Lorraine M. McElhinney

Current status of rabies and prospects for elimination

Summary Rabies is one of the most deadly infectious diseases, with a case-fatality rate approaching 100%. The disease is established on all continents apart from Antarctica; most cases are reported in Africa and Asia, with thousands of deaths recorded annually. However, the estimated annual figure of almost 60 000 human rabies fatalities is probably an underestimate. Almost all cases of human rabies result from bites from infected dogs. Therefore, the most cost-effective approach to elimination of the global burden of human rabies is to control canine rabies rather than expansion of the availability of human prophylaxis. Mass vaccination campaigns with parenteral vaccines, and advances in oral vaccines for wildlife, have allowed the elimination of rabies in terrestrial carnivores in several countries worldwide. The subsequent reduction in cases of human rabies in such regions advocates the multidisciplinary One Health approach to rabies control through the mass vaccination of dogs and control of canine populations.

European bat lyssaviruses: an emerging zoonosis.

In Europe, two bat lyssaviruses referred to as European bat lyssaviruses (EBLVs) types 1 and 2 (genotypes 5 and 6 respectively) which are closely related to classical rabies virus are responsible for an emerging zoonosis. EBLVs are host restricted to bats, and have been known to infect not only their primary hosts but also in rare circumstances, induce spillover infections to terrestrial mammals including domestic livestock, wildlife and man. Although spillover infections have occurred, there has been no evidence that the virus adapted to a new host. Since 1977, four human deaths from EBLVs have been reported. None of them had a record of prophylactic rabies immunization. Only fragmentary data exist about the effectiveness of current vaccines in cross-protection against EBLVs. It is clear that EBLV in bats cannot be eliminated using conventional strategies similar to the control programmes based on vaccine baits used for fox rabies in Europe during the 1980s. Due to the protected status of bats in Europe, our knowledge of EBLV prevalence and epidemiology is limited. It is possible that EBLV is under-reported and that the recorded cases of EBLV represent only a small proportion of the actual number of infected bats. For this reason, any interaction between man and bats in Europe must be considered as a possible exposure. Human exposure through biting incidents, especially unprovoked attacks, should be treated immediately with rabies post-exposure treatment and the bat, where possible, retained for laboratory analysis. Preventative measures include educating all bat handlers of the risks posed by rabies-infected animals and advising them to be immunized. This review provides a brief history of EBLVs, their distribution in host species and the public health risks.

MOLECULAR EPIDEMIOLOGY OF TERRESTRIAL RABIES IN THE FORMER SOVIET UNION

Fifty-five rabies virus isolates originating from different regions of the former Soviet Union (FSU) were compared with isolates originating from Eurasia, Africa, and North America according to complete or partial nucleoprotein (N) gene sequences. The FSU isolates formed five distinct groups. Group A represented viruses originating from the Arctic, which were similar to viruses from Alaska and Canada. Group B consisted of “Arctic-like” viruses, originating from the south of East Siberia and the Far East. Group C consisted of viruses circulating in the steppe and forest-steppe territories from the European part of Russia to Tuva and in Kazakhstan. These three phylogenetic groups were clearly different from the European cluster. Viruses of group D circulate near the western border of Russia. Their phylogenetic position is intermediate between group C and the European cluster. Group E consisted of viruses originating from the northwestern part of Russia and comprised a “northeastern Europe” group described earlier from the Baltic region. According to surveillance data, a specific host can be defined clearly only for group A (arctic fox; Alopex lagopus) and for the Far Eastern part of the group B distribution area (raccoon dog; Nyctereutes procyonoides). For other territories and rabies virus variants, the red fox (Vulpes vulpes) is the main virus reservoir. However, the steppe fox (Vulpes corsac), wolf (Canis lupus), and raccoon dog are also involved in virus circulation, depending on host population density. These molecular data, joined with surveillance information, demonstrate that the current fox rabies epizootic in the territory of the FSU developed independently of central and western Europe. No evidence of positive selection was found in the N genes of the isolates. In the glycoprotein gene, evidence of positive selection was strongly suggested in codons 156, 160, and 183. At these sites, no link between amino acid substitutions and phylogenetic placement or specific host species was detected.

Bats and Lyssaviruses

Numerous bat species have been identified as important reservoirs of zoonotic viral pathogens. Rabies and rabies-related viruses constitute one of the most important viral zoonoses and pose a significant threat to public health across the globe. Whereas rabies virus (RABV) appears to be restricted to bats of the New World, related lyssavirus species have not been detected in the Americas and have only been detected in bat populations across Africa, Eurasia, and Australia. Currently, 11 distinct species of lyssavirus have been identified, 10 of which have been isolated from bat species and all of which appear to be able to cause encephalitis consistent with that seen with RABV infection of humans. In contrast, whereas lyssaviruses are apparently able to cause clinical disease in bats, it appears that these lyssaviruses may also be able to circulate within bat populations in the absence of clinical disease. This feature of these highly encephalitic viruses, alongside many other aspects of lyssavirus infection in bats, is poorly understood. Here, we review what is known of the complex relationship between bats and lyssaviruses, detailing both natural and experimental infections of these viruses in both chiropteran and nonchiropteran models. We also discuss potential mechanisms of virus excretion, transmission both to conspecifics and spill-over of virus into nonvolant species, and mechanisms of maintenance within bat populations. Importantly, we review the significance of neutralizing antibodies reported within bat populations and discuss the potential mechanisms by which highly neurovirulent viruses such as the lyssaviruses are able to infect bat species in the absence of clinical disease.

Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century.

The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.

Ikoma Lyssavirus, Highly Divergent Novel Lyssavirus in an African Civet

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.

Phylogenetic comparison of the genus Lyssavirus using distal coding sequences of the glycoprotein and nucleoprotein genes

Summary. The phylogenetic relationships between all seven genotypes within the genus Lyssavirus were compared at the nucleotide level utilising two distal regions of the viral genome. The resulting analysis of each region produced similar, although not identical, phylogenetic results, suggesting that the evolutionary pressures on individual proteins within the genome vary. These differences are in part due to the increased variability observed within the glycoprotein sequence over the nucleoprotein sequence. Pair-wise comparison using the glycoprotein partial sequence between different isolates demonstrate that within genotypes, viruses show between 80 and 100% sequence identity, whilst between genotypes, viruses show between 50 and 75% identity. This provides a consistent guide to assigning new viruses to existing genotypes. Alignment of the amino acid sequence for the truncated glycoprotein sequence to the Pasteur Virus vaccine strain show significant residue variation between positions 139 and 170. However, residue variation tends to vary with genotype implying that these changes have not evolved due to immunological pressure from the host but have occurred following the separation of viruses into discrete groups. Comparison of the phylogenetic analysis for this partial region of the glycoprotein suggest that it gives comparable results to studies that have used larger regions of the Lyssavirus genome.

Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison

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.

Rabies emergence among foxes in Turkey.

Sixteen rabies isolates recently collected from mainland Turkey and two isolates held within a British archive were used to form a representative cohort from a range of vectors, and were analyzed to identify potential causes for an increase of rabies within the fox (Vulpes vulpes) population in Turkey. Each isolate was characterized by sequence analysis of the nucleoprotein gene and compared phylogenetically to the cohort, to isolates from neighboring countries and to isolates from continental Europe and Russia. From this analysis the isolates could be divided into three groups associated with geographic location. This included a western group, an eastern group, and one isolate that did not group with any other Turkish isolate. This observation was also found using the heteroduplex mobility assay as an alternative method for typing rabies virus isolates. Further comparison with isolates from neighboring countries suggests that this isolate was related to viruses present in Georgia and could represent a recent import to Turkey from that country. Within the two larger groups, sequence data were obtained from both infected dogs and foxes suggesting that there has been transmission of virus between these two species. The direction of transmission could not be identified by the phylogenetic analysis, although absence of rabies within the fox population in previous years suggests that this could represent a recent spillover from the domestic dog to the fox.

Bat Rabies Surveillance in Europe

Rabies is the oldest known zoonotic disease and was also the first recognized bat associated infection in humans. To date, four different lyssavirus species are the causative agents of rabies in European bats: the European Bat Lyssaviruses type 1 and 2 (EBLV‐1, EBLV‐2), the recently discovered putative new lyssavirus species Bokeloh Bat Lyssavirus (BBLV) and the West Caucasian Bat Virus (WCBV). Unlike in the new world, bat rabies cases in Europe are comparatively less frequent, possibly as a result of varying intensity of surveillance. Thus, the objective was to provide an assessment of the bat rabies surveillance data in Europe, taking both reported data to the WHO Rabies Bulletin Europe and published results into account. In Europe, 959 bat rabies cases were reported to the RBE in the time period 1977–2010 with the vast majority characterized as EBLV‐1, frequently isolated in the Netherlands, North Germany, Denmark, Poland and also in parts of France and Spain. Most EBLV‐2 isolates originated from the United Kingdom (UK) and the Netherlands, and EBLV‐2 was also detected in Germany, Finland and Switzerland. Thus far, only one isolate of BBLV was found in Germany. Published passive bat rabies surveillance comprised testing of 28 of the 52 different European bat species for rabies. EBLV‐1 was isolated exclusively from Serotine bats (Eptesicus serotinus and Eptesicus isabellinus), while EBLV‐2 was detected in 14 Daubenton′s bats (Myotis daubentonii) and 5 Pond bats (Myotis dasycneme). A virus from a single Natterer’s bat (Myotis nattereri) was characterized as BBLV. During active surveillance, only oral swabs from 2 Daubenton′s bats (EBLV‐2) and from several Eptesicus bats (EBLV‐1) yielded virus positive RNA. Virus neutralizing antibodies against lyssaviruses were detected in various European bat species from different countries, and its value and implications are discussed.