Emergence of Bluetongue virus serotype 5 in Sardinia- Italy, 2025.

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

Serum samples collected from 1,396 white-tailed deer (Odocoileus virginianus) in five areas of Georgia (USA) from 1989 to 1991 were tested for precipitating and serum neutralizing (SN) antibodies to the enzootic North American epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) serotypes. Precipitating antibodies to the EHDV or BTV serogroups, as detected by agar gel immunodiffusion (AGID) tests, were present in 35%, 29%, and 39% of deer sampled in 1989, 1990, and 1991, respectively. Significant differences (P < 0.05) in precipitating antibody prevalence were detected between physiographic regions during all years. Antibody prevalence consistently was highest in deer sampled from the Coastal Plain (77%), followed by the Piedmont (33%), Ridge and Valley (29%), Barrier Island (5%), and Blue Ridge (2%) regions. All AGID-positive samples were tested by SN tests for antibodies against all North American EHDV and BTV serotypes (EHDV serotypes 1 and 2, BTV serotypes 2, 10, 11, 13, and 17). Criteria for previous exposure to a specific serotype were either detection of monospecific results or clusters of positive results against that serotype. Serologic evidence of previous exposure to EHDV serotypes 1 and 2, and BTV serotypes 11 and 13 was detected during all years. Predominant serotypes varied among years. In general, evidence of exposure to EHDV serotype 2 appeared annually while exposure to BTV serotype 13 and EHDV serotype 1 decreased and increased, respectively. To determine serotype diversity prior to 1989, 134 AGID-positive white-tailed deer serum samples collected from 1967 to 1988 also were tested by SN. Evidence of exposure to EHDV serotypes 1 and 2 and BTV serotypes 11, 13, and 17 was detected.

To the Editor: Bluetongue is an arboviral disease of domestic and wild ruminants characterized by vascular injury that produces widespread edema and tissue necrosis (1). Bluetongue virus (BTV), the causative agent of bluetongue, is the prototype virus of the family Reoviridae and the genus Orbivirus (2). BTV occurs throughout temperate and tropical areas of the world coincident with the distribution of vector Culicoides spp. midges (3–5). Different midge species transmit different constellations of BTV serotypes in distinct global episystems (3,5). For example, C. sonorensis is the principal, if not exclusive, vector of BTV serotypes 10, 11, 13, and 17 in much of North America, whereas C. insignis is the major vector of multiple BTV serotypes (including BTV 1–4, 6, 8, 12, 17, 19, 20, and probably others) in the Caribbean basin, Central America, and South America. C. insignis is also found in the southeastern United States, and although this species might have recently expanded its range in the region, its distribution in North America remains poorly defined. Serotypes of BTV other than 10, 11, 13, and 17 are found in areas of the United States: BTV-2 was first reported in Florida in 1982. Since 1998, ten additional serotypes (BTV-1, 3, 5, 6, 9, 12, 14, 19, 22, and 24) have been identified in the southeastern United States (6). Approximately 26 BTV serotypes have been described and the global distribution of BTV has recently been altered (2,4). Coincident with the invasion of novel BTV serotypes into the southeastern United States (6), likely by extension from the adjacent Caribbean basin, multiple BTV serotypes have spread throughout much of continental Europe and parts of the British Isles and Scandinavia, precipitating an economically devastating epidemic (7). Similarly, ongoing surveillance has identified novel BTV serotypes in regions to which it historically has been endemic (e.g., Australia and the Middle East) (2). Climate change may have contributed to this dramatic recent expansion in global distribution of BTV, most notably in Europe (8). Bluetongue was first described in the late 19th century among sheep brought from Europe to South Africa, and later in North America in ≈1950 (4). Surveillance in western North America since that time has confirmed that only BTV-10, 11, 13 and 17 are present in this region, including our recent intensive surveillance of sentinel cattle on dairy farms throughout California, USA (9,10). However, during investigation of an outbreak of acute coronitis and ulcerative stomatitis among cattle at a dairy farm in the northern Sacramento Valley in California in August 2010, a blood sample from a heifer was found by using described methods (10) to be positive for BTV by serogroup-specific quantitative reverse transcription PCR (qRT-PCR) but negative by serotype-specific RT-qPCRs for BTV-10, 11, 13, and 17. Further analysis using additional serotype-specific RT-qPCRs identified virus in the blood sample as BTV-2. BTV was isolated in primary bovine endothelial cells from blood collected from the heifer. Sequence analysis of the serotype-specific L2 gene of the virus isolate confirmed it to be BTV-2 (2), and phylogenetic analyses showed it to be most closely related to a strain of BTV-2 isolated in Florida in 1999 (Figure). However, sequence analysis of the entire genome of the virus from California indicated that it is a reassortant that includes genes from BTV-6 and BTV-2. Specifically, genes encoding the viral protein 1 polymerase and viral protein 3 major core protein segregate with those of the US prototype strain of BTV-6 (isolated in 2006), but other genes are derived from BTV-2. BTV-2 and BTV-6 have been isolated only in the southeastern United States, which indicates translocation within the United States of reassortant BTV-2. Figure Cladogram comparing the L2 genes of different bluetongue virus (BTV) serotypes and global strains of BTV serotype 2 (BTV-2) with that of a virus isolated in northern California, USA (2-California-2010; GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:JQ822248-JQ822257,start_term:JQ822248,end_term:JQ822257,start_term_id:390980698,end_term_id:390980714}} ... How this virus spread to California is not known, and its distribution in the United States is uncertain because there is no comprehensive national BTV surveillance program. However, BTV-2 was not detected previously in California, suggesting that this serotype was recently introduced into the region or that it is uncommon. Identification of this novel BTV serotype in western North America emphasizes the need for ongoing entomologic and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infection (4,6,8).

The circulation of Bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD) in the Middle East has already been reported following serological analyses carried out since the 1980s, mostly on wild ruminants. Thus, an EHD virus (EHDV) strain was isolated in Bahrain in 1983 (serotype 6), and more recently, BT virus (BTV) serotypes 1, 4, 8 and 16 have been isolated in Oman. To our knowledge, no genomic sequence of these different BTV strains have been published. These same BTV or EHDV serotypes have circulated and, for some of them, are still circulating in the Mediterranean basin and/or in Europe. In this study, we used samples from domestic ruminant herds collected in Oman in 2020 and 2021 for suspected foot-and-mouth disease (FMD) to investigate the presence of BTV and EHDV in these herds. Sera and whole blood from goats, sheep and cattle were tested for the presence of viral genomes (by PCR) and antibodies (by ELISA). We were able to confirm the presence of 5 BTV serotypes (1, 4, 8, 10 and 16) and the circulation of EHDV in this territory in 2020 and 2021. The isolation of a BTV-8 strain allowed us to sequence its entire genome and to compare it with another BTV-8 strain isolated in Mayotte and with homologous BTV sequences available on GenBank.

In vitro and in vivo infections were conducted to determine if the epizootic hemorrhagic disease (EHD) and bluetongue (BT) viruses would replicate in peripheral blood mononuclear (PBM) cells of white-tailed deer (Odocoileus virginianus). All of the North American EHD and BT viruses (EHD virus serotypes 1 and 2, and BT virus serotypes 2, 10, 11, 13, and 17) replicated in vitro in cultures of white-tailed deer PBM cells. However, this replication appeared to be monocyte-dependent and was not enhanced by lymphocyte blastogenesis induced by the addition of concanavalin A. In white-tailed deer infected with either EHD virus serotype 2 or BT virus serotype 10, virus could be isolated consistently from PBM cells only from post-infection day 4 through 8, although they remained viremic through post-infection day 21. In deer, highest viral titers were associated with the erythrocyte fraction, and in no cases did viral titers detected in the platelet, PBM cell or polymorphonuclear cell fractions approach titers observed in whole blood. In the in vitro infections of white-tailed deer erythrocytes, the EHD and BT viruses were associated with pits in the erythrocyte membrane. This association may be important in the long-term viremia observed in deer.

Bluetongue (BT) is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV) serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT)) are slow (taking weeks, depend on availability of reference virus-strains or antisera) and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2) encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype) were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h) and reliable RT–PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype) were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT–PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

Bluetongue disease is caused by bluetongue virus (BTV) and BTV serotype 8 (BTV8) caused great economic damage in Europe during the last decade. From 1998 to 2007, in addition to BTV8, Europe had to face the emergence of BTV1, 2, 4, 9, and 16, spreading in countries where the virus has never been detected before. These unprecedented outbreaks trigger the need to evaluate and compare the clinical, virological and serological features of the European BTV serotypes in the local epidemiological context. In this study groups of calves were infected with one of the following European BTV serotypes, namely BTV1, 2, 4, 9 and 16. For each tested serotype, two groups of three male Holstein calves were used: one group vaccinated against BTV8, the other non-vaccinated. Clinical signs were quantified, viral RNA was detected in blood and organs and serological relationship was assessed. Calves were euthanized 35 days post-infection and necropsied. Most of the infected animals showed mild clinical signs. A partial serological cross reactivity has been reported between BTV8 and BTV4, and between BTV1 and BTV8. BTV2 and BTV4 viral RNA only reached low levels in blood, when compared to other serotypes, whereas in vitro growth assays could not highlight significant differences. Altogether the results of this study support the hypothesis of higher adaptation of some BTV strains to specific hosts, in this case calves. Furthermore, cross-protection resulting from a prior vaccination with BTV8 was highlighted based on cross-neutralization. However, the development of neutralizing antibodies is probably not totally explaining the mild protection induced by the heterologous vaccination.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015–2016

SUMMARY Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (btv) immunodiffusion test (idt) and virus-neutralization test (vnt), for 5 btv serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (ehdv) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the btv idt at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconverted as assessed by results of the btv idt. In the 16 seropositive cattle in 1984, neutralizing antibodies were detected to btv serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and ehdv serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to btv-2 or btv-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the idt. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to btv serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and ehdv serotypes 1 (n = 1) and 2 (n = 7). In 1985, no cattle seroconverted to btv-2. Clinical diseases attributable to btv or ehdv was not detected in these cattle in 1984 or 1985.

Complete nucleotide sequence of RNA segment 3 of bluetongue virus serotype 2 (Ona-A). Phylogenetic analyses reveal the probable origin and relationship with other orbiviruses

Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes.

Since 1998, Bluetongue virus (BTV) serotypes 1, 2, 4, 6, 8, 9, 11 and 16 have spread throughout Europe. In 2006, BTV serotype 8 (BTV‑8) emerged unexpectedly in Northern Europe, in countries such as Belgium, France, Germany, Luxembourg, and the Netherlands, to spread rapidly in the following year throughout the rest of Europe. In 2007, BTV‑1 spread in Southern Europe, in Spain and in South of France. In 2008, 2 more BTV serotypes were detected in Northern Europe: BTV‑6 in the Netherlands and in Germany, and BTV‑11 in Belgium. The European incursion of BTV has caused considerable economic losses, including direct losses from mortality and reduced production, as well as indirect losses generated by ensuing bans on trade of ruminants between infected and non-infected areas. Given the significance of the disease, all affected countries have established control and eradication measures that have evolved together with the availability of detection and prevention tools such as Polymerase Chain Reaction (PCR) tests and vaccines, respectively. This paper describes how the French National Reference Laboratory for BT has managed diagnosis during the fast and massive spread of BTV‑1 and 8 in 2007 and 2008.

A pair of novel primers for universal detection of the NS1 gene from various bluetongue virus serotypes

Exotic disease research in Australia

The epidemiological patterns of Bluetongue (BT) in North Africa and Mediterranean Basin (MB) dramatically changed by emergence of subsequent episodes of novel bluetongue virus (BTV) serotypes with highly pathogenic indexes and socio‐economic impacts. The objective of the study was to investigate the sero‐prevalence and serotype distribution of BTV in Libya. During 2015‐2016, a total of 826 serum samples were collected from domestic ruminants in Libya. All sera were assayed by competitive enzyme‐linked immunosorbent assays (c‐ELISA). C‐Elisa‐positive samples (43.3%; 173/400) were further analyzed by virus neutralization assay to identify BTV serotypes and determine the antibody titre of positive samples. An overall BTV sero‐prevalence was 48.4% (95% CI: 45.0%‐51.8%). Neutralizing antibodies were detected against the following BTV serotypes namely: BTV‐1, BTV‐2, BTV‐3, BTV‐4, BTV‐9 and BTV‐26. While BTV‐1, BTV‐2, BTV‐4 and BTV‐9 circulation was unsurprising as they have been responsible of the last year outbreaks in Northern African Countries, the detection of BTV‐3 and BTV‐26 was definitely new and concerning for the animal health of the countries facing the Mediterranean Basin. It is crucial that European and Northern African authorities collaborate in organizing common surveillance programmes to early detect novel strains or emerging serotypes in order to set up proper preventive measures, and, in case, develop specific vaccines and plan coordinated vaccination campaigns.