A sensitive and specific PCR/Southern blot assay for detection of bovine herpesvirus 4 in calves infected experimentally.
A PCR/Southern blot assay for detection of bovine herpesvirus 4 (BHV-4) in the background of bovine cellular DNA was developed. A BHV-4 specific sequence within the gene coding for the glycoprotein B (gB) was selected for primer sequences to guarantee the specificity of the assay. With a detection limit of six molecules BHV-4 DNA in the background of 1 microg of cellular DNA (equals about 150,000 bovine cells) this PCR/Southern blot assay represents a highly sensitive method for detection of BHV-4 DNA. At low concentrations of BHV-4 genomes, this assay also allows to estimate the copy number of BHV-4: a distinction between fewer than 6, 6-59 and more than 60 BHV-4 genomes/100 microl DNA suspension was possible. Tissue and blood samples of two calves, infected experimentally with BHV-4 were examined for the prevalence of BHV-4 DNA 130 days post infection. Ten days before taking samples, one of the calves was immuno-suppressed with dexamethasone. In both calves, BHV-4 DNA was detected in the leucocyte fraction of the blood, and beyond that in lower quantities in the spleen and the kidney of the immuno-suppressed calf. It is assumed that a latent BHV-4 infection was activated after application of dexamethasone and that the leucocyte fraction of the blood represents one site of latency of BHV-4 in cattle.
Boerner
B
B
Fachbereich Genetik/Gentechnik, Robert Koch-Institut, Berlin, Germany. boerner@lmb.uni-muenchen.de
Weigelt
W
W
Buhk
H J
HJ
Castrucci
G
G
Ludwig
H
H
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods / Q000706:statistics & numerical data; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005260:Female; D018142:Gammaherpesvirinae / Q000235:genetics* / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000662:veterinary* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D000199:Actins; D017931:DNA Primers; D004279:DNA, Viral