Bluetongue is an infectious, non-contagious, arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV), producing severe impacts on livestock. Historically, Southern Europe has suffered multiple incursions of different BTV serotypes with serious consequences. In 2021, BTV re-emerged in the Balearic Islands (Spain) after 16 years free of the disease, causing a large outbreak that mainly affected sheep, as well as cattle and goats. A novel emerging strain of BTV serotype 4 (BTV-4) was identified via preliminary molecular characterization as the etiological culprit of the epizootic. This study delineates the outbreak in the Balearic Islands in 2021, encompassing field-based clinical observations and laboratory findings. Additionally, an experimental infection was conducted in sheep using the novel BTV-4 strain to assess its virulence, pathogenicity, and laboratory diagnostic characteristics. The in vivo characterization was conducted concurrently with the virulent and widely disseminated BTV-4 RNM 2020 strain that has precipitated significant outbreaks in the Mediterranean region in recent years. Both strains exhibited analogous pathogenic potential in sheep and yielded equivalent outcomes in diagnostic parameters. Furthermore, the impact of the novel BTV-4 strain is discussed.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility–recumbency by days 9–10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9–10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

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.

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.

African horse sickness (AHS) is a disease affecting equids caused by the AHS virus (AHSV). The World Organisation for Animal Health (WOAH) includes AHS as a notifiable disease and, upon detection within the European Union, immediate control and eradication measures are mandated. Thus, validated diagnostic methods for rapid AHSV detection are essential. The Agüero 2008 and Guthrie 2013 rRT-PCR methods have been widely validated for detection of any AHSV strain and are included as reference rRT-PCRs in the WOAH manual. However, the WOAH Reference Laboratory for AHS in the Republic of South Africa (RSA) reported an AHSV variant undetected by the Agüero 2008 rRT-PCR. Therefore, a set of modified primers and probe, containing degenerate positions to avoid mismatches with the sequence of the new RSA strain, was developed. The modified-Agüero method was validated by the WOAH Reference Laboratories in Spain and the UK, employing a broad collection of AHSV strains and clinical samples as well as a synthetic RNA mimicking the target sequence of the new RSA AHSV variant (AHSV-sRNA-RSA). Comparative assessment of the modified-Agüero versus the WOAH reference rRT-PCRs showed that the modified method exhibited good diagnostic performance and enabled detection of the new RSA AHSV variant nucleic acid.

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral diseases of ruminants, producing severe impacts in livestock. It is caused by Bluetongue virus (BTV), a double-stranded (ds) RNA virus with segmented genome (10 segments), belonging to Seoreoviridae family, Orbivirus genus. Over the last 25 years, Europe has suffered multiple incursions of different BTV serotypes with serious consequences, which have been controlled mainly thanks to vaccination. In the case of Spain, since 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 especially on sheep but also in cattle and a multitude of outbreaks. Additionally, despite vaccination, also circulated serotype 4 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 BTV viruses 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 raises the question of whether the co-circulation of different BTV strains, an exceptional situation, could become the new normal in certain areas of Europe.