swabs of 16 patients were confirmed to be enterovirus by PCR directed to the 5' UTR of the genome. The isolates could not be neutralized by the Melnick intersecting pools, but were shown to be CV-A24 variant by limited sequencing within the VP1 and 3C regions of 12 strains. Phylogenetic analysis revealed that they were similar to the genotype III strains causing outbreaks in Korea 2002 and Malaysia 2003. The previous outbreak of conjunctivitis caused by CV-A24 in the Caribbean in the 1980s was also introduced from Asia, and disappeared after 3 years. This new introduction from Asia and its rapid spread into the Caribbean, where the infection disappeared after a few months, indicates that the CV-A24 variant has a different epidemiological pattern in this region compared to South East Asia, since it has not established an endemic infection. It had to be reintroduced from Asia, where it has been circulating since the 1970s.

Molecular identification and characterization of two proposed new enterovirus serotypes, EV74 and EV75.

Sequencing of the gene that encodes the capsid protein VP1 has been used as a surrogate for antigenic typing in order to distinguish enterovirus serotypes; three new serotypes were identified recently by this method. In this study, 14 enterovirus isolates from six countries were characterized as members of two new types within the species Human enterovirus B, based on sequencing of the complete capsid-encoding (P1) region. Isolates within each of these two types differed significantly from one another and from all other known enterovirus serotypes on the basis of sequences that encode either VP1 alone or the entire P1 region. Members of each type were > or =77.2 % identical to one another (89.5 % amino acid identity) in VP1, but members of the two different types differed from one another and from other enteroviruses by > or =31 % in nucleotide sequence (25 % amino acid sequence difference), indicating that the two groups represent separate new candidate enterovirus types. The complete P1 sequences differed from those of all other enterovirus serotypes by > or =31 % (26 % amino acid sequence difference), but were highly conserved within a serotype (<8 % amino acid sequence difference). Phylogenetic analyses demonstrated that isolates of the same serotype were monophyletic in both VP1 and the capsid as a whole, as shown previously for other enterovirus serotypes. This paper proposes that these 14 isolates should be classified as members of two new human enterovirus types, enteroviruses 74 and 75 (EV74 and EV75).

Diversity of enterovirus sequences detected in oysters by RT-heminested PCR.

Oysters harvested in western France, from five sites associated with outbreaks of food-borne norovirus gastroenteritis between February 2000 and March 2001, were assayed for enterovirus RNA by reverse transcriptase-heminested polymerase chain reaction (RT-heminested PCR). Forty percent (21/52) of shellfish samples (pool of seven oysters) were contaminated by enteroviruses. Infectious coxsackieviruses serotype A21 were isolated from three of these positive samples. Amplicons corresponding to 65 base sequences in the 5' untranslated region of the enteroviral genome were analyzed by direct sequencing. Interpretable results were obtained from 18 amplicons, but mixtures of sequences confused the results from 3 samples. Sequences isolated from samples from the different sites were different but similarities were observed between sequences detected in shellfish from two sites at different dates. Sequences were also compared to sequences of human, bovine and porcine enteroviruses. Both human and animal origins of enterovirus contamination of shellfish seemed likely.

Etiologic diagnosis of 204 pericardial effusions.

The etiologic evaluation of pericardial effusion is frequently unsuccessful when noninvasive methods are used. To determine the cause of the current episode, all patients with echographically identified pericardial effusion from May 1998 to December 2002 underwent noninvasive diagnostic testing of blood, throat, and stool samples. Patients with postpericardiotomy syndrome were excluded. To analyze the value of our tests, we tested randomly selected blood donors as negative controls. Among 204 included patients, 107 (52.4%) had a final etiologic diagnosis: the etiology of 52 was highly suspected at first examination and later confirmed (thyroid deficiency, 5 cases; systemic lupus erythematous, 7; rheumatoid arthritis, 7; scleroderma, 3; cancer, 25; and renal insufficiency, 5). A definite etiologic diagnosis was made in 11 patients from pericardial fluid analysis (cancer, 5 cases; tuberculosis, 3; Streptococcus pneumoniae, Citrobacter freundii, and Actinomyces, 1 case each). Among 141 patients considered to have idiopathic pericarditis, 44 (32.1%) gained an etiologic diagnosis by our systematic testing strategy. This included serologic evaluation of serum (Coxiella burnetii, 10 cases; Bartonella quintana, 1; Legionella pneumophila, 1; Mycoplasma pneumoniae, 4; influenza virus, 1), viral culture of throat swabs (enterovirus, 8 cases; and adenovirus, 1), high-level antinuclear antibodies (>1/400, 3 cases), and thyroid-stimulating hormone (15 abnormal results). Antibodies to Toxoplasma and cytomegalovirus, enterovirus recovered from rectal swabs, and low-level antinuclear antibodies were seen with equal frequency in patients and controls. Using our evaluation strategy, the number of pericardial effusions classified as idiopathic was less than in other series. Systematic testing for Q fever, Mycoplasma pneumoniae, thyroid abnormalities, and antinuclear antibodies, accompanied by viral throat cultures, frequently enabled us to diagnose diseases not initially suspected in patients with pericardial effusion.

Sequencing of 'untypable' enteroviruses reveals two new types, EV-77 and EV-78, within human enterovirus type B and substitutions in the BC loop of the VP1 protein for known types.

The N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 'untypable' isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19.4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15.7-18.2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24.2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.

Outbreak of aseptic meningitis in Djibouti in the aftermath of El Niño.

At the end of 1990s, an outbreak of enteroviral meningitis in Djibouti was associated with the cocirculation of multiple serotypes. This uncommon distribution was related to the dissemination of enterically transmitted agents in the aftermath of El Nino events disturbing the Horn of Africa. Both Djiboutians and expatriate residents were infected.

Encephalomyelitis due to human parechovirus type 1.

OBJECTIVES: The implication of a viral agent in encephalomyelitis has been reported for several years. In the present study we wanted to demonstrate the presence of human parechovirus type 1 (HPEV1) in a patient diagnosed with encephalomyelitis. STUDY DESIGN: Clinical samples (throat and rectal swabs, acute and convalescent sera, cerebrospinal fluid) were collected from a 10-month-old boy diagnosed with encephalomyelitis. The appropriated samples were tested for cytomegalovirus, varicella zona virus, mumps virus and enteroviruses with specific culture, and serological and molecular biological techniques. RESULTS: HPEV1 was isolated from the throat and its genome was additional detectable in the cerebrospinal fluid, throat swab and acute serum. The samples were negative for all other tested viruses. CONCLUSION: To our knowledge, this is the first reported case of HPEV1 infection related to encephalomyelitis. This emphasis that human parechoviruses can be responsible for severe central nervous system infections in children.

Specific RT-PCR procedure for the detection of human parechovirus type 1 genome in clinical samples.

Among the new genera of Parechoviruses, human parechovirus type 1, formerly ECHO virus 22, has recently been recognized on the basis of distinctive biological and molecular properties. This human pathogen generally causes mild gastro-enteritis, respiratory infections and is also responsible for central nervous system infections. To ensure reliable detection, these latter infections are diagnosed by using reverse transcription polymerase chain reaction (RT-PCR) procedures. Due to genetic differences, human parechovirus type 1 genome is not detected with an enterovirus specific RT-PCR procedure. Hence, a rapid, specific and sensitive RT-PCR assay was developed that can be used for the detection of human parechovirus type 1 in clinical samples.