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European bat lyssavirus 1 (EBLV-1, Lyssavirus hamburg ) is predominantly detected in serotine bats ( Eptesicus serotinus ) and is responsible for the majority of bat rabies cases in mainland Europe. A passive bat rabies surveillance scheme detected the virus in a serotine bat in the UK for the first time in October 2018. As of May 2024, 34 cases have been reported, 20 of which involved contact with an animal and 5 reported human contact. We investigated the emergence of EBLV-1 by undertaking comprehensive sequence analysis and Bayesian phylogenetics, based on complete virus genomes of 33 UK sequences and 108 sequences covering six countries in mainland Europe (1968-2023), including 21 French EBLV-1-positive RNA samples sequenced for this study. Sequence analysis revealed extreme similarity among UK EBLV-1 sequences (99.9%-100%), implying a single source of introduction rather than multiple independent introductions. Bayesian analysis revealed that the UK EBLV-1 sequences shared their most recent common ancestor with an EBLV-1 sequence from a serotine bat detected in Brittany, France, in 2001, with an estimated date of divergence of 1997. Within the UK sequences, the earliest divergence was estimated to occur in 2007. This study provides valuable insights into the molecular epidemiology of an emerging zoonotic pathogen and improved understanding of the risks posed to public and animal health., Competing Interests: None declared., (© Crown copyright 2024.)

Introduction: The awareness of scientists and policy makers regarding the requirement for an integrated One Health (OH) approach in responding to zoonoses has increased in recent years. However, there remains an overall inertia in relation to the implementation of practical cross-sector collaborations. Foodborne outbreaks of zoonotic diseases continue to affect the European population despite stringent regulations, evidencing the requirement for better 'prevent, detect and response' strategies. Response exercises play an essential role in the improvement of crisis management plans, providing the opportunity to test practical intervention methodologies in a controlled environment., Methods: The One Health European Joint Programme simulation exercise (OHEJP SimEx) aimed at practicing the OH capacity and interoperability across public health, animal health and food safety sectors in a challenging outbreak scenario. The OHEJP SimEx was delivered through a sequence of scripts covering the different stages of a Salmonella outbreak investigation at a national level, involving both the human food chain and the raw pet feed industry., Results: A total of 255 participants from 11 European countries (Belgium, Denmark, Estonia, Finland, France, Italy, Norway, Poland, Portugal, Sweden, the Netherlands) took part in national level two-day exercises during 2022. National evaluations identified common recommendations to countries aiming to improve their OH structure to establish formal communication channels between sectors, implement a common data sharing platform, harmonize laboratory procedures, and reinforce inter-laboratory networks within countries. The large proportion of participants (94%) indicated significant interest in pursuing a OH approach and desire to work more closely with other sectors., Discussion: The OHEJP SimEx outcomes will assist policy makers in implementing a harmonized approach to cross-sector health-related topics, by highlighting the benefits of cooperation, identifying gaps in the current strategies and suggesting actions required to better address foodborne outbreaks. Furthermore, we summarize recommendations for future OH simulation exercises, which are essential to continually test, challenge and improve national OH strategies., Competing Interests: The authors declare that the OHEJP SimEx project was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alves, Artursson, Bloch, Brisabois, Imberechts, Jokelainen, La Ragione, Lindblad, Forss, Marston, Parvizi, Tuominen and Omazic.)

Rabies infects all mammals; however, transmission cycles are only maintained in certain bat and carnivore species. The high incidence of rabies in Greater Kudu (Tragelaphus strepsiceros) observed in Namibia for over 40 years has led to postulation that independent virus transmission is occurring within this antelope population. We have analysed extensive experimental, epidemiological, phylogeographic and deep sequence data, which collectively refute maintenance of an independent rabies cycle in kudu. As rabies in kudu continues to have a negative impact on the Namibian agricultural sector, measures to protect kudu have been investigated, including the use of a third-generation oral rabies vaccine. Initial results show protection of kudu from rabies infection via the oral route, with an appropriate bait design, different application schedules and vaccination doses further enhancing the immune response. Rabies in kudu is a complex interplay at the wildlife-livestock interface and requires a concerted approach to successfully control., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Lyssaviruses are an important genus of zoonotic viruses which cause the disease rabies. The United Kingdom is free of classical rabies (RABV). However, bat rabies due to European bat lyssavirus 2 (EBLV-2), has been detected in Daubenton's bats ( Myotis daubentonii ) in Great Britain since 1996, including a fatal human case in Scotland in 2002. Across Europe, European bat lyssavirus 1 (EBLV-1) is commonly associated with serotine bats ( Eptesicus serotinus ). Despite the presence of serotine bats across large parts of southern England, EBLV-1 had not previously been detected in this population. However, in 2018, EBLV-1 was detected through passive surveillance in a serotine bat from Dorset, England, using a combination of fluorescent antibody test, reverse transcription-PCR, Sanger sequencing and immunohistochemical analysis. Subsequent EBLV-1 positive serotine bats have been identified in South West England, again through passive surveillance, during 2018, 2019 and 2020. Here, we confirm details of seven cases of EBLV-1 and present similarities in genetic sequence indicating that emergence of EBLV-1 is likely to be recent, potentially associated with the natural movement of bats from the near continent.

Background: Thermostable vaccines greatly improved the reach and impact of large-scale programmes to eliminate infectious diseases such as smallpox, polio, and rinderpest. A study from 2015 demonstrated that the potency of the Nobivac ® Rabies vaccine was not impacted following experimental storage at 30°C for 3 months. Whether the vaccine would remain efficacious following storage under more natural, fluctuating temperature conditions remains unknown. We carried out a randomised controlled non-inferiority trial to compare serological responses in dogs following vaccination with doses stored under cold chain conditions with those stored within a locally made Passive Cooling Device ("Zeepot") under fluctuating temperature conditions. Materials and Methods: Nobivac ® Rabies vaccine was stored under either cold-chain conditions or within the Zeepot for 2 months. Daily ambient temperatures and temperatures within the Zeepot were recorded every 3 h. Following storage, 412 domestic dogs were randomly assigned to receive either cold-chain or Zeepot stored Nobivac ® Rabies vaccine. Baseline and day 28-post vaccination blood samples were collected. Serological analysis using the Fluorescent Antibody Virus Neutralisation assay was carried out with a threshold of 0.5 IU/ml to determine seroconversion. In addition, the impact of dog Body Condition Score, sex, and age on seroconversion was examined. Results: The serological response of dogs vaccinated using Nobivac ® Rabies vaccine stored within the Zeepot was not inferior to the response of dogs vaccinated using cold-chain stored vaccine (z = 1.1, df = 313, p -value = 0.25). Indeed, the 28-day post-vaccination group geometric mean titre was 1.8 and 2.0 IU/ml for cold-chain vs. non-cold-chain storage, respectively. Moreover, the percentage of dogs that seroconverted in each arm was almost identical (85%). There was a positive linear trend between Body Condition Score (O.R. 2.2, 95% CI: 1.1-5.1) and seroconversion, suggesting dogs of poor condition may not respond as expected to vaccination. Conclusions: Our study demonstrated the potency of Nobivac ® Rabies vaccine is not impacted following storage under elevated fluctuating temperatures within a Zeepot. These results have potentially exciting applications for scaling up mass dog vaccination programmes in low-and-middle income countries, particularly for hard-to-reach populations with limited access to power and cold-chain vaccine storage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lugelo, Hampson, Czupryna, Bigambo, McElhinney, Marston, Kazwala and Lankester.)

Human fatalities caused by rabies are rarely reported in Jordan; however, domestic animals are more likely to fall victim to rabies compared to wild animals, at least this is the case in Jordan due to the presence of canine rabies. In this study, twelve brain samples from domestic and wild animals suspected of being infected with rabies virus from different regions of Jordan were collected during 2019. Seven of them tested positive using the fluorescent antibody test and real-time SYBR RT-PCR assay. Five specimens were from stray dogs and two from foxes. The whole genome sequences were obtained from the positive samples. Sequence analysis showed that one dog virus from Al Quwaysimah city located in Amman governorate, was closely related to an Israeli strain belonging to a Cosmopolitan ME1a clade. The genomes of the remaining six viruses (four from dogs and two from foxes) collected from different areas of Jordan were genetically-related to each other and clustered together with sequences from Iran and Turkey; all belong to Cosmopolitan ME2 clade. These sequences were analyzed with six other Jordanian rabies virus nucleoprotein (N) gene sequences available in the public database, five of them belong to ME1a clade and one belongs to ME1b clade. Rabies virus whole genome data is scarce across the Middle East. This study provides a better understanding of the molecular epidemiology of rabies virus in the region., Competing Interests: The authors have declared that no competing interests exist

European bat lyssavirus type 1 (EBLV-1) is the causative agent for almost all reported rabies cases found in European bats. In recent years, increasing numbers of available EBLV-1 full genomes and their phylogenetic analyses helped to further elucidate the distribution and genetic characteristics of EBLV-1 and its two subtypes, namely EBLV-1a and EBLV-1b. Nonetheless, the absence of full-genome sequences from regions with known detections of EBLV-1 still limit the understanding of the phylogeographic relations between viruses from different European regions. In this study, a set of 21 archived Danish EBLV-1 samples from the years 1985 to 2009 was processed for the acquisition of full-genome sequences using a high-throughput sequencing approach. Subsequent phylogenetic analysis encompassing all available EBLV-1 full genomes from databases revealed the Danish sequences belong to the EBLV-1a subtype and further highlighted the distinct, close phylogenetic relationship of Danish, Dutch and German isolates in this region. In addition, the formation of five putative groups nearly exclusively formed by Danish isolates and the overall increased resolution of the EBLV-1a branch indicate a higher genetic diversity and spatial segregation for this sublineage than was previously known. These results emphasize the importance of phylogenetic analyses of full-genome sequences of lyssaviruses for genetic geography.

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., Competing Interests: The authors have declared that no competing interests exist.

Emerging infectious diseases are of great concern to public health, as highlighted by the ongoing coronavirus disease 2019 (COVID-19) pandemic. Such diseases are of particular danger during mass gathering and mass influx events, as large crowds of people in close proximity to each other creates optimal opportunities for disease transmission. The Hashemite Kingdom of Jordan and the Kingdom of Saudi Arabia are two countries that have witnessed mass gatherings due to the arrival of Syrian refugees and the annual Hajj season. The mass migration of people not only brings exotic diseases to these regions but also brings new diseases back to their own countries, e.g., the outbreak of MERS in South Korea. Many emerging pathogens originate in bats, and more than 30 bat species have been identified in these two countries. Some of those bat species are known to carry viruses that cause deadly diseases in other parts of the world, such as the rabies virus and coronaviruses. However, little is known about bats and the pathogens they carry in Jordan and Saudi Arabia. Here, the importance of enhanced surveillance of bat-borne infections in Jordan and Saudi Arabia is emphasized, promoting the awareness of bat-borne diseases among the general public and building up infrastructure and capability to fill the gaps in public health preparedness to prevent future pandemics.

Rabies was first reported in ancient Iraqi civilizations, yet it remains a poorly quantified and important public health threat in the region. Efforts to control rabies in Iraq including dog population control, and vaccination of livestock and dogs, have increased since 2010. Officially reported data on human rabies, dog bites, and animal rabies cases between 2012 and 2017 are analysed here to assess the effect of existing control efforts, to inform future strategies, and to highlight gaps in surveillance and reporting. The results of molecular characterization of 32 viruses from animal cases from throughout Iraq are presented, to improve the understanding of rabies dynamics in the animal reservoir. Although annual numbers of reported human cases were lower in the period between 2012 and 2017 than prior to 2010, human cases continue. There was a distinct gender and age bias among human cases with nine cases in males for every one female and twice as many cases in children than adults. Spatial clustering analysis and phylogenetic evidence suggests rabies is endemic throughout the country, with no regional variation in risk, but better surveillance and reporting is required to underpin control strategies., Competing Interests: The authors have declared that no competing interests exist.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Brunker K et al.)

In South Africa, canid rabies virus (RABV) infection is maintained in domestic and wildlife species. The identification of rabies in African civets raised the question of whether this wildlife carnivore is a potential reservoir host of RABVs of direct and ancestral dog origin (dog-maintained and dog-derived origins) with an independent cycle of transmission. Genetic analyses of African civet nucleoprotein sequences for 23 African civet RABVs and historically published sequences demonstrated that RABVs from African civets have two origins related to dog and mongoose rabies enzootics. The data support observations of the interaction of civets with domestic dogs and wildlife mongooses, mostly in Northern South Africa and North-East Zimbabwe. Within each host species clade, African civet RABVs group exclusively together, implying intra-species virus transfer occurs readily. The canid RABV clade appears to support virus transfer more readily between hosts than mongoose RABVs. Furthermore, these data probably indicate short transmission chains with conspecifics that may be related to transient rabies maintenance in African civets. Hence, it is important to continue monitoring the emergence of lyssaviruses in this host. Observations from this study are supported by ongoing and independent similar cases, in which bat-eared foxes and black-backed jackal species maintain independent rabies cycles of what were once dog-maintained RABVs.

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

The recent discovery of novel alphacoronaviruses (alpha-CoVs) in European and Asian rodents revealed that rodent coronaviruses (CoVs) sampled worldwide formed a discrete phylogenetic group within this genus. To determine the evolutionary history of rodent CoVs in more detail, particularly the relative frequencies of virus-host co-divergence and cross-species transmission, we recovered longer fragments of CoV genomes from previously discovered European rodent alpha-CoVs using a combination of PCR and high-throughput sequencing. Accordingly, the full genome sequence was retrieved from the UK rat coronavirus, along with partial genome sequences from the UK field vole and Poland-resident bank vole CoVs, and a short conserved ORF1b fragment from the French rabbit CoV. Genome and phylogenetic analysis showed that despite their diverse geographic origins, all rodent alpha-CoVs formed a single monophyletic group and shared similar features, such as the same gene constellations, a recombinant beta-CoV spike gene, and similar core transcriptional regulatory sequences (TRS). These data suggest that all rodent alpha CoVs sampled so far originate from a single common ancestor, and that there has likely been a long-term association between alpha CoVs and rodents. Despite this likely antiquity, the phylogenetic pattern of the alpha-CoVs was also suggestive of relatively frequent host-jumping among the different rodent species.

In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Landscape heterogeneity plays an important role in disease spread and persistence, but quantifying landscape influences and their scale dependence is challenging. Studies have focused on how environmental features or global transport networks influence pathogen invasion and spread, but their influence on local transmission dynamics that underpin the persistence of endemic diseases remains unexplored. Bayesian phylogeographic frameworks that incorporate spatial heterogeneities are promising tools for analysing linked epidemiological, environmental and genetic data. Here, we extend these methodological approaches to decipher the relative contribution and scale-dependent effects of landscape influences on the transmission of endemic rabies virus in Serengeti district, Tanzania (area ~4,900 km 2 ). Utilizing detailed epidemiological data and 152 complete viral genomes collected between 2004 and 2013, we show that the localized presence of dogs but not their density is the most important determinant of diffusion, implying that culling will be ineffective for rabies control. Rivers and roads acted as barriers and facilitators to viral spread, respectively, and vaccination impeded diffusion despite variable annual coverage. Notably, we found that landscape effects were scale-dependent: rivers were barriers and roads facilitators on larger scales, whereas the distribution of dogs was important for rabies dispersal across multiple scales. This nuanced understanding of the spatial processes that underpin rabies transmission can be exploited for targeted control at the scale where it will have the greatest impact. Moreover, this research demonstrates how current phylogeographic frameworks can be adapted to improve our understanding of endemic disease dynamics at different spatial scales., (© 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

Lyssaviruses are a diverse range of viruses which all cause the disease rabies. Of the 16 recognized species, only rabies viruses (RABV) have multiple host reservoirs. Although lyssaviruses are capable of infecting all mammals, onward transmission in a new host population requires adaptation of the virus, in a number of stages with both host and virus factors determining the outcome. Due to an absence of recorded non-RABV host shifts, RABV data is extrapolated to draw conclusions for all lyssaviruses. In this article, we have focused on evidence of host shifts in the same insectivorous bat reservoir species in North America (RABV) and Europe (EBLV-1, EBLV-2 and BBLV). How RABV has successfully crossed species barriers and established infectious cycles in new hosts to be the global multi-host pathogen it is today, whilst other lyssaviruses appear restricted in host species is explored in this review. It hypothesized that RABV is the exception, rather than the rule, in this fascinating genus of viruses., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Rabies is caused by lyssaviruses, and is one of the oldest known zoonoses. In recent years, more than 21,000 nucleotide sequences of rabies viruses (RABV), from the prototype species rabies lyssavirus, have been deposited in public databases. Subsequent phylogenetic analyses in combination with metadata suggest geographic distributions of RABV. However, these analyses somewhat experience technical difficulties in defining verifiable criteria for cluster allocations in phylogenetic trees inviting for a more rational approach. Therefore, we applied a relatively new mathematical clustering algorythm named 'affinity propagation clustering' (AP) to propose a standardized sub-species classification utilizing full-genome RABV sequences. Because AP has the advantage that it is computationally fast and works for any meaningful measure of similarity between data samples, it has previously been applied successfully in bioinformatics, for analysis of microarray and gene expression data, however, cluster analysis of sequences is still in its infancy. Existing (516) and original (46) full genome RABV sequences were used to demonstrate the application of AP for RABV clustering. On a global scale, AP proposed four clusters, i.e. New World cluster, Arctic/Arctic-like, Cosmopolitan, and Asian as previously assigned by phylogenetic studies. By combining AP with established phylogenetic analyses, it is possible to resolve phylogenetic relationships between verifiably determined clusters and sequences. This workflow will be useful in confirming cluster distributions in a uniform transparent manner, not only for RABV, but also for other comparative sequence analyses.

Bat rabies cases in Europe are mainly attributed to two lyssaviruses, namely European Bat Lyssavirus 1 (EBLV-1) and European Bat Lyssavirus 2 (EBLV-2). Prior to the death of a bat worker in Finland in 1985, very few bat rabies cases were reported. Enhanced surveillance in the two subsequent years (1986-1987) identified 263 cases (more than a fifth of all reported cases to date). Between 1977 and 2016, 1183 cases of bat rabies were reported, with the vast majority (>97%) being attributed to EBLV-1. In contrast, there have been only 39 suspected cases of EBLV-2, of which 34 have been confirmed by virus typing and presently restricted to just two bat species; Myotis daubentonii and Myotis dasycneme . The limited number of EBLV-2 cases in Europe prompted the establishment of a network of European reference laboratories to collate all available viruses and data. Despite the relatively low number of EBLV-2 cases, a large amount of anomalous data has been published in the scientific literature, which we have here reviewed and clarified. In this review, 29 EBLV-2 full genome sequences have been analysed to further our understanding of the diversity and molecular evolution of EBLV-2 in Europe. Analysis of the 29 complete EBLV-2 genome sequences clearly corroborated geographical relationships with all EBLV-2 sequences clustering at the country level irrespective of the gene studied. Further geographical clustering was also observed at a local level. There are high levels of homogeneity within the EBLV-2 species with nucleotide identities ranging from 95.5-100% and amino acid identities between 98.7% and 100%, despite the widespread distribution of the isolates both geographically and chronologically. The mean substitution rate for EBLV-2 across the five concatenated genes was 1.65 × 10 -5 , and evolutionary clock analysis confirms the slow evolution of EBLV-2 both between and within countries in Europe. This is further supported by the first detailed EBLV-2 intra-roost genomic analysis whereby a relatively high sequence homogeneity was found across the genomes of three EBLV-2 isolates obtained several years apart (2007, 2008, and 2014) from M. daubentonii at the same site (Stokesay Castle, Shropshire, UK)., Competing Interests: The authors declare no conflict of interest.

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.

As part of further investigations into three linked haemorrhagic fever with renal syndrome (HFRS) cases in Wales and England, 21 rats from a breeding colony in Cherwell, and three rats from a household in Cheltenham were screened for hantavirus. Hantavirus RNA was detected in either the lungs and/or kidney of 17/21 (81%) of the Cherwell rats tested, higher than previously detected by blood testing alone (7/21, 33%), and in the kidneys of all three Cheltenham rats. The partial L gene sequences obtained from 10 of the Cherwell rats and the three Cheltenham rats were identical to each other and the previously reported UK Cherwell strain. Seoul hantavirus (SEOV) RNA was detected in the heart, kidney, lung, salivary gland and spleen (but not in the liver) of an individual rat from the Cherwell colony suspected of being the source of SEOV. Serum from 20/20 of the Cherwell rats and two associated HFRS cases had high levels of SEOV-specific antibodies (by virus neutralisation). The high prevalence of SEOV in both sites and the moderately severe disease in the pet rat owners suggest that SEOV in pet rats poses a greater public health risk than previously considered.

Passive surveillance for lyssaviruses in UK bats has been ongoing since 1987 and has identified 13 cases of EBLV-2 from a single species; Myotis daubentonii. No other lyssavirus species has been detected. Between 2005 and 2015, 10 656 bats were submitted, representing 18 species, creating a spatially and temporally uneven sample of British bat fauna. Uniquely, three UK cases originate from a roost at Stokesay Castle in Shropshire, England, where daily checks for grounded and dead bats are undertaken and bat carcasses have been submitted for testing since 2007. Twenty per cent of Daubenton's bats submitted from Stokesay Castle since surveillance began, have tested positive for EBLV-2. Phylogenetic analysis reveals geographical clustering of UK viruses. Isolates from Stokesay Castle are more closely related to one another than to viruses from other regions. Daubenton's bats from Stokesay Castle represent a unique opportunity to study a natural population that appears to maintain EBLV-2 infection and may represent endemic infection at this site. Although the risk to public health from EBLV-2 is low, consequences of infection are severe and effective communication on the need for prompt post-exposure prophylaxis for anyone that has been bitten by a bat is essential.

Canine distemper virus (CDV) has been implicated in population declines of wildlife, including many threatened species. Here we present the full genome of CDV from an Ethiopian wolf, Canis simensis , the world's rarest and most endangered canid., (© Crown copyright 2017.)

Background: In Europe, bat rabies is primarily attributed to European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) which are both strongly host-specific. Approximately thirty cases of infection with EBLV-2 in Daubenton's bats (Myotis daubentonii) and pond bats (M. dasycneme) have been reported. Two human cases of rabies caused by EBLV-2 have also been confirmed during the last thirty years, while natural spill-over to other non-flying mammals has never been reported. Rabies has never been diagnosed in mainland Norway previously., Case Presentation: In late September 2015, a subadult male Daubenton's bat was found in a poor condition 800 m above sea level in the southern part of Norway. The bat was brought to the national Bat Care Centre where it eventually displayed signs of neurological disease and died after two days. EBLV-2 was detected in brain tissues by polymerase chain reaction (PCR) followed by sequencing of a part of the nucleoprotein gene, and lyssavirus was isolated in neuroblastoma cells., Conclusions: The detection of EBLV-2 in a bat in Norway broadens the knowledge on the occurrence of this zoonotic agent. Since Norway is considered free of rabies, adequate information to the general public regarding the possibility of human cases of bat-associated rabies should be given. No extensive surveillance of lyssavirus infections in bats has been conducted in the country, and a passive surveillance network to assess rabies prevalence and bat epidemiology is highly desired.

A brain homogenate derived from a rabid dog in the district of Tojikobod, Republic of Tajikistan, was applied to a Flinders Technology Associates (FTA) card. A full-genome sequence of rabies virus (RABV) was generated from the FTA card directly without extraction, demonstrating the utility of these cards for readily obtaining genetic data., (© Crown copyright 2017.)

Background: The recommended screening of rabies in 'suspect' animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed., Results: Rabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases., Conclusions: Histopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

All lyssaviruses (family Rhabdoviridae) cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Using next-generation sequencing, the full-genome sequence for a novel lyssavirus, Lleida bat lyssavirus (LLEBV), from the original brain of a common bent-winged bat has been confirmed., (© Crown copyright 2017.)

Rabies virus kills tens of thousands of people globally each year, especially in resource-limited countries. Yet, there are genetically- and antigenically-related lyssaviruses, all capable of causing the disease rabies, circulating globally among bats without causing conspicuous disease outbreaks. The species richness and greater genetic diversity of African lyssaviruses, along with the lack of antibody cross-reactivity among them, has led to the hypothesis that Africa is the origin of lyssaviruses. This hypothesis was tested using a probabilistic phylogeographical approach. The nucleoprotein gene sequences from 153 representatives of 16 lyssavirus species, collected between 1956 and 2015, were used to develop a phylogenetic tree which incorporated relevant geographic and temporal data relating to the viruses. In addition, complete genome sequences from all 16 (putative) species were analysed. The most probable ancestral distribution for the internal nodes was inferred using three different approaches and was confirmed by analysis of complete genomes. These results support a Palearctic origin for lyssaviruses (posterior probability = 0.85), challenging the 'out of Africa' hypothesis, and suggest three independent transmission events to the Afrotropical region, representing the three phylogroups that form the three major lyssavirus clades., Competing Interests: The authors have declared that no competing interests exist.

A novel lyssavirus was isolated from brains of Indian flying foxes (Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.

Hantaviruses are emerging zoonotic viruses that cause human diseases. In this study, sera from 642 mammals from La Réunion and Mayotte islands (Indian Ocean) were screened for the presence of hantaviruses by molecular analysis. None of the mammals from La Réunion island was positive, but hantavirus genomic RNA was discovered in 29/160 (18 %) Rattus rattus from Mayotte island. The nucleoprotein coding region was sequenced from the liver and spleen of all positive individuals allowing epidemiological and intra-strain variability analyses. Phylogenetic analysis based on complete coding genomic sequences showed that this Murinae-associated hantavirus is a new variant of Thailand virus. Further studies are needed to investigate hantaviruses in rodent hosts and in Haemorrhagic Fever with Renal Syndrome (HFRS) human cases.

Using next-generation sequencing technologies, the first complete genome sequence of Rift Valley fever virus strain Lunyo is reported here. Originally reported as an attenuated antigenic variant strain from Uganda, genomic sequence analysis shows that Lunyo clusters together with other Ugandan isolates., (Copyright © 2016 Lumley et al.)

Ljungan virus (LV) (family Picornaviridae, genus Parechovirus) is a suspected zoonotic pathogen with associations to human disease in Sweden. LV is a single-stranded RNA virus with a positive sense genome. There are five published Ljungan virus strains, three isolated from Sweden and two from America, and are classified into four genotypes. A further two strains described here were isolated from wild bank voles (Myodes glareolus) caught in Västmanlands county, Sweden in 1994. These strains were sequenced using next generation pyrosequencing technology on the GS454flx platform. Genetic and phylogenetic analysis of the obtained genomes confirms isolates LV340 and LV342 as two new putative members of genotype 2 along with LV145SL, with 92% and 99% nucleotide identities respectively. Only two codon sites throughout the entire genome were identified as undergoing positive selection, both situated within the VP3 structural region, in or near to major antigenic sites. Whilst these two strains do not constitute new genotypes they provide evidence, though weakly supported, which suggests the evolution of Ljungan viruses to be relatively slow, a characteristic unlike other picornaviruses. Additional genomic sequences are urgently required for Ljungan virus strains, particularly from different locations or hosts, to fully understand the evolutionary and epidemiological properties of this potentially zoonotic virus., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

South African rabies viruses (RABVs) from dogs and jackals (canid viruses) are highly related and most likely originated from a single progenitor. RABV is the cause of most global human rabies cases. The complete genome sequences of 3 RABVs from South Africa and Zimbabwe are reported here., (Copyright © 2015 Sabeta et al.)

Many of the pathogens perceived to pose the greatest risk to humans are viral zoonoses, responsible for a range of emerging and endemic infectious diseases. Phylogeography is a useful tool to understand the processes that give rise to spatial patterns and drive dynamics in virus populations. Increasingly, whole-genome information is being used to uncover these patterns, but the limits of phylogenetic resolution that can be achieved with this are unclear. Here, whole-genome variation was used to uncover fine-scale population structure in endemic canine rabies virus circulating in Tanzania. This is the first whole-genome population study of rabies virus and the first comprehensive phylogenetic analysis of rabies virus in East Africa, providing important insights into rabies transmission in an endemic system. In addition, sub-continental scale patterns of population structure were identified using partial gene data and used to determine population structure at larger spatial scales in Africa. While rabies virus has a defined spatial structure at large scales, increasingly frequent levels of admixture were observed at regional and local levels. Discrete phylogeographic analysis revealed long-distance dispersal within Tanzania, which could be attributed to human-mediated movement, and we found evidence of multiple persistent, co-circulating lineages at a very local scale in a single district, despite on-going mass dog vaccination campaigns. This may reflect the wider endemic circulation of these lineages over several decades alongside increased admixture due to human-mediated introductions. These data indicate that successful rabies control in Tanzania could be established at a national level, since most dispersal appears to be restricted within the confines of country borders but some coordination with neighbouring countries may be required to limit transboundary movements. Evidence of complex patterns of rabies circulation within Tanzania necessitates the use of whole-genome sequencing to delineate finer scale population structure that can that can guide interventions, such as the spatial scale and design of dog vaccination campaigns and dog movement controls to achieve and maintain freedom from disease.

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., (Copyright © 2015 Atkinson et al.)

Genetic sequences of a highly pathogenic avian influenza (H5N8) virus in England have high homology to those detected in mainland Europe and Asia during 2014. Genetic characterization suggests this virus is an avian-adapted virus without specific affinity for zoonoses. Spatio-temporal detections of H5N8 imply a role for wild birds in virus spread.

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., (Copyright © 2015 Crown Copyright.)

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.

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., (Copyright © 2015 Marston et al.)

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.

Carcases received for rabies diagnosis are occasionally decomposed due to delays in finding, submitting or storing them. Positive diagnostic results from such samples are reliable but negative results may be invalid. Previous studies assessed the effect of decomposition on rabies detection using excised brains. To better reflect decomposition in the field, intact infected mouse carcases were stored at three temperatures for up to 70 days. The brains were then removed and tested using routine rabies diagnostic assays. Rabies virus was isolated using the Rabies Tissue Culture Inoculation Test (RTCIT) on days 18, 3 and 3 at 4°C, 25°C and 35°C, respectively. The Fluorescent Antibody Test (FAT) detected viral antigen on days 36, 12 and 3, whilst a rabies specific Hemi-nested RT-PCR detected viral RNA on days 70, 48 and 48 at 4°C, 25°C and 35°C, respectively. These findings suggest the persistence of infectious rabies virus in carcases left for 18 days at cold temperatures (4°C) and up to 3 days in temperatures reaching 35°C. The detection of viral RNA from a carcase decomposing at 35°C for 48 days supports the use of molecular assays to accompany OIE-prescribed rabies diagnostic tests particularly when decomposed samples are likely to be submitted. Count=199., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

The "gold standard" for post-mortem rabies diagnosis is the direct fluorescent antibody test (FAT). However, in the case of ante-mortem non-neural sample material or decomposed tissues, the FAT reaches its limit, and the use of molecular techniques can be advantageous. In this study, we developed and validated a reverse transcription PCR cascade protocol feasible for the classification of samples, even those for which there is no epidemiological background knowledge. This study emphasises on the most relevant European lyssaviruses. In a first step, two independent N- and L-gene based pan-lyssavirus intercalating dye assays are performed in a double-check application to increase the method's diagnostic safety. For the second step, characterization of the lyssavirus positive samples via two independent multiplex PCR-systems was performed. Both assays were probe-based, species-specific multiplex PCR-systems for Rabies virus, European bat lyssavirus type 1 and 2 as well as Bokeloh bat lyssavirus. All assays were validated successfully with a comprehensive panel of lyssavirus positive samples, as well as negative material from various host species. This double-check strategy allows for both safe and sensitive screening, detection and characterization of all lyssavirus species of humans and animals, as well as the rapid identification of currently unknown lyssaviruses in bats in Europe., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

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.

Background: Hantaviruses are single-stranded RNA viruses, which are transmitted to humans primarily via inhalation of aerosolised virus in contaminated rodent urine and faeces. Whilst infected reservoir hosts are asymptomatic, human infections can lead to two clinical manifestations, haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with varying degrees of clinical severity. The incidence of rodent and human cases of Seoul virus (SEOV) in Europe has been considered to be low, and speculated to be driven by the sporadic introduction of infected brown rats (Rattus norvegicus) via ports., Methods: Between October 2010 and March 2012, 128 brown rats were caught at sites across the Lyon region in France., Results: SEOV RNA was detected in the lungs of 14% (95% CI 8.01-20.11) of brown rats tested using a nested pan-hantavirus RT-PCR (polymerase gene). Phylogenetic analysis supports the inclusion of the Lyon SEOV within Lineage 7 with SEOV strains originating from SE Asia and the previously reported French & Belgian SEOV strains. Sequence data obtained from the recent human SEOV case (Replonges) was most similar to that obtained from one brown rat trapped in a public park in Lyon city centre. We obtained significantly improved recovery of virus genome sequence directly from SEOV infected lung material using a simple viral enrichment approach and NGS technology., Conclusions: The detection of SEOV in two wild caught brown rats in the UK and the multiple detection of SEOV infected brown rats in the Lyon region of France, suggests that SEOV is circulating in European brown rats. Under-reporting and difficulties in identifying the hantaviruses associated with HFRS may mask the public health impact of SEOV in Europe.

Bat rabies is an emerging disease of public health significance in the Americas. The Caribbean island of Trinidad experiences periodic outbreaks within the livestock population. We performed molecular characterisation of Trinidad rabies virus (RABV) and used a Bayesian phylogeographic approach to investigate the extent to which outbreaks are a result of in situ evolution versus importation of virus from the nearby South American mainland. Trinidadian RABV sequences were confirmed as bat variant and clustered with Desmodus rotundus (vampire bat) related sequences. They fell into two largely temporally defined lineages designated Trinidad I and II. The Trinidad I lineage which included sequences from 1997-2000 (all but two of which were from the northeast of the island) was most closely related to RABV from Ecuador (2005, 2007), French Guiana (1990) and Venezuela (1993, 1994). Trinidad II comprised sequences from the southwest of the island, which clustered into two groups: Trinidad IIa, which included one sequence each from 2000 and 2007, and Trinidad IIb including all 2010 sequences. The Trinidad II sequences were most closely related to sequences from Brazil (1999, 2004) and Uruguay (2007, 2008). Phylogeographic analyses support three separate RABV introductions from the mainland from which each of the three Trinidadian lineages arose. The estimated dates for the introductions and subsequent lineage expansions suggest periods of in situ evolution within Trinidad following each introduction. These data also indicate co-circulation of Trinidad lineage I and IIa during 2000. In light of these findings and the likely vampire bat origin of Trinidadian RABV, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology, in particular any movement between the mainland and Trinidad.

Louping ill virus (LIV) is a zoonotic virus causing fatal encephalitis in young sheep and grouse. We have recovered the complete genome sequence from a spinal cord sample prepared from a lamb that was naturally infected with LIV. This is only the second LIV genome sequence reported and the first prepared from a clinical sample.

Background: With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform., Results: As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers' minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed., Conclusions: The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.