Search Results (148)

Bluetongue virus (BTV), the prototype of the genus Orbivirus, infects livestock, causing high morbidity and mortality and impacting global trade. BTV is a non-enveloped, double-capsid virus, composed of seven structural proteins and a genome of 10 double-stranded RNA segments. This manuscript highlights our group’s recent findings on the molecular and structural mechanisms underlying BTV entry and assembly during replication. Viral entry is a stepwise, pH-dependent process. The outermost protein, VP2, attaches to sialic acids and senses the acidic pH of early endosomes, triggering their dissociation. Subsequently, the second outer capsid protein, VP5, undergoes major changes in late endosomes, forming a membrane-penetrating pore that releases the transcriptionally active inner core into the host cytoplasm. Core assembly also proceeds stepwise and requires the accurate packaging of 10 positive-sense RNA segments. These segments form an RNA–RNA interaction network independent of viral proteins, beginning with the smaller segments and guiding the complete genome assortment. The small capsid protein, VP6, interacts with VP3 to facilitate RNA encapsidation. While infectious cores assemble in vitro without non-structural proteins, NS2 is essential for the in vivo formation of viral inclusion bodies via liquid–liquid phase separation, concentrating viral components and promoting genome assembly. These comprehensive characterizations of BTV provide a foundation for future control strategies against related reoviruses. Full article

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral disease of ruminants, and has a severe impact on livestock. It is caused by Bluetongue virus (BTV), a double-stranded (ds) RNA virus with a segmented genome (10 segments), belonging to the Seoreoviridae family, Orbivirus genus. Over the last 25 years, Europe has suffered multiple incursions of different BTV serotypes with serious consequences, which have mainly been controlled thanks to vaccination. In the case of Spain, from 2000 to 2023, BTV serotypes 1, 2, 4 and 8 have caused epidemics, and, sporadically, BTV-1 and -4 were detected in the same area and period. In 2024, BTV serotypes 1, 3 and 8 circulated simultaneously in the southwest of the country, causing a severe clinical impact in sheep but also in cattle and a multitude of outbreaks. Additionally, despite vaccination, serotype 4 also circulated that year, especially in areas where the other serotypes were already circulating. Whole-genome sequencing and phylogenetic analyses allowed us to confirm that serotypes 1 and 4 were homologous to viruses circulating in the country since 2000s, while serotypes 3 and 8 were homologous to BTVs recently notified in neighboring countries. In this context, many BTV co-infections of two or more different serotypes were confirmed by serotype-specific RT-PCRs, both in farms and individual animals. An epidemic caused by four serotypes coinciding in space and time had never occurred before in Spain, being a challenge for the diagnosis and control of this disease. Moreover, it could have favored the appearance of reassortant viruses with an unknown virulence, posing an additional risk. The data presented here raise the question of whether the co-circulation of different BTV strains, an exceptional situation, could become the new normal in certain areas of Europe. Full article

Bluetongue virus (BTV), with a genome of ten double-stranded RNA segments (S1–S10), is an emerging animal pathogen causing major economic losses in livestock worldwide. BTV replication involves RNA-RNA and RNA–protein interactions, with RNA-binding proteins, VP6 and NS2 playing key roles in genome assembly and RNA packaging. To explore the dynamics of RNA segment interactions and the roles of VP6 and NS2 in RNA complex formation, we used RNA fluorescence in situ hybridization chain reaction (HCR), along with site-specific mutagenesis and reverse genetics. We found that RNA segments interact sequentially, from the smallest (S10) to the largest (S1), forming a single complex that includes the entire genome. This process is independent of VP6 or NS2, although NS2 enhances the assembly of larger segments. Additionally, we show that VP6 binds to +ssRNAs before their incorporation into viral assembly factories (inclusion bodies/VIBs). These findings reveal that RNA-RNA interactions, rather than primary replicase proteins, govern the sorting and recruitment of genome segments. Our data offer new insights into BTV RNA packaging, showing that genome segments destined for packaging and dsRNA synthesis are segregated through complex formation, distinct from +ssRNAs used in protein synthesis, including those encoding the replicase complex. Full article

To monitor the epidemiological situation of bluetongue virus (BTV) in Belgium, a national surveillance programme was conducted during the 2024–2025 winter season. The objective was to estimate the apparent seroprevalence of BTV-3 following the 2023–2024 epidemic and to prove the absence of active circulation of other BTV serotypes in mixed herds (cattle and sheep). A total of 2551 cattle and 1458 sheep were sampled across Belgium. Serological analyses were performed using ELISA, and molecular detection of BTV-3, BTV-8, and BTV-12 was conducted by RT-qPCR. The majority of cattle and sheep herds showed evidence of exposure to BTV-3, with a very high herd-level apparent seroprevalence (100%; 95% CI: 96.2–100% in cattle and 98.9%; 95% CI: 93.8–99.8% in sheep). Apparent within-herd seroprevalence was also high in cattle (94.6%; 95% CI: 91.8–96.5%) and sheep (85.5%; 95% CI: 80.4–89.5%). No evidence of active circulation of BTV-8 or BTV-12 was detected. A moderate significant positive correlation between Ct values and sampling date was observed both for bovine and ovine samples, consistent with a progressive decline in detectable BTV RNA during winter in the absence of vector activity. Full article

Water buffaloes in northern Australia occupy tropical wetlands where conditions favour the proliferation of arthropod vectors and the transmission of vector-borne livestock diseases. However, their role in maintaining economically important arboviruses such as Akabane virus (AKAV), bluetongue virus (BTV), and bovine ephemeral fever virus (BEFV) remains poorly understood. These three viruses cause significant production losses in cattle and pose ongoing surveillance challenges in remote areas. To assess exposure to these viruses, a convenience sample of feral water buffaloes from the Northern Territory, Australia, was collected. Commercial enzyme-linked immunosorbent assays (ELISAs) were used to detect antibodies against AKAV, BTV, and BEFV in 119 samples stored as dried blood on filter paper. Seroprevalence was 18.5% for AKAV, 66.4% for BTV, and 15.1% for BEFV. These results are consistent with previous serological studies in northern Australian cattle, confirming the circulation of these pathogens in the region. Our findings demonstrate that water buffaloes are exposed to these economically important arboviruses and may contribute to their maintenance, highlighting the need to consider feral buffalo populations in regional arbovirus surveillance strategies and livestock disease management. Full article

Bluetongue (BT) is a noncontagious, arthropod-borne viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), an arbovirus of the Orbivirus genus within the Sedoreoviridae family. At least 36 serotypes have been identified globally; recurrent circulation of BTV-1, -4, and -8, along with the recent emergence of BTV-3 in northern Europe, underscores a persistent incursion risk for Mediterranean herds. Key drivers include climate-driven expansion of Culicoides vector niches, windborne dispersal, animal movements, and subclinical reservoirs in cattle and goats. As no specific treatment is currently available, control of bluetongue disease still relies largely on vaccination. Live-attenuated vaccines and inactivated vaccines have reduced incidence, but important limitations persist: risk of reversion and the possibility of reassortment for LAVs; requirement for multiple doses and limited cross-protection for inactivated products; and the absence of DIVA capability for both. As an alternative, next-generation platforms are under active evaluation. Subunit formulations, often VP2 combined with VP5 and/or NS1/NS2 virus-like particles (VLPs), and viral-vectored constructs demonstrate favorable safety, strong humoral and cellular responses, inherent or engineered DIVA compatibility, and potential for rapid updating against emergent serotypes. This review synthesizes recent bluetongue activity across the Mediterranean Basin and provides a critical assessment of both existing and emerging vaccine strategies, with a focus on recommending next-generation platforms that emphasize DIVA-compliant, multiserotype, and adaptable vaccination approaches, supported by integrated surveillance and vector control in the region. Full article

Bluetongue virus (BTV) infects various ruminant species, posing significant threats to animal health and causing substantial economic losses to the livestock industry. Ovine type II alveolar epithelial cells (OAECIIs) play crucial roles in maintaining pulmonary structural integrity and modulating immune responses. Their dysfunction is closely associated with lung disease pathogenesis, making them important therapeutic targets. However, OAECIIs’ immunoregulatory functions and early response mechanisms during BTV infection remain unclear. To address this, we analyzed transcriptomic changes in OAECIIs following BTV-1 infection. RNA-seq revealed 1047 and 852 differentially expressed genes (DEGs) at 8 and 12 h post-infection (hpi), respectively, compared to uninfected controls. Bioinformatics analysis showed significant upregulation of nucleic acid-sensing receptors, interferon-stimulating factors, inflammatory mediators, and cytokines during early infection, mediated primarily through type I interferon signaling, TNF signaling, and cytosolic DNA-sensing pathways. We identified MAD5, ZNFX1, cGAS, OAS, PKR and ZBP1 as key pattern recognition receptors in OAECIIs during BTV infection. The IFN-β, MX1/2, RSAD2 and PLSCR1 pathways mediated antiviral responses, while IL-15, CXCL10, CCL2 triggered inflammatory responses, collectively causing structural alterations through AQP1/9 and tight junction protein modulation. These findings provide critical insights into early antiviral mechanisms and cellular structural changes in OAECIIs during BTV infection, establishing a foundation for understanding pneumonia pathogenesis and developing targeted BTV therapies. Full article

Bluetongue (BT) is an arthropod-borne viral disease primarily affecting domestic and wild ruminants. In recent years, several BTV serotypes and genotypes have been detected in Israel almost annually, raising questions about their origin and routes of introduction. Some BTV serotypes closely related to those first identified in Israel, including BTV-3, BTV-8, and BTV-12, were subsequently reported in Europe after a delay of several years. In this study, we sequenced the complete genomes of one representative strain of all newly identified Israeli BTV genotypes/serotypes—BTV-1, -4, -5, -8, and -11—first detected between 2021 and 2023. Additionally, complete sequences of enzootic Israeli BTV (2015) and eleven BTV-3 strains (2019–2023), with two representative strains for every year of isolation, except 2021 (three strains), were analyzed using phylogenetic, BLAST, and pairwise identity approaches. Genetic analyses revealed that recently identified Israeli and European BTV strains share common African ancestors, with some genomic “incursions” from Mayotte Island or the Arabian Peninsula. These incursions appeared more frequently in Israeli than in European strains. Nevertheless, nucleotide sequence differences of at least 2–3% across all genes indicate several years of independent evolution. The observed divergence suggests that no direct transmission of BTV occurred between Israel and Europe during the past decade. Full article

Bluetongue virus (BTV) is the causative agent of the significant livestock disease Bluetongue (BT), which causes severe economic losses associated with its considerable impact on the health and trade of ruminants. Background/Objectives: BTV infection and vaccination against the virus typically result in the induction of antibodies with the capacity to neutralize viral infection. Classic neutralization approaches resemble the methodology applied for neutralizing antibodies (NAbs) quantification. To improve long-standing and new-generation methodologies for the quantification of NAbs or evaluation of antivirals, we offer here the development of a new luciferase-based microneutralization approach as a proof-of-concept. Methods: Central to this innovative approach is the recently generated set of replication-competent reporter-expressing recombinant BTV, where the NanoLuc luciferase protein expression serves as a quantifiable readout for viral replication. After evaluating a set of heat-inactivated serum samples with neutralizing activity (measured via SNTs), these were incubated with 100 PFU of NLuc-expressing rBTV of serotype 1, 4 or 8 and Vero cells were infected with the serum–virus mixture. Then, the luminescent signal was measured at 48 h post-infection. Results: Using the proposed NLuc-based assay and the luminescent signal in the supernatant, we could detect neutralizing activity as soon as 48 h post-infection. Importantly, we were able to observe a strong correlation between NAbs titers measured by classic microneutralization assay and by our bioluminescent approach (BTV-1 Spearman r = 0.932901; p-value < 0.0001; BTV-4 Spearman r = 0.8070192; p-value < 0.0001; BTV-8 Spearman r = 0.9983; p-value < 0.0001). In addition, the NLuc-based assay displayed a serotype-specific character potentially equivalent to classic SNT methods. Conclusions: In summary, our reporter-based microneutralization assay provides a rapid and suitable method to quantify BTV-neutralizing antibodies in serum samples of natural hosts after vaccination or infection, with a serotype-specificity equivalent to classic SNT methods. Full article

Bluetongue virus serotype 3 (BTV-3) emerged in northwestern Europe in 2023–2024, raising concerns about its potential reproductive impact on rams, similar to previous outbreaks with BTV-8. This study assessed the effect of natural BTV-3 infection on the semen quality of 49 rams in Belgium using two cross-sectional sampling sessions during the 2024 outbreak. Semen and blood were tested for BTV RNA via RT-qPCR, and a composite semen quality score (SQS) was established based on key sperm parameters. On the first sampling date, 75% of rams were viremic, and 19% presented azoospermia. Rams with BTV RNA detectable in both semen and blood had significantly lower SQS and sperm concentrations than those with viral RNA in blood only or none at all. By the second sampling, 53 days later, semen quality had improved markedly, indicating a transient effect of infection. These findings confirm that BTV-3 can severely but temporarily impair ram fertility, particularly when viral replication occurs in the reproductive tract. Given the seasonal overlap between vector activity and breeding programs, these results underscore the importance of integrating reproductive health monitoring into outbreak response strategies. Full article

In July 2024, bluetongue virus serotype 3 (BTV-3) was first detected in southern Belgium, marking the onset of a major epidemic wave. This study documents, for the first time in Belgium, the ability of BTV-3 to cross the placental barrier in cattle, causing abortions and congenital central nervous system malformations. Abortion cases from January to December 2024 were monitored through the national abortion protocol, which mandates reporting and laboratory investigation (i.e., the year of emergence and the three previous years as the baseline data set). Among 5,751 reported abortions, 903 foetuses were tested by PCR, revealing widespread BTV-3 circulation. The first malformed PCR-positive foetus was recorded in mid-August, four weeks after a sharp increase in abortion rates. Lesions such as hydranencephaly were confirmed in PCR-positive foetuses, with a malformation rate of 32.24% in affected herds from weeks 36 to 52 (i.e., 22 times higher than in previous years). Gestational stage analysis indicated that congenital lesions were most frequent following infection between 70 and 130 days of gestation. Based on the observed gross lesions and the timing of abortion, it was deduced that the earliest maternal infections likely occurred in February–March 2024, implying low-level winter BTV-3 circulation before the official detection of the epidemic wave. These findings highlight the epidemiological value of systematic abortion monitoring as an early warning system tool and highlight the inadequacy of relying solely on clinical surveillance in adult ruminants. The abrupt emergence of BTV-3 across the territory without a gradual spatial spread underscores the need for anticipatory control strategies. Strategic, multivalent vaccination campaigns and enhanced abortion surveillance are critical to mitigate similar reproductive and economic losses in future bluetongue outbreaks. Full article

Background/Objectives: Bluetongue virus (BTV) is an emerging arbovirus causing significant economic losses in the ruminant industry. Current vaccines offer limited cross-protection against heterologous serotypes and do not enable differentiation between infected and vaccinated animals (DIVA). Subunit-based vaccines provide a potential DIVA-compatible solution. This study aimed to develop a vaccination protocol expressing BTV structural proteins VP7 or VP2 using antibiotic-resistance-free DNA plasmids and replication-defective adenovirus vectors. Methods: We evaluated homologous DNA prime–boost and heterologous DNA prime–adenovirus boost strategies in a murine model, assessing adaptive immune responses and protection against virulent BTV challenge. Results: The heterologous DNA–adenovirus prime–boost strategy expressing both antigens conferred full protection, preventing viremia, while homologous DNA-DNA prime–boost provided only partial protection. Both VP7 and VP2 elicited cellular and humoral immune responses, but the heterologous strategy significantly enhanced anti-BTV IgG, neutralizing antibody titers, and T cell activation. CD8⁺ T cell responses showed the strongest correlation with viral load reduction, suggesting that cellular immunity to conserved VP7 could serve as a platform for cross-protection against multiple BTV serotypes. Conclusions: These findings highlight the potential of heterologous DNA–adenovirus vaccination as an effective DIVA-compatible strategy for BTV control. By inducing strong and protective immune responses, this approach could improve disease surveillance and management, ultimately reducing the impact of BTV on livestock industries. Full article

Bluetongue virus belongs to the Reoviridae family, which causes morbidity in ruminants. Herein, we report the full-length genome sequence of a recombinant BTV strain of serotype 1 (YNDH/103/2013) isolated from sentinel cattle in Yunnan Province, China, in 2013. To identify and understand the characteristics of YNDH/103/2013, we screened closely related viruses using Simplot 3.5.1 software and conducted verification via phylogenetic analysis using MEGA 11.0.13 software. Phylogenetic analysis and segment examination showed that the YNDH/103/2013 strain likely originated from multiple recombination events involving prevalent strains such as Y863, NRT37/ABT/HSR, and YTS-4 in China and India. The genome of BTV-1/YNDH/103/2013 derives from at least three reassortments with these strains, showing recombination breakpoints mainly in segments 4, 6, 8, and 9. This study improves our understanding of the origin and spread of BTV-1 in China. Full article

Bovine viral diarrhea virus (BVDV) is a globally significant pathogen affecting both domestic livestock and wildlife, including white-tailed deer (WTD; Odocoileus virginianus). While experimental infections have demonstrated WTD susceptibility to BVDV, natural infections and associated reproductive outcomes remain scarcely documented. Here, we report the first confirmed case of naturally occurring BVDV-1 infection associated with fetal mummification in farmed WTD in Florida. A two-year-old doe experienced a stillbirth involving two mummified fetuses, which were submitted for necropsy and laboratory diagnostics. Gross findings included diarrhea and underdeveloped eyes in the fetuses, along with small white nodules indicative of amnion nodosum. While not harmful, this condition suggests underlying fetal compromise or intrauterine stress. Virus isolation using Vero E6 and bovine turbinate cell lines, along with a reverse transcription PCR (RT-PCR) assay specifically developed in this study, confirmed the presence of BVDV-1 (Pestivirus bovis) RNA in both maternal and fetal samples, suggesting vertical transmission. Sanger sequencing of RT-PCR amplicons further verified the virus species as BVDV-1. Differential diagnostics for other pathogens, including bluetongue virus, epizootic hemorrhagic disease virus, Mycobacterium spp., and Toxoplasma gondii, were negative. These findings underscore the importance of using biosecurity measures and including BVDV in the differential diagnosis of abortions to reduce the risk of BVDV transmission and potential outbreaks on deer farms, particularly those close to cattle operations. The molecular tools developed in this study provide a robust framework for improved detection and monitoring of BVDV in both wildlife and livestock populations. Full article

Bluetongue is a vector-borne viral infectious disease primarily affecting ruminants, transmitted by biting midges of the Culicoides species. The first bluetongue virus 3 (BTV-3) cases occurred in Hesse, Germany, in July 2024. From December 2024 onwards, field veterinarians observed calves born with neurological symptoms. A convenience sample of affected calves were admitted to the Veterinary Teaching Hospital at Justus-Liebig-University Giessen, Germany. A total of 13 calves from unvaccinated dams with pronounced neurological symptoms and positive PCR results for BTV-3 were studied. General and neurological examinations were performed and a blood sample was obtained for hematologic, blood biochemical and blood gas analysis. In 11 calves, magnetic resonance imaging (MRI) was performed. Due to the severe neurological lesions, all calves were euthanized and postmortem examinations were performed. The neurological examination of the calves revealed neurological indications consistent with diffuse forebrain disease. MRI revealed that all calves exhibited various stages of cortical parenchyma cell loss and secondary enlargement of the lateral ventricles. Postmortem examination revealed moderate to severe hydrocephalus internus or hydranencephaly. The results of clinical and neurological examinations, MRI, and postmortem examination, indicate severe impairment of brain development presumably associated with transplacental BTV-3 infection. Epidemiologic data and health records from the originating farms suggest that neurologic malformations developed after BTV-3 infection between 95 and 227 of the gestation period. Full article