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Abstract Following the first detection in the United Kingdom of Usutu virus (USUV) in wild birds in 2020, we undertook a multidisciplinary investigation that combined screening host and vector populations with interrogation of national citizen science monitoring datasets to assess the potential for population impacts on avian hosts. Pathological findings from six USUV-positive wild passerines were non-specific, highlighting the need for molecular and immunohistochemical examinations to confirm infection. Mosquito surveillance at the index site identified USUV RNA in Culex pipiens s.l. following the outbreak. Although the Eurasian blackbird (Turdus merula) is most frequently impacted by USUV in Europe, national syndromic surveillance failed to detect any increase in occurrence of clinical signs consistent with USUV infection in this species. Furthermore, there was no increase in recoveries of dead blackbirds marked by the national ringing scheme. However, there was regional clustering of blackbird disease incident reports centred near the index site in 2020 and a contemporaneous marked reduction in the frequency with which blackbirds were recorded in gardens in this area, consistent with a hypothesis of disease-mediated population decline. Combining results from multidisciplinary schemes, as we have done, in real-time offers a model for the detection and impact assessment of future disease emergence events.

Rabies virus (RABV) causes possibly the oldest disease and is responsible for an estimated >59,000 human fatalities/year. Post exposure prophylaxis (PEP), the administration of vaccine and rabies immunoglobulin, is a highly effective tool which is frequently unavailable in RABV endemic areas. Furthermore, due to the constraints of the blood-brain barrier, current PEP regimes are ineffective after the onset of clinical symptoms which invariably result in death. To circumvent this barrier, a live-attenuated recombinant RABV expressing a highly RABV-neutralising scFv antibody (62-71-3) linked to the fluorescent marker mCherry was designed. Once rescued, the resulting construct (named RABV-62scFv) was grown to high titres, its growth and cellular dissemination kinetics characterised, and the functionality of the recombinant 62-71-3 scFv assessed. Encouraging scFv production and subsequent virus neutralisation results demonstrate the potential for development of a therapeutic live-attenuated virus-based post-infection treatment (PIT) for RABV infection.

Abstract Background A number of zoonotic mosquito-borne viruses have emerged in Europe in recent decades. Batai virus (BATV), a member of the genus Orthobunyavirus, is one example of a relatively newly emerged mosquito-borne virus, having been detected in mosquitoes and livestock. We conducted vector competency studies on three mosquito species at a low temperature to assess whether Aedes and Culex mosquito species are susceptible to infection with BATV. Methods Colonised lines of Aedes aegypti and Culex pipiens and a wild-caught species, Aedes detritus, were orally inoculated with BATV strain 53.2, originally isolated from mosquitoes trapped in Germany in 2009. Groups of blood-fed female mosquitoes were maintained at 20 °C for 7 or 14 days. Individual mosquitoes were screened for the presence of BATV in body, leg and saliva samples for evidence of infection, dissemination and transmission, respectively. BATV RNA was detected by reverse transcription-PCR, and positive results confirmed by virus isolation in Vero cells. Results Aedes detritus was highly susceptible to BATV, with an infection prevalence of ≥ 80% at both measurement time points. Disseminated infections were recorded in 30.7–41.6% of Ae. detritus, and evidence of virus transmission with BATV in saliva samples (n = 1, days post-infection: 14) was observed. Relatively lower rates of infection for Ae. aegypti and Cx. pipiens were observed, with no evidence of virus dissemination or transmission at either time point. Conclusions This study shows that Ae. detritus may be a competent vector for BATV at 20 °C, whereas Ae. aegypti and Cx. pipiens were not competent. Critically, the extrinsic incubation period appears to be ≤ 7 days for Ae. detritus, which may increase the onward transmissibility potential of BATV in these populations. Graphical Abstract

Abstract Vaccine platforms enable fast development, testing, and manufacture of more affordable vaccines. Here, we evaluated Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles (OMVs) generated by genetically modified Gram‐negative bacteria, as a vaccine platform for viral pathogens. Influenza A virus hemagglutinin (HA), either physically mixed with GMMA (HA+STmGMMA mix), or covalently linked to GMMA surface (HA‐STmGMMA conjugate), significantly increased antigen‐specific humoral and cellular responses, with HA‐STmGMMA conjugate inducing further enhancement than HA+STmGMMA mix. HA‐STmGMMA conjugate protected mice from lethal challenge. The versatility for this platform was confirmed by conjugation of rabies glycoprotein (RABVG) onto GMMA through the same method. RABVG+STmGMMA mix and RABVG‐STmGMMA conjugate exhibited similar humoral and cellular response patterns and protection efficacy as the HA formulations, indicating relatively consistent responses for different vaccines based on the GMMA platform. Comparing to soluble protein, GMMA was more efficiently taken up in vivo and exhibited a B‐cell preferential uptake in the draining lymph nodes (LNs). Together, GMMA enhances immunity against viral antigens, and the platform works well with different antigens while retaining similar immunomodulatory patterns. The findings of our study imply the great potential of GMMA‐based vaccine platform also against viral infectious diseases.

Infection with the rabies virus (RABV) results in a 100% lethal neurological disease once symptoms develop. Post-exposure prophylaxis (PEP) consists of a combination of vaccination and anti-rabies immunoglobulins (RIGs); it is 100% effective if administered early after exposure. Because of its limited availability, alternatives for RIGs are needed. To that end, we evaluated a panel of 33 different lectins for their effect on RABV infection in cell culture. Several lectins, with either mannose or GlcNAc specificity, elicited anti-RABV activity, of which the GlcNAc-specific Urtica dioica agglutinin (UDA) was selected for further studies. UDA was found to prevent the entry of the virus into the host cell. To further assess the potential of UDA, a physiologically relevant RABV infection muscle explant model was developed. Strips of dissected swine skeletal muscle that were kept in a culture medium could be productively infected with the RABV. When the infection of the muscle strips was carried out in the presence of UDA, RABV replication was completely prevented. Thus, we developed a physiologically relevant RABV muscle infection model. UDA (i) may serve as a reference for further studies and (ii) holds promise as a cheap and simple-to-produce alternative for RIGs in PEP.

Summary: Biobanking infrastructures, which are crucial for responding early to new viral outbreaks, share pathogen genetic resources in an affordable, safe, and impartial manner and can provide expertise to address access and benefit-sharing issues. The European Virus Archive has had a crucial role in the global response to the COVID-19 pandemic by distributing EU-subsidised (free of charge) viral resources to users worldwide, providing non-monetary benefit sharing, implementing access and benefit-sharing compliance, and raising access and benefit-sharing awareness among members and users. All currently available SARS-CoV-2 material in the European Virus Archive catalogue, including variants of concern, are not access and benefit-sharing cases per se, but multilateral benefit-sharing has nevertheless occurred. We propose and discuss how a multilateral system enabling access and benefit-sharing from pathogen genetic resources, based on the European Virus Archive operational model, could help bridge the discrepancies between the current bilateral legal framework for pathogen genetic resources and actual pandemic response practices.

Rabies is a disease of antiquity and has a history spanning millennia ever since the first interactions between humans and dogs. The alarming fatalities caused by this disease have triggered rabies prevention strategies since the first century BC. There have been numerous attempts over the past 100 years to develop rabies vaccineswith the goal of preventing rabies in both humans and animals. Thepre-Pasteurian vaccinologists, paved the way for the actual history of rabies vaccines with the development of first generation vaccines. Further improvements for less reactive and more immunogenic vaccines have led to the expansion of embryo vaccines, tissue culture vaccines, cell culture vaccines, modified live vaccines, inactivated vaccines, and adjuvanted vaccines. The adventof recombinant technology and reverse genetics have given insight into the rabies viral genome and facilitated genome manipulations, which in turn led to the emergence of next-generation rabies vaccines, such as recombinant vaccines, viral vector vaccines, genetically modified vaccines, and nucleic acid vaccines. These vaccines were very helpful in overcoming the drawbacks of conventional rabies vaccines with increased immunogenicity and clinical efficacies. The path traversed in the development of rabies vaccines from Pasteur to the modern era vaccines, though, faced numerous challenges;these pioneering works have formed the cornerstone for the generation of thecurrent successful vaccines to prevent rabies. In the future, advancements in the scientific technologies and research focus will definitely lay the path for much more sophisticated vaccine candidates for rabies elimination.

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.

Abstract 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. Graphic abstract

Rabies is a neglected tropical disease. The prototype virus, the rabies virus, still causes tens of thousands of human fatalities annually. Rabies is one member of the genus Lyssavirus. The burden of other lyssaviruses is unclear. The continued emergence of novel lyssaviruses means that assessment of vaccine efficacy against these viruses is critical, as standard rabies vaccines are not efficacious against all lyssaviruses. Taiwan bat lyssavirus (TWBLV) was first reported in 2018 following isolation from Japanese house bats. Since the initial detection and genetic characterisation, no attempts have been made to antigenically define this virus. Due to the inaccessibility of the wildtype isolate, the successful generation of a live recombinant virus, cSN-TWBLV, is described, where the full-length genome clone of the RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of TWBLV. In vitro and in vivo characterization of cSN-TWBLV was undertaken and demonstrated evidence for cross-neutralisation of cSN-TWBLV with phylogroup I -specific sera and rabies virus standard sera. For neutralisation equivalent to 0.5 IU/mL of WHO and World Organisation of Animal Health (WOAH) sera against CVS, 0.5 IU/mL of WOAH sera and 2.5 IU/mL of WHO sera were required to neutralise cSN-TWBLV. In addition, specific sera for ARAV and EBLV-1 exhibited the highest neutralising antibody titres against cSN-TWBLV, compared to other phylogroup I-specific sera.

Abstract 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 × 107 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.

Correct mosquito species identification is essential for mosquito and disease control programs. However, this is complicated by the difficulties in morphologically identifying some mosquito species. In this study, variation of a partial sequence of the cytochrome c oxidase unit I (COI) gene was used for the molecular identification of British mosquito species and to facilitate the discovery of cryptic diversity, and monitoring invasive species. Three DNA extraction methods were compared to obtain DNA barcodes from adult specimens. In total, we analyzed 42 species belonging to the genera Aedes Meigen, 1818 (21 species), Anopheles Meigen, 1818 (7 species), Coquillettidia Theobald, 1904 (1 species), Culex Linnaeus, 1758 (6 species), Culiseta Felt, 1904 (7 species), and Orthopodomyia Theobald, 1904 (1 species). Intraspecific genetic divergence ranged from 0% to 5.4%, while higher interspecific divergences were identified between Aedes geminus Peus, 1971/Culiseta litorea (Shute, 1928) (24.6%) and Ae. geminus/An. plumbeus Stephens, 1828 (22.5%). Taxonomic discrepancy was shown between An. daciae Linton, Nicolescu & Harbach, 2004 and An. messeae Falleroni, 1828 indicating the poor resolution of the COI DNA barcoding region in separating these taxa. Other species such as Ae. cantans (Meigen, 1818)/Ae. annulipes (Meigen, 1830) showed similar discrepancies indicating some limitation of this genetic marker to identify certain mosquito species. The combination of morphology and DNA barcoding is an effective approach for the identification of British mosquitoes, for invasive mosquitoes posing a threat to the UK, and for the detection of hidden diversity within species groups.

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.

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.

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.

Abstract 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.

Abstract 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.

Abstract 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.

Urban (principally canine-mediated) rabies has been a public health risk for people living in Serbia for centuries. The first legal act in urban rabies prevention in Serbia was established in 1834 by introducing high taxes for pet dog owners. Five years later in 1839, the first set of literature describing rabies prevention was issued by the health department from The Serbian Ministry of Interior. An overview of cauterization of rabies wounds was presented as the principal method of rabies post exposure prophylaxis. In 1890, a human rabies vaccination was introduced in Serbia with the royal government directive which ordered patients to be treated at the Pasteur Institute in Budapest in receipt of rabies vaccination. Urban (canine) rabies was eliminated during the 1980s, but sylvatic (principally fox-mediated) rabies still prevailed. The last human rabies case was recorded in the Province of Kosovo and Metohija in 1980. Sylvatic rabies in Serbia is in the final stages of elimination by orally vaccinating foxes (Vulpes vulpes). The only published finding of a lyssavirus among Serbian bats was made in 1954 by Dr Milan Nikolić in the vicinity of Novi Sad. In 2006, a comprehensive two-year active surveillance program of lyssaviruses in bats in Serbia was undertaken. In this single study, all of the bats from Serbia tested negative for a lyssavirus.

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.

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

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.

Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne haemorrhagic disease that emerges to produce rapid large-scale outbreaks in livestock within sub-Saharan Africa. A range of mosquito species in Africa have been shown to transmit RVFV, and recent studies have assessed whether temperate mosquito species are also capable of transmission. In order to support vector competence studies, the ability to visualize virus localization in mosquito cells and tissue would enhance the understanding of the infection process within the mosquito body. Here, the application of in situ hybridization utilizing RNAscope® to detect RVFV infection within the mosquito species, Culex pipiens, derived from the United Kingdom was demonstrated. Extensive RVFV replication was detected in many tissues of the mosquito with the notable exception of the interior of ovarian follicles.

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.

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.

SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.

Abstract Background This field-based study examined the abundance and species complement of mosquitoes (Diptera: Culicidae) attracted to humans at four sites in the United Kingdom (UK). The study used a systematic approach to directly measure feeding by mosquitoes on humans at multiple sites and using multiple volunteers. Quantifying how frequently humans are bitten in the field by mosquitoes is a fundamental parameter in assessing arthropod-borne virus transmission. Methods Human landing catches were conducted using a standardised protocol by multiple volunteers at four rural sites between July and August 2013. Collections commenced two hours prior to sunset and lasted for a total of four hours. To reduce bias occurring due to collection point or to the individual attractiveness of the volunteer to mosquitoes, each collection was divided into eight collection periods, with volunteers rotated by randomised Latin square design between four sampling points per site. While the aim was to collect mosquitoes prior to feeding, the source of blood meals from any engorged specimens was also identified by DNA barcoding. Results Three of the four sites yielded human-biting mosquito populations for a total of 915 mosquitoes of fifteen species/species groups. Mosquito species composition and biting rates differed significantly between sites, with individual volunteers collecting between 0 and 89 mosquitoes (over 200 per hour) of up to six species per collection period. Coquillettidia richiardii (Ficalbi, 1889) was responsible for the highest recorded biting rates at any one site, reaching 161 bites per hour, whilst maximum biting rates of 55 bites per hour were recorded for Culex modestus (Ficalbi, 1889). Human-biting by Culex pipiens (L., 1758) form pipiens was also observed at two sites, but at much lower rates when compared to other species. Conclusions Several mosquito species are responsible for human nuisance biting pressure in southern England, although human exposure to biting may be largely limited to evening outdoor activities. This study indicates Cx. modestus can be a major human-biting species in the UK whilst Cx. pipiens f. pipiens may show greater opportunistic human-biting than indicated by earlier studies.

Abstract 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.

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.

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.

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 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.

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.

Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne viral disease that is transmitted by numerous species of ticks, which serve both as a reservoir and vector of CCHF virus (CCHFV). Molecular and serological tests were undertaken on hard ticks (Ixodidae spp.) and samples from livestock were collected in 2015 from Chabahar County in Southeast Iran. Using RT-PCR, the ticks were tested for the presence of CCHFV. In addition, seven livestock were serologically tested for the presence of IgG antibodies using an ELISA test. IgG antibodies against CCHFV were detected in one of 7 of the livestock that were tested. In total, 49 ticks including five species: Rhipicephalus sanguineus, Hyalomma anatolicum, Hy. asiaticum, Hy. dromedarii and Hy. marginatum with a prevalence of 46.9%, 32.7%, 4.1%, 4.1% and 2.1% respectively were identified. CCHFV was detected in three ticks among 49 collected ticks. The ticks infected with CCHFV belonged to the genus Hyalomma and Rhipicephalus. Phylogenetic analysis demonstrated that two sequences clustered in clade IV (Asia-1) and one sequence was located within clade IV (Asia-2). Most of the animal and human CCHF cases of the country are reported from Sistan and Baluchistan provinces. Regular monitoring programs in the tick population and livestock are needed in the future.

Abstract Background Determining the host feeding patterns of mosquitoes by identifying the origin of their blood-meals is an important part of understanding the role of vector species in current and future disease transmission cycles. Collecting large numbers of blood-fed mosquitoes from the field is difficult, therefore it is important to maximise the information obtained from each specimen. This study aimed to use mosquito genome sequence to identify the species within Anopheles maculipennis sensu lato (An. maculipennis s.l.), identify the vertebrate hosts of field-caught blood-fed An. maculipennis s.l. , and to test for the presence of myxoma virus (Poxviridae, genus Leporipoxvirus) in specimens found to have fed on the European rabbit (Oryctolagus cuniculus). Methods Blood-fed An. maculipennis s.l. were collected from resting sites at Elmley Nature Reserve, Kent, between June and September 2013. Hosts that An. maculipennis s.l. had fed on were determined by a PCR-sequencing approach based on the partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes were then identified to species by sequencing a region of the internal transcribed spacer-2. DNA extracts from all mosquitoes identified as having fed on rabbits were subsequently screened using PCR for the presence of myxoma virus. Results A total of 94 blood-fed Anopheles maculipennis s.l. were collected, of which 43 (46 %) provided positive blood-meal identification results. Thirty-six of these specimens were identified as Anopheles atroparvus, which had fed on rabbit (n = 33, 92 %) and cattle (n = 3, 8 %). Seven mosquitoes were identified as Anopheles messeae, which had fed on cattle (n = 6, 86 %) and dog (n = 1, 14 %). Of the 33 An. atroparvus that contained rabbit blood, nine (27 %) were positive for myxoma virus. Conclusions Results demonstrate that a single DNA extract from a blood-fed mosquito can be successfully used for molecular identification of members of the An. maculipennis complex, blood-meal identification, and for the targeted detection of a myxoma virus. This study shows that An. atroparvus has a strong feeding preference for both healthy and myxoma-infected rabbits, providing evidence that this species may play a significant role in the transmission of myxomatosis among wild rabbit populations in the United Kingdom (UK).

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.

Introduction: Grenada is a rabies endemic country, where terrestrial rabies is maintained in the small Indian mongoose (Herpestes auropunctatus). The role of bats in the epidemiology of rabies in Grenada is unknown. A 1974 report described one rabies virus positive Jamaican fruit bat (Artibeus jamaicensis), and a high seroprevalence in this species. In the current study, the natural exposure to rabies virus in Grenadian bats was re-evaluated. It is postulated that bats serve as a natural rabies reservoir, probably circulating a bat-specific rabies virus variant. Material and methods: Bats were trapped in 2015 in all six parishes of Grenada using mist- and hand nets. For the detection of rabies virus in brain tissue, the direct fluorescent antibody test (dFAT) and the reverse transcription polymerase chain reaction (RT-PCR) were used. Serum neutralizing antibodies were determined using the fluorescent antibody virus neutralization test (FAVN). Results and discussion: Brain tissue and sera from 111 insectivorous and frugivorous bats belonging to four species were tested (52 Artibeus jamaicensis, two Artibeus lituratus, 33 Glossophaga longirostris, 24 Molossus molossus). Rabies virus antigen and genomic RNA were not detected in brain tissues. Rabies virus neutralizing antibodies were detected in the sera of eight A. jamaicensis in four of the six parishes. Bats in Grenada continue to show natural exposure to rabies virus. As rabies virus was not isolated in this study, serology alone is not sufficient to determine the strain of rabies virus circulating in A. jamaicensis bats in Grenada. Conclusion: Artibeus jamaicensis appears to play a role as a reservoir bat species, which is of public health concern in Grenada. Dispersion of bats to neighboring islands is possible and serological bat surveys should be initiated in these neighboring states, especially in those areas that are free of rabies in terrestrial mammals.

The continued detection of zoonotic viral infections in bats has led to the microbial fauna of these mammals being studied at a greater level than ever before. Whilst numerous pathogens have been discovered in bat species, infection with lyssaviruses is of particular significance from a zoonotic perspective as, where human infection has been reported, it is invariably fatal. Here we review the detection of lyssaviruses within different bat species and overview what is understood regarding their maintenance and transmission following both experimental and natural infection. We discuss the relevance of these pathogens as zoonotic agents and the threat of newly discovered viruses to human populations.

Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. We hypothesize that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. On an evolutionary scale, this host–virus interaction might have resulted in the large diversity of zoonotic viruses in bats, possibly through bat viruses adapting to be more tolerant of the fever response and less virulent to their natural hosts.

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.

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.

Background: Crimean Congo hemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family and Nairovirus genus. It has a negative-sense, single stranded RNA genome approximately 19.2 kb, containing the Small, Medium, and Large segments. CCHFVs are relatively divergent in their genome sequence and grouped in seven distinct clades based on S-segment sequence analysis and six clades based on M-segment sequences. Our aim was to obtain new insights into the molecular epidemiology of CCHFV in Iran. Methods: We analyzed partial and complete nucleotide sequences of the S and M segments derived from 50 Iranian patients. The extracted RNA was amplified using one-step RT-PCR and then sequenced. The sequences were ana­lyzed using Mega5 software. Results: Phylogenetic analysis of partial S segment sequences demonstrated that clade IV-(Asia 1), clade IV-(Asia 2) and clade V-(Europe) accounted for 80 %, 4 % and 14 % of the circulating genomic variants of CCHFV in Iran respectively. However, one of the Iranian strains (Iran-Kerman/22) was associated with none of other sequences and formed a new clade (VII). The phylogenetic analysis of complete S-segment nucleotide sequences from selected Ira­nian CCHFV strains complemented with representative strains from GenBank revealed similar topology as partial sequences with eight major clusters. A partial M segment phylogeny positioned the Iranian strains in either associa­tion with clade III (Asia-Africa) or clade V (Europe). Conclusion: The phylogenetic analysis revealed subtle links between distant geographic locations, which we pro­pose might originate either from international livestock trade or from long-distance carriage of CCHFV by infected ticks via bird migration.

Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.