Juan Emilio Echevarría

Discovery of an Ebolavirus-Like Filovirus in Europe

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.

Rapid and sensitive diagnosis of human adenovirus infections by a generic polymerase chain reaction.

A new adenovirus specific nested polymerase chain reaction (PCR) method is described. It was designed inside the hexon protein gene of the adenovirus genome, and was able to detect DNA of all 47 human adenovirus types in a wide range of clinical samples. A sensitive internal control system able to assure proper analytical conditions for the amplification of as few as 100 molecules of a heterologous DNA was included to avoid false negative results. Sensitivity was estimated at about 10 molecules per tube of a plasmid containing an insert of the first amplification product. The method was able to detect adenovirus infection in 31/43 conjunctival scrapings from patients with acute kerato conjunctivitis 10/40 nasopharyngeal aspirates from patients admitted to hospital with acute respiratory disease and 2/26 urine samples from patients with haemorrhagic cystitis with better sensitivity than cell culture or rapid diagnosis by antigen detection by immunofluorescence (IF) in the case of respiratory specimens. Only two of 17 stools positive for a group F adenovirus specific latex immunoassay were PCR negative. The internal control system avoided a false negative result on another two stool samples. In conclusion, the method described below was shown to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection by IF.

Phylodynamics and human-mediated dispersal of a zoonotic virus

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.

Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs

ABSTRACT 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.

Novel lyssavirus in bat, Spain

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.

Enteroviruses in Spain: virological and epidemiological studies over 10 years (1988-97).

A total of 15,662 clinical samples were analysed for enterovirus (EV) isolation in cell cultures during a 10-year period (1988-97). Furthermore, 210 isolates of EV obtained in primary laboratories within Spain from patients with meningitis were characterized. The total number of EV typed was 758, including 727 non-polio EV and 31 Sabin-like (SL) polioviruses. Twenty-eight EV serotypes were represented. Echoviruses comprised 90% (653/727) of fully typed non-polio EV. The four most prevalent serotypes were echovirus 30, echovirus 9, echovirus 6 and echovirus 4. Echovirus 30 was the main serotype associated with meningitis. Echovirus 9 was the aetiological agent in 20 outbreaks of meningitis while the occurrence of echovirus 6 was localized in 1 year (1997). Coxsackieviruses A and B occurred in 3 and 7% of the non-polio EV respectively. Coxsackievirus B5 presented the relative greater abundance. This paper examines the epidemiology of EV in Spain to serotype level over a 10-year period with special attention to non-polio EV associated with meningitis.

Bat Rabies Surveillance in Europe

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.

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

ABSTRACT 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.

High Genetic Diversity of Measles Virus, World Health Organization European Region, 2005–2006

Importation of viruses from other continents caused prolonged circulation and large outbreaks in the WHO European Region.

Simultaneous Detection of Measles Virus, Rubella Virus, and Parvovirus B19 by Using Multiplex PCR

ABSTRACT 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 (TCID50) for measles virus and 0.04 TCID50 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.