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PubMed1989

Detection of enteroviruses using cDNA and synthetic oligonucleotide probes.

This study compares the detection of enterovirus RNA by cDNA probes prepared from both the 5' and 3' end of the genome of coxsackie A21 and B4 with the use of synthetic oligonucleotides prepared from short but highly conserved sequences in the 5' end non-coding region of the picornavirus genome. The cDNA probes detected enteroviruses with a variable level of sensitivity which presumably depended on the degree of genomic homology with the detecting probes. Generally probes from coxsackievirus A21 detected more enteroviruses than did similar probes from coxsackievirus B4. Probes from the 5' end of the genome of both viruses were more sensitive than 3' end probes. In contrast, synthetic oligonucleotides detected all enteroviruses efficiently suggesting that these probes could be useful as 'universal' probes to detect any enterovirus. This paper discusses the application of these probes in the diagnosis and differentiation of enteroviruses.

Base Sequence↗
PubMed1996

Sensitivity and specificity of mu-capture ELISA for detection of enterovirus IgM.

The sensitivity and specificity of an in-house mu-capture enzyme linked immunosorbent assay (ELISA) for enterovirus IgM in routine use was determined by analysing the results of 77 serum samples from 55 enterovirus culture-positive patients with aseptic meningitis and single serum samples from 140 patients with other infections. In addition, sera from 10 laboratory staff pre- and post-polio virus vaccination and 20 rheumatoid factor positive sera were tested for specificity. On testing the first serum specimen received, only 21 of 55 patients (38%) with aseptic meningitis yielded a positive result, rising to 33 of 55 (60%) on testing a second sample, where available. Out of 14 patients from whom multiple serum samples were tested and negative results obtained with the first serum, 12 were positive with the second sample (86%). Only patients with acute hepatitis A produced a significant number of false positives by the enterovirus ELISA (12 out of 20), but the reverse was not true: patients with enterovirus IgM did not produce false positive results in tests for hepatitis A IgM. Excluding samples positive for hepatitis A IgM, the number of non-enterovirus infections correctly reported as negative was 118 out of 120--a specificity of 98%. This test is probably the most useful serological test available at present for diagnosing recent enterovirus infection, although the limited sensitivity needs to be borne in mind.

Adolescent↗
PubMed1998

Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses.

To define the relationship between echovirus 23 (E23) and other human enteroviruses, we have determined the complete nucleotide sequence of a strain of E23 isolated from a child with high fever in Connecticut in 1986 and compared the nucleotide and deduced amino acid sequences with those of other enteroviruses representing each of the major enterovirus phylogenetic groups, poliovirus type 1, coxsackievirus A16, coxsackievirus B3, echovirus 22 (E22), and enterovirus 70. The genome of E23 (strain CT86-6760) was 7352 nucleotides in length, exclusive of the poly(A) tail, and the genome organization was typical of the picornaviruses. The nucleotide sequence and deduced amino acid sequences were most related to those of E22, a virus with which E23 shares many biological properties, and was quite divergent from the sequences of other enteroviruses (< 20% average amino sequence identity). These data lend further support to the suggestion that E22 and E23 are distinct from members of the Enterovirus genus and that they should be reclassified in a separate genus within the Picornaviridae.

Amino Acid Sequence↗
PubMed2002

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR.

BACKGROUND: Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES: In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN: A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS: With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS: The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.

Adult↗
PubMed2003

Impaired glutamate uptake and EAAT2 downregulation in an enterovirus chronically infected human glial cell line.

Rapid and efficient uptake of glutamate via the high-affinity glutamate transporter EAAT2 is important for limiting glutamate-mediated excitotoxicity involved in neuronal death. Furthermore, there is evidence of altered glutamate uptake and catabolism in motor neuron diseases. Such a defect has been reported in amyotrophic lateral sclerosis, the major motor neuron disease, and was associated with impairment in EAAT2 processing. We recently reported the presence of enterovirus genome specifically in the anterior horn of amyotrophic lateral sclerosis cases, suggesting the involvement of a chronic/persistent enterovirus infection in amyotrophic lateral sclerosis. To investigate a putative link between enterovirus infection and the glutamate-mediated excitotoxicity observed in amyotrophic lateral sclerosis, we developed an in vitro model consisting of a human glial cell line infected with ECHOvirus 6, one of the enteroviruses with sequences closely related to those detected in patients with amyotrophic lateral sclerosis. In these glial cells, an ECHOvirus 6 chronic infection was established, resulting in altered extracellular glutamate uptake. This correlated with an aberrant splicing of the EAAT2 pre-messenger ribonucleic acid and a significant loss of EAAT2 protein expression, similar to that observed in patients with amyotrophic lateral sclerosis. These results provide convincing evidence that an enterovirus chronic/persistent infection may alter glial glutamate uptake and catabolism. As enteroviruses are extremely common human pathogens, they may act as a trigger in the development of certain motor neuron diseases, such as amyotrophic lateral sclerosis.

Animals↗
PubMed2001

[Rational and efficient enterovirus diagnosis].

BACKGROUND AND OBJECTIVE: Demonstration of the causative pathogen by isolating the virus in cell culture is taken as the standard in the diagnosis of diseases caused by enterovirus. When diagnosing the virus in cerebrospinal fluid (CSF), isolation of the virus has been largely replaced by the rapid demonstration of the virus using the reverse transcriptase polymerase chain reaction (RT-PCR), because of its greater sensitivity. The serological diagnosis is mostly made with the complement binding reaction (CBR). A new enzyme immunoassay for demonstrating anti-enterovirus IGM antibodies (IgM-EIA) allows a more rapid diagnosis from a single serum sample. It was the aim of this study to compare the diagnostic value of these various tests. METHODS: Several methods for demonstrating virus from faeces, swabs and CSF (virus isolation in cell culture and RT-PCR) and of antibodies in serum (IgM-EIA and CBR) were compared. The clinical material was obtained largely from children under the age of 10 years, many of whom had serous meningitis, flu-like symptoms or enteritis. In one cohort (C1), only stool or throat swabs were available for each of 154 patients. In the other cohort (C2) of 164 patients, CSF and at least one serum sample were available in addition to occasional stool samples. RESULTS: From C1 enteroviruses were isolated from 32 patients. rotavirus twice from stool or throat swab and rotavirus once from stool or throat swab, and herpes simplex once from throat swab. RT-PCR was positive 55 times for enterovirus, five times false-negative when the virus had been isolated. In C2 enterovirus nucleic acid was demonstrated in 43 patients from CSF. Parallel serological tests gave positive IgM values for 15 patients, while CBR titres were raised in nine. CONCLUSIONS: Complementary tests of CSF, stool, swabs and serum samples by all possible combinations of viral isolation, RT-PCR and IgM-EIA improve the diagnosis of enterovirus-associated diseases. RT-PCR is the method of choice. The serological diagnosis should be confirmed by the demonstration of virus in stool or swab.

Adolescent↗
PubMed1998

No evidence for persistent enterovirus infection in patients with end-stage idiopathic dilated cardiomyopathy.

Persistence of enteroviruses in heart tissue has been implicated in the etiology of idiopathic dilated cardiomyopathy (IDC). Therefore, we determined the prevalence of enterovirus RNA in heart tissue from patients with end-stage IDC. During heart transplantation, 287 transmural biopsy specimens were aseptically collected from the explanted hearts of 38 patients with IDC and of 39 patients with cardiac failure of known cause. A nested polymerase chain reaction with general specificity for enteroviruses was used to screen for the presence of enterovirus RNA in the heart tissue samples. No enterovirus RNA was detected in any of the 287 heart biopsy specimens. These findings lead to a conclusion that enteroviruses do not persist in heart tissue from patients with end-stage IDC, nor with other heart diseases of known cause.

Adolescent↗
PubMed2005

No evidence of prolonged enterovirus excretion in HIV-seropositive patients.

Mutations frequently occur in oral poliovirus vaccine (OPV) strains upon replication in the human intestine. These strains occasionally revert to being neurovirulent. The more prolonged the excretion of OPV, the higher the risk of reversion. OPV strains can be secreted for several months in humans presenting humoral immune system deficiencies. The duration of excretion of OPV strains or other enteroviruses in individuals infected with the human immunodeficiency virus (HIV) is unknown. We investigated whether HIV infection, which is very prevalent in the Central African Republic, causes prolonged excretion of enteroviruses and, in particular, of OPV strains in adults. We studied 28 HIV-infected adults living with children who were immunized with OPV during national immunization days (NIDs). Blood samples were collected to confirm HIV status and to evaluate immunodeficiency before the NIDs. Stool samples for enterovirus isolation were also collected before the NIDs, between the two rounds of immunization and 2, 4 and 6 months after the second round of immunization. No poliovirus was isolated from any stool sample. Eight enteroviruses were isolated from eight adults (maximum one strain per patient). Enteroviruses were not more frequently isolated from severely immunodeficient patients. Thus, HIV-infected adults do not appear to be at high risk of infection with OPV strains and the excretion of enteroviruses (and thus of polioviruses) does not seem to be prolonged in HIV-infected adults.

Adult↗
PubMed1998

Multicenter evaluation of the Amplicor Enterovirus PCR test with cerebrospinal fluid from patients with aseptic meningitis. The European Union Concerted Action on Viral Meningitis and Encephalitis.

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the "gold standard" were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.

Cerebrospinal Fluid↗
PubMed2001

Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-PCR assay.

A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5' untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays.

Enterovirus↗
PubMed2002

Molecular identification of enterovirus by analyzing a partial VP1 genomic region with different methods.

VP1 is the most suitable region for use in the identification of enterovirus. Although VP1 sequencing methods may vary, it is necessary to agree on a common strategy of sequence analysis. Identification of a strain type may be achieved by three different approaches: pairwise sequence alignment, multiple-sequence alignment, and phylogenetic inference. Other methods are also available, but they are not simple enough to be performed at a virology laboratory. The performances of these methods were evaluated with nucleotide and protein sequences obtained from 32 original samples, 8 enterovirus isolates, and 64 GenBank sequences. Pairwise sequence alignment methods had very different results. The DNASTAR package identified only 28.8% of enterovirus strains, while the Genetics Computer Group package identified 50.0 or 72.1% of enterovirus strains when nucleotide or amino acid sequences were analyzed, respectively. Multiple-sequence alignment methods identified 94.2% (Clustal W program) or 92.3% (Pileup program) of the enterovirus strains, while the phylogenetic method increased this rate to 99.0%. Comparative evaluation of these analysis methods showed that the Clustal W program (version 1.81), a freely available multiple-sequence alignment program, presented one of the best performances when used with the correct criteria. Other commercial and expensive programs did not achieve the same performances, making them less suitable for molecular typing of enteroviruses. Finally, although phylogenetic inference is the most demanding method in terms of knowledge of the user, it remained the best option analyzed.

Algorithms↗
PubMed2004

Analysis of the serotype and genotype correlation of VP1 and the 5' noncoding region in an epidemiological survey of the human enterovirus B species.

The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5' noncoding region (5'NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to be particularly low. However, some substructure in the 5'NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.

Base Sequence↗
PubMed2003

Can enteroviruses cause type 1 diabetes?

Enterovirus infections have long been considered as one possible environmental trigger of type 1 diabetes. These viruses have been detected from diabetic patients more often than from control subjects and they can infect beta cells in cell culture and induce diabetes in animal models. Furthermore, a same kind of seasonality has been observed in both the onset of clinical diabetes and subclinical beta cell autoimmunity (appearance of autoantibodies) as in enterovirus infections. Recently, considerable new evidence has cumulated from prospective studies indicating the risk effect of enterovirus infections long before clinical diabetes was diagnosed. In addition, several studies have reported enterovirus genome sequences in diabetic patients more often than in control subjects. Currently, the evidence for the role of enteroviruses is stronger than for most other environmental agents, but still the final proof is lacking. The ongoing studies aim to prove the risk effect in different populations and to identify the underlying mechanisms. This research field is becoming more and more important because it could open up possibilities to prevent type 1 diabetes by an enterovirus vaccine.

Diabetes Mellitus, Type 1↗
PubMed2004

Detection of parvovirus B19, cytomegalovirus and enterovirus infections in cases of intrauterine fetal death.

AIMS: Maternal infections with parvovirus B19, cytomegalovirus (CMV) and enterovirus have been associated with intrauterine fetal death (IUFD), but the incidence of these infections is not clear. This prospective study was conducted to estimate this incidence. METHODS: A prospective study of 38 months was conducted on cases of IUFD referred to Huddinge University Hospital, Stockholm, Sweden. Placental biopsies, fetal blood and amniotic fluid were collected from cases of IUFD (n=52). Placental biopsies from normal pregnancies at term (n=53) were used as controls. These tissues were examined for parvovirus B19 DNA, CMV DNA and enterovirus RNA using polymerase chain reaction (PCR). Maternal viral serology was measured in 46 cases and virus isolation for enterovirus in maternal stool samples was performed in 31 cases. RESULTS: Viral nucleic acid was recovered in at least one tissue sample from six cases of fetal death (parvovirus B19 in two cases, CMV in three and enterovirus in one), while all placental biopsies from controls were found negative. Serological signs of primary maternal infection were found in two of the cases, and virus isolation for enterovirus was negative in all samples examined. CONCLUSION: Parvovirus B19, CMV and enterovirus may be considered as etiologic agents in cases of fetal death. PCR on placental and/or fetal tissue improves diagnostic accuracy for these infections.

Adult↗
PubMedDate not supplied

Enteroviruses isolated from patients with acute respiratory infections during seven years in Rio de Janeiro (1985-1991).

Enteroviruses were investigated in respiratory secretions collected from patients with acute respiratory infections (ARI) over a seven year period (1985-1991), as part of a longitudinal study of ARI aetiology. All the viruses that are most commonly associated with ARI were found in this study. Among the virus isolates, enteroviruses were only less frequent than respiratory syncytial viruses, adenoviruses and influenzaviruses. Forty five enterovirus samples were isolated from patients with either upper respiratory tract infections (URTI) or lower respiratory tract infections (LRTI). From these enterovirus isolates, thirty one samples were identified as poliovirus (n = 18) and non polio enterovirus (n = 13) by serum neutralization. Poliovirus were identified as type 1 and 2 and all of them were vaccinal strains. From thirteen non polio enterovirus, twelve were identified as echovirus serotypes 1, 2, 7, 11, 19 and 31. The remainder was identified as coxsackievirus B4.

Acute Disease↗
PubMed2002

Enterovirus surveillance--United States, 2000-2001.

Enteroviruses are common viruses associated with diverse clinical manifestations ranging from mild febrile illness to severe and potentially fatal syndromes including aseptic meningitis, encephalitis, neonatal systemic enteroviral disease, and paralytic poliomyelitis. A total of 64 enterovirus serotypes are recognized, including 61 nonpolio enteroviruses. Individual serotypes have different temporal patterns of circulation and often are associated with different clinical manifestations. This report describes temporal trends in reported enterovirus infections in the United States during 2000-2001, including widespread activity of two serotypes (echoviruses 13 and 18) that previously were detected rarely. Monitoring of circulating enterovirus serotypes helped identify these two agents as the primary causes of aseptic meningitis outbreaks in the United States in 2001. Further improvements in timeliness of reporting and geographic representation of the system are needed to allow more complete surveillance for enteroviruses.

Enterovirus↗
PubMed2003

[Enterovirus infections diagnosed in middle Norway during the period 1992-2001].

BACKGROUND: Enteroviruses comprise polioviruses, coxsackieviruses, ECHO viruses and the newer enteroviruses. Poliovirus is now declared eradicated from Europe, but viruses from the other three groups are still frequent causes of human diseases, ranging from mild upper respiratory infections to multi-organ failure. MATERIAL AND METHODS: We present a survey of 438 patients with enteroviral diseases diagnosed from 1992 to 2001. Enteroviral infections were diagnosed by polymerase chain reaction, isolation by tissue culture, and in a few cases by electron microscopy. Clinical information was gathered from referral forms and patient files. 172 of the isolates were typed. RESULTS: Meningitis, gastroenteritis, respiratory disease and fever were the most frequent diseases. ECHO virus 30, ECHO virus 6 and coxsackievirus A16 were the most common serotypes. 33 % of patients were younger than two years of age. INTERPRETATION: Our clinical data demonstrate the wide spectrum of diseases that enterovirus infections may cause. We have found a relatively high proportion of patients with encephalitis. The association between enterovirus and gastroenteritis is discussed. Enteroviruses are the most common cause of aseptic meningitis; enterovirus polymerase chain reaction should be performed routinely when dealing with this disease. Our data also illustrate the superior sensitivity of polymerase chain reaction compared to culture, though culturing is still needed as a source of supplementary information.

Adolescent↗
PubMed1978

Enterovirus survey before and after poliomyelitis vaccination in Kuala Lumpur, Malaysia.

Stool samples from healthy children mainly of the low income group aged 0 to 7 years of age from five Maternal and Child Health Centres in Kuala Lumpur were obtained for isolation of enteroviruses. The specimens were collected before and after the mass vaccination given in the face of polio type 1 epidemic which started in October, 1971. The prevelance rate of enteroviruses was 11.9% (3.0% polioviruses, 8.9% non-polio enteroviruses) before the vaccination and essentially the same after. Coxsackie A viruses predominated over the other enteroviruses in the pre- and post-vaccination phases. The highest isolation rate of enteroviruses was observed in children 0 to 2 years age. No significant differences in distribution by sex, race and month were noted. A sharp fall in the prevalence rates of total enteroviruses and polioviruses was noted shortly after the mass vaccination campaign However, the rates reverted to the pre-vaccination state during the next successive years.

Child↗