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Nonpolio enteroviruses can be reliably identified with molecular and computer tools for taxonomic, diagnostic and epidemiologic purposes. Seroneutralization tests can efficiently be replaced by genotyping assays using the VP1 capsid protein encoding gene to identify enterovirus strains isolated in cell cultures. Genotyping showed the close genetic relatedness between human enterovirus serotypes and animal enteroviruses and also rhinoviruses currently classified in a separate genus within the Picornaviridae family. Enterovirus genotyping can be done prospectively within 2 to 5 days in a greater number of meningitis patients, using cerebrospinal fluid specimens and hence can help in providing a prompt response to health alert. In the molecular epidemiology of human enteroviruses, recent advances were made by investigating genetic diversity within individual serotypes (genotypes, lineages) and the patterns of circulation and transmission of virus variants involved in epidemics (echovirus 30, enterovirus 71). The observation of epidemiologic features such as the frequent viral immigration of strains from different geographical origins speaks in favour of developing molecular identification of enteroviruses. Recombinant enterovirus strains can also be identified by genotyping. Homologous recombination is a major contributor to the genetic diversity in enteroviruses. Molecular signatures of recombination events are observed in circulating strains, suggesting the occurrence of frequent co-infections during their circulation within the general population. The role of genetic recombination in the emergence of virus variants and its involvement in the epidemiology of human enteroviruses should be investigated.

Human echovirus types 6 (E-6) and 30 (E-30) cause seasonal epidemics of aseptic meningitis. These two enteroviruses are frequently observed in co-circulation, an epidemiological pattern that is prerequisite for the occurrence of dual infections, which can lead to recombination between co-infecting virus strains. Viral sequences were determined at loci 1D (VP1 capsid protein) and 3CD (non structural proteins) in 49 E-6 strains recovered in a single geographical region in France from 1999 to 2007, during the epidemiological survey of enterovirus infections. They were compared with previously recorded sequences of E-30 strains to investigate their evolutionary histories and possible recombination patterns. Phylogenetic analyses identified two distinct E-6 populations and different subpopulations. Assuming a relaxed molecular clock model and a Bayesian skyline demographic model in coalescent analyses with the BEAST program, the substitution rate in E-6 was estimated at 8.597×10(-3) and 6.252×10(-3) substitution/site/year for loci 1D and 3CD respectively. Consistent estimates of divergence times (t(MRCA)) were obtained for loci 1D and 3CD indicating that two distinct E-6 populations originated in 1997 and 1999. Incongruent phylogenetic patterns inferred for the two loci were indicative of recombination events between the two populations. Phylogenies including the E-30 3CD sequences showed close genetic relationships between E-6 and discrete E-30 subpopulations. Recombination breakpoints were located with statistical significance in E-6 and E-30 genomes. Estimates of t(MRCA) of phylogenetic recombinant clades indicated directional genetic transfers from E-30 to E-6 populations and their co-divergence over the time period studied.

A comprehensive set of 443 1D gene sequences (encoding the VP1 capsid protein) was analyzed to investigate the phylogenetic relationships and evolutionary patterns among strains of human echovirus 30 (E30; genus Enterovirus, family Picornaviridae) characterized over 50 years. Maximum-likelihood (ML) phylogenetic trees of complete and nonredundant 1D gene sequences (total length = 876 nucleotides) showed evidence of distinct lineages related to the isolation period of virus strains. Virus transportation was confirmed as a major epidemiological factor in the appearance of epidemics since recurrence of aseptic meningitis outbreaks in a given geographic area was associated with distinct E30 variants detected earlier in distant regions. Detection of the codon changes associated with E30 evolution was investigated with methods implemented in the Datamonkey web server. Evolution of the 1D gene was dominated by continual negative (purifying) selection against nonsynonymous substitutions at most codon sites, as determined by dN/dS ratio. Amino acid polymorphism was maintained at a limited number of sites (10/292) in the VP1 protein (within loops connecting β strands and C-terminus). Amino acid changes are allowed at these sites because they are likely exposed on the virion particle and nonsynonymous substitutions are observed in the corresponding codons because negative selection is relaxed.

Enterovirus infections were investigated with special emphasis on performing rapid molecular identification of enterovirus serotypes responsible for aseptic meningitis directly in cerebrospinal fluid (CSF). Enterovirus genotyping was carried out directly with specimens tested for the diagnostic procedure, using two seminested PCR assays designed to amplify the complete and partial gene sequences encoding the VP1 and VP4/VP2 capsid proteins, respectively. The method was used for identifying the enterovirus serotypes involved in meningitis in 45 patients admitted in 2005. Enterovirus genotyping was achieved in 98% of the patients studied, and we obtained evidence of 10 of the most frequent serotypes identified earlier by genotyping of virus isolates. The method was applied for the prospective investigation of 54 patients with meningitis admitted consecutively in 2006. The enterovirus serotypes involved were identified with the cerebrospinal fluid (CSF) of 52 patients (96%) and comprised 13 serotypes within the human enterovirus B species and 1 within the human enterovirus A species. The three most common serotypes were echovirus 13 (E13; 24%), E6 (23%), and coxsackievirus B5 (11.5%), a pattern different from that observed in 2005. Genotyping of virus isolates was also performed in 35 patients in 2006 (meningitis, n = 31; other diseases, n = 4). By comparison, direct genotyping in CSF yielded a more complete pattern of enterovirus serotypes, thereby allowing the detection of rare serotypes: three less common serotypes (CB2, E21, and E27) were not detected by indirect genotyping alone. The study shows the feasibility of prospective enterovirus genotyping within 1 week in a laboratory setting.

Resume Les performances de deux trousses de detection rapide de l'antigene rotavirus ont ete comparees sur des selles recues au laboratoire de virologie du CHU de Clermont-Ferrand entre septembre 2002 et mai 2003. Cinq cent douze selles ont ete analysees par la trousse immunochromatographique Diarlex® MB (ICG) et par la trousse immunoenzymatique IDEIATM Rotavirus (EIA). Les echantillons donnant des resultats discordants ont ete observes en microscopie electronique (ME) et les dossiers cliniques correspondants reexamines. Sur 512 echantillons, 155 (30,3 %) etaient positifs et 332 (64,8 %) negatifs avec les deux trousses. Les resultats etaient contradictoires dans 25 cas (4,88 %), soient 24 enfants et un adulte, la trousse EIA donnant davantage de resultats positifs. L'examen en ME, possible retrospectivement pour 15/25 echantillons discordants, a confirme la presence de rotavirus dans 7/14 selles positives seulement en EIA, et un echantillon positif seulement en ICG. Le reexamen des 25 observations cliniques a montre la presence de signes de GEA dans tous les cas. L'analyse statistique montre une concordance excellente entre les deux trousses (kappa = 0,89, IC95 % = [0,85–0,93]) malgre un sous-depistage du test ICG par rapport au test EIA (p

Twenty-nine Enterococcus faecalis isolates from patients with endocarditis or bacteremia or from stools of healthy volunteers were investigated for their ability to adhere to Int-407 and Girardi heart cell lines and for the presence of known enterococcal virulence factors. Eight strains (27.6%) adhered predominantly to Int-407 cells. The adherence of enterococci was enhanced by proteolytic digestion, suggesting that some cell binding components become surface-exposed after treatment with trypsin. The occurrence of known potential virulence factors of enterococci among these strains was determined and was as follows: enterococcal surface protein (72.4%), gelatinase (58.6%), aggregation substance (48.3%) and cytolysin (17.2%). Bacterial adherence was not significantly associated with any of these virulence factors.

The seasonal incidence of enterovirus meningitis was analyzed in a prospective study of patients admitted for suspected meningitis from October 1, 1998 to April 30, 2000. In-house reverse transcription-polymerase chain reaction (RT-PCR) in cerebrospinal fluid (CSF) was used irrespective of cytological results. Fifty-two (45.2%) of the 115 patients had positive RT-PCR in CSF, including 44/86 children (51.2%) and 8/29 adults (27.6%). Six of the 52 (11.5%) had no pleocytosis. The numbers of CSF specimens with a predominance of lymphocytes or a predominance of neutrophils were closely similar. In 33 of the positive patients, an enterovirus, mainly echoviruses type 6 (48%) and 30 (24%), was recovered in one or more specimens. Sixteen cases of enteroviral meningitis were observed between November 1999 and March 2000 as against 2 cases between November 1998 and March 1999, showing that the disease persisted through the winter months of 1999-2000. During the same period, 96 enterovirus isolates were recovered from clinical specimens from other patients. The number of isolates was higher in the winter of 1999-2000 (P < 0.01) than in the winter of 1998-1999, indicating that the risk of enterovirus infection increased significantly in winter 1999-2000. Sixteen patients had aseptic meningitis, made a rapid recovery and had an enterovirus in throat swabs and stools (9/16) or in one of the two (7/16). RT-PCR was not requested. Nine patients were admitted during the cold months. The clinical management of both adult and child patients could be improved by year-round use of enterovirus generic RT-PCR.

Background: Enteroviruses are the most commonly identified cause of viral meningitis. Detection of the enterovirus genome in cerebrospinal fluid (CSF) using reverse-transcription polymerase chain reaction (PCR) has proved to be useful in diagnosis and is more rapid and sensitive than viral cultures. In routine practice, cytologic examination results of CSF are obtained swiftly and PCR indication is performed as a second step. Objectives: The aim of this study was to determine, by analysis of complete data from CSF results for 61 cases of proven enteroviral meningitis, whether cytologic CSF findings can be used to establish viral etiology and to indicate if PCR assay should be performed. Study design: From a prospective study of children admitted during 1997 for suspected enterovirus meningitis in which PCR and viral cultures of CSF were systematically performed, we selected 61 patients with proven enterovirus meningitis. We compared global white cell count (WCC), relative percentage of lymphocytes/neutrophils, PCR and culture for enterovirus, patient age, and clinical data. Results: 92% of patients (56/61) had positive PCR in CSF and in 48% (29/61) enterovirus was isolated in CSF. Nine patients (14.75%) had WCC 3 ; eight of them had positive PCR and two had positive culture. There were comparable numbers of CSF with a predominance of lymphocytes ( n =25) and CSF with a predominance of neutrophils ( n =22), and of positive PCR and positive cultures of CSF in the two groups. Results were not influenced by the age of the patients. Conclusion: Irrespective of other CSF parameters, it seems difficult to dispense with PCR assay for enterovirus genome detection. It should be introduced as a true rapid routine test. Early reporting of a positive PCR result could result in a considerable saving in health resources.

In echo virus type 25/JV-4 the shut off of host cell protein synthesis took significantly longer and the kinetics of the synthesis of viral proteins and viral RNA occurred much later than in the poliovirus. However, these characteristics impaired neither polyprotein processing nor virus production in the JV-4 strain. In contrast the two wild strains M.1262 and Th.222 had a lower virus yield than strain JV-4. The presence of a high Mr protein in the pattern of viral proteins of wild strains suggested that a defect in the polyprotein processing was responsible for the decreased virus yield. The infectious cycle of strain Th.222 differed from that of strains JV-4 and M.1262 in the rapid inhibition of host cell translation and the extent of viral protein synthesis. The sensitivity to actinomycin D was also investigated. Strain M.1262 was found to be insensitive. The virus yield of strains JV-4 and Th.222 was three- and fourfold lower respectively in the presence of actinomycin D. This sensitivity to the antibiotic was observed during viral RNA synthesis in strain JV-4 and during viral protein synthesis in strain Th.222. These results suggest that cellular factors are involved in the replication of echo virus type 25 strains in MRC5 cells.

The ability of two commercially available diagnosis rapid assays in detecting rotavirus antigen was compared in a prospective study conducted from September 2002 to May 2003. Five hundred and twelve faecal specimens were studied by IDEIA Rotavirus enzyme immunoassay test (EIA) and Diarlex MB immunochromatographic test (ICG). Specimens giving discrepant results were examined by electron microscopy (EM) and clinical data reconsidered. Out of 512 stool specimens, 155 (30.3%) were positive and 332 (64.8%) negative with the two assays. Discrepant results were obtained for 25 (4.88%) specimens (24 children, 1 adult), with EIA giving more positive results. The retrospective examination by EM, possible for fifteen stools on the 25 that gave discrepant results, confirmed the presence of rotavirus in 7/14 stools which were positive only by EIA and in the stool specimen that was found positive only by ICG. The 25 clinical observations re-examination showed the presence of GEA signs in all cases. The statistical analysis shows an excellent concordance between the EIA and the ICG tests (kappa = 0.89, IC(95%) = [0.85-0.93]) in spite of the underestimation of ICG test in comparison with EIA test (P0.0001).

Glutaraldehyde (GTA) is a potent virucidal disinfectant whose exact mode of action against enteroviruses is not understood. Earlier reports showed that GTA reacts preferentially with the VP1 capsid protein of echovirus 25 and poliovirus 1 and that GTA has affinity for exposed lysine residues on proteins. To investigate further the inactivation of enteroviruses by GTA, seven strains were selected on the basis of differences in their overall number and the positions of lysine residues in the amino acid sequences of the VP1 polypeptide. Inactivation kinetics experiments were performed with 0.10% GTA. The viruses grouped into three clusters and exhibited significantly different levels of sensitivity to GTA. The results were analyzed in the light of current knowledge of the three-dimensional structure of enteroviruses and the viral life cycle. The differences observed in sensitivity to GTA were related to the number of lysine residues and their locations in the VP1 protein. The overall findings suggest that the BC and DE loops, which cluster at the fivefold axis of symmetry and are the most exposed on the outer surface of the virions, are primary reactive sites for GTA.

The seasonal incidence of enterovirus meningitis was analyzed in a prospective study of patients admitted for suspected meningitis from October 1, 1998 to April 30, 2000. In-house reverse transcription-polymerase chain reaction (RT-PCR) in cerebrospinal fluid (CSF) was used irrespective of cytological results. Fifty-two (45.2%) of the 115 patients had positive RT-PCR in CSF, including 44/86 children (51.2%) and 8/29 adults (27.6%). Six of the 52 (11.5%) had no pleocytosis. The numbers of CSF specimens with a predominance of lymphocytes or a predominance of neutrophils were closely similar. In 33 of the positive patients, an enterovirus, mainly echoviruses type 6 (48%) and 30 (24%), was recovered in one or more specimens. Sixteen cases of enteroviral meningitis were observed between November 1999 and March 2000 as against 2 cases between November 1998 and March 1999, showing that the disease persisted through the winter months of 1999-2000. During the same period, 96 enterovirus isolates were recovered from clinical specimens from other patients. The number of isolates was higher in the winter of 1999-2000 (P0.01) than in the winter of 1998-1999, indicating that the risk of enterovirus infection increased significantly in winter 1999-2000. Sixteen patients had aseptic meningitis, made a rapid recovery and had an enterovirus in throat swabs and stools (9/16) or in one of the two (7/16). RT-PCR was not requested. Nine patients were admitted during the cold months. The clinical management of both adult and child patients could be improved by year-round use of enterovirus generic RT-PCR.

There have been conflicting studies of lymphocyte capping from patients with Duchenne Muscular Dystrophy. We have evaluated the proportion of capped fibroblasts of 10 patients with Duchenne Muscular Dystrophy. The results, compared with 15 normal controls, showed that the reduction of fibroblast capping is correlated to age patients. Additional studies, decreased number of retracted fibroblasts after colchicine incubation and ultrastructural observations, suggest that capping deficit would be the consequence of microtubular system alteration. This late phenomenon may be secondary to the primary membrane defect.

Hepatocytes from rats were isolated by treatment with trypsin and cultured. Plasma membranes at different culture stages were observed by electron microscopy. The activities of 5' nucleotidase and adenosinetriphosphatase on the plasma membranes were examined. The cell coat was also studied by use of the concanavalin A-peroxidase technique. The surfaces of single cells, covered with microvilli, are the site of adenosinetriphosphatase activity only and are devoid of 5'-nucleotidase activity. After a few h of culture, the cells are grouped together in tight clusters or long trails and are separated by an intercellular space of 250 A, partially permeable to lanthanum nitrate. The juxtaposed plasma membranes on which 5'-nucleotidase and adenosinetriphosphatase activities occur also delimit spaces similar to bile canaliculi. The formation of junction complexes and their permeability to lanthanum nitrate was also studied. No enzymatic activity is observed at the junctions. The numerous tight junctions, impervious to the tracer, are always accompanied by a profusion of microfilaments. Mature desmosomes are rare, and are present only in the form of "maculae adhaerentes diminutae." The gap junctions, nearly always permeable to the tracer, form rapidly and assume a variety of shapes (trail, bulge and ring-like), the significance of which is open to discussion. The use of concanavalin A permits localization of the free sugar sites on the surface of the cells, in the pinocytotic vesicles and in the internal space of the gap junctions.

Improvement in the method of heart cell cultures is described and justified in relation to environmental factors. The validity of such cardiac cell culture for cardiac research is discussed in light of particular cellular activities: differentiation of lipoprotein lipase, myoglobin biosynthesis, glucose and fatty acid metabolism pleiotypic responses (rotein, RNA, and DNA biosyntheses, substrates transports) to serum stimulation, and the architectonic growth of muscle and nonmuscle cells.