Epizootic hemorrhagic disease virus (EHDV) is transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) to numerous ruminant species and is a significant pathogen of white-tailed deer (WTD; Odocoileus virginianus). In North America, the only confirmed vector of EHDV is Culicoides sonorensis, although other Culicoides species are suspected vectors. Previous experimental infections have demonstrated Culicoides-to-WTD transmission of EHDV, but little is known about transmission efficiency. Our objective was to determine if the bite from as few as 5 EHDV-infected C. sonorensis is sufficient to infect a WTD. Colonized C. sonorensis were intrathoracically inoculated with EHDV-2, held for 10 days at 26 °C to allow for virus replication, and were then given the opportunity to feed on susceptible WTD (n = 2): deer A, 10 midges; deer B, 5 midges. After feeding, Culicoides were processed individually for virus. All Culicoides were positive by virus isolation except 1 midge from deer B, suggesting the inoculation failed in that midge. Deer were observed twice daily and sampled on 0, 4, 7, 11, and 14 days post feeding (dpf). Culicoides feeding was variable: deer A, only 1/10 midges fed and only took a partial blood meal but 4 additional probe sites were observed on the skin; deer B, 5/5 midges blood fed (1 partial, 4 to repletion) with a total of 5 bite sites observed. Both deer developed viremia and mild clinical disease. Infection of WTD with EHDV-2 by the bite of 1 to 4 C. sonorensis demonstrates EHDV-2 transmission efficiency and the potential importance of probing.

Heritable microbes shape host phenotypes and serve as important drivers of evolution. While interactions between insects and bacterial symbionts have been extensively studied, the prevalence and evolution of insect-viral symbioses remain poorly understood. We discovered multiple new species of iflaviruses in aphids, an important model for research on symbiosis, and found these microbes to be widespread across aphid species. We show that iflaviruses are persistently maintained in asexual host lines without apparent fitness costs while being transmitted vertically from mothers to offspring. Using field data and phylogenetic evidence, we found that aphid iflaviruses move horizontally among host species, but laboratory experiments showed that horizontal transmission does not result in persistent infections. Using quantitative PCR and immunohistochemistry, we discovered that viral infections localize in the host fat bodies and developing embryos. Surprisingly, we also found viral infections inside bacteria-housing cells called bacteriocytes, along with a positive correlation between viral and bacterial symbiont density. Together, our work suggests that iflaviruses are widespread heritable symbionts in aphids. IMPORTANCE:In recent years, the rise of metatranscriptome sequencing has led to the rapid discovery of novel viral sequences in insects. However, few studies have carefully investigated the dynamics of insect-virus interactions to produce a general understanding of viral symbiosis. Aphids are an important model for understanding the evolution and molecular basis of symbiosis, but whether viruses are forming persistent symbiotic relationships with aphids remains unclear. Here, we show that heritable iflaviruses are a widespread but previously unrecognized part of the aphid heritable microbiome. Aphid iflaviruses are transmitted alongside bacteria from mothers to offspring, potentially via specialized bacteriocytes that house symbiotic microbes. Our findings suggest that aphids establish persistent relationships with iflaviruses and are likely coevolving with these viral symbionts.

Vesicular stomatitis (VS) is a viral disease that affects horses, cattle, and swine that is transmitted by direct contact and hematophagous insects. In 2023, a multi-state outbreak of vesicular stomatitis New Jersey virus (VSNJV) occurred in California, Nevada, and Texas, infecting horses, cattle, and rhinoceros. To identify possible insect vectors, we conducted insect surveillance at various locations in San Diego County, CA, including at a wildlife park. CO2 baited traps set from mid-May to mid-August 2023 collected 2357 Culicoides biting midges and 1215 Simulium black flies, which are insect genera implicated in VSNJV transmission. Insects were pooled by species, location, and date, then tested for viral RNA. Nine RNA-positive pools of Culicoides spp. and sixteen RNA-positive pools of Simulium spp were detected. Infectious virus was detected by cytopathic effect in 96% of the RNA-positive pools. This is the first report of VSNJV in wild-caught C. bergi, C. freeborni, C. occidentalis, S. argus, S. hippovorum, and S. tescorum. The vector competency of these species for VSNJV has yet to be determined but warrants examination. Active vector surveillance and testing during disease outbreaks increases our understanding of the ecology and epidemiology of VS and informs vector control efforts.

Vesicular stomatitis virus (VSV) is an arthropod-borne virus affecting livestock. In the United States, sporadic outbreaks result in significant economic losses. During epizootics, Culicoides biting midges are biological vectors and key to the geographic expansion of outbreaks. Additionally, Culicoides may play a role in VSV overwintering because females and males are capable of highly efficient venereal transmission, despite their relatively low virus titers. We hypothesized that VSV propagated within a midge has increased fitness for subsequent midge infections. To evaluate the potential host-specific fitness increase, we propagated three viral isolates of VSV in porcine skin fibroblasts and Culicoides cell lines. We then evaluated the viral infection dynamics of the different cell-source groups in Culicoides sonorensis. Our results indicate that both mammalian- and insect-derived VSV replicate well in midges inoculated via intrathoracic injection, thereby bypassing the midgut barriers. However, when the virus was required to infect and escape the midgut barrier to disseminate after oral acquisition, the insect-derived viruses had significantly higher titers, infection, and dissemination rates than mammalian-derived viruses. Our research suggests that VSV replication in Culicoides cells increases viral fitness, facilitating midge-to-midge transmission and subsequent replication, and further highlights the significance of Culicoides midges in VSV maintenance and transmission dynamics.

Bluetongue (BT) is a Culicoides midge-borne hemorrhagic disease affecting cervids and ruminant livestock species, resulting in significant economic losses from animal production and trade restrictions. Experimental animal infections using the α/β interferon receptor knockout IFNAR mouse model and susceptible target species are critical for understanding viral pathogenesis, virulence, and evaluating vaccines. However, conducting experimental vector-borne transmission studies with the vector itself are logistically difficult and experimentally problematic. Therefore, experimental infections are induced by hypodermic injection with virus typically derived from baby hamster kidney (BHK) cells. Unfortunately, for many U.S. BTV serotypes, it is difficult to replicate the severity of the disease seen in natural, midge-transmitted infections by injecting BHK-derived virus into target host animals. Using the IFNAR BTV murine model, we compared the virulence of traditional BHK cell-derived BTV-17 with C. sonorensis midge (W8) cell-derived BTV-17 to determine whether using cells of the transmission vector would provide an in vitro virulence aspect of vector-transmitted virus. At both low and high doses, mice inoculated with W8-BTV-17 had an earlier onset of viremia, earlier onset and peak of clinical signs, and significantly higher mortality compared to mice inoculated with BHK-BTV-17. Our results suggest using a Culicoides W8 cell-derived inoculum may provide an in vitro vector-enhanced infection to more closely represent disease levels seen in natural midge-transmitted infections while avoiding the logistical and experimental complexity of working with live midges.

Retrospective serological and case diagnostic data of endemic bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) provide evidence of viral transmission among livestock and wildlife from 2016 in Kansas and Nebraska. Serological testing of mature cattle in nine distinct regional zones of Kansas revealed 76% to 100% had detectable antibodies to BTV and/or EHDV. Specimens tested in the Kansas Veterinary Diagnostic Laboratory (55 submissions) were 51% test positive for antibodies to BTV and/or EHDV. Specimens tested in the Nebraska Veterinary Diagnostic Center (283 submissions) were 25% test positive for antibodies to BTV and/or EHDV. Low disease incidence in white-tailed deer and other susceptible wild ungulates was observed during 2016. However, there were no confirmed reports of disease in livestock in either state. The reasons for emergence of significant clinical disease in livestock and wildlife populations remain undefined.

Reemerging and notifiable diseases of cattle and bison continue to pose potential risks to their health and lives and affecting production and the livelihoods of producers. It is essential to understand the clinical presentation of these diseases to watch for possible incursions and infections and to immediately report your suspicions to your State and Federal Animal Health Officials. Three of these reemerging and notifiable diseases of cattle and bison, malignant catarrhal fever, bluetongue virus, and New World screwworm, are presented in this article for increased awareness to consider as a differential if examinations present suggestive clinical signs.

A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxTr. The cell line is heterogeneous, composed predominantly of small, round cells, and spindle-shaped cells with a doubling time of approximately 52-60 h. The identity of the cell line was verified as Cx. tarsalis by sequencing of cytochrome oxidase I and the cells were found to be free of contaminating cells, bacteria, fungi, and mycoplasma. The permissiveness of CxTr cells to arbovirus infection was investigated with vaccine and wildtype arboviruses from four viral families: Flaviviridae (Japanese encephalitis virus), Phenuiviridae (Rift Valley fever phlebovirus), Rhabdoviridae (vesicular stomatitis virus), and Togaviridae (Mayaro virus). All viruses were able to infect and replicate within CxTr cells.

Vector-borne disease prevalence is increasing at a time when surveillance capacity in the United States is decreasing. One way to address this surveillance deficiency is to utilize established infrastructure, such as zoological parks, to investigate animal disease outbreaks and improve our epidemiological understanding of vector-borne pathogens. During fall 2020, an outbreak of epizootic hemorrhagic disease (EHD) at the Minnesota Zoo resulted in morbidity and seroconversion of several collection animals. In response to this outbreak, insect surveillance was conducted, and the collected insects were tested for the presence of epizootic hemorrhagic disease virus (EHDV) by RT-qPCR to better understand the local transmitting vector populations responsible for the outbreak. Six pools of Culicoides biting midges were positive for EHDV, including three pools of Culicoides sonorensis, two pools of Culicoides variipennis, and a pool of degraded C. variipennis complex midges. All three endemic serotypes of EHDV (1, 2, and 6) were detected in both animals and midge pools from the premises. Despite this outbreak, no EHDV cases had been reported in wild animals near the zoo. This highlights the importance and utility of using animal holding facilities, such as zoos, as sentinels to better understand the spatio-temporal dynamics of pathogen transmission.

Culicoides sonorensis midges vector multiple livestock arboviruses, resulting in significant economic losses worldwide. Due to the tight association between virus transmission, blood feeding, and egg development, understanding midge physiology is paramount to limiting pathogen transmission. Previous studies have demonstrated the importance of small non-coding RNAs (ncRNAs), specifically microRNAs (miRNAs), in multiple aspects of vector physiology. These small ncRNAs regulate gene expression at the post-transcriptional level and display differential expression during pathogen infection. Due to the lack of annotated miRNAs in the biting midge and associated expression profiles, we used small RNA-Seq and miRDeep2 analyses to determine the Culicoides miRNAs in whole females and midgut tissues in response to blood feeding. Our analyses revealed 76 miRNAs within C. sonorensis composed of 73 orthologous and three candidate novel miRNAs, as well as conserved miRNA clusters. miRNA conservation suggests an interesting evolutionary relationship between miRNA expression and hematophagy in the infraorder Culicomorpha. We also identified multiple blood meal-regulated and tissue-enriched miRNAs. Lastly, we further identified miRNAs with expression patterns potentially associated with virus infection by probing publicly available datasets. Together, our data provide a foundation for future ncRNA work to untangle the dynamics of gene regulation associated with midge physiology.

Vesicular stomatitis virus (VSV) primarily infects livestock and is transmitted by direct contact and vectored by Culicoides midges (Diptera: Ceratopogonidae). Endemic to Central and South America, specific VSV lineages spread northward out of endemic regions of Mexico and into the U.S. sporadically every five to ten years. In 2012, a monophyletic epidemic lineage 1.1 successfully spread northward into the U.S. In contrast, the closest endemic ancestor, lineage 1.2, remained circulating exclusively in endemic regions in Mexico. It is not clear what roles virus-animal interactions and/or virus-vector interactions play in the ability of specific viral lineages to escape endemic regions in Mexico and successfully cause outbreaks in the U.S., nor the genetic basis for such incursions. Whole-genome sequencing of epidemic VSV 1.1 and endemic VSV 1.2 revealed significant differences in just seven amino acids. Previous studies in swine showed that VSV 1.1 was more virulent than VSV 1.2. Here, we compared the efficiency of these two viral lineages to infect the vector Culicoides sonorensis (Wirth and Jones) and disseminate to salivary glands for subsequent transmission. Our results showed that midges orally infected with the epidemic VSV 1.1 lineage had significantly higher infection dissemination rates compared to those infected with the endemic VSV 1.2 lineage. Thus, in addition to affecting virus-animal interactions, as seen with higher virulence in pigs, small genetic changes may also affect virus-vector interactions, contributing to the ability of specific viral lineages to escape endemic regions via vector-borne transmission.

Culicoides midges play an important role in vesicular stomatitis virus (VSV) transmission to US livestock. After VSV-blood feeding, blood digestion followed by oviposition occurs while ingested virus particles replicate and disseminate to salivary glands for transmission during subsequent blood-feeding events. Changes to environmental temperature may alter the feeding–oviposition–refeeding cycles, midge survival, VSV infection, and overall vector capacity. However, the heterothermic midge may respond rapidly to environmental changes by adjusting their thermal behavior to resting in areas closer to their physiological range. Here we investigated the effects of four constant environmental temperatures (20, 25, 30, and 35 °C) on C. sonorensis survival, oviposition, and VSV infection, as well as resting thermal preferences after blood-feeding. We found that most midges preferred to rest in areas at 25–30 °C. These two constant temperatures (25 and 30 °C) allowed an intermediate fitness performance, with a 66% survival probability by day 10 and oviposition cycles occurring every 2–3 days. Additionally, VSV infection rates in bodies and heads with salivary glands were higher than in midges held at 20 °C and 35 °C. Our results provide insight into the implications of temperature on VSV–Culicoides interactions and confirm that the range of temperature preferred by midges can benefit both the vector and the arbovirus.

Culicoides midges are hematophagous insects that transmit arboviruses of veterinary importance. These viruses include bluetongue virus (BTV) and epizootic hemorrhagic fever virus (EHDV). The endosymbiont Wolbachia pipientis Hertig spreads rapidly through insect host populations and has been demonstrated to inhibit viral pathogen transmission in multiple mosquito vectors. Here, we have demonstrated a replication inhibitory effect on BTV and EHDV in a Wolbachia (wAlbB strain)-infected Culicoides sonorensis Wirth and Jones W8 cell line. Viral replication was significantly reduced by day 5 for BTV and by day 2 for EHDV as detected by real-time polymerase chain reaction (RT-qPCR) of the non-structural NS3 gene of both viruses. Evaluation of innate cellular immune responses as a cause of the inhibitory effect showed responses associated with BTV but not with EHDV infection. Wolbachia density also did not play a role in the observed pathogen inhibitory effects, and an alternative hypothesis is suggested. Applications of Wolbachia-mediated pathogen interference to impact disease transmission by Culicoides midges are discussed.

Abstract Vesicular stomatitis (VS) is a vector‐borne viral disease that causes lesions in livestock, premises, county and state quarantines, and important economic losses. We investigated vector–habitat characteristics for vectors associated with VS in regions of recurrent disease within the western United States (US) that consistently lead to the environment where vector, host, and pathogen populations intersect to enable pathogen transmission. We analyzed the habitats of previously identified insect vectors, including black flies (BFs) (Simulium vittatum complex), biting midges (BMs) (Culicoides variipennis complex, which includes Culicoides sonorensis), and sand flies (SFs) (Lutzomyia shannoni) in six regions of interest (ROIs) containing hotspots of VS ranging from Texas (TX) to Wyoming. This analysis broadened the understanding of (1) how regions of reoccurring VS differ from the broader western US, (2) how geographically separated regions and hotspots are similar across time, and (3) how vector–environment habitat a priori knowledge relates to observed hotspots. Analysis of watershed factors (livestock densities, land‐cover proportions, stream and lake densities, and irrigation methods) indicated a complex system separating areas with high, recurring VS from the broader western US. Although no single characteristic separated the six ROIs from other areas, we found two distinct emerging groups (northern ROI and TX). Hotspots, estimated from monthly VS concentrations, evolved northward throughout the year and most hotspots were closer to flowing water and agricultural land than the broader ROI. All ROIs contained environmental conditions suitable for multiple vectors at some point in the year, but BFs had the highest suitability scores, whereas BM scores were lower and varied annually with higher suitability in summer. SFs had the lowest suitability score in all ROIs, consistent with their low likelihood of being vectors. BM habitat patches were often orders of magnitude smaller than BF patches, and hotspot patches reinforce the likelihood that BF may be the most critical vector in northern ROI, whereas both BM and BF have similar likelihood in southern ROI. Given limited existing vector data, this analysis provides an alternate pathway for using habitat information to associate likely vectors responsible for transmission. Results could support early warning and mitigation efforts to reduce the incidence of VS.

Bacteria are part of the insect gut system and influence many physiological traits of their host. Gut bacteria may even reduce or block the transmission of arboviruses in several species of arthropod vectors. Culicoides biting midges are important arboviral vectors of several livestock and wildlife diseases, yet limited information is available on their gut bacterial communities. Addressing this gap will help inform how these communities can be manipulated and ultimately used as novel tools to control pathogens. To assess how bacterial communities change during the life stages of lab-reared C. nubeculosus and C. sonorensis, endosymbiotic bacteria were identified using Illumina sequencing of 16S rRNA and taxonomically characterised. Analyses were conducted to determine how gut bacterial communities in adults are influenced by species identity and geographic distance among biting midge populations. Communities of the two lab-reared Culicoides species significantly changed after pupation and with maturation into 6-day-old adults. Pseudomonas, Burkholderiaceae and Leucobacter bacteria were part of a core community that was trans-stadially transmitted and found throughout their life cycle. Among field-collected biting midges, the bacterial communities were unique for almost each species. Cardinium, Rickettsia and Wolbachia were some of the most abundant bacteria in midges collected from wetlands. Only Pseudomonas was present in high relative abundance in all field-collected species. In this study, species identity, as well as geographic distance, influenced the gut bacterial communities and may partly explain known inter- and intra-species variability in vector competence. Additionally, stably associated bacterial species could be candidates for paratransgenic strategies to control vector-borne pathogens.

Vesicular stomatitis (VS) is a reportable viral disease which affects horses, cattle, and pigs in the Americas. Outbreaks of vesicular stomatitis virus New Jersey serotype (VSV-NJ) in the United States typically occur on a 5–10-year cycle, usually affecting western and southwestern states. In 2019–2020, an outbreak of VSV Indiana serotype (VSV-IN) extended eastward into the states of Kansas and Missouri for the first time in several decades, leading to 101 confirmed premises in Kansas and 37 confirmed premises in Missouri. In order to investigate which vector species contributed to the outbreak in Kansas, we conducted insect surveillance at two farms that experienced confirmed VSV-positive cases, one each in Riley County and Franklin County. Centers for Disease Control and Prevention miniature light traps were used to collect biting flies on the premises. Two genera of known VSV vectors, Culicoides biting midges and Simulium black flies, were identified to species, pooled by species, sex, reproductive status, and collection site, and tested for the presence of VSV-IN RNA by RT-qPCR. In total, eight positive pools were detected from Culicoides sonorensis (1), Culicoides stellifer (3), Culicoides variipennis (1), and Simulium meridionale (3). The C. sonorensis- and C. variipennis-positive pools were from nulliparous individuals, possibly indicating transovarial or venereal transmission as the source of virus. This is the first report of VSV-IN in field caught C. stellifer and the first report of either serotype in S. meridionale near outbreak premises. These results improve our understanding of the role midges and black flies play in VSV epidemiology in the United States and broadens the scope of vector species for targeted surveillance and control.

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.

Vesicular stomatitis (VS) is the most common vesicular livestock disease in North America. Transmitted by direct contact and by several biting insect species, this disease results in quarantines and animal movement restrictions in horses, cattle and swine. As changes in climate drive shifts in geographic distributions of vectors and the viruses they transmit, there is considerable need to improve understanding of relationships among environmental drivers and patterns of disease occurrence. Multidisciplinary approaches integrating pathology, ecology, climatology, and biogeophysics are increasingly relied upon to disentangle complex relationships governing disease. We used a big data model integration approach combined with machine learning to estimate the potential geographic range of VS across the continental United States (CONUS) under long-term mean climate conditions over the past 30 years. The current extent of VS is confined to the western portion of the US and is related to summer and winter precipitation, winter maximum temperature, elevation, fall vegetation biomass, horse density, and proximity to water. Comparison with a climate-only model illustrates the importance of current processes-based parameters and identifies regions where uncertainty is likely to be greatest if mechanistic processes change. We then forecast shifts in the range of VS using climate change projections selected from CMIP5 climate models that most realistically simulate seasonal temperature and precipitation. Climate change scenarios that altered climatic conditions resulted in greater changes to potential range of VS, generally had non-uniform impacts in core areas of the current potential range of VS and expanded the range north and east. We expect that the heterogeneous impacts of climate change across the CONUS will be exacerbated with additional changes in land use and land cover affecting biodiversity and hydrological cycles that are connected to the ecology of insect vectors involved in VS transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged coronavirus that is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 in humans is characterized by a wide range of symptoms that range from asymptomatic to mild or severe illness including death. SARS-CoV-2 is highly contagious and is transmitted via the oral–nasal route through droplets and aerosols, or through contact with contaminated fomites. House flies are known to transmit bacterial, parasitic and viral diseases to humans and animals as mechanical vectors. Previous studies have shown that house flies can mechanically transmit coronaviruses, such as turkey coronavirus; however, the house fly’s role in SARS-CoV-2 transmission has not yet been explored. The goal of this work was to investigate the potential of house flies to mechanically transmit SARS-CoV-2. For this purpose, it was determined whether house flies can acquire SARS-CoV-2, harbor live virus and mechanically transmit the virus to naive substrates and surfaces. Two independent studies were performed to address the study objectives. In the first study, house flies were tested for infectivity after exposure to SARS-CoV-2-spiked medium or milk. In the second study, environmental samples were tested for infectivity after contact with SARS-CoV-2-exposed flies. During both studies, samples were collected at various time points post-exposure and evaluated by SARS-CoV-2-specific RT-qPCR and virus isolation. All flies exposed to SARS-CoV-2-spiked media or milk substrates were positive for viral RNA at 4 h and 24 h post-exposure. Infectious virus was isolated only from the flies exposed to virus-spiked milk but not from those exposed to virus-spiked medium. Moreover, viral RNA was detected in environmental samples after contact with SARS-CoV-2 exposed flies, although no infectious virus was recovered from these samples. Under laboratory conditions, house flies acquired and harbored infectious SARS-CoV-2 for up to 24 h post-exposure. In addition, house flies were able to mechanically transmit SARS-CoV-2 genomic RNA to the surrounding environment up to 24 h post-exposure. Further studies are warranted to determine if house fly transmission occurs naturally and the potential public health implications of such events.