Genome-scale molecular and phylogenetic characterization of Middle Point orbiviruses from Australia.

Middle Point orbivirus (MPOV) is an Australian arbovirus, belongs to the Yunnan orbivirus species found in China. First detected and reported from Beatrice Hill, Northern Territory (NT), MPOV has to date, only been exclusively reported from the NT, Australia. Whilst genetic characterization of MPOV has been previously described, only restricted to sequence information for segments 2 and 3 coding core protein VP2 and outer capsid protein VP3, respectively. This study presents for the first time nearly full-length genome sequences of MPOV, which represent 24 isolates collected over a span of more than 20 years from 1997 to 2018. Whilst the majority of isolates were sampled at Beatrice Hill, NT where MPOV is most frequently isolated, this report also describes the first two isolations of MPOV from Queensland (QLD), Australia. One of which is the first non-bovine isolate obtained from the mosquito vector Aedes vittiger. We further compared these MPOV sequences with known sequences of the Yunnan orbivirus and other known orbivirus sequences of mosquito origin found in Australia. The phylogenetic analyses indicate the Australian MPOV sequences are more closely related to each other than other known sequences of Yunnan orbivirus. Furthermore, MPOV sequences are closely related to sequences from the Indonesian isolate JKT-8650. The clustering of Australian sequences in the phylogenetic tree suggests the monophyletic lineage of MPOV circulating in Australia. Further, ongoing surveillance is required to assess the existence and prevalence of this or other yet undetected lineages of MPOV and other orbiviruses in Australia.

Characterization of a novel reassortment Tibet orbivirus isolated from Culicoides spp. in Yunnan, PR China.

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.

Orbiviruses in biting midges and mosquitoes from the Zambezi region, Namibia.

The genus Orbivirus includes a variety of pathogenic viruses that are transmitted by biting midges, mosquitoes and ticks. Some of the economically most relevant orbiviruses are endemic to Namibia, like the livestock-pathogenic Bluetongue and African horse sickness viruses. Here, we assessed the diversity of orbiviruses circulating in the Zambezi region of north-eastern Namibia. A total of 10 250 biting midges and 10 206 mosquitoes were collected and screened for orbivirus infections. We identified Palyam virus (PALV) in a pool of biting midges (Culicoides sp.) sampled in the Wuparo Conservancy and three strains of Corriparta virus (CORV) in Culex sp. mosquitoes sampled in Mudumu National Park and the Mashi Conservancy. This is, to our knowledge, the first detection of PALV and CORV in Namibia. Both viruses infect vertebrates but only PALV has been reported to cause disease. PALV can cause foetal malformations and abortions in ruminants. Furthermore, a novel orbivirus, related to Kammavanpettai virus from India and Umatilla virus from North America, was discovered in biting midges (Culicoides sp.) originating from Mudumu National Park and tentatively named Mudumu virus (MUMUV). Complete genomes of PALV, CORV and MUMUV were sequenced and genetically characterized. The Namibian CORV strain showed 24.3 % nucleotide divergence in its subcore shell gene to CORV strains from Australia, indicating that African CORV variants vary widely from their Australian relatives. CORV was isolated in cell culture and replicated to high titres in mosquito and duck cells. No growth was found in rodent and primate cells. The data presented here show that diverse orbiviruses are endemic to the Zambezi region. Further studies are needed to assess their effects on wildlife and livestock.

Isolation of Tibet Orbivirus from Culicoides jacobsoni (Diptera, Ceratopogonidae) in China.

BACKGROUND: Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. METHODS: Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. RESULTS: Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoides jacobsoni was identified as TIBOV, while the other two viruses from C. orientalis and C. tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. CONCLUSIONS: The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known.

Genomic analysis of putative novel serotypes of Tibet orbivirus isolated in Japan.

Tibet orbivirus (TIBOV) was initially isolated in Tibet in 2009 and subsequently in Guangdong, Hunan, and Yunnan, China. We document the first isolation of TIBOV outside of China: two TIBOV isolates from Culicoides collected in 2009 and 2010 in Kagoshima, Japan. Their complete genome sequences were also determined. Our results suggest that the two virus isolates are of novel serotypes, evident by variability within genome segment 2 encoding VP2. These new putative TIBOV serotypes will help with future virus surveillance and with the evaluation of its potential to cause disease in domestic ruminants.

Isolation and characterization of Wad Medani virus obtained in the tuva Republic of Russia.

Wad Medani virus (WMV) belongs to the genus Orbivirus and is a poorly studied arbovirus with unclear medical significance. Presently, a limited number of WMV strains are characterized and available in NCBI GenBank, some isolated many years ago. A new WMV strain was isolated in 2012 from Dermacentor nuttalli ticks collected from sheep in the Tuva Republic, Russia, and sequenced using high-throughput methods. Complete coding sequences were obtained revealing signs of multiple intersegment reassortments. These point to a high variability potential in WMV that may lead to the formation of strains with novel properties. These new data on WMV can promote better understanding of: ecological features of its circulation; relationships within the genus Orbivirus; and the medical significance of the virus.

Identification and characterization of a novel orbivirus, Yonaguni orbivirus, isolated from cattle on the westernmost island of Japan.

A novel orbivirus (genus Orbivirus, family Reoviridae), designated Yonaguni orbivirus (YONOV), was isolated from bovine blood collected on a subtropical island of Japan in 2015. The YONOV genome (20,054 nucleotides in total) has a coding arrangement similar to those of mosquito-borne orbiviruses. YONOV has a close genetic relationship to mosquito-borne orbiviruses, especially to Mobuck virus (MBV), which was isolated in North America. However, YONOV and MBV share less than 74% nucleotide sequence identity in the major subcore protein (T2) coding sequence, which satisfies the criterion for species demarcation. It is still uncertain whether YONOV should be assigned to a novel species in the genus Orbivirus.

Tracking Community Timing: Pattern and Determinants of Seasonality in Culicoides (Diptera: Ceratopogonidae) in Northern Florida.

Community dynamics are embedded in hierarchical spatial-temporal scales that connect environmental drivers with species assembly processes. Culicoides species are hematophagous arthropod vectors of orbiviruses that impact wild and domestic ruminants. A better sense of Culicoides dynamics over time is important because sympatric species can lengthen the seasonality of virus transmission. We tested a putative departure from the four seasons calendar in the phenology of Culicoides and the vector subassemblage in the Florida panhandle. Two years of weekly abundance data, temporal scales, persistence and environmental thresholds were analyzed using a tripartite Culicoides ß-diversity based modeling approach. Culicoides phenology followed a two-season regime and was explained by stream flow and temperature, but not rainfall. Species richness fit a nested pattern where the species recruitment was maximized during spring months. Midges were active year-round, and two suspected vectors species, Culicoides venustus and Culicoides stellifer, were able to sustain and connect the seasonal modules. Persistence suggests that Orbivirus maintenance does not rely on overwintering and that viruses are maintained year-round, with the seasonal dynamics resembling subtropical Culicoides communities with temporal-overlapping between multivoltine species. Viewing Culicoides-borne orbiviruses as a time-sensitive community-based issue, our results help to recommend when management operations should be delivered.

Genomic and biological features of a novel orbivirus isolated from mosquitoes, in China.

A novel orbivirus had been identified as a member of the Orbivirus genus, which was isolated from pooled Culex fatigans mosquitoes in Guangdong of China, named as the Fengkai virus (FKOV). The cytopathic effects (CPEs) on both Aedes albopictus cells (C6/36) and mammalian cell lines (Vero and BHK-21) emerged in the cell cultures inoculated above virus in. Experimental confirmation as the Orbivirus genus was conducted by the Real-time PCR and based on Ion Torrent Next-Generation in sequencing. The Identities of VP1, VP2 and VP3 in amino acid sequences between the Tibet orbivirus (TIBOV) and this strain were 98.6%, 42.9%, and 99.9%, respectively, which indicated that this strain shares the same genus (VP1, Pol) and species (VP3, T2) with TIBOV but was greatly different in VP2 and VP5 (10.3%) of TIBOV. The VP2 and VP5 diversities of both TIBOV and FKOV strains suggested both serotypes are distinct with each other. As natural evolution and circulation, this strain might expand its host ranges and infect human beings as a potential and severe pathogen.

Genomic characterization of Changuinola viruses from Panama: evidence for multiple genome segment reassortment.

The complete coding sequences of five divergent strains of Changuinola virus (CGLV), collected over a 16-year period in Panama, were determined, using viral metagenomics. Each strain had 10 RNA segments that encoded structural and non-structural proteins with amino acid identities ranging from 33 to 99% with sequences of other 15 members of the Changuinola virus (Reoviridae: Orbivirus) species group. Genetic analyses of the five Panamanian virus strains revealed probable reassortment among multiple segments of the viruses.

Letea Virus: Comparative Genomics and Phylogenetic Analysis of a Novel Reassortant Orbivirus Discovered in Grass Snakes (Natrix natrix).

The discovery and characterization of novel arthropod-borne viruses provide valuable information on their genetic diversity, ecology, evolution and potential to threaten animal or public health. Arbovirus surveillance is not conducted regularly in Romania, being particularly very scarce in the remote and diverse areas like the Danube Delta. Here we describe the detection and genetic characterization of a novel orbivirus (Reoviridae: Orbivirus) designated as Letea virus, which was found in grass snakes (Natrix natrix) during a metagenomic and metatranscriptomic survey conducted between 2014 and 2017. This virus is the first orbivirus discovered in reptiles. Phylogenetic analyses placed Letea virus as a highly divergent species in the Culicoides-/sand fly-borne orbivirus clade. Gene reassortment and intragenic recombination were detected in the majority of the nine Letea virus strains obtained, implying that these mechanisms play important roles in the evolution and diversification of the virus. However, the screening of arthropods, including Culicoides biting midges collected within the same surveillance program, tested negative for Letea virus infection and could not confirm the arthropod vector of the virus. The study provided complete genome sequences for nine Letea virus strains and new information about orbivirus diversity, host range, ecology and evolution. The phylogenetic associations warrant further screening of arthropods, as well as sustained surveillance efforts for elucidation of Letea virus natural cycle and possible implications for animal and human health.

Ixodes persulcatus/pavlovskyi natural hybrids in Siberia: Occurrence in sympatric areas and infection by a wide range of tick-transmitted agents.

Ixodes persulcatus and Ixodes pavlovskyi ticks, two closely related species of the I. ricinus - I. persulcatus group, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatus and I. pavlovskyi ticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyi hybrids in several locations with different ratios of parental tick species and to investigate the prevalence and genetic variability of a wide range of infectious agents in these hybrids compared to the parental tick species. Natural hybrids of I. persulcatus and I. pavlovskyi ticks were identified in all examined locations in Altai and Novosibirsk, Western Siberia, Russia. The abundance of hybrids varied from 7% to 40% in different locations and was maximal in a location with similar proportions of I. persulcatus and I. pavlovskyi ticks. For the first time, it was shown that hybrids can be infected with the same agents as their parental tick species: tick-borne encephalitis and Kemerovo viruses, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Rickettsia raoultii, Rickettsia sibirica, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis", and Babesia microti. The prevalence of most bacterial agents in hybrids was intermediate compared to their parental tick species. Most genetic variants of the identified agents have been previously found in the parental tick species. Wide distribution of I. persulcatus/pavlovskyi natural hybrids implies that I. persulcatus, I. pavlovskyi and their hybrids coexist in all I. persulcatus - I. pavlovskyi sympatric areas.

Phylogenetic Characterization of the Palyam Serogroup Orbiviruses.

The Palyam serogroup orbiviruses are associated with abortion and teratogenesis in cattle and other ruminants. Of the 13 different serotypes that have been identified, the full genome sequence of only one, Kasba, has been published. We undertook to perform Next Generation Sequencing (NGS) and phylogenetic analysis on 12 Palyam serotypes plus field isolates of the African serotypes in our possession. The Palyam serogroup was found to be most closely related to the African horse sickness virus group and showed the most distant evolutionary relationship to the equine encephalosis viruses (EEV). Amino acid sequence analysis revealed that the gene encoding VP7 was the most conserved within serotypes and VP2 and VP5 showed the highest degree of variation. A high degree of sequence identity was found for isolates from the same geographical region. The phylogenetic analysis revealed two clades where the African serotypes were all very closely related in one clade and the other clade contained the Australian and Asian serotypes and one African serotype, Petevo. It was evident from the sequence data that the geographical origin of Palyam serogroup viruses played an important role in the development of the different serotypes.

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

Discovery and Characterization of Bukakata orbivirus (Reoviridae:Orbivirus), a Novel Virus from a Ugandan Bat.

While serological and virological evidence documents the exposure of bats to medically-important arboviruses, their role as reservoirs or amplifying hosts is less well-characterized. We describe a novel orbivirus (Reoviridae:Orbivirus) isolated from an Egyptian fruit bat (Rousettus aegyptiacus leachii) trapped in 2013 in Uganda and named Bukakata orbivirus. This is the fifth orbivirus isolated from a bat, however genetic information had previously only been available for one bat-associated orbivirus. We performed whole-genome sequencing on Bukakata orbivirus and three other bat-associated orbiviruses (Fomede, Ife, and Japanaut) to assess their phylogenetic relationship within the genus Orbivirus and develop hypotheses regarding potential arthropod vectors. Replication kinetics were assessed for Bukakata orbivirus in three different vertebrate cell lines. Lastly, qRT-PCR and nested PCR were used to determine the prevalence of Bukakata orbivirus RNA in archived samples from three populations of Egyptian fruit bats and one population of cave-associated soft ticks in Uganda. Complete coding sequences were obtained for all ten segments of Fomede, Ife, and Japanaut orbiviruses and for nine of the ten segments for Bukakata orbivirus. Phylogenetic analysis placed Bukakata and Fomede in the tick-borne orbivirus clade and Ife and Japanaut within the Culicoides/phlebotomine sandfly orbivirus clade. Further, Bukakata and Fomede appear to be serotypes of the Chobar Gorge virus species. Bukakata orbivirus replicated to high titers (106⁻107 PFU/mL) in Vero, BHK-21 [C-13], and R06E (Egyptian fruit bat) cells. Preliminary screening of archived bat and tick samples do not support Bukakata orbivirus presence in these collections, however additional testing is warranted given the phylogenetic associations observed. This study provided complete coding sequence for several bat-associated orbiviruses and in vitro characterization of a bat-associated orbivirus. Our results indicate that bats may play an important role in the epidemiology of viruses in the genus Orbivirus and further investigation is warranted into vector-host associations and ongoing surveillance efforts.

Quantitative RT-PCR assays for identification and typing of the Equine encephalosis virus.

Equine encephalosis (EE) is an acute, arthropod-borne, noncontagious, febrile disease of equids. The clinical signs of EE are similar to milder forms of African horse sickness (AHS) and the two diseases can be easily confused. The Equine encephalosis virus (EEV) is a distinct virus species within the genus Orbivirus, family Reoviridae, with ten linear segments of dsRNA genome. Seven distinct serotypes of EEV have been recognised on the basis of sequence analyses of Seg-2. The need for differential diagnosis of similar forms of EE and AHS warranted the development of molecular diagnostic methods for specific detection and identification of EEV. We report the development of quantitative real-time RT-PCR assay for detection of any member of the EEV species targeting the highly conserved EEV Seg-9. Similar serotype-specific qRT-PCR assays were designed for each of the seven EEV serotypes targeting genome Seg-2, encoding the serotype determining VP2 protein. These assays were evaluated using different EEV serotypes and other closely related orbiviruses. They were shown to be EEV virus species-specific, or EEV type-specific capable of detecting 1 to 13 copies of viral RNA in clinical samples. The assays failed to detect RNA from closely related orbiviruses, including AHSV and Peruvian horse sickness virus (PHSV) isolates.

Skunk River virus, a novel orbivirus isolated from Aedes trivittatus in the United States.

The genomic organization and in vitro host range of a novel mosquito-associated orbivirus, designated Skunk River virus, is described. The virus was isolated from Aedes trivittatus collected in Iowa in the United States. Three recognized viruses were also recovered: Culex flavivirus (family Flaviviridae), Houston virus (family Mesoniviridae) and Umatilla virus (family Reoviridae). The genome of Skunk River virus contains 10 segments and its organization is characteristic of viruses in the genus Orbivirus (family Reoviridae). The coding region of each segment was fully sequenced, revealing that the greatest nucleotide identity was to the corresponding regions of Big Cypress orbivirus and Sathuvachari virus, two recently described mosquito-associated orbiviruses. The phylogenetic inference is in agreement with these findings. In vitro host range experiments revealed that Aedes, Anopheles and Culex cell lines, and select lepidopteran and rodent cell lines, are permissive to Skunk River virus replication. In conclusion, we provide evidence of a novel mosquito-associated orbivirus in Iowa.

[Results of investigation of ticks in Volga river delta (Astrakhan region, 2017) for Crimean-Congo hemorrhagic fever virus (Nairoviridae, Orthonairovirus, CCHFV) and other tick-borne arboviruses.]

INTRODUCTION: There are natural foci of Crimean-Congo hemorrhagic fever (CCHF) that vectored by Hyalomma marginatum ticks in Volga river delta (Astrakhan region, South of Russia). The circulation of Dhori virus (DHOV) (Thogotovirus: Orthomyxoviridae) has been also shown here. We hypothesized that other tick-borne arboviruses are also likely to circulate in the region. In particular, Bhanja virus (Phlebovirus: Phenuiviridae), Wad Medani virus (Orbivirus: Reoviridae), and Tamdy virus (Orthonairovirus: Nairoviridae), which were found to circulate in neighboring regions and are vectored by Haemaphysalis spp., Dermacenter spp., and Hyalomma spp. ticks. OBJECTIVES: The aim of the study was to examine ixodid ticks in Volga river delta for the presence of CCHFV, DHOV, Bhanja virus, Wad Medani virus, and Tamdy virus. MATERIAL AND METHODS: Ticks were collected in Volga river delta in 2017. We used molecular genetic methods for the detection and analysis of nucleic acids (PCR, sequencing, phylogenetic analysis). RESULTS: We detect CCHFV and DHOV RNA in H. marginatum ticks. The rate of infected H. marginatum ticks was 1.98% for CCHFV and 0.4% for DHOV. The results of genetic analysis showed that found DHOV strains are almost identical (99-100% in the M gene) and forms a separate genetic lineage alongside of Batken virus from Central Asia. At the same time, Bhanja virus, Wad Medani virus, and Tamdy virus were not found in ticks, collected in this region. CONCLUSIONS: DHOV is circulating in the natural foci of CCHF in the Volga river delta. The ratio of infection of H. marginatum with CCHFV and DHOV was determined for the first time.

Full genome sequence of a Sathuvachari virus strain isolated in the southwestern-most archipelago of Japan.

Two virus strains, tentatively designated as ON-6/P/05 and ON-7/E/05, were isolated from blood samples of healthy cattle in the Yaeyama Islands, located in the southwestern-most region of Japan, in 2005. Ultrastructural observations of infected baby hamster (BHK-21) cells revealed that the viruses had features consistent with those of orbivirus. As with other orbiviruses, the viral genome consists of 10 double-stranded RNA segments. The full genome sequence of ON-6/P/05 was determined and shared high nucleotide and amino acid identities (90.07-98.22% nucleotide identity; 96.16-99.72% amino acid identity) with that of Sathuvachari virus (SVIV), a member of the species Sathuvachari virus of the genus Orbivirus, originally isolated from starlings collected in southern India in 1963. The sequence of segment two of ON-7/E/05 was identical to that of ON-6/P/05. The isolation of SVIV from cattle also indicated that the virus has a wider host range than previously thought. The potential pathogenicity of SVIV in domestic animals should be considered in future disease surveillance within its distribution range.

Laboratory studies on the oviposition stimuli of Culicoides stellifer (Diptera: Ceratopogonidae), a suspected vector of Orbiviruses in the United States.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) exert a significant impact on animal agriculture worldwide because they transmit bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) to ruminants. Without effective vaccines, BTV/EHDV vector management strategies are needed, particularly in commercial white-tailed deer (WTD) facilities. However, detailed information on the ecology of midge immatures in/around cervid operations is currently lacking. Towards filling this knowledge gap, we conducted two-choice oviposition experiments with field-collected Culicoides stellifer Coquillett (a suspected vector of BTV/EHDV in the USA) under laboratory conditions to examine which natural source from the larval habitat is relatively more attractive for midge oviposition. METHODS: Field-collected C. stellifer females (CDC-UV light traps) were given a blood meal from live chicken and examined for their oviposition preferences for individual (or mixed) potential larval habitat oviposition stimuli in two-choice bioassays. Substrates included mud from C. stellifer habitat, mud from allopatric site, vegetation (Sphagnum spp. mosses), field water, WTD manure and de-ionized water (control). RESULTS: The majority of midges (91%) oviposited in only one dish, with few females (9%) ovipositing in both the dishes. Gravid females demonstrated an overall oviposition preference for substrates with mud and vegetation from the larval habitat, depositing a significantly higher proportion of eggs on mud (52.3%) and vegetation (81.8%) than on controls (≤ 18.2%) (P ≤ 0.0320). Moreover, greater number of eggs per female were deposited on mud (29.5-40.7 depending on trial) and vegetation (38.2) than on controls (≤ 5.8). WTD manure, field water and mud from allopatric site were not found to be more attractive than controls for oviposition. Combining individual substrates (mud + WTD manure; mud + moss + WTD manure + field water) did not elicit greater oviposition responses than mud or moss alone. CONCLUSIONS: Management strategies to discourage C. stellifer oviposition in/around commercial cervid facilities should likely focus on mud and/or vegetation, rather than WTD manure. However, further studies are needed to examine whether the spatial distributions of C. stellifer and Sphagnum spp. moss are correlated, and to determine whether targeting vegetation in/around cervid facilities can contribute to reductions in local midge densities.