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Background: 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., Methods: 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., Results: 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., Conclusions: 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.

First appearing in 2011 in Northern Europe, Schmallenberg virus (SBV), an Orthobunyavirus of the Simbu serogroup, is associated with clinical disease mainly in ruminants such as cattle, sheep and goats. The clinical signs are characterized by abortion and congenital deformities in newborns. The virus is transmitted by Culicoides midges of the Obsoletus complex. SBV infection induces a solid protective immunity that persists for at least 4 or 6 years in sheep and cattle, respectively. SBV infection can be diagnosed directly by real-time RT-qPCR and virus isolation or indirectly by serological assays. Three vaccines are commercially available in Europe. This article provides a comprehensive literature review on this emerging disease regarding pathogenesis, transmission, diagnosis, control and prevention. This review also highlights that although much has been learned since SBV's first emergence, there are still areas that require further study to devise better mitigation strategies.

Epizootic hemorrhagic disease viruses (EHDVs) are arboviral pathogens of white-tailed deer and other wild and domestic ruminants in North America. Transmitted by various species of Culicoides , EHDVs circulate wherever competent vectors and susceptible ruminant host populations co-exist. The impact of variation in the level and duration of EHDV viremia in white-tailed deer ( Odocoileus virginianus ) on Culicoides infection prevalence is not well characterized. Here we examined how infection prevalence in a confirmed North American vector of EHDV-2 ( Culicoides sonorensis ) varies in response to fluctuations in deer viremia. To accomplish this, five white-tailed deer were experimentally infected with EHDV-2 and colonized C. sonorensis were allowed to feed on deer at 3, 5, 7, 10, 12, 14, 18, and 24 days post infection (dpi). Viremia profiles in deer were determined by virus isolation and titration at the same time points. Blood-fed Culicoides were assayed for virus after a 10-day incubation (27 °C) period. We found that increases in deer EHDV blood titers significantly increased both the likelihood that midges would successfully acquire EHDV and the proportion of midges that reached the titer threshold for transmission competence. Unexpectedly, we identified four infected midge samples (three individuals and one pool) after feeding on one deer 18 and 24 dpi, when viremia was no longer detectable by virus isolation. The ability of ruminants with low-titer viremia to serve as a source of EHDV for blood-feeding Culicoides should be explored further to better understand its potential epidemiological significance.

This review covers the basic biology of the West Nile virus and the host-vector-pathogen interactions that result in significant disease in wild birds, horses and humans. The review describes the basic properties of the virus, cellular infection and the pathogenesis of the disease, and the ecology of virus maintenance, amplification and transmission. Disease epidemiology and risk estimation strategies that are currently in use are also examined, and host immune responses and vaccination practices described. The principles of vector control, exposure control and long-term risks caused by climatic and habitat factors are also included.

To determine which arthropods should be targeted for control should Rift Valley fever virus (RVFV) be detected in North America, we evaluated Culex erraticus (Dyar and Knab), Culex erythrothorax Dyar, Culex nigripalpus Theobald, Culex pipiens L., Culex quinquefasciatus Say, Culex tarsalis Coquillett, Aedes dorsalis (Wiedemann), Aedes vexans (Meigen), Anopheles quadrimaculatus Say, and Culicoides sonorensis Wirth and Jones from the western, midwestern, and southern United States for their ability to transmit RVFV. Female mosquitoes were allowed to feed on adult hamsters inoculated with RVFV, after which engorged mosquitoes were incubated for 7-21 d at 260C, then allowed to refeed on susceptible hamsters, and tested to determine infection, dissemination, and transmission rates. Other specimens were inoculated intrathoracically, held for 7 d, and then allowed to feed on a susceptible hamster to check for a salivary gland barrier. When exposed to hamsters with viremias > or =10(8.8) plaque-forming units/ml blood, Cx. tarsalis transmitted RVFV efficiently (infection rate = 93%, dissemination rate = 56%, and estimated transmission rate = 52%). In contrast, when exposed to the same virus dose, none of the other species tested transmitted RVFV efficiently. Estimated transmission rates for Cx. erythrothorax, Cx. pipiens, Cx. erraticus, and Ae. dorsalis were 10, 8, 4, and 2%, respectively, and for the remaining species were < or = 1%. With the exception of Cx. tarsalis and Cx. pipiens, all species tested had moderate to major salivary gland barriers. None of the C. sonorensis became infected and none of the An. quadrimaculatus tested transmitted RVFV by bite, even after intrathoracic inoculation, indicating that these species would not be competent vectors of RVFV. Although Ae. vexans from Florida and Louisiana were relatively efficient vectors of RVFV, specimens of this species captured in Colorado or California were virtually incompetent, illustrating the need to evaluate local population for their ability to transmit a pathogen. In addition to laboratory vector competence, factors such as seasonal density, host feeding preference, longevity, and foraging behavior should be considered when determining the potential role that these species could play in RVFV transmission.

To determine the vector competence of Culicoides sonorensis Wirth & Jones midges for vesicular stomatitis virus (VSV)-New Jersey, insects were experimentally infected per os and sampled over time. Viral replication, as determined by in situ hybridization, was seen in epithelial, neural, and hemolymph cell types throughout the insect. Spatial and temporal distribution of virus was determined by immunohistochemical examination of sequentially sampled insects. Tissues of the alimentary canal were infected in a temporal pattern that paralleled the route of digestion/absorption: foregut and midgut by day 1, surrounding hemolymph and Malpighian tubules by day 3, and finally the midgut/ hindgut junction, hindgut, and rectal region by day 5. The circulation of virus in the hemolymph by day 3 coincided with infection of the dermis and fat bodies, the salivary glands, eyes, cerebral and subthoracic ganglia, and the ovaries. Oviduct epithelium and ovarial sheaths were infected by day 3, followed by infection of the developing oocytes by day 5. Interestingly, neural infections were seen in the subabdominal ganglia innervating the midgut in 33% of insects by 1 d postfeeding in the absence of positive staining in the hemolymph or surrounding tissues. A retrograde axonal transport infection route for these ganglia is discussed. The disseminated, productive, noncytolytic infection in Culicoides is consistent with that of an efficient biological vector for VSV. Virus readily replicated throughout the insect, passing both midgut and salivary gland infection barriers and reaching transmission-related organs in 3 d. Establishing the competence of this insect vector for VSV provides the foundation for animal transmission studies in the future. The possibility of horizontal, transovarial, and mechanical transmission is discussed.

Numerous Culicoides spp. are important vectors of livestock or human disease pathogens. Transcriptome information from midguts and salivary glands of adult female Culicoides sonorensis provides new insight into vector biology. Of 1719 expressed sequence tags (ESTs) from adult serum-fed female midguts harvested within 5 h of feeding, twenty-eight clusters of serine proteases were derived. Four clusters encode putative iron binding proteins (FER1, FERL, PXDL1, PXDL2), and two clusters encode metalloendopeptidases (MDP6C, MDP6D) that probably function in bloodmeal catabolism. In addition, a diverse variety of housekeeping cDNAs were identified. Selected midgut protease transcripts were analysed by quantitative real-time PCR (q-PCR): TRY1&#95;115 and MDP6C mRNAs were induced in adult female midguts upon feeding, whereas TRY1&#95;156 and CHYM1 were abundant in midguts both before and immediately after feeding. Of 708 salivary gland ESTs analysed, clusters representing two new classes of protein families were identified: a new class of D7 proteins and a new class of Kunitz-type protease inhibitors. Additional cDNAs representing putative immunomodulatory proteins were also identified: 5' nucleotidases, antigen 5-related proteins, a hyaluronidase, a platelet-activating factor acetylhydrolase, mucins and several immune response cDNAs. Analysis by q-PCR showed that all D7 and Kunitz domain transcripts tested were highly enriched in female heads compared with other tissues and were generally absent from males. The mRNAs of two additional protease inhibitors, TFPI1 and TFPI2, were detected in salivary glands of paraffin-embedded females by in situ hybridization.

Bluetongue viruses (BTVs) are economically important arboviruses that affect sheep and cattle. The overwintering mechanism of BTVs in temperate climates has eluded researchers for many years. Many arboviruses overwinter in their invertebrate vectors. To test the hypothesis that BTVs overwinter in their vertically infected insect vectors, Culicoides sonorensis larvae were collected from long-term study sites in northern Colorado, USA, and assayed for the presence of BTV RNA by nested RT-PCR. Sequences from BTV RNA segment 7 were detected in 30 % (17/56) of pools composed of larvae and pupae collected in 1998 and in 10 % (31/319) of pools composed of adults reared from larvae collected in 1996. BTV was not isolated from the insects. Additionally, Culicoides cell-culture lines derived from material collected at one of the sites, or derived from insect samples collected during a BTV outbreak, contained BTV RNA segment 7. In contrast, segment 2 RNA was detected at half the rate of segment 7 RNA in the field-collected larvae and was only detected in the Culicoides cell lines with one of two primer sets. These data suggest that BTVs could overwinter in the insect vector and that there is reduced expression of the outer capsid genes during persistent infection.

Vesicular stomatitis (VS) is an economically devastating disease of livestock in the Americas. Despite strong circumstantial evidence for the role of arthropods in epizootics, no hematophagous vector explains the field evidence. Based on the spatiotemporal association of grasshopper outbreaks and VS epizootics, we investigated the potential role of these insects as vectors and reservoirs of the disease. The critical steps in the grasshopper-bovine transmission cycle were demonstrated, including 1) 62% of grasshoppers [Melanoplus sanguinipes (F.)] fed vesicular stomatitis virus (VSV) from cell culture became infected, with titers reaching 40,000 times the inoculative dose; 2) 40% of grasshoppers that cannibalized VSV-infected grasshopper cadavers became infected, amplifying virus up to 1,000-fold; 3) one of three cattle consuming VSV-infected grasshopper cadavers contracted typical VS and shed virus in saliva; and 4) 15% of grasshoppers became infected when fed saliva from this infected cow. The ecological conditions and biological processes necessary for these transmissions to occur are present throughout much of the Americas. Field studies will be required to show these findings are relevant to the natural epidemiology of VSV.

Two polymerase chain reaction tests for the detection of bluetongue viral (BLU) RNA in the principal North American insect vector, Culicoides variipennis, were developed. The BLU serogroup specific test used the highly expressed non-structural protein 1 gene as the target gene and two amplification steps. First a 1228 base pair product was amplified using an outer primer pair, then a second amplification using a nested or internal primer pair produced a 930 base pair product. This nested PCR test was found to be very sensitive detecting an equivalent to 1 plaque-forming unit of BLU viral RNA extracted from infected biting midges. The serotype specific test used a multiplex PCR approach in which five different primer pairs were used simultaneously. Each pair was based on the variable outer capsid protein VP2 gene of the five US serotypes generating specific product which were easily identified by size difference. The sensitivity of the multiplex PCR was less sensitive than the nested-PCR but sufficient for use with field collected samples. These tests provide valuable tools for epidemiologic studies of BLU disease.

Nucleic acid hybridization was used to detect epizootic hemorrhagic disease (EHD) virus serotype 1 and serotype 2 in Culicoides variipennis (Coquillett). Adult females were inoculated intrathoracically with virus, then were assayed daily for the presence of viral RNA for 2 wk, at which time maximum virus replication is likely to occur. Viral RNA of EHD serotypes 1 and 2 was first detected by hybridization on days 9 and 7 after infection, respectively, and then for up to 14 days after infection. EHD serotype 1 viral RNA was detected by hybridization in infected flies fixed in ethanol at room temperature for 7 d and in unfixed (frozen) infected flies. However, weak false positives diminished prospects for application of this method.