Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene.
Anguillid herpesvirus (AnHV, also known as Herpesvirus anguillae or HVA) is found in both Japanese and European eels. Based on restriction enzyme analysis a small number of differences were found between AnHV isolated from Japanese eels and from European eels. The total genome size of both is about 245 kb, which is confirmed by alternating-field electrophoresis. Using a set of degenerate primers based on conserved regions within DNA-directed DNA polymerase coding regions, a 463 base pair fragment was isolated from both Japanese and European AnHV. Nucleotide sequence analysis showed that the cloned regions of both viruses have identical sequences. Based on this part of the DNA-polymerase sequence, primers were selected and used to develop a sensitive PCR to detect AnHV DNA in eel tissue samples. To avoid false negative results and to estimate the number of AnHV genome copies found in tissues, 100 copies of an internal control plasmid were added to the tissue samples. This semi-quantitative AnHV PCR can be used for both the European and Japanese isolates of AnHV, detects as few as 10 genome copies and is 100 times more sensitive than standard virus isolation.
Rijsewijk
Frans
F
Virus Discovery Unit, Wageningen University and Research Centre, Animal Sciences Group (ASG), Lelystad, The Netherlands. Frans.Rijsewijk@wur.nl
Pritz-Verschuren
Sylvia
S
Kerkhoff
Sonja
S
Botter
Annemiek
A
Willemsen
Madelon
M
van Nieuwstadt
Ton
T
Haenen
Olga
O
D000595:Amino Acid Sequence; D000811:Anguilla / Q000821:virology*; D000818:Animals; D004279:DNA, Viral / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D016679:Genome, Viral; D006564:Herpesviridae / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase