title: Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene.
abstract: 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.
authors.affiliation: Virus Discovery Unit, Wageningen University and Research Centre, Animal Sciences Group (ASG), Lelystad, The Netherlands. Frans.Rijsewijk@wur.nl
authors.forename: Frans
authors.initials: F
authors.lastname: Rijsewijk
authors.forename: Sylvia
authors.initials: S
authors.lastname: Pritz-Verschuren
authors.forename: Sonja
authors.initials: S
authors.lastname: Kerkhoff
authors.forename: Annemiek
authors.initials: A
authors.lastname: Botter
authors.forename: Madelon
authors.initials: M
authors.lastname: Willemsen
authors.forename: Ton
authors.initials: T
authors.lastname: van Nieuwstadt
authors.forename: Olga
authors.initials: O
authors.lastname: Haenen
mesh_terms: 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*
chemical_list: D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase