Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.     

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.     

Molecular epidemiological study of HEV-B enteroviruses involved in the increase in meningitis cases occurred in Spain during 2006

Human enteroviruses are one of the main etiological agents of aseptic meningitis and other central nervous system infections, particularly the serotypes included in the enterovirus B species. Molecular methods have proved useful to identify serotypes in clinical samples, facilitating the epidemiological study of these viruses. In the spring of 2006, there was a significant increase in meningitis cases caused by enteroviruses in Spain. In the present study, 138 enteroviruses directly detected in clinical samples of patients with aseptic meningitis (n = 116) and other neurological pathologies (n = 22) received by the National Center for Microbiology during the year, were genotyped by amplification and sequencing part of the VP1 region and phylogenetic analysis. Echovirus 30 was the most frequent serotype, followed in decreasing order by echovirus 6, 9, 13, 18, enterovirus 75, coxsackievirus A9, echovirus 11, 14, 29, 4, and coxsackievirus B4 and B5. Phylogenetic analysis with all Spanish echovirus 30 strains detected in 2006 and other reported echovirus 30 sequences, demonstrated that Spanish strains formed a new lineage, different from others previously described. In conclusion, echovirus 30 is the most commonly reported enterovirus serotype associated with aseptic meningitis in Spain. Direct molecular typing of clinical samples also allows rapid identification of the serotypes involved in an epidemic alert and phylogenetic analysis in the 3'-VP1 region is useful to study viral epidemiology.     

Laboratory investigation and phylogenetic analysis of enteroviruses involved in an aseptic meningitis outbreak in Greece during the summer of 2007

BackgroundAseptic meningitis is the most commonly observed CNS infection and is mainly attributed to Non-Polio Enteroviruses (EV).ObjectiveIdentification and genetic analysis of the EV involved in the recent aseptic meningitis outbreak which occurred in Greece, during the summer of 2007.Study designIn total, 213 CSF and faecal samples were examined for EV presence by culture, while enteroviral RNA detection was performed by nucleic acid sequence-based amplification assay (NASBA). EV strains were typed by seroneutralization, as well as nested RT-PCR followed by VP1-2A gene partial sequencing. Phylogenetic analysis was carried out for the identification of the genetic relatedness among the isolated EV strains.ResultsEV detection rate in CSF and faecal samples was 43.9% and 70.8%, respectively. EV serotyping and VP1 region analysis revealed the predominance of echovirus 4 (ECV4) serotype and the circulation of ECV6, 9, 14, 25, Coxsackie A6, A15, A24 and Coxsackie B1 serotypes. All ECV4 isolates presented a 98.7% similarity in nucleotide sequence, with a Spanish ECV4 strain, isolated during a meningitis outbreak in 2006.ConclusionsIt is the first time that ECV4 is associated with an aseptic meningitis outbreak in Greece, during which 9 different EV serotypes were co-circulating. All Greek ECV4 isolates were closely related to the Spanish ECV4 strain. Genetic analysis of the VP1 gene can significantly contribute to the revelation of the endemic EV strains circulation pattern and their phylogenetic relationship with enteroviruses involved in epidemics of distant geographical areas at different time periods.     

Molecular identification of human enteroviruses in children with neurological infections from the central region of Argentina

In the central area of Argentina, epidemiological and molecular characteristics of human enterovirus infections are still unknown. RT-nested PCR of the highly conserved 5′NCR was used to detect enteroviruses in 168 samples of cerebrospinal fluid from hospitalized patients with suspected infection of the central nervous system (2007–2008), and 13 (7.7%) were positive. Molecular typing was performed by sequencing of the 3′-half VP1 region. Echovirus 30 was the predominant type detected, followed by coxsackie viruses A9 and B4. All echovirus 30 strains of 2007 clustered in lineage H, whereas the echovirus 30 isolate obtained in 2008 was more distantly related, possibly representing a new lineage.     

Molecular identification of enteroviruses associated with aseptic meningitis in children from India

We identified and characterized enteroviruses associated with aseptic meningitis in children between April 2009 and March 2010. Enterovirus RNA was detected in 51 (45.5 %) of 112 CSF samples. Molecular typing by RT-PCR and sequencing of a partial VP1 region revealed the predominance of echovirus (ECV) 32 (n = 20), followed by ECV 11 (n = 10), ECV 13 and ECV 14 (n = 5 each), coxsackievirus (CV) B3 and CV B6 (n = 3 each), CV A2, CV A10 and ECV 30 (n = 1 each). Phylogenetic analysis of ECV 32 showed 0 to 4 % sequence divergence among strains of the present study and 20-23 % from the prototype Puerto Rico strain at the nucleotide level. This is the first report of ECV 32 associated with an aseptic meningitis epidemic and identification of seven different enterovirus serotypes (CV A2, CV A10, CV B3, CV B6, ECV 13, ECV 14 and ECV 32) in meningitis cases from India.     

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.     

Molecular characterization of enteroviruses detected in Gyeong-Ju and Po-Hang provinces of Korea in 2003

We identified and characterized enteroviruses by amplifying the partial VP1 gene from pediatric patients with aseptic meningitis and other enterovirus-related diseases from the Gyeong-Ju and Po-Hang provinces of Korea in 2003. We identified two strains each of coxsackievirus A (CA) 6, CA9, and CA10; three enterovirus 71 (EV71) strains; and six echovirus 30 (E30) strains. The three EV71 strains were characterized as genogroup C4. These results are the first documentation reporting the occurrence of CA10 and EV71 genogroup C4.     

Picornaviruses in cerebrospinal fluid of children with meningitis in Luanda, Angola

Human enteroviruses are the most common cause of viral meningitis. Viral-bacterial interaction may affect the clinical course and outcome of bacterial meningitis. In Africa, viruses might be responsible for 14-25% of all meningitis cases. However, only few studies from Africa have reported detection of viruses in the cerebrospinal fluid (CSF) or mixed viral-bacterial infections of the central nervous system (CNS). The aim of the present study was to investigate the presence of picornaviruses in the CSF of children suffering from meningitis in Luanda, Angola. The study included 142 consecutive children enrolled in a prospective study of bacterial meningitis in Luanda between 2005 and 2006, from whom a CSF sample was available. CSF samples were obtained at hospital admission, stored in a deep-freeze, and transported to Finland for testing by real-time PCR for picornaviruses. Enteroviruses were detected in 4 (3%) of 142 children with presumed bacterial meningitis. A 5-month-old girl with rhinovirus and Haemophilus influenzae meningitis recovered uneventfully. An 8-year-old girl with human enterovirus and pneumococcal meningitis developed no sequelae. A 2-month-old girl with human enterovirus and malaria recovered quickly. A 7-month-old girl with human enterovirus was treated for presumed tuberculous meningitis and survived with severe sequelae. Mixed infections of the CNS with picornaviruses and bacteria are rare. Detection of an enterovirus does not affect the clinical picture and outcome of bacterial meningitis.     

Sequence comparison of echovirus type 30 isolates to other enteroviruses in the 5′noncoding region

The genetic relationship between echovirus type 30 (E30) isolates were characterised by means of amplification of a part of the 5′noncoding region by polymerase chain reaction and direct sequencing. Later, the E30 sequences were compared with other enteroviruses. Homology between recent clinical E30 isolates from different years exceeds 98% at the nucleotide level. Comparing the E30 sequence with other enteroviruses, homology varied between 68% (coxsackievirus A24) and 93% (coxsackievirus B3). E30 appeared to have coxsackie B-like characteristics in this genomic part. It is considered that E30 and coxsackie B viruses belong to the same enterovirus subgroup. © 1995 Wiley-Liss, Inc.     

Rapid enterovirus genotyping in cerebrospinal fluids: a two-year prospective study in a virology laboratory setting

Enterovirus (EV - 68 serotypes) infections comprise a wide spectrum of clinical presentations including infections of the central nervous system. In severe clinical presentation or epidemics, the precise identification of the involved serotype is necessary. OBJECTIVES: To perform enterovirus genotyping directly in cerebrospinal fluid (CSF) samples, and to assess its feasibility in a laboratory setting. METHODS: Enterovirus genotyping was carried out directly with CSF specimens tested for the diagnostic procedure by amplifying the complete 1D gene encoding the VP1 protein of the HEV-B serotypes (the most frequent) - providing results in two days. Secondly, sequences 1A/1B encoding the VP4/VP2 capsid proteins, respectively, were analysed (results in five days). RESULTS: Direct enterovirus genotyping allowed the identification of enterovirus involved in 77 out of 81 (95%) meningitis cases between January 2006 and December 2007. In combination with the indirect genotyping of enterovirus isolates, identification of the type was achieved in 94 out of 97 (96.9%) patients included in the study. The most frequent serotypes were echovirus 6 (E6) and 13 in 2006, coxsackievirus B2 and E30 in 2007. Four children presented an EV71 associated meningitis. CONCLUSION: When prospectively applied in a laboratory setting, direct enterovirus genotyping in CSF samples allows the identification of the involved enterovirus in two to five days. This time frame is relevant for an optimal patient management, the rapid identification of a new enterovirus variant or in the context of an epidemic alert.     

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.     

Prospective identification of enteroviruses involved in meningitis in 2006 through direct genotyping in cerebrospinal fluid

Enterovirus infections were investigated with special emphasis on performing rapid molecular identification of enterovirus serotypes responsible for aseptic meningitis directly in cerebrospinal fluid (CSF). Enterovirus genotyping was carried out directly with specimens tested for the diagnostic procedure, using two seminested PCR assays designed to amplify the complete and partial gene sequences encoding the VP1 and VP4/VP2 capsid proteins, respectively. The method was used for identifying the enterovirus serotypes involved in meningitis in 45 patients admitted in 2005. Enterovirus genotyping was achieved in 98% of the patients studied, and we obtained evidence of 10 of the most frequent serotypes identified earlier by genotyping of virus isolates. The method was applied for the prospective investigation of 54 patients with meningitis admitted consecutively in 2006. The enterovirus serotypes involved were identified with the cerebrospinal fluid (CSF) of 52 patients (96%) and comprised 13 serotypes within the human enterovirus B species and 1 within the human enterovirus A species. The three most common serotypes were echovirus 13 (E13; 24%), E6 (23%), and coxsackievirus B5 (11.5%), a pattern different from that observed in 2005. Genotyping of virus isolates was also performed in 35 patients in 2006 (meningitis, n = 31; other diseases, n = 4). By comparison, direct genotyping in CSF yielded a more complete pattern of enterovirus serotypes, thereby allowing the detection of rare serotypes: three less common serotypes (CB2, E21, and E27) were not detected by indirect genotyping alone. The study shows the feasibility of prospective enterovirus genotyping within 1 week in a laboratory setting.     

Molecular characterization of human enteroviruses in clinical samples: comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.     

Prospective identification of HEV-B enteroviruses during the 2005 outbreak

Enteroviruses (EVs) represent the main etiological agents of epidemics of viral meningitis and especially the serotypes related to the human enterovirus B species. Genetic typing by sequencing a PCR-amplified portion of the genome has proved to be useful for identifying EVs and is more rapid than standard seroneutralization tests. However, prospective genotyping has not been reported in routine practice within a clinical diagnostic laboratory. A genetic typing assay using two sets of primers was developed for the amplification and sequencing of the VP1 coding sequence of the HEV-B serotypes. Identification was carried out by sequence comparisons with EV sequences in GenBank using the BLAST search tool and confirmed by phylogenetic analysis. This method was used to identify prospectively the 48 enteroviruses isolated in patients with either enterovirus-proved meningitis (n = 41) or other clinical manifestations (n = 7) admitted to the University Hospital of Clermont-Ferrand (France) in 2005. The assay was also used to type retrospectively EVs isolated in cerebrospinal fluid specimens of 25 patients admitted to the Trousseau Paediatric Hospital in Paris (France) between February and August 2005. In both prospective and retrospective investigations of meningitis, echovirus 30 (E30) was the most frequent serotype, followed in decreasing order by E18, E13, coxsackievirus B5, B3, E6, E4, E7, E11, E33, and coxsackievirus A9. In patients with other manifestations, coxsackievirus B3, B5, and E3 were each identified twice, and E2 once. In E30 infected patients, nine different lineages were demonstrated by phylogenetic analysis. Genetic typing allowed the prospective, effective and rapid identification of all EV isolates involved in the 2005 outbreak. Molecular typing in combination with phylogenetic analysis will be a reliable means to confirm the emergence of new EV variants, and is of interest of both individual patients and public health.     

Echovirus 30 meningitis epidemic followed by an outbreak-specific RT-qPCR

BackgroundAn outbreak of enteroviral aseptic meningitis emerged in Southwestern Finland in August 2009. The same enterovirus reappeared with increasing incidence of meningitis in other parts of Finland in 2010.ObjectivesTo identify the incidence and molecular epidemiology of enteroviral meningitis outbreak.Study designThe causative agent was identified as echovirus 30 (E-30) by sequencing partial viral protein 1 capsid genome, and a virus type-specific RT-qPCR was set up for sensitive detection of the virus in cerebrospinal fluid specimens. Enterovirus positive CSF specimens were subjected to the E-30-specific assay to investigate this unusual occurrence of aseptic meningitis and facilitate case confirmation during the outbreaks between August 2009 and September 2010.ResultsE-30 was detected in 106 (72%) enterovirus positive cerebrospinal fluid specimens. All the meningitis cases in 2009 and most of them in 2010 were among adolescents and several were members of sport teams.ConclusionsBetween August 2009 and September 2010, E-30 caused an extensive outbreak with two peaks in Finland. Type-specific RT-PCR allowed rapid diagnostic follow-up of the epidemic.