Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage

• Published in: Diseases of aquatic organisms 2012-08, Vol.100 (1), p.11-18
• Main Authors: Yuasa, Kei, Kurita, Jun, Kawana, Morihiko, Kiryu, Ikunari, Oseko, Norihisa, Sano, Motohiko
• Format: Article
• Language: English
• Subjects: Animals; Carps - virology; Cyprinus carpio; Fish Diseases - virology; Freshwater; Gene Expression Regulation, Viral - physiology; Herpesviridae - classification; Herpesviridae - isolation & purification; Herpesviridae - physiology; Herpesviridae Infections - diagnosis; Herpesviridae Infections - veterinary; Herpesviridae Infections - virology; Hot Temperature; koi herpesvirus; Marine; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - veterinary; RNA, Messenger - isolation & purification; RNA, Viral - isolation & purification; Sensitivity and Specificity; Virus Replication - physiology
• ISSN: 0177-5103; 0171-8630; 1616-1580
• DOI: 10.3354/dao02499

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.