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Novel Bluetongue Virus Serotype from Kuwait

Year: 2011

Type: article

Abstract: 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 identifi ed KUW2010/02 as a novel BTV serotype. The StudyDuring 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.KUW2010/02 RNA analyzed by agarose gel electrophoresis generated a genome segment migration pattern (electropherotype) typical of BTV (Figure 1).Although KUW2010/02 was negative by BTV-specifi c rRT-PCR selective for Seg-1 (6), it had C t values of 16.8 in Seg-10-specifi c assays (8).Identifi cation of KUW2010/02 as BTV was confi rmed by indirect antigen-sandwich ELISA selective for core protein VP7(T13) (10) with optical density at 490 nm values >0.15 (equivalent BTV titer of 6.75 log 10 50% tissue culture infective dose/mL).RNA from KUW2010/01 and KUW2010/02, extracted by using TRIzol (Invitrogen, Carlsbad, CA, USA) (11), gave uniformly negative results by typespecifi c rRT-PCRs targeting Seg-2 of BTV serotypes

Source: Emerging infectious diseases

DOI: 10.3201/eid1705.101742