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1.  Bat rabies surveillance in Finland 
Background
In 1985, a bat researcher in Finland died of rabies encephalitis caused by European bat lyssavirus type 2 (EBLV-2), but an epidemiological study in 1986 did not reveal EBLV-infected bats. In 2009, an EBLV-2-positive Daubenton’s bat was detected. The EBLV-2 isolate from the human case in 1985 and the isolate from the bat in 2009 were genetically closely related. In order to assess the prevalence of EBLVs in Finnish bat populations and to gain a better understanding of the public health risk that EBLV-infected bats pose, a targeted active surveillance project was initiated.
Results
Altogether, 1156 bats of seven species were examined for lyssaviruses in Finland during a 28–year period (1985–2012), 898 in active surveillance and 258 in passive surveillance, with only one positive finding of EBLV-2 in a Daubenton’s bat in 2009. In 2010–2011, saliva samples from 774 bats of seven species were analyzed for EBLV viral RNA, and sera from 423 bats were analyzed for the presence of bat lyssavirus antibodies. Antibodies were detected in Daubenton’s bats in samples collected from two locations in 2010 and from one location in 2011. All seropositive locations are in close proximity to the place where the EBLV-2 positive Daubenton’s bat was found in 2009. In active surveillance, no EBLV viral RNA was detected.
Conclusions
These data suggest that EBLV-2 may circulate in Finland, even though the seroprevalence is low. Our results indicate that passive surveillance of dead or sick bats is a relevant means examine the occurrence of lyssavirus infection, but the number of bats submitted for laboratory analysis should be higher in order to obtain reliable information on the lyssavirus situation in the country.
doi:10.1186/1746-6148-9-174
PMCID: PMC3846527  PMID: 24011337
EBLV; Lyssavirus; Rabies; Seroprevalence
2.  Experimental infection of Foxes with European bat Lyssaviruses type-1 and 2 
Background
Since 1954, there have been in excess of 800 cases of rabies as a result of European Bat Lyssaviruses types 1 and 2 (EBLV-1, EBLV-2) infection, mainly in Serotine and Myotis bats respectively. These viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in Denmark, a stone marten in Germany and a cat in France. The purpose of this study was to investigate the susceptibility of foxes to EBLVs using silver foxes (Vulpes vulpes) as a model.
Results
Our experimental studies have shown that the susceptibility of foxes to EBLVs is low by the intramuscular (IM) route, however, animals were sensitive to intracranial (IC) inoculation. Mortality was 100% for both EBLV-1 (~4.5 logs) and EBLV-2 (~3.0 logs) delivered by the IC route. Virus dissemination and inflammatory infiltrate in the brain were demonstrated but virus specific neutralising antibody (VNA) was limited (log(ED50) = 0.24–2.23 and 0.95–2.39 respectively for specific EBLV-1 and EBLV-2). Foxes were also susceptible, at a low level, to peripheral (IM) infection (~3.0 logs) with EBLV-1 but not EBLV-2. Three out of 21 (14.3%) foxes developed clinical signs between 14 and 24 days post-EBLV-1 infection. None of the animals given EBLV-2 developed clinical disease.
Conclusion
These data suggest that the chance of a EBLV spill-over from bat to fox is low, but with a greater probability for EBLV-1 than for EBLV-2 and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal infection.
doi:10.1186/1746-6148-5-19
PMCID: PMC2694770  PMID: 19454020
3.  Phylogeography, Population Dynamics, and Molecular Evolution of European Bat Lyssaviruses 
Journal of Virology  2005;79(16):10487-10497.
European bat lyssaviruses types 1 and 2 (EBLV-1 and EBLV-2) are widespread in Europe, although little is known of their evolutionary history. We undertook a comprehensive sequence analysis to infer the selection pressures, rates of nucleotide substitution, age of genetic diversity, geographical origin, and population growth rates of EBLV-1. Our study encompassed data from 12 countries collected over a time span of 35 years and focused on the glycoprotein (G) and nucleoprotein (N) genes. We show that although the two subtypes of EBLV-1—EBLV-1a and EBLV-1b—have both grown at a low exponential rate since their introduction into Europe, they have differing population structures and dispersal patterns. Furthermore, there were strong constraints against amino acid change in both EBLV-1 and EBLV-2, as reflected in a low ratio of nonsynonymous to synonymous substitutions per site, particularly in EBLV-1b. Our inferred rate of nucleotide substitution in EBLV-1, approximately 5 × 10−5 substitutions per site per year, was also one of the lowest recorded for RNA viruses and implied that the current genetic diversity in the virus arose 500 to 750 years ago. We propose that the slow evolution of EBLVs reflects their distinctive epidemiology in bats, where they occupy a relatively stable fitness peak.
doi:10.1128/JVI.79.16.10487-10497.2005
PMCID: PMC1182613  PMID: 16051841
4.  Ecological Factors Associated with European Bat Lyssavirus Seroprevalence in Spanish Bats 
PLoS ONE  2013;8(5):e64467.
Bats have been proposed as major reservoirs for diverse emerging infectious viral diseases, with rabies being the best known in Europe. However, studies exploring the ecological interaction between lyssaviruses and their natural hosts are scarce. This study completes our active surveillance work on Spanish bat colonies that began in 1992. Herein, we analyzed ecological factors that might affect the infection dynamics observed in those colonies. Between 2001 and 2011, we collected and tested 2,393 blood samples and 45 dead bats from 25 localities and 20 bat species. The results for dead confirmed the presence of EBLV-1 RNA in six species analyzed (for the first time in Myotis capaccinii). Samples positive for European bat lyssavirus-1 (EBLV-1)–neutralizing antibodies were detected in 68% of the localities sampled and in 13 bat species, seven of which were found for the first time (even in Myotis daubentonii, a species to date always linked to EBLV-2). EBLV-1 seroprevalence (20.7%) ranged between 11.1 and 40.2% among bat species and seasonal variation was observed, with significantly higher antibody prevalence in summer (July). EBLV-1 seroprevalence was significantly associated with colony size and species richness. Higher seroprevalence percentages were found in large multispecific colonies, suggesting that intra- and interspecific contacts are major risk factors for EBLV-1 transmission in bat colonies. Although bat-roosting behavior strongly determines EBLV-1 variability, we also found some evidence that bat phylogeny might be involved in bat-species seroprevalence. The results of this study highlight the importance of life history and roost ecology in understanding EBLV-1–prevalence patterns in bat colonies and also provide useful information for public health officials.
doi:10.1371/journal.pone.0064467
PMCID: PMC3659107  PMID: 23700480
5.  Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs 
Journal of Clinical Microbiology  2001;39(10):3678-3683.
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.
doi:10.1128/JCM.39.10.3678-3683.2001
PMCID: PMC88406  PMID: 11574590
6.  Temporal Dynamics of European Bat Lyssavirus Type 1 and Survival of Myotis myotis Bats in Natural Colonies 
PLoS ONE  2007;2(6):e566.
Many emerging RNA viruses of public health concern have recently been detected in bats. However, the dynamics of these viruses in natural bat colonies is presently unknown. Consequently, prediction of the spread of these viruses and the establishment of appropriate control measures are hindered by a lack of information. To this aim, we collected epidemiological, virological and ecological data during a twelve-year longitudinal study in two colonies of insectivorous bats (Myotis myotis) located in Spain and infected by the most common bat lyssavirus found in Europe, the European bat lyssavirus subtype 1 (EBLV-1). This active survey demonstrates that cyclic lyssavirus infections occurred with periodic oscillations in the number of susceptible, immune and infected bats. Persistence of immunity for more than one year was detected in some individuals. These data were further used to feed models to analyze the temporal dynamics of EBLV-1 and the survival rate of bats. According to these models, the infection is characterized by a predicted low basic reproductive rate (R0 = 1.706) and a short infectious period (D = 5.1 days). In contrast to observations in most non-flying animals infected with rabies, the survival model shows no variation in mortality after EBLV-1 infection of M. myotis. These findings have considerable public health implications in terms of management of colonies where lyssavirus-positive bats have been recorded and confirm the potential risk of rabies transmission to humans. A greater understanding of the dynamics of lyssavirus in bat colonies also provides a model to study how bats contribute to the maintenance and transmission of other viruses of public health concern.
doi:10.1371/journal.pone.0000566
PMCID: PMC1892799  PMID: 17593965
7.  New Lyssavirus Genotype from the Lesser Mouse-eared Bat (Myotis blythi), Kyrghyzstan 
Emerging Infectious Diseases  2003;9(3):333-337.
The Aravan virus was isolated from a Lesser Mouse-eared Bat (Myotis blythi) in the Osh region of Kyrghyzstan, central Asia, in 1991. We determined the complete sequence of the nucleoprotein (N) gene and compared it with those of 26 representative lyssaviruses obtained from databases. The Aravan virus was distinguished from seven distinct genotypes on the basis of nucleotide and amino acid identity. Phylogenetic analysis based on both nucleotide and amino acid sequences showed that the Aravan virus was more closely related to genotypes 4, 5, and—to a lesser extent—6, which circulates among insectivorus bats in Europe and Africa. The Aravan virus does not belong to any of the seven known genotypes of lyssaviruses, namely, rabies, Lagos bat, Mokola, and Duvenhage viruses and European bat lyssavirus 1, European bat lyssavirus 2, and Australian bat lyssavirus. Based on these data, we propose a new genotype for the Lyssavirus genus.
doi:10.3201/eid0903.020252
PMCID: PMC2958534  PMID: 12643828
Lyssavirus; genotype; N gene; phylogenetic analysis; bat; central Asia; research
8.  European Bat Lyssavirus in Scottish Bats 
Emerging Infectious Diseases  2005;11(4):572-578.
Daubenton bats may roost infrequently in human dwellings, so risk for human contact is low.
We report the first seroprevalence study of the occurrence of specific antibodies to European bat lyssavirus type 2 (EBLV-2) in Daubenton's bats. Bats were captured from 19 sites across eastern and southern Scotland. Samples from 198 Daubenton's bats, 20 Natterer's bats, and 6 Pipistrelle's bats were tested for EBLV-2. Blood samples (N = 94) were subjected to a modified fluorescent antibody virus neutralization test to determine antibody titer. From 0.05% to 3.8% (95% confidence interval) of Daubenton's bats were seropositive. Antibodies to EBLV-2 were not detected in the 2 other species tested. Mouth swabs (N = 218) were obtained, and RNA was extracted for a reverse transcription–polymerase chain reaction (RT-PCR). The RT-PCR included pan lyssavirus-primers (N gene) and internal PCR control primers for ribosomal RNA. EBLV-2 RNA was not detected in any of the saliva samples tested, and live virus was not detected in virus isolation tests.
doi:10.3201/eid1104.040920
PMCID: PMC3320325  PMID: 15829196
Lyssavirus; EBLV-2; seroprevalence; Daubenton bats; Scotland; research
9.  Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein 
The Journal of General Virology  2013;94(Pt 2):284-292.
European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (104 f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (103 f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G–matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.
doi:10.1099/vir.0.048827-0
PMCID: PMC3709617  PMID: 23100360
10.  Phylogeny of European Bat Lyssavirus 1 in Eptesicus isabellinus Bats, Spain 
Emerging Infectious Diseases  2011;17(3):520-523.
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.
doi:10.3201/eid1703100894
PMCID: PMC3166003  PMID: 21392449
Lyssavirus; bats; phylogeny; rabies; EBLV-1; Eptesicus isabellinus; viruses; Spain; dispatch
11.  Antigenic and molecular characterization of bat rabies virus in Europe. 
Journal of Clinical Microbiology  1992;30(9):2419-2426.
The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.
Images
PMCID: PMC265516  PMID: 1401009
12.  Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison 
The Journal of General Virology  2008;89(Pt 9):2204-2213.
Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag–pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40 % of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100 % concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or β-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 μl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies.
doi:10.1099/vir.0.2008/000349-0
PMCID: PMC2886951  PMID: 18753230
13.  Intergenotypic Replacement of Lyssavirus Matrix Proteins Demonstrates the Role of Lyssavirus M Proteins in Intracellular Virus Accumulation ▿  
Journal of Virology  2009;84(4):1816-1827.
Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.
doi:10.1128/JVI.01665-09
PMCID: PMC2812392  PMID: 19955305
14.  Rabies-Related Knowledge and Practices Among Persons At Risk of Bat Exposures in Thailand 
Background
Rabies is a fatal encephalitis caused by lyssaviruses. Evidence of lyssavirus circulation has recently emerged in Southeast Asian bats. A cross-sectional study was conducted in Thailand to assess rabies-related knowledge and practices among persons regularly exposed to bats and bat habitats. The objectives were to identify deficiencies in rabies awareness, describe the occurrence of bat exposures, and explore factors associated with transdermal bat exposures.
Methods
A survey was administered to a convenience sample of adult guano miners, bat hunters, game wardens, and residents/personnel at Buddhist temples where mass bat roosting occurs. The questionnaire elicited information on demographics, experience with bat exposures, and rabies knowledge. Participants were also asked to describe actions they would take in response to a bat bite as well as actions for a bite from a potentially rabid animal. Bivariate analysis was used to compare responses between groups and multivariable logistic regression was used to explore factors independently associated with being bitten or scratched by a bat.
Findings
Of 106 people interviewed, 11 (10%) identified bats as a potential source of rabies. A history of a bat bite or scratch was reported by 29 (27%), and 38 (36%) stated either that they would do nothing or that they did not know what they would do in response to a bat bite. Guano miners were less likely than other groups to indicate animal bites as a mechanism of rabies transmission (68% vs. 90%, p = 0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p = 0.003). Guano mining, bat hunting, and being in a bat cave or roost area more than 5 times a year were associated with history of a bat bite or scratch.
Conclusions
These findings indicate the need for educational outreach to raise awareness of bat rabies, promote exposure prevention, and ensure appropriate health-seeking behaviors for bat-inflicted wounds, particularly among at-risk groups in Thailand.
Author Summary
Rabies is a fatal encephalitis caused by lyssaviruses. Evidence of lyssavirus circulation has recently emerged in Southeast Asian bats. We surveyed persons regularly exposed to bats and bat habitats in Thailand to assess rabies‐related knowledge and practices. Targeted groups included guano miners, bat hunters, game wardens, and residents/personnel at Buddhist temples where mass bat roosting occurs. Of the 106 people interviewed, 11 (10%) identified bats as a source of rabies. History of a bat bite/scratch was reported by 29 (27%), and 38 (36%) expressed either that they would do nothing or that they did not know what they would do in response to a bat bite. Guano miners were less likely than other groups to indicate animal bites as a mechanism of transmission (68% vs. 90%, p=0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p=0.003). These findings indicate a need for educational outreach in Thailand to raise awareness of bat rabies, promote exposure prevention, and ensure health‐seeking behaviors for bat‐inflicted wounds, particularly among at‐risk groups.
doi:10.1371/journal.pntd.0001054
PMCID: PMC3125144  PMID: 21738801
15.  Endemic Circulation of European Bat Lyssavirus Type 1 in Serotine Bats, Spain 
Emerging Infectious Diseases  2008;14(8):1263-1266.
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.
doi:10.3201/1408.080068
PMCID: PMC2600403  PMID: 18680651
lyssavirus; bats; surveillance; rabies; dispatch
16.  European Bat Lyssavirus Infection in Spanish Bat Populations 
Emerging Infectious Diseases  2002;8(4):413-420.
From 1992 to 2000, 976 sera, 27 blood pellets, and 91 brains were obtained from 14 bat species in 37 localities in Spain. Specific anti-European bat lyssavirus 1 (EBL1)-neutralizing antibodies have been detected in Myotis myotis, Miniopterus schreibersii, Tadarida teniotis, and Rhinolophus ferrumequinum in the region of Aragon and the Balearic Islands. Positive results were also obtained by nested reverse transcription-polymerase chain reaction on brain, blood pellet, lung, heart, tongue, and esophagus-larynx-pharynx of M. myotis, Myotis nattereri, R. ferrumequinum, and M. schreibersii. Determination of nucleotide sequence confirmed the presence of EBL1 RNA in the different tissues. In one colony, the prevalence of seropositive bats over time corresponded to an asymmetrical curve, with a sudden initial increase peaking at 60% of the bats, followed by a gradual decline. Banded seropositive bats were recovered during several years, indicating that EBL1 infection in these bats was nonlethal. At least one of this species (M. schreibersii) is migratory and thus could be partially responsible for the dissemination of EBL1 on both shores of the Mediterranean Sea.
doi:10.3201/eid0804.010263
PMCID: PMC2730232  PMID: 11971777
Lyssavirus; polymerase chain reaction; serology; Spain
17.  Susceptibility and Pathogenesis of Little Brown Bats (Myotis lucifugus) to Heterologous and Homologous Rabies Viruses 
Journal of Virology  2013;87(16):9008-9015.
Rabies virus (RABV) maintenance in bats is not well understood. Big brown bats (Eptesicus fuscus), little brown bats (Myotis lucifugus), and Mexican free-tailed bats (Tadarida brasiliensis) are the most common bats species in the United States. These colonial bat species also have the most frequent contact with humans and domestic animals. However, the silver-haired bat (Lasionycteris noctivagans) RABV is associated with the majority of human rabies virus infections in the United States and Canada. This is of interest because silver-haired bats are more solitary bats with infrequent human interaction. Our goal was to determine the likelihood of a colonial bat species becoming infected with and transmitting a heterologous RABV. To ascertain the potential of heterologous RABV infection in colonial bat species, little brown bats were inoculated with a homologous RABV or one of two heterologous RABVs. Additionally, to determine if the route of exposure influenced the disease process, bats were inoculated either intramuscularly (i.m.) or subcutaneously (s.c.) with a homologous or heterologous RABV. Our results demonstrate that intramuscular inoculation results in a more rapid progression of disease onset, whereas the incubation time in bats inoculated s.c. is significantly longer. Additionally, cross protection was not consistently achieved in bats previously inoculated with a heterologous RABV following a challenge with a homologous RABV 6 months later. Finally, bats that developed rabies following s.c. inoculation were significantly more likely to shed virus in their saliva and demonstrated increased viral dissemination. In summary, bats inoculated via the s.c. route are more likely to shed virus, thus increasing the likelihood of transmission.
doi:10.1128/JVI.03554-12
PMCID: PMC3754046  PMID: 23741002
18.  A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories 
PLoS ONE  2013;8(3):e58372.
Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory.
The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result.
Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing.
doi:10.1371/journal.pone.0058372
PMCID: PMC3592807  PMID: 23520505
19.  Bat Rabies in Canada 1963-1967 
Six hundred and twenty-eight insectivorous bats originating from seven provinces were submitted to this Institute for rabies diagnosis between August 1, 1963 and December 31, 1967. Brain tissue was examined by the fluorescent antibody technique and the mouse infectivity test was carried out with brain, salivary gland, interscapular adipose tissue and kidney samples. Rabies virus was detected in 44 bats, 29 of which were from Ontario, 12 from British Columbia and three from Manitoba. Most of the positive cases were diagnosed in summer months. Seven species were represented among the specimens found to be rabid; there were 32 big brown bats, three hoary bats, three silver-haired bats, two little brown bats, one eastern pipistrelle, one Keen myotis and one red bat. Another bat which was not identified also proved to be infected with rabies.
PMCID: PMC1319378  PMID: 4242773
20.  A universal real-time assay for the detection of Lyssaviruses 
Journal of Virological Methods  2011;177(1-24):87-93.
Highlights
► Universal real-time PCR primer pair demonstrated to hybridize to and detect each of the known Lyssaviruses (including Rabies virus) with greater sensitivity than a standard pan-Lyssavirus hemi-nested RT-PCR typically used. ► Target sequences of bat derived virus species unavailable for analysis (Aravan-, Khujand-, Irkut-, West Caucasian bat- and Shimoni bat virus) were synthesized to produce oligonucleotides and the synthetic DNA was used as a target for primer hybridization.
Rabies virus (RABV) is enzootic throughout most of the world. It is now widely accepted that RABV had its origins in bats. Ten of the 11 Lyssavirus species recognised, including RABV, have been isolated from bats. There is, however, a lack of understanding regarding both the ecology and host reservoirs of Lyssaviruses. A real-time PCR assay for the detection of all Lyssaviruses using universal primers would be beneficial for Lyssavirus surveillance. It was shown that using SYBR® Green, a universal real-time PCR primer pair previously demonstrated to detect European bat Lyssaviruses 1 and 2, and RABV, was able to detect reverse transcribed RNA for each of the seven virus species available to us. Target sequences of bat derived virus species unavailable for analysis were synthesized to produce oligonucleotides. Lagos Bat-, Duvenhage- and Mokola virus full nucleoprotein gene clones enabled a limit of 5–50 plasmid copies to be detected. Five copies of each of the synthetic DNA oligonucleotides of Aravan-, Khujand-, Irkut-, West Caucasian bat- and Shimoni bat virus were detected. The single universal primer pair was therefore able to detect each of the most divergent known Lyssaviruses with great sensitivity.
doi:10.1016/j.jviromet.2011.07.002
PMCID: PMC3191275  PMID: 21777619
Lyssavirus; Rabies; Bat; SYBR Green; Real-time PCR; Synthetic DNA
21.  Host immunity to repeated rabies virus infection in big brown bats 
The Journal of General Virology  2010;91(Pt 9):2360-2366.
Bats are natural reservoirs for the majority of lyssaviruses globally, and are unique among mammals in having exceptional sociality and longevity. Given these facets, and the recognized status of bats as reservoirs for rabies viruses (RABVs) in the Americas, individual bats may experience repeated exposure to RABV during their lifetime. Nevertheless, little information exists with regard to within-host infection dynamics and the role of immunological memory that may result from abortive RABV infection in bats. In this study, a cohort of big brown bats (Eptesicus fuscus) was infected intramuscularly in the left and right masseter muscles with varying doses [10−0.1–104.9 median mouse intracerebral lethal doses (MICLD50)] of an E. fuscus RABV variant isolated from a naturally infected big brown bat. Surviving bats were infected a second time at 175 days post-(primary) infection with a dose (103.9–104.9 MICLD50) of the same RABV variant. Surviving bats were infected a third time at either 175 or 305 days post-(secondary) infection with a dose (104.9 MICLD50) of the same RABV variant. When correcting for dose, similar mortality was observed following primary and secondary infection, but reduced mortality was observed following the third and last RABV challenge, despite infection with a high viral dose. Inducible RABV-neutralizing antibody titres post-infection were ephemeral among infected individuals, and dropped below levels of detection in several bats between subsequent infections. These results suggest that long-term repeated infection of bats may confer significant immunological memory and reduced susceptibility to RABV infection.
doi:10.1099/vir.0.020073-0
PMCID: PMC3052523  PMID: 20519458
22.  Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010 
Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar.
doi:10.4061/2011/727821
PMCID: PMC3170745  PMID: 21991442
23.  Isolation of Irkut Virus from a Murina leucogaster Bat in China 
Background and objectives
Bats are recognized as a major reservoir of lyssaviruses; however, no bat lyssavirus has been isolated in Asia except for Aravan and Khujand virus in Central Asia. All Chinese lyssavirus isolates in previous reports have been of species rabies virus, mainly from dogs. Following at least two recent bat-associated human rabies-like cases in northeast China, we have initiated a study of the prevalence of lyssaviruses in bats in Jilin province and their public health implications. A bat lyssavirus has been isolated and its pathogenicity in mice and genomic alignment have been determined.
Results
We report the first isolation of a bat lyssavirus in China, from the brain of a northeastern bat, Murina leucogaster. Its nucleoprotein gene shared 92.4%/98.9% (nucleotide) and 92.2%/98.8% (amino acid) identity with the two known Irkut virus isolates from Russia, and was designated IRKV-THChina12. Following intracranial and intramuscular injection, IRKV-THChina12 produced rabies-like symptoms in adult mice with a short inoculation period and high mortality. Nucleotide sequence analysis showed that IRKV-THChina12 has the same genomic organization as other lyssaviruses and its isolation provides an independent origin for the species IRKV.
Conclusions
We have identified the existence of a bat lyssavirus in a common Chinese bat species. Its high pathogenicity in adult mice suggests that public warnings and medical education regarding bat bites in China should be increased, and that surveillance be extended to provide a better understanding of Irkut virus ecology and its significance for public health.
Author Summary
The Lyssavirus genus presently comprises 12 species and two unapproved species with different antigenic characteristics. Rabies virus is detectable worldwide; Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus, and Ikoma lyssavirus circulate in Africa; European bat lyssavirus types 1 and 2, Irkut virus, West Caucasian bat virus, and Bokeloh bat lyssavirus are found in Europe; and Australian bat lyssavirus has been isolated in Australia. Only Aravan and Khujand viruses have been identified in central Asia. Bats are recognized as the most important reservoirs of lyssaviruses. In China, all lyssavirus isolates in previous reports have been rabies virus, mainly from dogs; none has been from bats. Recently, however, at least two bat-associated human rabies or rabies-like cases have been reported in northeast China. Therefore, we conducted a search for bat lyssaviruses in Jilin province, close to where the first bat-associated human rabies case was recorded. We isolated a bat lyssavirus, identified as an Irkut virus isolate with high pathogenicity in experimental mice. Our findings suggest that public warnings and medical education regarding bat bites in China should be increased, and that surveillance should be extended to provide a better understanding of Irkut virus ecology and its significance for public health.
doi:10.1371/journal.pntd.0002097
PMCID: PMC3591329  PMID: 23505588
24.  The epidemiology of bat rabies in New York State, 1988-92. 
Epidemiology and Infection  1994;113(3):501-511.
In 1993 New York and Texas each reported a human rabies case traced to a rare variant of rabies virus found in an uncommon species of bat. This study examined the epidemiology of bat rabies in New York State. Demographic, species, and animal-contact information for bats submitted for rabies testing from 1988-92 was analysed. The prevalence of rabies in 6810 bats was 4.6%. Nearly 90% of the 308 rabid bats identified to species were the common big brown bat (Eptesicus fuscus), which comprised 62% of all submissions. Only 25 submissions were silver-haired bats (Lasionycterus noctivagans), the species associated with the two 1993 human cases of rabies, and only two of these bats were positive. Rabies was most prevalent in female bats, in bats submitted because of human [corrected] contact, and in animals tested during September and October. These results highlight the unusual circumstances surrounding the recent human rabies cases in the United States. A species of bat rarely encountered by humans, and contributing little to the total rabies cases in bats, has been implicated in the majority of the indigenously acquired human rabies cases in the United States. The factors contributing to the transmission of this rare rabies variant remain unclear.
Images
PMCID: PMC2271321  PMID: 7995360
25.  Alphacoronaviruses in New World Bats: Prevalence, Persistence, Phylogeny, and Potential for Interaction with Humans 
PLoS ONE  2011;6(5):e19156.
Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.
doi:10.1371/journal.pone.0019156
PMCID: PMC3093381  PMID: 21589915

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