María Cabrerizo

Viral infections of the central nervous system in Spain: A prospective study

The aim of the study was to determine the incidence of viruses causing aseptic meningitis, meningoencephalitis, and encephalitis in Spain. This was a prospective study, in collaboration with 17 Spanish hospitals, including 581 cases (CSF from all and sera from 280): meningitis (340), meningoencephalitis (91), encephalitis (76), febrile syndrome (7), other neurological disorders (32), and 35 cases without clinical information. CSF were assayed by PCR for enterovirus (EV), herpesvirus (herpes simplex [HSV], varicella‐zoster [VZV], cytomegalovirus [CMV], Epstein–Barr [EBV], and human herpes virus‐6 [HHV‐6]), mumps (MV), Toscana virus (TOSV), adenovirus (HAdV), lymphocytic choriomeningitis virus (LCMV), West Nile virus (WNV), and rabies. Serology was undertaken when methodology was available. Amongst meningitis cases, 57.1% were characterized; EV was the most frequent (76.8%), followed by VZV (10.3%) and HSV (3.1%; HSV‐1: 1.6%; HSV‐2: 1.0%, HSV non‐typed: 0.5%). Cases due to CMV, EBV, HHV‐6, MV, TOSV, HAdV, and LCMV were also detected. For meningoencephalitis, 40.7% of cases were diagnosed, HSV‐1 (43.2%) and VZV (27.0%) being the most frequent agents, while cases associated with HSV‐2, EV, CMV, MV, and LCMV were also detected. For encephalitis, 27.6% of cases were caused by HSV‐1 (71.4%), VZV (19.1%), or EV (9.5%). Other positive neurological syndromes included cerebellitis (EV and HAdV), seizures (HSV), demyelinating disease (HSV‐1 and HHV‐6), myelopathy (VZV), and polyradiculoneuritis (HSV). No rabies or WNV cases were identified. EVs are the most frequent cause of meningitis, as is HSV for meningoencephalitis and encephalitis. A significant number of cases (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) still have no etiological diagnosis. J. Med. Virol. 85:554–562, 2013.

Transmission Networks and Population Turnover of Echovirus 30

ABSTRACT Globally, echovirus 30 (E30) is one of the most frequently identified enteroviruses and a major cause of meningitis. Despite its wide distribution, little is known about its transmission networks or the dynamics of its recombination and geographical spread. To address this, we have conducted an extensive molecular epidemiology and evolutionary study of E30 isolates collected over 8 years from a geographically wide sample base (11 European countries, Asia, and Australia). 3Dpol sequences fell into several distinct phylogenetic groups, interspersed with other species B serotypes, enabling E30 isolates to be classified into 38 recombinant forms (RFs). Substitutions in VP1 and 3Dpol regions occurred predominantly at synonymous sites (ratio of nonsynonymous to synonymous substitutions, 0.05) with VP1 showing a rapid substitution rate of 8.3 × 10−3 substitutions per site per year. Recombination frequency was tightly correlated with VP1 divergence; viruses differing by evolutionary distances of >0.1 (or 6 years divergent evolution) almost invariably (>97%) had different 3Dpol groups. Frequencies of shared 3Dpol groups additionally correlated with geographical distances, with Europe and South Asia showing turnover of entirely distinct virus populations. Population turnover of E30 was characterized by repeated cycles of emergence, dominance, and disappearance of individual RFs over periods of 3 to 5 years, although the existence and nature of evolutionary selection underlying these population replacements remain unclear. The occurrence of frequent “sporadic” recombinants embedded within VP1 groupings of other RFs and the much greater number of 3Dpol groups than separately identifiable VP1 lineages suggest frequent recombination with an external diverse reservoir of non-E30 viruses.

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. J. Med. Virol. 80:1018–1024, 2008.

Evolutionary Dynamics and Temporal/Geographical Correlates of Recombination in the Human Enterovirus Echovirus Types 9, 11, and 30

ABSTRACT The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.

Enteroviruses in Spain over the decade 1998-2007: virological and epidemiological studies.

BACKGROUND Human enteroviruses (HEV) are the commonest cause of viral meningitis as well as other pathologies, therefore HEV characterization is important both in patient management and epidemiological investigation. OBJECTIVES A 10-year study of patients with enteroviral infection was carried out in Spain to determine the underlying etiology. STUDY DESIGN HEV were fully typed by microneutralisation tests and/or molecular methods. RESULTS A collection of 86404 clinical samples were studied in several Spanish laboratories. These were collected from patients with different syndromes, mainly aseptic meningitis (AM), fever, respiratory diseases and acute flaccid paralysis. Of these, 6867 HEV were obtained. At the National Poliovirus Laboratory 2814 were serotypically characterised. Among non-polio enteroviruses, the eight main serotypes were Echovirus 30 (25%), Echovirus 6 (12.4%), Echovirus 13 (8.3%), Echovirus 11 (7.4%) and Echovirus 9 (4.7%), followed by Coxsackievirus B5 (4.2%) and Echovirus 7 and Coxsackievirus A9 (3.7%) each. In AM cases, Echovirus 30 was identified in 39% of them, followed by Echovirus 6 (14%). However, Echovirus 6 was mainly associated with respiratory disease (17%), followed by Echovirus 11 (10%). On the other hand, Echovirus 30, Echovirus 11 and Echovirus 6 contributed equally with 12% of each serotype in the cases of fever. CONCLUSIONS The present report complements previous data (Trallero et al.(13)), with the results of HEV incidence in Spain from 1998 to 2007. The surveillance described in this study provided valuable information as to which serotypes are in circulation, the emergence of new HEV and association with clinical manifestations.

The Association of Recombination Events in the Founding and Emergence of Subgenogroup Evolutionary Lineages of Human Enterovirus 71

ABSTRACT Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.

Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain

Hand, foot and mouth disease (HFMD) is a childhood illness frequently caused by genotypes belonging to the enterovirus A species, including coxsackievirus (CV)-A16 and enterovirus (EV)-71. Between 2010 and 2012, several outbreaks and sporadic cases of HFMD occurred in different regions of Spain. The objective of the present study was to describe the enterovirus epidemiology associated with HFMD in the country. A total of 80 patients with HFMD or atypical rash were included. Detection and typing of the enteroviruses were performed directly in clinical samples using molecular methods. Enteroviruses were detected in 53 of the patients (66%). CV-A6 was the most frequent genotype, followed by CV-A16 and EV-71, but other minority types were also identified. Interestingly, during almost all of 2010, CV-A16 was the only causative agent of HFMD but by the end of the year and during 2011, CV-A6 became predominant, while CV-A16 was not detected. In 2012, however, both CV-A6 and CV-A16 circulated. EV-71 was associated with HFMD symptoms only in three cases during 2012. All Spanish CV-A6 sequences segregated into one major genetic cluster together with other European and Asian strains isolated between 2008 and 2011, most forming a particular clade. Spanish EV-71 strains belonged to subgenogroup C2, as did most of the European sequences circulated. In conclusion, the recent increase of HFMD cases in Spain and other European countries has been due to a larger incidence of circulating species A enteroviruses, mainly CV-A6 and CV-A16, and the emergence of new genetic variants of these viruses.

Onychomadesis after a hand, foot, and mouth disease outbreak in Spain, 2009.

Few reports exist regarding the association between onychomadesis and an enterovirus infection presenting clinically as hand, foot, and mouth disease (HFMD). In February 2009, an outbreak of HFMD occurred in a Spanish nursery school, followed by onychomadesis 36-69 days later. Twelve of 17 children with HFMD developed nail shedding; enterovirus was detected in stool samples from eight (47%) of the 17. However, in only three of the children could an enterovirus serotype coxsackievirus B1 be identified. The epidemiological results of this study confirm onychomadesis as a complication in HFMD. In future outbreaks, molecular characterization of enterovirus from appropriate clinical samples should be studied.

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. J. Med. Virol. 86:857–864, 2014.

Paralysis case and contact spread of recombinant vaccine-derived poliovirus, Spain.

To the Editor: The World Health Organization Polio Eradication Initiative has reduced to 4 the number of countries with endemic transmission of wild polioviruses and has reported the widespread circulation of polioviruses that have evolved from attenuated vaccine (oral poliovirus vaccine [OPV]), so-called vaccine-derived polioviruses (VDPVs) (1,2). VDPVs can reportedly replicate in immunocompromised persons, in whom they produce paralysis, and can spread to contacts and produce paralytic polio in immunocompetent but incompletely immunized persons; these viruses can even cause some polio outbreaks in areas with a low level of vaccination coverage (2). When VDPVs are detected, a careful follow-up of VDPV cases and contacts is essential if spread is to be avoided. Because point mutations or recombination events have been associated with reversion to neurovirulence transmission and thus a greater probability of outbreaks (2), meticulous molecular studies of the detected strains are recommended. Spain progressively adopted vaccination with OPV for children in 2004. As a consequence, the incidence of vaccine-like poliovirus detection in recently vaccinated children, which was relatively common up to that time (51 isolates in 2003) (3), began to decrease (15 isolates in 2004 and none in 2005 except the case described here). In July 2005, a 14-month-old boy from Morocco with residual paralysis and major histocompatibility class II immunodeficiency was reported through the Spanish Acute Flaccid Paralysis Surveillance System. The patient had received 2 OPV doses at birth and at 6 months of age in Morocco; 8 months later, meningoencephalitis developed. The case was immediately considered suspicious and was therefore monitored at least monthly until the boy died. Sampling was conducted, coinciding with his visits to the hospital to receive therapy with immunoglobulin (γ globulin 0.5 g/kg). His contacts were studied, environmental surveillance was conducted, and molecular analysis of all detected viruses was performed. Laboratory methods for virus detection and characterization, including 10 new reverse-transcription–PCRs designed to cover the entire genome, are detailed in the Table. Table Laboratory methods used for study of vaccine-derived poliovirus case, Spain, 2005* Serotype 2 VDPVs were detected in all 10 stool samples of the patient with residual paralysis for 6 months, until he died, and in 3 of the 7 family contacts analyzed (father and 2 brothers, 11 and 13 years of age, none with confirmed previous vaccination). One of the contacts, considered immunocompetent, shed virus for 216 days (5 fecal samples in which 5 complete genomes were obtained and 1 additional fecal sample in which virus capsid protein l [VP1] could be amplified); a stool sample collected on day 284 was negative. Technical problems delayed sewage sampling. When sewage from the area in which the patient and positive contacts lived was sampled on February 8, 2006, no polioviruses were detected; however, an echovirus 30 was detected. Poliovirus viral load fluctuated (106–109 copies/mL in the paralysis-affected person), decreasing after each immunoglobulin therapy dose (Figure 1 in the Technical Appendix). The corresponding level was <105 in the contacts. The highest value of viral load was recorded in the patient’s final sample, taken before he died. Homology of the VP1 gene with respect to the original vaccine PV2 fluctuated from 97.8% to 98.6% in the case samples but remained constant (98.4%) in the contact samples (Figure 1 in the Technical Appendix). All studied polioviruses featured the following nucleotide substitutions in the 5′ untranslated region: G309A, T344C, T355C, T398C, A481G, T500C, and T743C (Figure 2 in the Technical Appendix). Furthermore, the final sample from the patient had A476C, G505T, T588A, and A738C. Several nucleotide substitutions detected in VP1–4 were common to all samples (Figure 2 in the Technical Appendix); 5 resulted in amino acid changes, including T2909C (VP1 I143T) and G3277A (VP1 V266I). All samples contained 2 noncontiguous recombination fragments Sabin 2/Sabin 1 in the nonstructural genes, including the entire 3C gene and the 3′ half of the 3D-pol (Figure 2 in the Technical Appendix) as in other reports (7–10). Both fragments, when compared with C species enterovirus, were closely related to Sabin 1 (99.6% and 97.9%, respectively). Specific nucleotide and amino acid comparisons among the isolates are detailed in Figure 3 in the Technical Appendix. According to the proposed classification (2), all the detected viruses were iVDPVs (isolated from immunocompromised patients) that spread only to close contacts because they were not detected in local sewage. If we assume that the greater the amount of viral excretion in feces that occurs, the higher number of replicating polioviruses (as well as the potential for greater genetic diversity), the patient had a more active infection (that responded to the therapy) than did the contacts. Fluctuation in homology to the parental OPV strain might be due not only to the calculation method (calculation was made on the basis of the majority-base call at each chromatogram position, and case sequences presented many mixed nucleotide positions) but also to immunotherapy. Treatment appeared to have decreased virus replication, probably by its action mainly on species with greater fitness and higher replication rates (those that were more similar to the original Sabin strain). As a consequence, treatment might produce a bottleneck that unmasked more divergent species. Both the case and contact strains had intertypic Sabin 1/Sabin 2 recombination in nonstructural genes and also shared most of the nucleotide and amino acid substitutions. However, pathologic changes occurred only in the patient whose immunologic mechanisms were affected and whose viral load was consequently much higher. A recent report (1) suggests that VDPVs can emerge in any country that uses OPV with insufficient vaccine coverage. In a polio-free IPV-user country, poliomyelitis can arise and spread to contacts who are not properly vaccinated. In the case we present here, the high level of vaccination coverage in Spain and the rapid control of close contacts achieved through the surveillance and control programs prevented virus spread. In the global pre-eradication phase, countries are recommended to change vaccination from OPV to IPV. However, IPV-adopting countries commonly share borders with OPV-adopting countries and residents may travel back and forth; thus, although the probability of VDPV circulation decreases, it does not reduce to zero. Therefore, active surveillance, rapid classification of isolates, and molecular characterization of the virus are essential.