A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Phylogenetic evidence for a recent spread of two populations of human enterovirus 71 in European countries.

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.

Impact of rapid enterovirus molecular diagnosis on the management of infants, children, and adults with aseptic meningitis.

Enteroviruses (EV) are the main etiological agents of aseptic meningitis. Diagnosis is made by detecting the genome using RT-PCR. The aim of the study was to evaluate the impact of a positive diagnosis on the management of infants, children, and adults. During 2005, 442 patients were admitted to hospital with suspected meningitis. Clinical and laboratory data and initial treatment were recorded for all patients with enteroviral meningitis. The turnaround time of tests and the length of hospital stay were analyzed. The results showed that EV-PCR detected EV in 69 patients (16%), 23% (16/69) were adults. About 18% of CSF samples had no pleocytosis. After positive PCR results, 63% of children were discharged immediately (mean 2 hr 30 min) and 95% within 24 hr. Infants and adults were discharged later (after 1.8 and 2 days, respectively). The use of antibiotics was significantly lower in children than in infants and adults. The PCR results allowed discontinuation of antibiotics in 50-60% of all patients treated. Patients received acyclovir in 16% of cases (7% children vs. 50% adults) and 23% (11% vs. 69%) underwent a CT scan. Clinical data were compared between patients whose positive EV-PCR results were available within 24 hr (n = 32) and those whose results were available > 24 hr after collection of CSF (n = 14). Duration of antibiotic treatment (difference: 2.3 days; P = 0.05) was reduced between the two groups. No statistical difference in the length of stay was observed. The EV-PCR assay should be performed daily in hospital laboratory practice and considered as part of the initial management of meningitis.

An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.

The aim of this study was to assess the presence and seasonal frequency of various enteric viruses in wastewater treatment. The detection of astrovirus, norovirus, enterovirus, hepatitis A virus (HAV) and rotavirus was carried out by molecular analyses in concentrated water samples collected over 18 months at the entrance and exit of an activated sludge sewage treatment plant. The reverse transcriptase-polymerase chain reaction (RT-PCR) results were confirmed by sequencing, and comparative phylogenetic analysis was performed on the isolated strains. Genomes of human astrovirus and human rotavirus were identified in 26/29 and 11/29 samples of raw sewage, respectively, and in 12/29 and 13/29 treated effluent samples, respectively. Some rotavirus sequences detected in environmental samples were very close to those of clinical strains. Noroviruses, enteroviruses and HAV were not detected during the study period. This could be related to the small sample volume, to the sensitivity of the detection methods or to local epidemiological situations. Frequent detection of viral RNA, whether infectious or not, in the exit effluent of sewage treatment indicates wide dispersion of enteric viruses in the environment. Consequently, viral contamination resulting from the use of these treated waters is a risk that needs to be addressed.

[Genotyping and molecular epidemiology of non polyomyelitic enteroviruses]. / Génotypage et épidémiologie moléculaire des entérovirus non poliomyélitiques.

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.

The epidemiology and virology of hepatitis C virus genotype 5 in central France.

BACKGROUND: We previously reported high prevalence of hepatitis C virus genotype 5a (HCV 5) (14%) in Central France. AIM: To identify the risk factors associated with HCV5 infection and to characterize local HCV5 lineages. METHOD: A case-control study and phylogenetic analysis were conducted. RESULTS: In all, 131 HCV5 and 343 HCV non 5 infected patients were enrolled. No HCV5 patient was born in sub-Saharan Africa and only two were injection drug user. HCV5 contamination was associated with living in a rural area called Vic le Comte (VLC) in non-transfused patients (OR = 17.7), with transfusion in patients living outside VLC (OR = 3.8) and with receiving injections in patients from VLC (OR = 3.1). More than 80% of the patients from outside VLC were contaminated by transfusion and those from VLC mainly by an iatrogenic factor - injections performed before 1972 by the local physician. Phylogenetic analysis of HCV5 isolates evidenced no distinct genetic cluster, but close relationships between the isolates of spouse pairs and between blood donors and recipients. CONCLUSIONS: Our results suggest that HCV5 spread in our district by iatrogenic route before 1972 and then via transfusion to the whole district. Collaborative studies are underway to study viral sequences from different parts of Africa and Europe to estimate the origin of our HCV 5a strains.

[From prospective molecular diagnosis of enterovirus meningitis...to the prevention of antibiotic resistance]. / Du diagnostic moléculaire initial prospectif des méningites à entérovirus...à la lutte contre l'antibiorésistance.

Meningitis initially presents with intense manifestations that are not generally specific to a given etiology. The first major question for the physician is to decide whether to initiate a probabilistic treatment. Enteroviruses are a major cause of aseptic meningitis, which is benign in immunocompetent patients. Molecular diagnosis is now becoming the gold standard and its prospective use at the time of patient admission, on the sole basis of clinical suspicion of meningitis, has yielded more reliable data. Cytological and biochemical data from CSF analyses are of low predictive value to influence the initial decision to treat with antibiotics. In addition, cases of meningitis during winter are not uncommon. Adults are concerned in about 25% of cases. Thus, if molecular diagnostic tools are not rapidly available, patient management may be inconsistent, leading to unnecessary scans, laboratory investigations and treatment (including overconsumption of antibiotics). Current progress in the automation and practicability of viral genomic detection yields the result within a few hours after admission. Rapid molecular viral diagnosis of a benign disease that does not require treatment but which is initially worrying is of unquestionable advantage. It is of benefit to both the patient and the community because of its input on health economics, the needless consumption of drugs and, as a result, resistance to antibiotics. The diagnosis of meningitis can no longer remain a retrospective diagnosis after elimination of all the possible causes, since not prescribing unnecessary laboratory tests and not treating are true therapeutic decisions.

Variations in the recognition of echovirus type 11 strains by a group-specific anti-VP1 monoclonal antibody.

BACKGROUND: Mab 5-D8/1 is a monoclonal antibody (Mab) that was shown to be directed towards a conserved epitope of the capsid protein VP1 among the genus enterovirus. The use of this Mab for the routine detection of enteroviruses in clinical specimens led to the observation that several strains of echovirus type 11 (EV-11) could not be detected on spontaneously detached cells from 26-h cultures using a two-step immunofluorescence (IF) assay. Conversely, these strains were detected positive with the same Mab when tested on adherent or trypsinizated cells. OBJECTIVES: The aim of this study was to understand the misrecognition of some strains of EV-11 by this Mab. STUDY DESIGN AND RESULTS: IF tests at different times of the viral cycle brought evidence that the detection of a variant strain of EV-11 decreased rapidly with time, becoming undetectable 26 h post-infection, since the reference strain remained positive up to 46 h post-infection. The infective titres of the variant strains were shown to be high in comparison with those of well-recognised strains. Sequencing the Mab binding epitope confirmed that the variant strains exhibited no antigenic shift. CONCLUSION: These results suggest that the poor recognition of some strains of EV-11 by Mab 5-D8/1 is due to a rapid decrease of the expression of the binding epitope in the cell, maybe in relation with the high lytic power of these strains. From a practical point of view, our data indicate that a negative result when Mab 5-D8/1 is used for enterovirus typing must be interpreted cautiously with highly replicative strains and that detached cells should not be used for enterovirus identification under these circumstances.

Prospective analysis of 61 cases of enteroviral meningitis: interest of systematic genome detection in cerebrospinal fluid irrespective of cytologic examination results.

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<10/mm(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.

Use of non-neutralizing monoclonal antibodies in an ELISA for intratypic differentiation of 28 echovirus type 25 clinical isolates.

Three non-neutralizing monoclonal antibodies were produced and selected against echovirus type 25 JV-4 prototype strain. They were used in an ELISA to investigate the intratypic differentiation of 28 wild isolates. Clinical isolates fell into seven different groups according to their reactivity patterns in ELISA. Two of the non-neutralizing monoclonal antibodies, 9E4 and 6D3, were highly specific, while the third, 6C9, may recognize an epitope common to other types of echoviruses. In contrast, mouse polyclonal antiserum exhibited large cross-reactivities among echovirus serotypes. The reactivity patterns and the geographical origin of the isolates were generally not correlated and, in the same area, four major antigenic variants sometimes coexisted, especially in the south of France. Moreover, reactivity patterns found with ELISA were hardly ever correlated with those observed in a previous study when neutralization tests were used. These results again underline the non-correlation between structure and biological function in the Picornavirus family.

An outbreak due to echovirus type 30 in a neonatal unit in France in 1997: usefulness of PCR diagnosis.

Between February and August 1997, 53 patients with enterovirus meningitis were hospitalized in Clermont-Ferrand, France. All but one were children. Echovirus type 30 was involved in 70% of cases with identified serotype. The outbreak ceased on August 8. Two months later, a neonate was admitted to the neonatal unit with an echovirus type 30 meningitis thought to be acquired at delivery. Twenty days later a nosocomial outbreak of echovirus type 30 involving five neonates occurred. Two of them presented with meningitis and two with febrile seizure; One was asymptomatic. The retrospective examination of the maternal sera in a neutralization test, using the index case strain as a source of antigen, showed that none of the neonates was passively immunized before hospitalization. The use of genome detection in cerebrospinal fluid allowed rapid diagnosis and infection was contained by re-inforcing hygiene measures. Prospective examination of stools in the neonatal and paediatric units showed no further occurrences of the disease. No sporadic case was observed in the general population. Hence, nosocomial infections can occur a long time after an outbreak in the general population; rapid diagnosis with molecular tools is useful both for a definite diagnosis in patients already hospitalized, and to act as a rapid alert, even in intervals between seasonal outbreaks.

[Salmonella aortic valve endocarditis presenting with rupture of a femoral artery mycotic aneurysm]. / Anévrisme mycotique fémoral rompu, révélateur d'une endocardite aortique à salmonelle.

The incidence of Salmonella enteritidis infections has greatly increased over the last few years. Cardiovascular are amongst the most severe extra-digestive complications. The authors report a case of Salmonella enteritidis presenting with rupture of a femoral artery mycotic aneurysm in a chronic alcoholic patient. Salmonella enteritidis was isolated from blood cultures and the operation specimen after the obligatory limb amputation. The outcome was finally favourable after appropriate antibiotic therapy with a residual, stable grade 3 aortic regurgitation. This rare condition is generally observed in immuno-compromised subjects and carries a high mortality (40 to 70% of cases). The initial infectious signs may be masked, and, in these cases, rupture of an aneurysm is often the mode of presentation. Rapid treatment is essential with, ideally, resection of the aneurysm with reestablishment of arterial continuity and adapted, prolonged antibiotic therapy.

Outbreak of hand, foot and mouth disease/herpangina associated with coxsackievirus A6 and A10 infections in 2010, France: a large citywide, prospective observational study.

Hand, foot and mouth disease (HFMD) and herpangina (HA) are frequently caused by several distinct serotypes belonging to the human enterovirus A species (HEVA). Enterovirus 71 is considered as a significant public health threat because of rare but fatal neurological complications. A sentinel surveillance system involving paediatricians from Clermont-Ferrand (France) was set up to determine the clinical and epidemiological characteristics of HFMD/HA associated with enterovirus infections. A standardized report form was used to collect demographic and clinical data. Throat or buccal specimens were obtained prospectively and tested for the presence of enteroviruses. The frequency of HEVA serotypes was determined by genotyping. Phylogenetic relationships were analysed to identify potential new virus variants. From 1 April to 31 December 2010, a total of 222 children were enrolled. The predominant clinical presentation was HA (63.8%) and this was frequently associated with clinical signs of HFMD (48%). An enterovirus infection was diagnosed in 143 (64.4%) patients and serotype identification was achieved in 141/143 (98.6%). The predominant serotypes were coxsackievirus A10 (39.9%) and A6 (28%), followed by coxsackievirus A16 (17.5%) and enterovirus 71 (6.3%). Fever was observed in 115 (80.4%) children. No patient had neurological complications. Coxsackievirus A10 and A6 strains involved in the outbreak were consistently genetically related with those detected earlier in Finland and constituted distinct European lineages. Although several enterovirus serotypes have been involved in HFMD/HA cases, the outbreak described in this population survey was caused by coxsackievirus A6 and coxsackievirus A10, the third dual outbreak in Europe in the last 3 years.

Repeated genomic transfers from echovirus 30 to echovirus 6 lineages indicate co-divergence between co-circulating populations of the two human enterovirus serotypes.

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.

Phylogeography of circulating populations of human echovirus 30 over 50 years: nucleotide polymorphism and signature of purifying selection in the VP1 capsid protein gene.

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 beta 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.

[Rapid enterovirus genotyping in cerebrospinal fluids: a two-year prospective study in a virology laboratory setting]. / Génotypage direct rapide des entérovirus dans le liquide céphalorachidien : étude prospective de deux ans au sein d'un laboratoire de virologie clinique.

UNLABELLED: Enterovirus (EV - 68 serotypes) infections comprise a wide spectrum of clinical presentations including infections of the central nervous system. In severe clinical presentation or epidemics, the precise identification of the involved serotype is necessary. OBJECTIVES: To perform enterovirus genotyping directly in cerebrospinal fluid (CSF) samples, and to assess its feasibility in a laboratory setting. METHODS: Enterovirus genotyping was carried out directly with CSF specimens tested for the diagnostic procedure by amplifying the complete 1D gene encoding the VP1 protein of the HEV-B serotypes (the most frequent) - providing results in two days. Secondly, sequences 1A/1B encoding the VP4/VP2 capsid proteins, respectively, were analysed (results in five days). RESULTS: Direct enterovirus genotyping allowed the identification of enterovirus involved in 77 out of 81 (95%) meningitis cases between January 2006 and December 2007. In combination with the indirect genotyping of enterovirus isolates, identification of the type was achieved in 94 out of 97 (96.9%) patients included in the study. The most frequent serotypes were echovirus 6 (E6) and 13 in 2006, coxsackievirus B2 and E30 in 2007. Four children presented an EV71 associated meningitis. CONCLUSION: When prospectively applied in a laboratory setting, direct enterovirus genotyping in CSF samples allows the identification of the involved enterovirus in two to five days. This time frame is relevant for an optimal patient management, the rapid identification of a new enterovirus variant or in the context of an epidemic alert.

Comparative sensitivity of the echovirus type 25 JV-4 prototype strain and two recent isolates to glutaraldehyde at low concentrations.

The sensitivity of two recently isolated antigenic variants of echovirus type 25 (Montpellier 76-1262 and Thionville 86-222) to glutaraldehyde (GTA) at low concentrations was compared with that of the JV-4 prototype strain. The purified viruses were treated under the same conditions with GTA at concentrations ranging from 0.002 to 0.10%. The wild strains exhibited significantly lower sensitivity to GTA than did the prototype strain; with 0.10% GTA, a 2 log10 unit reduction was obtained in 5 min for JV-4 and in 60 and 80 min for Montpellier 76-1262 and Thionville 86-222, respectively. A comparison with previous results obtained with poliovirus type 1 showed that the inactivation rates of echovirus type 25 wild strains were fivefold lower than those of the poliovirus type 1 Sabin strain. The comparative electrophoretic and immunoblot analyses showed differences in the results of GTA binding with capsid proteins of the viruses. Unlike in the poliovirus type 1 Mahoney strain and in the echovirus type 25 JV-4 reference strain, GTA produced only minor intermolecular cross-linkings in the viral particles of the two wild strains of echovirus type 25. Our results suggest that there are both intertypic and intratypic differences in the GTA sensitivities of enterovirus strains. They are of relevance to disinfection procedures in digestive endoscopy and to the choice of the enterovirus strain used for evaluating the efficacy of disinfectants.

Activity of glutaraldehyde at low concentrations against capsid proteins of poliovirus type 1 and echovirus type 25.

The activity of glutaraldehyde (GTA) against capsid proteins of poliovirus type 1 and echovirus type 25 was studied to understand the mode of action of this reagent against enteroviruses. The viruses were treated with GTA concentrations ranging from 0.005 to 0.10%. In the poliovirus particles, high-molecular-weight products were formed by 0.05% GTA, whereas in the echovirus particles, they were formed at 0.005% GTA. These products consist of complexes composed essentially of VP1 and VP3. There seemed to be differences in the composition of the complexes in the two viruses. Cross-linkings between the two polypeptides of the poliovirus capsid may be due to the accessibility to GTA of lysine residues on the loops of VP1 and VP3, which twist out from the surface of the shell.

Heterogeneity of capsid proteins of echovirus type 25 wild-type strain and prototype strain, studied by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were used to compare the capsid proteins of 19 antigenic variants of echovirus type 25 wild-type strains isolated in France between 1976 and 1987 with those of the prototype JV-4 reference strain isolated in 1957. Immunoblots were developed by using polyclonal sera from rabbits and mice immunized with the reference strain. Immunoblotting patterns revealed reactivity only against viral protein VP1 for sera from both animals. Comparative immunoblotting patterns showed differences in the electrophoretic mobilities of viral protein VP1, especially for the Montpellier 76.1262 wild-type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of [35S]methioinine-labeled viral polypeptides revealed that the two variant strains, Montpellier 76.1262 and Thionville 86.222, exhibited significant and reproducible shifts in the relative mobilities of VP1 and VP3 and, to a lesser extent, in those of VP0 and VP2. The relative mobility of VP4 seemed very similar for the JV-4 reference strain and the two variants. Interestingly, the structural differences in VP1 and VP3 of Montpellier 76.1262 were not correlated with the pattern of neutralization by monoclonal antibodies, unlike in our previous study, in which this strain differed from the prototype strain in only two epitopes. We concluded that, in addition to the heterogeneity of their biological and antigenic properties that we observed previously, echovirus type 25 wild-type strains may exhibit differences in their structural proteins.

Replication of echo virus type 25 JV-4 reference strain and wild type strains in MRC5 cells compared with that of poliovirus type 1.

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.