Molecular epidemiology of enterovirus and parechovirus infections according to patient age over a 4-year period in Spain.

The epidemiology and clinical association of enterovirus (EV) and parechovirus (HPeV) infections, as well as the type-distribution-according-to-age, were determined during a 4-year study period in Spain. During 2010-2013, a total of 21,832 clinical samples were screened for EV and the detection frequency was 6.5% (1,430). Of the total EV-negative samples, only 1,873 samples from 2011 to 2013 were available for HPeV testing. HPeV was detected in 42 (2%) of them. Positive samples were genotyped using PCR and sequencing. EV infections occurred in all age groups of patients: neonates (17%), children 28 days to 2 years (29%), children 2-14 years (40%), and adults (14%). Thirty-four different EV types were identified. HPeV infections were detected exclusively in infants <8 m (70% neonates, P < 0.05). All but one HPeV were HPeV-3. Differences in type frequency detection were found according to age and clinical manifestation. Coxsackievirus (CV)-B4 (61%), CV-B5 (83%), and HPeV-3 (64%) were more frequent in neonates than in older patients (P < 0.05). Echovirus (E)-3 (60%), E-18 (47%), E-25 (62%), CV-A6 (61%), CV-A16 (72%), and EV-71 (75%) were mainly detected in children 28 days to 2 years (P < 0.05), whereas, E-6 (79%), E-20 (88%), and E-30 (85%) were predominant in children >2 years and adults (P < 0.05). Clinically, meningitis was associated with EV (P < 0.01) whereas, encephalitis was more frequent in HPeV-infected patients. CV-B types were associated with myocarditis (90%; P < 0.05) and EV species A with hand-foot-mouth-disease/atypical exanthema (88%; P < 0.05). J. Med. Virol. 89:435-442, 2017. © 2016 Wiley Periodicals, Inc.

Molecular epidemiology of coxsackievirus B3 infection in Spain, 2004-2014.

Epidemiological and clinical characteristics of coxsackievirus B3 infections in Spain were investigated. This enterovirus (EV) type was detected mainly in young children (<6 months) and was associated with neurological (78 %) and respiratory diseases (10 %) but also with myo/pericarditis (10 %). Two myocarditis cases were fatal. Phylogenetic analysis of the VP1 region showed that genotype III circulated in the country between 2004 and 2008 and was replaced by genotype V in 2010. Furthermore, phylogenetic analysis of the 3D region indicated that recombination events have occurred and contributed to the genetic evolution of this EV type.

Clinical characteristics and molecular epidemiology of Enterovirus infection in infants <3 months in a referral paediatric hospital of Barcelona.

UNLABELLED: Enterovirus (EV) infection is common in infants, but the information with regard to the molecular epidemiology and the associations between types and clinical variables is very scarce. This study includes 195 children <3 months old with fever, attended from March 2010 to December 2012 in an emergency department of a tertiary paediatric hospital in whom EV infection was confirmed by real-time PCR in blood and/or cerebrospinal fluid. Clinical and epidemiological data was prospectively collected. In 152 (77.9 %) patients, EVs could be typed. The most common type was Echovirus-5 (E5; 32, 21.1 %), followed by Echovirus-11 (E11; 18, 11.8 %), Echovirus-21 and Echovirus-25 (E21, E25; 11 each one, 7.2 %) and Coxsackievirus-B4 (CVB4; 6, 6.6 %). The majority of types appeared in spring, but E5 and E25 were found mainly during summer (p < 0.01). E21 was associated with high-grade fever (p < 0.01); E5 with exanthema (p = 0.03) and CVB4 tended to cause meningitis more often than the other types (p = 0.07). CONCLUSION: The most common EV types were Echovirus-5 and Echovirus-11. Some significant associations between types and epidemiologic and clinical findings were observed. What is Known-What is New • Enteroviruses cause a normally benign illness in young infants, except in some cases. • The molecular epidemiology of Enterovirus infection is not well known in European countries. • This study describes a large number of infants with Enterovirus infection and shows the seasonality of different types, and their associations with epidemiologic and clinical variables.

Comparison of epidemiology and clinical characteristics of infections by human parechovirus vs. those by enterovirus during the first month of life.

UNLABELLED: Human parechoviruses (HPeV) have been recently recognized as important viral agents in paediatric infections. The aims of this study were to investigate the HPeV infection prevalence in infants <1 month in Spain and, secondly, to analyse the clinical and epidemiological characteristics of the infected patients compared with those infected by enterovirus (EV). Infants <1 month with neurological or systemic symptoms were included in a multicentre prospective study. EV and HPeV detection by RT-PCR and genotyping were performed in cerebrospinal fluids (CSF), sera or throat swabs. Out of the total of 84 infants studied during 2013, 32 were EV positive (38 %) and 9 HPeV positive (11 %). HPeV-3 was identified in eight cases and HPeV-5 in one. Mean age of HPeV-positive patients was 18 days. Diagnoses were fever without source (FWS) (67 %), clinical sepsis (22 %) and encephalitis (11 %). Leukocytes in blood and CSF were normal. Pleocytosis (p = 0.03) and meningitis (p = 0.001) were significantly more frequent in patients with EV infections than with HPeV. CONCLUSIONS: Although HPeV-3 infections were detected less frequently than EV, they still account for approximately 10 % of the cases analysed in infants younger than 1 month. HPeV-3 was mainly associated with FWS and without leukocytosis and pleocytosis in CSF. In these cases, HPeV screening is desirable to identify the aetiologic agent and prevent unnecessary treatment and prolonged hospitalization.

Recombination and evolutionary dynamics of human echovirus 6.

Enterovirus (EV) infections are associated with a wide array of often severe disease presentations including aseptic meningitis, encephalitis, and acute flaccid paralysis. Surveillance for polioviruses and other EVs is therefore important as a public health measure both for patient management and epidemiological studies. From 1988 to 2008, echovirus (E) 30 was the predominant genotype in Spain (33.7% of the total typed EVs). E6 was also endemic throughout this period although isolated less frequently (12.5%). In 2009, however, a substantial increase in the incidence of E6 was detected (60%), displacing E30 type (2%). To investigate the evolution and recombination in the epidemiology and transmission of E6 in Spain, a genetic analysis in VP1 and 3Dpol regions of 67 Spanish strains collected during the period 2004-2010 was performed. All VP1 sequences clustered monophyletically in the assigned genogroup C, subgroup 9, currently the predominant circulating strains identified in Europe and elsewhere in the last 10 years. 3Dpol sequences were interspersed with other species B EVs resulting from several recombination events that generated at least 12 different recombinant forms (RFs) among study samples. These showed typically minimal divergence in VP1. The co-circulation of different lineages of E6 in the same geographical area associated with its mainly endemic pattern of transmission may have contributed to the extremely short estimated half-life of E6 RFs (0.87 years). This pattern contrasts markedly with other species B EVs and EV71 where VP1 lineage expansion and extinction occurred in step with defined recombination events and periodic changes in incidence.

Combining cell lines to optimize isolation of human enterovirus from clinical specimens: report of 25 years of experience.

Cell culture is still the gold standard for the diagnosis of human enteroviruses (HEVs) although molecular techniques are required for detection of some serotypes. Due to the diversity of HEVs, a single cell line is not susceptible to all serotypes, and several lines are required to optimize the isolation of HEVs. In this study, the results of HEV isolation during the last 25 years are reported. A total of 1,192 HEVs were isolated and isolation rates varied depending on the cell line used. MRC5 cells yielded the best results (70.7%), followed by A549 cells (52.6%), RD cells (37.5%), and HEp-2 cells (29.7%). A total of 521 HEVs were characterized, and HEV-B was the most frequent species (81%). Polioviruses (PV) and HEV-A were isolated less frequently (17% and 1%, respectively). None of the cell lines detected all the enteroviruses. MRC5 cells were the most susceptible for isolation of echoviruses (85.7%) and PVs (85.4%), whereas HEp2 was the most susceptible for Coxsackieviruses B (82.6%). Some serotypes were isolated in one cell line only. 40.5% of echoviruses were isolated in MRC5 cells whereas 42.3% and 23.9% of Coxsackieviruses B were isolated in RD cells and HEp2 cells, respectively. Although A549 cells did not achieve the best performance for any enterovirus serotypes, they isolated 52.6% of the total HEVs. In view of these results, MRC5 cells, A549 cells, and RD cells should be combined to optimize isolation of HEVs.

Molecular identification of an enterovirus 99 strain in Spain.

Enterovirus 99 is a recently described genotype of virus belonging to the species Human enterovirus C. So far, only a few sequences of this enterovirus type have been available. In 2010, during Spanish enterovirus surveillance, an enterovirus 99 strain was found in an acute flaccid paralysis patient. The virus was detected and typed in the clinical samples using molecular methods. Phylogenetic analysis in the 3Dpol region revealed recombination events with other species-C enteroviruses. This is the first finding of this unusual type in Spain.

Molecular characterization of a coxsackievirus A24 variant that caused an outbreak of acute haemorrhagic conjunctivitis in Spain, 2004.

BACKGROUND: Coxsackievirus A24 variant is one of the major etiological agents involved in acute haemorrhagic conjunctivitis. STUDY DESIGN: An outbreak of acute hemorrhagic conjunctivitis occurred in the Southeast of Spain between September and November 2004. Cellular and molecular methods were used to identify and characterize the viral agent associated with the epidemic. RESULTS: Enterovirus was detected in the conjunctival swabs of 35 patients. None of the viruses isolated could be typed by conventional neutralization assays; however, amplification and sequencing of the 3'-end VP1 region of 19 of the samples identified coxsackievirus A24 variant as the serotype causing the outbreak. Phylogenetic analysis of the 5'-half VP1 region of the genome revealed that Spanish sequences, like other strains isolated during outbreaks of hemorrhagic conjunctivitis in American and African countries in 2003 and 2004, were closely related to the isolates detected in Korea (2002), thus proving their worldwide spread. CONCLUSIONS: This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Spain. Molecular typing in combination with phylogenetic analysis is useful to study the enterovirus epidemiology associated with epidemics.

[Lymphocytic meningitis in Spain: a possible epidemic situation in 2000]. / Meningitis linfocitaria en España: posible situación epidémica en el año 2000.

UNLABELLED: In order to clarify the unexpected increase in 2000 of lymphocyticmeningitis analyzed in the Centro Nacional de Microbiología,Instituto de Salud Carlos III, Madrid, here we present their microbiological features. PATIENTS AND METHOD: Samples of 732 patients were tested by virological and serological methods to detect infections by neurotrophic agents. RESULTS: Infections were identified in 375 (51%) cases. Nopolio enteroviruses were identified in 90% cases. Echovirus 30 and 13 were mainly characterized, the latter for the first time in Spain. CONCLUSIONS: The emergence of Echovirus 13 may explain the past epidemic situation.

Design and validation of a real-time RT-PCR for the simultaneous detection of enteroviruses and parechoviruses in clinical samples.

Human enteroviruses (EVs) and parechoviruses (HPeVs) are important etiological agents causing infections such as meningitis, encephalitis and sepsis-like disease in neonates and young children. We have developed a real-time RT-PCR for simultaneous detection of EV and HPeV in clinical samples. Primers and probe sets were designed from the conserved 5'-noncoding region of the genomes. The sensitivity, specificity and reproducibility of the technique were measured using a set of 25 EV and 6 HPeV types. All EVs but no HPeVs were detected with the EV primers-probe set. The HPeV primers-probe set detected only the 6 HPeV types. The lower detection limit was found to be 4 and 40CCID50/ml for HPeV and EV respectively, demonstrating high sensitivity of the technique for both viruses. The threshold cycle values were highly reproducible on repeat testing of positive controls among assay runs. The assay was evaluated in 53 clinical samples of suspected meningitis, sepsis or febrile syndromes from children under 3 years. In 11 of these (21%) EVs were detected, while 4, i.e. 7.5%, were HPeV positive. Molecular typing was carried out for 73% of the viruses. In summary, the RT-PCR method developed demonstrated effectively both EV and HPeV detection, which can cause similar clinical symptoms in infants.

[Role of the National Poliovirus Laboratory for the Program of eradication and poliomyelitis surveillance]. / Papel del Laboratorio Nacional de Poliovirus en el Programa de erradicación y vigilancia de la poliomielitis.

The Spanish acute flaccid paralysis surveillance network is coordinated by the National Poliovirus Laboratory (NPL), which, since 1998, carries out polioviruses (PV) and other enteroviruses detected characterization by cell culture and molecular techniques. A total of 110,725 (70046+40679) samples were studied between 1998-2012 and enteroviruses were detected in 8% of these. Among these enteroviruses 241 PV were characterized as PV Sabin-like, except samples belong to an imported poliomyelitis case, all of which were characterised as vaccine derived PV type 2. The NPL has carried out the serotyping and the intratypic differentiation of all the isolated PV in Spain of any syndrome. It is shown that wild PV has not circulated in our country during the 15 years studied and that has led to the signing of the Act of the "eradication of poliomyelitis in Spain" by WHO in 2001, and the /"certification of the eradication of wild PV free for European countries" on 21 June 2002. Currently only 3 countries have endemic transmission of wild PV (Pakistan, Afghanistan and Nigeria). Until a complete worldwide eradication, was achieved, Spain will actively continue to participate in the maintenance of the poliomyelitis eradication infrastructure by monitoring and vaccination as well as the wild PV containment plan to avoid the spread of wild PV.

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.

[Enterovirus 75, a new pathogenic virus in Granada province (Spain)]. / Enterovirus 75, un nuevo virus patógeno en nuestro medio.

INTRODUCTION: Members of the genus Enterovirus are usually investigated for their etiological role in neurological syndromes. However, they are often associated with other syndromes such as febrile illness, acute respiratory infection and exanthema. In this study, clinical and epidemiological data from five subjects with infection by the recently described enterovirus 75 were analyzed in the province of Granada (Spain). METHODS: Diagnosis at the genus level was carried out by viral culture in MRC-5 and rhabdomyosarcoma cell lines. Isolate serotypes were determined by RT-PCR of a fragment of the VP1 region and subsequent sequencing of the PCR products. RESULTS: Among the five enterovirus 75 isolated, two were detected in children with aseptic meningitis (1 month and 12 years old) and three in subjects with non-neurological syndromes, i.e. acute respiratory infection, febrile illness and gastroenteritis (all were aged less than one year). The five cases were detected between December 2005 and May 2006. All patients recovered without sequelae. CONCLUSION: These data demonstrate that enterovirus 75 circulates in the south of Spain and indicate that this enterovirus serotype may be implicated in less severe non-neurological syndromes, particularly in younger children, and mainly during the cold months of the year.

[Laboratory Network within the Polio Eradication Initiative (1998-2003): six years of surveillance for acute flaccid paralysis in Spain]. / Red de Laboratorios del Plan de Erradicación de la Poliomielitis (1998-2003): 6 años de vigilancia de parálisis flácida aguda en España.

INTRODUCTION: Worldwide poliomyelitis eradication was proposed in 1988 by the World Health Organization (WHO), based on reaching and maintaining high vaccination coverage and on implementing effective poliovirus infection surveillance systems. METHODS: In Spain the surveillance system focuses on active searching for acute flaccid paralysis cases through a nine-laboratory network, coordinated by the National Poliovirus Laboratory (NPL) in the National Center of Microbiology, and supported by Autonomous Community epidemiologists. Additionally, the Network sends enterovirus isolation data from other syndromes. The Laboratory Network is responsible for the primary virological study, while the NPL characterizes all polioviruses and the most epidemiologically relevant non-polio enteroviruses. RESULTS: A total of 54,533 samples were studied during the six-year period, and enteroviruses were detected in 9%. All the polioviruses isolated (n = 196), were characterized as Sabin-like (poliovirus vaccine), and among the non-polio enteroviruses, the most frequent was Echovirus 30. A total of 3% of the samples studied corresponded to the 268 acute flaccid paralysis cases or their contacts. DISCUSSION: According to the results and the WHO virological classification, Spain can be considered polio-free. However, the geographic situation of our country may facilitate the introduction of wild polioviruses that can give rise to imported poliomyelitis. Hence, the laboratory network should actively continue to participate in all the proposed WHO strategies, particularly maintenance of the poliomyelitis eradication infrastructures, and continuing monitoring and vaccination.

Molecular analysis of echovirus 13 isolates and aseptic meningitis, Spain.

Echovirus 13 (EV13), considered rare, was reported worldwide in 2000, mostly related to aseptic meningitis outbreaks. In Spain, 135 EV13 isolates were identified. The genetic relationships between 64 representative strains from Spain and other reported isolates from the United States, Germany, Italy, Japan, and Sweden were described by analyzing the partial sequence of the major capsid protein (VP1) gene. The strains from Spain were clearly identified as EV13 (79.5% similarity with the EV13 reference strain) and were grouped phylogenetically into two different clusters (by origination on either the Iberian Peninsula or Canary Islands). Isolates from Germany from 2000 clustered with the Canary Islands group. The isolates from other countries obtained before 2000 were genetically distant. Changes in EV13 coding sequence involved several differences in the C-terminal extreme of the VP1 protein. Part of the neutralizing antigenic site III has been described in this genome region in poliovirus and swine vesicular disease virus.

[Poliomyelitis eradication initiative. Search for and control of wild poliovirus stored at Spanish laboratories]. / Erradicación de la poliomielitis. Búsqueda y control de poliovirus salvajes almacenados en los laboratorios españoles.

INTRODUCTION: An initiative to achieve global eradication of poliomyelitis was proposed in 1988. Since then, World Health Organization (WHO) efforts have been focused on reaching and maintaining high vaccination coverage and on implementing effective surveillance systems. Additionally, since 1998 the WHO has urged health authorities from countries over the world to locate and control the wild polioviruses stored in laboratories as well as potentially infectious materials that could contain these viruses or originate them under particular circumstances. METHODS: In Spain the Wild Poliovirus Containment Plan was initiated in 1999. All Spanish laboratories were inventoried and a specially designed questionnaire was distributed to them. The responses were analyzed. RESULTS: A total of 3209 laboratories were surveyed and 10 of them were found to contain infectious or potentially infectious material with wild poliovirus. The heads of these laboratories were informed about the Containment Plan and were requested to destroy this material under proper conditions. The laboratories that did not destroy the viruses adopted the BSL2/polio bio-safety measures. CONCLUSION: Despite its complexity, the search process was completed within the schedule established by the WHO, thanks to the efforts of health authorities from the Autonomous Communities. Spain reported its results to the WHO Regional Commission for the Certification of Poliomyelitis Eradication in May 2002.

[Epidemic outbreak of meningitis due to Echovirus type 13 on the island of Gran Canaria (Spain)]. / Brote epidémico de meningitis por virus Echo serotipo 13 en la isla de Gran Canaria.

INTRODUCTION: Since the implementation of routine vaccination against mumps, enteroviruses have become the commonest cause of viral meningitis. In Spain the most frequently isolated echoviruses are serotypes 30, 9, 6 and 4.Objectives. Determine the clinical-epidemiological characteristics of a meningitis outbreak due to echovirus type 13. METHODS: We studied 152 cases of enteroviral meningitis diagnosed in the year 2000. Viral isolation was performed on fibroblast MRC-5 and human rhabdomyosarcoma cells. The viruses were identified with monoclonal antibodies and serotyped by neutralization. RESULTS: Echovirus was isolated in 131 out of 152 CSF samples (86.2%). In 21 patients with negative CSF culture, the diagnosis was made by echovirus isolation from upper respiratory tract and/or feces samples. The cytopathic effect was seen in all cases in monolayers of rhabdomyosarcoma cells. The average age of the patients was 67 months (range: 1-350) and the male:female ratio was 2:1. Most of the patients had fever, headache and other signs of meningitis. Hospitalization was required in 52.6%. All patients were discharged without sequelae. The highest incidence was recorded from April to June. CONCLUSIONS: There had been no record of the presence of echovirus 13 in Spain in recent years until the emergence of this outbreak. Isolation of the virus from sites other than CSF is often helpful in establishing the diagnosis and patient management. Enterovirus isolates must be specifically serotyped to determine enteroviral activity in the population, which can be sporadic or epidemic.