Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus.

Mosquito-borne viruses cause various infectious diseases in humans and animals. Tibet orbivirus (TIBOV), a newly identified arbovirus, efficiently replicates in different types of vertebrate and mosquito cells, with its neutralizing antibodies detected in cattle and goats. However, despite being isolated from Culicoides midges, Anopheles, and Culex mosquitoes, there has been a notable absence of systematic studies on its vector competence. Thus, in this study, Aedes aegypti and Culex pipiens pallens were reared in the laboratory to measure vector susceptibility through blood-feeding infection. Furthermore, RNA sequencing was used to examine the overall alterations in the Ae. aegypti transcriptome following TIBOV infection. The results revealed that Ae. aegypti exhibited a high susceptibility to TIBOV compared to Cx. p. pallens. Effective replication of the virus in Ae. aegypti midguts occurred when the blood-feeding titer of TIBOV exceeded 105 plaque-forming units mL-1. Nevertheless, only a few TIBOV RNA-positive samples were detected in the saliva of Ae. aegypti and Cx. p. pallens, suggesting that these mosquito species may not be the primary vectors for TIBOV. Moreover, at 2 dpi of TIBOV, numerous antimicrobial peptides downstream of the Toll and Imd signaling pathways were significantly downregulated in Ae. aegypti, indicating that TIBOV suppressed mosquitos' defense to survive in the vector at an early stage. Subsequently, the stress-activated protein kinase JNK, a crucial component of the MAPK signaling pathway, exhibited significant upregulation. Certain genes were also enriched in the MAPK signaling pathway in TIBOV-infected Ae. aegypti at 7 dpi.IMPORTANCETibet orbivirus (TIBOV) is an understudied arbovirus of the genus Orbivirus. Our study is the first-ever attempt to assess the vector susceptibility of this virus in two important mosquito vectors, Aedes aegypti and Culex pipiens pallens. Additionally, we present transcriptome data detailing the interaction between TIBOV and the immune system of Ae. aegypti, which expands the knowledge about orbivirus infection and its interaction with mosquitoes.

Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Genome and evolution of Tibet orbivirus, TIBOV (genus Orbivirus, family Reoviridae).

Tibet orbivirus (TIBOV) was first isolated from Anopheles maculatus mosquitoes in Xizang, China, in 2009. In recent years, more TIBOV strains have been isolated in several provinces across China, Japan, East Asia, and Nepal, South Asia. Furthermore, TIBOVs have also been isolated from Culex mosquitoes, and several midge species. Additionally, TIBOV neutralizing antibodies have been detected in serum specimens from several mammals, including cattle, sheep, and pigs. All of the evidence suggests that the geographical distribution of TIBOVs has significantly expanded in recent years, with an increased number of vector species involved in its transmission. Moreover, the virus demonstrated infectivity towards a variety of animals. Although TIBOV is considered an emerging orbivirus, detailed reports on its genome and molecular evolution are currently lacking. Thus, this study performed the whole-genome nucleotide sequencing of three TIBOV isolates from mosquitoes and midges collected in China in 2009, 2011, and 2019. Furthermore, the genome and molecular genetic evolution of TIBOVs isolated from different countries, periods, and hosts (mosquitoes, midges, and cattle) was systematically analyzed. The results revealed no molecular specificity among TIBOVs isolated from different countries, periods, and vectors. Meanwhile, the time-scaled phylogenetic analysis demonstrated that the most recent common ancestor (TMRCA) of TIBOV appeared approximately 797 years ago (95% HPD: 16-2347) and subsequently differentiated at least three times, resulting in three distinct genotypes. The evolutionary rate of TIBOVs was about 2.12 × 10-3 nucleotide substitutions per site per year (s/s/y) (95% HPD: 3.07 × 10-5, 9.63 × 10-3), which is similar to that of the bluetongue virus (BTV), also in the Orbivirus genus. Structural analyses of the viral proteins revealed that the three-dimensional structures of the outer capsid proteins of TIBOV and BTV were similar. These results suggest that TIBOV is a newly discovered and rapidly evolving virus transmitted by various blood-sucking insects. Given the potential public health burden of this virus and its high infectious rate in a wide range of animals, it is significant to strengthen research on the genetic variation of TIBOVs in blood-feeding insects and mammals in the natural environment and the infection status in animals.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

The Study of Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) Circulation and Vectors at the Municipal Parks and Zoobotanical Foundation of Belo Horizonte, Minas Gerais, Brazil (FPMZB-BH).

Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) are Orbiviruses primarily transmitted by their biological vector, Culicoides spp. Latreille, 1809 (Diptera: Ceratopogonidae). These viruses can infect a diverse range of vertebrate hosts, leading to disease outbreaks in domestic and wild ruminants worldwide. This study, conducted at the Belo Horizonte Municipal Parks and Zoobotany Foundation (FPMZB-BH), Minas Gerais, Brazil, focused on Orbivirus and its vectors. Collections of Culicoides spp. were carried out at the FPMZB-BH from 9 December 2021 to 18 November 2022. A higher prevalence of these insects was observed during the summer months, especially in February. Factors such as elevated temperatures, high humidity, fecal accumulation, and proximity to large animals, like camels and elephants, were associated with increased Culicoides capture. Among the identified Culicoides spp. species, Culicoides insignis Lutz, 1913, constituted 75%, and Culicoides pusillus Lutz, 1913, 6% of the collected midges, both described as competent vectors for Orbivirus transmission. Additionally, a previously unreported species in Minas Gerais, Culicoides debilipalpis Lutz, 1913, was identified, also suspected of being a transmitter of these Orbiviruses. The feeding preferences of some Culicoides species were analyzed, revealing that C. insignis feeds on deer, Red deer (Cervus elaphus) and European fallow deer (Dama dama). Different Culicoides spp. were also identified feeding on humans, raising concerns about the potential transmission of arboviruses at the site. In parallel, 72 serum samples from 14 susceptible species, including various Cervids, collected between 2012 and 2022 from the FPMZB-BH serum bank, underwent Agar Gel Immunodiffusion (AGID) testing for BTV and EHDV. The results showed 75% seropositivity for BTV and 19% for EHDV. Post-testing analysis revealed variations in antibody presence against BTV in a tapir and a fallow deer and against EHDV in a gemsbok across different years. These studies confirm the presence of BTV and EHDV vectors, along with potential virus circulation in the zoo. Consequently, implementing control measures is essential to prevent susceptible species from becoming infected and developing clinical diseases.

Culicoides-borne Orbivirus epidemiology in a changing climate.

Orbiviruses are of significant importance to the health of wildlife and domestic animals worldwide; the major orbiviruses transmitted by multiple biting midge (Culicoides) species include bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus. The viruses, insect vectors, and hosts are anticipated to be impacted by global climate change, altering established Orbivirus epidemiology. Changes in global climate have the potential to alter the vector competence and extrinsic incubation period of certain biting midge species, affect local and long-distance dispersal dynamics, lead to range expansion in the geographic distribution of vector species, and increase transmission period duration (earlier spring onset and later fall transmission). If transmission intensity is associated with weather anomalies such as droughts and wind speeds, there may be changes in the number of outbreaks and periods between outbreaks for some regions. Warmer temperatures and changing climates may impact the viral genome by facilitating reassortment and through the emergence of novel viral mutations. As the climate changes, Orbivirus epidemiology will be inextricably altered as has been seen with recent outbreaks of bluetongue, epizootic hemorrhagic disease, and African horse sickness outside of endemic areas, and requires interdisciplinary teams and approaches to assess and mitigate future outbreak threats.

Orbivirus NS4 Proteins Play Multiple Roles to Dampen Cellular Responses.

Non-structural protein 4 (NS4) of insect-borne and tick-borne orbiviruses is encoded by genome segment 9, from a secondary open reading frame. Though a protein dispensable for bluetongue virus (BTV) replication, it has been shown to counter the interferon response in cells infected with BTV or African horse sickness virus. We further explored the functional role(s) of NS4 proteins of BTV and the tick-borne Great Island virus (GIV). We show that NS4 of BTV or GIV helps an E3L deletion mutant of vaccinia virus to replicate efficiently in interferon-treated cells, further confirming the role of NS4 as an interferon antagonist. Our results indicate that ectopically expressed NS4 of BTV localised with caspase 3 within the nucleus and was found in a protein complex with active caspase 3 in a pull-down assay. Previous studies have shown that pro-apoptotic caspases (including caspase 3) suppress type I interferon response by cleaving mediators involved in interferon signalling. Our data suggest that orbivirus NS4 plays a role in modulating the apoptotic process and/or regulating the interferon response in mammalian cells, thus acting as a virulence factor in pathogenesis.

Occurrence and surveillance of Taiwanese bovine arboviruses using hematophagous insects in dairy farms during 2012-2019.

Culicoides-borne viruses are an important arbovirus group causing bovine diseases. During 2012-2019, 2,525 pools consisting of 108,937 specimens of vectors were subjected to PCR detection of bovine arbovirus belonging to Orthobunyavirus, Orbivirus, and Ephemerovirus. Twelve virus RNAs, of which 6, that is, Shuni virus, Shamonda virus, and Sathuperi virus in Orthobunyavirus and Sathuvachari virus and epizootic hemorrhagic disease virus serotypes 4 and 7 in Orbivirus were detected for the first time in the area. Potential vector species were evaluated by the minimum infection rate, and the population abundance of Culicoides oxystoma, Culex tritaeniorhynchus, and Anopheles sinensis indicated that they were the main potential vector species in dairy farms in Taiwan.

Two putative novel serotypes of Tibet orbivirus isolated from Culicoides spp. in Yunnan, China.

Tibet orbivirus (TIBOV) was identified as a novel orbivirus in 2014. Antibodies against TIBOV were detected in cattle, Asian buffalo, and goats, while all the sequenced TIBOV strains were isolated from mosquitos and Culicoides. The known TIBOV strains have been classified into four putative serotypes. In this study, two TIBOV strains isolated from Culicoides spp. in Shizong County of Yunnan Province, China, were fully sequenced. The phylogenetic analysis of outer capsid protein 2 (VP2) indicated that these two viral strains belong to two novel putative serotypes of TIBOV. The updated putative serotypes may help in an investigation of the distribution and virulence of TIBOV.

Identification of Orbivirus Non-Structural Protein 5 (NS5), Its Role and Interaction with RNA/DNA in Infected Cells.

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.

Characterization of a Novel Orbivirus from Cattle Reveals Active Circulation of a Previously Unknown and Pathogenic Orbivirus in Ruminants in Kenya.

Arboviruses are among emerging pathogens of public and veterinary health significance. However, in most of sub-Saharan Africa, their role in the aetiologies of diseases in farm animals is poorly described due to paucity of active surveillance and appropriate diagnosis. Here, we report the discovery of a previously unknown orbivirus in cattle collected in the Kenyan Rift Valley in 2020 and 2021. We isolated the virus in cell culture from the serum of a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. High-throughput sequencing revealed an orbivirus genome architecture with 10 double-stranded RNA segments and a total size of 18,731 bp. The VP1 (Pol) and VP3 (T2) nucleotide sequences of the detected virus, tentatively named Kaptombes virus (KPTV), shared maximum similarities of 77.5% and 80.7% to the mosquito-borne Sathuvachari virus (SVIV) found in some Asian countries, respectively. Screening of 2,039 sera from cattle, goats, and sheep by specific RT-PCR identified KPTV in three additional samples originating from different herds collected in 2020 and 2021. Neutralizing antibodies against KPTV were found in 6% of sera from ruminants (12/200) collected in the region. In vivo experiments with new-born and adult mice induced body tremors, hind limb paralysis, weakness, lethargy, and mortality. Taken together, the data suggest the detection of a potentially disease-causing orbivirus in cattle in Kenya. Its impact on livestock, as well as its potential economic damage, needs to be addressed in future studies using targeted surveillance and diagnostics. IMPORTANCE The genus Orbivirus contains several viruses that cause large outbreaks in wild and domestic animals. However, there is little knowledge on the contribution of orbiviruses to diseases in livestock in Africa. Here, we report the identification of a novel presumably disease-causing orbivirus in cattle, Kenya. The virus, designated Kaptombes virus (KPTV), was initially isolated from a clinically sick cow aged 2 to 3 years, presenting signs of lethargy. The virus was subsequently detected in three additional cows sampled in neighboring locations in the subsequent year. Neutralizing antibodies against KPTV were found in 10% of cattle sera. Infection of new-born and adult mice with KPTV caused severe symptoms and lead to death. Together, these findings indicate the presence of a previously unknown orbivirus in ruminants in Kenya. These data are of relevance as cattle represents an important livestock species in farming industry and often is the main source of livelihoods in rural areas of Africa.

Genetic Characterization of DH13M98, Umatilla Virus, Isolated from Culex tritaeniorhynchus Giles in Yunnan Province, China.

Background: In August 2013, a virus strain (DH13M98) was isolated from Culex tritaeniorhynchus Giles collected in Mangshi, the southwestern border area of Yunnan Province, China. The virus replicated and caused cytopathic effects (CPE) in Aedes albopictus (C6/36) cells, but not in baby hamster Syrian kidney (BHK-21) cells. Materials and Methods: Agarose gel electrophoresis (AGE) analysis revealed that the DH13M98 virus was a 10-segment double-stranded RNA (dsRNA) virus, with a "1-1-1-2-1-1-2-1" pattern. The full genome of the DH13M98 virus was sequenced by full-length amplification of complementary DNAs (FLAC). Results: Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), major subcore-shell (T2), and major core-surface (T13) protein showed that DH13M98 clustered with Umatilla virus (UMAV), and the amino acid (aa) sequences of DH13M98 shared more than 89.5% (Pol), 95% (T2), and 91.1% (T13) identity with UMAV. However, the aa identity of outer capsid protein one (OC1) of DH13M98 with other UMAV was 57.1-79.2%, suggesting that DH13M98 was UMAV, but distinct from other strains of UMAV from the United States, Japan, and Germany at OC1, and it may be a high variant strain of UMAV, even a new serotype. Conclusion: This is the first isolation of UMAV in China, which enriches the resources of virus species in China and provides new insights into the genetic diversity and geographical distribution of the virus.

Full-genome characterisation of a putative novel serotype of Yonaguni orbivirus isolated from cattle in Yunnan province, China.

In July 2019, a novel viral strain (JH2019C603) was isolated from sentinel cattle in Jinghong City, in the subtropical region of Yunnan Province, China. The virus replicated and caused cytopathological effects in both Aedes albopictus (C6/36) and Baby Hamster Syrian Kidney (BHK-21) cells. Agarose gel electrophoresis analysis revealed a viral genome comprised of 10 segments of double-stranded RNA, with a 1-2-2-1-1-1-1-1 migration pattern. Complete genome sequences of the JH2019C603 virus were determined through full-length cDNA amplification. Phylogenetic analysis based on the amino acid (aa) sequences of RNA-dependent RNA Polymerase (Pol), Major subcore (T2) and Major core-surface (T13) showed that JH2019C603 clustered with Yonaguni orbivirus (YONOV) from Japan, with aa identities relative to YONOV of 97.7% (Pol), 99.0% (T2) and 98.5% (T13). However, phylogenetic analysis based on the aa sequences of the outer capsid protein one and two (OC1 and OC2) showed that JH2019C603 formed an independent branch in the phylogenetic tree, and its aa identity with YONOV was only 55.4% (OC1) and 80.8% (OC2), respectively. Compared with the prototype of YONOV, a notable sequence deletion was observed in the 3' non-coding region of NS1, with the NS1 of JH2019C603 encoded within segment 7 (Seg-7), in contrast to YONOV, which contains NS1 in Seg-6. These results indicate that JH2019C603 belongs to the YONOV lineage and might be a novel serotype or a highly variant strain of YONOV. These findings will facilitate the identification of new isolates and clarify their geographical distribution, epidemiology, genetic diversity and possible disease associations.

Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

Comparative Genome Analysis of All Nine African Horse Sickness Serotypes Isolated From Equine Fatalities in Kenya and South Africa.

African horse sickness (AHS) is a viral disease of equids, caused by a virus of the genus Orbivirus, family Reoviridae. The African horse sickness virus (AHSV) genome is made up of ten double-stranded RNA (dsRNA) segments that together code for seven structural and four nonstructural proteins. AHS is endemic in sub-Saharan countries. The efficacy and safety of inactivated AHS vaccines containing all nine serotypes, produced at the Central Veterinary Research Laboratory (CVRL) in Dubai, United Arab Emirates have been proven in the past. All nine AHSV serotypes were isolated from 102 samples collected in the last 20 years from horse fatalities in seven different area of Kenya, Africa. CVRL inactivated AHS vaccines are used in a few African countries defining the importance of this present study to compare the genome sequences of the nine AHSV serotypes isolated from horse fatalities in Kenya and nine AHSV serotypes isolated in South Africa. The hypothesized serotypes of the newly sequenced AHSV field strains from Kenya were likewise confirmed in this investigation, and they show substantial sequence homologies with recently isolated AHSV field strains.

Isolation and characterization of two novel serotypes of Tibet orbivirus from Culicoides and sentinel cattle in Yunnan Province of China.

Tibet orbivirus (TIBOV), a new candidate of Orbivirus genus, was initially isolated from mosquitoes in Tibet in 2009 and subsequently from both Culicoides and mosquitoes in several provinces of China and Japan. Little is known about the origin, genetic diversity, dissemination and pathogenicity of TIBOV, although its potential threat to animal health has been acknowledged. In this study, two viruses, V290/YNSZ and V298/YNJH, were isolated from the Culicoides and sentinel cattle in Yunnan Province. Their genome sequences, cell tropism in mammalian and insect cell lines along with pathogenicity in suckling mice were determined. Genome phylogenetic analyses confirmed their classification as TIBOV species; however, OC1 proteins of the V290/YNSZ and V298/YNJH shared maximum sequence identities of 31.5% and 33.9% with other recognized TIBOV serotypes (TIBOV-1 to TIBOV-4) and formed two monophyletic branches in phylogenetic tree, indicating they represented two novel TIBOV serotypes which were tentatively designated as TIBOV-5 and TIBOV-6. The viruses replicated robustly in BHK, Vero and C6/36 cells and triggered overt clinical symptoms in suckling mice after intracerebral inoculation, causing mortality of 100% and 25%. Cross-sectional epidemiology analysis revealed silent circulation of TIBOV in Yunnan Province with overall prevalence of 16.4% (18/110) in cattle, 10.8% (13/120) in goats and 5.5% (6/110) in swine. The prevalence patterns of four investigated TIBOV serotypes (TIBOV-1, -2, -5 and 6) differed from each one another, with their positive rates ranging from 8.2% (9/110) for TIBOV-2 in cattle to 0.9% (1/110) for TIBOV-1 and TIBOV-5 in cattle and swine. Our findings provided new insights for diversity, pathogenicity and epidemiology of TIBOV and formed a basis for future studies addressing the geographical distribution and the zoonotic potential of TIBOV.

[Dwarf bat's (Pipistrellus pipistrellus) lung diploid cell strains and their permissivity to orbiviruses (Reoviridae: Orbivirus) - pathogens of vector-borne animal diseases].

INTRODUCTION: Bat cell cultures are a popular model both for the isolation of vector-borne disease viruses and for assessing the possible role of these mammalian species in forming the natural reservoirs of arbovirus infection vectors. The goal of the research was to obtain and characterize strains of diploid lung cells of the bat (Pipistrellus pipistrellus) and evaluate their permissivity to bluetongue, African horse sickness (AHS), and epizootic hemorrhagic disease of deer (EHD) viruses. MATERIALS AND METHODS: Cell cultures of the dwarf bat's lung were obtained by standard enzymatic disaggregation of donor tissue and selection of cells for adhesive properties. The permissivity of cell cultures was determined to bluetongue, AHL, and EHD orbiviruses. RESULTS: Diploid cell strains (epithelium-like and fibroblast-like types) retaining cytomorphological characteristics and karyotype stability were obtained from tissue of the bat's lung. Their permissivity to viruses of the genus Orbivirus of the Reoviridae family, pathogens of transmissible animal diseases, has been established. DISCUSSION: The permissivity of the obtained strains of bat's lung cells to bluetongue, AHL, and EHD viruses is consistent with the isolation of orbiviruses in bats of the species Pteropus poliocephalus, Pteropus hypomelanus, Rousettus aegyptiacus leachii, Syconycteris crassa, Myotis macrodactylus, and Eidolon helvum. CONCLUSION: Strains of diploid lung cells of the dwarf bat are permissive to orbiviruses of bluetongue, AHS, and EHD, which allows us to recommend them for the isolation of these viruses, and the species Pipistrellus pipistrellus to be considered as a potential natural reservoir and carrier of pathogens of these vector-borne diseases.

Field Comparison of Removed Substrate Sampling and Emergence Traps for Estimating Culicoides Orbivirus Vectors in Northern Florida.

The larval ecology of Culicoides (Diptera: Ceratopogonidae) influences their spatial distributions and the pathogens they transmit. These features are of special concern for deer farmers in Florida where epizootic hemorrhagic disease virus (EHDV) is a major source of mortality in captive herds. Rarity of larval morphological expertise leads many researchers to study larval ecology by quantifying emergence, either with field emergence traps or removing substrate from the field for observation under laboratory conditions. We investigated the comparability of these methods in Florida seepages where two recently implicated EHDV vectors, Culicoides stellifer Coquillett and Culicoides venustus Hoffman, are common. We compared the abundance and composition of emerging Culicoides collected from emergence traps with removed substrate samples (soil plugs) at three seepages. Soil plugs were sampled adjacent to the emergence trap and from underneath the trap footprint, and then monitored under laboratory conditions for 11-13 wk to compare the methods and to assess the role of incubation period for removed substrate samples. Emergence traps and removed substrate sampling largely agreed on community compositions and trends within different seepages. However, comparatively large numbers of C. stellifer emerged later than expected and well into the incubation period with emergence still occurring after 13 wk (90 d). Removed substrate samples were more similar to emergence traps at shorter incubation times. The importance of time for the capture of Culicoides in removed substrate sampling was more pronounced than we anticipated and is important from both a methodological and biological perspective.

Evaluation of Vector Competence of Ixodes Ticks for Kemerovo Virus.

Tick-borne viruses are responsible for various symptoms in humans and animals, ranging from simple fever to neurological disorders or haemorrhagic fevers. The Kemerovo virus (KEMV) is a tick-borne orbivirus, and it has been suspected to be responsible for human encephalitis cases in Russia and central Europe. It has been isolated from Ixodes persulcatus and Ixodes ricinus ticks. In a previous study, we assessed the vector competence of I. ricinus larvae from Slovakia for KEMV, using an artificial feeding system. In the current study, we used the same system to infect different tick population/species, including I. ricinus larvae from France and nymphs from Slovakia, and I. persulcatus larvae from Russia. We successfully confirmed the first two criteria of vector competence, namely, virus acquisition and trans-stadial transmission, for both tick species that we tested. The estimated infection rates of engorged and moulted ticks suggest specificities between viral strains and tick species/developmental stages.

Evaluation of two artificial infection methods of live ticks as tools for studying interactions between tick-borne viruses and their tick vectors.

Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR-/- or BALB/c). Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.