The Emergence of Bluetongue Virus Serotype 3 in Portugal (2024)

1. Introduction

Bluetongue fever (BT) is a viral disease that affects domestic and wild ruminants [1,2] caused by a member of the genus Orbivirus in the family Reoviridae. Although infections in other species namely in carnivores have been occasionally reported [1,3,4,5], the primary hosts remain ruminants. The disease is transmitted by blood-feeding insects of the genus Culicoides, which facilitate the transmission of the virus from infected animals in the viremia stage to healthy individuals [6,7]. Therefore, in affected regions, BT exhibits a seasonal distribution, with increased incidence during the summer months, when rising temperatures promote the reproduction and persistence of Culicoides vectors in the environment.

BT is generally more severe in sheep, which typically exhibit high fever, depression, mucosal lesions in the mouth and nose, congestion of the coronary band, hypersalivation, nasal discharge, lameness and mortality [2]. In contrast, cattle usually present with asymptomatic or mild clinical manifestations [8]. The severity of the disease in both species, however, can vary significantly depending on the infecting viral strain. Certain serotypes of bluetongue virus (BTV) have been identified as more aggressive, inducing severe clinical signs and high mortality rates. Such is the case of the newly recognised BTV-3, which emerged in the Netherlands in 2023 [9].

Historically two clusters of BTV-3 have been identified: one comprising the Western topotypes of Africa, Mediterranean Basin and North America, and the other the Eastern topotypes of Japan, India and Australia [10]. Although there was a limited number of BTV-3 outbreaks in Italy between 2017 and 2022, the onset of BT in the Netherlands on 17 June 2023 was attributed to a new BTV-3 given its rapid geographic spread and higher pathogenicity. This new BTV-3 also belongs to the Western topotype. The emergence of new BTV-3 in Europe has been documented in 2023 across the Netherlands [9] and Belgium in September 2023, Germany in October 2023 and Great Britain in November 2023 (WOAH disease reports). Notably, in the summer of 2024, BTV-3 was also detected in Denmark and France in July, in Luxembourg, Norway and Switzerland in August, and in The Czech Republic in early September (WOAH disease reports).

In summary, the history of BTV in Portugal has involved the emergence and management of various serotypes over the decades. Serotype 10 was the first identified in 1956, prompting significant concern, but it was successfully eradicated by 1960 through the implementation of live attenuated vaccination programs. Serotype 4 emerged in 2004 and remains endemic, posing ongoing challenges for livestock health and management in the region. Serotype 2 was detected sporadically between 2004 and 2006, but it has not been observed circulating since then, indicating a limited impact on local livestock [11]. Serotype 1 was first reported in 2007 (OIE reference: 6248/September/2007) and has since spread extensively across the country. To mitigate the risks associated with these serotypes, vaccination of sheep against BTV-1 and BTV-4 has been mandatory in Portugal.

Here we report the first detections of BTV-3 in mainland Portugal in September 2024.

2. Materials and Methods

2.1. Case Descriptions

The first case was an adult female crossbred sheep (Sheep 1) that died 4-5 days after sampling for laboratory investigation. The animal was part of a flock of 130 individuals originated from Évora District, that had been vaccinated against BTV-1 and BTV-4 in April 2024. Clinical signs were first observed on 1 September in 15 animals, including prostration, severe dyspnoea, wheezing, rhinorrhoea, and oedema of the head. At that time, blood samples from Sheep-1 were taken and submitted to the national reference laboratory on 12 September 2024 with a suspected diagnosis of BTV or epizootic haemorrhagic disease virus infection (EHDV). In this farm, there was no importation of animals, nor was there any introduction of new animals into the herd, reinforcing the hypothesis of localized transmission, potentially mediated by vectors endemic to the area.

The second case involved a male ovine (Sheep 2) also from Évora District, which developed fever (41.5ºC), rhinorrhoea and arthritis prior to its death on September 7, at the time with suspicion of contagious ecthyma (parapoxvirus). The onset of symptoms in the flock occurred on the September 4 affecting also other sheep that died in the same day.

This herd encompassed around 3000 animals, of which 150 died until October 2, 2024 representing a mortality of 5%. The last introduction of animals, comprising a batch of female lambs, occurred in mid-August 2024 from Azaruja (Évora), raising the possibility that asymptomatic animals in the early stages of infection may have played a role in the outbreak, alongside potential vector-mediated transmission.

2.2. Nucleic Acid Extraction

Lung, spleen, liver and intestine samples from the dead sheep were homogenised at 20% (w/v) with phosphate-buffered saline by mechanical homogenization with 0.5 mm zirconium beads (Sigma-Aldrich, St. Louis, Missouri, EUA) using four cycles of 15 seconds at 3000 rpm with an interval of 10 seconds (Precellys^® Evolution, Paris, France) and then clarified at 3,000g for 5 min. Total nucleic acid extraction was carried out from 200 μl of the clarified supernatants, or from 200 μl blood samples using the IndiMag^® Pathogen Kit (Indical, Leipzig, Germany) in a KingFisher Flex extractor (ThermoFisher Scientific, Waltham, USA), following the manufacturer’s protocol. Nucleic acids were preserved at -20°C until use. Extractions were validated with an 18S rDNA qPCR [12] and a RT-qPCR for the detection of spiked synthetic RNA (VLP-RNA EXTRACTION, Meridian Life Science, Memphis, USA).

2.3. Molecular Investigations

Molecular investigations were conducted for bluetongue virus (BTV) and its serotypes BTV-1, BTV-3, BTV-4, and BTV-8, as well as for epizootic hemorrhagic disease virus (EHDV), catarrhal malignant fever virus (CMFV), and contagious ecthyma virus (ORFV) as part of the differential diagnosis. The methodologies employed, as well as PCR kits used, are detailed in Table 1.

Amplifications were carried out in a Bio-Rad CFX96™ Thermal Cycler (Bio-Rad Laboratories Srl, Redmond, USA), or T100 Thermal Cycler (Bio-Rad Laboratories Srl, Redmond, USA) for real time and conventional PCR, respectively.

2.4. Sanger Sequencing Analysis in the EURL

Nucleic acid was extracted from 200 μL of EDTA blood and tissue homogenate using a BioSprint^® 96 DNA Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Nucleid acid was eluted in a final volume of 50 μL of nuclease-free water. Three gel-based RT-PCR methods targeted to segments 2 (in house), 5 (Katz et al., 1993) and 10 (Rijn et al., 2012) were performed. The PCR products were visualised in 2% horizontal electrophoresis agarose gel, purified using the QIAquick^® PCR Purification Kit (Qiagen), and directly sequenced using the ABI Prism BigDye Terminator v3.1 Cycle sequencing kit on a 3730 Genetic Analyser (Applied Biosystems, Foster City, CA, U.S.A). Nucleotide sequences were analysed and assembled into consensus sequences using the DNA Sequencing Analysis Software Version 6.0 and SeqScape v.3.0 de Applied Biosystems.

3. Results

Both animals (Sheep 1 and Sheep 2) tested positive for bluetongue virus (BTV). Serotype-specific testing confirmed the presence of BTV-3, while both samples were negative for BTV-1, BTV-4, and BTV-8. Additionally, the blood sample from Sheep-1 tested negative for epizootic hemorrhagic disease virus (EHDV).

Sheep 2 tested negative for contagious ecthyma virus, a member of the genus Parapoxvirus within the subfamily Chordopoxvirinae and family Poxviridae. However, it was positive for catarrhal malignant fever virus (CMFV), caused by ovine herpesvirus-2, which belongs to the Gammaherpesvirinae subfamily of the Herpesviridae family.

As an emerging disease in the country and following European Commission directives, a whole blood sample from Sheep 1 and an organ macerate from Sheep 2 were sent to the European Union Reference Laboratory (EURL) for Bluetongue (BT). The laboratory confirmed the presence of the new BTV-3 strain through serogroup- and serotype-specific RT-qPCR testing. Sanger sequencing further identified a 100% sequence match with the BTV-3 strain from northern Europe (BTV-3/NET2023, GenBank accession numbers OR603993.1, OR603996.1, and OR604001.1) across three genome fragments of 1,133, 229, and 219 nucleotides in segments 2, 5, and 10, respectively. Notable genetic differences were observed when compared to BTV-3 SAR 2018 and other BTV strains.

4. Discussion

In mid-September 2024, BTV-3 was detected for the first time in Portugal, in sheep, specifically in the Évora district, marking a significant event, as at the time no prior cases had been reported in the Iberian Peninsula. Shortly after, at the end of September 2024, Spain confirmed its first outbreak of BTV-3, occurring approximately two weeks after the initial detection in Portugal. Despite these detections, the origin of the BTV-3 virus remains undetermined. The geographic distance from the first reported case to the border with Spain, combined with the absence of any reported case of BTV-3 in Spain at the time, suggests that the virus is unlikely to have originated from the neighboring country. Although the partial sequences of three viral genome segments (2, 5 and 10) suggest a similarity to BTV-3 circulating in northern Europe countries, and shows differences to BTV-3 SAR 2018 , there is no documented history of animal imports from countries affected by BTV-3 from northern Europe countries. To better understand the origin of the virus, full genomic sequencing of the BTV-3 strain is being conducted. This analysis will help identify how the Portuguese strain relates to other known BTV-3 strains and may provide insights into how the virus arrived in the region.

Interestingly, Sheep 2 was also positive to CMFV, a finding integral to the differential diagnosis process. CMFV is known to cause subclinical infections in sheep, which may reactivate during various disease processes. In this case, the reactivation of CMFV may have exacerbated the clinical severity associated with BTV-3 infection.

Approximately one month after this initial detection, several additional cases were reported in sheep and cattle, not only in the Évora district but also in neighbouring districts to the north (Castelo Branco), south (Beja), and east (Setúbal). At that time BTV-3 infection was confirmed on five cattle farms, with one case linked to clinical signs and increased neonatal mortality—a pattern rarely observed in other serotypes, except BTV-8. The increased virulence of the newly identified BTV-3, along with its apparent enhanced ability to infect carnivores [9] and its rapid geographical spread, raises significant concerns about its potential to cross species barriers and infect other hosts. In response, vaccination against BTV-3 with SYVAZUL BTV3 inactivated vaccine (Syva, Spain) is being implemented, reflecting the country’s commitment to controlling the disease and safeguarding its agricultural economy.