Increase of Diversity of Mumps Virus Genotype G SH Variants Circulating Among a Highly Immunized Population: Spain, 2007-2019.

MuV caused three epidemic waves in Spain since genotype G emerged in 2005, despite high vaccination coverage. SH gene sequencing according to WHO protocols allowed the identification of seven relevant variants and 88 haplotypes. While the originally imported MuVi/Sheffield.GBR/1.05/-variant prevailed during the first two waves, it was subsequently replaced by other variants originated by either local evolution or importation, according to the additional analysis of hypervariable NCRs. The time of emergence of the MRCA of each MuV variant clade was concordant with the data of the earliest sequence. The analysis of Shannon entropy showed an accumulation of variability on six particular positions as the cause of the increase on the number of circulating SH variants. Consequently, SH gene sequencing needs to be complemented with other more variable markers for mumps surveillance immediately after the emergence of a new genotype, but the subsequent emergence of new SH variants turns it unnecessary.

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

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

Molecular characterization of enteroviruses associated with neurological infections in Spain, 2008.

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.

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

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

Mumps virus diagnosis and genotyping using a novel single RT-PCR.

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.

Circulation of mumps virus genotypes in Spain from 1996 to 2007.

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.

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

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

Phylogenetic analysis of rubella virus strains from an outbreak in Madrid, Spain, from 2004 to 2005.

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.

Asymptomatic rhabdovirus infection in meridional serotine bats (Eptesicus isabellinus) from Spain.

Different rhabdoviruses have been found in healthy bats, suggesting asymptomatic infection. The aim of this study was to focus on the epidemiology and pathogenesis of EBLV1 infection in the meridional serotine bat (Eptesicus isabellinus), as well as to search for other rhabdoviruses in this bat, which is the responsible for more than 95% of cases of human exposure to lyssaviruses in Europe. RT-PCR on oropharyngeal swabs was used together with antibody detection by the Rapid Fluorescent Focus Inhibition Test (RFFIT) to investigate EBLV1 circulation in 19 natural colonies of meridional serotine bats in Andalusia (Spain) from 1998 to 2003. The survey was based on 1,227 different captures of 1,033 individuals that were ring banded, sampled and released. Individuals that were repeatedly captured were always found in the same colony, despite the fact that some colonies were less than five km apart. Viral circulation was detected in ten colonies either by RT-PCR, serology or both. Each colony showed a different temporal pattern of viral circulation suggesting independent endemic circulation. Some positive individuals were captured healthy in following campaigns providing evidence for survival after viral infection. RNA from two apparently new Dimarhabdoviruses was also found in the pharyngeal cavity of two healthy bats.

Adenovirus transmission in a nursing home: analysis of an epidemic outbreak of keratoconjunctivitis.

BACKGROUND: An epidemic outbreak of keratoconjunctivitis occurred in a nursing home in Madrid from August to December 2005. OBJECTIVE: This article reports the outbreak, the infection control measures taken, and risk factors for keratoconjunctivitis. METHODS: A cohort study was conducted on the nursing home staff and residents. Specific attack rates and relative risks with their 95% confidence intervals were estimated. A multivariate analysis (logistic regression) was performed proving odds ratios (OR) of becoming ill. Conjunctival swab samples were taken and tested for viral infection. More stringent infection control measures were implemented following the occurrence of the initial cases. RESULTS: Forty-six cases were identified in the nursing home (infection rates of 30.5% in residents and 8.3% in workers). Total duration of the outbreak was 120 days. Corneal ulcer occurred in 3 cases. The factors appearing as independent risk factors were age (OR = 5.7 in people aged >or=90 years compared to those aged <80 years), cognitive impairment (OR = 2.64) and nursing home floor (OR = 2.74 for the first floor, where the outbreak started). Adenoviral DNA was amplified in 10 samples, and 8 of them could be typed as adenovirus serotype 8. CONCLUSIONS: Early adoption of adequate hygiene measures is essential to control these outbreaks. In nursing homes with a high number of people with cognitive impairment, an additional effort should be made when the first cases occur to provide such people an increased and improved care and monitoring.

A measles outbreak in children under 15 months of age in La Rioja, Spain, 2005-2006.

This paper describes a measles outbreak in La Rioja, Spain, which began in December 2005 and mainly affected children under 15 months of age who were not yet immunised with MMR vaccine. The measles cases were detected by the mandatory reporting system, under which laboratories must report every confirmed measles case. Cases were classified in accordance with the National Measles Elimination Plan: suspected and laboratory-confirmed. In the period 14 December 2005 to 19 February 2006, 29 suspected cases of measles were investigated, and 18 were confirmed. The mean incubation period was 13.8 days (range: 9 to 18). Of the 18 confirmed cases, only two were in adults. MMR vaccination was recommended for all household contacts, as well as for children aged 6 to 14 months who attended the daycare centres where the cases had appeared. At these centres, the second dose of MMR was administered ahead of schedule for children under three years of age. It was recommended that the first dose of MMR vaccine be administered ahead of schedule for all children aged 9 to 14 months. During an outbreak of measles, children aged 6 months or older, who have not previously been vaccinated against measles, mumps and rubella, should receive a first dose as soon as possible, and those who have had a first dose should receive a second dose as soon as possible, provided that a minimum of one month has elapsed between the two doses.

EBLV1 circulation in natural bat colonies of Eptesicus serotinus: a six year survey.

The serotine bat (Eptesicus serotinus) accounts for 95 % of cases of human exposition to EBLV1. The aim of this study was to focus on the epidemiology and pathogenesis of EBLV1 infection in the serotine bat. Our first objective was the development of an RT-PCR technique for the specific detection of EBLV1 RNA in oro-pharyngeal swabs. This technique showed better performance than the classical immunofluorescence (IF) on brain in detecting EBLV1 in healthy flying bats. We have used this technique together with antibody detection by the fluorescent focus inhibition test (FFIT) to investigate EBLV1 circulation in 19 natural colonies of serotine bats in Andalusia (Spain) from 1998 to 2003. The survey was based on 1223 different captures of 1080 individuals that were ring banded, sampled and released. Individuals that were repeatedly captured were always found in the same colony even though some colonies were less than five Km apart. Viral circulation was detected in nine colonies either by RT-PCR, serology or both. Each colony showed a different temporal pattern of viral circulation suggesting independent endemic circulation. Some positive individuals were captured healthy in following campaigns evidencing survival after viral infection.

RT-PCR for detection of all seven genotypes of Lyssavirus genus.

The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

Virological analysis in the diagnosis of sudden children death: a medico-legal approach.

Infections are considered to be an important cause of unexpected death in children. It has also been assumed that respiratory viruses are involved in the genesis of sudden infant death syndrome (SIDS). The Spanish National Institute of Toxicology and Forensic Sciences act as the forensic reference centre for Spain. We analyse the experience of this centre in the virological study of 64 cases of sudden children death where viral serology, virological cultures, herpesviruses polymerase chain reaction (PCR) and electron microscopy were performed. According to pathological findings, death could only be attributed to an adenovirus infection in one amygdalitis with upper airways stenosis and asphyxia. Human herpes virus 6 (HHV-6) was detected by PCR in one case with pathological findings characteristic of SIDS. Recent infection by respiratory syncytial virus (RSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were also detected. Meanwhile, 85.9% of the cases yielded negative viral results. Twenty-eight infants were finally categorised as SIDS. Pathological findings of infection were detected in 12 patients despite the negativity of viral analyses. Although viral infection is an uncommon cause of sudden children death, a complete microbiological investigation will help to solve the puzzle of SIDS. Definitive guidelines for microbiological analyses need to be updated whilst new pathogens are discovered or new techniques are implemented in order to clarify unsolved cases.

A measles outbreak in children under 15 months of age in La Rioja, Spain, 2005-2006.

This paper describes a measles outbreak in La Rioja, Spain, which began in December 2005 and mainly affected children under 15 months of age who were not yet immunised with MMR vaccine. The measles cases were detected by the mandatory reporting system, under which laboratories must report every confirmed measles case. Cases were classified in accordance with the National Measles Elimination Plan: suspected and laboratory-confirmed. In the period 14 December 2005 to 19 February 2006, 29 suspected cases of measles were investigated, and 18 were confirmed. The mean incubation period was 13.8 days (range: 9 to 18). Of the 18 confirmed cases, only two were in adults. MMR vaccination was recommended for all household contacts, as well as for children aged 6 to 14 months who attended the daycare centres where the cases had appeared. At these centres, the second dose of MMR was administered ahead of schedule for children under three years of age. It was recommended that the first dose of MMR vaccine be administered ahead of schedule for all children aged 9 to 14 months. During an outbreak of measles, children aged 6 months or older, who have not previously been vaccinated against measles, mumps and rubella, should receive a first dose as soon as possible, and those who have had a first dose should receive a second dose as soon as possible, provided that a minimum of one month has elapsed between the two doses.

Molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region.

Here we present a system for adenovirus detection and genotyping based on PCR amplification and phylogenetic analysis of a conserved hexon gene fragment. The system was validated using 157 sequences (86 previously typed and 71 clinical samples) and correctly identified species and serotype in 100% and 84% of sequences, respectively. Known associations between specific serotypes and clinical syndromes are verified. Possible new associations are described to allow further independent testing.

A human case of travel-related rabies in Austria, September 2004.

A young male Austrian tourist, aged 23 years and unvaccinated against rabies, was bitten by a dog in Morocco in July 2004. One month later he was hospitalised in Ceuta with symptoms compatible with rabies. He died on 23 September in an Austrian hospital after a diagnosis of rabies was confirmed by FAT, IHC and RT-PCR (including sequencing) of the neck skin and the RT-PCR (including sequencing) of the pharyngeal swab. This Austrian case of laboratory confirmed rabies highlights the urgent need for reinforcement of the international recommendations for travel vaccinations.

Molecular identification of mumps virus genotypes from clinical samples: standardized method of analysis.

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.

A human case of travel-related rabies in Austria, September 2004.

A young male Austrian tourist, aged 23 years and unvaccinated against rabies, was bitten by a dog in Morocco in July 2004. One month later he was hospitalised in Ceuta with symptoms compatible with rabies. He died on 23 September in an Austrian hospital after a diagnosis of rabies was confirmed by FAT, IHC and RT-PCR (including sequencing) of the neck skin and the RT-PCR (including sequencing) of the pharyngeal swab. This Austrian case of laboratory confirmed rabies highlights the urgent need for reinforcement of the international recommendations for travel vaccinations.

Screening of active lyssavirus infection in wild bat populations by viral RNA detection on oropharyngeal swabs.

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.