PCR detection of North American and Central African isolates of epizootic hemorrhagic disease virus (EHDV) based on genome segment 10 of EHDV serotype 1

Imadeldin E. Aradaib, William C. Wilson, Caroline E. Schore, Mohammed E H Mohammed, Tilahun Yilma, James S Cullor, Bennie Osburn

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2604-2608
Number of pages5
JournalJournal of Clinical Microbiology
Volume36
Issue number9
StatePublished - Sep 1998

Fingerprint

Epizootic hemorrhagic disease virus
African Americans
Genome
Polymerase Chain Reaction
RNA
Deer
Serogroup
Virus Diseases
Cell Culture Techniques
Oligoribonucleotides
Bluetongue virus

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

PCR detection of North American and Central African isolates of epizootic hemorrhagic disease virus (EHDV) based on genome segment 10 of EHDV serotype 1. / Aradaib, Imadeldin E.; Wilson, William C.; Schore, Caroline E.; Mohammed, Mohammed E H; Yilma, Tilahun; Cullor, James S; Osburn, Bennie.

In: Journal of Clinical Microbiology, Vol. 36, No. 9, 09.1998, p. 2604-2608.

Research output: Contribution to journalArticle

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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.",
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