Novel Bluetongue Virus Serotype from Kuwait

Sheep and goats sampled in Kuwait during February 2010 were seropositive for bluetongue virus (BTV). BTV isolate KUW2010/02, from 1 of only 2 sheep that also tested positive for BTV by real-time reverse transcription–PCR, caused mild clinical signs in sheep. Nucleotide sequencing identified KUW2010/02 as a novel BTV serotype.

B luetongue virus (BTV) infects ruminants, camelids, and occasionally large carnivores. Clinical signs of bluetongue disease (BT) are usually more severe in sheep or white-tailed deer, particularly in populations previously unexposed to the virus; cattle and goats are often asymptomatic (1). Initial diagnosis of BT based on clinical signs can be confi rmed by virus isolation and characterization or identifi cation of viral RNA by reverse transcription PCR. BTV particles contain 3 concentric protein layers surrounding 10 linear double-stranded RNA genome segments, identifi ed as segment-1 to segment-10 (Seg-1 to Seg-10) in order of decreasing size (from 3,954 bp to 822 bp) (2). Twenty-fi ve BTV serotypes have been identifi ed on the basis of the specifi city of reactions with neutralizing antibodies generated by their mammalian hosts (3). Consequently, BTV outer capsid proteins, particularly viral protein (VP) 2 (encoded by Seg-2), show sequence variations that determine virus serotype (4). Other BTV proteins, including subcore shell protein VP3(T2) encoded by Seg-3, are more highly conserved (2). Phylogenetic comparisons of Seg-3 sequences have been used to identify different BTV topotypes and distinguish different Orbivirus species (4).
BTV has been reported in several Middle Eastern countries (Egypt, Jordan, Syria, Turkey, Cyprus, and Iraq) since 1951 (5). In 2008, Egypt reported the absence of BT, and Egypt is the only country in the region to have prohibited BTV vaccination (5). Iran reported outbreaks of BT in 2008, and Saudi Arabia reported infection without clinical signs, although the serotype(s) were not identifi ed (5). Multiple serotypes were detected in Israel during 2008 (5) and Oman in 2009 (S. Maan et al., unpub. data). We report characterization of a novel BTV serotype identifi ed in Kuwait in 2010.

The Study
During February 2010, sheep and goats in the Abdali region of Kuwait, close to the Iraq border, showed the following clinical signs consistent with BT: lameness, coughing, mouth lesions, stillbirth, congenital abnormalities, pneumonia, enteritis, and hepatitis. Fortysix of 48 serum samples were positive for BTV-specifi c antibodies by competitive ELISA (Investcare-Vet, London, UK) at the Veterinary Diagnostic Laboratory and Animal Research Center in Kuwait, or by double antigenrecognition ELISA (ID Vet, Montpellier, France) at the Institute for Animal Health in the United Kingdom.
Twenty-six EDTA-treated blood samples, 4 spleens, and 1 liver sent for analysis for BTV to the World Organisation for Animal Health reference laboratory at the Institute for Animal Health (Woking, UK) all gave negative results by real-time reverse transcription-PCR (rRT-PCR) targeting either BTV Seg-1 (6) or Seg-1 and Seg-5 (7). However, 2 blood samples (from animals 364 and 374) were positive in assays selective for Seg-10 (8), with cycle threshold (C t ) values of 35 and 28, respectively. Previous attempts to isolate BTV from blood samples with C t values >32 were usually unsuccessful, and no further work was done with animal 364. Washed blood from animal 374 (reference collection sample KUW2010/01) (9) was injected into embryonated chicken eggs. Although no hemorrhages were detected, the virus was passaged twice in BHK-21 cells (isolate KUW2010/02), producing atypical cytopathic effects.

Conclusions
Most serum samples tested from sheep and goats in Kuwait showing clinical signs of disease were seropositive for BTV-specifi c antibodies. However, BTV RNA was detected in only 2 sheep serum samples (animals 364 and 374), suggesting that the clinical signs were not caused by ongoing BTV infection. All samples were also tested for peste des petits ruminants virus by rRT-PCR (14), but results were uniformly negative.
BTV RNA was detected by using a BTV Seg-10specifi c rRT-PCR (8) previously used to detect BTV-25 in Switzerland (15). However, BTV Seg-1-or Seg-1-and Seg-5-specifi c assays (6,7) failed to detect KUW2010/02, identifying it is an unusual or atypical BTV strain. Alignment of the Seg-1-specifi c and Seg-5-specifi c primers and probes with KUW2010/02 sequences identifi ed numerous mismatches that would prevent detection of the viral RNA (data not shown). However, the probe and primers designed by Orrù et al. (8) showed a perfect match with Seg-10 of KUW2010/02, demonstrating the need for appropriate rRT-PCR protocols to detect this virus. Agarose gel electrophoresis analysis of KUW2010/02 genome segments generated a migration pattern typical of BTV (Figure 1). Levels of nucleotide/amino acid identity of Seg-3 (up to 76.6%/89%) of KUW2010/02 with other BTV isolates also identifi ed it as BTV.
At peak viremia, a previously unexposed sheep infected with a virulent BTV strain could be expected to show C t values ≈20. The C t of 28 obtained with blood of animal 374 (KUW2010/01) indicates a low viremia, suggesting that the severe clinical signs observed were not caused by BTV. Experimental infection of previously unexposed sheep with KUW2010/02 also caused only mild clinical signs (data not shown).
Different isolates of a single BTV serotype show >68.4%/72.6% nt/aa identity in Seg-2/VP2, with 40.5%/ 22.1% to 71.5%/77.8% identity between different sero-  types (4). KUW2010/02 showed only 42.8%/28.3% to 63.9%/61.5% identity in Seg-2/VP2 with recognized BTV serotypes, consistent with membership of a novel 26th BTV type and Seg-2 nucleotype (L) (4). These conclusions were supported by virus neutralization test results. The sequence data presented here will help support development of new diagnostic tools (RT-PCR-based typing assays) to determine the incidence and distribution of this novel serotype, as well as natural reservoir(s) and insect vectors.