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BackgroundIn the Netherlands, echovirus type 6 (E6) is identified through clinical and environmental enterovirus surveillance (CEVS and EEVS). AimWe aimed to identify E6 transmission clusters and to assess the role of EEVS in surveillance and early warning of E6. MethodsWe included all E6 strains from CEVS and EEVS from 2007 through 2016. CEVS samples were from patients with enterovirus illness. EEVS samples came from sewage water at pre-specified sampling points. E6 strains were defined by partial VP1 sequence, month and 4-digit postcode. Phylogenetic E6 clusters were detected using pairwise genetic distances. We identified transmission clusters using a combined pairwise distance in time, place and phylogeny dimensions. ResultsE6 was identified in 157 of 3,506 CEVS clinical episodes and 92 of 1,067 EEVS samples. Increased E6 circulation was observed in 2009 and from 2014 onwards. Eight phylogenetic clusters were identified; five included both CEVS and EEVS strains. Among these, identification in EEVS did not consistently precede CEVS. One phylogenetic cluster was dominant until 2014, but genetic diversity increased thereafter. Of 14 identified transmission clusters, six included both EEVS and CEVS; in two of them, EEVS identification preceded CEVS identification. Transmission clusters were consistent with phylogenetic clusters, and with previous outbreak reports. ConclusionAlgorithms using combined time-place-phylogeny data allowed identification of clusters not detected by any of these variables alone. EEVS identified strains circulating in the population, but EEVS samples did not systematically precede clinical case surveillance, limiting EEVS usefulness for early warning in a context where E6 is endemic.

Echovirus 30 (E30) is a major pathogen associated with aseptic meningitis. In the summer of 2014, a family clustering aseptic meningitis outbreak occurred in urban-rural fringe of Ningbo city in Zhejiang Province in China. To identify the etiologic agent, specimens were tested by cell culture and reverse transcriptase-polymerase chain reaction. Pathogenic examination confirmed that the outbreak is caused by E30. The first case is a 6-year-old child, who studied in kindergarten in local, suffered from headache and fever. Same symptoms appeared in his parents, aunts, and other six relatives continuously. Meanwhile, vomiting occurred in majority of the patients and diarrhea in parts of them. White blood cells in cerebrospinal fluid (CSF) exceeded normal range in all patients. Protein levels in CSF were above normal range in half of the patients. Glucose levels in CSF were within normal range in all patients. We isolated six strains E30 in the stool specimens of patients, and carried out sequencing analysis to VP1 region. Sequencing results showed that 100% sequence identity was seen in both nucleotide and amino acid levels. Phylogenetic analysis discovered that isolate in this study was grouped into sublineage D2 together with sequences isolated from other areas of China in the 2000s and 2010s. Our study is the first family clustering outbreak of aseptic meningitis caused by E30 in Zhejiang Province in China. It is essential to establish an enterovirus molecular surveillance system in China to prevent mass outbreaks in Zhejiang.

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We detected enteroviral RNA and cultured infectious virus from a series of banked breast milk samples from the mother of an infant with neonatal sepsis; sequencing of the enterovirus isolate identified it as echovirus type 18. In this case, it is possible that enterovirus transmission occurred through the breast milk.

From July to October 2001, 215 cases of aseptic meningitis occurred among the inhabitants of the German city of Kassel and neighbouring counties. A matched case-control study identified bathing in a public, nature-like pond during the beginning of the outbreak as a risk factor for disease [matched odds ratio (mOR) 44.8, 95% confidence interval (CI) 3.9-515.6]. Among bathers, patients with meningitis spent more time in the water (mOR 18.8, 95% CI 2.0-174.1) and swallowed water more frequently (mOR = 7.3, 95% CI 0.7-81.8). Of 30 cerebrospinal fluid samples tested, echovirus 30 was cultured from 16, and echovirus 13 from seven. An echovirus 30 sequence obtained from one pond water sample showed a 99% nucleotide and 100% amino-acid homology with patient isolates. This outbreak demonstrates the potential of nature-like swimming ponds to cause widespread community infection with substantial public health impact.

Aims/hypothesis: Neonatal diabetes mellitus is rare, and it has not been associated with beta-cell autoimmunity. Enteroviral infections during pregnancy have been implicated as a risk factor for the later development of Type I (insulin-dependent) diabetes mellitus. We now report of a baby girl who was born severely growth-retarded with neonatal insulin-deficient diabetes, and look for evidence of intrauterine enteroviral infections and beta-cell targeted autoimmunity., Methods: Diabetes-associated autoimmunity was studied by measurement of several types of islet cell reactive autoantibodies. The infant's T-cell responses to insulin and enterovirus antigens were recorded and enterovirus antibodies were measured both from the mother and the child., Results: Several types of diabetes-associated autoantibodies were detected postnatally, including insulin autoantibodies, conventional islet cell autoantibodies and glutamic acid decarboxylase antibodies, whereas no autoantibodies were observed in the mother. The infant's T-cells showed reactivity to insulin and purified enterovirus particles. Based on serological studies, the pathogenetic process could have been triggered by an echovirus 6 infection during pregnancy. The patient's diabetes has been permanent, although there were signs of endogenous insulin production for several months. Exocrine pancreatic insufficiency was diagnosed at the age of 1 year., Conclusion/interpretation: These observations suggests that enteroviral infections may induce beta-cell autoimmunity even in utero.

We investigated six cases of enterovirus infection in a neonatal unit. The index patient, a 5-day-old boy, was admitted with aseptic meningitis due to echovirus 30 (E30). Secondary infections with E30 occurred in five babies. Comparison of the complete VP1 sequences showed that the isolates recovered from the index patient and his mother were closely related to those recovered from the five babies with secondary infections, demonstrating a nosocomial transmission of the virus. In the phylogenetic tree reconstructed from the VP1 sequences, the isolates formed a monophyletic cluster related to an E30 strain collected in June 1997 during an outbreak of aseptic meningitis.

Objective: A report of five cases of viral meningitis among adults with children enrolled in a child care center prompted an investigation of risk factors for viral transmission from children to adult household members., Methods: To determine recent echovirus 30 (E30) infections, the authors conducted a serologic survey. To determine risk factors for infection among adult household members, they conducted a retrospective cohort study using written questionnaires., Results: Recent E30 infections were found in 84% of children tested, 57% of adult household members tested, and 47% of staff members tested. Infected adults were more likely than infected children to have clinical meningitis. Among adult household members, changing diapers was a risk factor for recent infection. Women who changed > or = 90 diapers per month had a higher infection rate than women who changed fewer diapers; in contrast, men who changed > or = 90 diapers per month had a lower infection rate than men who changed fewer diapers. Handwashing was protective: there was a negative correlation between handwashing after diaper changes and E30 infection among adults with infected children in diapers., Conclusions: Because child care centers can be a source of enteroviral infections among adult household members, adults with viral meningitis should be questioned about their children's day care or preschool attendance. The importance of handwashing should be stressed to adults with children in day care.

Forty-six people became ill with vomiting, diarrhoea and headache within days of an outdoor swimming pool opening for the summer season in a small seaside village. During the weekend of the outbreak, 185 tickets to the pool had been sold. It was found that 34 bathers were ill, and one subject had vomited into the pool. All other cases arose after this incident. The risk of infection was greatest among those who swallowed pool water (24/28 versus 10/17, p = 0.07). Echovirus 30 was isolated from the case who vomited into the pool and from six other cases. Normal chlorine levels had not been adequate to contain the infection risk from vomitus, and, in future, pool attendants witnessing such incidents should consider closing the pool to the public and seeking advice on superchlorination.

In July 1992, 13 parents with children attending a child care center (CCC) developed aseptic meningitis (AM) due to echovirus 30 (E30). To determine the extent of illness and risk factors for transmission, survey and blood specimens were collected from CCC families and teachers and from adult and pediatric controls. Infection was defined as the presence of anti-E30 IgM antibodies. CCC parents (60%, 67/111) and children (75%, 79/105) had significantly higher infection rates than did teachers (14%, 3/22), adult controls (24%, 10/41), and pediatric controls (24%, 17/70). Infected CCC parents had more severe illness (18% [12/65] had AM; 11% [7/65] were hospitalized) than did infected CCC children (3% [2/79] had AM and 1% [1/79] were hospitalized). More frequent handwashing among teachers compared with parents and among mothers of toddlers was associated with significantly lower rates of infection (P < or = .05). Education of parents about good handwashing practices may reduce transmission of E30 and other infectious agents from children to adults.

Objectives: The aim of this study was to define the major features of enterovirus infections in the neonatal period based on our own experience., Methods: Epidemiology, clinical manifestations and laboratory investigations concerning 21 neonates having experienced a Coxsackie B or an Echovirus infection between 1987 and 1991, were retrospectively reviewed. Aetiological diagnosis was made by classical viral isolation and/or by evidencing Coxsackie B-specific IgM antibodies with an immunocapture enzyme immunoassay., Results: In 13 neonates the infection occurred between June and September. The onset of clinical signs ranged from day 1 to day 25 after birth with two separate periods: before 7 days of age, suggesting a perinatal transmission of the virus, or beyond this date, more likely connected with a postnatal transmission. Clinical manifestations included hyperthermia, gastroenteritis, meningitis, encephalitis, pneumonia and myocarditis, with a diphasic pattern in 6 cases. Most of the neonates improved gradually and developed normally. The Coxsackie B-specific IgM assay was the most rapid method whereas viral isolation, even though it took more time, was the most sensitive technique to establish the aetiological diagnosis in neonates., Conclusions: Enterovirus infections in neonates are difficult to diagnose and to differentiate from bacterial infections. A viral-like illness in the environment of the neonate allows the clinician to anticipate the clinical signs and a possibly fatal disease. Identification of the causal virus should be performed by both viral isolation and search for specific IgM antibodies. Treatment and prophylaxis are so far disappointing.

A prospective study of 78 pregnant women was undertaken to detect maternal enterovirus infection. Maternal faecal specimens and blood samples, placental and fetal tissue were taken for viral study, electron microscopy, histochemistry, and morphological examination. We present the post-mortem findings in three fetuses whose maternal infection was detected before delivery by isolation of ECHO virus type 33 and type 27 from faecal specimens and/or placental and fetal tissues. The morphological aspects were similar in all cases and included an acute infection of the placenta and hypoxic/hypotensive injury to fetal organs. In one case, viral particles were detected by electron microscopy of the fetal liver. This series of cases of intrauterine ECHO virus infection confirms the potential gravity of such infection during pregnancy and the need to prevent enteroviral disease.

Four cases of fatal echovirus 11 disease occurred in premature infants during a community outbreak of enteroviral disease in Massachusetts in 1979. Each infant developed nonspecific symptoms and jaundice at 4 to 6 days of age, and subsequent progressive hepatic failure and generalized bleeding. Only one infant survived longer than six days. Virus was recovered from multiple sites premortem, and from virtually all tissue cultured at autopsy. Myocarditis was not present clinically or pathologically. Clinical and laboratory evidence implicated perinatal transmission of virus from mother to infant. Three mothers experienced a febrile illness with abdominal pain within the last five days of pregnancy. In two, the illness led to a false diagnosis of abruptio placenta and interruption of pregnancy by cesarian section. Review of case reports of this syndrome caused by other echovirus serotypes revealed that many had similar perinatal events. Each mother ultimately developed neutralizing antibody to echovirus 11. However, all four infants were born without passively acquired antibody, probably because they were delivered prior to the appearance of specific maternal IgG. Although laboratory studies by others have shown other factors may be responsible for the ability of enterovirus to cause overwhelming disease in neonates, uncontrolled data from these four infants and their mothers suggest that timing of maternal illness in relation to delivery of the infant may also be important.

Between September 23 and November 30, 1981, 1,032 children from Panamá City, Republic of Panamá were hospitalized with aseptic meningitis. Forty-four per cent of the cases were in the age group 5-9 years old; the disease was mild and self-limiting with an average hospital stay of five days. Echovirus 4 isolates were obtained from 48 of 160 patients. To identify risk factors associated with the epidemic, the authors randomly selected 10 per cent of hospitalized cases and conducted a family-based seroepidemiologic study. The closest neighboring house with at least one child younger than 15 years was similarly studied as a control. In total, 182 households and 1,083 of 1,177 residents were included. No risk factors ascertained by the study were associated with aseptic meningitis; however, several factors were related to recent echovirus 4 infection. Overall, 56 per cent of cases had echovirus 4 antibody, as did 29 per cent of their family members and 19 per cent of control family members. Children who attended kindergarten or primary school were more likely to have antibody than other household members and, within case families, individuals involved in child-care had an excess risk for infection.

A two-part study of echovirus-12 was done in volunteers. In the first part the human infectious dose of the virus was determined in 149 healthy adults with undetectable serum antibody, each of whom drank 0-330,000 plaque-forming units (pfu) of virus in 100 ml of nonchlorinated water. Infection was defined as fecal shedding of virus or significant (fourfold or greater) increases in serum antibody titer. The HID50 (i.e., the dose required for infection of 50% of the volunteers) was 919 pfu. Through statistical analysis of the data by probit transformation, a 1% human-infectious dose of 17 pfu was predicted. These results were used in the second portion of the study to determine the effect of previous infection on the infectious dose. Previously infected volunteers (those with neutralizing serum antibody) were given a dose of echovirus-12 (1,500 pfu) that had been found to infect 60% of persons with undetectable serum antibody. The presence of serum antibody caused no significant change in the percentage of volunteers infected by this dose. Furthermore, the concentration of serum antibody did not affect the rate of infection or the duration of viral shedding. These results indicate that previous infection with echovirus-12 does not provide lasting protection against reinfection.

Epidemiology, clinical features and laboratory results of a nosocomial outbreak of Echovirus 7 infections are described. After the admission of the index case with presumably perinatal infection to the neonatology unit, 3 additional babies from the total of 16 patients on the ward were infected. A 5th baby in another unit was also infected; this baby shared in part the nursing staff with the other group. Clinical symptoms of the infection were fever (5 children), gastrointestinal (5) and neurological symptoms (4), apnea and bradycardia (1), and a fine macular rash (1). Five of the 24 persons of the staff suffered from a febrile gastroenteritis during this period. The virus was isolated from stool swabs, throat swabs, urine, blood and CFS. An augmented percentage of immature neutrophils in the peripheral blood count was the only laboratory value found to be abnormal in all of the children. We measured neutralizing antibodies against this virus in an immunoglobulin preparation for i.v. use (Sandoglobulin), to get information about its prophylactic value in these cases. We could not detect any neutralizing antibodies.

A review of literature published before June 1985 revealed 61 reported cases of neonatal echovirus infection at a nonmucosal site, including 43 cases (70%) due to echovirus 11. Onset of disease occurred between the third and fifth days of life in 63% of cases, indicating that most infections are acquired in the immediate perinatal period rather than in utero. Mortality was higher in infants with severe hepatitis (83%) than in infants with infection of the central nervous system (19%). Acute illness occurred within one week before delivery in 68% of the mothers of nonnosocomially infected infants. There was a trend (P = .11) towards a higher mortality rate for infants born by cesarian section than for those delivered vaginally. In the 11 nosocomially acquired cases, the onset of infection was later and the mortality rate lower. In 16 outbreaks in nurseries, 206 infants developed illness attributed to echovirus infection. Attack rates of clinical disease were 22%-52% and illness was generally mild. In four outbreaks, six index cases were identified as infants who had acquired infection from their mothers; five of these infants had severe disease and three died. The 24 infants subsequently infected by nosocomial spread in these outbreaks had milder disease; three (12%) died. Thus, whereas acute illness in the mother before birth often precedes neonatal echovirus infection and infections transmitted vertically from mother to infant may be severe, postnatal transmission of the same serotype results in milder disease.

Two babies in a neonatal unit presented on the same day with meningitis due to echovirus 11, which was thought to have been introduced by staff. At this time echovirus 11 was also isolated from the stools of eight other babies; five of them did not have signs of infection. No intervention was made except to emphasise the importance of handwashing. There was evidence of secondary spread to two babies who were both clinically well. The attack rate was twelve (29%) of forty-one babies exposed. Seven of the twelve infected babies were born before 30 weeks' gestation and would have had little or no maternal antibody, yet only two of the seven babies had signs of infection. Despite lack of special measures, all babies recovered. Most cases of horizontally acquired neonatal echovirus infection are mild: extreme measures in the management of outbreaks are unnecessary.

Enteroviral infections late in pregnancy are common, especially during periods of high prevalence of community infection. Most of these infections, however, are not associated with significant maternal or neonatal disease. Conversely, as many as 65 per cent of women who give birth to infants with proven enteroviral infection have symptomatic disease during the perinatal period. Maternal echovirus or coxsackievirus B infections are not associated with an increased risk of spontaneous abortions, but stillbirths late in pregnancy have been described. Although a slightly increased risk for congenital heart defects and urogenital anomalies has been reported for the offspring of women who seroconverted to the group B coxsackievirus during pregnancy, these data are highly tentative. Transmission of enteroviruses from mother to infant is relatively common (30-50 per cent) and may occur through contact with maternal secretions during vaginal delivery, blood, or upper respiratory tract secretions. Intrauterine transmission has been documented, but its frequency is unknown. Postnatal transmission from maternal or nonmaternal sources also occurs regularly. Neonatal disease may range from inapparent infection to overwhelming systemic illness and death. Common clinical syndromes associated with neonatal enteroviral infections are meningoencephalitis, pneumonia, myocarditis, and hepatitis. The severity and outcome of perinatally acquired enteroviral infection is influenced by several factors, including the virus strain involved, mode of transmission, and presence of passively acquired serotype-specific maternal antibody. Newborn nursery outbreaks of nonpolio enteroviral infections usually coincide with seasonal peaks of enteroviral disease in the community. These outbreaks have been due mostly to echovirus 11 or group B coxsackievirus serotypes 1 to 5 and are associated with attack rates of up to 50 per cent.

A continuing surveillance on enterovirus infection in healthy infants was conducted from October 1971 through February 1973 in urban areas in Ghana, West Africa. About 40 infants were visited in every two months for collection of faecal specimens and examined for infection. Enteroviruses were recovered in tissue culture. The overall isolation rate of enteroviruses was approximately 44%, and there was no seasonal difference between rainy and dry seasons. The rate of virus isolation in urban areas was significantly higher than in rural areas. Within the urban areas, however, no difference in the rate of virus isolation was detected between densely populated and sparsely populated areas. The results of virus identification revealed that all three types of Poliovirus, many types of Echovirus and a few Coxsackieviruses were isolated during the course of the study. It was observed that improvement of sanitary facilities decreased the frequency of virus infection among infants, but the condition of water supply did not influence the virus infection rate. Neither the age of infants nor the size of siblings showed any relation to the virus isolation rate. It was suggested from the results that many types of enteroviruses have been circulation continuously in the tropical urban community throughout the year.

The case described herein represents the first laboratory-confirmed case indicating intrauterine infection due to echovirus type II. The virus was recovered from the vagina of the mother and from the blood from the umbilical cord and nasopharynx of an apathetic newborn (all cultures were taken within 60 minutes of birth in the delivery room) with a generalized maculopapular exanthem. When the infant was 15 days of age, results of all laboratory tests and physical examinations were normal.

After an 8-day-old child had died with clinical signs of septicemia, 6 other newborns fell ill. Virus was isolated from various sites from all the 6 children (28 isolations). The agent was identified by cross neutralization tests as an antigenic variant of Echovirus 11. The agent could not be isolated from mothers or nursery staff (49 people). We therefore tried to trace the path of infection by isolating specific IgM and IgG antibodies. A laboratory infection by the agent isolated enabled the time pattern of the serologic immune response to be roughly determined. The data collected indicate that the infection spread through close contact between the affected newborns and nurses working in the newborn room. Rigorous hygienic and isolation measures, initiated immediately, appeared to interrupt the spread of infection.

One type of vinyl and seven types of latex gloves without visual defects were tested with respect to their barrier function against high concentrations of three viruses of varying size: herpes simplex virus type 1 (HSV-1, 180 nm), human immunodeficiency virus type 1 (HIV-1, 100 nm), and echovirus type 9 (Echo 9, 25 nm). Viral suspensions of HSV-1 (10(8) TCD50/ml), HIV-1 (10(5) TCD50/ml), and echovirus type 9 (10(7.5)TCD 50/ml) were placed in an inverted glove finger immersed in media and maintained for 3 h at room temperature with sampling performed from outside the glove at 10 min, 30 min, 1 h, 2 h, and 3 h. No cytopathic effect (CPE) was identified after inoculation onto Vero cells or RhMK cells for HSV-1 and Echo 9, respectively, and reverse transcriptase activity was not detected in Hut 78 cells after inoculation of HIV-1 during any of the time intervals. Stretching of a glove finger for 18 h with repetition of the procedure with Echo 9 revealed no CPE. We conclude that under these experimental conditions, intact gloves act as effective barriers to the transmission of viral particles, including HIV in the health care setting.

During a community outbreak of enterovirus infection, seven of 194 pregnant women (3.6 per cent) were found to be excreting a prime strain of echovirus 11 at term. Each of the seven women possessed serum neutralizing echovirus 11 antibody in titers ranging from 1:20 to 1:320, and the cord serum of their seven infants had antibody in titers of 1:10 to 1:640. None of these seven infants became ill, but four were shedding virus from the respiratory or gastrointestinal tract by three days of age. In a previous study, four infants who died of generalized infection due to the same strain of echovirus 11 had no detectable antibody in cord serum. None of the infants of virus-negative mothers became infected, according to cultures at hospital discharge (151 infants) or at two weeks of age (135 infants). We conclude that passive transplacental acquisition of antibody prevents severe, systemic echovirus disease but does not prevent mucosal infection in the perinatally infected infant.