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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.

There are ~240 species of Culicidae in Mexico, of which some are vectors of arthropod-borne viruses such as Zika virus, dengue virus, chikungunya virus, and West Nile virus. Thus, the identification of mosquito feeding preferences is paramount to understanding of vector-host-pathogen interactions that, in turn, can aid the control of disease outbreaks. Typically, DNA and RNA are extracted separately for animal (insects and blood meal hosts) and viral identification, but this study demonstrates that multiple organisms can be analyzed from a single RNA extract. For the first time, residual DNA present in standard RNA extracts was analyzed by DNA barcoding in concert with Sanger and next-generation sequencing (NGS) to identify both the mosquito species and the source of their meals in blood-fed females caught in seven sylvan communities in Chiapas State, Mexico. While mosquito molecular identification involved standard barcoding methods, the sensitivity of blood meal identification was maximized by employing short primers with NGS. In total, we collected 1,634 specimens belonging to 14 genera, 25 subgenera, and 61 morphospecies of mosquitoes. Of these, four species were new records for Mexico ( Aedes guatemala, Ae. insolitus, Limatus asulleptus, Trichoprosopon pallidiventer ), and nine were new records for Chiapas State. DNA barcode sequences for >300 bp of the COI gene were obtained from 291 specimens, whereas 130 bp sequences were recovered from another 179 specimens. High intraspecific divergence values (>2%) suggesting cryptic species complexes were observed in nine taxa: Anopheles eiseni (5.39%), An. pseudopunctipennis (2.79%), Ae. podographicus (4.05%), Culex eastor (4.88%), Cx. erraticus (2.28%), Toxorhynchites haemorrhoidalis (4.30%), Tr. pallidiventer (4.95%), Wyeomyia adelpha/Wy. guatemala (7.30%), and Wy. pseudopecten (4.04%). The study increased the number of mosquito species known from 128 species to 138 species for Chiapas State, and 239 for Mexico as a whole. Blood meal analysis showed that Aedes angustivittatus fed on ducks and chicken, whereas Psorophora albipes fed on humans. Culex quinquefasciatus fed on diverse hosts including chicken, human, turkey, and Mexican grackle. No arbovirus RNA was detected by reverse transcriptase-polymerase chain reaction in the surveyed specimens. This study demonstrated, for the first time, that residual DNA present in RNA blood meal extracts can be used to identify host vectors, highlighting the important role of molecular approaches in both vector identification and revealing host-vector-pathogen interactions., 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. The reviewer MH declared a shared affiliation with the authors SP, PH, and NN to the handling editor at time of review., (Copyright © 2021 Hernández-Triana, Garza-Hernández, Ortega Morales, Prosser, Hebert, Nikolova, Barrero, de Luna-Santillana, González-Alvarez, Mendez-López, Chan-Chable, Fooks and Rodríguez-Pérez.)

Mosquitoes are commonly identified to species level using morphological traits, but complementary methods for identification are often necessary when specimens are collected as immature stages, stored inadequately, or when delineation of species complexes is problematic. DNA-barcoding using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene is one such tool used for the morphological identification of species. A comprehensive entomological survey of mosquito species in Mexico State identified by COI DNA barcoding and morphology is documented in this paper. Specimens were collected from all the physiographic provinces in Mexico State between 2017 and 2019. Overall, 2,218 specimens were collected from 157 localities representing both subfamilies Anophelinae and Culicinae. A species checklist that consists of 6 tribes, 10 genera, 20 subgenera, and 51 species, 35 of which are new records for Mexico State, is provided. Three hundred and forty-two COI sequences of 46 species were analysed. Mean intraspecific and interspecific distances ranged between 0% to 3.9% and from 1.2% to 25.3%, respectively. All species groups were supported by high bootstraps values in a Neighbour-Joining analysis, and new COI sequences were generated for eight species: Aedes chionotum Zavortink, Ae. vargasi Schick, Ae. gabriel Schick, Ae. guerrero Berlin, Ae. ramirezi Vargas and Downs, Haemagogus mesodentatus Komp and Kumm, Culex restrictor Dyar and Knab, and Uranotaenia geometrica Theobald. This study provides a detailed inventory of the Culicidae from Mexico State and discusses the utility of DNA barcoding as a complementary tool for accurate mosquito species identification in Mexico., (Copyright © 2020. Published by Elsevier B.V.)

Background: Aedes aegypti mosquito-borne viruses including Zika (ZIKV), dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) have emerged and re-emerged globally, resulting in an elevated burden of human disease. Aedes aegypti is found worldwide in tropical, sub-tropical, and temperate areas. The characterization of mosquito blood meals is essential to understand the transmission dynamics of mosquito-vectored pathogens., Methodology/principal Findings: Here, we report Ae. aegypti and Culex quinquefasciatus host feeding patterns and arbovirus transmission in Northern Mexico using a metabarcoding-like approach with next-generation deep sequencing technology. A total of 145 Ae. aegypti yielded a blood meal analysis result with 107 (73.8%) for a single vertebrate species and 38 (26.2%) for two or more. Among the single host blood meals for Ae. aegypti, 28.0% were from humans, 54.2% from dogs, 16.8% from cats, and 1.0% from tortoises. Among those with more than one species present, 65.9% were from humans and dogs. For Cx. quinquefasciatus, 388 individuals yielded information with 326 (84%) being from a single host and 63 (16.2%) being from two or more hosts. Of the single species blood meals, 77.9% were from dogs, 6.1% from chickens, 3.1% from house sparrows, 2.4% from humans, while the remaining 10.5% derived from other 12 host species. Among those which had fed on more than one species, 11% were from dogs and humans, and 89% of other host species combinations. Forage ratio analysis revealed dog as the most over-utilized host by Ae. aegypti (= 4.3) and Cx. quinquefasciatus (= 5.6) and the human blood index at 39% and 4%, respectively. A total of 2,941 host-seeking female Ae. aegypti and 3,536 Cx. quinquefasciatus mosquitoes were collected in the surveyed area. Of these, 118 Ae. aegypti pools and 37 Cx. quinquefasciatus pools were screened for seven arboviruses (ZIKV, DENV 1-4, CHIKV, and West Nile virus (WNV)) using qRT-PCR and none were positive (point prevalence = 0%). The 95%-exact upper limit confidence interval was 0.07% and 0.17% for Ae. aegypti and Cx. quinquefasciatus, respectively., Conclusions/significance: The low human blood feeding rate in Ae. aegypti, high rate of feeding on mammals by Cx. quinquefasciatus, and the potential risk to transmission dynamics of arboviruses in highly urbanized areas of Northern Mexico is discussed., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

The pandemic of Zika virus in 2016 and other arboviruses prompted La Rioja Government in Spain to implement an entomological surveillance program of mosquitoes (Diptera; Culicidae) in the region of La Rioja. The morphological identification was supported by genetic analysis using the COI (cytochrome c oxidase subunit I) and the ITS2 (internal transcribed spacer 2) genes. In total, we identified 24 species arranged in 6 genera: Aedes (7 species), Anopheles (4 species), Coquillettidia (1 species), Culex (7 species), Culiseta (4 species), and Uranotaenia (1 species). Aedes sticticus and Aedes geniculatus are newly reported for La Rioja region. In total, 465 COI sequences were analyzed for Culicinae and Anophelinae and 54 ITS2 sequences for Anophelinae; all individuals identified as the same species clustered together in the Neighbor Joining trees. The levels of sequence divergence based on COI ranged between 0% and 2.62%, while the interspecific genetic divergence ranged from 3.05% to 20.07%. Within the genus Culiseta, certain specimens of Culiseta annulata , Culiseta litorea, and Culiseta subochrea were morphologically misidentified due to variation in the main diagnostic characters. The interspecific genetic divergence based on the ITS2 ranged from 0% to 2.98%. An accurate identification of mosquito vectors is the first step to establish a vector surveillance program for preventing pathogen transmission.

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.

In August 2020, as part of a long-term disease surveillance programme, Usutu virus was detected in five Eurasian blackbirds ( Turdus merula ) and one house sparrow ( Passer domesticus ) from Greater London, England. This was initially detected by reverse transcription-PCR and was confirmed by virus isolation and by immunohistochemical detection of flavivirus in tissues. Phylogenetic analysis identified Usutu virus African 3.2 lineage, which is prevalent in the Netherlands and Belgium, suggesting a potential incursion from mainland Europe.

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.

West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus able to cause severe neurological disease in humans, horses and various avian species. The more severe pathological changes of neurotropic WNV infection are caused by virus neuroinvasion and/or the immunological response in the central nervous system (CNS). The extent in which inflammatory cell trafficking orchestrated by chemokines is involved in the pathogenesis of CNS lesions has not been entirely elucidated. To understand the sequence of pro-inflammatory chemokine induction during WNV encephalitis, a murine intranasal inoculation model was used. The relationship between lesional patterns in the mice CNS, the viral antigen distribution and the expression of pro-inflammatory chemokine (CCL2, CCL5 and CXCL10) were evaluated. Viral antigen was first observed in olfactory tract and pyriform cortex neurons, suggesting a retrograde neuronal infection from the olfactory nerve. A spatio-temporal association between WNV antigen and perivascular cuffs development was observed. Chemokine immunostaining was widely distributed in the brain from early stages. CCL2 immunolabelling was localised in neurons, astrocytes, microglia and endothelial cells as well as mononuclear leucocytes within perivascular cuffs. In contrast, CCL5 and CXCL10 immunostaining were mainly observed in astroglia and neurons, respectively. A strong correlation was demonstrated between the presence of perivascular cuffs and CCL2 and CCL5 expression in most brain areas, while CXCL10 was only associated with inflammatory lesions in few specific regions. Importantly, a strong correlation between WNV and CCL5 distribution was observed. However, no correlation was observed between CXCL10 and viral antigen. Neurons were confirmed as the main target cells of WNV, as well as one of the sources of CCL2, CCL5 and CXCL10. This study shows the sequence and comparative distribution pattern between histological lesions, WNV antigen and chemokine expression over the infection process. Furthermore, it identifies potential targets for immune intervention to suppress damaging chemokine responses., (© 2019 Crown copyright. Transboundary and Emerging Diseases © 2019 Blackwell Verlag GmbH.)

Many high-consequence human and animal pathogens persist in wildlife reservoirs. An understanding of the dynamics of these pathogens in their reservoir hosts is crucial to inform the risk of spill-over events, yet our understanding of these dynamics is frequently insufficient. Viral persistence in a wild bat population was investigated by combining empirical data and in-silico analyses to test hypotheses on mechanisms for viral persistence. A fatal zoonotic virus, European Bat lyssavirus type 2 (EBLV-2), in Daubenton's bats (Myotis daubentonii) was used as a model system. A total of 1839 M. daubentonii were sampled for evidence of virus exposure and excretion during a prospective nine year serial cross-sectional survey. Multivariable statistical models demonstrated age-related differences in seroprevalence, with significant variation in seropositivity over time and among roosts. An Approximate Bayesian Computation approach was used to model the infection dynamics incorporating the known host ecology. The results demonstrate that EBLV-2 is endemic in the study population, and suggest that mixing between roosts during seasonal swarming events is necessary to maintain EBLV-2 in the population. These findings contribute to understanding how bat viruses can persist despite low prevalence of infection, and why infection is constrained to certain bat species in multispecies roosts and ecosystems.

As a neglected zoonotic disease, rabies causes approximately 5.9 × 10 4 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., Competing Interests: The authors declare no conflict of interest.

Here, we report the first complete genome of a bovine ephemeral fever virus (BEFV) isolate from an infected bovine in Israel. The genome shares 95.3% identity with a Turkish genomic sequence but contains α3 and γ open reading frames that are truncated compared to those of existing BEFV genome sequences., (© Crown copyright 2019.)

During the 2013-2016 Ebola outbreak in West Africa an expert panel was established on the instructions of the UK Prime Minister to identify priority pathogens for outbreak diseases that had the potential to cause future epidemics. A total of 13 priority pathogens were identified, which led to the prioritisation of spending in emerging diseases vaccine research and development from the UK. This meeting report summarises the process used to develop the UK pathogen priority list, compares it to lists generated by other organisations (World Health Organisation, National Institutes of Allergy and Infectious Diseases) and summarises clinical progress towards the development of vaccines against priority diseases. There is clear technical progress towards the development of vaccines. However, the availability of these vaccines will be dependent on sustained funding for clinical trials and the preparation of clinically acceptable manufactured material during inter-epidemic periods., (Copyright © 2019.)

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., (Copyright © 2018. Published by Elsevier Ltd.)

The use of the rabies vaccine for post-exposure prophylaxis started as early as 1885, revealing a safe and efficient tool to prevent human rabies cases. Preventive vaccination is the basis for the control of canine-mediated rabies, which has already been eliminated from extensive parts of the world, including Europe. Plans to eliminate canine-mediated human rabies by 2030 have been agreed upon by international organisations. However, rabies vaccines are not efficacious against some divergent lyssaviruses. The presence in European indigenous bats of recently described lyssaviruses, which are not neutralised by antibody responses to existing vaccines, as well as the declaration of an imported case of an African lyssavirus, which also escapes vaccine-derived protection, leaves the European health authorities unable to provide efficacious protective vaccines to some potential situations of human exposure. All these circumstances highlight the need for a universal pan-lyssavirus rabies vaccine, able to prevent human rabies in all circumstances.

There is no antiviral treatment available once clinical disease following rabies virus infection has initiated. Considered a neglected tropical disease, >60,000 human rabies deaths are estimated each year despite the availability of pre- and post-exposure prophylaxis for pre-immunisation or administration following a potential exposure before the onset of clinical disease. Such post-exposure treatments include administration of rabies immunoglobulin (RIG) and vaccination at a distant site to prime a humoral immune response. However, current therapeutic options are limited. Regardless there is a need for molecules that target virus infection following the onset of clinical disease where the outcome of infection is invariably fatal. Numerous molecules have been assessed as potential antivirals against rabies virus (RABV) but with little promise. Favipiravir (T-705) is a broad-spectrum RNA polymerase inhibitor, which has been shown to have antiviral activity against a range of RNA viruses including some against RABV. In the present study, the utility of T-705 has been reassessed in vitro as well as in vivo in a murine model using intraperitoneal administration to investigate any immune protective effect of the molecule. In vitro T-705 effectively reduces RABV replication. However, in vivo, following assessment of various applications of the molecule in both pre- and post-exposure scenarios, the effect was limited. T-705 treatment delayed the onset of clinical signs when virus was delivered intramuscularly at a higher dose (10 6.8  TCID 50 /ml) and reduced the number of mice that developed clinical signs when virus was delivered at a lower dose (10 5.8  TCID 50 /ml) during the observation period. The day at which treatment commenced did not appear to have a statistically significant effect on the results in either experiment. The use of T-705 as a single biological entity may be limited, however, further work is required to assess the synergistic effect of T-705 as a component of a multi-drug therapy for treating human rabies infections., (Copyright © 2017. Published by Elsevier Ltd.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Rabies causes more than 60,000 human deaths annually in areas where the virus is endemic. Importantly, rabies is one of the few pathogens for which there is no treatment following the onset of clinical disease with the outcome of infection being death in almost 100% of cases. Whilst vaccination, and the combination of vaccine and rabies immunoglobulin treatment for post-exposure administration are available, no tools have been identified that can reduce or prevent rabies virus replication once clinical disease has initiated. The search for effective antiviral molecules to treat those that have already developed clinical disease associated with rabies virus infection is considered one of the most important goals in rabies research. The current study assesses a single chain antibody molecule (ScFv) based on a monoclonal antibody that potently neutralises rabies in vitro as a potential therapeutic candidate. The recombinant ScFv was generated in Nicotiana benthamiana by transient expression, and was chemically conjugated (ScFv/RVG) to a 29 amino acid peptide, specific for nicotinic acetylcholine receptor (nAchR) binding in the CNS. This conjugated molecule was able to bind nAchR in vitro and enter neuronal cells more efficiently than ScFv. The ability of the ScFv/RVG to neutralise virus in vivo was assessed using a staggered administration where the molecule was inoculated either four hours before, two days after or four days after infection. The ScFv/RVG conjugate was evaluated in direct comparison with HRIG and a potential antiviral molecule, Favipiravir (also known as T-705) to indicate whether there was greater bioavailability of the ScFv in the brains of treated mice. The study indicated that the approach taken with the ScFv/RVG conjugate may have utility in the design and implementation of novel tools targetting rabies virus infection in the brain., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Over the last 20 years, natural peptides playing a key role in defense mechanisms and innate immunity have been isolated from unicellular organisms. Amphibian skin secretes dermaseptins, 24-34 amino acids in length that have a wide antimicrobial spectrum incorporating yeast, fungi, protozoa, bacteria and enveloped viruses. The anti-rabies virus (RABV) activity of dermaseptins S3 (30aa) and S4 (28aa) from Phyllomedusa sauvagei has been investigated, and further dissected its molecular basis by comparing punctual mutation or deletion of S4 analogues. The results showed that: (1) S4 is more active than S3 against RABV infection, 89% versus 38% inhibition at 7.5 μM; (2) the 5 NH2-aa of S4 are crucial for its inhibitory potential (S4 6 - 28 lost any inhibition) but the COOH terminus stabilizes the inhibitory potential (S4 1 - 16 showed only 23% inhibition at 7.5 μM); (3) there is a correlation between viral inhibition and dermaseptin cytotoxicity, which remains however moderated for BSR cells (≤12% at 10 μM). A single mutation in position 4 (S4 M4K ) slightly reduced cytotoxicity while keeping its antiviral activity, 97% at 7.5 μM. S4 and S4 M4K showed an antiviral activity in vitro when provided 1 h after infection. In vivo experiments in mice by intramuscular injection of non-toxic doses of dermaseptin S4 M4K 1 h post-infection by a lethal dose of RABV at the same site allowed more than 50% improvement in mice survival. This study highlights the potential interest of dermaseptins as non-expansive alternatives to rabies immunoglobulins for the treatment of rabies that continues to claim about 60,000 human lives per year worldwide, almost exclusively in developing countries., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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.

In February 2019, following the annual taxon ratification vote, the order Mononegavirales was amended by the addition of four new subfamilies and 12 new genera and the creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

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.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

: 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., Competing Interests: The authors declare no conflict of interest.

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.

Disease surveillance in wildlife populations presents a logistical challenge, yet is critical in gaining a deeper understanding of the presence and impact of wildlife pathogens. Erinaceus coronavirus (EriCoV), a clade C Betacoronavirus, was first described in Western European hedgehogs (Erinaceus europaeus) in Germany. Here, our objective was to determine whether EriCoV is present, and if it is associated with disease, in Great Britain (GB). An EriCoV-specific BRYT-Green® real-time reverse transcription PCR assay was used to test 351 samples of faeces or distal large intestinal tract contents collected from casualty or dead hedgehogs from a wide area across GB. Viral RNA was detected in 10.8% (38) samples; however, the virus was not detected in any of the 61 samples tested from Scotland. The full genome sequence of the British EriCoV strain was determined using next generation sequencing; it shared 94% identity with a German EriCoV sequence. Multivariate statistical models using hedgehog case history data, faecal specimen descriptions and post-mortem examination findings found no significant associations indicative of disease associated with EriCoV in hedgehogs. These findings indicate that the Western European hedgehog is a reservoir host of EriCoV in the absence of apparent disease.

The lyssaviruses are an important group of viruses that cause a fatal encephalitis termed rabies. The prototypic lyssavirus, rabies virus, is predicted to cause more than 60 000 human fatalities annually. The burden of disease for the other lyssaviruses is undefined. The original reports for the recently described highly divergent Lleida bat lyssavirus were based on the detection of virus sequence alone. The successful isolation of live Lleida bat lyssavirus from the carcass of the original bat and in vitro characterization of this novel lyssavirus are described here. In addition, the ability of a human rabies vaccine to confer protective immunity following challenge with this divergent lyssavirus was assessed. Two different doses of Lleida bat lyssavirus were used to challenge vaccinated or naïve mice: a high dose of 100 focus-forming units (f.f.u.) 30 µl -1 and a 100-fold dilution of this dose, 1 f.f.u. 30 µl -1 . Although all naïve control mice succumbed to the 100 f.f.u. 30 µl -1 challenge, 42 % (n=5/12) of those infected intracerebrally with 1 f.f.u. 30 µl -1 survived the challenge. In the high-challenge-dose group, 42 % of the vaccinated mice survived the challenge (n=5/12), whilst at the lower challenge dose, 33 % (n=4/12) survived to the end of the experiment. Interestingly, a high proportion of mice demonstrated a measurable virus-neutralizing antibody response, demonstrating that neutralizing antibody titres do not necessarily correlate with the outcome of infection via the intracerebral route. Assessing the ability of existing rabies vaccines to protect against novel divergent lyssaviruses is important for the development of future public health strategies.

Ticks are one of the principal arthropod vectors of human and animal infectious diseases. Whereas the prevalence of tick-borne encephalitis virus in ticks in Europe is well studied, there is less information available on the prevalence of the other tick-borne viruses (TBVs) existing worldwide. The aim of this study was to improve the epidemiological survey tools of TBVs by the development of an efficient high-throughput test to screen a wide range of viruses in ticks.In this study, we developed a new high-throughput virus-detection assay based on parallel real-time PCRs on a microfluidic system, and used it to perform a large scale epidemiological survey screening for the presence of 21 TBVs in 18 135 nymphs of Ixodes ricinus collected from five European countries. This extensive investigation has (i) evaluated the prevalence of four viruses present in the collected ticks, (ii) allowed the identification of viruses in regions where they were previously undetected.In conclusion, we have demonstrated the capabilities of this new screening method that allows the detection of numerous TBVs in a large number of ticks. This tool represents a powerful and rapid system for TBVs surveillance in Europe and could be easily customized to assess viral emergence.

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., Competing Interests: AVSH and ADD are named inventors on a patent application relating to the development of the ChAdOx2 vector. AVSH and SJD are named inventors on a patent relating to the use of the intron-containing promoter used in ChAdOx2 RabG. The University of Oxford and the Wistar Institute have entered into a partnership to share any future revenue from development of the ChAdOx2 RabG vaccine.

Competing Interests: Competing interests: None declared.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Blackflies have negative impact on public and animal health due to the haematophagous habit of females. In recent times, in some regions in Spain, blackfly outbreaks are becoming more and more frequent, threatening the public health. However, there is still a paucity of data concerning the Spanish blackfly fauna. Correct identification of species is of paramount importance in order to provide correct information on species distribution, biology and behaviour, so that control measures could be implemented appropriately., Methods: Blackflies specimens (larvae, pupae, reared adults and biting females) were collected in the period 2015-2017 in and near rivers and streams from different regions in Spain. A modified Hotshot technique was used for the DNA extraction and the cox1 DNA barcoding region of the cytochrome c oxidase subunit 1 was sequenced from the specimens collected., Results: In total, we collected 239 specimens representing 22 species. Of these, six species are new records for the Aragón region: P. tomosvaryi, S. bertrandi, S. galloprovinciale, S. lineatum, S. rubzovianum and S. xanthinum. Cox1 DNA barcode sequences for 21 species were recovered, including four species of the genus Prosimulium and 17 species of the genus Simulium [Boophthora (1 species), Eusimulium (1 species), Nevermannia (4 species), Simulium (s.s.) (6 species), Trichodagmia (1 species) and Wilhelmia (4 species)]. For the first time the complete DNA barcodes for five species (P. tomosvaryi, S. carthusiense, S. brevidens, S. monticola and S. sergenti) were registered. Most of the specimens belonging to the same recognized species were clustered together in the neighbour-joining tree, except for S. argyreatum, S. monticola and S. variegatum. The overall genetic distance in the dataset was 0.14%. The average of the intraspecific genetic divergence within the different taxa was 1.47% (0.05-3.96%). In contrast, the interspecific divergence varied between 2.50-22.0%., Conclusions: In this study we assessed the use of the cox1 DNA barcoding region for the identification of species of blackflies in Spain. Our results showed that combining DNA barcoding with morphology enhanced our taxonomic rationale in identifying the blackflies in the country.

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.

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.

West Nile virus (WNV) is the aetiological agent of the mosquito-borne zoonotic disease West Nile fever. The virus, first isolated in Uganda in 1937, evolved into two distinct lineages in sub-Saharan Africa (SSA) that subsequently spread to most continents where the virus has evolved further as evident through phylogenetic analysis of extant genomes. Numerous published reports from the past 70 years from countries in SSA indicate that the virus is endemic across the region. However, due in part to the limited availability of diagnostic methods across large areas of the continent, the human burden of WNV is poorly understood. So too are the drivers for translocation of the virus from countries south of the Sahara Desert to North Africa and Europe. Migratory birds are implicated in this translocation although the transient viraemia, measured in days, and the time taken to migrate, measured in weeks, suggest a more complex mechanism is in play. This review considers the evidence for the presence of WNV across SSA and the role of migratory birds in the emergence of the virus in other continents.

Pathogens causing acute disease and death or lasting immunity require specific spatial or temporal processes to persist in populations. Host traits, such as maternally-derived antibody (MDA) and seasonal birthing affect infection maintenance within populations. Our study objective is to understand how viral and host traits lead to population level infection persistence when the infection can be fatal. We collected data on African fruit bats and a rabies-related virus, Lagos bat virus (LBV), including through captive studies. We incorporate these data into a mechanistic model of LBV transmission to determine how host traits, including MDA and seasonal birthing, and viral traits, such as incubation periods, interact to allow fatal viruses to persist within bat populations. Captive bat studies supported MDA presence estimated from field data. Captive bat infection-derived antibody decayed more slowly than MDA, and while faster than estimates from the field, supports field data that suggest antibody persistence may be lifelong. Unobserved parameters were estimated by particle filtering and suggest only a small proportion of bats die of disease. Pathogen persistence in the population is sensitive to this proportion, along with MDA duration and incubation period. Our analyses suggest MDA produced bats and prolonged virus incubation periods allow viral maintenance in adverse conditions, such as a lethal pathogen or strongly seasonal resource availability for the pathogen in the form of seasonally pulsed birthing., Competing Interests: The authors have declared that no competing interests exist.

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 10 6 or 10 7 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 10 7 PFU/ml compared to 10 6 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.

Lyssaviruses constitute a diverse range of viruses with the ability to cause fatal encephalitis known as rabies. Existing human rabies vaccines and post exposure prophylaxes (PEP) are based on inactivated preparations of, and neutralising antibody preparations directed against, classical rabies viruses, respectively. Whilst these prophylaxes are highly efficient at neutralising and preventing a productive infection with rabies virus, their ability to neutralise other lyssaviruses is thought to be limited. The remaining 15 virus species within the lyssavirus genus have been divided into at least three phylogroups that generally predict vaccine protection. Existing rabies vaccines afford protection against phylogroup I viruses but offer little to no protection against phylogroup II and III viruses. As such, work involving sharps with phylogroup II and III must be considered of high risk as no PEP is thought to have any effect on the prevention of a productive infection with these lyssaviruses. Whilst rabies virus itself has been characterised in a number of different animal models, data on the remaining lyssaviruses are scarce. As the lyssavirus glycoprotein is considered to be the sole target of neutralising antibodies we generated a vaccine strain of rabies using reverse genetics expressing highly divergent glycoproteins of West Caucasian Bat lyssavirus and Ikoma lyssavirus. Using these recombinants, we propose that recombinant vaccine strain derived lyssaviruses containing heterologous glycoproteins may be a suitable surrogate for wildtype viruses when assessing vaccine protection for the lyssaviruses., Competing Interests: The authors declare no conflict of interest.

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.