Vaccines against bluetongue in Europe

After the incursion of bluetongue virus (BTV) into European Mediterranean countries in 1998, vaccination was used in an effort to minimize direct economic losses to animal production, reduce virus circulation and allow safe movements of animals from endemic areas. Vaccination strategies in different countries were developed according to their individual policies, the geographic distribution of the incurring serotypes of BTV and the availability of appropriate vaccines. Four monovalent modified live virus (MLV) vaccines were imported from South Africa and subsequently used extensively in both cattle and sheep. MLVs were found to be immunogenic and capable of generating strong protective immunity in vaccinated ruminants. Adverse side effects were principally evident in sheep. Specifically, some vaccinated sheep developed signs of clinical bluetongue with fever, facial oedema and lameness. Lactating sheep that developed fever also had reduced milk production. More severe clinical signs occurred in large numbers of sheep that were vaccinated with vaccine combinations containing the BTV-16 MLV, and the use of the monovalent BTV-16 MLV was discontinued as a consequence. Abortion occurred in <0.5% of vaccinated animals. The length of viraemia in sheep and cattle that received MLVs did not exceed 35 days, with the single notable exception of a cow vaccinated with a multivalent BTV-2, -4, -9 and -16 vaccine in which viraemia persisted at least 78 days. Viraemia of sufficient titre to infect Culicoides insects was observed transiently in MLV-vaccinated ruminants, and natural transmission of MLV strains has been confirmed. An inactivated vaccine was first developed against BTV-2 and used in the field. An inactivated vaccine against BTV-4 as well as a bivalent vaccine against serotypes 2 and 4 were subsequently developed and used in Corsica, Spain, Portugal and Italy. These inactivated vaccines were generally safe although on few occasions reactions occurred at the site of inoculation. Two doses of these BTV inactivated vaccines provided complete, long-lasting immunity against both clinical signs and viraemia, whereas a single immunization with the BTV-4 inactivated vaccine gave only partial reduction of viraemia in vaccinated cattle when challenged with the homologous BTV serotype. Additional BTV inactivated vaccines are currently under development, as well as new generation vaccines including recombinant vaccines.     

Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria

This study reports on an outbreak of disease that occurred in central Algeria during July 2006. Sheep in the affected area presented clinical signs typical of bluetongue (BT) disease. A total of 5245 sheep in the affected region were considered to be susceptible, with 263 cases and thirty-six deaths. Bluetongue virus (BTV) serotype 1 was isolated and identified as the causative agent. Segments 2, 7 and 10 of this virus were sequenced and compared with other isolates from Morocco, Italy, Portugal and France showing that they all belong to a ‘western’ BTV group/topotype and collectively represent a western Mediterranean lineage of BTV-1.     

Description of the outbreak of bluetongue in Corsica in 2003, and lessons for surveillance

Since 1999, several serotypes of bluetongue virus (btv) have been isolated in the western part of the Mediterranean basin, and since 2000, Corsica has been exposed to three different serotypes: BTV serotype 2 in 2000, BTV serotype 4 (BTV-4) in 2003 and BTV serotype 16 in 2004. In 2000 there were no surveillance systems for bluetongue, but in 2003, active surveillance of the circulation of BTV and its vector Culicoides species, aided by a raised level of awareness in farmers and veterinarians, made it possible to study the introduction of BTV-4. The monitoring and analysis of the seroconversions of sentinel herds of goats, clinical signs and meteorological variables showed that the serotype had been present in the island since May that year, but clinical signs were first observed only in October. Moreover, the weather conditions and wind patterns were suitable for the transport of Culicoides species from Sardinia in May. These observations suggest that btv had been transported on air currents from a southern infected area, and that it could have spread without causing clinical signs of disease for a few months.     

VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean basin during the 1998-2003 outbreak

The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.     

Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle

The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV-2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow-up, and challenge. Thirty-two 4-month-old calves were randomly allocated into 2 groups of 16 animals each. One group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged with 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in the study and used as sentinels to confirm that no BTV was circulating locally. At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4. However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response in terms of both the number of virus neutralization (VN) positive animals and antibody titres. After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in the circulating blood of all unvaccinated animals, commencing on day 3 post-challenge and lasting 16 days. It is concluded that administration of the bivalent BTV-2 and BTV-4 inactivated vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged with high doses of BTV-2 or BTV-4.     

Differentiation between field and vaccine strain of bluetongue virus serotype 16

In August 2000, bluetongue virus (BTV) appeared for the first time in Sardinia and, since then, the infection spread across Sicily and into the mainland of Italy involving at the beginning serotypes 2 and 9 and then, from 2002, 4 and 16. To reduce direct losses due to disease and indirect losses due to new serotype circulation, the 2004 Italian vaccination campaign included the modified-live vaccines against BTV-4 and 16 produced by Onderstepoort Biological Product (OBP), South Africa. Few months after the end of the campaign, BTV-16 was reported broadly in the country and the need of differentiating field from the BTV-16 vaccine isolate became crucial. In this study, the gene segments 2, 5, 6 and 10 of both the Italian and vaccine BTV-16 strains were sequenced and their molecular relationship determined. As sequences of segment 5 were those showing the highest differences (17.3%), it was possible to develop a new diagnostic tool able to distinguish the Italian BTV-16 NS1 gene from that of the homologous vaccine strain. The procedure based on the use of a RT-PCR and the subsequent sequencing of the amplified product showed a high degree of sensitivity and specificity when samples from either BTV-16 vaccinated or infected sheep were tested.     

Bluetongue disease in Swiss sheep breeds: clinical signs after experimental infection with bluetongue virus serotype 8

Clinical disease of bluetongue (BT) in sheep may differ depending on breed, age and immunity of infected sheep and may also vary between serotype and strain of BT virus (BTV). Since there are no data available on the susceptibility of Swiss sheep breeds for BT, we performed experimental infection of the 4 most common Swiss sheep breeds and the highly susceptible Poll Dorset sheep with the BTV serotype 8 (BTV-8) circulating in Northern Europe since 2006. Clinical signs were assessed regarding severity, localisation, progression and time point of their appearance. The results clearly show that the Swiss sheep breeds investigated were susceptible to BTV-8 infection. They developed moderate, BT-characteristic symptoms, which were similar to those observed in Poll Dorset sheep. Regardless of breed, the majority of infected animals showed fever, swelling of the head as well as erosions of the mouth and subcutaneous haemorrhages.     

Bluetongue virus serotype 26: infection kinetics and pathogenesis in Dorset Poll sheep

Bluetongue virus serotype 26 (BTV-26) has recently been isolated from sheep in Kuwait. The aim of this study was to assess the pathogenicity and infection kinetics of BTV-26 in Dorset Poll sheep. Six sheep were experimentally infected with BTV-26 and samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and two group specific ELISAs. Five of the six sheep showed mild clinical signs characteristic of bluetongue including conjunctivitis, reddening of the mouth mucosal membranes, slight oedema of the face and nasal discharge. Viral RNA was detected in 5 of the 6 sheep by real time RT-PCR, however the levels of viral RNA detected in the samples were lower and of shorter duration than seen with other field strains of BTV. Virus was isolated from the blood of infected animals at the peak of viraemia at around 9 dpi. Antibodies against BTV were first detected by 7 dpi using the early detection BTV ELISA and a little later (7-14 dpi) using a BTV specific competitive ELISA. Four of the five remaining sheep developed neutralising antibodies to BTV-26, measured by a serum neutralisation test (SNT), with titres (log(10)) ranging from 1.40 to 2.08.     

Genetic variation of bluetongue virus serotype 11 isolated from host (sheep) and vector (Culicoides variipennis) at the same site

Genetic relatedness of 2 strains of bluetongue virus (BTV) serotype 11 that were isolated from the same geographic site--one from host (sheep) and the other from the vector Culicoides variipennis during an enzootic of bluetongue at Bruneau, Idaho, in August 1973--was determined by comparing the oligonucleotide fingerprint analyses of the individual double-stranded RNA segments of the genomes. It was observed that the 2 strains of BTV-11 exhibit considerable differences in their genotypes, the percentage of diversity being different for each of the corresponding RNA species of the 2 strains of BTV-11. These results indicate that more than one genotype of BTV can circulate in juxtaposition in a given geographic site. The observed genotypic diversity might be due to the accumulation of point mutations on specific RNA species or antecedent reassortment of RNA segments between different BTV in nature or both.     

Analysis of the genomes of bluetongue virus serotype 10 vaccines and a recent BTV-10 isolate from Washington

The 10 double-stranded RNA gene segments of 2 vaccinal strains of bluetongue virus (BTV) serotype 10 that are used in the United States (BTV CA8 California and BT-8 Colorado), and a BTV-10 isolate recently obtained from infected sheep in Washington (state) were characterized by oligonucleotide fingerprint analyses. It was determined that although the 2 BTV-10 vaccinal strains are genotypically distinct, they are closely related both to each other and to the United States prototype BTV-10 virus. The BTV-10 field isolate appears to be a naturally occurring reassortment virus with genome segments derived from both United States prototype BTV-10 and BTV-11 viruses. However, one RNA segment of the isolate was totally unlike the corresponding segments of United States prototype BTV-10, -11, -13 and -17 viruses.     

T lymphocyte subset alterations following bluetongue virus infection in sheep and cattle

To determine potential mechanisms of differential disease expression in ruminants infected with bluetongue virus (BTV), clinically normal, BTV-seronegative, yearling sheep and cattle were infected subcutaneously with a standardized insect-source inoculum of BTV serotype 17 (BTV-17) (three infected and one contact control each) or animal adapted BTV serotype 10 (BTV-10) (three sheep only). BTV was isolated from peripheral blood cell components of infected sheep and cattle and all infected animals showed evidence of seroconversion by 14 days post infection (PI). Sheep infected with both serotypes of BTV developed pyrexia, oral lesions, and leukopenia which were most severe on days 7-8 PI. Analysis of peripheral blood mononuclear leukocytes with specific monoclonal antibodies and flow cytometry revealed panlymphocytopenia on day 7 PI. This response was further characterized by an increase in the CD4/CD8 ratio (greater than 3) resultant from a greater decrease in absolute numbers of circulating SBU-T8(CD8+) ("cytotoxic/suppressor") lymphocytes compared to SBU-T4 (CD4)+ ("helper") lymphocytes. SBU-T19+ lymphocytes were also decreased below baseline values on days 5-14 post infection. On day 14 PI there were increased CD8+ lymphocytes and decreased CD4/CD8 ratios (approximately 0.6) in these sheep. Clinical and hematologic changes in cattle infected with BTV-17 were minimal and consisted of mild pyrexia (rectal temperature 103 degrees F) on day 9 PI in two of three infected animals and mild leukopenia on several days PI in one animal. This leukopenia was the result of a pan T lymphocytopenia with CD4/CD8 ratios in the expected range (1-2). Similar to infected sheep, infected cattle did have a shift (decrease, approximately 0.8) in the peripheral CD4/CD8 ratio associated with an increase in circulating BoT8 (CD8)+ lymphocytes on day 14 post infection. Lymphocytes in the peripheral blood of all sheep and cattle infected with BTV-17 proliferated in vitro in response to purified BTV-17. These results confirm and extend those of previous studies that indicate species differences in the hematologic response to an equivalent BTV infection in domestic ruminants.     

Transplacental transmission of field and rescued strains of BTV-2 and BTV-8 in experimentally infected sheep

Transplacental transmission of bluetongue virus has been shown previously for the North European strain of serotype 8 (BTV-8) and for tissue culture or chicken egg-adapted vaccine strains but not for field strains of other serotypes. In this study, pregnant ewes (6 per group) were inoculated with either field or rescued strains of BTV-2 and BTV-8 in order to determine the ability of these viruses to cross the placental barrier. The field BTV-2 and BTV-8 strains was passaged once in Culicoides KC cells and once in mammalian cells. All virus inoculated sheep became infected and seroconverted against the different BTV strains used in this study. BTV RNA was detectable in the blood of all but two ewes for over 28 days but infectious virus could only be detected in the blood for a much shorter period. Interestingly, transplacental transmission of BTV-2 (both field and rescued strains) was demonstrated at high efficiency (6 out of 13 lambs born to BTV-2 infected ewes) while only 1 lamb of 12 born to BTV-8 infected ewes showed evidence of in utero infection. In addition, evidence for horizontal transmission of BTV-2 between ewes was observed. As expected, the parental BTV-2 and BTV-8 viruses and the viruses rescued by reverse genetics showed very similar properties to each other. This study showed, for the first time, that transplacental transmission of BTV-2, which had been minimally passaged in cell culture, can occur; hence such transmission might be more frequent than previously thought.     

A two year BTV-8 vaccination follow up: molecular diagnostics and assessment of humoral and cellular immune reactions

The compulsory vaccination campaign against Bluetongue virus serotype eight (BTV-8) in Germany was exercised in the state of Bavaria using three commercial monovalent inactivated vaccines given provisional marketing authorisation for emergency use. In eleven Bavarian farms representing a cross sectional area of the state the immune reactions of sheep and cattle were followed over a two year period (2008-2009) using cELISA, a serum neutralisation test (SNT) and interferon gamma (IFN-γ) ELISPOT. For molecular diagnostics of BTV genome presence two recommended real time quantitative RT-PCR protocols were applied. The recommended vaccination scheme led to low or even undetectable antibody titers (ELISA) in serum samples of both cattle and sheep. A fourfold increase of the vaccine dose in cattle, however, induced higher ELISA titers and virus neutralising antibodies. Accordingly, repeated vaccination in sheep caused an increase in ELISA-antibody titers. BTV-8 neutralising antibodies occurred in most animals only after multiple vaccinations in the second year of the campaign. The secretion of interferon gamma (IFN-γ) in ELISPOT after in vitro re-stimulation of PBMC of BTV-8 vaccinated animals with BTV was evaluated in the field for the first time. Sera of BTV-8 infected or vaccinated animals neutralising BTV-8 could also neutralise an Italian BTV serotype 1 cell culture adapted strain and PBMC of such animals secreted IFN-γ when stimulated with BTV-1.     

Clinical syndromes associated with the circulation of multiple serotypes of bluetongue virus in dairy cattle in Israel

From 2008 to 2011, seven distinct bluetongue virus (BTV) serotypes (BTV-2, BTV-4, BTV-5, BTV-8, BTV-15, BTV-16 and BTV-24) have been identified to be circulating in diseased sheep and cattle in Israel. This paper describes the array of clinical manifestations caused by BTV in cattle in Israel. Each set of clinical manifestations has been categorised as a syndrome and six distinct clinical syndromes have been observed in dairy cattle: 'footrot-like syndrome', 'sore nose syndrome', 'subcutaneous emphysema syndrome', 'red/rough udder syndrome', 'bluetongue/epizootic haemorrhagic disease systemic syndrome' and 'maladjustment syndrome'.     

Recombinant DNA probe for serotype-specific identification of bluetongue virus 17

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.     

Bluetongue virus: European Community inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods

European Community national reference laboratories participated in two inter-laboratory comparison tests in 2006 to evaluate the sensitivity and specificity of their 'in-house' ELISA and RT-PCR assays for the detection of bluetongue virus (BTV) antibodies and RNA. The first ring trial determined the ability of laboratories to detect antibodies to all 24 serotypes of BTV. The second ring trial, which included both antisera and EDTA blood samples from animals experimentally infected with the northern European strain of BTV-8, determined the ability of laboratories to detect BTV-8 antibodies and RNA, as well as the diagnostic sensitivity of the assays. A total of six C-ELISAs, six real-time RT-PCR and three conventional RT-PCR assays were used. All C-ELISAs were capable of detecting the BTV serotypes currently circulating in Europe (BTV-1, 2, 4, 8, 9 and 16), however some assays displayed inconsistencies in the detection of other serotypes, particularly BTV-19. All C-ELISAs detected BTV-8 antibodies in cattle and sheep by 21 dpi, while the majority of assays detected antibodies by 9 dpi in cattle and 8 dpi in sheep. All the RT-PCR assays were able to detect BTV-8, although the real-time assays were more sensitive compared to the conventional assays. The majority of real-time RT-PCR assays detected BTV RNA as early as 2 dpi in cattle and 3 dpi in sheep. These two ring trails provide evidence that national reference laboratories within the EC are capable of detecting BTV antibodies and RNA and provide specificity and sensitivity information on the detection methods currently available.     

Evidence of genome segment 5 reassortment in bluetongue virus field isolates.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.     

Multiple bluetongue virus-cloned genetic probes: application to diagnostics and bluetongue virus genetic relationships

A shotgun-cloning method incorporating all 10 bluetongue virus genome segments can simultaneously produce complete and partial copies of any of the genome segments. We report here 4 different cloned probes derived from 3 genome segments and individually defined by different hybridization recognition capabilities. One probe hybridized strongly with all 5 United States prototype strains of the 5 different bluetongue virus (BTV) serotypes existing in the United States and, as such, is a strong candidate for a broad BTV diagnostic probe in the United States. Another probe derived from genome segment 2 of BTV-17 hybridized only with the BTV-17 prototypic serotype, thereby demonstrating serospecific hybridization diagnostic potential. The implications for diagnostic and genetic relationship studies on BTV, using various genetic probes, are discussed.     

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.     

Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle,sheep, and deer

OBJECTIVE: To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). SAMPLE POPULATION: Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. PROCEDURE: Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. RESULTS: All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. CONCLUSIONS AND CLINICAL RELEVANCE: There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.