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Resumen Introduccion En Espana, al igual que en otros paises donde el sarampion endemico ha sido eliminado, es necesario utilizar de forma rutinaria las herramientas diagnosticas que confirmen los casos para su prevencion y control de la diseminacion. Se describen los diferentes ensayos microbiologicos utilizados para su diagnostico durante un brote de sarampion en 2019 en la provincia de Guadalajara (Espana). Metodos Las pruebas serologicas y moleculares se realizaron en el laboratorio de Microbiologia del Hospital Universitario de Guadalajara y en el Centro Nacional de Microbiologia del Instituto de Salud Carlos III (Majadahonda, Espana). Los datos de los pacientes se obtuvieron del sistema epidemiologico de vigilancia. Resultados Se diagnosticaron de sarampion un total de 43 pacientes por metodos microbiologicos: 29 casos por PCR (exudado faringeo u orina) junto con IgM especifica positiva, 11 pacientes solamente por PCR, y 3 pacientes exclusivamente por presencia de IgM. El genotipo D8 fue identificado en 35 pacientes y el genotipo A en 2 casos descartados como postvacunal. La PCR en suero fue positiva en 11 de 14 pacientes con ausencia de IgM en su primera muestra recogida de suero. Once casos confirmados habian recibido una o 2 dosis de la vacuna. Doce adultos fueron ingresados, todos diagnosticados de hepatitis. Conclusiones La combinacion de pruebas moleculares y la presencia de IgG e IgM especificas son necesarias para un diagnostico correcto y la clasificacion de los pacientes como fallo vacunal (primario o secundario). El genotipado es una herramienta fundamental para la correcta clasificacion de los pacientes en el contexto de un programa de eliminacion del sarampion.

Given sustained high vaccination coverage and enhanced surveillance for measles, Spain has been free of endemic measles transmission since 2014, achieving elimination certification from the World Health Organization in 2017. In November 2017, measles was introduced through an imported case travelling to the Valencian Community, causing an interregional outbreak. Here, we describe the outbreak using data reported to the national epidemiological surveillance network. The outbreak involved 154 cases (67 males, 87 females) notified in four regions; 148 were laboratory-confirmed and six epidemiologically linked. Most cases were adults aged 30–39 (n = 62, 40.3%) years. Sixty-two cases were hospitalised (40.3%) and 35 presented complications (22.7%). Two thirds of the cases (n = 102) were unvaccinated including 11 infants (≤ 1 year) not yet eligible for vaccination. The main route of transmission was nosocomial; at least six healthcare facilities and 41 healthcare workers and support personnel were affected. Sequencing of the viral nucleoprotein C-terminus (N450) identified genotype B3, belonging to the circulating MVs/Dublin.IRL/8.16-variant. Control measures were implemented, and the outbreak was contained in July 2018. The outbreak highlighted that raising awareness about measles and improving the vaccination coverage in under-vaccinated subgroups and personnel of healthcare facilities are key measures for prevention of future outbreaks.

In Spain, like in other countries where endemic measles has been eliminated, there is a need for available diagnostic tools for confirming any cases in order to prevent and control its transmission. We describe the different microbiological tests used for the diagnosis of measles during an outbreak that occurred in 2019 in the province of Guadalajara (Spain).Serological and molecular tests were performed at the Microbiology laboratory of the Guadalajara University Hospital and at the National Center for Microbiology of the Carlos III Health Institute (Majadahonda, Spain). Patient data were obtained from the surveillance system.A total of 43 patients had a laboratory diagnosis of measles: 29 cases by PCR (pharyngeal exudate or urine) and positive specific IgM, 11 cases by PCR, and 3 cases only by a positive IgM. Genotype D8 was identified in 35 confirmed cases and genotype A in 2 that were discarded as post-vaccination cases. PCR was positive in the acute sera of 11 out of 14 patients with a negative IgM. Eleven confirmed cases had recieved one or 2 vaccine doses. Twelve adult patients were hospitalizated, all of them with a diagnostic of hepatitis.The combination of molecular tests and the presence of specific IgG and IgM are necessary for a correct diagnosis of measles and also to classify patients with a breakthrough infection or vaccine failures (primary or secondary). Genotyping is essential for the correct classification of the patients in the context of a measles elimination program.

En la primavera de 2019 se ha producido en la Comunidad de Madrid un aumento de casos de sarampión. El objetivo de este estudio es describir el tipo de muestras clínicas estudiadas y los resultados obtenidos en este brote. Entre el 1 de abril y el 25 de junio de 2019 se procesaron 193 muestras biológicas correspondientes a 109 sospechas clínicas de sarampión. Las determinaciones serológicas (IgM e IgG frente a sarampión y rubéola) se llevaron a cabo por ELISA indirecto. La amplificación de ácidos nucleicos de los virus del sarampión y la rubéola se efectuó mediante RT-PCR. Se confirmaron 44 casos de sarampión. Dos de ellos habían sido vacunados muy recientemente y ambos presentaban el genotipo vacunal A. Los amplificados de todas las cepas salvajes (21) eran D8. En esta serie, el tiempo medio observado entre recepción de muestras y obtención del resultado positivo fue de 0,5 días. En 32 casos confirmados se dispuso de muestras tanto de exudado faríngeo para RT-PCR como de sangre para serología y en 32 la RT-PCR resultó positiva (sensibilidad 100%, IC95% 86,7- 99,7) mientras que en 22 la IgM fue positiva (sensibilidad 68,8%, IC95% 49,9- 83,3). Inversamente, en 49 sospechas descartadas se dispuso de muestras de exudado faríngeo para RT-PCR como de sangre para serología y en 49 la RT-PCR resultó negativa (especificidad 100%, IC95% 90,9- 99,8). En 1 muestra de una sospecha descartada la IgM fue positiva (especificidad 98,0%, IC95% 87,8- 99,9). En la etapa de eliminación del sarampión y la rubéola los estándares de vigilancia deben ir dirigidos a detectar todos los posibles casos. Para ello es preciso la obtención en tiempos correctos de las muestras biológicas adecuadas (suero y especialmente exudado faríngeo).

España presenta una situación peculiar Europa, ya que nunca se vio afectada por la epizootia europea de rabia vulpina, pero mantiene un tráfico intenso de bienes y personas con territorios altamente endémicos del norte de África, e incluso fronteras terrestres enCeuta y Melilla, lo que nos coloca en una situación vulnerable en cuanto a importación de casos. Por otra parte, sabemos de la presencia de dos de los lisavirus de murciélago europeos en nuestro territorio. El laboratorio es un elemento esencial en la vigilancia que se hace imprescindible ante esta situación. El Centro Nacional de Microbiología lleva más de treinta años actuando de laboratorio nacional de referencia en apoyo de los laboratorios autonómicos existentes y haciendo diagnóstico para el resto del territorio, además de representar a España en la red europea de laboratorios RABNET. Así, en el CNM se han diagnosticado y caracterizado la gran mayoría de los casos declarados en España en este período, tanto de rabia de murciélagos, como de rabia importada animal y humana. De manera complementaria a esta actividad de vigilancia, se realizan investigaciones tanto sobre rabia canina como de murciélagos, que, desde hace años, se han ampliado a otros agentes víricos asociados a murciélagos, Spain shows peculiarities regarding rabies in Europe. It was not affected by the European vulpine epizootic but keeps an intense traffic of goods and people with highly endemic territories in North Africa and even has terrestrial boundaries in the cities of Ceuta and Melilla. This makes Spain particularly vulnerable to importation of rabies cases. On the other hand,we know the presence in Spain of two of the five bat lyssaviruses endemic from Europe. The laboratory is an essential tool for the surveillance that is strictly necessary to keep in order to handle this situation. The National Center of Microbiology (CNM)provides support to autonomic laboratories, makes primary diagnosis for the rest of the territory and represents Spain in the European laboratory net RABNET from more than thirty years to now, according to its role of National Reference Laboratory. Duringthis period, the CNM has diagnosed and characterized most rabies cases declared in Spain, including both bat rabies and imported animal and human cases. Associated to these surveillance activities, the CNM makes research in the field of canine and bat rabies, which has been extended to other bat-associated viruses in the last years.

Rabies is enzootic in over one hundred countries worldwide. In the European Union/European Economic Area (EU/EEA), the vast majority of human rabies cases are travellers bitten by dogs in rabies-enzootic countries, mostly in Asia and Africa. Thus, EU/EEA travellers visiting rabies enzootic countries should be aware of the risk of being infected with the rabies virus when having physical contact with mammals. They should consider pre-exposure vaccination following criteria recommended by the World Health Organization and if unvaccinated, immediately seek medical attention in case of bites or scratches from mammals. As the majority of the EU/EEA countries are free from rabies in mammals, elimination of the disease (no enzootic circulation of the virus and low number of imported cases) has been achieved by 2020. However, illegal import of potentially infected animals, mainly dogs, poses a risk to public health and might threaten the elimination goal. Additionally, newly recognised bat lyssaviruses represent a potential emerging threat as the rabies vaccine may not confer protective immunity. To support preparedness activities in EU/EEA countries, guidance for the assessment and the management of the public health risk related to rabies but also other lyssaviruses, should be developed.

Adenoviruses are double-strained DNA viruses found in a great number of vertebrates, including humans. In order to understand their transmission dynamics, it is crucial, even from a human health perspective, to investigate how host traits influence their prevalence. Bats are important reservoirs for adenoviruses, and here we use the results of recent screenings in Western Europe to evaluate the association between characteristic traits of bat species and their probability of hosting adenoviruses, taking into account their phylogenetic relationships. Across species, we found an important phylogenetic component in the presence of adenoviruses and mating strategy as the most determinant factor conditioning the prevalence of adenoviruses across bat species. Contrary to other more stable mating strategies (e.g. harems), swarming could hinder transmission of adenoviruses since this strategy implies that contacts between individuals are too short. Alternatively, bat species with more promiscuous behavior may develop a stronger immune system. Outstandingly high prevalence of adenoviruses was reported for the Iberian species Pipistrellus pygmaeus, P. kuhlii and Nyctalus lasiopterus and we found that in the latter, males were more likely to be infected by adenoviruses than females, due to the immunosuppressing consequence of testosterone during the mating season. As a general trend across species, we found that the number of adenoviruses positive individuals was different across localities and that the difference in prevalence between populations was correlated with their geographic distances for two of the three studied bat species (P. pygmaeus and P.kuhlii). These results increase our knowledge about the transmission mechanisms of adenoviruses.

Resumen Introduccion En la fase de eliminacion del sarampion, reviste especial importancia la investigacion de cada caso sospechoso, especialmente en personas vacunadas, pues en ellas la clinica es mas leve, y los resultados de laboratorio mas dificiles de interpretar. Nuestro estudio se centro en la epidemiologia del sarampion en personas vacunadas. Metodos Se realizo un estudio longitudinal de los casos de sarampion en personas vacunadas notificados en Espana entre 2003 y 2014. Resultados Se observaron 138 casos confirmados de sarampion en personas vacunadas con 2 dosis, 90 de ellos confirmados por laboratorio. La mediana de tiempo entre la ultima dosis de vacuna recibida y la aparicion del exantema mostro una tendencia lineal creciente (p Conclusion Nuestros resultados evidencian la importancia de cumplir el protocolo diagnostico para confirmar o descartar casos de sarampion, especialmente en los vacunados con 2 dosis. Ante una IgM negativa en muestras precoces, seria necesario obtener una nueva muestra y realizar un nuevo test de IgM, asi como la prueba de PCR.

During the final phase of measles elimination rigorous investigation of each individual case becomes fundamental to confirm or discard cases, particularly among vaccinated people, since they experience a milder disease, and laboratory diagnosis is more complex. Our study focused in the epidemiology of measles in vaccinated people.Longitudinal study on measles cases in two dose vaccinated people in Spain from 2003 to 2014.We confirmed 138 measles cases (90 of them, laboratory confirmed) in people with two doses of vaccine. The median of time from last vaccination to rash onset showed a lineal trend (P.001), in parallel with the number of doses of vaccine received (0, 1, 2 doses). Among confirmed cases, the hospitalization risk decreased inversely proportional to the number of administered vaccine doses (linear trend, P.001). Only in 23.9% of confirmed cases and 50% of discarded cases the guidelines about sample taking were fulfilled. 50% of samples in two dose vaccinated people were taken without fulfilling time delay criteria. 16.7% (36/215) of discarded cases with a negative IgM result did correspond to samples taken early (first 72h after rash) and could represent false negatives.Our results highlight the importance of fulfilling properly the guidelines for laboratory diagnosis in order to confirm or discard every measles case, especially in two dose vaccinated people. When a negative IgM result is obtained in early samples a new IgM test should be practiced, as well as a PCR test, in order to avoid infra-detection of cases.

Introduction The aim of this study was to evaluate a cut-off point of the titration of IgG by ELISA in the diagnosis of mumps. Methods A study was made of serum samples from 85 mumps cases (confirmed by PCR in saliva) and 2351 controls of the general population of the Region of Madrid. Results The IgM detection was positive in 21 cases (sensitivity of 24.7%). The best cut-off point corresponded to IgG titres ≥ 4900 (sensitivity of 64.7% and specificity of 86.1%). Among 42 patients immunised with at least one dose of measles mumps, rubella vaccine IgM was detected in 4 cases. However, the detection of IgG ≥ 4900 was positive in 29 (sensitivity of 69.0%). Conclusions An IgG result of ≥4900 was almost 5 times more probable in a patient with mumps than in a non-infected patient. The detection of high titres of IgG against mumps could improve the IgM results in vaccinated people.

Resumen Introduccion El objetivo del estudio fue determinar un punto de corte de la titulacion de IgG mediante ELISA en el diagnostico de parotiditis. Metodos Se estudiaron sueros de 85 casos de parotiditis (confirmados por PCR en saliva) y de 2.351 controles de la poblacion de la Comunidad de Madrid. Resultados La deteccion de IgM fue positiva en 21 casos (sensibilidad del 24,7). El mejor punto de corte de IgG correspondia a titulos ≥ 4.900 (sensibilidad del 64,7% y especificidad del 86,1%). De 42 pacientes vacunados con ≥ 1 dosis de triple virica se detecto IgM en 4, mientras que la deteccion de IgG ≥ 4.900 fue positiva en 29 (sensibilidad del 69,0%). Conclusiones Un resultado de IgG ≥ 4.900 fue casi 5 veces mas probable en un paciente con parotiditis que en otro sujeto no infectado. La deteccion de titulos elevados de IgG frente a parotiditis puede mejorar el rendimiento diagnostico de la IgM en vacunados.

Adenoviruses are double-strained DNA viruses found in a great number of vertebrates, including humans. In order to understand their transmission dynamics, it is crucial, even from a human health perspective, to investigate how host traits influence their prevalence. Bats are important reservoirs for Adenoviruses, and here we use the results of recent screenings in Western Europe to evaluate the association between characteristic traits of bat species and their probability of hosting Adenoviruses, taking into account their phylogenetic relationships. Across species, we found an important phylogenetic component in the presence of Adenoviruses and mating strategy as the most determinant factor conditioning the prevalence of Adenoviruses across bat species. Contrary to other more stable mating strategies (e.g. harems), swarming could hinder transmission of Adenoviruses since this strategy implies that contacts between individuals are too short. Alternatively, bat species with more promiscuous behavior may develop a stronger immune system. Outstandingly high prevalence of Adenoviruses was reported for the Iberian species Pipistrellus pygmaeus, P. kuhlii and Nyctalus lasiopterus and we found that in the latter, males were more likely to be infected by Adenoviruses than females, due to the immunosuppressing consequence of testosterone during the mating season. As a general trend across species, we found that the number of Adenoviruses positive individuals was different across localities and that the difference in prevalence between populations was correlated with their geographic distances (P. pygmaeus). These results increase our knowledge about the transmission mechanisms of Adenoviruses.Author SummaryAdenoviruses are DNA viruses with a wide range of vertebrate hosts, including humans, causing ocular, respiratory and gastrointestinal diseases. Here, we focus on the prevalence of Adenoviruses in bats, which are known to be natural reservoir of many viruses, using the results of recent screenings for prevalence of these viruses in 33 European bat species. Our aim is to find association between Adenoviruses prevalence and biological and behavioral host traits, considering the heterogeneity both between and within species in order to have a deeper understanding of mechanisms of viral transmission.Our results highlight the importance of mating strategy: bats species using swarming as mating strategy are less likely to be infected by Adenoviruses. Moreover, we found that locality of capture can explain a higher prevalence of Adenovirus within species. However, no general pattern has been found in the analysis at individual level, suggesting a strong species specificity and complex viral transmission dynamics.

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber&rsquo, s Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber&rsquo, s Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP2 in all of Schreiber&rsquo, s Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber&rsquo, s Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs.

With a highly immunized population, rubella infection in Spain is so low that the WHO has declared the elimination of rubella. Rubella in pregnant women is also very rare. The objective of this study is to describe the last cases of congenital rubella syndrome reported and recommend actions to maintain the status of the disease as eliminated. The CRS cases reported to the Spanish National Epidemiological Surveillance Network between 1997 and 2016 were studied, and the epidemiological, clinical, diagnostic and maternal characteristics of newborns with CRS described. The incidence of CRS was calculated using Birth Statistics from the Spanish National Statistics Agency (INE). Twenty-three cases of CRS were reported, 70% of which were associated with rubella outbreaks. The most common clinical conditions were heart disease (52.2%), deafness (39.1%) and cataracts (30.4%); 91.3% of cases were confirmed by laboratory testing. 70.0% were born from a non-vaccinated foreign mother, resident in Spain (cumulative rate incidence (CR): 1.1/100,000 births), with mothers coming from Africa (36.0%), Latin America (29.0%), Eastern Europe (21.0%) and Asia (14.0%). Six were born to Spanish mothers (CR: 0.08/ 100,000 births), the last of which were in 2005. The majority of CRS cases were born to unvaccinated immigrant women infected in Spain during rubella outbreaks. Universal vaccination in childhood is the most efficient strategy to prevent rubella. The limited circulation of the virus will, however, quickly lead to a loss of awareness about rubella among clinicians and epidemiologists. It is necessary to maintain protocols capable of identifying signs consistent with rubella in pregnant women and signs suggestive of congenital rubella in newborns. This work is the result of the stay that Elina M. Seppälä, a student at the University of Tampere (Finland) and granted an Erasmus + scholarship, held at the National Epidemiology Centre (ISCIII) between February and April 2017

Introduction and objectives The MMR vaccine was included in the official vaccination schedule in Spain in 1981. Currently, most women of childbearing age are vaccinated and have not been naturally infected. Several studies have shown that vaccinated women have a lower antibody concentration than that achieved after natural infection, and a shorter duration of transplacentally acquired antibodies in their children. The objective of this study was to determine the antibody titer in mothers and their infants at birth and throughout the first year of life under current epidemiological circumstances. Material and methods Single-center, observational, descriptive and prospective study conducted between October 2013 and December 2014. One sample of serum and another of a dried blood spot on filter paper were taken from each mother. Dried blood spot samples on filter paper were taken from the children at birth, and at 3, 6, 9 and 12 months. In all the samples, levels of antibodies to the measles, rubella and mumps viruses were measured using standardized quantitative assays. Results 146 mother-child pairs were included. 78.4%, 86.9% and 67.1% of mothers had antibodies to measles, rubella and mumps, respectively. A decrease in the antibody titer in children was observed after 3 months, and no antibodies against the three diseases were detected by the age of 6 months. Comparisons revealed no statistically significant differences between the antibody titers of children of mothers born before or after 1981 during the first year of their life. Discussion The rapid loss of transplacentally acquired antibodies against measles, rubella and mumps, under current epidemiological conditions, suggests that bringing the MMR vaccination forward to 9 months might be justified. Larger population studies are needed to confirm these results.

We report the first case, to our knowledge, of a child who presented with severe zonal hepatic necrosis after allogeneic hematopoietic stem cell transplantation secondary to infection with the rubella virus RA27/3 vaccine strain.

International audience; In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

In recent decades, vaccination has substantially reduced the number of measles cases to levels close to the elimination stage. However, major measles outbreaks occurred in Europe during 2010-2012, after the introduction of the D4-Enfield lineage. We have performed a molecular characterization of 75 measles virus genotype D4 strains from patients infected in Spain between 2004 and 2012 by sequencing the N-450 region and the M-F non-coding region (M-F NCR) in order to identify genetic features of these viruses. The analysis of the N-450 region confirmed that all samples obtained since 2008 belonged to variants or sets of identical sequences of the D4-Enfield lineage, including a new one named MVs/Madrid.ESP/46.10/. Analysis of the M-F NCR showed insertions and deletions associated with previously described, uncommon non-standard genome length measles viruses. This genetic feature was identified in the D4-Enfield lineage viruses, but not in the other D4 viruses that were circulating in Spain before 2008, suggesting that these non-standard length M-F NCR sequences are characteristic of the D4-Enfield lineage. The results of the phylogenetic analysis of Spanish M-F NCRs suggest higher resolution in discriminating strains than did the N-450 analysis. In addition, the results of the analysis of the M-F NCR on the MVs/Madrid.ESP/46.10/ sub-lineage seem to support the potential utility of this region as a tool for epidemiological surveillance complementary to the N-450 region, as previously suggested. Further investigation on this question, as well as the surveillance of new potentially emerging strains with non-standard length M-F NCR are strongly recommended as part of future strategies for measles elimination. This work was supported by the Fondo de Investigación Sanitaria (FIS PI12/02006) y el Instituto de Salud Carlos III (PI15CIII/00023). AFG was funded by CIBER de Epidemiología y Salud Pública (ESPG13). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

BACKGROUND: Despite childhood universal VZV immunization was introduced in 2015, there are no data on VZV clade distribution in Spain. OBJECTIVES: To characterize the varicella-zoster virus strains circulating in Spain between 1997 and 2016. STUDY DESIGN: In this retrospective study, we determined the VZV clades in 294 patients with different pathologies (mainly encephalitis, zoster and varicella) by sequencing three fragments within ORF 22, ORF 21 and ORF 50 and, subsequently analyzing 7 relevant SNPs. RESULTS: Among these 294 patients, 132(44.9%) patients were infected by clade 1, 42(14.3%) patients by clade 3, 19(6.5%) by clade 5, 29(9.9%) by clade VI and 3(1%) by clade 4. Four patients (1.4%) were infected by clade 2 vOKA strains, who received one dose of live-attenuated varicella vaccine. Putative recombinant clade 1/3 was identified in 6 cases (2.0%). Results obtained from partial sequences were assigned to clade 1 or 3 in 56(19%) patients and clade 5 or VI in 3(1.0%) patients. In the multivariate analysis, encephalitis was independently associated with clades 1 and 3 and age >14y.o. (P = 0.035 and P = 0.021, respectively). Additionally, Madrid had significant fewer cases of encephalitis compared with the rest of regions analyzed (P = 0.001). CONCLUSIONS: Higher prevalence of clades 1 and 3 and their relation with encephalitis and age >14y.o. suggest earlier introduction of this clades in Spain. Putative interclade 1 and 3 recombinants are circulating in patients with encephalitis, herpes zoster and varicella. Several cases were related to vOKA vaccination but vaccine strains do not seem to circulate in the general population. This work was supported by a grant from Instituto de Salud Carlos III. Project code MPY1372/12. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

In the context of long-term screening for viruses on Western Palaearctic bats, we tested for the presence of adenovirus 1392 oropharyngeal swabs and 325 stool samples taken from 27 bat species. Adenoviruses were detected in 12 species of the Vespertilionidae and the Rhinolophidae families. Fifty positive respiratory and 26 positive stool samples were studied. Phylogenetic analyses of partial hexon protein and partial DNA-dependent DNA polymerase genes indicate that all these bat adenoviruses belong to the genus Mastadenovirus but without constituting a monophyletic cluster. According to genetic identities, the new groups are distinct to the previously described Bat mastadenovirus A and B species and contribute with potentially new members. Our data support that diversity of bat mastadenovirus is host-dependent and increase the knowledge of potentially pathogenic virus from bats. Due to the active role of bats as viral reservoirs, the characterization of these viruses is relevant for Public Health., This project was financially supported by an agreement between the Public Health Department of the Spanish Ministry of Health and the Instituto de Salud Carlos III for the development of “Rabies Surveillance in Spain” and by projects SAF 2006-12784-C02-01, SAF 2006-12784-C02-02, SAF 2009-09172 and SAF2013-47194-P of the General Research Programme of the Spanish Ministry of Science and Education. We thank the Genomics Unit of the Instituto de Salud Carlos III for the sequencing of amplified products. We thank all members of the Spanish Bat Conservation Society (SECEMU) that generously contributed with their samples to the present study.

BackgroundSince mumps vaccination was introduced in 1981 in Spain, the incidence of the disease has dropped significantly. However, cyclic epidemic waves and outbreaks still occur, despite high vaccination coverage. The World Health Organization (WHO) recommends genotyping to trace the pattern of mumps virus (MuV) circulation. Genotype H was predominant in Spain, but was replaced in 2005 by genotype G which has subsequently remained dominant. Of the small hydrophobic protein gene sequences, 78% are identical and belong to the MuVi/ Sheffield.GBR.1.05/[G]-variant. Aim: Our study aimed to investigate whether the circulation of MuV strains in Spain was continuous after the emergence of genotype G in 2005. Method: We obtained 46 samples from Spanish patients infected with MuVi/Sheffield.GBR.1.05/[G] during two epidemic waves and analysed them using new molecular markers based on genomic non-coding regions (NCRs) that discriminate subvariants of this virus strain. Results: Phylogenetic analyses of the nucleoprotein-phosphoprotein and matrix protein-fusion protein NCR indicated strain replacement after a drop in incidence in 2009, which had not been detectable by SH sequencing. Clustering of sequences from patients epidemiologically linked in the same outbreak suggests a potential use for these NCRs in outbreak characterisation. Conclusion: We suggest to consider their use in conjunction with the SH gene in the future WHO recommendations for MuV epidemiological surveillance. We would like to thank the Genomic Unit of the CNM-ISCIII for their technical assistance with sequencing. This work was supported by the “Fondo de Investigación Sanitaria” (FIS PI12/02006) and the “Instituto de Salud Carlos III” (PI15CIII/00023). Aurora Fernández García was funded by CIBER de Epidemiología y Salud Pública (CIBERESP), ISCIII. Sí

Previous studies have shown that EBLV-1 strains exclusively hosted by Eptesicus isabellinus bats in the Iberian Peninsula cluster in a specific monophyletic group that is related to the EBLV-1b lineage found in the rest of Europe. More recently, enhanced passive surveillance has allowed the detection of the first EBLV-1 strains associated to Eptesicus serotinus south of the Pyrenees. The aim of this study is the reconstruction of the EBLV-1 phylogeny and phylodynamics in the Iberian Peninsula in the context of the European continent. We have sequenced 23 EBLV-1 strains detected on nine E. serotinus and 14 E. isabellinus. Phylogenetic analyses were performed on the first 400-bp-5’ fragment of the Nucleoprotein (N) gene together with other 162 sequences from Europe. Besides, fragments of the variable region of the phosphoprotein (P) gene and the glycoprotein-polymerase (G-L) intergenic region were studied on Spanish samples. Phylogenies show that two of the new EBLV-1a strains from Iberian E. serotinus clustered together with French strains from the North of the Pyrenees, suggesting a recent expansion southwards of this subtype. The remaining seven Iberian strains from E. serotinus grouped, instead, within the cluster linked, so far, to E. isabellinus, indicating that spatial distribution prevails over species specificity in explaining rabies distribution and supporting interspecific transmission. The structure found within the Iberian Peninsula for EBLV-1b is in concordance with that described previously for E. isabellinus. Finally, we have found that the current EBLV-1 European strains could have emerged only 175 years ago according to our evolutionary dynamics analyses., Author summary Rabies is caused by at least fourteen different viruses of the genus Lyssavirus. Although the classical rabies virus transmitted by the dog accounts for most human cases, most lyssaviruses are hosted by bats, which are able to transmit the disease to humans. The European bat lyssaviruses 1 (EBLV-1) and 2 (EBLV-2) have caused human rabies in Europe, while more than 90% of the bat rabies cases have been reported from serotine bats (Eptesicus serotinus) infected by EBLV-1. These cases clearly accumulate in certain areas of Europe, but the factors driving this distribution are unknown. The evolutionary relationships among these viral strains provide valuable information, however, they are only partially known. The south of the Iberian Peninsula is the only location within Europe with an additional reservoir for EBLV-1, the isabelline serotine bat (Eptesicus isabellinus) which is present also in North Africa. In this study we have reconstructed the EBLV-1 phylogeny and phylodynamics in the Iberian Peninsula in the context of the European continent. Our results suggest that some lineages have longer evolutionary history in their distribution areas, than others which seem to be in the process of a geographical expansion.

The mumps vaccine (Jeryl-Lynn-strain) was introduced in Spain in 1981, and a vaccination policy which included a second dose was added in 1995. From 1992-1999, a Rubini-strain based vaccine was administered in many regions but later withdrawn due to lack of effectiveness. Despite high levels of vaccination coverage, epidemics have continued to appear. We characterized the three epidemic waves of mumps between 1998 and 2014, identifying major changes in susceptible populations using Poisson regression. For the period 1998-2003 (P1), the most affected group was from 1 to 4years old (y) [Incidence Rate (IR)=71.7 cases/100,000 population]; in the periods 2004-2009 (P2) and 2010-2014 (P3) IR ratio (IRR) increased among 15-24y (P2=1.46; P3=2.68) and 25-34y (P2=2.17; P3=4.05). Hospitalization rate (HR), complication rate (CR) and neurological complication rate (NR) among hospitalized subjects decreased across the epidemics, except for 25-34y which increased: HR ratio (HRR) (P2=2.18; P3=2.16), CRR (P3=2.48), NRR (P3=2.41). In Spain mumps incidence increased, while an overall decrease of hospitalizations and severe complications occurred across the epidemics. Cohorts born during periods of low vaccination coverage and those vaccinated with Rubini-strain were the most affected populations, leading to a shift in mumps cases from children to adolescents and young adults; this also reveals the waning immunity provided by the mumps vaccine. Despite not preventing all mumps cases, the vaccine appears to prevent serious forms of the disease. This study was funded in part by Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (PI12/02006) and by Acción Estratégica de Salud, Instituto de Salud Carlos III (PI15CIII/00023).

Research Letter; International audience; We detected Bartonella in 11 of 109 insectivorous bats from France and 1 of 26 bats from Spain. These genetic variants are closely related to bat-associated Bartonella described in Finland and the United Kingdom and to B. mayotimonensis, the agent of a human endocarditis case in the United States.

All lyssaviruses (family Rhabdoviridae) cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Using next-generation sequencing, the full-genome sequence for a novel lyssavirus, Lleida bat lyssavirus (LLEBV), from the original brain of a common bent-winged bat has been confirmed. This project received funding from Defra, the Scottish Government, and the Welsh Government through grants SE0427 and SE0431 and from the EU H2020-funded Research Infrastructure Grant “European Virus Archive Global (EVAg)” (H2O20—grant agreement no. 653316). Sí

Resumen Introduccion El sarampion es una infeccion casi erradicada que en los ultimos anos esta reemergiendo en Espana y en Europa. El objetivo del estudio fue describir las caracteristicas microbiologicas y clinico-epidemiologicas de un brote de sarampion ocurrido en la provincia de Guadalajara de junio a agosto de 2012. Metodos Estudio descriptivo y retrospectivo. Se analizaron 117 muestras (suero, orina y exudado faringeo) de 52 casos sospechosos de sarampion. Resultados Se confirmaron 50 casos de sarampion, 41 por laboratorio y 9 por vinculo epidemiologico, agrupados en 4 brotes comunitarios. No se observaron casos importados. La IgM y la PCR fueron positivas en 25 pacientes, solo PCR en 11 y solo IgM en 5. El genotipo fue D4 en 13/14 cepas y genotipo A en un caso posvacunal. Los grupos de edades mas afectados fueron adultos entre 20 y 34 anos (38%) y menores de 15 meses (26%). El 88% de pacientes no estaban vacunados (43% etnia gitana, 27% menores de 15 meses, 11% razones ideologicas), y el 6% habia recibido una dosis. La clinica fue exantema y fiebre (100%), tos (82%) y conjuntivitis (50%). El 32% requirio hospitalizacion y el 28% presento complicaciones. Conclusion Es de especial importancia intensificar la vigilancia epidemiologica en infecciones en fase de eliminacion. El aumento de la incidencia de sarampion estuvo asociado a bolsas de no vacunados que representan un desafio para la salud publica, que debera elaborar estrategias para conseguir una elevada cobertura vacunal y alcanzar la erradicacion del sarampion.

Producción Científica, 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.

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. Grant sponsor: Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness; Grant number: PI07/90154 Sí

A thorough search for bat herpesviruses was carried out in oropharyngeal samples taken from most of the bat species present in the Iberian Peninsula from the Vespertilionidae, Miniopteridae, Molossidae and Rhinolophidae families, in addition to a colony of captive fruit bats from the Pteropodidae family. By using two degenerate consensus PCR methods targeting two conserved genes, distinct and previously unrecognized bat-hosted herpesviruses were identified for the most of the tested species. All together a total of 42 potentially novel bat herpesviruses were partially characterized. Thirty-two of them were tentatively assigned to the Betaherpesvirinae subfamily while the remaining 10 were allocated into the Gammaherpesvirinae subfamily. Significant diversity was observed among the novel sequences when compared with type herpesvirus species of the ICTV-approved genera. The inferred phylogenetic relationships showed that most of the betaherpesviruses sequences fell into a well-supported unique monophyletic clade and support the recognition of a new betaherpesvirus genus. This clade is subdivided into three major clades, corresponding to the families of bats studied. This supports the hypothesis of a species-specific parallel evolution process between the potentially new betaherpesviruses and their bat hosts. Interestingly, two of the betaherpesviruses' sequences detected in rhinolophid bats clustered together apart from the rest, closely related to viruses that belong to the Roseolovirus genus. This suggests a putative third roseolo lineage. On the contrary, no phylogenetic structure was detected among several potentially novel bat-hosted gammaherpesviruses found in the study. Remarkably, all of the possible novel bat herpesviruses described in this study are linked to a unique bat species

The National Plan for the Elimination of Rubella was implemented in Spain in 2008 using the logistics of the National Plan for the Elimination of Measles that have been employed since year 2000. Molecular characterization of rubella virus (RUBV) is important for disease surveillance and for monitoring elimination of the disease throughout the world. We describe the first complete series of data regarding the circulation of RUBV genotypes in Spain. The 739-nucleotide fragment designated by the WHO for RUBV genotyping was sequenced in 88 selected cases collected from 1998 to 2014. Five genotypes were identified: 1E, 2B, 1J, 1I, and 1a. Genotype 1E was predominant between 1998 and 2003 but was replaced by genotype 2B, which was detected in sporadic cases in 2004, 2006, 2008, 2012, 2013 and 2014. There was an outbreak of genotype 2B in Algeciras (Andalusia) in 2008. Genotype 1J caused an outbreak in Madrid in 2004/2005 and sporadic cases in 2005 and 2007. Genotype 1I was found to have infected an immune-suppressed patient with neurological symptoms in 2008. Finally, vaccine strain RA 27/3 was detected in three sporadic cases, two of them immune-suppressed and without a recent history of vaccination. This suggests that during these years there were a series of imported sporadic cases and outbreaks, confirming the findings of epidemiological data analysis. The importation sources were generally consistent with our geographic and cultural ties, mainly with Europe (genotypes 1E, 2B, 1I) and Latin America (1J). This work has received financial support from the fellowship for Ph.D. Study by Republic of Panama and Acuerdo de Encomienda de Gestión entre la Dirección General de Salud Pública del Ministerio de Sanidad y Consumo y el Instituto de Salud Carlos III. N° expediente: DGVI-1429/05-3, and Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (IP12/02006). Also, has been developed under the Project FIS PI12/02006 funded by the Strategic Action on Health of the Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Rabies is the oldest known zoonotic disease and was also the first recognized bat associated infection in humans. To date, four different lyssavirus species are the causative agents of rabies in European bats: the European Bat Lyssaviruses type 1 and 2 (EBLV-1, EBLV-2), the recently discovered putative new lyssavirus species Bokeloh Bat Lyssavirus (BBLV) and the West Caucasian Bat Virus (WCBV). Unlike in the new world, bat rabies cases in Europe are comparatively less frequent, possibly as a result of varying intensity of surveillance. Thus, the objective was to provide an assessment of the bat rabies surveillance data in Europe, taking both reported data to the WHO Rabies Bulletin Europe and published results into account. In Europe, 959 bat rabies cases were reported to the RBE in the time period 1977-2010 with the vast majority characterized as EBLV-1, frequently isolated in the Netherlands, North Germany, Denmark, Poland and also in parts of France and Spain. Most EBLV-2 isolates originated from the United Kingdom (UK) and the Netherlands, and EBLV-2 was also detected in Germany, Finland and Switzerland. Thus far, only one isolate of BBLV was found in Germany. Published passive bat rabies surveillance comprised testing of 28 of the 52 different European bat species for rabies. EBLV-1 was isolated exclusively from Serotine bats (Eptesicus serotinus and Eptesicus isabellinus), while EBLV-2 was detected in 14 Daubenton's bats (Myotis daubentonii) and 5 Pond bats (Myotis dasycneme). A virus from a single Natterer's bat (Myotis nattereri) was characterized as BBLV. During active surveillance, only oral swabs from 2 Daubenton's bats (EBLV-2) and from several Eptesicus bats (EBLV-1) yielded virus positive RNA. Virus neutralizing antibodies against lyssaviruses were detected in various European bat species from different countries, and its value and implications are discussed.

Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2). Between 1977 and 2011, 961 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV-1. There have been 25 suspected cases of EBLV-2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in south-west Russia in 2002 (West Caucasian bat virus) and in Germany in 2010 (Bokeloh bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV-1 and EBLV-2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV-2 cases for which viral sequences (N gene, 400 nucleotides) are available (n = 21). Oropharyngeal swabs collected from two healthy Myotis daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV-2). Despite the relatively low number of EBLV-2 cases, a surprisingly large amount of anomalous data has been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV-1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV-1 isolates from Spain and the EBLV-2 isolates to provide support for the existence of sublineages.

This article describes the development and validation with clinical samples of an internally controlled multiplex quantitative real-time PCR (QRT-PCR) for human polyomaviruses BK (BKV) and JC (JCV). Blood and urine samples from renal transplant recipients with suspected nephropathy, and cerebrospinal fluid (CSF) specimens from AIDS, natalizumab-treated and HIV-negative patients with suspected progressive multifocal leukoencephalopathy, previously checked for BKV and JCV by conventional PCR, were tested by QRT-PCR. All samples positive by conventional PCR were confirmed by QRT-PCR. Four cases of JCV-associated neurological infection, including all those detected in natalizumab-treated patients, and one case of BKV-related neurological infection were only identified by QRT-PCR. BKV was quantified in the CSF of neurological patients for the first time. Analyses of the Quality Control for Molecular Diagnostics 2010 panel were "highly satisfactory" for BKV and "satisfactory" for JCV. The QRT-PCR is specific and reproducible. It improves the sensitivity of conventional PCR for the diagnosis of BKV and JCV infection in various diseases.

The Laboratory is a fundamental component on the surveillance of measles and rubella. Cases need to be properly confirmed to ensure an accurate estimation of the incidence. Strains should be genetically characterized to know the transmission pattern of these viruses and frequently, outbreaks and transmission chains can be totally discriminated only after that. Finally, the susceptibility of the population is estimated on the basis of sero-prevalence surveys. Detection of specific IgM response is the base of the laboratory diagnosis of these diseases. It should be completed with genomic detection by RT-PCR to reach an optimal efficiency, especially when sampling is performed early in the course of the disease. Genotyping is performed by genomic sequencing according to reference protocols of the WHO. Laboratory surveillance of measles and rubella in Spain is organized as a net of regional laboratories with different capabilities. The National Center of Microbiology as National Reference Laboratory (NRL), supports regional laboratories ensuring the availability of all required techniques in the whole country and watching for the quality of the results. The NRL is currently working in the implementation of new molecular techniques based on the analysis of genomic hypervariable regions for the strain characterization at sub-genotypic levels and use them in the surveillance.

Background IgM detection is considered as the gold standard for mumps diagnosis. Currently, most cases in developed countries occur in highly vaccinated populations due to secondary vaccine failure. In these patients, pre-existing vaccine-induced antibodies are not able to neutralise the virus, but prevent the typical primary response, so that specific IgM is not always elicited. Consequently, acute infection has to be demonstrated by direct detection of the virus by viral isolation or genomic amplification. RT-PCR allows a diagnosis with the maximum sensitivity to be made and also forms the basis for genotype characterisation by sequencing the SH gene, according to WHO recommendations. However, none of the RT-PCR techniques properly evaluated for the diagnosis of acute mumps infection yields an amplification fragment useful for genotyping, and none of the amplification techniques described for genotyping has proved to be sensitive enough for diagnosis. Objectives Development of a RT-PCR for the mumps virus diagnosis and genotyping, properly evaluated in comparison with serological gold-standard technique. Study design 195 suspected mumps cases and six wild type MuV genotypes were studied. Results Our method was able to detect 0.001 TCID 50 of mumps virus. Fifty-eight of these showed positive results, of which 54 (93.3%) showed mumps RNA in saliva, while only 20 (34.5%) had mumps IgM in serum. Genotypes G1, G2, H1, H2, D1 and C were identified in positive samples. Conclusions The technique described could be a very useful tool for mumps surveillance, management and control.

Bat coronaviruses (CoV) are putative precursors of the severe acute respiratory syndrome (SARS) CoV and other CoV that crossed the species barrier from zoonotic reservoirs into the human population. To determine the presence and distribution of CoV in Iberian bats, 576 individuals of 26 different bat species were captured in 13 locations in Spain. We report for the first time the presence of 14 coronaviruses in 9 Iberian bat species. Phylogenetic analysis of a conserved CoV genome region (RdRp gene) shows a wide diversity and distribution of alpha and betacoronavirus in Spain. Interestingly, although some of these viruses are related to other European BatCoV, or to Asian CoV, some of the viruses found in Spain cluster in new groups of α and β CoV.

The infections of the central nervous system are associated with high morbidity and mortality. Several agents including bacteria, viruses, fungi and protozoa can invade the CNS. They are different clinical presentations of these infections: meningitis, encephalitis, brain and epidural abscesses and cerebrospinal fluid shunt infections. The clinical course could be acute, subacute or chronic depending on the infecting agent and the location of the infection. The travelling entails a risk of infection by exotic agents of meningo-encephalitis such as robovirus and arbovirus, which require new diagnostic and therapeutic methods. Despite some progress in the treatment of the CNS infections, the mortality is usually high. Rapid diagnosis and emergent interventions are necessary to improve the outcome of those patients, and early and targeted antimicrobial treatment and support measures are of paramount importance for a favourable clinical patietn outcome. The antigen detection techniques and particularly those of genetic diagnosis by amplification (PCR and others) have advanced, and improved the diagnostic of those diseases. In this paper the clinical signs and symptoms and diagnostic procedures of CNS infections are presented.

Mumps virus (MuV) was detected in the cerebrospinal fluid (CSF) of 6 of 158 patients with meningitis or encephalitis in absence of clinical mumps in the context of mumps epidemics. Our results suggest the need for the study of MuV RNA in the CSF of neurological patients in this context.