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1.  Antigenic and genetic characterization of a divergent African virus, Ikoma lyssavirus 
The Journal of General Virology  2014;95(Pt 5):1025-1032.
In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between the genome of IKOV and those of other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spillover event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone, however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in vitro and in vivo characterization of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains and (3) poorly neutralized by sera from humans and animals immunized against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.
doi:10.1099/vir.0.061952-0
PMCID: PMC3983756  PMID: 24496827
2.  Complete Genomic Sequence of Issyk-Kul Virus 
Genome Announcements  2015;3(4):e00662-15.
Issyk-Kul virus (ISKV) is an ungrouped virus tentatively assigned to the Bunyaviridae family and is associated with an acute febrile illness in several central Asian countries. Using next-generation sequencing technologies, we report here the full-genome sequence for this novel unclassified arboviral pathogen circulating in central Asia.
doi:10.1128/genomeA.00662-15
PMCID: PMC4490839  PMID: 26139711
3.  Complete Genome Sequence of Ikoma Lyssavirus 
Journal of Virology  2012;86(18):10242-10243.
Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.
doi:10.1128/JVI.01628-12
PMCID: PMC3446578  PMID: 22923801
4.  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
5.  Canine Distemper in Endangered Ethiopian Wolves 
Emerging Infectious Diseases  2015;21(5):824-832.
Investigation into mortalities within endangered species can direct conservation efforts.
The Ethiopian wolf (Canis simensis) is the world’s rarest canid; ≈500 wolves remain. The largest population is found within the Bale Mountains National Park (BMNP) in southeastern Ethiopia, where conservation efforts have demonstrated the negative effect of rabies virus on wolf populations. We describe previously unreported infections with canine distemper virus (CDV) among these wolves during 2005–2006 and 2010. Death rates ranged from 43% to 68% in affected subpopulations and were higher for subadult than adult wolves (83%–87% vs. 34%–39%). The 2010 CDV outbreak started 20 months after a rabies outbreak, before the population had fully recovered, and led to the eradication of several focal packs in BMNP’s Web Valley. The combined effect of rabies and CDV increases the chance of pack extinction, exacerbating the typically slow recovery of wolf populations, and represents a key extinction threat to populations of this highly endangered carnivore.
doi:10.3201/eid2105.141920
PMCID: PMC4412237  PMID: 25898177
canine distemper; morbillivirus; wolves; endangered; Ethiopian wolves; Canis simensis; Bale Mountains National Park; Ethiopia; canid species; conservation; viruses; epizootics; rabies; vaccination
6.  Ikoma Lyssavirus, Highly Divergent Novel Lyssavirus in an African Civet1 
Emerging Infectious Diseases  2012;18(4):664-667.
Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.
doi:10.3201/eid1804.111553
PMCID: PMC3309678  PMID: 22469151
Tanzania; African civet; rabies virus; West Caucasian bat virus; rabies virus; viruses; Lyssavirus; lyssaviruses; Ikoma lyssavirus; novel rabies virus; novel lyssavirus
7.  Flavivirus-induced antibody cross-reactivity 
The Journal of General Virology  2011;92(Pt 12):2821-2829.
Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.
doi:10.1099/vir.0.031641-0
PMCID: PMC3352572  PMID: 21900425
8.  Complete Genomic Sequence of Rabies Virus from an Ethiopian Wolf 
Genome Announcements  2015;3(2):e00157-15.
Ethiopian wolves are the rarest canid in the world, with only 500 found in the Ethiopian highlands. Rabies poses the most immediate threat to their survival, causing epizootic cycles of mass mortality. The complete genome sequence of a rabies virus (RABV) derived from an Ethiopian wolf during the most recent epizootic is reported here.
doi:10.1128/genomeA.00157-15
PMCID: PMC4384137  PMID: 25814597
9.  Complex Epidemiology of a Zoonotic Disease in a Culturally Diverse Region: Phylogeography of Rabies Virus in the Middle East 
PLoS Neglected Tropical Diseases  2015;9(3):e0003569.
The Middle East is a culturally and politically diverse region at the gateway between Europe, Africa and Asia. Spatial dynamics of the fatal zoonotic disease rabies among countries of the Middle East and surrounding regions is poorly understood. An improved understanding of virus distribution is necessary to direct control methods. Previous studies have suggested regular trans-boundary movement, but have been unable to infer direction. Here we address these issues, by investigating the evolution of 183 rabies virus isolates collected from over 20 countries between 1972 and 2014. We have undertaken a discrete phylogeographic analysis on a subset of 139 samples to infer where and when movements of rabies have occurred. We provide evidence for four genetically distinct clades with separate origins currently circulating in the Middle East and surrounding countries. Introductions of these viruses have been followed by regular and multidirectional trans-boundary movements in some parts of the region, but relative isolation in others. There is evidence for minimal regular incursion of rabies from Central and Eastern Asia. These data support current initiatives for regional collaboration that are essential for rabies elimination.
Author Summary
Despite being one of the oldest recognised infectious diseases, rabies continues to cause thousands of preventable human deaths per year. As a zoonotic disease, control of infection in the reservoir has been proven the most efficient route to reduction of human cases. In some regions, the epidemiology is well understood, with either dogs or wildlife known to be the primary reservoir and with little or no movement from, or into other regions. This is not the case in the Middle East, where rabies is underreported in animals and humans, there is little laboratory confirmation of infection, and the extent of rabies spread from country to country is not known. Previous studies have demonstrated trans-boundary movement of rabies but have been limited by a low number of available samples from some countries, and the direction of spread has been difficult to estimate. Here we use rabies virus partial genome sequences of 183 viruses from over 20 countries, combined with geographical and temporal information, to reconstruct the evolution of rabies viruses circulating in the Middle East. The results reveal an apparent barrier to spread between some regions but regular movement between others. These analyses will support policy on rabies control by indicating the relative importance of local control and animal movement restrictions when allocating resources.
doi:10.1371/journal.pntd.0003569
PMCID: PMC4374968  PMID: 25811659
10.  Defining the Chemokine Basis for Leukocyte Recruitment during Viral Encephalitis 
Journal of Virology  2014;88(17):9553-9567.
ABSTRACT
The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent and virulent Semliki Forest virus (SFV) as well as West Nile virus infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC chemokines in all models. On this basis, we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is the most active; CCR2 is also active but less so, and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually and the resulting suppression of leukocyte recruitment to the infected brain have no effect on viral titer or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain.
IMPORTANCE Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of encephalitis is the recruitment of inflammatory leukocytes to the infected brain, a process driven by members of the chemokine family. Here we provide an in-depth analysis of the chemokines involved in leukocyte recruitment to the virally infected brain and demonstrate that simultaneous blockade of two of these receptors, namely, CXCR3 and CCR2, does not alter viral titers within the brain but markedly reduces inflammatory leukocyte recruitment and enhances survival in a murine model of lethal viral encephalitis. Our results therefore highlight chemokine receptors as plausible therapeutic targets in treating viral encephalitis.
doi:10.1128/JVI.03421-13
PMCID: PMC4136330  PMID: 24899190
11.  Complete Genomic Sequence of European Bat Lyssavirus 1, Isolated from Eptesicus isabellinus in Spain 
Genome Announcements  2015;3(1):e01518-14.
All members of the lyssavirus genus cause the disease rabies. European bat lyssavirus 1 (EBLV-1) viruses are divided genetically into three groups according to geographic location and host reservoir. We report here the first genome sequence for an EBLV-1 isolated from Eptesiscus isabellinus in the Iberian Peninsula, Spain.
doi:10.1128/genomeA.01518-14
PMCID: PMC4333652  PMID: 25676752
12.  Enhanced West Nile virus surveillance in the North Kent marshes, UK 
Parasites & Vectors  2015;8:91.
Background
As part of efforts to more fully understand the potential risks posed by West Nile virus (WNV) and Usutu virus (USUV) in the UK, and following on from previous reports of a potential bridge vector Culex modestus for these viruses, at wetland sites in North Kent, mosquito surveillance was undertaken more widely across the Isle of Sheppey, the Hoo Peninsula and the Kent mainland.
Methods
Larval surveys were conducted and Mosquito Magnet® adult traps were used to collect adult mosquitoes. Pools of female mosquitoes were tested for the presence of WNV using real-time reverse transcriptase polymerase chain reaction. A subset of samples was tested for USUV.
Results
Culex modestus was found in both the pre-imaginal and imago stage at all five locations surveyed, accounting for 90% of adult mosquitoes collected. WNV or USUV were not detected in any sample.
Conclusions
Although no mosquitoes have been shown to be virus positive, the field survey data from this study demonstrated the dominance of an important bridge vector species for WNV in this region. Its wide geographical distribution highlights the need to update risk assessments on WNV introduction, and to maintain vigilance for WNV in the South East of England.
doi:10.1186/s13071-015-0705-9
PMCID: PMC4342892  PMID: 25884920
Culex modestus; United Kingdom; West Nile virus; Usutu virus; Culicidae; Mosquito; Surveillance
13.  Public Health Threat of New, Reemerging, and Neglected Zoonoses in the Industrialized World 
Improving our capacity to respond to these pathogens is essential.
Microbiologic infections acquired from animals, known as zoonoses, pose a risk to public health. An estimated 60% of emerging human pathogens are zoonotic. Of these pathogens, >71% have wildlife origins. These pathogens can switch hosts by acquiring new genetic combinations that have altered pathogenic potential or by changes in behavior or socioeconomic, environmental, or ecologic characteristics of the hosts. We discuss causal factors that influence the dynamics associated with emergence or reemergence of zoonoses, particularly in the industrialized world, and highlight selected examples to provide a comprehensive view of their range and diversity.
doi:10.3201/eid1601.081467
PMCID: PMC2874344  PMID: 20031035
Zoonoses; bacteria; viruses; parasites; infectious diseases; arthropod-borne disease; new zoonoses; emerging diseases; reemerging infections; synopsis
14.  Emergence of West Nile Virus Lineage 2 in Europe: A Review on the Introduction and Spread of a Mosquito-Borne Disease 
West Nile virus (WNV) is transmitted by mosquitoes and causes fever and encephalitis in humans, equines, and occasionally wild birds. The virus was first isolated in sub-Saharan Africa where it is endemic. WNV lineage 1 has been responsible for repeated disease outbreaks in the countries of the Mediterranean basin over the past 50 years. This lineage was also introduced into North America in 1999 causing widespread human, equine, and avian mortality. WNV lineage 2, the first WNV lineage to be isolated, was believed to be restricted to sub-Saharan Africa causing a relatively mild fever in humans. However, in 2004, an investigation in Hungary of a case of encephalitis in a wild goshawk (Accipiter gentiles) resulted in the isolation of WNV lineage 2. During the summer of 2004, and in subsequent years, the virus appeared to spread locally throughout Hungary and into neighboring Austria. Subsequently, WNV lineage 2 emerged in Greece in 2010 and in Italy in 2011, involving outbreaks on the Italian mainland and Sardinia. Further spread through the Balkan countries is also suspected. Whole genome sequencing has confirmed that the virus responsible for the outbreaks in Greece and Italy was almost identical to that isolated in Hungary. However, unlike the outbreaks in Hungary, the burden of disease in Mediterranean countries has fallen upon the human population with numerous cases of West Nile fever and a relatively higher mortality rate than in previous outbreaks. The emergence of WNV lineage 2 in Europe, its over-wintering and subsequent spread over large distances illustrates the repeated threat of emerging mosquito-borne diseases. This article will review the emergence of WNV lineage 2 in Europe; consider the pathways for virus spread and the public health implications for the continent.
doi:10.3389/fpubh.2014.00271
PMCID: PMC4258884  PMID: 25538937
West Nile virus; lineage; emergence; encephalitis; Europe
15.  Achieving Population-Level Immunity to Rabies in Free-Roaming Dogs in Africa and Asia 
Canine rabies can be effectively controlled by vaccination with readily available, high-quality vaccines. These vaccines should provide protection from challenge in healthy dogs, for the claimed period, for duration of immunity, which is often two or three years. It has been suggested that, in free-roaming dog populations where rabies is endemic, vaccine-induced protection may be compromised by immuno-suppression through malnutrition, infection and other stressors. This may reduce the proportion of dogs that seroconvert to the vaccine during vaccination campaigns and the duration of immunity of those dogs that seroconvert. Vaccination coverage may also be limited through insufficient vaccine delivery during vaccination campaigns and the loss of vaccinated individuals from populations through demographic processes. This is the first longitudinal study to evaluate temporal variations in rabies vaccine-induced serological responses, and factors associated with these variations, at the individual level in previously unvaccinated free-roaming dog populations. Individual-level serological and health-based data were collected from three cohorts of dogs in regions where rabies is endemic, one in South Africa and two in Indonesia. We found that the vast majority of dogs seroconverted to the vaccine; however, there was considerable variation in titres, partly attributable to illness and lactation at the time of vaccination. Furthermore, >70% of the dogs were vaccinated through community engagement and door-to-door vaccine delivery, even in Indonesia where the majority of the dogs needed to be caught by net on successive occasions for repeat blood sampling and vaccination. This demonstrates the feasibility of achieving population-level immunity in free-roaming dog populations in rabies-endemic regions. However, attrition of immune individuals through demographic processes and waning immunity necessitates repeat vaccination of populations within at least two years to ensure communities are protected from rabies. These findings support annual mass vaccination campaigns as the most effective means to control canine rabies.
Author Summary
Canine-mediated rabies is a horrific disease that claims tens of thousands of human lives every year, particularly in Asia and Africa. The disease can be effectively controlled through mass vaccination of dogs with high-quality vaccines; however, questions remain over the effectiveness of vaccination where the health status of free-roaming dogs may be compromised and the life expectancy and access to these dogs may be limited. This study evaluated rabies-vaccine induced immune responses and vaccine delivery in previously unvaccinated, free-roaming dog populations in two rabies endemic regions in Asia and Africa, to better understand the effectiveness of vaccination campaigns. We found that the majority of dogs seroconverted to the vaccine regardless of health status. Excellent vaccination coverage was achieved through community engagement and door-to-door vaccine delivery, even where the majority of the dogs needed to be caught by net for vaccination. However, attrition of immune individuals through demographic processes and waning immunity reinforces the importance of frequent and regular vaccination campaigns to ensure effective vaccination coverage is maintained.
doi:10.1371/journal.pntd.0003160
PMCID: PMC4230884  PMID: 25393023
16.  The Phylogeography of Rabies in Grenada, West Indies, and Implications for Control 
In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.
Author Summary
Rabies, a fatal disease of animals and humans has been endemic in Grenada, West Indies, since the early 1900s. The small Indian mongoose, an introduced animal, is the most likely rabies reservoir, with spillover into domestic animals and humans. To control rabies, large numbers of mongooses were killed in the 1960s/1970s, but this effort did not alter long-term rabies dynamics. Vaccination of dogs, cats and livestock is efficient in protecting these animals, yet is not regularly undertaken. Post-exposure prophylaxis (PEP) in humans is routinely done and no human has died of rabies in Grenada since 1970. However, the threat of rabies and potential to adversely affect the tourism industry, are a burden on the Grenadian government and public. This study has re-evaluated the role of the mongoose in the maintenance of rabies in Grenada, and for the first time, the rabies virus circulating in Grenada has been described. Grenada offers optimal conditions for an oral rabies vaccination (ORV) program, being an island with strict live animal import controls, and a single wildlife rabies reservoir. Although further work is needed before an ORV campaign could be implemented, elimination of rabies from Grenada seems a realistic goal.
doi:10.1371/journal.pntd.0003251
PMCID: PMC4199513  PMID: 25330178
17.  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
18.  Antibodies against Lagos Bat Virus in Megachiroptera from West Africa 
Emerging Infectious Diseases  2008;14(6):926-928.
To investigate the presence of Lagos bat virus (LBV)–specific antibodies in megachiroptera from West Africa, we conducted fluorescent antibody virus neutralization tests. Neutralizing antibodies were detected in Eidolon helvum (37%), Epomophorus gambianus (3%), and Epomops buettikoferi (33%, 2/6) from Ghana. These findings confirm the presence of LBV in West Africa.
doi:10.3201/eid1406.071421
PMCID: PMC2600291  PMID: 18507903
Lagos Bat Virus; rabies; megachiroptera; bat; Lyssavirus; dispatch
19.  Phylogenetic Analysis of West Nile Virus Genome, Iran 
Emerging Infectious Diseases  2014;20(8):1419-1421.
doi:10.3201/eid2008.131321
PMCID: PMC4111181  PMID: 25061976
West Nile virus; viruses; arbovirus; phylogenetic analysis; genome; lineage 2; encephalitis; Iran
20.  Comparative studies on the genetic, antigenic and pathogenic characteristics of Bokeloh bat lyssavirus 
The Journal of General Virology  2014;95(Pt 8):1647-1653.
Bokeloh bat lyssavirus (BBLV), a novel lyssavirus, was isolated from a Natterer’s bat (Myotis nattererii), a chiropteran species with a widespread and abundant distribution across Europe. As a novel lyssavirus, the risks of BBLV to animal and human health are unknown and as such characterization both in vitro and in vivo was required to assess pathogenicity and vaccine protection. Full genome sequence analysis and antigenic cartography demonstrated that the German BBLV isolates are most closely related to European bat lyssavirus type 2 (EBLV-2) and Khujand virus and can be characterized within phylogroup I. In vivo characterization demonstrated that BBLV was pathogenic in mice when inoculated peripherally causing clinical signs typical for rabies encephalitis, with higher pathogenicity observed in juvenile mice. A limited vaccination-challenge experiment in mice was conducted and suggested that current vaccines would afford some protection against BBLV although further studies are warranted to determine a serological cut-off for protection.
doi:10.1099/vir.0.065953-0
PMCID: PMC4103065  PMID: 24828330
21.  Lyssaviruses and Bats: Emergence and Zoonotic Threat  
Viruses  2014;6(8):2974-2990.
The continued detection of zoonotic viral infections in bats has led to the microbial fauna of these mammals being studied at a greater level than ever before. Whilst numerous pathogens have been discovered in bat species, infection with lyssaviruses is of particular significance from a zoonotic perspective as, where human infection has been reported, it is invariably fatal. Here we review the detection of lyssaviruses within different bat species and overview what is understood regarding their maintenance and transmission following both experimental and natural infection. We discuss the relevance of these pathogens as zoonotic agents and the threat of newly discovered viruses to human populations.
doi:10.3390/v6082974
PMCID: PMC4147683  PMID: 25093425
rabies; bats; lyssaviruses; emergence; zoonoses
22.  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
23.  Continent-wide panmixia of an African fruit bat facilitates transmission of potentially zoonotic viruses 
Nature communications  2013;4:10.1038/ncomms3770.
The straw-coloured fruit bat, Eidolon helvum, is Africa’s most widely distributed and commonly hunted fruit bat, often living in close proximity to human populations. This species has been identified as a reservoir of potentially zoonotic viruses, but uncertainties remain regarding viral transmission dynamics and mechanisms of persistence. Here we combine genetic and serological analyses of populations across Africa, to determine the extent of epidemiological connectivity among E. helvum populations. Multiple markers reveal panmixia across the continental range, at a greater geographical scale than previously recorded for any other mammal, whereas populations on remote islands were genetically distinct. Multiple serological assays reveal antibodies to henipaviruses and Lagos bat virus in all locations, including small isolated island populations, indicating that factors other than population size and connectivity may be responsible for viral persistence. Our findings have potentially important public health implications, and highlight a need to avoid disturbances which may precipitate viral spillover.
doi:10.1038/ncomms3770
PMCID: PMC3836177  PMID: 24253424
24.  Bat Flight and Zoonotic Viruses 
Emerging Infectious Diseases  2014;20(5):741-745.
High metabolism and body temperatures of flying bats might enable them to host many viruses.
Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. We hypothesize that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. On an evolutionary scale, this host–virus interaction might have resulted in the large diversity of zoonotic viruses in bats, possibly through bat viruses adapting to be more tolerant of the fever response and less virulent to their natural hosts.
doi:10.3201/eid2005.130539
PMCID: PMC4012789  PMID: 24750692
bats; body temperature; Chiroptera; emerging zoonotic viruses; fever; flight; metabolic rate; mammals
25.  A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? 
Bats are the natural reservoirs of a number of high-impact viral zoonoses. We present a quantitative analysis to address the hypothesis that bats are unique in their propensity to host zoonotic viruses based on a comparison with rodents, another important host order. We found that bats indeed host more zoonotic viruses per species than rodents, and we identified life-history and ecological factors that promote zoonotic viral richness. More zoonotic viruses are hosted by species whose distributions overlap with a greater number of other species in the same taxonomic order (sympatry). Specifically in bats, there was evidence for increased zoonotic viral richness in species with smaller litters (one young), greater longevity and more litters per year. Furthermore, our results point to a new hypothesis to explain in part why bats host more zoonotic viruses per species: the stronger effect of sympatry in bats and more viruses shared between bat species suggests that interspecific transmission is more prevalent among bats than among rodents. Although bats host more zoonotic viruses per species, the total number of zoonotic viruses identified in bats (61) was lower than in rodents (68), a result of there being approximately twice the number of rodent species as bat species. Therefore, rodents should still be a serious concern as reservoirs of emerging viruses. These findings shed light on disease emergence and perpetuation mechanisms and may help lead to a predictive framework for identifying future emerging infectious virus reservoirs.
doi:10.1098/rspb.2012.2753
PMCID: PMC3574368  PMID: 23378666
trait-based approaches; zoonoses; viral richness; reservoir host; spillover; Chiroptera

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