Search

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.

Rabies virus causes an invariably fatal encephalitis following the onset of clinical disease. Despite the availability of safe and effective vaccines, the clinical stages of rabies encephalitis remain untreatable, with few survivors being documented. A principal obstacle to the treatment of rabies is the neurotropic nature of the virus, with the blood-brain barrier size exclusion limit rendering the delivery of antiviral drugs and molecules to the central nervous system inherently problematic. This review focuses on efforts to try and overcome barriers to molecule delivery to treat clinical rabies and overviews current progress in the development of experimental live rabies virus vaccines that may have future applications in the treatment of clinical rabies, including the attenuation of rabies virus vectors through either the duplication or mutation of existing genes or the incorporation of non-viral elements within the genome. Rabies post-infection treatment (PIT) remains the holy grail of rabies research.

Rabies remains endemic in more than 150 countries. In 99% of human cases, rabies virus is transmitted by dogs. The disease, which is nearly always fatal, is preventable by vaccines given either before and/or after exposure to a rabid animal. Numerous factors including the high cost of vaccines, the relative complexity of post-exposure vaccination protocols requiring multiple doses of vaccine, which in cases of severe exposure have to be combined with a rabies immune globulin, lack of access to health care, and insufficient surveillance contribute to the estimated 59,000 human deaths caused by rabies each year. New, less expensive and more immunogenic rabies vaccines are needed together with improved surveillance and dog rabies control to reduce the death toll of human rabies. Here, we discuss new rabies vaccines that are in clinical and pre-clinical testing and evaluate their potential to replace current vaccines.

Rabies is a deadly viral disease with an extremely high fatality rate in humans. Previously, it was suggested that an enhancement of the blood-brain barrier (BBB) permeability, which allows immune cells and/or antibodies to enter the central nervous system (CNS) tissue, is critical to clear the infection. In this study, we utilised mannitol to increase BBB permeability in mice infected with highly pathogenic silver-haired bat rabies virus (SHBRV). We found that intraperitoneal injection of mannitol causes a slight, transient increase of BBB permeability in the treated mice. SHBRV-infected mice were treated with intraperitoneally administered mannitol daily from day 3 or day 4 post-infection, but no effect of this treatment on the time of disease onset, clinical signs or survival was observed. This data indicates that the increase of BBB permeability by mannitol is not efficient in promoting CNS virus clearance in SHBRV-infected mice.

Infection with the rabies virus (RABV) causes fatal encephalitis and paralysis in humans and animals. Post-exposure prophylaxis (PEP) consists of vaccination and the injection of anti-rabies immunoglobulins (RIGs) around the (bite) wound. This is 100% effective in preventing disease if administered in a timely manner. However, the costs, the required cold chain for storage and transport and the limited availability of RIGs makes the treatment challenging. Cheaper and easier to produce alternatives are urgently needed. To aid in the discovery and development of such alternative therapeutics, we developed a physiological relevant infection model. Strips of freshly dissected swine skeletal muscle were placed under tension in culture medium and infected with RABV. Viral antigens were produced in the muscle explants and the virus production increased significantly over time, indicating that RABV infects and replicates in the muscle explants. Subsequently, in a search for inhibitors of RABV entry in muscle cells, we first screened a panel of 34 different lectins in a RABV / BHK-21J cell assay. The Urtica Dioica (stinging nettle) Agglutinin (UDA; a N-acetyl-D-glucosamine specific agglutinin) was found to be able to completely inhibit infection of cells with the RABV (EC50 8.2 μg/mL) by preventing binding of the virus to the host cell. When the infection of the muscle strips was carried out in the presence of UDA, infection of the tissue was completely prevented. We thus developed a physiological relevant RABV muscle infection model and identified an easy to produce component that (i) may serve as a reference for further studies and (ii) holds promise as an alternative for RIGs in PEP.

Rabies is a fatal encephalitis caused by an important group of viruses within the Lyssavirus genus. The prototype virus, rabies virus, is still the most commonly reported lyssavirus and causes approximately 59,000 human fatalities annually. The human and animal burden of the other lyssavirus species is undefined. The original reports for the novel lyssavirus, Kotalahti bat lyssavirus (KBLV), were based on the detection of viral RNA alone. In this report we describe the successful generation of a live recombinant virus, cSN-KBLV, where the full-length genome clone of RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of KBLV. Subsequent in vitro characterisation of cSN-KBLV is described here. In addition, the ability of a human rabies vaccine to confer protective immunity in vivo following challenge with this recombinant virus was assessed. Naïve or vaccinated mice were infected intracerebrally with a dose of 100 focus-forming units/30 µL of cSN-KBLV, all naïve mice and 8% (n = 1/12) of the vaccinated mice succumbed to the challenge, whilst 92% (n = 11/12) of the vaccinated mice survived to the end of the experiment. This report provides strong evidence for cross-neutralisation and cross-protection of cSN-KBLV using purified Vero cell rabies vaccine.

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., Author summary In this study, we performed whole genome sequencing (WGS) for rabies virus (RABV) isolates from seven samples, five of which were of stray dogs, and the other two were from foxes. Specimens were collected from animals across Jordan, including Balqa, Amman, Irbid, Tafilah, and Madaba governorates. Six out of the seven isolates were belonging to the Cosmopolitan ME2 clade, which related to the Iranian and Turkish sequences. This is not the case previously, where the majority of the Jordanian isolates belong to Cosmopolitan ME1a clade and closely related to the sequences from Israel. This shift might be due to the applied regulations across borders between Jordan and Israel. Besides the growth in travel and trade movement between Jordan and Turkey, where the latter is a border country with Iran. These collected data, where such studies are not common in the Middle East countries, will enhance our understanding of the RABV evolution and epidemiology in the region for rapid and effective response for rabies virus outbreaks.

Rabies is one of most ancient and feared viral pathogens with productive infection causing an invariably fatal encephalitis responsible for over 59,000 human deaths annually. Rabies is zoonotic being transmitted to humans in almost all cases through the bite of an infected dog. The virus is highly neurotropic and replication in the central nervous system represents a key barrier to treatment. A multifaceted approach for human rabies eradication that involves government support, disease awareness, vaccination of at-risk human populations and, most importantly, dog rabies control, is necessary to achieve the WHO goal of eliminating dog-mediated human rabies by 2030.

Funder: Royal Society Wolfson Research Merit award, 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.

Background Bovine ephemeral fever virus (Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined. Methods Colony lines of biting midges (Culicoides sonorensis) and mosquitoes (Aedes aegypti, Culex pipiens and Culex quinquefasciatus) were infected with a strain of BEFV originating from Israel by feeding on blood–virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves. Results There was no evidence of BEFV replication within mosquitoes fed on blood/virus suspensions for mosquitoes of any species tested for each of the three colony lines. In 170 C. sonorensis fed on the blood/virus suspension, BEFV RNA was detected in the bodies of 13 individuals and in the heads of two individuals, indicative of fully disseminated infections and an oral susceptibility rate of 1.2%. BEFV RNA replication was further demonstrated in all C. sonorensis that were inoculated by the intrathoracic route with virus after 5, 6 or 7 days post-infection. Despite this, transmission of BEFV could not be demonstrated when infected C. sonorensis were allowed to feed on calves. Conclusions No evidence for infection or dissemination of BEFV (bovine/Israel/2005-6) in mosquitoes of three different species was found. Evidence was found for infection of C. sonorensis by the oral route. However, attempts to transmit BEFV to calves from infected C. sonorensis failed. These results highlight the challenge of defining the natural vector of BEFV and of establishing an in vivo transmission model. The results are discussed with reference to the translation of laboratory-based studies to inference of vector competence in the field. Graphical abstract

Rabies has shaped humanity for centuries and continues to be one of the few pathogens that have a near 100% case fatality rate following the onset of clinical disease. Rabies virus, as with all related viruses within the Lyssavirus genus, is spread via the mechanistic action of the bite of an infected animal. Lyssaviruses are strongly neurotropic and as such most often require the dermal barrier to be breached to enable access to and infection of the nervous system. The domestic dog represents the principal reservoir of rabies virus globally and 99% of human cases involve the bite of an infected dog. Lyssaviruses are predominantly shed through saliva and so although a bite appears to be the most efficient mechanism of transmission rare alternative routes, including organ transplantation, have been reported. Despite the near 100% case fatality rate, post-exposure intervention can prevent the development of clinical disease and resulting fatality. Vaccines against rabies have been available, in various forms, for over 100 years and alongside the observation that passive immunisation with rabies immunoglobulin can completely prevent disease when administered pre-clinically the disease is entirely preventable. However, in endemic regions the cost and availability of post-exposure vaccines and immunoglobulins often precludes their use and rabies develops with the concomitant high fatality reported in endemic countries. The need for efficacious and yet cheaper pan-lyssavirus vaccines and biologicals to both prevent and treat rabies remains an important issue for future development. Furthermore, a lack of adequate reporting systems means that rabies is grossly underreported and that the burden of disease may be considerably higher. This chapter considers the basis of rabies classification, epidemiology and pathogenesis is reviewed highlighting areas for potential improvement in our understanding of this important group of viruses.

International audience; As a neglected zoonotic disease, rabies causes approximately 5.9 × 104 human deaths annually, primarily affecting low- and middle-income countries in Asia and Africa. In those regions, insufficient surveillance is hampering adequate medical intervention and is driving the vicious cycle of neglect. Where resources to provide laboratory disease confirmation are limited, there is a need for user-friendly and low-cost reliable diagnostic tools that do not rely on specialized laboratory facilities. Lateral flow devices (LFD) offer an alternative to conventional diagnostic methods and may strengthen control efforts in low-resource settings. Five different commercially available LFDs were compared in a multi-centered study with respect to their diagnostic sensitivity and their agreement with standard rabies diagnostic techniques. Our evaluation was conducted by several international reference laboratories using a broad panel of samples. The overall sensitivities ranged from 0% up to 62%, depending on the LFD manufacturer, with substantial variation between the different laboratories. Samples with high antigen content and high relative viral load tended to test positive more often in the Anigen/Bionote test, the latter being the one with the best performance. Still, the overall unsatisfactory findings corroborate a previous study and indicate a persistent lack of appropriate test validation and quality control. At present, the tested kits are not suitable for in-field use for rabies diagnosis, especially not for suspect animals where human contact has been identified, as an incorrect negative diagnosis may result in human casualties. This study points out the discrepancy between the enormous need for such a diagnostic tool on the one hand, and on the other hand, a number of already existing tests that are not yet ready for use.

International audience; Domestic dogs are responsible for nearly all the »59,000 global human rabies deaths that occur annually. Numerous control measures have been successful at eliminating dog-mediated human rabies deaths in upper-income countries, including dog population management, parenteral dog vaccination programs, access to human rabies vaccines, and education programs for bite prevention and wound treatment. Implementing these techniques in resource-poor settings can be challenging; perhaps the greatest challenge is maintaining adequate herd immunity in free-roaming dog populations. Oral rabies vaccines have been a cornerstone in rabies virus elimination from wildlife populations; however, oral vaccines have never been effectively used to control dog-mediated rabies. Here, we convey the perspectives of the World Organisation for Animal Health Rabies Reference Laboratory Directors, the World Organisation for Animal Health expert committee on dog rabies control, and World Health Organization regarding the role of oral vaccines for dogs. We also issue recommendations for overcoming hesitations to expedited field use of appropriate oral vaccines.

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., Author summary Rabies was first reported from the region that is modern day Iraq, yet the disease remains a poorly quantified and important public health threat there. Efforts to control rabies in Iraq including culling and vaccinating dogs, have been increased since 2010, but their effect on the disease is unknown. Officially reported data on human rabies, reported 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 virus typing of 32 viruses from animal cases from throughout Iraq are presented, to improve understanding of which strains of the virus are circulating in the country. Results of the analysis show that although annual numbers of reported human cases were lower after 2012 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 in females and twice as many cases in children than adults. Investigating the distribution of cases among governates, and reconstructing the evolutionary history of the viruses using virus typing data suggests rabies is endemic throughout the country, with no differences in risk between areas. These data together provide useful information for prioritizing control efforts, but demonstrate that better surveillance and reporting is required.

Since antiquity, rabies has remained one of the deadliest infectious diseases known to humankind, with a case fatality rate approaching 100% following the onset of clinical disease. It is present on all continents where terrestrial mammals exist, with the majority of animal and human cases being reported in the resourcelimited countries of Africa and Asia, with thousands of human rabies deaths being recorded annually. It is likely, however, that the global figure of approximately 59,000 annual human rabies fatalities is an underestimate. The impact of the disease has been reduced substantially across vast regions of the globe since the development of effective rabies vaccines. The success of different vaccines and vaccination policies in the defined 'at risk' populations has been born out of scientific innovation. Mass vaccination campaigns of animals, using parenteral vaccines to immunise companion animals, and advances in oral vaccines for wildlife, have allowed the elimination of rabies in terrestrial carnivores in several regions worldwide, including Western Europe and much of North America. In addition, human vaccines, largely used for post-exposure treatments, have reduced the burden of rabies in endemic areas.

Background Rift Valley fever phlebovirus (RVFV) is a mosquito-borne arbovirus causing severe disease in humans and livestock. It is endemic in Africa and spread to the Arabian Peninsula in 2000 raising concerns it could emerge in Europe. The ability of temperate mosquitoes from the United Kingdom (UK) to support replication and transmission of RVFV is unknown. Methods In this study, two colonised lines of Culex pipiens, wild-caught Aedes detritus and Ae. rusticus from the UK were infected with pathogenic strains of RVFV to assess their vector competence. Mosquitoes were offered artificial blood-meals containing 106 or 107 plaque forming units (PFU)/ml RVFV, simulating natural peak viraemia in young ruminants, and maintained at 20 °C or 25 °C for up to 21 days. Bodies, legs and saliva were collected and tested for the presence of viral RNA and infectious virus to determine the infection, dissemination and transmission potential. Results Across temperatures, doses and strains the average infection, dissemination and transmission rates were: 35, 13 and 5% (n = 91) for Cx. pipiens (Caldbeck); 23, 14 and 5% (n = 138) for Cx. pipiens (Brookwood); 36, 28 and 7% (n = 118) for Ae. detritus. However, despite 35% (n = 20) being susceptible to infection, Ae. rusticus did not transmit RVFV. Survival of Aedes species was negatively affected by maintenance at 25 °C compared to the more representative peak average British summer temperature of 20 °C. Increased mortality was also observed with some species infected with 107 PFU/ml compared to 106 PFU/ml. Conclusions It can be concluded that temperate mosquito species present in the UK demonstrate a transmission potential for RVFV in the laboratory but, even at high temperatures, this occurred at low efficiency. Electronic supplementary material The online version of this article (10.1186/s13071-018-2884-7) contains supplementary material, which is available to authorized users.

SUMMARYEffective methods to increase awareness of preventable infectious diseases are key components of successful control programmes. Rabies is an example of a disease with significant impact, where public awareness is variable. A recent awareness campaign in a rabies endemic region of Azerbaijan provided a unique opportunity to assess the efficacy of such campaigns. A cluster cross-sectional survey concerning rabies was undertaken following the awareness campaign in 600 households in 38 randomly selected towns, in districts covered by the campaign and matched control regions. This survey demonstrated that the relatively simple awareness campaign was effective at improving knowledge of rabies symptoms and vaccination schedules. Crucially, those in the awareness campaign group were also 1·4 times more likely to report that they had vaccinated their pets, an essential component of human rabies prevention. In addition, low knowledge of appropriate post-exposure treatment and animal sources of rabies provide information useful for future public awareness campaigns in the region and other similar areas.

The Biological Standards Commission of the World Organisation for Animal Health (OIE) oversees the preparation and validation of OIE-approved International Reference Standards for use in serological assays for detecting infectious diseases of animals or the adequacy of their immune response following vaccination against those diseases. The principal use of OIE-approved International Reference Standards is to harmonise serological testing and to promote the mutual recognition of test results for international trade. In the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, the organisation recommends the use of the OIE anti-rabies positive reference serum of dog origin to titrate serum samples in international units (IU)/ml for use in rabies serological tests. The first batch of OIE reference serum of dog origin was produced in1991 and was used internationally until the beginning of 2010. The preparation of the new batch began in 2012 and, in contrast to the previous batch, three commercial inactivated rabies vaccines based on the most frequently used vaccine strains (Pasteur Virus and Flury Low Egg Passage) were selected for the immunisation of dogs in accordance with OIE guidelines. In 2013, calibration was completed through an inter-laboratory test involving five OIE Reference Laboratories for Rabies with the Second World Health Organization (WHO) International Standard for Anti-Rabies Immunoglobulin being used as a reference standard in this calibration. After statistical analysis of the results, the consensus titre was established as 5.59 IU/ml. The technical and statistical data were submitted to the OIE for assessment. In February 2014, the OIE Biological Standards Commission adopted this serum as an OIE-approved standard reagent for rabies serology.

Lagos bat lyssavirus (LBV) comprising four lineages (A, B, C and D) can potentially cause the fatal disease rabies. Although LBV-B was initially isolated in Nigeria in 1956, there is no information on LBV lineages circulating in Nigeria. This study was undertaken for the first time to measure the neutralizing antibodies against four lineages of LBVs in straw-colored fruit bats (Eidolon helvum) in Makurdi, Nigeria. Serum samples (n = 180) collected during two periods (November 2017–March 2018 and November 2018–March 2019) from terminally bled bats captured for human consumption were tested using a modified fluorescent antibody virus neutralization (mFAVN) assay. A high proportion of bat sera (74%) neutralized at least one lineage of LBV (with reciprocal titers from 9 to >420.89) and most of them neutralized LBV-A (63%), followed by LBV-D (49%), LBV-C (45%) and LBV-B (24%). The majority of positive sera (75%, n = 100) neutralized multiple LBV lineages while the remaining 25% (n = 33) neutralized only a single lineage, i.e., LBV-A (n = 23), LBV-D (n = 8) and LBV-C (n = 2). None exclusively neutralized LBV-B. The results suggest that exposure to LBV is common in E. helvum and that LBV-A (but not LBV-B) is likely to be circulating in this region of Nigeria.

Rift Valley fever virus (RVFV) is a mosquito-borne arbovirus causing severe disease in humans and ruminants. Spread of RVFV out of Africa has raised concerns that it could emerge in Europe or the USA. Virus persistence is dependent on successful infection of, replication in, and transmission to susceptible vertebrate and invertebrate hosts, modulated by virus-host and vector-virus interactions. The principal accepted theory for the long-term maintenance of RVFV involves vertical transmission (VT) of virus to mosquito progeny, with the virus surviving long inter-epizootic periods within the egg. This VT hypothesis, however, is yet to be comprehensively proven. Here, evidence for and against the VT of RVFV is reviewed along with the identification of factors limiting its detection in natural and experimental data. The observations of VT for other arboviruses in the genera Alphavirus, Flavivirus and Orthobunyavirus are discussed within the context of RVFV. The review concludes that VT of RVFV is likely but that current data are insufficient to irrefutably prove this hypothesis.

Background The United Kingdom is considered free of autochthonous transmission of canine babesiosis although cases are reported in dogs associated with recent travel abroad. During the winter months of 2015/16, a cluster of cases of disease in dogs with signs suggestive of canine babesiosis were reported in Harlow, Essex. Methods Babesia species were detected in dog blood samples by Giemsa staining of blood smears and by pan-piroplasm PCRs. Babesia species were also detected in extracts of tick DNA using pan-piroplasm PCRs. DNA sequencing and phylogenetic analysis was used to confirm the species of Babesia present in dog blood and tick samples. Tick species were identified by PCR-sequencing based on amplification of the cytochrome c oxidase subunit one (cox1) gene. Dermacentor reticulatus ticks were sampled from field sites in England and Wales. Results Blood smear analysis on samples taken from some of the affected dogs confirmed the presence of a large Babesia species within erythrocytes. A tick recovered from one of these cases was identified as Dermacentor reticulatus, a species with a limited distribution in England and Wales, but a known vector of canine babesiosis in continental Europe. Babesia canis was subsequently identified in blood samples obtained from three clinical cases (all dogs) within the area and from ticks associated with these dogs. A field survey detected 17 adult D. reticulatus ticks from one area visited by the affected dogs. Fourteen of these ticks were shown to be positive for the B. canis parasite, implicating them as a potential source for babesiosis in Harlow. In order to assess whether the parasite is present in more than one tick population, D. reticulatus ticks from across England and Wales were screened for the presence of Babesia species. In addition to the Harlow site, a further five locations where D. reticulatus is present were screened for Babesia species. Babesia was not detected from most sites tested but one tick from a single location in Wales was positive for B. canis. Conclusions Infection with B. canis was confirmed in a number of dogs in Harlow, Essex, with no history of travel outside of the country. The same pathogen was identified in field-caught D. reticulatus ticks in the same area and is considered the likely source of infection. This highlights the need for vigilance by veterinary surgeons for future outbreaks of tick-borne disease in dogs.

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.

Background The range of vertebrate hosts on which species of mosquito blood-feed is an important parameter for identifying potential vectors and in assessing the risk of incursion and establishment of vector-borne pathogens. In the United Kingdom, studies of mosquito host range have collected relatively few specimens and used techniques that could only broadly identify host species. This study conducted intensive collection and analysis of mosquitoes from a grazing marsh environment in southeast England. This site provides extensive wetland habitat for resident and migratory birds and has abundant human nuisance biting mosquitoes. The aim was to identify the blood-feeding patterns of mosquito species present at the site which could contribute to the transmission of pathogens. Methods Twice-weekly collections of mosquitoes were made from Elmley Nature Reserve, Kent, between June and October 2014. Mosquitoes were collected using resting boxes, by aspiration from man-made structures and using a Mosquito Magnet Pro baited with 1-octen-3-ol. Blood-fed specimens were classified according to the degree of blood meal digestion using the Sella scale and vertebrate origin determined using sequencing of a fragment of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes that were morphologically cryptic were identified to species level using multiplex PCR and sequencing methods. Results A total of 20,666 mosquitoes of 11 species were collected, and 2,159 (10.4%) were blood-fed (Sella scale II-VI); of these 1,341 blood-fed specimens were selected for blood meal analysis. Vertebrate origin was successfully identified in 964 specimens (72%). Collections of blood-fed individuals were dominated by Anopheles maculipennis complex (73.5%), Culiseta annulata (21.2%) and Culex pipiens form pipiens (10.4%). Nineteen vertebrate hosts comprising five mammals and 14 birds were identified as hosts for mosquitoes, including two migratory bird species. Feeding on birds by Culex modestus and Anopheles atroparvus populations in England was demonstrated. Conclusions This study expands the vertebrate host range of mosquitoes in the Thames estuary region of the UK. Feeding on both resident and migratory bird species by potential arbovirus vectors including Cx. pipiens f. pipiens and Cx. modestus indicates the potential for enzootic transmission of an introduced arbovirus between migratory and local bird species by native mosquito species. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2098-4) contains supplementary material, which is available to authorized users.

Japanese encephalitis virus (JEV) is a significant cause of neurological disease in humans throughout Asia causing an estimated 70,000 human cases each year with approximately 10,000 fatalities. The virus contains a positive sense RNA genome within a host-derived membrane and is classified within the family Flaviviridae. Like many flaviviruses, it is transmitted by mosquitoes, particularly those of the genus Culex in a natural cycle involving birds and some livestock species. Spill-over into domestic animals results in a spectrum of disease ranging from asymptomatic infection in some species to acute neurological signs in others. The impact of JEV infection is particularly apparent in pigs. Although infection in adult swine does not result in symptomatic disease, it is considered a significant reproductive problem causing abortion, still-birth and birth defects. Infected piglets can display fatal neurological disease. Equines are also infected, resulting in non-specific signs including pyrexia, but occasionally leading to overt neurological disease that in extreme cases can lead to death. Veterinary vaccination is available for both pigs and horses. This review of JEV disease in livestock considers the current diagnostic techniques available for detection of the virus. Options for disease control and prevention within the veterinary sector are discussed. Such measures are critical in breaking the link to zoonotic transmission into the human population where humans are dead-end hosts.

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.

Background: A number of mosquito-borne viruses such as dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV) are autochthonously transmitted in Europe and six invasive mosquito species have been detected in this temperate region. This has increased the risk for the emergence of further mosquito-borne diseases. However, there is a paucity of information on whether European populations of invasive mosquito species are competent to transmit arboviruses. In this study, the susceptibility of Aedes albopictus originating from Spain and a laboratory-adapted colony of Aedes aegypti, was assessed for infection with, and transmission of Zika virus (ZIKV). Vertical transmission in both species was also assessed. Methods: Aedes albopictus colonised from eggs collected in Spain and an existing colony of Ae. aegypti were fed infectious blood meals containing ZIKV (Polynesian strain) at 1.6 × 10 7 PFU/ml. Blood-fed mosquitoes were separated and maintained at 20 °C or 25 °C. Legs, saliva and bodies were sampled from specimens at 7, 14 and 21 days post-infection (dpi) in order to determine infection, dissemination and transmission rates. All samples were analysed by real-time RT-PCR using primers targeting the ZIKV NS1 gene. Results: At 14 dpi and 21 dpi, ZIKV RNA was detected in the bodies of both species at both temperatures. However, live virus only was detected in the saliva of Ae. aegypti at 25 °C with a transmission rate of 44%. No evidence for virus expectoration was obtained for Ae. albopictus under any condition. Notably, ZIKV RNA was not detectable in the saliva of Ae. aegypti at 20 °C after 21 days. No vertical transmission of ZIKV was detected in this study. Conclusions: Experimental infection of Ae. albopictus colonized from Spain with ZIKV did not result in expectoration of virus in saliva in contrast to results for Ae. aegypti. No evidence of vertical transmission of virus was observed in this study. This suggests that this strain of Ae. albopictus is not competent for ZIKV transmission under the conditions tested. © 2019 The Author(s).

International audience

The lyssavirus genus is a diverse group of viruses all capable of causing an invariably fatal disease known as rabies, most commonly caused by the prototype species rabies virus (RABV). Alongside RABV the lyssavirus genus currently contains 15 other viruses capable of causing rabies. These viruses are broadly categorised into phylogroups according to the predicted level of vaccine protection, with protection from current vaccines and therapeutics afforded against phylogroup I but not II or III. Current evidence suggests that for a protective neutralising antibody response against RABV a neutralising antibody titre of 0.5 IU ml−1 is sufficient. This arbitrary value has been developed and promoted as a serological cut-off based on the reactivity of defined sera with a standardised dose of RABV. Studies using cross protection assays, have suggested that for protection against more divergent members of the genus, even those in phylogroup I, 10-fold or greater than the 0.5 IU ml−1 antibody titres are required. The continued discovery of novel lyssaviruses globally warrants an in-depth assessment of the protective titre required to protect against all the lyssaviruses to inform occupationally high-risk groups (e.g., scientists, bat workers and speleologists). Based on live virus neutralization assays, a minimum of 7 distinct lyssavirus glycoprotein antigens would have to be included in any pan-lyssavirus vaccine. Certainly, representative immunogens from all lyssavirus species characterized in phylogroups II and III are required to stimulate a pan-lyssavirus response.

Bats in the EU have been associated with several zoonotic viral pathogens of significance to both human and animal health. Virus discovery continues to expand the existing understating of virus classification, and the increased interest in bats globally as reservoirs or carriers of zoonotic agents has fuelled the continued detection and characterisation of new lyssaviruses and other viral zoonoses. Although the transmission of lyssaviruses from bat species to humans or terrestrial species appears rare, interest in these viruses remains, through their ability to cause the invariably fatal encephalitis—rabies. The association of bats with other viral zoonoses is also of great interest. Much of the EU is free of terrestrial rabies, but several bat species harbor lyssaviruses that remain a risk to human and animal health. Whilst the rabies virus is the main cause of rabies globally, novel related viruses continue to be discovered, predominantly in bat populations, that are of interest purely through their classification within the lyssavirus genus alongside the rabies virus. Although the rabies virus is principally transmitted from the bite of infected dogs, these related lyssaviruses are primarily transmitted to humans and terrestrial carnivores by bats. Even though reports of zoonotic viruses from bats within the EU are rare, to protect human and animal health, it is important characterise novel bat viruses for several reasons, namely: (i) to investigate the mechanisms for the maintenance, potential routes of transmission, and resulting clinical signs, if any, in their natural hosts; (ii) to investigate the ability of existing vaccines, where available, to protect against these viruses; (iii) to evaluate the potential for spill over and onward transmission of viral pathogens in novel terrestrial hosts. This review is an update on the current situation regarding zoonotic virus discovery within bats in the EU, and provides details of potential future mechanisms to control the threat from these deadly pathogens.

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.

Despite the availability of safe and effective human vaccines, rabies remains a global threat, with an estimated 60,000 human deaths annually attributed to rabies. Pre-exposure prophylaxis against rabies infection is recommended for travelers to countries where rabies is endemic, and also for those with a higher risk of exposure. In this study, the rabies-specific neutralising antibody responses in a cohort of rabies-vaccinated recipients over a period of twenty years have been assessed. In particular, the antibody response to primary vaccinations and boosters, and the waning of antibody post primary vaccination and post booster were investigated. The significance of gender, age at vaccination, vaccine manufacturer and vaccination intervals were also evaluated. These data confirm that rabies vaccination can elicit a neutralising antibody response that can remain at detectable levels for a number of years, without additional booster vaccinations. The antibody response following both primary vaccination and booster was significantly influenced by the gender of the subject (p=0.002 and 0.03 respectively), with supportive data that suggests an effect by the make of vaccine administered following primary vaccination, with significantly higher VNA titres observed for one vaccine manufactured prior to 2006 (p

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.

In Nepal, most dogs are free to roam and may transmit diseases to humans and animals. These dogs often suffer from malnutrition and lack basic health care. Minimal information is available about their demographics and about public attitudes concerning dogs and diseases. We carried out a study in Chitwan District (central Nepal), to collect baseline data on free-roaming owned dog demographics, assess knowledge, attitudes and practices of dog owners concerning dogs and rabies, evaluate rabies vaccination coverage and anthelmintic treatment of dogs, measure dogs' response to rabies vaccination and assess dog health through body condition scores and parasites. We conducted household interviews with owners of free-roaming female dogs (n = 60) and administered dogs with rabies vaccination and anthelmintics. Dog owners regularly fed free-roaming dogs but provided minimal health care; 42% of respondents did not claim ownership of the dog for which they provided care. We collected skin, faecal and blood samples for parasite identification and for measuring rabies virus-specific antibodies. Ninety-two per cent of dog owners were aware of the routes of rabies virus transmission, but only 35% described the correct post-exposure prophylaxis (PEP) following a dog bite. Twenty-seven per cent of the dogs had measurable rabies virus-specific antibody titres and 14% had received anthelmintics in the previous year. Following rabies vaccination, 97% of dogs maintained an adequate antibody titre for ≥6 months. Most dogs appeared healthy, although haemoprotozoans, endoparasites and ectoparasites were identified in 12%, 73% and 40% of the dogs, respectively. Poor skin condition and parasite load were associated. Seventy-four per cent of the females had litters in 1 year (mean litter size = 4.5). Births occurred between September and February; we estimated 60% mortality in puppies. We concluded that vaccination coverage, PEP awareness and anthelmintic treatment should be emphasized in educational programmes focussed on animal welfare, veterinary and public health.

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.

There is a public concern of rabies re-introduction to the UK, given the recent changes in pet trade with parts of Eastern Europe and an increase in the movement of puppies. A previously developed quantitative risk assessment (QRA) for rabies introduction into the UK was modified in order to assess the risk from only Eastern European Union member states. The model estimates the annual probability of rabies entering the UK and also the expected number of years between rabies introductions. The change in risk between the original model and the updated model is then assessed. While the risk has increased compared to the previous assessment, the risk still remains low, with a case expected every 317 years (5th and 95th percentile, 193 and 486 years, respectively) and an annual risk of 3.41 × 10−3 (5th and 95th percentile, 2.05 × 10−3 and 5.17 × 10−3, respectively).

Widespread deaths recently devastated the smallest known population of Ethiopian wolves. Of 7 carcasses found, all 3 tested were positive for rabies. Two wolves were subsequently vaccinated for rabies; 1 of these later died from canine distemper. Only 2 of a known population of 13 wolves survived.

Background: Crimean - Congo hemorrhagic fever (CCHF) is a severe viral disease. Slaughterhouses are potentially high risk working environments for CCHF infection due to close contact of livestock and humans. Objectives: The current study aimed at conducting a serosurvey among abattoir workers and evaluating different factors affecting the transmission of CCHF. Methods: A serosurvey was conducted to determine the frequency of Crimean-Congo haemorrhagic fever virus (CCHFV) IgG antibodies among abattoir workers in Mashhad, Northeastern Iran. Sera were collected from 136 slaughterhouse workers and assessed by the enzyme - linked immunosorbent assay (ELISA) for IgG CCHFV antibodies. In addition, a questionnaire was used to evaluate the risk factors involving in the transmission of the virus to the workers. Results: Serological evidence was observed in 39 out of 136 (29%) participants. The infection rate did not correlate with the work experience, type of livestock, and the permanent use of available personal protection equipment (PPE). However, standard hand disinfectants had a significant role in decreasing CCHFV IgG seropositivity (OR = 0.2, P = 0.004). Two out of 39 seropositive cases reported the history of hospitalization and CCHF infection diagnosis. Conclusions: The results of the study demonstrated that almost one-third of the investigated slaughterhouse workers were exposed to CCHFV, though the clinical manifestations were less than those of nosocomial transmissions. The currently used PPE could not protect workers against CCHFV infection; therefore, the need for effective preventive strategies for workers in the livestock industry should be emphasized.

Background: Crimean-Congo hemorrhagic fever (CCHF) is asymptomatic in infected animals, yet the virus poses a serious threat to humans causing a symptomatic, hemorrhagic disease with a high case-fatality rate. Numerous genera of ticks serve as both vectors and reservoirs of the Crimean-Congo hemorrhagic fever virus (CCHFV). Objectives: The aim of the present study is to determine the CCHFV prevalence in ticks from northeast Iran to establish a phylogenetic relationship of the tick-derived CCHFV strains circulating in Iran. Methods: During April to June 2015, a total of 93 hard ticks were collected from different animals in the Damghan district. The S-segment of positive samples was fully sequenced using the Sanger technique. A total of 142 CCHFV sequences comprised full-length of CCHFV sequences obtained in this study were aligned using the MAFFT algorithm, then phylogenetic tree was constructed using Geneious v 7.1.8. Results: The identified tick species included Hyalomma marginatum (6.5%), Hy. dromedarii (21.5%), Hy. anatolicum (15.1%), Hy. asiaticum (3.2%) and Hy. schulzei (2.2%), as well as Rhipicephalus sanguineus (47.3%). The CCHFV RNA was detected in 4 samples of 93 tick samples (4.3%) by RT-PCR. A total of 4 CCHFV sequences were obtained in this study clustered within clade IV (Asia-1 and Asia-2). Conclusions: We demonstrated that 4 species of hard ticks could be a vector for CCHFV in Iran. In addition, our findings indicate the circulation of CCHFV clade IV strain in the northeast of Iran and provide a solid base for more targeted surveillance and prevention programs in Iran.

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., Author summary Rabies is a neurologic disease that causes severe suffering and is almost always fatal. The disease is caused by infection with a virus of the genus Lyssavirus, of which 16 species are known. These viruses replicate in neurons, are excreted in the mouth, and are transmitted by bites. Dogs are the most important source of rabies for humans, but recently there is a relative increase in people contracting the disease from bats. To better understand the development of human rabies caused by these bat-acquired viruses, we need to study this disease in its bat host under controlled circumstances. To do so, we chose a naturally occurring lyssavirus–host combination: Lagos bat virus in straw-colored fruit bats. We compared three available strains of Lagos bat virus (all isolated from brains of this bat species) for their ability to mimic a natural infection. We used intracranial inoculation to ensure infection of the brain. All three strains infected brain neurons, resulting in fatal neurologic disease, however only two of the strains showed the ability to reach the site of excretion—the mouth—and were considered a suitable virus to use for further studies of this disease in bats.

Background Estimates of current global rabies mortality range from 26,000 to 59,000 deaths per annum. Although pre-exposure prophylaxis using inactivated rabies virus vaccines (IRVs) is effective, it requires two to three doses and is regarded as being too expensive and impractical for inclusion in routine childhood immunization programmes. Methodology/ Principal findings Here we report the development of a simian-adenovirus-vectored rabies vaccine intended to enable cost-effective population-wide pre-exposure prophylaxis against rabies. ChAdOx2 RabG uses the chimpanzee adenovirus serotype 68 (AdC68) backbone previously shown to achieve pre-exposure protection against rabies in non-human primates. ChAdOx2 differs from AdC68 in that it contains the human adenovirus serotype 5 (AdHu5) E4 orf6/7 region in place of the AdC68 equivalents, enhancing ease of manufacturing in cell lines which provide AdHu5 E1 proteins in trans. We show that immunogenicity of ChAdOx2 RabG in mice is comparable to that of AdC68 RabG and other adenovirus serotypes expressing rabies virus glycoprotein. High titers of rabies virus neutralizing antibody (VNA) are elicited after a single dose. The relationship between levels of VNA activity and rabies virus glycoprotein monomer-binding antibody differs after immunization with adenovirus-vectored vaccines and IRV vaccines, suggesting routes to further enhancement of the efficacy of the adenovirus-vectored candidates. We also demonstrate that ChAdOx2 RabG can be thermostabilised using a low-cost method suitable for clinical bio-manufacture and ambient-temperature distribution in tropical climates. Finally, we show that a dose-sparing effect can be achieved by formulating ChAdOx2 RabG with a simple chemical adjuvant. This approach could lower the cost of ChAdOx2 RabG and other adenovirus-vectored vaccines. Conclusions/ Significance ChAdOx2 RabG may prove to be a useful tool to reduce the human rabies death toll. We have secured funding for Good Manufacturing Practice- compliant bio-manufacture and Phase I clinical trial of this candidate., Author summary Rabies was, after smallpox, the second human disease for which an efficacious vaccine was developed, by Pasteur in 1885. Although it is eminently preventable, with highly efficacious vaccines available for both humans and animals, it still causes considerable mortality in low and middle-income countries. It is a particular problem in areas with the weakest healthcare and veterinary infrastructure, where achieving prompt post-exposure prophylaxis or high-coverage dog vaccination are challenging. Here, we report the development of a new candidate rabies vaccine, designed to enable low-cost single-dose pre-exposure human rabies prophylaxis in such settings. ChAdOx2 RabG is based upon a simian adenovirus-vectored candidate previously shown to achieve protection after a single dose in non-human primates, now modified to allow clinical-grade bio-manufacture. We show that it induces a potent immune response in mice, that this response can be further enhanced by a clinically-relevant adjuvant, and that we can stabilise it such that it can withstand temperatures of up to 45 °C for a month. We will be performing a clinical trial of this candidate in the near future.

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).

Investigation into mortalities within endangered species can direct conservation efforts., The Ethiopian wolf (Canis simensis) is the world’s rarest canid; ≈500 wolves remain. The largest population is found within the Bale Mountains National Park (BMNP) in southeastern Ethiopia, where conservation efforts have demonstrated the negative effect of rabies virus on wolf populations. We describe previously unreported infections with canine distemper virus (CDV) among these wolves during 2005–2006 and 2010. Death rates ranged from 43% to 68% in affected subpopulations and were higher for subadult than adult wolves (83%–87% vs. 34%–39%). The 2010 CDV outbreak started 20 months after a rabies outbreak, before the population had fully recovered, and led to the eradication of several focal packs in BMNP’s Web Valley. The combined effect of rabies and CDV increases the chance of pack extinction, exacerbating the typically slow recovery of wolf populations, and represents a key extinction threat to populations of this highly endangered carnivore.

Ownership of companion animals or pets is popular throughout the world. Unfortunately, such animals are susceptible to and potential reservoirs of zoonotic pathogens. Close proximity to and contact with pets can lead to human infections. The distribution of zoonotic diseases associated with companion animals such as dogs and cats is not uniform around the world, and moving animals between regions, countries, and continents carries with it the risk of relocating the pathogens they might harbor. Critical among these zoonotic diseases are rabies, echinococcosis, and leishmania. In addition, the protozoan parasites, Toxoplasma gondii and Giardia duodenalis, are also significant agents for human disease of pet origin. Considerable effort is applied to controlling movements of companion animals, particularly dogs, into the European Union. However, free movement of people and their pets within the European Union is a risk factor for the translocation of diseases and their vectors. This review considers the current distribution of some of these diseases, the risks associated with pet travel, and the controls implemented within Europe to prevent the free movement of zoonotic pathogens.

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.

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.

Bats are key species for ecological function, but they are also reservoirs of zoonotic agents, such as lyssaviruses that cause rabies. Little is known about the maintenance and transmission of lyssaviruses in bats, although the observation of clinically sick bats, both in experimental studies and wild bats, has at least demonstrated that lyssaviruses are capable of causing clinical disease in bat species. Despite this, extensive surveillance for diseased bats has not yielded lyssaviruses, whilst serological surveys demonstrate that bats must be exposed to lyssavirus without developing clinical disease. We hypothesize that there is endemic circulation of Lagos bat virus (LBV) in the straw-coloured fruit bat (Eidolon helvum) in Ghana, West Africa. To investigate this further, longitudinal blood sampling was undertaken quarterly between 2012 and 2014 on wild E. helvum at two sites in Ghana. Serum samples were collected and tested for LBV-neutralizing antibodies using a modified flourescent antibody virus neutralisation (FAVN) assay (n = 294) and brains from moribund or dead bats were tested for antigen and viral RNA (n = 55). Overall, 44.7% of the 304 bats sampled had LBV-neutralising antibodies. None of the brain samples from bats contained lyssavirus antigen or RNA. Together with the results of an earlier serological study, our findings demonstrate that LBV is endemic and circulates within E. helvum in Ghana even though the detection of viral infection in dead bats was unsuccessful. Confirmation that LBV infection is endemic in E. helvum in Ghana is an important finding and indicates that the potential public health threats from LBV warrant further investigation.

Rabies viruses have the highest case fatality rate of any known virus and are responsible for an estimated 60,000 deaths each year. This is despite the fact that there are highly efficacious vaccines and postexposure prophylaxis available. However, while it is assumed these biologics provide protection against all rabies virus isolates, there are certain subdivisions of RABV lineages, such as within the Arctic-like RABV (AL rabies virus lineage, where data is limited and thus the potency of existing biologics has not been thoroughly assessed. By fusing the Arctic-like rabies virus envelope glycoprotein ecto- and transmembrane domains with the vesicular stomatitis virus cytoplasmic domain, a high titre (7.7 x 105− 6.1 x 106RLU/ml) pseudotyped virus was generated that was subsequently used in a pseudotyped virus neutralisation assay. These results showed that Arctic-like rabies viruses are neutralised to human, canine and feline vaccines and human post-exposure prophylaxis and this was not influenced by the swapping of the cytoplasmic domains (CVS-11 vs CVS-11etmVSVc;r= 0.99,p< 0.0001). This study supports the concept that rabies virus vaccines and newly identified mAbs are able to neutralise rabies virus variants that cluster in a monophyletic clade, referred to as phylogroup I lyssaviruses.

Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. Vector competence is a component of vectorial capacity and depends on genetic determinants affecting the ability of a vector to transmit a pathogen. These determinants affect traits such as tick-host-pathogen and susceptibility to pathogen infection. Therefore, the elucidation of the mechanisms involved in tick-pathogen interactions that affect vector competence is essential for the identification of molecular drivers for tick-borne diseases. In this review, we provide a comprehensive overview of tick-pathogen molecular interactions for bacteria, viruses, and protozoa affecting human and animal health. Additionally, the impact of tick microbiome on these interactions was considered. Results show that different pathogens evolved similar strategies such as manipulation of the immune response to infect vectors and facilitate multiplication and transmission. Furthermore, some of these strategies may be used by pathogens to infect both tick and mammalian hosts. Identification of interactions that promote tick survival, spread, and pathogen transmission provides the opportunity to disrupt these interactions and lead to a reduction in tick burden and the prevalence of tick-borne diseases. Targeting some of the similar mechanisms used by the pathogens for infection and transmission by ticks may assist in development of preventative strategies against multiple tick-borne diseases., Part of the research included in this review was supported by the Ministerio de Economia y Competitividad (Spain) grant BFU2016-79892-P and the European Union (EU) Seventh Framework Programme (FP7) ANTIGONE project number 278976. SA and AD would like to acknowledge FCT for funds to GHTM - UID/Multi/04413/2013. MV was supported by the Research Plan of the University of Castilla-La Mancha (UCLM), Spain.

An on-going surveillance program on Crimean-Congo haemorrhagic fever virus (CCHFV) in Iran has been launched since 2000. An outbreak of CCHF occurred in northern Iran between June and July 2015. Three cases were involved in this outbreak. One patient died after admission to hospital, and the others were treated successfully. Phylogenetic analysis showed that three sequences obtained from Iranian patients grouped within clade IV (Asia-1), clade V (Europe) and clade VI (Greece). The partial sequence of the strain Noshahr59 (KT588642) showed the highest similarity with other strains isolated from Russia, Kosovo and Turkey (Clade V, Europe). The genome sequence of the strain Chalous65 (KT588640) showed 100% homology to the strain AP29 isolated from Greece (DQ211638). The genome sequence of the strain Noshahr43 (KT588641) showed 88% similarity to the Pakistani and previously reported Iranian strains (AF527810, AJ538198, AY366379 and AY366373). These data support previous studies, which showed a distinct similarity between Iranian S segments of CCHFV strains with other strains within clade IV (Asia-1) and clade V (Europe). In addition, clade VI was detected for the first time in Iran. Moreover, strain Chalous65 with similar genetic characteristics to strain AP29 from Greece was isolated from a fatal human case.