Echovirus Type Research Articles

Echovirus type 30 infection associated with aseptic meningitis in Nassau County, New York, USA.

An outbreak of echovirus type 30, primarily affecting infant under 1 year old, was documented in Nassau County, N.Y., USA. Twenty-four of the 40 infected patients were definitively diagnosed with aseptic meningitis. A positive correlation was found between the proportion of polymorphonuclear leukocytes and the overall cerebrospinal fluid white cell count.

Epidemic of aseptic meningitis with echovirus type 30 in Gifu Prefecture

During July to December in 1991, an epidemic of aseptic meningitis occurred in Gifu Prefecture in children. Epidemiological, virological and serological investigations were performed. The results are as follows: 1) Cases involved ranged from 0 to 13 years of age, and 75.9% of them were in the 3 to 7 year of age group. 2) Three types of enteroviruses were isolated from 83 of 133 cases (62.4%), and the echovirus type 30 was recovered from 71 cases (85.5%). 3) In 1991 with the cross neutralizing test between the prototype and isolated strains of echovirus type 30, a remarkable antigenic variation could not be found. 4) In serological studies, the positive rates of neutralizing antibodies to echovirus type 30 in infant sera aged 0-9 years in 1992-1993, was 51.9% with the prototype strain, but 34.0% with an isolated strain in 1991.

An Outbreak of Aseptic Meningitis in Okinawa in 1993.

In the summer of 1993, an epidemic of aseptic meningitis had broken out in Okinawa island, the southernmost area of Japan. It started in June (or probably in May), reached a peak early in July and subsided in August. A total of 216 specimens (cerebrospinal fluids, throat swabs and sera) was collected from the patients with aseptic meningitis and viruses were recovered from 83 specimens (38.4%). Five isolates randomly selected were examined for the identification of the virus. The virus was identified as echovirus type 30 (echo-30) by plaque reduction neutralization test using enterovirus antiserum pools and monospecific antiserum. Large-plaque and small-plaque phenotypes of echo-30 were isolated. The two were neutralized by anti-echo-30 sera. For the plaque assay of echo-30 in RD-18S cells, the use of tragacanth gum for the overlay medium could reduce the incubation period to 25-30 hr.

Epidemiological Features of Type 22 Echovirus Infection

During a 25-year observation period, isolates of type 22 echovirus were obtained from 109 patients. 92% of the patients were < 2 years old. Echovirus type 22 was isolated with peaks both during late summer and autumn, as enterovirus infections, and during the winter months and early spring, as respiratory viruses. Diarrhea was the most common symptom, followed by obstructive bronchitis and, less often, CNS symptoms. Nosocomial infections were common. In a noticeable number of the children, maternal neutralizing antibodies were most probably present at the time of infection. The epidemiologic features of type 22 echovirus infections with regard to age and seasonal distribution, contagiousness and a relative lack of protection by neutralizing antibodies differed from most enterovirus infections.

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.

Detection of enteroviral RNA by polymerase chain reaction in faecal samples from patients with aseptic meningitis.

An assay based on the polymerase chain reaction (PCR) for detection of enteroviral RNA in stool samples was carried out using specimens from 74 patients with aseptic meningitis. The primer pair and probe were derived from the highly conserved 5' non-coding enterovirus genomic region. Enteroviral RNA was detected in faeces of all 36 patients in whom an enterovirus was isolated from stool. The PCR assay yielded positive results in additionally 3/6 cases where enterovirus diagnoses were obtained by virus isolation from cerebrospinal fluid and/or serological tests. Thus, the positive outcome of the PCR assay was 39 (93%) among the 42 patients with enterovirus diagnoses. Furthermore, 7/19 (37%) cases with an etiology that was not established by other means were positive in the test indicating that the PCR assay may give considerable additional etiological information in patients with aseptic meningitis. The limit of RNA detectability in the PCR assay was about 100 TCID50 when highly cytopathogenic enterovirus types (coxsackievirus type B5 and echovirus type 11) were tested. The PCR was negative in all 13 patients with non-enterovirus diagnoses except in one case with a herpes simplex virus type 2 infection. Since enterovirus-specific IgM antibodies could be detected in this case a dual infection seemed probable. All the negative controls, included in the study, were PCR-negative and no contamination was encountered. This study proves the usefulness of the PCR assay for detection of enteroviral RNA in stool samples and suggests that the test may be an alternative to virus isolation for rapid enterovirus diagnosis in patients with aseptic meningitis.

Placental phagocytic cells infected with herpes simplex type 2 and echovirus type 19: virological and ultrastructural aspects.

Placental macrophage cells were kept in a short-term culture and infected with herpes simplex type 2 virus and echovirus type 19. These were observed under optical and electron microscopy. Immunofluorescence, virus titration and autoradiographic technique were used to determine if the virus was replicating in the system. The results showed that the placental phagocytic cells do not allow virus growth and that the virus particles are destroyed right after virus uptake, within 4 h post-infection. The increase of lipid bodies and other cellular alterations suggested the intensive action of these cells against viruses.

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.

Enteroviral aseptic meningitis in Japan, 1981-1991. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

In Japan, aseptic meningitis cases due to enterovirus infections increase every summer in various degrees with an incidence peak usually in July. During the past 11 years from 1981 through 1991, a total of 8,595 enterovirus isolations from aseptic meningitis cases were reported from 54 participating laboratories. Eight enterovirus types caused large epidemics; more than 100 isolations of each type from aseptic meningitis cases were reported for every epidemic year of the respective type. They were coxsackievirus (C) types B3 and B5, echovirus (E) types 4, 6, 7, 9, 18 and 30. Among these, the highest meningitis-associating frequency was reported for E30, representing 82.6% of the total isolations reported for the type during this period, followed by E4, 71.1%. The frequencies of E9, E7, E6 and CB5 were in a range from 54.5% to 44.4%, while that of E18 was 37.7% and that of CB3 21.0%. During the epidemics, enterovirus-associated meningitis was most frequently reported among children of 4-7 years of age. High frequencies were also shown in infants less than 1-year of age in some types. A total of 4,240 enteroviruses were isolated from cerebrospinal fluid of aseptic meningitis cases, representing 49.3% of the cases with enterovirus isolation.

General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections.

The aim of this study was to determine the applicability of the polymerase chain reaction (PCR) for routine diagnostic use and for the detection of persistent enteroviral infections. To this end, general primers were selected in the highly conserved part of the 5'-noncoding region of the enteroviral genome. They were tested on 66 different enterovirus serotypes. A specific fragment was amplified from 60 of 66 serotypes. An amplification product was not observed from coxsackievirus types A11, A17, and A24 and echovirus types 16, 22, and 23. Enteroviral RNA was detected by the PCR in routinely collected throat swabs and stool specimens that were found to be positive for enterovirus by isolation in tissue culture. Enteroviral RNA was detected in one of five myocardial biopsy specimens from patients with dilated cardiomyopathy, implicating virus persistence. No amplification product was obtained from eight control samples. Our results demonstrate the significance of the PCR for the detection of enteroviral RNA and, in particular, for the demonstration of persistent enteroviral infections.

The relationship of clinical manifestations and age distribution in enterovirus infections--viral isolation and seroepidemiology in Aichi Prefecture

We studied the epidemiology of enterovirus infection in Aichi Prefecture from 1985 to 1989. We examined the age distribution of aseptic meningitis patients (AM) and exanthematous disease patients (Ex) and a seroepidemiological study of echovirus type 7 (E7), E9, E18 and group A coxsackievirus type 9 (CA9), was performed. The results is as follows: 1) E7, E9, E18 and CA9 were isolated from AM and Ex but E6, E11 and group B Coxsackie viruses (CB) were isolated in fewer cases from Ex. 2) The AM was consistently increased from June to August. Whereas the Ex was seen in all seasons but a slight increase was noted between June to July, and enteroviruses (EV) isolation was increased in this season. 3) The AM occurred in 0 year olds and 4 year olds whereas the Ex was seen in 0 to 1 years. EV was mainly isolated from 0-1 year olds. 4) The relationship of clinical manifestations and age was very clear in E9 and E18, a higher proportion of children at 1 years or under were those of the Ex and most children of the latter part of 4 years were those of the AM. The Ex had the same results with E7 and CA9 but AM was increased in 0 years and 4 year olds. 5) We studied the age distributions of neutralizing antibodies against E7, E9, E18 and CA9. The positive rate of neutralizing antibody after prevalence rose between 2-5 years of age. There were few patients among the 2 to 3 year olds but the neutralizing antibody was raised in this age. I considered that reason the enteroviruses infected mainly the 2 to 6 year olds showing no clinical symptoms where as some of the 3 year olds had aseptic meningitis and some under 1 year had symptoms of exanthematous diseases.

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.

Comparative Inactivation of Hepatitis A Virus and Other Enteroviruses in Water by Iodine

Iodine is used as a disinfectant of small and field water supplies, but little is known about its ability to inactivate important waterborne viruses such as hepatitis A virus (HAV). In laboratory studies we determined the inactivation of purified, aggregated HAV, poliovirus type 1 and echovirus type 1 by 8 and 16 mg/l doses of iodine in both phosphate buffered, iodine demand-free (clean) water and the same water containing 10 mg/l of a 1:1 mixture of humic and fulvic acids and 5 NTU of bentonite clay turbidity (dirty water). Virus inactivation studies in clean water were done at pH 4.5, 7.0 and 9.5 and at 5 and 25 C, and in dirty water they were done at the same pH levels but at 5°C only. Iodine doses of 8 and 16 mg/l in clean water produced 99.99% or 4 log10 inactivation of HAV by 60 minutes or less. Four log10 inactivation of polio 1 and echo 1 was not achieved in 60 minutes by 8 mg/l iodine at 5°C ani pH 7.0 and 4.5 or by 16 mg/l iodine at 5°C and pH 4.5. HAV was inactivated more efficiently by iodine than were the other two test viruses, and the overall order of virus inactivation was: HAV &amp;gt; echo 1 &amp;gt; polio 1. Virus inactivation was generally more effective at higher pH, in cleaner water, at higher temperature and at higher iodine dose.

1986年の北九州市におけるエコーウイルス7型の流行について

In 1986, under the Infectious Disease Surveillance system in Fukuoka Prefecture, no isolation of echovirus type 7 from aseptic meningitis patients were reported. It was unclear that epidemics of this virus occurred in Fukuoka Prefecture, including Kitakyushu City. We tried to isolate enteric viruses from the sewage (activated sludges) in Kitakyushu City in 1986. Outbreaks of isolations of echovirus type 7 started in September and continued until in December. This result suggested that echovirus type 7 caused epidemic during this period in Kitakyushu City. Sera were collected from children in 1989, and were examined neutralizing antibody titer against echovirus type 7. Antibody titers were positive in the sera of about half of children above 1 year of age in 1986. From results of virus isolations from the sewage and serological study, it was confirmed that echovirus type 7, reported no isolation in Fukuoka Prefectural surveillance system, caused epidemics in Kitakyushu City in 1986.

Protein analysis of newly isolated variants of echovirus type 18 by electrophoresis and Western blotting

The protein patterns of two newly isolated antigenic variants of echovirus type 18 were compared with those of the prototype strain. Electrophoretic mobilities of VP1 and VP2 of the variants differed from those of the prototype strain. The antiserum against the prototype strain reacted to VP0 and VP2 in the Western blotting experiment. In addition, the variants differed from the prototype strain in growth behavior in the cultured cells.

Impact on routine diagnosis of echovirus infections of intratypic differentiation and antigenic variation in echovirus type 25 studied by using monoclonal antibodies

We studied the biological and antigenic properties of wild strains of echovirus type 25 isolated in France between 1982 and 1987 and compared them with the JV-4 prototype strains isolated in 1957. The wild strains differed from the prototype strain in their cellular tropism. The prototype strain grew readily in five cell lines (MRC5, MA 104, Vero, BGM, and HT 29-18), while for wild strains MRC5 and HT 29-18 cells were the most sensitive and supported growth to high titres (between 4.5 and 7.4 50% tissue culture infective doses per 0.05 ml). Plaques produced by wild strains were larger (6.05 +/- 0.94 mm in diameter [mean +/- standard deviation]) than those of the prototype strain (2.3 +/- 0.97 mm in diameter) and heterogeneous, even after cloning by three terminal dilution passages, which suggested heterogeneous virus populations. Virus neutralization with polyclonal monovalent sera showed that wild strains were significantly less neutralized by two reference immune sera than the prototype strain was. Monoclonal antibodies were raised against the echovirus type 25 JV-4 prototype strain. Nine clones with neutralizing activity were identified. Heterologous neutralizations of 14 clinical isolates revealed highly conserved, moderately conserved, and poorly conserved epitopes. The natural isolates differed from the prototype strain in two to four epitopes and can be classified into four different groups. We concluded that echovirus type 25, like coxsackie- and polioviruses, consists of heterogeneous viral populations with respect to biological and antigenic properties. In term of viral diagnosis, it may become increasingly difficult to identify recently isolated strains because of their antigenic variation.

Detection and differentiation of picornaviruses in clinical samples following genomic amplification

A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5' non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.

Detection of immunoglobulin G, M, and A antibodies to enterovirus structural proteins by immunoblot technique in echovirus type 4-infected patients

Paired serum specimens from 24 patients with echovirus (EV) type 4 infection by virus isolation were tested by the immunoblot technique for the presence of IgG, IgM, and IgA antibodies to EV4 structural proteins. Single sera from 20 patients without neutralizing enterovirus IgM were used as controls. All the sera from EV4-infected patients had IgG antibodies to VP1 of EV4 but also 13 out of the 20 controls. 23 out of 24 EV4-infected patients elicited IgM and IgA specific antibodies to VP1, a pattern highly significant as compared with controls ( 3 20 for IgM and 8 20 for IgA). In 16 out of the 24 EV4-infected patients, the IgM antibodies were also directed against VP2 (versus 2 out of 20 in the control group). Anti-VP2 IgA were detected in 4 out of the 24 EV4 patients (versus 0 in controls). The 24 paired sera from EV4-infected subjects were also tested by immunoblot technique against three other enteroviruses: EV21, coxsackievirus A9 and poliovirus 1. Cross-reactivities were observed to a large extent against VP1 and VP2 proteins with the three classes of antibodies. These results confirm the data of previous studies on the reactivity of IgM antibodies to various structural proteins that IgG antibodies react exclusively to VP1. Furthermore, this study demonstrates the occurrence of circulating IgA antibodies directed to VP1 and sometimes VP2 in the course of enterovirus infection. The potential interest of this latter finding for diagnosis requires further investigation.

Isolation and preliminary characterization of antigenic variant of echovirus type 11

Nosocomial infection with echovirus type 11 resulting in aseptic meningitis occurred among newborn babies in a hospital neonatal room at Fukui city. The virus was identified as a variant of echovirus type 11 by cross-neutralization tests with antisera against the prototype Gregory strain and the current Fukui isolate. Fukui isolates expressed strain specific antigen(s) in addition to type specific common antigen(s), but lacked a certain antigen(s) which was present in the prototype strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virus polypeptides revealed that the capsid proteins VP2 and VP3 of Fukui strain migrated more rapidly than the Gregory strain, while both strains had the same migration pattern of VP1 protein on which the antigenic determinants responsible for virus neutralization were present. The current strain produced large plaques and was more thermoresistant, suggesting some alterations in the structural proteins of the virus.

Specific detection of enteroviruses in clinical samples by molecular hybridization using poliovirus subgenomic riboprobes

Enteroviruses were specifically detected in crude clinical specimens or in cell cultures in which the viruses were amplified by dot hybridization by using poliovirus type 1-derived, subgenomic radiolabeled cRNA probes (riboprobes). The sensitivity of this test varied from 2.5 to 33%, when clinical specimens without cell culture were examined, and was about 85% in cell culture lysates. The specificity of the test was 90 to 100%. The riboprobe corresponding to the 5'-noncoding sequence specifically detected the majority of enteroviruses (56 of 57 tested); the riboprobe derived from the VPI capsid region hybridized with the three poliovirus serotypes and with some coxsackieviruses type A and with echovirus type 7. Echovirus 22 did not hybridize with any riboprobe. In stool specimens, nasal aspirates, and cerebrospinal fluids from patients with meningitis, only one type of virus was identified in different clinical samples from the same patient by the seroneutralization test. Hybridization allowed the detection of enteroviral RNAs easily in stool specimens and nasal aspirates but with a low efficiency in cerebrospinal fluids without amplification of the viruses in cell cultures.