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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.

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.

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.

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

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

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.

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.

Although the WHO recommends the use of genotyping as a tool for epidemiological surveillance for mumps, limited data on mumps virus (MV) genotype circulation that may be used to trace the patterns of virus spread are available. We describe the first complete series of data from Spain. The small hydrophobic region was sequenced from 237 MV-positive samples from several regions of Spain collected between 1996 and 2007. Six different genotypes were identified: A, C, D (D1), G (G1, G2), H (H1, H2), and J. Genotype H1 was predominant during the epidemic that occurred from 1999 to 2003 but was replaced by genotype G1 as the dominant genotype in the epidemic that occurred from 2005 to 2007. The same genotype G1 strain caused concomitant outbreaks in different parts of the world (the United States, Canada, and the United Kingdom). The remaining genotypes (genotypes A, C, D, and J) appeared in sporadic cases or small limited outbreaks. This pattern of circulation seems to reflect continuous viral circulation at the national level, despite the high rates of vaccine coverage.

To determine the presence of European bat lyssavirus type 1 in southern Spain, we studied 19 colonies of serotine bats (Eptesicus isabellinus), its main reservoir, during 1998–2003. Viral genome and antibodies were detected in healthy bats, which suggests subclinical infection. The different temporal patterns of circulation found in each colony indicate independent endemic circulation.

All members of the lyssavirus genus cause the disease rabies. European bat lyssavirus 1 (EBLV-1) viruses are divided genetically into three groups according to geographic location and host reservoir. We report here the first genome sequence for an EBLV-1 isolated from Eptesiscus isabellinus in the Iberian Peninsula, Spain. This work was supported by grants from the Department for Environment, Food and Rural Affairs (SE0423 and SE0427) and by the European Union FP7-funded Research Infrastructure Grant European Virus Archive (19 228292). Sí

The aim of the study was to evaluate the usefulness of serological detection of mumps IgM and titration of IgG in patients with acute parotitis according to their vaccination status. The detection of mumps virus RNA in saliva by RT-PCR was used as reference. 116 patients (109 of them previously vaccinated) with mumps RT-PCR-negative results and 21 (19 vaccinated) with mumps RT-PCR-positive results were studied. Mumps-specific IgM and IgG were assayed by EIA (Enzygnost, Dade Behring, Germany). IgM results were expressed as positive or negative. For IgG, several cut-offs were calculated using receiver operating characteristic (ROC) curves. Seven RT-PCR-positive and five RT-PCR-negative patients showed IgM-positive results (sensitivity 33.3% and specificity 95.7%). Among vaccinated patients, the sensitivity and specificity of IgM were 26.3% (5/19) and 99.1% (108/109). For IgG, a titer of 5,000 in all the patients showed a sensitivity of 76.2% (16/21) and a specificity of 83.6% (97/116). In vaccinated patients, the corresponding figures for this cut-off were 84.2% (16/19) and 83.5% (91/109), respectively. Although IgM detection against mumps is highly specific, its sensitivity is very low in immunized subjects. In this group, the titration of IgG could serve as an additional diagnostic tool.

Clinical research suggests a role for viral load measurement in predicting and monitoring Epstein-Barr virus (EBV)-associated diseases. The aim of this study was to assess the performance of the recently commercially available quantitative assays for EBV based on real-time PCR: the RealArt EBV LC PCR kit and the LightCycler EBV quantification kit. A total of 87 samples were analyzed: 67 samples were obtained from transplant recipients and patients with EBV-associated diseases, 8 samples were obtained from the Quality Control for Molecular Diagnostics 2002 EBV Proficiency Program, and 12 negative qualitative nested PCR samples were used as negative controls. Inter- and intra-assay variabilities were determined by running replicates of two samples. All samples were run in a LightCycler instrument. The differences between positive and negative results were not considered statistically significant ( P = 0.5355). There were no false-positive results using either method for nested PCR negative-control samples. The difference in viral load values using the two different methods was considered statistically significant ( P < 0.01). The logarithmic linear correlation for both assays was low ( r = 0.449) but significant ( P < 0.01). The LightCycler EBV quantification kit showed a wider dispersal in results but produced substantially more-accurate melting temperature profile curves. The bias towards lower measurements was considerable in comparison with higher viral load. The differences in PCR efficiency and the presence of mutations could explain the disparity between the two methods. It was concluded that confidence intervals would be required to report the results rather than plain absolute values of viral load for patient monitoring.

A sensitive nested reverse transcription-PCR assay, targeting a short fragment of the gene encoding the small hydrophobic protein (SH gene), was developed to allow rapid characterization of mumps virus in clinical samples. The sensitivity and specificity of the assay were established using representative genotypes A, B, C, D, E, and F. Mumps virus RNA was characterized directly from cerebrospinal fluid (CSF) samples and in extracts of mumps virus isolates from patients with various clinical syndromes. Direct sequencing of products and subsequent phylogenetic analysis enabled genetic classification. A simple web-based system of sequence analysis was established. The study also allowed characterization of mumps virus strains from Argentina as part of a new subgenotype. This PCR assay for characterization of mumps infections coupled to a web-based analytical program provides a rapid method for identification of known and novel strains. Fil: Palacios, Gustavo. Columbia University. Jerome L. and Dawn Greene Infectious Disease Laboratory; Estados Unidos. Fil: Jabado, O. Columbia University. Jerome L. and Dawn Greene Infectious Disease Laboratory; Estados Unidos. Fil: Cisterna, Daniel. Instituto de Salud Carlos III. Services of Diagnostic Microbiology and Virology; España. Fil: de Ory, F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Virología. División Neurovirosis; Argentina. Fil: Renwick, N. Columbia University. Jerome L. and Dawn Greene Infectious Disease Laboratory; Estados Unidos. Fil: Echevarria, J. E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Virología. División Neurovirosis; Argentina. Fil: Castellanos, A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Virología. División Neurovirosis; Argentina. Fil: Mosquera, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Virología. División Neurovirosis; Argentina. Fil: Freire, María Cecilia. tituto de Salud Carlos III. Services of Diagnostic Microbiology and Virology; España. Fil: Campos, Rodolfo. Universidad de Buenos Aires. Cátedra de Virología; Argentina. Fil: Lipkin, W. Ian. Columbia University. Jerome L. and Dawn Greene Infectious Disease Laboratory; Estados Unidos.

In order to investigate the etiology of viral neurological infections in Spain, a national study was performed in 2008. The results obtained have been published. Enteroviruses were the most frequent cause of the aseptic meningitis and infant febrile syndromes. The present report supplements the previous study with the genotyping of the detected enteroviruses. Typing was by amplification of partial VP1 region and sequencing in 70 (53%) of the 132 available cerebrospinal fluid samples positive for enteroviruses. Twelve different genotypes within the B species were identified. Echovirus 4 was predominant (24%), followed by echovirus 30 (19%), echovirus 9 (17%), and echovirus 6 (14%). In summary, a co-circulation of several enterovirus types associated with meningitis in children under 15 years old was observed. Although infrequently detected, echovirus 4 was the predominant genotype identified due to an aseptic meningitis outbreak which occurred in the Canary Islands in 2008. J Med. Virol. 85:1975–1977, 2013. © 2013 Wiley Periodicals, Inc.

A new tentative lyssavirus, Lleida bat lyssavirus, was found in a bent-winged bat (Miniopterus schreibersii) in Spain. It does not belong to phylogroups I or II, and it seems to be more closely related to the West Causasian bat virus, and especially to the Ikoma lyssavirus.

Molecular characterization of measles virus is important for disease surveillance and for monitoring elimination of the virus throughout the world. Furthermore, knowledge of genotype distribution in as many countries as possible, is useful for tracing the origin of a strain, especially in countries without endemic measles disease, where most cases are imported. Data on genotypes circulating in Spain from 1970 to 1997 showed the prevalence of genotypes C1, C2, and D6, with subsequent replacement of each other. After the establishment of the Spanish Measles Elimination Plan, genotyping with a new retrotranscriptase polymerase chain reaction (RT-PCR) was undertaken directly on 92 specimens, corresponding to 90 patients, which were positive for measles by a different diagnostic RT-PCR. Genotypes B3, D4, D8, A, C2, H1, and D7 were found in different autonomous communities (Madrid, Balearic Islands, Valencia Community, Extremadura, Andalusia, Canary Islands and Murcia) between 2001 and 2003 with none of these genotypes being prevalent. After the introduction of the vaccine in 1978, the incidence of the disease decreased from 150,000 cases in 1977 to 64 in 2002. This could be the reason for the change observed in the pattern of measles genotype circulation, since this pattern was reported in countries at an advanced stage of eradication of measles. This report considers that Spain is on the way to eradicating measles.

Background: Compelling arguments exist for a role of infectious agent in giant cell arteritis (GCA). Parvovirus B19 and several herpesviruses have focussed the attention in recent years, but the few studies to date have yielded inconsistent results. Objectives: To study the relationship between the presence of parvovirus B19 DNA or major known herpesviruses and the histopathological features of GCA. Study design: Between January 1997 and March 2002, 147 consecutive temporal artery biopsies were performed in our center because of a clinical suspicion of GCA. Using polymerase chain reaction (PCR) procedures validated by the World Health Organization and employed routinely by our laboratory, we examined the paraffin-embedded specimens for DNA from parvovirus B19, herpes simplex viruses (HSV) 1 and 2, Epstein–Barr virus (EBV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and human herpesvirus 6 (HHV-6). We investigated positive results further with immunohistochemistry studies. Results: Fifty of the 147 temporal artery biopsies (34%) showed histological features of GCA. Three biopsies (2.5%) were initially PCR positive for parvovirus B19. None of the herpesvirus PCR assays were positive. Upon repeat testing by both PCR and immunohistochemistry, none of the three initially positive parvovirus B19 assays were confirmed. The results of both positive and negative control assays in these studies validated these findings. We confirmed the presence of amplifiable DNA in the temporal artery biopsy specimens using PCR primers for beta-globin and indoleamine 2,3-dioxygenase (IDO). Conclusions: The results of our study do not support a role in the etiopathogenesis of GCA for either parvovirus B19 or any of these six herpesviruses.

We describe here a multiplex reverse transcription-PCR (RTMNPCR) assay designed to detect and differentiate measles virus, rubella virus, and parvovirus B19. Serial dilution experiments with vaccine strains that compared cell culture isolation of measles in B95 cells and rubella in RK13 cells showed sensitivity rates of 0.004 50% tissue culture infective dose (TCID 50 ) for measles virus and 0.04 TCID 50 for rubella virus. This RTMNPCR can detect as few as 10 molecules for measles virus and rubella virus and one molecule for parvovirus B19 in dilution experiments with plasmids containing inserts of the primary reaction amplification products. Five pharyngeal exudates from measles patients and 2 of 15 cerebrospinal fluid samples from measles-related encephalitis were found to be positive for measles virus by this RTMNPCR. A total of 3 of 27 pharyngeal exudates from vaccinated children and 2 pharyngeal exudates, plus one urine sample from a case of congenital rubella syndrome, were found to be positive for rubella virus by RTMNPCR, whereas 16 of 19 sera from patients with erythema infectiosum were determined to be positive for parvovirus B19 by RTMNPCR. In view of these results, we can assess that this method is a useful tool in the diagnosis of these three viruses and could be used as an effective surveillance tool in measles eradication programs.

The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lübeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V-IgM and -IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V-specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non-infected, and 29 from other viral recent infections (Epstein-Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well-established Biotrin EIAs. Sí

Brain analysis cannot be used for the investigation of active lyssavirus infection in healthy bats because most bat species are protected by conservation directives. Consequently, serology remains the only tool for performing virological studies on natural bat populations; however, the presence of antibodies merely reflects past exposure to the virus and is not a valid marker of active infection. This work describes a new nested reverse transcription (RT)-PCR technique specifically designed for the detection of the European bat virus 1 on oropharyngeal swabs obtained from bats but also able to amplify RNA from the remaining rabies-related lyssaviruses in brain samples. The technique was successfully used for surveillance of a serotine bat (Eptesicus serotinus) colony involved in a case of human exposure, in which 15 out of 71 oropharyngeal swabs were positive. Lyssavirus infection was detected on 13 oropharyngeal swabs but in only 5 brains out of the 34 animals from which simultaneous brain and oropharyngeal samples had been taken. The lyssavirus involved could be rapidly identified by automatic sequencing of the RT-PCR products obtained from 14 brains and three bat oropharyngeal swabs. In conclusion, RT-PCR using oropharyngeal swabs will permit screening of wild bat populations for active lyssavirus infection, for research or epidemiological purposes, in line not only with conservation policies but also in a more efficient manner than classical detection techniques used on the brain

Reverse transcription (RT)-PCR assays have been widely described for use in the diagnosis of human parainfluenza viruses (HPIVs) and other respiratory virus pathogens. However, these assays are mostly monospecific, requiring separate amplifications for each HPIV type. In the present work, we describe multiplex RT-PCR assays that detect and differentiate HPIV serotypes 1, 2, and 3 in a combined reaction. Specifically, a mixture of three pairs of primers to conserved regions of the hemagglutinin-neuraminidase gene of each HPIV serotype was used for primary amplification, yielding amplicons with similar sizes. For typing, a second amplification was performed with a mixture of nested primers, yielding amplicons with sizes easily differentiated by agarose gel electrophoresis. A modified single-amplification RT-PCR assay with fluorescence-labeled nested primers, followed by analysis of the labeled products on an automated sequencing gel, was also evaluated. Fifteen temporally and geographically diverse HPIV isolates from the Centers for Disease Control and Prevention archives and 26 of 30 (87%) previously positive nasopharyngeal specimens (8 of 10 positive for HPIV serotype 1 [HPIV1], 9 of 10 positive for HPIV2, and 9 of 10 positive for HPIV3) were positive and were correctly typed by both assays. Negative results were obtained with naso- or oropharyngeal specimens and/or culture isolates of 33 unrelated respiratory tract pathogens, including HPIV4, enterovirus, rhinovirus, respiratory syncytial virus, adenovirus, influenza virus, and Streptococcus pneumoniae . Our multiplex RT-PCR assays provide sensitive, specific, and simplified tools for the rapid diagnosis of HPIV infections.

Rhabdoviruses infect a variety of hosts, including mammals, birds, reptiles, fish, insects and plants. As bats are the natural host for most members of the genus Lyssavirus, the specificity of the amplification methods used for active surveillance is usually restricted to lyssaviruses. However, the presence of other rhabdoviruses in bats has also been reported. In order to broaden the scope of such methods, a new RT-PCR, able to detect a diverse range of rhabdoviruses, was designed. The method detected 81 of 86 different rhabdoviruses. In total, 1488 oropharyngeal bat swabs and 38 nycteribiid samples were analysed, and 17 unique rhabdovirus-related sequences were detected. Phylogenetic analysis suggested that those sequences detected in bats did not constitute a monophyletic group, even when originating from the same bat species. However, all of the sequences detected in nycteribiids and one sequence obtained from a bat did constitute a monophyletic group with Drosophila melanogaster sigma rhabdovirus. © 2013 Crown.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5′ noncoding region of the enterovirus genome were designed. The RT-PCR product size was-500 bp (479 bp for Poliovirus, 500 bp for Cox-sackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR wer eaccomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis. © Wiley-Liss, Inc.

Data concerning the HBsAg subtype distribution in Spain are out-of-date and confined to a restricted geographical area. Furthermore, the complex distribution observed in the countries surrounding Spain prevents any prediction. To obtain further data on HBsAg subtype distribution among Spanish HBsAg carriers, subtyping analysis (d and y determinants) was performed in 670 samples from subjects belonging to various epidemiological risk groups and coming from different geographical areas of the country. Similar frequencies were found for both mutually exclusive d/y subtype determinants among non-risk, normal HBsAg carriers from almost all geographical areas studied. In contrast, the ay subtype was clearly predominant (79-87%) among intravenous drug users, irrespective of their geographical origin. Thirteen different institutions for mentally retarded patients behaved as closed communities for HBV circulation, showing independent subtype distributions. Thus, no significant geographical variations were found for HBsAg subtype distribution in Spain. The prevalence of each particular subtype is mainly dependent on the epidemiological characteristics of the carriers studied. Subtype distribution was independent of the presence of HBeAg or HDV infection serum markers when homogeneous groups were considered separately. Atypical HBsAg phenotypes, either with coexistence or absence of both subtype determinants, were found in some cases.

BK polyomavirus (BKPyV) has recently been postulated as an emerging opportunistic pathogen of the human central nervous system (CNS), but it is not known whether specific strains are associated with the neurotropic character of BKPyV. The presence of BKPyV large T-antigen DNA was examined in 2406 cerebrospinal fluid (CSF) samples from neurological patients with suspected JC polyomavirus infection. Twenty patients had a large T-antigen DNA-positive specimen. The non-coding control region (NCCR) of the BKPyV strains amplified from CSF from these 20 patients, strains circulating in renal and bone marrow transplant recipients and from healthy pregnant women was sequenced. The archetypal conformation was the most prevalent in all groups and 14 of the neurological patients harboured archetypal strains, while the remaining six patients possessed BKPyV with rearranged NCCR similar to previously reported variants from non-neurological patients. Transfection studies in Vero cells revealed that five of six early and four of six late rearranged promoters of these CSF isolates showed significantly higher activity than the corresponding archetypal promoter. From seven of the neurological patients with BKPyV DNA-positive CSF, paired serum samples were available. Five of them were negative for BKPyV DNA, while serum from the remaining two patients harboured BKPyV strains with archetypal NCCR that differed from those present in their CSF. Our results suggest that NCCR rearrangements are not a hallmark for BKPyV neurotropism and the dissemination of a rearranged NCCR from the blood may not be the origin of BKPyV CNS infection.

HBsAg subtypes were defined by means of adsorbed polyclonal antisera; however, HBsAg subtyping is currently usually carried out with monoclonal antibodies (Mab). We developed a complementary subtyping method based on the polymerase chain reaction. Reference samples belonging to all known HBsAg subtypes could be detected and grouped into four different categories (ayw1/ayw4/ayr, ayw2/ayw3, adw2/adrq+/adrq-, adw4). Thirteen HBsAg-positive serum samples previously subtyped as ad by means of monoclonal antibodies fell into the adw2/adrq+/adrq- group, as well as 13 ay samples into the ayw2/ayw3 group. These results could be confirmed by means of reference polyclonal antisera in nine ad cases (all adw2) and in seven ay cases (all ayw3); the remaining seven were below the detection limit of the polyclonal assay. Four samples which were not recognized by any of the d/y subtype-specific Mab were shown to contain ayw2/ayw3 sequences. Only one contained sufficient HBsAg to be confirmed as ayw3 by means of reference antisera. Three of five sera showing simultaneous reactivity both for d and y-specific Mab were classified as adw4 by PCR, as was one by reference polyclonal antisera. The y-specific monoclonal antibody cross-reacted with the adw4 subtype. Single adw2 sequences were amplified in one of the remaining two cases, as well as single ayw2/ayw3 sequences in the other, suggesting that they showed true coexistence of two strains of different subtype, only one of which was in active replication state. It is concluded that the method described is useful in the solution of some undefined cases obtained with the monoclonal-based assays.

While BK virus (BKV) is frequently associated with pathological conditions in bone marrow and renal transplant recipients, BKV infection in neurological individuals has been rarely reported. As a result of a BKV, JCV, and SV40 large T antigen-specific multiplex PCR on 2,062 cerebrospinal fluid (CSF) samples from neurological patients suspicious of JCV infection, we identified 20 subjects with at least 1 CSF specimen positive for BKV large T antigen DNA. Because VP1 protein has been suggested to influence the biological/pathological properties of BKV, we tried to sequence the entire VP1 gene in the BKV-positive neurological patients and succeeded in 14 of the 20 neurological patients. To compare the VP1 sequence of the BKV neurological strains with that of non-neurotropic strains in other clinical situations, full-length VP1 DNA was sequenced in 15 renal and 6 bone marrow transplant recipients positive to BKV-viremia, and in 8 pregnant women as non-pathological controls. An increased (respectively, decreased) tendency for mutations in the BC loop (respectively, EF loop) was observed, and no mutations were detected in the CD, GH, and HI loops. Subtype I was predominant (93%) and compared to archetypal BKV (WW), amino acid substitutions were detected in 4/14 neurological patients, 10/15 renal transplant recipients, 3/6 bone marrow transplant patients, and in all the pregnant women. Each patient group had distinctive VP1 mutations, but these unique substitutions were not present in all patients of this group. However, molecular modeling simulations of the VP1 mutants predicted changes in protein surface properties which might affect the VP1–receptor interaction. J. Cell. Physiol. 227: 136–145, 2012. © 2011 Wiley Periodicals, Inc.

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To better understand the epidemiology of European bat lyssavirus 1 (EBLV-1) in Europe, we phylogenetically characterized Lyssavirus from Eptesicus isabellinus bats in Spain. An independent cluster of EBLV-1 possibly resulted from geographic isolation and association with a different reservoir from other European strains. EBLV-1 phylogeny is complex and probably associated with host evolutionary history.

Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain., Author Summary A novel filovirus, provisionally named Lloviu virus (LLOV), was detected during the investigation of Miniopterus schreibersii die-offs in Cueva del Lloviu in southern Europe. LLOV is genetically distinct from other marburgviruses and ebolaviruses and is the first filovirus detected in Europe that was not imported from an endemic area in Africa. Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification of genetically distinct filovirus in dead insectivorous bats in caves in Spain.

A new approach to simultaneous detection and typing of related agents by the multiplex polymerase chain reaction (PCR) is described. The reaction was been applied to human herpesviruses by nested amplification of fragments of the DNA polymerase genes. During the first amplification, primers were used as two equimolar mixtures of non-degenerate oligonucleotides, aligning the 3'-ends with selected consensus regions, and their 5'-ends with the non-related sequences of each herpesvirus to be amplified. The specific fragments obtained were the substrate for a second, multiplex reaction for which primers were designed to produce different-size fragments for each related virus. The results showed high specificity for the detection and typing of the human herpesviruses with known sequences and no amplification of human DNA, in spite of the presence of the same consensus regions within human DNA polymerase alpha. It is concluded that this new approach would be useful for the differential diagnosis of herpesviruses, as well as for other groups of agents with conserved regions in their genomes and causing similar syndromes.

Understanding the role of humans in the dispersal of predominately animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geo-political boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens., Author Summary At least 15 million doses of anti-rabies post-exposure prophylaxis are administered annually worldwide, and an estimated 55,000 people die of rabies every year. Over 99% of these deaths occur in developing countries, predominantly in Asia and in Africa where rabies is endemic in domestic dogs. Despite the global health burden due to rabies, little is known about the patterns of the spread of dog rabies in these endemic regions. We used recently developed Bayesian analytical methods to unravel the dynamics and determinants of the spatial diffusion of dog rabies viruses in North Africa based on viral genetic data. Our analysis reveals a combination of restricted spread across administrative borders, the occasional long-distance movement of rabies viruses, and a strong fit between spatial spread of the virus and road distances between localities. Together, these data indicate that by transporting dogs, humans have played a key role in the dispersal of a major animal pathogen. Our studies therefore provide essential new information on the transmission dynamics of rabies in Africa, and in doing so will greatly assist in future intervention strategies.

The aim of the study was to compare RNA amplification using multiplex RT-PCR and IgM detection by means of indirect and capture ELISAs for the diagnosis of measles and rubella. A total of 229 cases of maculopapular rash with serum and throat swab samples were included. Specific serological IgM to measles and rubella was determined by Enzygnost (Siemens) and Platelia (Bio-Rad). Both viruses were researched using multiplex RT-PCR performed on throat samples. Criteria for inclusion of measles or rubella cases were a positive RT-PCR result for one virus and negative for the other; and/or a positive IgM result for one virus by both ELISAs and negative RT-PCR for the other virus. A total of 74 cases were classified as measles and 54 as rubella. In measles, sensitivity and specificity were 93.2% and 100% for RT-PCR, 97.3% and 98.1% for Enzygnost, and 90.5% and 95.5% for Platelia. For rubella, these values were 42.6% and 100% for RT-PCR, 100% and 97.1% for Enzygnost, and 94.4% and 98.3% for Platelia. Enzygnost and Platelia are useful techniques for detecting IgM against measles and rubella. RNA amplification by RT-PCR was both sensitive and specific for the diagnosis of measles; however, for rubella, the sensitivity of this technique must be improved.

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. (C) 2009 Published by Elsevier B.V.

An outbreak of rubella affected 460 individuals in 2004 and 2005 in the community of Madrid, Spain. Most of the patients were nonvaccinated Latin American immigrants or Spanish males. This study presents the first data on rubella virus genotypes in Spain. Forty selected clinical samples (2 urine, 5 serum, 3 blood, 2 saliva, and 28 pharyngeal exudate samples) from 40 cases were collected. The 739-nucleotide sequence recommended by the World Health Organization obtained from viral RNA in these samples was analyzed by using the MEGA v4.0 software. Seventeen isolates were obtained from 40 clinical samples from the outbreak, including two isolated from congenital rubella syndrome cases. Only viral RNA of genotype 1j was detected in both isolates and clinical specimens. Two variations in amino acids, G253C and T394S, which are involved in neutralization epitopes arose during the outbreak, but apparently there was no positive selection of either of them. The origin of the outbreak remains unknown because of poor virologic surveillance in Latin America and the African countries neighboring Spain. On the other hand, this is the first report of this genotype in Europe. The few published sequences of genotype 1j indicate that it comes from Japan and the Philippines, but there are no epidemiological data supporting this as the origin of the Madrid outbreak.