Seasonal change and influence of environmental variables on host-seeking activity of the biting midge Culicoides sonorensis at a southern California dairy, USA.

BACKGROUND: As a primary vector of bluetongue virus (BTV) in the US, seasonal abundance and diel flight activity of Culicoides sonorensis has been documented, but few studies have examined how time of host-seeking activity is impacted by environmental factors. This knowledge is essential for interpreting surveillance data and modeling pathogen transmission risk. METHODS: The diel host-seeking activity of C. sonorensis was studied on a California dairy over 3 years using a time-segregated trap baited with CO2. The relationship between environmental variables and diel host-seeking activity (start, peak, and duration of activity) of C. sonorensis was evaluated using multiple linear regression. Fisher's exact test and paired-sample z-test were used to evaluate the seasonal difference and parity difference on diel host-seeking activity. RESULTS: Host-seeking by C. sonorensis began and reached an activity peak before sunset at a higher frequency during colder months relative to warmer months. The time that host-seeking activity occurred was associated low and high daily temperature as well as wind speed at sunset. Colder temperatures and a greater diurnal temperature range were associated with an earlier peak in host-seeking. Higher wind speeds at sunset were associated with a delayed peak in host-seeking and a shortened duration of host-seeking. Parous midges reached peak host-seeking activity slightly later than nulliparous midges, possibly because of the need for oviposition by gravid females before returning to host-seeking. CONCLUSIONS: This study demonstrates that during colder months C. sonorensis initiates host-seeking and reaches peak host-seeking activity earlier relative to sunset, often even before sunset, compared to warmer months. Therefore, the commonly used UV light-baited traps are ineffective for midge surveillance before sunset. Based on this study, surveillance methods that do not rely on light trapping would provide a more accurate estimate of host-biting risk across seasons. The association of environmental factors to host-seeking shown in this study can be used to improve modeling or prediction of host-seeking activity. This study identified diurnal temperature range as associated with host-seeking activity, suggesting that Culicoides may respond to a rapidly decreasing temperature by shifting to an earlier host-seeking time, though this association needs further study.

Spatial Transmission Characteristics of the Bluetongue Virus Serotype 3 Epidemic in The Netherlands, 2023.

A devastating bluetongue (BT) epidemic caused by bluetongue virus serotype 3 (BTV-3) has spread throughout most of the Netherlands within two months since the first infection was officially confirmed in the beginning of September 2023. The epidemic comes with unusually strong suffering of infected cattle through severe lameness, often resulting in mortality or euthanisation for welfare reasons. In total, tens of thousands of sheep have died or had to be euthanised. By October 2023, more than 2200 locations with ruminant livestock were officially identified to be infected with BTV-3, and additionally, ruminants from 1300 locations were showing BTV-associated clinical symptoms (but not laboratory-confirmed BT). Here, we report on the spatial spread and dynamics of this BT epidemic. More specifically, we characterized the distance-dependent intensity of the between-holding transmission by estimating the spatial transmission kernel and by comparing it to transmission kernels estimated earlier for BTV-8 transmission in Northwestern Europe in 2006 and 2007. The 2023 BTV-3 kernel parameters are in line with those of the transmission kernel estimated previously for the between-holding spread of BTV-8 in Europe in 2007. The 2023 BTV-3 transmission kernel has a long-distance spatial range (across tens of kilometres), evidencing that in addition to short-distance dispersal of infected midges, other transmission routes such as livestock transports probably played an important role.

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo.

Segmented RNA viruses are a taxonomically diverse group that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during coinfection of a host by a process termed "reassortment." Reassortment enables rapid evolutionary change, but where transmission involves a biological arthropod vector, this change is constrained by the selection pressures imposed by the requirement for replication in two evolutionarily distant hosts. In this study, we use an in vivo, host-arbovirus-vector model to investigate the impact of reassortment on two phenotypic traits, virus infection rate in the vector and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV comprises 10 linear segments of dsRNA, and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4, and BTV-8) were isolated from naturally infected ruminants in Europe and ancestral/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to replicate in vector Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the ancestral virus strains, which allowed inferences of the genome segments conferring this phenotypic trait. IMPORTANCE Reassortment between virus strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses, with consequences for spread, persistence, and impact. The ability of these pathogens to adapt rapidly to their environment through this mechanism presents a major challenge in defining the conditions under which emergence can occur. Utilizing a representative mammalian host-insect vector infection and transmission model, we provide direct evidence of this phenomenon in closely related ancestral and reassortant strains of BTV. Our results demonstrate that efficient infection of Culicoides observed for one of three ancestral BTV strains was also evident in two reassortant strains that had subsequently emerged in the same ecosystem.

Oviposition of Culicoides insignis (Diptera: Ceratopogonidae) under laboratory conditions with notes on the developmental life history traits of its immature stages.

BACKGROUND: Culicoides insignis is a confirmed vector of bluetongue virus (BTV) throughout the American tropics and a possible vector of epizootic hemorrhagic disease virus (EHDV) in Florida. Despite its importance, fundamental information on the biology and ecology of this vector species is lacking. In this study, we examined the oviposition of C. insignis under laboratory conditions, monitored the development of immature stages and attempted colonization of this species. METHODS: Live C. insignis females were collected from the field using CDC-UV-LED traps, allowed to blood-feed on live chicken and given various natural substrates for oviposition in two-choice assays. The eggs deposited were transferred to 0.3% agar slants, and the hatched larvae were provided a diet of Panagrellus redivivus Linnaeus nematodes and the development of all immature stages was monitored. RESULTS: Culicoides insignis females exhibited an overall oviposition preference for dishes containing mud from their larval habitat as gravid females deposited a significantly higher number of eggs on these dishes (35.3 eggs/female) than on controls (17.7 eggs/female). The ovipositing females also deposited a higher percentage of eggs on substrates with habitat mud and other organically enriched muds (≥ 75.2%) compared to controls (31.0%). The larvae developed successfully to adulthood on the nematode diet, exhibiting high overall larval survival rates (85.0%). Sex ratios of the F1 generation were male biased, approximately 3:1 (male:female). Captive mating could not be induced in the F1 adults. CONCLUSIONS: Mud from the larval habitat and other organically enriched muds provide strong oviposition cues to C. insignis under laboratory conditions. Further studies will be needed to identify the key biotic/abiotic factors influencing midge oviposition in the field. The agar/nematode method is effective for the rearing of C. insignis larvae. However, further studies will be needed to address the issue of male-biased sex ratios in the progeny and to examine the mating habits/cues of C. insignis in nature, which may provide clues towards inducing captive mating in the F1 adults.

Modelling bluetongue risk in Kazakhstan.

BACKGROUND: Bluetongue is a serious disease of ruminants caused by the bluetongue virus (BTV). BTV is transmitted by biting midges (Culicoides spp.). Serological evidence from livestock and the presence of at least one competent vector species of Culicoides suggests that transmission of BTV is possible and may have occurred in Kazakhstan. METHODS: We estimated the risk of transmission using a mathematical model of the reproduction number R0 for bluetongue. This model depends on livestock density and climatic factors which affect vector density. Data on climate and livestock numbers from the 2466 local communities were used. This, together with previously published model parameters, was used to estimate R0 for each month of the year. We plotted the results on isopleth maps of Kazakhstan using interpolation to smooth the irregular data. We also mapped the estimated proportion of the population requiring vaccination to prevent outbreaks of bluetongue. RESULTS: The results suggest that transmission of bluetongue in Kazakhstan is not possible in the winter from October to March. Assuming there are vector-competent species of Culicoides endemic in Kazakhstan, then low levels of risk first appear in the south of Kazakhstan in April before spreading north and intensifying, reaching maximum levels in northern Kazakhstan in July. The risk declined in September and had disappeared by October. CONCLUSION: These results should aid in surveillance efforts for the detection and control of bluetongue in Kazakhstan by indicating where and when outbreaks of bluetongue are most likely to occur. The results also indicate where vaccination efforts should be focussed to prevent outbreaks of disease.

Predicting temperature-dependent transmission suitability of bluetongue virus in livestock.

The transmission of vector-borne diseases is governed by complex factors including pathogen characteristics, vector-host interactions, and environmental conditions. Temperature is a major driver for many vector-borne diseases including Bluetongue viral (BTV) disease, a midge-borne febrile disease of ruminants, notably livestock, whose etiology ranges from mild or asymptomatic to rapidly fatal, thus threatening animal agriculture and the economy of affected countries. Using modeling tools, we seek to predict where the transmission can occur based on suitable temperatures for BTV. We fit thermal performance curves to temperature-sensitive midge life-history traits, using a Bayesian approach. We incorporate these curves into S(T), a transmission suitability metric derived from the disease's basic reproductive number, [Formula: see text] This suitability metric encompasses all components that are known to be temperature-dependent. We use trait responses for two species of key midge vectors, Culicoides sonorensis and Culicoides variipennis present in North America. Our results show that outbreaks of BTV are more likely between 15[Formula: see text] C and [Formula: see text], with predicted peak transmission risk at 26 [Formula: see text] C. The greatest uncertainty in S(T) is associated with the following: the uncertainty in mortality and fecundity of midges near optimal temperature for transmission; midges' probability of becoming infectious post-infection at the lower edge of the thermal range; and the biting rate together with vector competence at the higher edge of the thermal range. We compare three model formulations and show that incorporating thermal curves into all three leads to similar BTV risk predictions. To demonstrate the utility of this modeling approach, we created global suitability maps indicating the areas at high and long-term risk of BTV transmission, to assess risk and to anticipate potential locations of disease establishment.

Spatio-temporal modelling of Culicoides Latreille (Diptera: Ceratopogonidae) populations on Reunion Island (Indian Ocean).

BACKGROUND: Reunion Island regularly faces outbreaks of bluetongue and epizootic hemorrhagic diseases, two insect-borne orbiviral diseases of ruminants. Hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae) are the vectors of bluetongue (BTV) and epizootic hemorrhagic disease (EHDV) viruses. In a previous study, statistical models based on environmental and meteorological data were developed for the five Culicoides species present in the island to provide a better understanding of their ecology and predict their presence and abundance. The purpose of this study was to couple these statistical models with a Geographic Information System (GIS) to produce dynamic maps of the distribution of Culicoides throughout the island. METHODS: Based on meteorological data from ground weather stations and satellite-derived environmental data, the abundance of each of the five Culicoides species was estimated for the 2214 husbandry locations on the island for the period ranging from February 2016 to June 2018. A large-scale Culicoides sampling campaign including 100 farms was carried out in March 2018 to validate the model. RESULTS: According to the model predictions, no husbandry location was free of Culicoides throughout the study period. The five Culicoides species were present on average in 57.0% of the husbandry locations for C. bolitinos Meiswinkel, 40.7% for C. enderleini Cornet & Brunhes, 26.5% for C. grahamii Austen, 87.1% for C. imicola Kieffer and 91.8% for C. kibatiensis Goetghebuer. The models also showed high seasonal variations in their distribution. During the validation process, predictions were acceptable for C. bolitinos, C. enderleini and C. kibatiensis, with normalized root mean square errors (NRMSE) of 15.4%, 13.6% and 16.5%, respectively. The NRMSE was 27.4% for C. grahamii. For C. imicola, the NRMSE was acceptable (11.9%) considering all husbandry locations except in two specific areas, the Cirque de Salazie-an inner mountainous part of the island-and the sea edge, where the model overestimated its abundance. CONCLUSIONS: Our model provides, for the first time to our knowledge, an operational tool to better understand and predict the distribution of Culicoides in Reunion Island. As it predicts a wide spatial distribution of the five Culicoides species throughout the year and taking into consideration their vector competence, our results suggest that BTV and EHDV can circulate continuously on the island. As further actions, our model could be coupled with an epidemiological model of BTV and EHDV transmission to improve risk assessment of Culicoides-borne diseases on the island.

Spatial and temporal distribution of Culicoides species in the New England region of New South Wales, Australia between 1990 and 2018.

Culicoides are one of the smallest hematophagous flies measuring 1-5 mm in size with only females seeking blood for egg development. The present study investigated spatio-temporal distribution of Culicoides species trapped between 1990 and 2018 at 13 sites in the New England region of NSW, Australia using automated light traps. Trapping locations were divided into three subregions (tablelands, slopes and plains). Nineteen Culicoides species were identified. Culicoides marksi and C. austropalpalis were the most abundant and widespread species. Culicoides brevitarsis, the principal vector of livestock diseases in New South Wales comprised 2.9% of the total catch and was detected in 12 of the 13 locations in the study. Abundance as determined by Log10 Culicoides count per trapping event for the eight most abundant species did not vary significantly with season but trended towards higher counts in summer for C. marksi (P = 0.09) and C. austropalpalis (P = 0.05). Significant geographic variation in abundance was observed for C. marksi, C. austropalpalis and C. dycei with counts decreasing with increasing altitude from the plains to the slopes and tablelands. Culicoides victoriae exhibited the reverse trend in abundance (P = 0.08). Greater abundance during the warmer seasons and at lower altitudes for C. marksi and C. austropalpalis was indicative of temperature and rainfall dependence in this region with moderate summer dominance in rainfall. The Shannon-Wiener diversity index of species was higher on the tablelands (H = 1.59) than the slopes (H = 1.33) and plains (H = 1.08) with evenness indices of 0.62, 0.46 and 0.39 respectively. Culicoides species on the tablelands were more diverse than on the slopes and plains where C. marksi and C. austropalpalis dominated. The temporal and spatial variation in abundance, diversity and evenness of species reported in this diverse region of Australia provides additional insight into Culicoides as pests and disease vectors and may contribute to future modelling studies.

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

High dispersal capacity of Culicoides obsoletus (Diptera: Ceratopogonidae), vector of bluetongue and Schmallenberg viruses, revealed by landscape genetic analyses.

BACKGROUND: In the last two decades, recurrent epizootics of bluetongue virus and Schmallenberg virus have been reported in the western Palearctic region. These viruses affect domestic cattle, sheep, goats and wild ruminants and are transmitted by native hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae). Culicoides dispersal is known to be stratified, i.e. due to a combination of dispersal processes occurring actively at short distances and passively or semi-actively at long distances, allowing individuals to jump hundreds of kilometers. METHODS: Here, we aim to identify the environmental factors that promote or limit gene flow of Culicoides obsoletus, an abundant and widespread vector species in Europe, using an innovative framework integrating spatial, population genetics and statistical approaches. A total of 348 individuals were sampled in 46 sites in France and were genotyped using 13 newly designed microsatellite markers. RESULTS: We found low genetic differentiation and a weak population structure for C. obsoletus across the country. Using three complementary inter-individual genetic distances, we did not detect any significant isolation by distance, but did detect significant anisotropic isolation by distance on a north-south axis. We employed a multiple regression on distance matrices approach to investigate the correlation between genetic and environmental distances. Among all the environmental factors that were tested, only cattle density seems to have an impact on C. obsoletus gene flow. CONCLUSIONS: The high dispersal capacity of C. obsoletus over land found in the present study calls for a re-evaluation of the impact of Culicoides on virus dispersal, and highlights the urgent need to better integrate molecular, spatial and statistical information to guide vector-borne disease control.

Detection of bluetongue virus in Culicoides spp. in southern Yunnan Province, China.

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) are vectors for many arboviruses. At least 20 species are considered as vectors or potential vectors of bluetongue virus (BTV) which cause bluetongue disease in ruminants. A BTV prevalence of 30-50% among cattle and goats in tropical southern Yunnan Province, China, prompted an investigation of the potential BTV vectors in this area. METHODS: Culicoides were collected by light trapping at three sites in the tropical region of Yunnan Province. Species were identified based on morphology and DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in these specimens. Phylogenetic analysis was used to analyze the cox1 sequences of Culicoides specimens infected with BTV. RESULTS: Approximately 67,000 specimens of Culicoides were collected, of which 748 were tested for the presence of BTV. Five specimens, including two of Culicoides jacobsoni, one of C. tainanus and two of C. imicola, were identified as infected with BTV. No specimens of C. (subgenus Trithecoides) or C. oxystoma tested were positive for BTV infection. CONCLUSIONS: To our knowledge this is the first report of C. jacobsoni as a potential BTV vector and the fourth report of an association between C. tainanus and BTV, as well as the first direct evidence of an association between BTV and C. imicola in Asia. A fourth potential cryptic species within C. tainanus was identified in this study. Further analysis is required to confirm the importance of C. jacobsoni and C. tainanus in BTV epidemiology in Asia.

Biting midge dynamics and bluetongue transmission: a multiscale model linking catch data with climate and disease outbreaks.

Bluetongue virus (BTV) serotype 8 has been circulating in Europe since a major outbreak occurred in 2006, causing economic losses to livestock farms. The unpredictability of the biting activity of midges that transmit BTV implies difficulty in computing accurate transmission models. This study uniquely integrates field collections of midges at a range of European latitudes (in Sweden, The Netherlands, and Italy), with a multi-scale modelling approach. We inferred the environmental factors that influence the dynamics of midge catching, and then directly linked predicted midge catches to BTV transmission dynamics. Catch predictions were linked to the observed prevalence amongst sentinel cattle during the 2007 BTV outbreak in The Netherlands using a dynamic transmission model. We were able to directly infer a scaling parameter between daily midge catch predictions and the true biting rate per cow per day. Compared to biting rate per cow per day the scaling parameter was around 50% of 24 h midge catches with traps. Extending the estimated biting rate across Europe, for different seasons and years, indicated that whilst intensity of transmission is expected to vary widely from herd to herd, around 95% of naïve herds in western Europe have been at risk of sustained transmission over the last 15 years.

Environmental Drivers of Adult Seasonality and Abundance of Biting Midges Culicoides (Diptera: Ceratopogonidae), Bluetongue Vector Species in Spain.

Bluetongue is a viral disease affecting wild and domestic ruminants transmitted by several species of biting midges Culicoides Latreille. The phenology of these insects were analyzed in relation to potential environmental drivers. Data from 329 sites in Spain were analyzed using Bayesian Generalized Linear Mixed Model (GLMM) approaches. The effects of environmental factors on adult female seasonality were contrasted. Obsoletus complex species (Diptera: Ceratopogonidae) were the most prevalent across sites, followed by Culicoides newsteadi Austen (Diptera: Ceratopogonidae). Activity of female Obsoletus complex species was longest in sites at low elevation, with warmer spring average temperatures and precipitation, as well as in sites with high abundance of cattle. The length of the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) female adult season was also longest in sites at low elevation with higher coverage of broad-leaved vegetation. Long adult seasons of C. newsteadi were found in sites with warmer autumns and higher precipitation, high abundance of sheep. Culicoides pulicaris (Linnaeus) (Diptera: Ceratopogonidae) had longer adult periods in sites with a greater number of accumulated degree days over 10°C during winter. These results demonstrate the eco-climatic and seasonal differences among these four taxa in Spain, which may contribute to determining sites with suitable environmental circumstances for each particular species to inform assessments of the risk of Bluetongue virus outbreaks in this region.

Development and lifespan of Culicoides obsoletus s.s. (Meigen) and other livestock-associated species reared at different temperatures under laboratory conditions.

Culicoides Latreille (Diptera: Ceratopogonidae) transmit arboviruses affecting wild and domestic ruminants such as bluetongue (BTV) and Schmallenberg virus (SBV). The sub-adult development and lifespan of Culicoides obsoletus s.s. (Meigen), Culicoides circumscriptus Kieffer and Culicoides paolae Boorman were examined at three different temperatures under laboratory conditions. Insects were collected from field between spring and autumn 2015 in two livestock farms located in Majorca (Spain). Gravid females were held individually at 18, 25 or 30 °C. Low temperatures increased the adult lifespan, time to oviposit and rate of development, whereas high temperatures increased the number of eggs, successful pupation and adult emergence as well as the larvae growth rate. The results showed that C. obsoletus s.s. have optimum development at 18 °C, whereas the optimal rearing temperature for C. circumscriptus and C. paolae was under warmer conditions of 25-30 °C. Variations in temperature/humidity and assays with different materials and substrates for oviposition should be considered in future studies. Understanding the requirements of the different species of Culicoides optimizing the results should be of special interest for predicting environmental change effects on these species, in addition to determining the rearing conditions for candidate Culicoides vectors.

Global emergence and evolutionary dynamics of bluetongue virus.

Bluetongue virus (BTV) epidemics are responsible for worldwide economic losses of up to US$ 3 billion. Understanding the global evolutionary epidemiology of BTV is critical in designing intervention programs. Here we employed phylodynamic models to quantify the evolutionary characteristics, spatiotemporal origins, and multi-host transmission dynamics of BTV across the globe. We inferred that goats are the ancestral hosts for BTV but are less likely to be important for cross-species transmission, sheep and cattle continue to be important for the transmission and maintenance of infection between other species. Our models pointed to China and India, countries with the highest population of goats, as the likely ancestral country for BTV emergence and dispersal worldwide over 1000 years ago. However, the increased diversification and dispersal of BTV coincided with the initiation of transcontinental livestock trade after the 1850s. Our analysis uncovered important epidemiological aspects of BTV that may guide future molecular surveillance of BTV.

Identifying Spanish Areas at More Risk of Monthly BTV Transmission with a Basic Reproduction Number Approach.

Bluetongue virus (BTV) causes a disease that is endemic in Spain and its two major biological vector species, C. imicola and the Obsoletus complex species, differ greatly in their ecology and distribution. Understanding the seasonality of BTV transmission in risk areas is key to improving surveillance and control programs, as well as to better understand the pathogen transmission networks between wildlife and livestock. Here, monthly risk transmission maps were generated using risk categories based on well-known BTV R0 equations and predicted abundances of the two most relevant vectors in Spain. Previously, Culicoides spp. predicted abundances in mainland Spain and the Balearic Islands were obtained using remote sensing data and random forest machine learning algorithm. Risk transmission maps were externally assessed with the estimated date of infection of BTV-1 and BTV-4 historical outbreaks. Our results highlight the differences in risk transmission during April-October, June-August being the period with higher R0 values. Likewise, a natural barrier has been identified between northern and central-southern areas at risk that may hamper BTV spread between them. Our results can be relevant to implement risk-based interventions for the prevention, control and surveillance of BTV and other diseases shared between livestock and wildlife host populations.

The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner.

Genome segmentation is mainly thought to facilitate reassortment. Here, we show that segmentation can also allow differences in segment abundance in populations of bluetongue virus (BTV). BTV has a genome consisting in 10 segments, and its cycle primarily involves periodic alternation between ruminants and Culicoides biting midges. We have developed a reverse transcription-quantitative PCR (RT-qPCR) approach to quantify each segment in wild BTV populations sampled in both ruminants and midges during an epizootic. Segment frequencies deviated from equimolarity in all hosts. Interestingly, segment frequencies were reproducible and distinct between ruminants and biting midges. Beyond a putative regulatory role in virus expression, this phenomenon could lead to different evolution rates between segments.IMPORTANCE The variation in viral gene frequencies remains a largely unexplored aspect of within-host genetics. This phenomenon is often considered to be specific to multipartite viruses. Multipartite viruses have segmented genomes, but in contrast to segmented viruses, their segments are each encapsidated alone in a virion. A main hypothesis explaining the evolution of multipartism is that, compared to segmented viruses, it facilitates the regulation of segment abundancy, and the genes the segments carry, within a host. These differences in gene frequencies could allow for expression regulation. Here, we show that wild populations of a segmented virus, bluetongue virus (BTV), also present unequal segment frequencies. BTV cycles between ruminants and Culicoides biting midges. As expected from a role in expression regulation, segment frequencies tended to show specific values that differed between ruminants and midges. Our results expand previous knowledge on gene frequency variation and call for studies on its role and conservation beyond multipartite viruses.

The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission.

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV's alternating-host transmission cycle on the virus's genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV's genome. To understand the role of alternating hosts in BTV's genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

Update of the Culicoides (Diptera: Ceratopogonidae) species checklist from Algeria with 10 new records.

BACKGROUND: The Culicoides fauna of Algeria has been historically investigated, leading to the description of many new species by Kieffer in the 1920s, Clastrier in the 1950s or Callot in the 1960s and to a comprehensive inventory by Szadziewski in the 1980s. The emergence of bluetongue in the late 1990s enhanced Culicoides collections made in the country over the last two decades, but information remained mostly unpublished. The aim of this study is therefore to provide a comprehensive and updated checklist of Culicoides biting midge species in Algeria. METHODS: The literature (published and grey, in French and in English) from 1920 to date on Culicoides collections in Algeria was collected and analyzed in the light of the current taxonomic and systematic knowledge and methods. Fresh Culicoides material was also analyzed using light/suction trap collections carried out from November 2015 to September 2018 in nine localities of the 'wilayah' of Tiaret (northwestern Algeria). Slide mounted specimens were identified morphologically using the interactive identification key IIKC and original descriptions. Specimens were then compared with non-type material originating from different countries and partly with type material. RESULTS: A total of 13,709 Culicoides, belonging to at least 36 species within 10 subgenera, were examined leading to 10 new records in Algeria, including C. chiopterus, C. dewulfi, C. navaiae, C. grisescens, C. paradoxalis, C. shaklawensis, C. simulator, C. univittatus, C. achrayi and C. picturatus. These new records and all previous records provided by the literature review were discussed. CONCLUSIONS: We propose a Culicoides checklist for the Algerian fauna of 59 valid species, including species mainly with a large Palaearctic distribution and a specific Mediterranean distribution, and only a few species from the Afrotropical region. Among them, several species, mainly of the subgenera Avaritia and Culicoides, are confirmed or probable vectors of arboviruses important in animal health.

Species composition and relative abundance of the genus Culicoides (Diptera: Ceratopogonidae) in Romania.

BACKGROUND: Culicoides biting midges are vectors involved in the biological transmission cycle of important animal diseases such as bluetongue and African horse sickness. In Romania, the first outbreaks of bluetongue were reported in 2014, leading to increased activities within the existing entomological surveillance network. The main goals of the surveillance activities were the establishment of the vector free period in relation to animal trade and the identification of Culicoides species involved in the transmission of the pathogen. This study was conducted on the composition and relative abundance of the species belonging to the genus Culicoides (Diptera: Ceratopogonidae) in certain regions of Romania and provided the opportunity to update the existing checklist of Culicoides species of this country. METHODS: The study was conducted in 33 of the 42 administrative units (counties), including a total of 659 catches, in 102 locations. The collections were carried out with UV blacklight suction traps (OVI type). The collected insects were preserved in 70% ethanol. Morphological insect identification was carried out using a stereomicroscope, according to established identification keys. In ten localities the relative abundance of the cryptic species of the Obsoletus complex was determined by multiplex PCR assay based on the ITS2 segment. The identification of the Culicoides chiopterus (Meigen) species by morphological examination was confirmed by PCR assay based on the ITS1 segment. RESULTS: Eleven species were identified using morphological and PCR tools. The rest of the individuals were separated into five taxa. The species of the Obsoletus complex (grouping Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle) were the most abundant, accounting for 59% of the total number of captured Culicoides spp. Three of the identified species are mentioned, according to our knowledge, for the first time in Romania: Culicoides newsteadi Austen, Culicoides flavipulicaris Dzhafarov and Culicoides bysta Sarvasová, Kocisová, Candolfi & Mathieu. CONCLUSIONS: Our study demonstrates that the Culicoides species most commonly cited as being involved in the transmission of arboviruses in Europe (i.e. bluetongue and Schmallenberg viruses) make up a high proportion of adult Culicoides trapped in Romania.