Nosocomial transmission of echovirus 30: molecular evidence by phylogenetic analysis of the VP1 encoding sequence

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.     

A neonatal echovirus 11 outbreak in an obstetric clinic.

An echovirus 11 outbreak occurred among neonates in an obstetric clinic in November 2003. Thirteen neonates were transferred to our medical center, and all were found to have echovirus 11 infection. Viral studies were performed for 32 other infants born in the clinic during the same period, including 30 asymptomatic neonates and 2 febrile infants transferred to another hospital. Two of the asymptomatic infants had echovirus 11 isolated from rectal swabs. The first patient transferred to our medical center developed extensive hemorrhage and died 6 days later. Three family members of this infant were also proved to have echovirus 11 infections. One other infant had a fulminant course and had residual hepatic impairment. The other infants had no complications. Viral studies in the 24 nursery staff were all negative. This outbreak shows how a neonatal enterovirus outbreak can occur in a nursery, starting from an infected infant in the incubation period. Early recognition and prompt management of an outbreak is important to prevent further spread of the infection.     

A study of enterovirus isolations in Glasgow from 1977 to 1997.

The number and range of enteroviruses isolated in the Regional Virus Laboratory, Glasgow during 1977-1997 was determined. Over this period, 3,039 enterovirus isolations were reported. The echoviruses represented 67% of isolations with echovirus 4 (due to an outbreak in 1990), echovirus 30 and echovirus 11 being the most frequently isolated types. The pattern of prevalence of non-polio enteroviruses had changed from the previous 20-year period with echovirus types isolated more often (77% vs. 55.4%) and coxsackieviruses isolated less often (23% vs. 44.6%). The polymerase chain reaction (PCR) introduced into the routine diagnostic service in 1996 increased the detection of enteroviruses from cerebrospinal fluid samples compared with traditional cell culture methods. Finally, the 5' nontranslated region (NTR, bases 63-475) and the VP4/VP2 region (bases 581-1199) of selected echovirus 30 and coxsackie B3 isolates were sequenced. These represented endemic and epidemic types respectively and were shown to be closely related within their type, but different from the published sequences. The current echovirus 30 strains differed from 1966 isolates by 16-20% in both the 5' NTR and VP4/VP2 regions. The coxsackie B3 isolates, predominant in 1997 after 5 years of absence, were also dissimilar from previously isolated strains, causing a small outbreak.     

A possible new enterovirus serotype isolated in ontario

Summary From a collection of viruses isolated over a period of years from clinical material and not identifiable by routine procedures, three were selected for this investigation. All three isolates were recovered in 1958 from cases of aseptic meningitis. Cross neutralization tests revealed that they were antigenically indistinguishable. One of these, 275/58, isolated from the cerebrospinal fluid of a 3 year old child, was chosen for detailed study. The general characteristics of this virus were found to be compatible with those of enteroviruses as specified by the Committee on Enteroviruses. Human origin of this virus was established by neutralization tests with patient's serum, pooled human gamma globulin and human sera submitted during enterovirus seasons from cases suspected to have virus infections. The 275/58 virus was not neutralized either by intersecting pools or individual antisera prepared against all known enteroviruses, nor by WHO enterovirus reference antisera. Conversely, antiserum to 275/58 virus failed to neutralize poliovirus types 1–3, echovirus types 1–33, Coxsackie virus B 1B 6 and Coxsackie virus A 9. The virus was found to be non-pathogenic for infant mice.It is suggested that these three isolates represent a new enterovirus serotype, possibly belonging to the echovirus group.     

Epidemiologic aspects and laboratory features of enterovirus infections in Western Germany, 2000-2005

From 2000 to 2005, a total of 1,096 enterovirus infections were diagnosed either by isolation of virus from cell culture or by RT-PCR (5'non-coding region (NCR)). Typing of viruses (n = 674) was carried out by immunofluorescence with monoclonal antibodies, neutralization test or molecular methods. Seasons with high enterovirus activity were characterized by high prevalence of echovirus 30 (62.2% in 2000, 25.5% in 2001) and echovirus 13 (34.5% in 2001). In contrast, in the 2003 season, which had very low enterovirus activity, these types were rare. During this season, cell culture sensitivity (human colonic carcinoma cells and human embryonic lung fibroblasts (HEL)) was exceptionally low. In order to determine the type of "non-cultivable" enteroviruses, purified RNA from selected stool samples was subjected to direct molecular typing. VP1/2A-specific fragments were amplified by RT-PCR, cloned and sequenced. The predominant virus identified was coxsackie A. Consequently, rhabdomyosarcom cells were introduced into the daily routine, which improved the isolation of enteroviruses. Echovirus 30 was again most commonly isolated during seasons 2004 and 2005 with increasing enterovirus activity. In conclusion, high prevalence of echovirus 30 and 13 is indicative of seasons with high enterovirus activity. The type of circulating enteroviruses may influence isolation of enterovirus from cell culture. RT-PCR (VP1/2A) combined with cloning and sequencing of amplicons is a useful tool for viral typing directly from stool samples. In cases of severe enterovirus infection, virological diagnosis should not solely rely on virus isolation from cell culture.     

Determination of enterovirus serotype inferred from sequence analysis of PCR products.

BACKGROUND: Enterovirus infections are common in neonates. Virus isolation is the only diagnostic method to confirm enterovirus serotype infections, however, is not always successful. OBJECTIVES: A new approach for the diagnosis of enterovirus infections was performed, using the reference strain inferred from sequence analysis of PCR products. STUDY DESIGN: Virus isolation, enterovirus RT-PCR and sequence analysis were performed from clinical samples or stored sera from two neonates with fever and rash. Neutralizing test (NT) antibodies against prototype reference virus were measured in paired sera. RESULTS: Virus isolation was negative in both patients but the enterovirus genome was amplified in the acute phase sera obtained from the two patients. From the results of sequence analysis of 109 nucleotides located in the 5'-noncoding of the conserved region of enteroviruses, a high homology to echovirus types 25 and 30 was found. More than a 4-fold increase in NT antibodies against reference viruses was demonstrated in the acute and convalescent phase sera. They were confirmed as echovirus type 25 and 30 infection, respectively. CONCLUSIONS: These virological examinations are practical and useful for clinical settings for a diagnosis of enterovirus infections because of an insufficient positive rate in virus isolation.     

ECHO-II virus, the pathogen of the third outbreak of uveitis in children in Kransnoyarsk, 1986

The third outbreak of an acute infection accompanied in 3 cases by inflammation of the uveal membrane (uveitis) was recorded among infants in Krasnoyarsk in 1986. From the infants with the eye involvement as well as from those in contact with the former, 36 strains of ECHO-11 virus were isolated, and seroconversion to this virus was demonstrated. All the infants in contact with the eye patients were given gamma-globulin which seemed to limit the spread of the infection. The third outbreak of uveitis, 1986, was similar to the 1st (1980-1981) and 2nd (1982) outbreaks of uveitis in Krasnoyarsk in such aspects as the clinical eye disease (anterior uveitis-iridocyclitis), symptoms of general infection, age of the infants (3-8 months), the nosocomial pattern of infection, enterovirus etiology, marked ophthalmotropism of ECHO-11 virus isolates in monkeys.     

Disseminated neonatal echovirus 11 disease following antenatal maternal infection with a virus-positive cervix and virus-negative gastrointestinal tract

An infant girl was born apparently well one week after her mother had had a mild illness with chills, fever, and diarrhea. On the third day of life, the infant became ill and died four days later with necrotizing hepatitis. On the same day, echovirus type 11 was recovered from the throat, rectum, and buffy coat of the infant and from the cervix of the mother. At this time, the mother had an IgM neutralizing antibody titer to echovirus type 11 and 1:128, but no IgG antibodies. The infant had no echovirus type 11 antibodies. The virus was also isolated from the baby's liver and adrenal at autopsy. These findings raise the possibility of enterovirus infection at delivery from a contaminated cervix.     

Echovirus 7 associated encephalomyelitis.

BACKGROUND: Hand, foot, and mouth disease (HFMD) is endemic in Malaysia. In 1997, a large outbreak of enterovirus 71 (EV-71) associated HFMD resulted in 41 deaths due to severe left ventricular dysfunction and central nervous system infection with extensive damage to the medulla and pons. The clinical presentation in all these patients were rapid cardio-respiratory decompensation leading to cardiac arrest. Another large outbreak of HFMD with 55 fatal cases and a similar clinical picture was reported in Taiwan in 1998. In 2000, an outbreak of HFMD resulted in the deaths of three children who had rapid cardio-respiratory decompensation and one child who survived a central nervous system infection. OBJECTIVES: We set out to study the etiologic agent and mechanism involved in three children who presented to our hospital, two of whom died and one survived a central nervous system infection. STUDY DESIGN: The clinical course of the disease was described. Throat, rectal swab and cerebrospinal fluid samples were subjected to viral isolation and viral isolates were identified by immunofluorescence, micro-neutralisation using human rhabdomyosarcoma (RD) cells, and reverse transcritpase polymerase chain reaction. Magnetic resonance imaging was performed on two of the patients. RESULTS: Echovirus 7 was the sole pathogen isolated from three cases of acute encephalomyelitis, two of which were fatal due to severe left ventricular dysfunction resistant to inotropic support. The survivor had residual bulbar palsy, but is considered to have had a good neurological outcome. CONCLUSION: Echovirus 7 infection associated with encephalomyelitis could be fatal due to indirect involvement of the heart resulting in severe left ventricular dysfunction. In addition one of the children presented with hand, foot, and mouth disease, a syndrome that has not been previously associated with echovirus 7 infection.     

Intratypic genome variability of echovirus type 30 in part of the VP4/VP2 coding region

Summary The genetic relationship of 33 echovirus type 30 (E30) isolates associated with three different outbreaks of meningitis in Norway and one outbreak in USA was assessed using direct sequencing of amplicons derived from a region covering part of the capsid proteins VP4 and VP2. The E30 sequences were compared to each other, and to other enteroviruses. Less sequence variation was observed between the isolates from a single outbreak (2–3%) than between groups of isolates from different outbreaks (4–9%). All observed nucleotide substitutions were amino acid silent. Homology between enteroviruses obtained from GenEMBL and the nucleotide consensus sequence generated from the E30 isolates varied between 44.8% (coxsackievirus A24) and 72.6% (coxsackievirus A9). Comparing the E30 sequences in this part of the genome with other enteroviruses, E30 clearly belongs to the coxsackie B-like virus group.     

Molecular analysis of an echovirus 3 strain isolated from an individual concurrently with appearance of islet cell and IA-2 autoantibodies

Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type 1 diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3), Human enterovirus B (HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction.     

Variation in enterovirus receptor genes

The increased incidence of a enterovirus infections observed in patients with type 1 diabetes preceding the development of the clinical disease could be partially explained by variation in the genes coding for enterovirus receptors. We carried out sequence analysis of the most common enterovirus receptor molecules in 21 diabetic children and 20 healthy adults. DNA was isolated from the leukocytes, and gene regions known to code for virus-recognizing domains in major enterovirus receptors were amplified and sequenced. Heterozygous single-nucleotide polymorphism (SNP), Ala 67 (GCG) --> Thr (ACG), was detected in the poliovirus receptor gene in four individuals in the diabetes group, but not in the control group. However, serological studies could not confirm that this substitution would convey different susceptibility to poliovirus infection. A heterozygous SNP, Lys 29 (AAG) --> Met (ATG), was found in the intracellular adhesion molecule-1 (ICAM-1) (receptor for rhinoviruses and some coxsackie A viruses) in one individual in both groups. A silent SNP in the alpha2 integrin subunit gene (echovirus 1 receptor) was frequently found in both groups, a silent heterozygotic SNP in coxsackievirus-adenovirus receptor (coxsackie B virus receptor) gene was seen in one individual in the diabetes group, whereas no variation was found in the DAF (echovirus receptor) and beta3 integrin subunit sequences (receptor for coxsackievirus A9) studied. In conclusion, both synonymous and nonsynonymous sequence variability of genes coding for enterovirus and rhinovirus receptors was shown to occur, but no pattern directly specific for type 1 diabetes was found. =     

Isolation and molecular identification of echovirus 13 isolated from patients of aseptic meningitis in Korea, 2002

During 2002, several epidemics of aseptic meningitis were attributed to echovirus 13 in Korea. The causative agents of these outbreaks were isolated and identified using rhabdosarcoma cells, HEp-2 and Buffalo green monkey kidney cells, and a neutralization test using monospecific antiserum. Fifty-four echovirus 13 isolates were isolated from patients with aseptic meningitis in the provinces, Seoul, Kyonggi, Gwangju, Jeonju, Busan, and Ulsan. Symptoms associated with aseptic meningitis infection in patients included the occurrence of headaches and mild fever. Molecular characterization of echovirus 13 samples was achieved by sequence and phylogenetic analyses on partial VP1 sequences from 20 Korean isolates and 10 foreign isolates listed in Genbank. Minor variation was observed among the Korean isolates, which formed a unique cluster with isolates of German and Japanese origin. The marked similarities between isolates could be attributed to a relatively recent arrival of the virus in Korea. This is the first such investigation of aseptic meningitis caused by echovirus 13 on the Korean peninsula.     

An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22

An outbreak of acute flaccid paralysis in Jamaica in 1986 associated with echovirus type 22 is described. Six patients aged 1 to 27 years developed acute onset of severe flaccid paralysis, with inability to walk. Three cases had facial weakness, four required intensive care with assisted ventilation, and two died. Echovirus type 22 was isolated from the stool of two patients who showed a significant increase in antibody titre. Echovirus type 22 was also isolated from the stool of another patient who had aseptic meningitis without any neurological deficit. There was no evidence of poliovirus infection in any of these patients, most of whom were fully immunized. Of the four surviving cases with flaccid paralysis, three had residual weakness in their lower limbs and walked with an abnormal gait 3 years after the acute paralytic attack. This is the first report in the literature of acute flaccid paralysis associated with type 22 echovirus.     

Method for selecting optimal cells for enterovirus isolation as determined in an outbreak of echovirus type 33 meningitis

An outbreak of echovirus type 33-induced meningitis which occurred in Belgium in 1982 is reported. To identify the causative agent, titers of an early isolate were measured on a variety of cells in order to select the optimal cells. A human rhabdomyosarcoma cell line was found to be the substrate of choice due to its proficient isolation of the virus. This method of determining infectious titres is recommended for improving enterovirus isolation in other epidemics.     

Isolation and identification of an enterovirus 77 recovered from a refugee child from Kosovo, and characterization of the complete virus genome

The complete nucleotide sequence of an enterovirus 77 isolate is reported. The virus designated FR/CF496-99 (France/Clermont-Ferrand 496-1999) was recovered from the feces of a 4-year-old child hospitalized for Salmonella gastroenteritis. The virus was identified by a molecular typing assay based on the genomic sequence encoding the VP1 capsid protein. The phylogenetic analysis based on the VP1 sequence demonstrated that the enterovirus isolated in the child clustered with viruses included in the human enterovirus B species (HEV-B) and was most closely related to enterovirus 77. A sliding window analysis of the complete genome showed an overall nucleotide similarity >80% between the P3 genomic region of the FR/CF496-99 isolate and that of the echovirus 30 prototype strain. A comparative analysis based on partial 3D(pol) sequences showed that the FR/CF496-99 virus was more closely related to recent enteroviruses from different serotypes and different geographical areas than to the prototype strains collected in the 1950s. This suggests that, in this enterovirus, the 3D(pol) encoding sequence is of recent origin.     

Outbreak of enteroviral uveitis in children in Omsk in 1987-1988

From May, 1987, to July, 1988, 56 cases of acute enterovirus infection complicated by uveitis were reported in Omsk, Western Siberia, USSR. Infants aged from 15 days to 19 months were involved. The infection was nosocomial, the peak of the incidence occurring in January 1988. Severe and moderately severe cases of uveitis (43 out of 56 infants) were prevalent running a course with destruction of the iris, formation of posterior synechias, deformation of the ciliary body, lenticular opacity, appearance of prelental films, retardation of the development of the affected eye, hemorrhages in the fundus of the eye. The causative agent of uveitis outbreak was ECHO-11 virus: 40 strains of ECHO-11 virus were isolated from 27 out of 33 examined infants with uveitis; in 32 out of 33 infants a high titre of antibodies (1:256-1:16384) to the endemic ECHO-11 virus was demonstrated. Uveitis was reproduced in monkeys infected with Omsk strains of ECHO-11 virus. The uveitis outbreak in Omsk is similar in its etiology, epidemiology, and clinical pattern to three previous outbreaks of enterovirus uveitis observed in Krasnoyarsk (central Siberia) observed in 1980-1986.