Background: Bluetongue virus serotype 3 (BTV‐3) was detected for the first time in cattle and sheep in southern England in 2023, the first UK BTV incursion for more than 15 years. Clinical signs were not observed, yet severe clinical disease and mortality were reported during recent BTV‐3 outbreaks in northern Europe. Methods: To investigate the clinical disease and infection kinetics associated with this UK BTV‐3 strain, five British sheep were infected with a UK BTV‐3 isolate using Culicoides biting midges. Clinical signs, pathology, infection dynamics, immune responses and Culicoides infection rates were assessed. Results: All sheep were infected with BTV‐3 and developed mild to moderate clinical bluetongue disease, characterised by fever, haemorrhagic diarrhoea, lameness, depression and widespread petechial haemorrhage. Three sheep reached clinical humane endpoints and were euthanased. Clinical signs/severity, infection kinetics and immune responses were highly variable. Infectious BTV‐3 was isolated from sheep blood up to 28 days postinfection. Limitations: The impact of BTV‐3 infection on British cattle and infection rate in UK Culicoides require investigation to fully determine the risk of this strain to UK livestock. Conclusions: This study confirms the potential impact of a BTV‐3 incursion/outbreak on the UK sheep population, highlighting the need for an effective vaccine.

Since the 2000s, the distribution of bluetongue virus (BTV) has changed, leading to numerous epidemics and economic losses in Europe. Previously, we found a BTV-4 field strain with a higher infection rate of a Culicoides vector than a BTV-1 field strain has. We reverse-engineered parental BTV-1 and BTV-4 strains and created BTV-1/BTV-4 reassortants to elucidate the influence of individual BTV segments on BTV replication in both C. sonorensis midges and in KC cells. Substitution of segment 2 (Seg-2) with Seg-2 from the rBTV-4 significantly increased vector infection rate in reassortant BTV-1_4S2 (30.4%) in comparison to reverse-engineered rBTV-1 (1.0%). Replacement of Seg-2, Seg-6 and Seg-7 with those from rBTV-1 in reassortant BTV-4_1S2S6S7 (2.9%) decreased vector infection rate in comparison to rBTV-4 (30.2%). However, triple-reassorted BTV-1_4S2S6S7 only replicated to comparatively low levels (3.0%), despite containing Seg-2, Seg-6 and Seg-7 from rBTV-4, indicating that vector infection rate is influenced by interactions of multiple segments and/or host-mediated amino acid substitutions within segments. Overall, these results demonstrated that we could utilize reverse-engineered viruses to identify the genetic basis influencing BTV replication within Culicoides vectors. However, BTV replication dynamics in KC cells were not suitable for predicting the replication ability of these virus strains in Culicoides midges.

Introduction: Bluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host.Methods: In this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4⁺, CD8⁺, or WC1⁺ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies.Results: The absence of cytotoxic CD8⁺ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4⁺ and WC1⁺ γδ T cells both resulted in an increased clinical severity. The absence of CD4⁺ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4⁺ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7.Discussion: This study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

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.