Search Results (17)

During the 2023 surveillance of mosquito-borne viruses in Ningxia Hui Autonomous Region, a strain of Umatilla virus (UMAV) was isolated from a pool of Culex pipiens pallens (NX23166) collected in Xiji County and cultured in C6/36 cells. Electron microscopy revealed that NX23166-infected mosquito cells showed approximately 70-nm virus particles, typical of the genus Orbivirus. Through next-generation sequencing, 10 double-stranded RNA (dsRNA) segments of the virus were obtained. Phylogenetic and homology analyses based on these sequences revealed that this strain was most closely related to the first Chinese isolate from Yunnan in 2013 (DH13M98) and an Australian isolate from 2015 (M4941_15). However, the VP3 protein of this strain showed the closest evolutionary relationship to a German isolate from 2019 (ED-I-205-19), with an amino acid sequence identity of 94.00%. In contrast, the identity of the VP3 protein to that of other strains ranged only from 47.38% to 51.49%, suggesting that these two strains may belong to the same serotype. Nevertheless, this hypothesis needs to be further verified by a serum neutralization test. Furthermore, transcriptome sequencing analysis showed that infection with the Ningxia isolate of UMAV induced significant temporal transcriptomic reprogramming in C6/36 cells. This reprogramming was characterized by early activation of innate immune responses such as the Toll signaling pathway and autophagy, followed by significant suppression of metabolic pathways, including oxidative phosphorylation in the mid to late stages of infection, demonstrating a molecular phenotype of coordinated immune activation and metabolic suppression. These results provide new insights into the genetic diversity and geographic distribution of the species UMAV. Full article

A newly formatted enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bluetongue virus (BTV) was developed and validated for bovine and ovine sera and plasma. Validation of the new sandwich ELISA (sELISA) was achieved with 949 negative bovine and ovine sera from BTV endemic and non-endemic areas of Australia and 752 BTV positive (field and experimental) sera verified by VNT and/or PCR. The test diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 99.70% and 99.20%, respectively, for bovine sera, and 97.80% and 99.50%, respectively, for ovine sera. Comparable diagnostic performances were noted for the sELISA compared to four competition ELISAs. While the sensitivity of the sELISA remained unaffected by BTV-15 positive sera, the cELISAs were not as sensitive. BTV-15 is endemic to Australia, and early warning depends on sensitive diagnoses of all serotypes: endemic or incurring. The sELISA failed to discriminate against epizootic hemorrhagic disease virus (EHDV) antibodies, the most serologically related orbivirus to BTV. The ACDP cELISA and the IDEXX kit showed cross-reactivity with some EHDV serotypes, with the least cross-reactive being the VMRD and the IDVet kits. Cross-reactivities, however, were also detected in sera raised experimentally from 10 isolates of the 21 known non-BTV orbiviruses. In this case, the sELISA was the least affected, followed equally by the VMRD and IDVet kits, and the IDEXX kit and the ACDP cELISA were the least discriminatory. In addition to exclusivity assessment of the ELISAs, an inclusivity assessment was made for all ELISAs using well characterized reference sera positive for antibodies to all serotypes BTV-1 to BTV-24. Full article

The bluetongue virus (BTV), transmitted by biting midges, poses a significant threat to livestock globally. This orbivirus induces bluetongue disease, leading to substantial economic losses in the agricultural sector. The current control measures have limitations, necessitating the development of novel, efficient vaccines. In this study, an immunoinformatics approach is employed to design a multi-epitope subunit vaccine for Ovis aries targeting six BTV serotypes. Focusing on the VP2 capsid protein, the vaccine incorporates B-cell, helper-T lymphocytes (HTL), and cytotoxic T-cell lymphocytes (CTL) epitopes. Molecular docking reveals stable interactions with TLR2 and TLR4 receptors, suggesting the stability of the complex, indicating the potential viability of the multi-epitope vaccine. The computational approach offers a rapid and tailored strategy for vaccine development, highlighting potential efficacy and safety against BTV outbreaks. This work contributes to understanding BTV and presents a promising avenue for effective control. Full article

Bluetongue virus (BTV, Sedoreoviridae: Orbivirus) causes an economically important disease, namely, bluetongue (BT), in domestic and wild ruminants worldwide. BTV is endemic to South India and has occurred with varying severity every year since the virus was first reported in 1963. BT can cause high morbidity and mortality to sheep flocks in this region, resulting in serious economic losses to subsistence farmers, with impacts on food security. The epidemiology of BTV in South India is complex, characterized by an unusually wide diversity of susceptible ruminant hosts, multiple vector species biting midges (Culicoides spp., Diptera: Ceratopogonidae), which have been implicated in the transmission of BTV and numerous co-circulating virus serotypes and strains. BT presence data (1997–2011) for South India were obtained from multiple sources to develop a presence/absence model for the disease. A non-linear discriminant analysis (NLDA) was carried out using temporal Fourier transformed variables that were remotely sensed as potential predictors of BT distribution. Predictive performance was then characterized using a range of different accuracy statistics (sensitivity, specificity, and Kappa). The top ten variables selected to explain BT distribution were primarily thermal metrics (land surface temperature, i.e., LST, and middle infrared, i.e., MIR) and a measure of plant photosynthetic activity (the Normalized Difference Vegetation Index, i.e., NDVI). A model that used pseudo-absence points, with three presence and absence clusters each, outperformed the model that used only the recorded absence points and showed high correspondence with past BTV outbreaks. The resulting risk maps may be suitable for informing disease managers concerned with vaccination, prevention, and control of BT in high-risk areas and for planning future state-wide vector and virus surveillance activities. Full article

African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated the ability of this assay to identify all nine AHSV serotypes, with detection limits ranging from 1 to 277 genome copies/µL depending on the AHSV serotype. An evaluation of diagnostic sensitivity and specificity revealed a sensitivity of 88% and specificity of 100%. This method can serotype up to 42 samples per run and can be completed in approximately 4–6 h. It provides a powerful tool to enhance the rapidity and efficiency of AHSV serotype detection, thereby facilitating the generation of epidemiological data that can help understand and control the incidence of AHSV worldwide. Full article

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. Full article

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct—cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution. Full article

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform. Full article

Sedoreoviridae is a family of viruses belonging to the order Reovirales and comprises six genera, two of which, Orbivirus and Seadornavirus, contain arboviruses that cause disease in humans and livestock. Areas such as Yunnan Province in southwestern China, have high arboviral activity due in part to warm and wet summers, which support high populations of biting flies such as mosquitoes and Culicoides. Three viral isolates previously obtained from Culicoides collected at cattle farms in Shizong County of Yunnan Province, China, between 2019 and 2020 were completely sequenced and identified as Banna virus (BAV) genotype A of Seadornavirus and serotypes 1 and 7 of epizootic hemorrhagic disease virus (EHDV) of Orbivirus. These results suggest that Culicoidestainanus and C. orientalis are potential vectors of BAV and EHDV, respectively, and represent the first association of a BAV with C. tainanus and of an arbovirus with C. orientalis. Analysis using VP9 generally agreed with the current groupings within this genus based on VP12, although the classification for some strains should be corrected. Furthermore, the placement of Kadipiro virus (KDV) and Liao ning virus (LNV) in Seadornavirus may need confirmation as phylogenetic analysis placed these viruses as sister to other species in the genus. Full article

Equine encephalosis (EE) is an arthropod-borne, noncontagious, febrile disease of horses. It is caused by EE virus (EEV), an Orbivirus of the Reoviridae family transmitted by Culicoides. Within the EEV serogroup, seven serotypes (EEV-1–7) have been identified to date. This virus was first isolated from a horse in South Africa in 1967 and until 2008 was believed to be restricted to southern Africa. In 2008–2009, isolation of EEV in an outbreak reported from Israel demonstrated the emergence of this pathogen into new niches. Indeed, testing in retrospect sera samples revealed that EEV had already been circulating outside of South Africa since 2001. Although EEV normally does not cause severe clinical disease, it should be considered important since it may indicate the possible spread of other related, much more pathogenic viruses, such as African horse sickness virus (AHSV). The spread of EEV from South Africa to central Africa, the Middle East and India is an example of the possible emergence of new pathogens in new niches, as was seen in the case of West Nile virus, and should be a reminder not to limit the differential list when facing a possible outbreak or a cluster of clinical cases. This review summarizes current knowledge regarding EEV structure, pathogenesis, clinical significance, and epidemiology. Full article

Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1–S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA–RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention. Full article

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three ‘novel’ serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same ‘VP2 nucleotype’. Full article

Epizootic hemorrhagic disease virus (EHDV; family Reoviridae, genus Orbivirus) is an arthropod-borne virus of ungulates, primarily white-tailed deer in North America. Culicoides sonorensis, the only confirmed North American vector of EHDV, is rarely collected from Florida despite annual virus outbreaks. Culicoides insignis is an abundant species in Florida and is also a confirmed vector of the closely related Bluetongue virus. In this study, oral challenge of C. insignis was performed to determine vector competence for EHDV serotype-2. Field-collected female midges were provided bovine blood spiked with three different titers of EHDV-2 (5.05, 4.00, or 2.94 log₁₀PFUe/mL). After an incubation period of 10 days or after death, bodies and legs were collected. Saliva was collected daily from all females from 3 days post feeding until their death using honey card assays. All samples were tested for EHDV RNA using RT-qPCR. Our results suggest that C. insignis is a weakly competent vector of EHDV-2 that can support a transmissible infection when it ingests a high virus titer (29% of midges had virus positive saliva when infected at 5.05 log₁₀PFUe/mL), but not lower virus titers. Nevertheless, due to the high density of this species, particularly in peninsular Florida, it is likely that C. insignis plays a role in the transmission of EHDV-2. Full article

Bluetongue virus (BTV), the prototype member of the genus Orbivirus (family Reoviridae), is the causative agent of an important livestock disease, bluetongue (BT), which is transmitted via biting midges of the genus Culicoides. To date, up to 29 serotypes of BTV have been described, which are classified as classical (BTV 1–24) or atypical (serotypes 25–27), and its distribution has been expanding since 1998, with important outbreaks in the Mediterranean Basin and devastating incursions in Northern and Western Europe. Classical vaccine approaches, such as live-attenuated and inactivated vaccines, have been used as prophylactic measures to control BT through the years. However, these vaccine approaches fail to address important matters like vaccine safety profile, effectiveness, induction of a cross-protective immune response among serotypes, and implementation of a DIVA (differentiation of infected from vaccinated animals) strategy. In this context, a wide range of recombinant vaccine prototypes against BTV, ranging from subunit vaccines to recombinant viral vector vaccines, have been investigated. This article offers a comprehensive outline of the live viral vectors used against BTV. Full article

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines that utilize viral vectors or are based on reverse genetics or virus-like particle technologies. This review summarizes the current understanding of AHSV structure and the viral replication cycle and also evaluates existing and potential vaccine strategies that may be applied to prevent or control the disease. Full article