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PubMed2006

Enterovirus surveillance--United States, 1970-2005.

PROBLEM: Enteroviruses are common human viruses associated with various clinical syndromes, from minor febrile illness to severe, potentially fatal conditions (e.g., aseptic meningitis, paralysis, myocarditis, and neonatal enteroviral sepsis). Multiple enterovirus serotypes exist. Individual serotypes have different temporal patterns of circulation and often are associated with different clinical manifestations. Changes in circulating serotypes might be accompanied by large-scale outbreaks. REPORTING PERIOD COVERED: 1970-2005. DESCRIPTION OF SURVEILLANCE SYSTEM: The National Enterovirus Surveillance System (NESS) is a voluntary, passive surveillance system that has monitored trends in circulating enteroviruses since 1961. Enterovirus detections by serotype with specimen type, collection date, and demographic information are reported monthly by participating laboratories to CDC, which summarizes the data and disseminates the results. For this analysis, the available data set for 1970-1982 included only information on serotype and state for each report; complete records were available for 1983-2005. RESULTS: During 1970-2005, a total of 52,812 enterovirus detections were reported to NESS (29,772 of them during 1983-2005). Laboratory participation and the numbers of reports declined throughout the 1990s, but they increased again after 2000. The 15 most commonly reported enteroviruses accounted for 83.5% of reports with known serotype, and the five most commonly reported serotypes (echoviruses [E] 9, 11, 30, and 6, and coxsackievirus B5) accounted for 48.1%. Predominant serotypes and ranking of individual enteroviruses changed over time. Long-term circulation patterns for individual serotypes varied but were consistent with epidemic (e.g., E9, E13, E30, and coxsackievirus B5) or endemic patterns (e.g., coxsackieviruses A9, B2, B4, and enterovirus 71). Children aged <1 year accounted for 44.2% of reports with known age. Male predominance was present among patients aged <20 years, but not among those aged >/=20 years (male/female ratio: 1.4 and 0.9, respectively). Enterovirus detections had prominent summer-fall seasonality, with June-October accounting for 77.9% of reports with known month of specimen collection. Cerebrospinal fluid was the most common specimen type, followed by respiratory and fecal specimens (49.8%, 26.9%, and 13.6%, respectively). Death was reported for 3.3% of detections with known outcome. Infections with coxsackievirus B4 (odds ratio [OR] = 3.3; 95% confidence interval [CI] = 1.7-6.0), and human parechovirus 1 (formerly E22) (OR = 3.7; CI = 1.7-7.6) were associated with higher risk for death, and infections with E9 were associated with lower risk for death (OR = 0.1; CI = 0-0.4) than infections with other enteroviruses. INTERPRETATION: NESS data allowed identification and description of a core group of consistently circulating enteroviruses that probably determine the disease burden associated with enterovirus infections. These data also are helpful in guiding outbreak investigations and identifying targets for development of diagnostic assays and antivirals. Efforts to update the reporting system initiated in the early 2000s (i.e., simplification of reporting forms and transition to electronic reporting) resulted in a substantial increase in reporting compared with the late 1990s. PUBLIC HEALTH ACTION: Efforts to increase laboratory participation in NESS should continue to allow for more complete and accurate surveillance for enteroviruses. Further improvement in the timeliness of feedback through the development of a NESS website to allow access to historic data and to the information on circulating serotypes can provide additional incentives to public health laboratories to participate in NESS.

Enterovirus↗
PubMed1995

Myocardial enterovirus infection with left ventricular dysfunction: a benign disease compared with idiopathic dilated cardiomyopathy.

OBJECTIVES: Endomyocardial biopsy samples from patients with idiopathic dilated cardiomyopathy were screened for the presence of enterovirus genome. Patients with enterovirus-positive samples were further studied with regard to disease course, histologic variables and response to interferon-alpha treatment. BACKGROUND: Studies of patients with idiopathic dilated cardiomyopathy have reported widely divergent clinical outcomes, suggesting that there is no unique underlying pathogenetic mechanism. METHODS: Five left ventricular endomyocardial biopsy samples were screened for the presence of the enterovirus genome by an established in situ hybridization technique in combination with a histologic, histomorphometric and immunohistologic workup. The course of the disease was then prospectively followed for up to 50 months. Virus-positive patients whose condition deteriorated were treated with interferon-alpha. RESULTS: Of 77 patients, 20 (26%) had enterovirus-positive and 57 (74%) enterovirus-negative biopsy samples. During a mean follow-up period of 25.8 +/- 13.7 months, 1 patient in the enterovirus-positive group and 11 in the enterovirus-negative group died. Four patients in the enterovirus-negative group underwent heart transplantation (p < 0.05). The surviving 19 enterovirus-positive patients had a decrease in mean left ventricular end-diastolic diameter from 66 to 61 mm (p < 0.05) and a mean increase in left ventricular ejection fraction from 0.35 to 0.43 (p < 0.05). In contrast, enterovirus-negative patients had no significant change in end-diastolic diameter or left ventricular ejection fraction. Four patients in the enterovirus-positive group whose condition deteriorated were treated with a 6-month course of subcutaneous interferon-alpha (3 x 10(6) U every second day). This treatment induced hemodynamic improvement in all four patients and eliminated the persistent enteroviral infection in two. CONCLUSIONS: Enterovirus-positive patients have a better heart transplantation-free survival rate and hemodynamic course, with fewer histologic changes, than do enterovirus-negative patients. In addition, enterovirus-positive patients respond favorably to interferon-alpha treatment. These observations indicate that myocardial enteroviral infection with associated left ventricular dysfunction is a distinct disease entity with a benign course.

Cardiac Catheterization↗
PubMed1992

Serum IgA, IgG, and IgM responses to different enteroviruses as measured by a coxsackie B5-based indirect ELISA.

An enterovirus-specific indirect ELISA, based on a single local isolate of coxsackie B5 as antigen, was used to study the IgA, IgG, and IgM responses in 19 patients with a recent or current enterovirus infection. Twelve different enterovirus serotypes were isolated from 15 patients. Paired serum samples were available from 10 and a single serum from 5 of these 15 patients. In addition, 4 patients diagnosed by a significant titer rise of complement fixing antibodies to enterovirus were included. A serological diagnosis, defined as an increase in titer of enterovirus IgG and/or presence of enterovirus IgM, were established in all 14 patients with paired sera. Enterovirus IgM was present in either a single serum or in both sera in 13 of them. Out of 5 patients with a single serum sample only, enterovirus IgA or enterovirus IgM was found in 4. Specific IgA was present in either a single serum or in both sera in 14 of the 19 patients. Seven of the 10 enterovirus isolate patients with paired sera had a significant titer rise of complement fixing antibodies; however, all 10 were diagnosed by ELISA. Among 64 healthy controls 2 had enterovirus IgA and none had enterovirus IgM. In conclusion, the use of a single antigen-based ELISA was found to be reliable for the diagnosis of recent and current enterovirus infections.

Adolescent↗
PubMed2002

Use of molecular assay in diagnosis of hand, foot and mouth disease caused by enterovirus 71 or coxsackievirus A 16.

Hand, foot and mouth disease is a common illness in children and is usually caused by coxsackievirus A 16 and enterovirus 71. It has been noted that enterovirus 71 infection is more severe with significantly greater frequency of serious complications and fatality than coxsackievirus A 16. Therefore, it is important to develop a rapid and specific assay for discriminating coxsackievirus A 16 and enterovirus 71 in hand, foot and mouth disease outbreaks. In this study we designed two sets of RT-PCR primers specific for coxsackievirus A 16 and enterovirus 71. One hundred and eighty-nine viruses were evaluated for this molecular diagnosis assay. Among 110 enterovirus 71 strains, the enterovirus 71 specific primers gave clear signal for 107 clinical enterovirus 71 isolates and three reference enterovirus 71 strains. None of coxsackievirus A 16, other enteroviruses or non-enteroviruses show signal for enterovirus 71-specific primers. On the other hand, among 28 coxsackievirus A 16 strains, the coxsackievirus A 16-specific primers detect 27 clinical isolates and one reference strain but show no cross-reaction with other viruses. The molecular assay developed in this study provides a sensitive and specific way to distinguish coxsackievirus A 16 and enterovirus 71 induced hand, foot and mouth disease, which will be a useful rapid diagnostic method in future outbreaks.

Enterovirus↗
PubMed1998

Use of monoclonal antibodies to identify serotypes of enterovirus isolates.

Nonpoliovirus enteroviruses cause a variety of diseases that are common in young children and adults. The "gold standard" for laboratory diagnosis of enteroviruses is cell culture isolation, followed by serotype identification by neutralization assay. These procedures are time-consuming and expensive. Rapid serotype identification of enteroviruses is important in differentiating nonpoliovirus enterovirus pathogens from vaccine strain polioviruses that can be shed for some time after vaccination. In the present investigation, we evaluated a rapid method for serotype identification of enteroviruses by indirect immunofluorescence assay (IFA) using commercially available monoclonal antibodies for polioviruses, coxsackieviruses type B, and six serotypes of commonly circulating echoviruses. Of 291 isolates of enteroviruses included in the study, 95 were polioviruses and 196 were nonpoliovirus enteroviruses. Two hundred thirty-four of these (38 polioviruses and 196 nonpoliovirus enteroviruses) were consecutively grown in the laboratory over a 5-year period. IFA identified the serotypes of 74% of the consecutive isolates and 71% of all enterovirus isolates by yielding a positive staining result. The levels of agreement in the identification of the enterovirus group between IFA and neutralization tests were 92% for consecutively grown isolates and 85% for all enterovirus isolates. The sensitivity of the IFA for the detection of viruses for which specific monoclonal antibodies were applied was 73% for polioviruses, 85% for coxsackieviruses type B, and 94% for echoviruses. Specificity was near 100% for polioviruses and coxsackieviruses type B and 94% for echoviruses. We conclude that IFA can be helpful as a preliminary test for serotype identification of enteroviruses. The results are most accurate when the test identifies the isolate as a poliovirus.

Antibodies, Monoclonal↗
PubMedDate not supplied

Role of recombination in evolution of enteroviruses.

Enteroviruses, members of the Picornaviridae family, comprise a large (over 70 serotypes) group of viruses that are ubiquitous in nature, infect different species and cause a wide range of diseases. Human enteroviruses were recently classified into five species, human enterovirus A-D and poliovirus. Recombination has long been known to be an important property of poliovirus genetics. Recently, several publications demonstrated that recombination is extremely frequent also in non-polio enteroviruses, and allows independent evolution of enterovirus genome fragments even on a microevolutionary scale. Prototype enterovirus strains were shown to have complex phylogenetic relations, and almost all modern enterovirus isolates turned out to be recombinants compared with the prototype strains. Recombination takes place strictly between members of the same species, and usually spares the capsid-encoding genome region. Therefore, it can be concluded that the enterovirus species exist as a worldwide reservoir of genetic material comprising a limited quantity of capsid gene sets defining a finite number of serotypes and a range of non-structural genes that recombine frequently to produce new virus variants. This new model of enterovirus genetics helps to explain the failure of previous attempts to connect serotype and disease profile in non-polio enteroviruses, and seriously questions existing typing approaches that are based solely on the capsid-encoding genome region. It remains to be determined what role recombination plays in the emergence of new enterovirus variants and in the macroevolution of animal enteroviruses and viruses of the picorna-like supergroup.

Enterovirus↗
PubMed1996

Serological and molecular evidence of enterovirus infection in patients with end-stage dilated cardiomyopathy.

OBJECTIVE: To study the relative diagnostic value of enterovirus-specific molecular biological and serological assays in patients with end-stage dilated cardiomyopathy, and to investigate the possible role of other cardiotropic viruses in dilated cardiomyopathy. DESIGN: Analysis of recipient myocardial tissue and serum from patients with dilated cardiomyopathy and controls undergoing cardiac transplantation for end-stage cardiac disease. SETTING: University virology department and transplantation unit. METHODS: Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis of myocardial RNA and DNA; enterovirus-specific in situ hybridization; enterovirus-specific immunoglobulin M detection. RESULTS: Enterovirus RNA was detected in myocardial tissue from only a small proportion of (five of 75) hearts. However, although enterovirus-specific immunoglobulin M responses were detected in 22 (28%) of 39 controls patients, a significantly higher prevalence was observed among patients with dilated cardiomyopathy (22 (56%) of 39 patients; P < 0.005). All enteroviruses detected in myocardium showed greatest nucleotide sequence homology with coxsackievirus type B3. Detection of enterovirus RNA in myocardium by the polymerase chain reaction and by in situ hybridisation gave comparable results. Other potentially cardiotropic virus genomes, including human cytomegalovirus, influenzaviruses, and coronaviruses were not detected in myocardium. CONCLUSION: This study found that enterovirus-specific immunoglobulin M responses provided the strongest evidence of enterovirus involvement in patients with end-stage dilated cardiomyopathy. However, the high background prevalence of these responses limits their diagnostic value. The finding that enteroviruses detected in myocardium were coxsackievirus type B3 accords with recent findings in patients with acute myocarditis, and indicates that this serotype is the major cardiotropic human enterovirus.

Antibodies, Viral↗
PubMed2002

Development and evaluation of a nucleic acid sequence based amplification (NASBA) protocol for the detection of enterovirus RNA in cerebrospinal fluid samples.

A nucleic acid sequence based (NASBA) assay for the generic detection of enterovirus RNA in cerebrospinal fluid (CSF) samples was developed and compared with an established reverse transcription/nested polymerase chain reaction (PCR) protocol. The sensitivity of NASBA followed by detection of amplicons with a biotinylated oligonucleotide probe was < or = ten copies of enterovirus RNA, indicating that enterovirus NASBA achieves a similar sensitivity as nested PCR. Moreover, NASBA detected a panel of 22 different serotypes of the species poliovirus, human enterovirus A, human enterovirus B and human enterovirus C completely. For evaluating NASBA as a diagnostic tool, 61 CSF samples of patients suffering from aseptic meningitis were tested in parallel with NASBA and nested PCR. NASBA detected enterovirus RNA in four CSF samples, two of these were also positive by nested PCR and two other CSF samples were positive only by nested PCR (in total six positive samples). All other 55 of 61 CSF samples were concordantly enterovirus negative by both methods. In conclusion, the more simple to handle 'one step' NASBA is as sensitive as nested PCR and may be used as an alternative method for the detection of enterovirus RNA in CSF samples. Enterovirus NASBA is a 'one step' RNA amplification procedure that is less prone to cross-contamination compared to a three step nested PCR.

Base Sequence↗
PubMed1998

Molecular epidemiology of enteroviruses with special reference to their potential role in the etiology of insulin-dependent diabetes mellitus (IDDM). A review.

BACKGROUND: Several lines of evidence suggest that enterovirus infections may be involved in the etiology of the insulin-dependent diabetes mellitus (IDDM). Often in the literature, a reference is given to specifically diabetogenic strains of enterovirus but there is no systematic assessment about the generation of such strains in the course of evolution or about their abundance among the 64 enterovirus serotypes pathogenic to man. If enteroviruses truly are involved in the etiology of IDDM, a possibility to prevent the disease with enterovirus vaccines might become feasible. In such a situation it would be important to know which serotypes and strains are the most important ones, and whether there would be differences between the strains as regards the pathogenetic mechanisms involved. OBJECTIVE: To present a brief summary of the basic biology of enteroviruses, on existing data of genetic variation of enteroviruses, and on molecular epidemiology of human enteroviruses with special reference to the different epidemiological modes of their putative involvement in the pathogenesis of IDDM. CONCLUSIONS: Like RNA viruses in general, enteroviruses exist as a quasispecies, a mixture of genetic microvariants with a vast potential to adapt to new environments. This means that specifically beta cell-tropic and potentially diabetogenic variants could, in theory, emerge sporadically during systemic infection of any individual. The patterns of genetic diversification of enteroviruses, cocirculation of separate genetic lineages in the human populations, and the assumed geographical restrictions of endemic transmission of the lineages, allow one to hypothesize that populations with a high persisting IDDM incidence might be endemically infected by some specific strains of enteroviruses. However, so far, there is no systematically collected data supporting this hypothesis.

Amino Acid Sequence↗
PubMed1983

Prevalence of infection with nonpolio enteroviruses among healthy children from a children's home and in a summer camp.

The sera obtained at 6 samplings between June 1981 and May 1982 from 30 children in a children's home were tested for neutralizing antibodies to coxsackieviruses B1 through B5, echovirus serotypes 1, 2, 4, 5, 6, 7, 11, 19, 24, 30 and to enterovirus type 71. Another group of examined children comprised 19 individuals from the children's home and 63 children from families who enrolled as participants of a summer camp recreation. Samples of their sera were obtained at the beginning and the end of camping in July and August of 1981. Virus isolations were attempted in 150 stool specimens collected at various times during the observation period, and in specimens of summer camp swimming pool water collected 2-3 times a week. Overall, 10 strains of enterovirus (CB2, E2, E19, E24) were isolated from stool, but none from swimming pool water. Serologically, fourfold or greater increases in titre of antibody to at least one of the enteroviruses tested were observed 17 children, of whom 4 children showed antibody titre increases simultaneously to two enterovirus serotypes. None of the present viruses showed a tendency to mass spread. Some of the examined children showed the presence of serum antibody to enterovirus before its isolation from stool. Eight of the ten children positive for enterovirus in stool did not react by significant increases in titre of antibodies. Enteroviruses were more frequently isolated in children's home in autumn than in summer camp. Clinically, all virologically or serologically demonstrated enteroviral infections were asymptomatic. Children's home children had a significantly higher prevalence of antibodies to enteroviruses than children from families. Presumably, asymptomatic infections with nonpolio enteroviruses among children appear to be far more frequent than the results of neutralization tests in the study indicated. Moreover, enteroviruses may apparently circulate among humans who are seropositive for the respective enterovirus serotype.

Adolescent↗
PubMed1997

Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5'- and 3'-nontranslated regions.

Comparative genomics of viruses in evolutionary and phylogenetic studies is well established. Previous nucleic acid sequence analyses have demonstrated that enteroviruses and rhinoviruses of the family Picornaviridae exhibit a similar structure of the 5'-nontranslated region (NTR) differing significantly from the 5'-NTR of cardiovirus, aphthovirus, hepatovirus, and echovirus 22 (provisionally parechovirus 1). Available nucleotide sequence information of the 5'- and 3'-nontranslated regions of more than 70 serotypes of enteroviruses, bovine enteroviruses and rhinoviruses has been compared and correlated with previous findings obtained after analysis of the coding and noncoding genome regions. As a result, the 5'- and 3'-NTRs of all three virus groups are characterized by group-specific nucleotide sequences. Focusing on bovine enterovirus (BEV) serotypes, unique characteristics in all secondary structures of the NTRs were observed. These features clearly separate the BEVs from the human enteroviruses and rhinoviruses. Concerning the 5'-NTR, the most remarkable property is an insertion of about 110 nucleotides between the putative cloverleaf structure at the very 5'-end of the viral genome and the IRES element. This insertion was demonstrated for BEV 1 and 2 and has a predicted folding pattern which is very similar to the 5'-cloverleaf structure. One stem-loop of this second cloverleaf is almost identical to the 3CDpro-binding domain of rhinoviral 5'-cloverleafs. It was also demonstrated that the IRES elements and the 3'-NTRs of both, enteroviruses and rhinoviruses, have group-specific features which differ significantly from the corresponding genome regions of BEV. These results suggest that bovine enteroviruses hold an exceptional taxonomic position besides the established genera Enterovirus and Rhinovirus. Within the Enterovirus and Rhinovirus genera, the existence of virus clusters representing subgenera was previously proposed. Whereas the 5'-NTRs of the four human enterovirus clusters fall into two groups, all four clusters have characteristic secondary structures at the 3'-NTR supporting the concept of enterovirus clusters. For rhinoviruses, the existence of two virus clusters was confirmed.

Animals↗
PubMed2000

Serotype-specific identification of enterovirus 71 by PCR.

BACKGROUND: Enterovirus 71 and coxsackievirus A16 are closely related genetically and are causative agents of hand foot and mouth disease. Because enterovirus 71 is more often associated with severe neurological disease, there is a need to rapidly discriminate between enterovirus 71 and coxsackievirus A16 during hand, foot, and mouth disease outbreaks. OBJECTIVES: Our goal was to develop and evaluate a serotype-specific reverse transcription-polymerase chain reaction (RT/PCR)-based typing method for enterovirus 71. STUDY DESIGN: Two sets of PCR primers were designed to match conserved amino acid intervals of enterovirus 71. One diagnostic primer pair contains deoxyinosine at sites of 4-fold codon degeneracy. A second primer pair was designed for use in sequencing and molecular epidemiology studies. Primer pairs were tested on strains encountered in routine diagnostic samples. RESULTS: Using both sets of primers on a panel of 61 prototype enteroviral strains, both primer pairs gave strong positive signals for only enterovirus 71. These primers amplified all enterovirus 71 isolates tested and discriminated between enterovirus 71 and the most closely related enterovirus, coxsackievirus A16. CONCLUSIONS: Our RT-PCR assay can be used for specific identification of enterovirus 71 clinical isolates. Furthermore, the 484-bp product of one primer pair has proven useful in sequencing studies to identify distinct genotypes of enterovirus 71.

Amino Acid Sequence↗
PubMed2000

[Evaluation of different clinical samples and cell lines in the isolation of enterovirus in pediatric patients].

BACKGROUND: To evaluate prospectively the efficacy of different clinical samples and cellular lines in the isolation of Enteroviruses from pediatric patients. METHODS: In the period july-1997 to july-1999 we analyze the samples of 102 pediatric patients (< 2 years old) with an unknown febrile etiology syndrome. After the process of decontamination the samples were inoculated in the MRC-5, Hep-2 and Vero cell lines by the shell-vial assay. After 2-3 days of incubation the monolayers were stained by a monoclonal antibody against VP1; then the strains were identified as Poliovirus, ECHO-virus or Coxsacke by specific antibodies. RESULTS: 96 clinical samples were studied (45 pharyngeal swabs, 28 feces, 13 cerebrospinal fluid, 5 bloods, 4 urines and one bronchoalveolar lavage). Enteroviruses were isolated in 48 patients (47%), corresponding to 60 clinical samples (62.5%). The enteroviruses were isolated in 75.5% of pharyngeal swabs, 71.4% of feces, 30.7% of cerebrospinal fluid, one blood (20%) and the bronchoalveolar lavage. 28 patients presented simultaneously both pharyngeal and rectal swabs; in this group the use of both samples permitted the isolation of Enteroviruses in 26 cases (92.8%). Of 60 Enteroviruses, 59 (98.3%) were isolated in the MRC-5 cell line, 23 (38.3%) in the Hep-2, and 14 (23.3%) in the Vero; the MRC-5 demonstrated a significant difference in the isolation of Enteroviruses compared with the other cell lines. The Enteroviruses were identified as 30 (50%) ECHO-virus, 15 (25%) as Poliovirus vaccine, 12 (20%) could not to be identified and in 3 cases (5%) no growth of virus was detected in the culture. The MRC-5 cell line was significatively superior to the other in the isolation of the different Enteroviruses. CONCLUSIONS: To obtain the maximum diagnostic efficacy in front a patient with a possible infection by Enteroviruses, is necessary to use different clinical samples, specially the more related to the infection focci (cerebrospinal fluid or blood). The MRC-5 cell line has been the most efficace in the isolation of Enteroviruses independently of the type of clinical sample or the viral genus. The shell-vial assay is a good method for the isolation and identification of Enteroviruses isolated from clinical samples.

Animals↗
PubMed1997

Enterovirus heart disease of adults: a persistent, limited organ infection in the presence of neutralizing antibodies.

Detection of enterovirus RNA in endomyocardial biopsies (EMB) by reverse transcription/polymerase chain reaction (RT-PCR) is currently the preferred diagnostic procedure in suspected enterovirus heart disease (EHD), which can present clinically as myocarditis, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). EMB and peripheral blood mononuclear cells (PBMC) of 44 patients with suspected EHD were examined by nested RT-PCR to investigate whether the myocardial enterovirus infection is limited to the heart or is generalized. Enterovirus RNA was detected in EMB, but not in PBMC, of 8 patients (3 of these suffered from ARVC), whereas EMB of 16 controls and PBMC of 45 controls were negative. In addition, enterovirus RNA was demonstrated in PBMC, but not in EMB, of a single patient with suspected EHD. A high sequence homology of the amplicons to coxsackievirus B3 was demonstrated in 7 patients, and to coxsackievirus B2 in two patients. In order to evaluate whether the myocardial enterovirus infection was acute or persistent, neutralization and complement fixation tests were performed for antibodies against the serotypes indicated by the nucleic acid sequences. Neutralizing antibodies were detected in the sera of all 9 patients, but complement fixing antibodies were demonstrated only in one EHD patient and in the patient positive for enterovirus RNA in PBMC. In conclusion, the molecular and serological data demonstrate that CVB3 predominates as cardiotropic enterovirus, and that the enterovirus replication is limited to the heart in EHD. Serological results support the hypothesis of myocardial enterovirus RNA persistence in spite of neutralizing antibodies.

Adult↗
PubMed2004

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).

Capsid Proteins↗
PubMed1988

Comparison of cell cultures for rapid isolation of enteroviruses.

Cell culture isolation is still the most reliable method for the detection of enteroviruses from clinical specimens. Rapid diagnosis of enterovirus infection affects patient management. To increase yield and enhance the rapidity of enterovirus isolation in cell cultures, we used Buffalo green monkey kidney (BGM) cells and subpassages of primary human embryonic kidney (HEK) cells in addition to the human diploid fibroblast (MRC-5) cells and primary cynomolgus or rhesus monkey kidney (MK) cells routinely used for enterovirus culturing. Growth characteristics of enteroviruses from 421 specimens were studied. All specimens were cultured in MRC-5, MK, and BGM cells, and 204 of these specimens were also cultured in HEK cells. Forty-two percent of the enteroviruses became positive within 3 days, and 85% did so within 7 days. MRC-5 cells provided the highest yield of enteroviruses overall and were the best cell type for the recovery of poliovirus and echovirus. MK cells provided the second best yield but were more useful than MRC-5 cells for coxsackievirus. BGM cells supported the growth of additional isolates of coxsackievirus and enhanced the speed of isolation. HEK cells supported the growth of additional isolates of both coxsackievirus and echovirus, but subculturing was always required for definite enterovirus cytopathic effects. The recovery rate increased 11% when two additional cell lines were used. The use of two tubes of MK cells significantly increased the yield of all enterovirus types. We conclude that the use of multiple appropriate cell lines increases yield and enhances the rapidity of enterovirus isolation.

Animals↗
PubMed2001

Enterovirus antibody levels during the first two years of life in prediabetic autoantibody-positive children.

AIMS/HYPOTHESIS: We evaluated the role of enterovirus infections in the pathogenesis of Type I (insulin-dependent) diabetes mellitus by monitoring enterovirus antibody levels in prediabetic children who turned positive for diabetes-associated autoantibodies in a prospective birth cohort study. METHODS: Serial serum samples taken during prospective observation starting at birth were analysed for IgG and IgA class antibodies against enterovirus antigens including purified coxsackievirus B4, echovirus 11, poliovirus 1 and a synthetic enterovirus peptide antigen using enzyme immunoassay. Maternal samples taken at the end of the third month of pregnancy were also studied. Analyses were done from 21 childen who developed autoantibodies and from 104 autoantibody-negative control children who were matched for the time of birth, gender and HLA susceptibility alleles. For comparison, adenovirus antibodies were also analysed from all samples collected. RESULTS: IgG class enterovirus antibody levels were high in maternal samples and in cord blood in both case and control children. After birth the IgG levels decreased reaching a nadir at the age of 6 months. No IgA class antibodies were detected at birth but started to emerge postnatally. Antibody levels did not differ between the autoantibody positive and the control children during the first 6 months of life. From 6 months to 24 months of age, the autoantibody positive children had higher IgG and IgA levels against coxsackievirus B4, echovirus 11 and the synthetic enterovirus peptide antigens than control children but poliovirus 1 and adenovirus antibodies were closely similar in the two groups. The difference between children with autoantibodies and control children was predominantly seen among boys and among those with the HLA-DQB1*0302/x genotype. CONCLUSIONS/INTERPRETATION: Our data show that children who seroconverted for diabetes-associated auto-antibodies develop stronger humoral immune responses to coxsackievirus B4, echovirus 11 and a synthetic enterovirus peptide antigen than children who remained negative for autoantibodies. Poliovirus antibodies induced by uniform vaccinations did not differ between the prediabetic and control children suggesting that the regulation of antibody responses to enteroviruses is not disturbed. Accordingly, the results imply a stronger enterovirus exposure in prediabetic children supporting the role of enteroviruses in the pathogenesis of Type I diabetes.

Antibodies, Viral↗
PubMedDate not supplied

RT-nested PCR for the detection of enterovirus in biological samples from patients with suspected enteroviral infections.

In this study, we have tested a reverse transcription (RT) nested polymerase chain reaction (nPCR) for detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF), serum samples, and conjunctival swabs (CS) from patients with suspected enterovirus infections. A specific 113-bp fragment was amplified using primers designed based on 5' non coding region of the enterovirus genome. The enterovirus RT-nPCR was able to detect 0.001 plaque forming unit (pfu)/ml. Since no PCR product was detected in each of the CSF, CS and serum samples from patients with proven-non-enterovirus viral infections, this method was found to be specific. EV RNA was detected in all 30 culture-confirmed CSF samples and yielded positive results in 5 out of 7 additional cases of culture-negative CSF samples with other evidences of enterovirus infection. Overall, EV RNA was detected in 95% of the patients with clinical diagnosis of viral central nervous system (CNS) disease and confirmed enterovirus infection. Furthermore, we were able to detect EV RNA in 24 (47%) out of 51 CSF samples from patients with clinical diagnosis of viral CNS disease and negative laboratory evidence of viral infection. The percentage of positive EV RNA detection in paired CSF and serum samples from 11 patients with an enterovirus isolate in CSF was 100% (11 of 11) and 73% (8 of 11), respectively. In addition, EV-specific IgM was detected in 64% (7 of 11) of the sera tested. The method was also tested against 136 samples of CS from patients with clinical diagnosis of acute hemorrhagic conjunctivitis. Ninety nine of them resulted positive (73%), while only 27 (20%) had been positive for viral culture. In summary, our study shows the importance of enterovirus RT-nPCR for the diagnosis of enterovirus associated disease in different kind of biological samples and different types of diseases.

Acute Disease↗