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ABSTRACT: PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.
Journal of Clinical Microbiology 10/1998; 36(9):2604-8. · 4.15 Impact Factor
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ABSTRACT: The potential use of the recently reported polymerase chain reaction (PCR) protocol for detection of United States epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-1) and serotype 2 (EHDV-2) ribonucleic acid in cell culture and clinical specimens was evaluated for detection of Sudanese EHDV strains. EHDV serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped) designated (EHDV-318), recovered from sentinel calves at the Khartoum University farm (Sudan) were studied. RNA from EHDV-5 and EHDV-318 and a number of EHDV field isolates, propagated in cell cultures, were detected by the described PCR-based assay. The specific 387 bp PCR products were visualized on ethidium-bromide stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with non-radiolabeled internal probe. Amplification product was not detected when the PCR-based assay was applied to RNA from blutongue virus (BTV) prototypes serotypes 2, 10, 11, 13, 16 and 17; total nucleic acid extracts from uninfected BHK-21 cells. The results of this study indicated that the previously described EHDV-PCR assay could be applied for detection of Sudanese as well as United States strains of EHDV serogroup. In addition, the described EHDV-PCR assay could be used as a supportive diagnostic assay to the current conventional virus isolation procedures used for detection of EHDV infection in susceptible ruminants.
Comparative Immunology Microbiology and Infectious Diseases 07/1997; 20(3):211-8. · 2.34 Impact Factor
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ABSTRACT: Epizootic hemorrhagic disease virus (EHDV), isolate 318 (EHDV-318), an untyped virus recovered from a sentinel calf herd at the Khartoum University farm in central Sudan, was characterized using molecular biological techniques. With dot blot hybridization technique, a cDNA probe derived from genome segment 6 of EHDV-2 (Alberta strain) hybridized with RNA from EHDV-318. Application of serogroup-specific EHDV polymerase chain reaction (PCR) to EHDV-318 RNA resulted in specific amplification of a 387 bp PCR product. Amplification product was visualized on ethidium bromide-stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with a non-radiolabelled internal probe. No amplification product or hybridization signal was detected when the serotype-specific EHDV-1 or EHDV-2 PCR-based assays were applied to RNA from EHDV-318. The scientific data presented in this study indicated that cDNA probes and serogroup-specific PCR-based assay can be used to classify the virus as a member of EHDV serogroup, and as serotypically distinct from EHDV-1 and EHDV-2.
Veterinary Microbiology 11/1996; 52(3-4):201-8. · 3.33 Impact Factor
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ABSTRACT: The seasonal incidence of bluetongue virus (BTV) in Central Sudan is related primarily to fluctuations in the prevalence of the vector, Culicoides imicola. Population densities of this midge begin to rise with the onset of precipitation and peak during October, before falling sharply at the end of the rainy season in November. These are also the months of BTV transmission. Populations of C. schultzei, the commonest midge in Central Sudan, are also related to the rainy season but this species does not seem to be involved with BTV transmission. BTV serotype 2 was isolated from C. imicola confirming the status of this midge as a known vector but a second isolate of the same serotype was made from a mixed pool of Culicoides not including C. imicola. This suggests that BTV transmission in the Sudan may involve more than one species of Culicoides. Epizootic haemorrhagic disease virus (EHDV) serotype 4 and a palyam virus were isolated from C. schultzei which indicates that this species may be involved in the transmission of BT-related viruses. Seven further virus isolates from sentinel calves at Shambat (Khartoum) confirmed the presence of BTV serotypes 1, 4 and 16, and an untyped EHDV (designated 318) in the Sudan. All of the viruses isolated and identified during the course of this work are recorded from the Sudan for the first time.
Epidemiology and Infection 01/1991; 105(3):619-32. · 2.84 Impact Factor
