Tick-Borne Encephalitis Virus, United Kingdom.

During February 2018-January 2019, we conducted large-scale surveillance for the presence and prevalence of tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) in sentinel animals and ticks in the United Kingdom. Serum was collected from 1,309 deer culled across England and Scotland. Overall, 4% of samples were ELISA-positive for the TBEV serocomplex. A focus in the Thetford Forest area had the highest proportion (47.7%) of seropositive samples. Ticks collected from culled deer within seropositive regions were tested for viral RNA; 5 of 2,041 ticks tested positive by LIV/TBEV real-time reverse transcription PCR, all from within the Thetford Forest area. From 1 tick, we identified a full-length genomic sequence of TBEV. Thus, using deer as sentinels revealed a potential TBEV focus in the United Kingdom. This detection of TBEV genomic sequence in UK ticks has important public health implications, especially for undiagnosed encephalitis.

Integration of shared-pathogen networks and machine learning reveals the key aspects of zoonoses and predicts mammalian reservoirs.

Diseases that spread to humans from animals, zoonoses, pose major threats to human health. Identifying animal reservoirs of zoonoses and predicting future outbreaks are increasingly important to human health and well-being and economic stability, particularly where research and resources are limited. Here, we integrate complex networks and machine learning approaches to develop a new approach to identifying reservoirs. An exhaustive dataset of mammal-pathogen interactions was transformed into networks where hosts are linked via their shared pathogens. We present a methodology for identifying important and influential hosts in these networks. Ensemble models linking network characteristics with phylogeny and life-history traits are then employed to predict those key hosts and quantify the roles they undertake in pathogen transmission. Our models reveal drivers explaining host importance and demonstrate how these drivers vary by pathogen taxa. Host importance is further integrated into ensemble models to predict reservoirs of zoonoses of various pathogen taxa and quantify the extent of pathogen sharing between humans and mammals. We establish predictors of reservoirs of zoonoses, showcasing host influence to be a key factor in determining these reservoirs. Finally, we provide new insight into the determinants of zoonosis-sharing, and contrast these determinants across major pathogen taxa.

Potential for Zika virus transmission by mosquitoes in temperate climates.

Mosquito-borne Zika virus (ZIKV) transmission has almost exclusively been detected in the tropics despite the distributions of its primary vectors extending farther into temperate regions. Therefore, it is unknown whether ZIKV's range has reached a temperature-dependent limit, or if it can spread into temperate climates. Using field-collected mosquitoes for biological relevance, we found that two common temperate mosquito species, Aedes albopictus and Ochlerotatus detritus, were competent for ZIKV. We orally exposed mosquitoes to ZIKV and held them at between 17 and 31°C, estimated the time required for mosquitoes to become infectious, and applied these data to a ZIKV spatial risk model. We identified a minimum temperature threshold for the transmission of ZIKV by mosquitoes between 17 and 19°C. Using these data, we generated standardized basic reproduction number R0-based risk maps and we derived estimates for the length of the transmission season for recent and future climate conditions. Our standardized R0-based risk maps show potential risk of ZIKV transmission beyond the current observed range in southern USA, southern China and southern European countries. Transmission risk is simulated to increase over southern and Eastern Europe, northern USA and temperate regions of Asia (northern China, southern Japan) in future climate scenarios.

Tissue Tropisms and Transstadial Transmission of a Rickettsia Endosymbiont in the Highland Midge, Culicoides impunctatus (Diptera: Ceratopogonidae).

Rickettsia is a genus of intracellular bacteria which can manipulate host reproduction and alter sensitivity to natural enemy attack in a diverse range of arthropods. The maintenance of Rickettsia endosymbionts in insect populations can be achieved through both vertical and horizontal transmission routes. For example, the presence of the symbiont in the follicle cells and salivary glands of Bemisia whiteflies allows Belli group Rickettsia transmission via the germ line and plants, respectively. However, the transmission routes of other Rickettsia bacteria, such as those in the Torix group of the genus, remain underexplored. Through fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) screening, this study describes the pattern of Torix Rickettsia tissue tropisms in the highland midge, Culicoides impunctatus (Diptera: Ceratopogonidae). Of note is the high intensity of infection of the ovarian suspensory ligament, suggestive of a novel germ line targeting strategy. Additionally, localization of the symbiont in tissues of several developmental stages suggests transstadial transmission is a major route for ensuring maintenance of Rickettsia within C. impunctatus populations. Aside from providing insights into transmission strategies, the presence of Rickettsia bacteria in the fat body of larvae indicates potential host fitness and vector capacity impacts to be investigated in the future.IMPORTANCE Microbial symbionts of disease vectors have garnered recent attention due to their ability to alter vectorial capacity. Their consideration as a means of arbovirus control depends on symbiont vertical transmission, which leads to spread of the bacteria through a population. Previous work has identified a Rickettsia symbiont present in several species of biting midges (Culicoides spp.), which transmit bluetongue and Schmallenberg arboviruses. However, symbiont transmission strategies and host effects remain underexplored. In this study, we describe the presence of Rickettsia in the ovarian suspensory ligament of Culicoides impunctatus Infection of this organ suggests the connective tissue surrounding developing eggs is important for ensuring vertical transmission of the symbiont in midges and possibly other insects. Additionally, our results indicate Rickettsia localization in the fat body of Culicoides impunctatus As the arboviruses spread by midges often replicate in the fat body, this location implies possible symbiont-virus interactions to be further investigated.

Distinct spread of DNA and RNA viruses among mammals amid prominent role of domestic species.

AIM: Emerging infectious diseases arising from pathogen spillover from mammals to humans constitute a substantial health threat. Tracing virus origin and predicting the most likely host species for future spillover events are major objectives in One Health disciplines.We assessed patterns of virus sharing among a large diversity of mammals, including humans and domestic species. LOCATION: Global. TIME PERIOD: Current. MAJOR TAXA STUDIED: Mammals and associated viruses. METHODS: We used network centrality analysis and trait-based Bayesian hierarchical models to explore patterns of virus sharing among mammals. We analysed a global database that compiled the associations between 1,785 virus species and 725 mammalian host species as sourced from automatic screening of meta-data accompanying published nucleotide sequences between 1950 and 2019. RESULTS: We show that based on current evidence, domesticated mammals hold the most central positions in networks of known mammal-virus associations. Among entire host-virus networks, Carnivora and Chiroptera hold central positions for mainly sharing RNA viruses, whereas ungulates hold central positions for sharing both RNA and DNA viruses with other host species. We revealed strong evidence that DNA viruses were phylogenetically more host specific than RNA viruses. RNA viruses exhibited low functional host specificity despite an overall tendency to infect phylogenetically related species, signifying high potential to shift across hosts with different ecological niches. The frequencies of sharing viruses among hosts and the proportion of zoonotic viruses in hosts were larger for RNA than for DNA viruses. MAIN CONCLUSIONS: Acknowledging the role of domestic species in addition to host and virus traits in patterns of virus sharing is necessary to improve our understanding of virus spread and spillover in times of global change. Understanding multi-host virus-sharing pathways adds focus to curtail disease spread.

Global risk model for vector-borne transmission of Zika virus reveals the role of El Niño 2015.

Zika, a mosquito-borne viral disease that emerged in South America in 2015, was declared a Public Health Emergency of International Concern by the WHO in February of 2016. We developed a climate-driven R0 mathematical model for the transmission risk of Zika virus (ZIKV) that explicitly includes two key mosquito vector species: Aedes aegypti and Aedes albopictus The model was parameterized and calibrated using the most up to date information from the available literature. It was then driven by observed gridded temperature and rainfall datasets for the period 1950-2015. We find that the transmission risk in South America in 2015 was the highest since 1950. This maximum is related to favoring temperature conditions that caused the simulated biting rates to be largest and mosquito mortality rates and extrinsic incubation periods to be smallest in 2015. This event followed the suspected introduction of ZIKV in Brazil in 2013. The ZIKV outbreak in Latin America has very likely been fueled by the 2015-2016 El Niño climate phenomenon affecting the region. The highest transmission risk globally is in South America and tropical countries where Ae. aegypti is abundant. Transmission risk is strongly seasonal in temperate regions where Ae. albopictus is present, with significant risk of ZIKV transmission in the southeastern states of the United States, in southern China, and to a lesser extent, over southern Europe during the boreal summer season.

Detection of new endemic focus of tick-borne encephalitis virus (TBEV), Hampshire/Dorset border, England, September 2019.

The presence of tick-borne encephalitis virus (TBEV) was detected in a questing tick pool in southern England in September 2019. Hitherto, TBEV had only been detected in a limited area in eastern England. This southern English viral genome sequence is distinct from TBEV-UK, being most similar to TBEV-NL. The new location of TBEV presence highlights that the diagnosis of tick-borne encephalitis should be considered in encephalitic patients in areas of the United Kingdom outside eastern England.

Dairy Heifers Naturally Exposed to Fasciola hepatica Develop a Type 2 Immune Response and Concomitant Suppression of Leukocyte Proliferation.

Fasciola hepatica is a parasitic trematode of global importance in livestock. Control strategies reliant on anthelmintics are unsustainable due to the emergence of drug resistance. Vaccines are under development, but efficacies are variable. Evidence from experimental infection suggests that vaccine efficacy may be affected by parasite-induced immunomodulation. Little is known about the immune response to F. hepatica following natural exposure. Hence, we analyzed the immune responses over time in calves naturally exposed to F. hepatica infection. Cohorts of replacement dairy heifer calves (n = 42) with no prior exposure to F. hepatica, on three commercial dairy farms, were sampled over the course of a grazing season. Exposure was determined through an F. hepatica-specific serum antibody enzyme-linked immunosorbent assay (ELISA) and fluke egg counts. Concurrent changes in peripheral blood leukocyte subpopulations, lymphocyte proliferation, and cytokine responses were measured. Relationships between fluke infection and immune responses were analyzed by using multivariable linear mixed-effect models. All calves from one farm showed evidence of exposure, while cohorts from the remaining two farms remained negative over the grazing season. A type 2 immune response was associated with exposure, with increased interleukin-4 (IL-4) production, IL-5 transcription, and eosinophilia. Suppression of parasite-specific peripheral blood mononuclear cell (PBMC) proliferation was evident, while decreased mitogen-stimulated gamma interferon (IFN-γ) production suggested immunomodulation, which was not restricted to parasite-specific responses. Our findings show that the global immune response is modulated toward a nonproliferative type 2 state following natural challenge with F. hepatica This has implications in terms of the timing of the administration of vaccination programs and for host susceptibility to coinfecting pathogens.

Development and validation of a protocol to identify and recruit participants into a large scale study on liver fluke in cattle.

BACKGROUND: Liver fluke infection caused by the parasite Fasciola hepatica is a major cause of production losses to the cattle industry in the UK. To investigate farm-level risk factors for fluke infection, a randomised method to recruit an appropriate number of herds from a defined geographical area into the study was required. The approach and hurdles that were encountered in designing and implementing this study are described. The county of Shropshire, England, was selected for the study because of the variation between farms in exposure to fluke infection observed in an earlier study. RESULTS: From a sampling list of 569 holdings in Shropshire randomly drawn from the RADAR cattle population dataset, 396 (69.6%) holdings were successfully contacted by telephone and asked if they would be interested in taking part in the study. Of 296 farmers who agreed to receive information packs by post, 195 (65.9%) agreed to take part in the study. Over the period October 2014 - April 2015 visits were made to 100 dairy and 95 non-dairy herds. During the farm visits 40 faecal samples +/- bulk-tank milk samples were collected and a questionnaire administered. Composite faecal samples were analysed for the presence of F. hepatica eggs by sedimentation and bulk tank milk samples were tested with an antibody ELISA for F. hepatica. Forty-five (49%) of non-dairy herds were positive for liver fluke infection as determined by the finding of one or more fluke eggs, while 36 (36%) dairy herds had fluke positive faecal samples and 41 (41%) dairy herds were positive for F. hepatica antibody. Eighty-seven (45.8%) farmers said that they monitored their cattle for liver fluke infection and 118 (62.1%) reported that they used flukicide drugs in their cattle. CONCLUSIONS: Using a protocol of contacting farmers directly by telephone and subsequently sending information by post, 79% of the target sample size was successfully recruited into the study. A dataset of farm-specific information on possible risk factors for liver fluke infection and corresponding liver-fluke infection status was generated for the development of statistical models to identify risk factors for liver fluke infection at the farm-level.

Torix group Rickettsia are widespread in Culicoides biting midges (Diptera: Ceratopogonidae), reach high frequency and carry unique genomic features.

There is increasing interest in the heritable bacteria of invertebrate vectors of disease as they present novel targets for control initiatives. Previous studies on biting midges (Culicoides spp.), known to transmit several RNA viruses of veterinary importance, have revealed infections with the endosymbiotic bacteria, Wolbachia and Cardinium. However, rickettsial symbionts in these vectors are underexplored. Here, we present the genome of a previously uncharacterized Rickettsia endosymbiont from Culicoides newsteadi (RiCNE). This genome presents unique features potentially associated with host invasion and adaptation, including genes for the complete non-oxidative phase of the pentose phosphate pathway, and others predicted to mediate lipopolysaccharides and cell wall modification. Screening of 414 Culicoides individuals from 29 Palearctic or Afrotropical species revealed that Rickettsia represent a widespread but previously overlooked association, reaching high frequencies in midge populations and present in 38% of the species tested. Sequence typing clusters the Rickettsia within the Torix group of the genus, a group known to infect several aquatic and hematophagous taxa. FISH analysis indicated the presence of Rickettsia bacteria in ovary tissue, indicating their maternal inheritance. Given the importance of biting midges as vectors, a key area of future research is to establish the impact of this endosymbiont on vector competence.

The potential role of Wolbachia in controlling the transmission of emerging human arboviral infections.

PURPOSE OF REVIEW: Wolbachia is a genus of Gram-negative intracellular bacteria that is naturally found in more than half of all arthropod species. These bacteria cannot only reduce the fitness and the reproductive capacities of arthropod vectors, but also increase their resistance to arthropod-borne viruses (arboviruses). This article reviews the evidence supporting a Wolbachia-based strategy for controlling the transmission of dengue and other arboviral infections. RECENT FINDINGS: Studies conducted 1 year after the field release of Wolbachia-infected mosquitoes in Australia have demonstrated the suppression of dengue virus (DENV) replication in and dissemination by mosquitoes. Recent mathematical models show that this strategy could reduce the transmission of DENV by 70%. Consequently, the WHO is encouraging countries to boost the development and implementation of Wolbachia-based prevention strategies against other arboviral infections. However, the evidence regarding the efficacy of Wolbachia to prevent the transmission of other arboviral infections is still limited to an experimental framework with conflicting results in some cases. There is a need to demonstrate the efficacy of such strategies in the field under various climatic conditions, to select the Wolbachia strain that has the best pathogen interference/spread trade-off, and to continue to build community acceptance. SUMMARY: Wolbachia represents a promising tool for controlling the transmission of arboviral infections that needs to be developed further. Long-term environmental monitoring will be necessary for timely detection of potential changes in Wolbachia/vector/virus interactions.

Infectious bronchitis vaccine virus detection and part-S1 genetic variation following single or dual inoculation in broiler chicks.

An investigation was undertaken of the extent of genetic variation occurring within infectious bronchitis virus (IBV) vaccine strains following vaccination of day-old broiler chicks. Chicks were divided into seven groups, with two groups receiving single Massachusetts (Mass) vaccinations while the other four were inoculated with combinations of different IBV serotypes; Mass, 793B, D274 and Arkansas (Ark). The remaining group was maintained as an unvaccinated control. Following vaccination, swabs and tissues collected at intervals were pooled and RNA was extracted for detection of IBV by reverse transcription polymerase chain reaction. Positive amplicons were sequenced for the part-S1 gene and compared to the original vaccine strain sequences. Single nucleotide polymorphisms, amino acid variations and hydrophobicity changes were identified and recorded for each sampling point. A total of 106 single nucleotide polymorphisms were detected within 28 isolates. The average single nucleotide polymorphism counts of swab isolates were greater than those found in tissue samples. This translated into 64 amino acid changes; however only six resulted in a change to the hydrophobicity properties. All hydrophobic alterations occurred within swab isolates and the majority were recovered at 3 days post vaccination suggesting such changes to be detrimental to early virus survival. Nucleotide deletions were seen only in the group given the combination of Mass and Ark. Of the 16 sequenced samples in this group, 13 contained the same AAT deletion at position 1033 1035 in the Ark strains. Findings presented in this study demonstrate alteration in the S1 nucleotide sequence following co-administration of live IBV vaccines.

Potential impact of climate change on emerging vector-borne and other infections in the UK.

Climate is one of several causes of disease emergence. Although half or more of infectious diseases are affected by climate it appears to be a relatively infrequent cause of human disease emergence. Climate mostly affects diseases caused by pathogens that spend part of their lifecycle outside of the host, exposed to the environment. The most important routes of transmission of climate sensitive diseases are by arthropod (insect and tick) vectors, in water and in food. Given the sensitivity of many diseases to climate, it is very likely that at least some will respond to future climate change. In the case of vector-borne diseases this response will include spread to new areas. Several vector-borne diseases have emerged in Europe in recent years; these include vivax malaria, West Nile fever, dengue fever, Chikungunya fever, leishmaniasis, Lyme disease and tick-borne encephalitis. The vectors of these diseases are mosquitoes, sand flies and ticks. The UK has endemic mosquito species capable of transmitting malaria and probably other pathogens, and ticks that transmit Lyme disease. The UK is also threatened by invasive mosquito species known to be able to transmit West Nile, dengue, chikungunya and Zika, and sand flies that spread leishmaniasis. Warmer temperatures in the future will increase the suitability of the UK's climate for these invasive species, and increase the risk that they may spread disease. While much attention is on invasive species, it is important to recognize the threat presented by native species too. Proposed actions to reduce the future impact of emerging vector-borne diseases in the UK include insect control activity at points of entry of vehicles and certain goods, wider surveillance for mosquitoes and sand flies, research into the threat posed by native species, increased awareness of the medical profession of the threat posed by specific diseases, regular risk assessments, and increased preparedness for the occurrence of a disease emergency.

Climate-driven changes to the spatio-temporal distribution of the parasitic nematode, Haemonchus contortus, in sheep in Europe.

Recent climate change has resulted in changes to the phenology and distribution of invertebrates worldwide. Where invertebrates are associated with disease, climate variability and changes in climate may also affect the spatio-temporal dynamics of disease. Due to its significant impact on sheep production and welfare, the recent increase in diagnoses of ovine haemonchosis caused by the nematode Haemonchus contortus in some temperate regions is particularly concerning. This study is the first to evaluate the impact of climate change on H. contortus at a continental scale. A model of the basic reproductive quotient of macroparasites, Q0 , adapted to H. contortus and extended to incorporate environmental stochasticity and parasite behaviour, was used to simulate Pan-European spatio-temporal changes in H. contortus infection pressure under scenarios of climate change. Baseline Q0 simulations, using historic climate observations, reflected the current distribution of H. contortus in Europe. In northern Europe, the distribution of H. contortus is currently limited by temperatures falling below the development threshold during the winter months and within-host arrested development is necessary for population persistence over winter. In southern Europe, H. contortus infection pressure is limited during the summer months by increased temperature and decreased moisture. Compared with this baseline, Q0 simulations driven by a climate model ensemble predicted an increase in H. contortus infection pressure by the 2080s. In northern Europe, a temporal range expansion was predicted as the mean period of transmission increased by 2-3 months. A bimodal seasonal pattern of infection pressure, similar to that currently observed in southern Europe, emerges in northern Europe due to increasing summer temperatures and decreasing moisture. The predicted patterns of change could alter the epidemiology of H. contortus in Europe, affect the future sustainability of contemporary control strategies, and potentially drive local adaptation to climate change in parasite populations.

Heterologous live infectious bronchitis virus vaccination in day-old commercial broiler chicks: clinical signs, ciliary health, immune responses and protection against variant infectious bronchitis viruses.

Groups of one-day-old broiler chicks were vaccinated via the oculo-nasal route with different live infectious bronchitis virus (IBV) vaccines: Massachusetts (Mass), 793B, D274 or Arkansas (Ark). Clinical signs and gross lesions were evaluated. Five chicks from each group were humanely killed at intervals and their tracheas collected for ciliary activity assessment and for the detection of CD4+, CD8+ and IgA-bearing B cells by immunohistochemistry (IHC). Blood samples were collected at intervals for the detection of anti-IBV antibodies. At 21 days post-vaccination (dpv), protection conferred by different vaccination regimes against virulent M41, QX and 793B was assessed. All vaccination programmes were able to induce high levels of CD4+, CD8+ and IgA-bearing B cells in the trachea. Significantly higher levels of CD4+ and CD8+ expression were observed in the Mass2 + 793B2-vaccinated group compared to the other groups (subscripts indicate different manufacturers). Protection studies showed that the group of chicks vaccinated with Mass2 + 793B2 produced 92% ciliary protection against QX challenge; compared to 53%, 68% and 73% ciliary protection against the same challenge virus by Mass1 + D274, Mass1 + 793B1 and Mass3 + Ark, respectively. All vaccination programmes produced more than 85% ciliary protection against M41 and 793B challenges. It appears that the variable levels of protection provided by different heterologous live IBV vaccinations are dependent on the levels of local tracheal immunity induced by the respective vaccine combination. The Mass2 + 793B2 group showed the worst clinical signs, higher mortality and severe lesions following vaccination, but had the highest tracheal immune responses and demonstrated the best protection against all three challenge viruses.

Japanese encephalitis: on the One Health agenda.

Japanese encephalitis (JE) is one of the most well studied arthropod zoonotic diseases with human and animal research and their integration spanning 6-7 decades. JE research and policy in some Asian countries has epitomized the 'One Health' strategy of attainment of optimal health for people, animals, and the environment. However, despite significant mitigation of JE in some Asian countries primarily due to vaccination programs and infrastructural development, JE continues to be a major disease burden in the Asian region. Arthropod-borne zoonotic infections such as JE present some of the greatest challenges to animal and human health globally. Their emergence involves a complex interplay of vectors, hosts, environment, climate, and anthropogenic factors. Therefore, the integrated management of infectious agents that affect both humans and animals is perhaps the most highly coveted strategy that public health policy makers aspire to attain in the twenty-first century. This is in response to the seemingly growing challenges of controlling the burden of emerging infectious diseases such as shrinking financial budgets and resources, increasing demand for public health deliverables, demographic shifts and mobility, global trade economies, and climate and landscape changes. Thus, while JE research and policy is an excellent example of the One Health strategy in action, further work is required to address the obstinate burden of transmission.

Evaluation of Flinders Technology Associates cards for storage and molecular detection of avian metapneumoviruses.

The feasibility of using Flinders Technology Associates (FTA) cards for the molecular detection of avian metapneumovirus (aMPV) by reverse transcriptase-polymerase chain reaction (RT-PCR) was investigated. Findings showed that no virus isolation was possible from aMPV-inoculated FTA cards, confirming viral inactivation upon contact with the cards. The detection limits of aMPV from the FTA card and tracheal organ culture medium were 10(1.5) median ciliostatic doses/ml and 10(0.75) median ciliostatic doses/ml respectively. It was possible to perform molecular characterization of both subtypes A and B aMPV using inoculated FTA cards stored for up to 60 days at 4 to 6°C. Tissues of the turbinate, trachea and lung of aMPV-infected chicks sampled either by direct impression smears or by inoculation of the tissue homogenate supernatants onto the FTA cards were positive by RT-PCR. However, the latter yielded more detections. FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization.

Surveillance of Culex spp. vectors and zoonotic arboviruses at a zoo in the United Kingdom.

The emergence of several zoonotic mosquito-borne pathogens in Europe, including West Nile virus, Sindbis virus and Usutu virus, has emphasised the importance of consistent surveillance. Considerable fieldwork effort is usually needed to detect low-prevalence pathogens in mosquitoes and screening vertebrate hosts and reservoirs is rarely done simultaneously with mosquito sampling. Zoological gardens offer an opportunity for the surveillance of pathogens, mosquitoes, hosts, and reservoirs concurrently; thus, the aim of this study was undertaking integrated surveillance for mosquito-borne pathogens of wild birds and mosquitoes in Chester Zoo (Cheshire) in the United Kingdom. Mosquitoes were collected in September 2020 and tested for zoonotic bird-hosted arboviruses (i.e., West Nile virus, Usutu virus and Sindbis virus) using RT-qPCRs. Of the 3316 mosquitoes trapped, 98% were identified as Culex spp. The average minimum prevalence of the viruses found in the literature was used to calculate the sample size needed for detecting these viruses with 99% confidence. The testing of 2878 Culex females found no evidence of presence of the three viruses. Significant differences were found in mosquito abundance per sampling site and collection date; furthermore, important sources of immature and resting mosquitoes were found near aviaries. Eighteen wild birds belonging to 11 species were found dead in the zoo from May to December 2020 and were RT-qPCR tested for West Nile virus and Usutu virus; all samples resulted negative for viral infection. It is unlikely that these viruses were present in the zoo during the sampling period; however, since they circulate in Europe and Usutu virus has been isolated in the United Kingdom and may overwinter here, continued monitoring of mosquitoes and wild birds is recommended as virus introduction and dissemination are possible. This study highlights the importance of regular and integrated arboviral surveillance of zoonotic pathogens in zoos providing baseline information to that end.

Field-based assessments of the seasonality of Culex pipiens sensu lato in England: an important enzootic vector of Usutu and West Nile viruses.

BACKGROUND: Usutu virus (USUV), which is closely related to West Nile virus (WNV), sharing a similar ecology and transmission cycle, was first reported in the UK in the southeast of England in 2020. Both USUV and WNV are emerging zoonotic viruses hosted by wild birds. The 2020 finding of USUV in England raised awareness of this virus and highlighted the importance of understanding the seasonality of Culex pipiens sensu lato (Cx. pipiens s.l.), the main enzootic vector of these viruses. Zoos are prime locations for trapping mosquitoes because of their infrastructure, security, and range of vertebrate hosts and aquatic habitats. METHODS: Three independent zoo-based case studies at four locations that cover the seasonality of Cx. pipiens s.l. in England were undertaken: (i) London Zoo (Zoological Society London [ZSL]) and surrounding areas, London; (ii) Chester Zoo (Cheshire); (ii) Twycross Zoo (Leicestershire); and (iv) Flamingo Land (zoo; North Yorkshire). Various adult mosquito traps were used to catch adult Cx. pipiens s.l. across seasons. RESULTS: High yields of Cx. pipiens s.l./Culex torrentium were observed in Biogents-Mosquitaire and Center for Disease Control and Prevention Gravid traps in all studies where these traps were used. Mosquito counts varied between sites and between years. Observations of adult Cx. pipiens s.l./Cx. torrentium abundance and modelling studies demonstrated peak adult abundance between late July and early August, with active adult female Cx. pipiens s.l./Cx. torrentium populations between May and September. CONCLUSIONS: The information collated in this study illustrates the value of multiple mosquito monitoring approaches in zoos to describe the seasonality of this UK vector across multiple sites in England and provides a framework that can be used for ongoing and future surveillance programmes and disease risk management strategies.

Mitochondrial and microbial diversity of the invasive mosquito vector species Culex tritaeniorhynchus across its extensive inter-continental geographic range.

Background: Culex (Cx.) tritaeniorhynchus is an invasive mosquito species with an extensive and expanding inter-continental distribution, currently reported across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary pathogens which cause significant morbidity and mortality in human and animal populations. Across regions endemic for Japanese encephalitis virus (JEV), Cx. tritaeniorhynchus is considered the major vector and has also been shown to contribute to the transmission of several other zoonotic arboviruses including Rift Valley fever virus (RVFV) and West Nile virus (WNV). Methods: In this study, we used laboratory vector competence experiments to determine if Cx. tritaeniorhynchus from a Southern European population were competent JEV vectors. We also obtained samples from multiple geographically dispersed Cx. tritaeniorhynchus populations from countries within Europe, Africa, Eurasia and Asia to perform phylogenetic analysis to measure the level of mitochondrial divergence using the cytochrome oxidase subunit 1 ( CO1) gene. We also undertook bacterial 16S rRNA gene amplicon sequencing to determine microbial diversity and used multi-locus sequence typing (MLST) to determine any evidence for the presence of strains of the naturally occurring endosymbiotic bacterium Wolbachia. Results: Cx. tritaeniorhynchus from a Greek population were shown be be competent vectors of JEV with high levels of virus present in saliva. We found a signficant level of mitochondrial genetic diversity using the mosquito CO1 gene between geographically dispersed populations. Furthermore, we report diverse microbiomes identified by 16S rRNA gene amplicon sequencing within and between geographical populations. Evidence for the detection of the endosymbiotic bacteria Wolbachia was confirmed using Wolbachia-specific PCR and MLST. Conclusions: This study enhances our understanding of the diversity of Cx. tritaeniorhynchus and the associated microbiome across its inter-continental range and highlights the need for greater surveillance of this invasive vector species in Europe.

The mosquito species Culex (Cx.) tritaeniorhynchus is expanding its range and is now present in over 50 countries across Asia, Africa, the Middle East, Europe and now Australasia. It can transmit human and animal pathogens, resulting in significant morbidity and mortality. This species transmits Japanese encephalitis virus in endemic areas of Asia, and it has also been shown to contribute to the transmission of several other viruses that can infect humans, including Rift Valley fever virus and West Nile virus. In this study, we firstly undertook some lab experiments to show that Cx. tritaeniorhynchus from a Southern European population are competent vectors of Japanese encephalitis virus. We also obtained field mosquitoes from countries within Europe, Africa, Eurasia and Asia and used phylogenetic analysis to demonstrate a high level of mitochondrial divergence within and between populations. In addition, we analysed the bacteria present within mosquitoes and found a high level of microbial diversity. Finally, we present evidence for the presence of Wolbachia endosymbiotic bacteria in some populations of this mosquito species. This study highlights the need for greater surveillance of this invasive vector species ­ particularly in Europe.