Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats

BACKGROUND: In the frame of active lyssavirus surveillance in bats, oropharyngeal swabs from German (N = 2297) and Danish (N = 134) insectivorous bats were investigated using a newly developed generic pan-lyssavirus real-time reverse transcriptase PCR (RT-qPCR). FINDINGS: In total, 15 RT-qPCR positive swabs were detected. Remarkably, sequencing of positive samples did not confirm the presence of bat associated lyssaviruses but revealed nine distinct novel rhabdovirus-related sequences. CONCLUSIONS: Several novel rhabdovirus-related sequences were detected both in German and Danish insectivorous bats. The results also prove that the novel generic pan-lyssavirus RT-qPCR offers a very broad detection range that allows the collection of further valuable data concerning the broad and complex diversity within the family Rhabdoviridae

Renewed Public Health Threat from Emerging Lyssaviruses

Pathogen discovery contributes to our knowledge of bat-borne viruses and is linked to the heightened interest globally in bats as recognised reservoirs of zoonotic agents. The transmission of lyssaviruses from bats-to-humans, domestic animals, or other wildlife species is uncommon, but interest in these pathogens remains due to their ability to cause an acute, progressive, invariably fatal encephalitis in humans. Consequently, the detection and characterisation of bat lyssaviruses continues to expand our knowledge of their phylogroup definition, viral diversity, host species association, geographical distribution, evolution, mechanisms for perpetuation, and the potential routes of transmission. Although the opportunity for lyssavirus cross-species transmission seems rare, adaptation in a new host and the possibility of onward transmission to humans requires continued investigation. Considering the limited efficacy of available rabies biologicals it is important to further our understanding of protective immunity to minimize the threat from these pathogens to public health. Hence, in addition to increased surveillance, the development of a niche pan-lyssavirus vaccine or therapeutic biologics for post-exposure prophylaxis for use against genetically divergent lyssaviruses should be an international priority as these emerging lyssaviruses remain a concern for global public health

Genetic analysis of a rabies virus host shift event reveals within-host viral dynamics in a new host

Host shift events play an important role in epizootics as adaptation to new hosts can profoundly affect the spread of the disease and the measures needed to control it. During the late 1990s, an epizootic in Turkey resulted in a sustained maintenance of rabies virus (RABV) within the fox population. We used Bayesian inferences to investigate whole genome sequences from fox and dog brain tissues from Turkey to demonstrate that the epizootic occurred in 1997 (±1 year). Furthermore, these data indicated that the epizootic was most likely due to a host shift from locally infected domestic dogs, rather than an incursion of a novel fox or dog RABV. No evidence was observed for genetic adaptation to foxes at consensus sequence level and dN/dS analysis suggested purifying selection. Therefore, the deep sequence data were analysed to investigate the sub-viral population during a host shift event. Viral heterogeneity was measured in all RABV samples; viruses from the early period after the host shift exhibited greater sequence variation in comparison to those from the later stage, and to those not involved in the host shift event, possibly indicating a role in establishing transmission within a new host. The transient increase in variation observed in the new host species may represent virus replication within a new environment, perhaps due to increased replication within the CNS, resulting in a larger population of viruses, or due to the lack of host constraints present in the new host reservoir

Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland

There has been limited knowledge on spatio-temporal epidemiology of zoonotic arctic fox rabies among countries bordering the Arctic, in particular Greenland. Previous molecular epidemiological studies have suggested the occurrence of one particular arctic rabies virus (RABV) lineage (arctic-3), but have been limited by a low number of available samples preventing in-depth high resolution phylogenetic analysis of RABVs at that time. However, an improved knowledge of the evolution, at a molecular level, of the circulating RABVs and a better understanding of the historical perspective of the disease in Greenland is necessary for better direct control measures on the island. These issues have been addressed by investigating the spatio-temporal genetic diversity of arctic RABVs and their reservoir host, the arctic fox, in Greenland using both full and partial genome sequences. Using a unique set of 79 arctic RABV full genome sequences from Greenland, Canada, USA (Alaska) and Russia obtained between 1977 and 2014, a description of the historic context in relation to the genetic diversity of currently circulating RABV in Greenland and neighboring Canadian Northern territories has been provided. The phylogenetic analysis confirmed delineation into four major arctic RABV lineages (arctic 1-4) with viruses from Greenland exclusively grouping into the circumpolar arctic-3 lineage. High resolution analysis enabled distinction of seven geographically distinct subclades (3.I - 3.VII) with two subclades containing viruses from both Greenland and Canada. By combining analysis of full length RABV genome sequences and host derived sequences encoding mitochondrial proteins obtained simultaneously from brain tissues of 49 arctic foxes, the interaction of viruses and their hosts was explored in detail. Such an approach can serve as a blueprint for analysis of infectious disease dynamics and virus-host interdependencies. The results showed a fine-scale spatial population structure in Greenland arctic foxes based on mitochondrial sequences, but provided no evidence for independent isolated evolutionary development of RABV in different arctic fox lineages. These data are invaluable to support future initiatives for arctic fox rabies control and elimination in Greenland

Estimating the global burden of endemic canine rabies

Background: Rabies is a notoriously underreported and neglected disease of lowincome countries. This study aims to estimate the public health and economic burden of rabies circulating in domestic dog populations, globally and on a country-by-country basis, allowing an objective assessment of how much this preventable disease costs endemic countries.<p></p> Methodology/Principal Findings: We established relationships between rabies mortality and rabies prevention and control measures, which we incorporated into a model framework. We used data derived from extensive literature searches and questionnaires on disease incidence, control interventions and preventative measures within this framework to estimate the disease burden. The burden of rabies impacts on public health sector budgets, local communities and livestock economies, with the highest risk of rabies in the poorest regions of the world. This study estimates that globally canine rabies causes approximately 59,000 (95% Confidence Intervals: 25- 159,000) human deaths, over 3.7 million (95% CIs: 1.6-10.4 million) disability-adjusted life years (DALYs) and 8.6 billion USD (95% CIs: 2.9-21.5 billion) economic losses annually. The largest component of the economic burden is due to premature death (55%), followed by direct costs of post-exposure prophylaxis (PEP, 20%) and lost income whilst seeking PEP (15.5%), with only limited costs to the veterinary sector due to dog vaccination (1.5%), and additional costs to communities from livestock losses (6%).<p></p> Conclusions/Significance: This study demonstrates that investment in dog vaccination, the single most effective way of reducing the disease burden, has been inadequate and that the availability and affordability of PEP needs improving. Collaborative investments by medical and veterinary sectors could dramatically reduce the current large, and unnecessary, burden of rabies on affected communities. Improved surveillance is needed to reduce uncertainty in burden estimates and to monitor the impacts of control efforts.<p></p&gt

Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species.

Rabies is a fatal neurologic disease caused by lyssavirus infection. Bats are important natural reservoir hosts of various lyssaviruses that can be transmitted to people. The epidemiology and pathogenesis of rabies in bats are poorly understood, making it difficult to prevent zoonotic transmission. To further our understanding of lyssavirus pathogenesis in a natural bat host, an experimental model using straw-colored fruit bats (Eidolon helvum) and Lagos bat virus, an endemic lyssavirus in this species, was developed. To determine the lowest viral dose resulting in 100% productive infection, bats in five groups (four bats per group) were inoculated intramuscularly with one of five doses, ranging from 100.1 to 104.1 median tissue culture infectious dose (TCID50). More bats died due to the development of rabies after the middle dose (102.1 TCID50, 4/4 bats) than after lower (101.1, 2/4; 101.1, 2/4) or higher (103.1, 2/4; 104.1, 2/4) doses of virus. In the two highest dose groups, 4/8 bats developed rabies. Of those bats that remained healthy 3/4 bats seroconverted, suggesting that high antigen loads can trigger a strong immune response that abrogates a productive infection. In contrast, in the two lowest dose groups, 3/8 bats developed rabies, 1/8 remained healthy and seroconverted and 4/8 bats remained healthy and did not seroconvert, suggesting these doses are too low to reliably induce infection. The main lesion in all clinically affected bats was meningoencephalitis associated with lyssavirus-positive neurons. Lyssavirus antigen was detected in tongue epithelium (5/11 infected bats) rather than in salivary gland epithelium (0/11), suggesting viral excretion via the tongue. Thus, intramuscular inoculation of 102.1 TCID50 of Lagos bat virus into straw-colored fruit bats is a suitable model for lyssavirus associated bat rabies in a natural reservoir host, and can help with the investigation of lyssavirus infection dynamics in bats

Pathogenesis of bat rabies in a natural reservoir: Comparative susceptibility of the straw-colored fruit bat (Eidolon helvum) to three strains of Lagos bat virus.

Rabies is a fatal neurologic disease caused by lyssavirus infection. People are infected through contact with infected animals. The relative increase of human rabies acquired from bats calls for a better understanding of lyssavirus infections in their natural hosts. So far, there is no experimental model that mimics natural lyssavirus infection in the reservoir bat species. Lagos bat virus is a lyssavirus that is endemic in straw-colored fruit bats (Eidolon helvum) in Africa. Here we compared the susceptibility of these bats to three strains of Lagos bat virus (from Senegal, Nigeria, and Ghana) by intracranial inoculation. To allow comparison between strains, we ensured the same titer of virus was inoculated in the same location of the brain of each bat. All bats (n = 3 per strain) were infected, and developed neurological signs, and fatal meningoencephalitis with lyssavirus antigen expression in neurons. There were three main differences among the groups. First, time to death was substantially shorter in the Senegal and Ghana groups (4 to 6 days) than in the Nigeria group (8 days). Second, each virus strain produced a distinct clinical syndrome. Third, the spread of virus to peripheral tissues, tested by hemi-nested reverse transcriptase PCR, was frequent (3 of 3 bats) and widespread (8 to 10 tissues positive of 11 tissues examined) in the Ghana group, was frequent and less widespread in the Senegal group (3/3 bats, 3 to 6 tissues positive), and was rare and restricted in the Nigeria group (1/3 bats, 2 tissues positive). Centrifugal spread of virus from brain to tissue of excretion in the oral cavity is required to enable lyssavirus transmission. Therefore, the Senegal and Ghana strains seem most suitable for further pathogenesis, and for transmission, studies in the straw-colored fruit bat

A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories

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