Prevention of human rabies: a challenge for the European Union and the European Economic Area.

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

Isolation, antigenicity and immunogenicity of Lleida bat lyssavirus.

The lyssaviruses are an important group of viruses that cause a fatal encephalitis termed rabies. The prototypic lyssavirus, rabies virus, is predicted to cause more than 60 000 human fatalities annually. The burden of disease for the other lyssaviruses is undefined. The original reports for the recently described highly divergent Lleida bat lyssavirus were based on the detection of virus sequence alone. The successful isolation of live Lleida bat lyssavirus from the carcass of the original bat and in vitro characterization of this novel lyssavirus are described here. In addition, the ability of a human rabies vaccine to confer protective immunity following challenge with this divergent lyssavirus was assessed. Two different doses of Lleida bat lyssavirus were used to challenge vaccinated or naïve mice: a high dose of 100 focus-forming units (f.f.u.) 30 µl-1 and a 100-fold dilution of this dose, 1 f.f.u. 30 µl-1. Although all naïve control mice succumbed to the 100 f.f.u. 30 µl-1 challenge, 42 % (n=5/12) of those infected intracerebrally with 1 f.f.u. 30 µl-1 survived the challenge. In the high-challenge-dose group, 42 % of the vaccinated mice survived the challenge (n=5/12), whilst at the lower challenge dose, 33 % (n=4/12) survived to the end of the experiment. Interestingly, a high proportion of mice demonstrated a measurable virus-neutralizing antibody response, demonstrating that neutralizing antibody titres do not necessarily correlate with the outcome of infection via the intracerebral route. Assessing the ability of existing rabies vaccines to protect against novel divergent lyssaviruses is important for the development of future public health strategies.

Taxonomy of the order Mononegavirales: update 2018.

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.

Genomic non-coding regions reveal hidden patterns of mumps virus circulation in Spain, 2005 to 2015.

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.

Potentially Zoonotic Bartonella in Bats from France and Spain.

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.

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.

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.

Utility of MF-non coding region for measles molecular surveillance during post-elimination phase, Spain, 2017-2020.

Background: In countries entering the post-elimination phase for measles, the study of variants by sequencing of 450 nucleotides of the N gene (N450) does not always allow the tracing of chains of transmission. Indeed, between 2017 and 2020, most measles virus sequences belonged to either the MVs/Dublin.IRL/8.16 (B3-Dublin) or the MVs/Gir Somnath.IND/42.16 (D8-Gir Somnath) variants. We evaluated the additional use of a non-coding region (MF-NCR) as a tool to enhance resolution and infer case origin, chains of transmission and characterize outbreaks. Methods: We obtained 115 high-quality MF-NCR sequences from strains collected from Spanish patients infected with either B3-Dublin or D8-Gir Somnath variants between 2017 and 2020, performed epidemiological, phylogenetic and phylodynamic analyses and applied a mathematical model to determine relatedness among identified clades. Results: Applying this model allowed us to identify phylogenetic clades potentially derived from concomitant importations of the virus rather than single chain of transmission, inferred based on only N450 and epidemiology data. In a third outbreak, we found two related clades that corresponded to two chains of transmission. Discussion: Our results show the ability of the proposed method to improve identification of simultaneous importations in the same region which could trigger enhanced contact tracing. Moreover, the identification of further transmission chains indicates that the size of import-related outbreaks was smaller than previously found, supporting the interpretation that endemic measles transmission was absent in Spain between 2017 and 2020. We suggest considering the use of the MF-NCR region in conjunction with the study of N450 variants in future WHO recommendations for measles surveillance.

Comparison of circulation patterns of mumps virus in the Netherlands and Spain (2015-2020).

Background: Mumps is a viral infection mainly characterized by inflammation of the parotid glands. Despite of vaccination programs, infections among fully vaccinated populations were reported. The World Health Organization (WHO) recommends molecular surveillance of mumps based on sequencing of the small hydrophobic (SH) gene. The use of hypervariable non-coding regions (NCR) as additional molecular markers was proposed in multiple studies. Circulation of mumps virus (MuV) genotypes and variants in different European countries were described in the literature. From 2010 to 2020, mumps outbreaks caused by genotype G were described. However, this issue has not been analyzed from a wider geographical perspective. In the present study, sequence data from MuV detected in Spain and in The Netherlands during a period of 5 years (2015- March 2020) were analyzed to gain insights in the spatiotemporal spread of MuV at a larger geographical scale than in previous local studies. Methods: A total of 1,121 SH and 262 NCR between the Matrix and Fusion protein genes (MF-NCR) sequences from both countries were included in this study. Analysis of SH revealed 106 different haplotypes (set of identical sequences). Results: Of them, seven showing extensive circulation were considered variants. All seven were detected in both countries in coincident temporal periods. A single MF-NCR haplotype was detected in 156 sequences (59.3% of total), and was shared by five of the seven SH variants, as well as three minor MF-NCR haplotypes. All SH variants and MF-NCR haplotypes shared by both countries were detected first in Spain. Discussion: Our results suggest a transmission way from south to north Europe. The higher incidence rate of mumps in Spain in spite of similar immunization coverage in both countries, could be associated with higher risk of MuV exportation. In conclusion, the present study provided novel insights into the circulation of MuV variants and haplotypes beyond the borders of single countries. In fact, the use of MF-NCR molecular tool allowed to reveal MuV transmission flows between The Netherlands and Spain. Similar studies including other (European) countries are needed to provide a broader view of the data presented in this study.

Investigating Local Patterns of Mumps Virus Circulation, Using a Combination of Molecular Tools.

Mumps is a vaccine-preventable disease caused by the mumps virus (MuV). However, MuV has re-emerged in many countries with high vaccine coverage. The World Health Organization (WHO) recommends molecular surveillance based on sequencing of the small hydrophobic (SH) gene. Additionally, the combined use of SH and non-coding regions (NCR) has been described in different studies, proving to be a useful complement marker to discriminate general patterns of circulation at national and international levels. The aim of this work is to test local-level usefulness of the combination of SH and MF-NCR sequencing in tracing hidden transmission clusters and chains during the last epidemic wave (2015-2020) in Spain. A database with 903 cases from the Autonomous Community of Madrid was generated by the integration of microbiological and epidemiological data. Of these, 453 representative cases were genotyped. Eight different SH variants and thirty-four SH haplotypes were detected. Local MuV circulation showed the same temporal pattern previously described at a national level. Only two of the thirteen previously identified outbreaks were caused by more than one variant/haplotype. Geographical representation of SH variants allowed the identification of several previously undetected clusters, which were analysed phylogenetically by the combination of SH and MF-NCR, in a total of 90 cases. MF-NCR was not able to improve the discrimination of geographical clusters based on SH sequencing, showing limited resolution for outbreak investigations.

BK virus-associated infection in cerebrospinal fluid of neurological patients and mutation analysis of the complete VP1 gene in different patient groups.

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.

BK polyomavirus with archetypal and rearranged non-coding control regions is present in cerebrospinal fluids from patients with neurological complications.

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.

Phylodynamics and human-mediated dispersal of a zoonotic virus.

Understanding the role of humans in the dispersal of predominantly 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 geopolitical 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.

Phylogeny of European bat Lyssavirus 1 in Eptesicus isabellinus bats, Spain.

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.

Detection of alpha and betacoronaviruses in multiple Iberian bat species.

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 ß CoV.

Mating strategy is determinant of adenovirus prevalence in European bats.

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.

Current Rabies Vaccines Do Not Confer Protective Immunity against Divergent Lyssaviruses Circulating in Europe.

The use of the rabies vaccine for post-exposure prophylaxis started as early as 1885, revealing a safe and efficient tool to prevent human rabies cases. Preventive vaccination is the basis for the control of canine-mediated rabies, which has already been eliminated from extensive parts of the world, including Europe. Plans to eliminate canine-mediated human rabies by 2030 have been agreed upon by international organisations. However, rabies vaccines are not efficacious against some divergent lyssaviruses. The presence in European indigenous bats of recently described lyssaviruses, which are not neutralised by antibody responses to existing vaccines, as well as the declaration of an imported case of an African lyssavirus, which also escapes vaccine-derived protection, leaves the European health authorities unable to provide efficacious protective vaccines to some potential situations of human exposure. All these circumstances highlight the need for a universal pan-lyssavirus rabies vaccine, able to prevent human rabies in all circumstances.

Varicella-zoster virus clades circulating in Spain over two decades.

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.

Last cases of rubella and congenital rubella syndrome in Spain, 1997-2016: The success of a vaccination program.

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

Endemic circulation of European bat lyssavirus type 1 in serotine bats, Spain.

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.