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

Abstract

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.

Cyprinid Herpesvirus 3

“…Assays developed for research applications include a primer probe designed against an exonic mRNA-coding sequence that allows the detection of replicating CyHV-3 (Yuasa, Kurita, et al, 2012). IF and ISH methods, performed on separated fish leucocytes obtained by nondestructive (nonlethal) techniques, have also been used in research applications for detection or identification of CyHV-3 (Bergmann, Lutze, et al, 2010;Bergmann et al, 2009).…”

Detection and application of circular (concatemeric) DNA as an indicator of koi herpesvirus infection

“…5a, bottom). Detection of expressed viral mRNA by mRNA-specific RT-PCR is therefore a useful method for confirming virus replication in the host (Yuasa et al 2012). On the other hand, the DR-PCR method developed in this study is useful for determining whether the host is infected by the virus.…”

“…DNA template was used for amplification by DR-PCR, Sph-PCR, and real-time PCR (Gilad et al 2004). KHV mRNA was detected using the RT-PCR method of Yuasa et al (2012). The viral titer of cell culture supernatant was determined using the 50% tissue culture infectious dose (TCID 50 ) method as described by Reed & Muench (1938).…”

“…We then verified efficacy of the PCR assay for identifying virus infection of CCB cells using a time-course experiment. We also performed RT-PCR to detect viral mRNA (Yuasa et al 2012), TaqMan real-time PCR to measure the copy numbers of viral genome (Gilad et al 2004), and quantitative isolation of progeny viruses by deter mining 50% tissue culture infectious dose (TCID 50 ). Finally, we clarified the life cycle of KHV in vitro.…”

Cyprinid herpesvirus 3: an interesting virus for applied and fundamental research

“…CyHV-3 genome can also be detected and quantified in environmental water by real-time TaqMan PCR after viral concentration [85]. Recently, a mRNA-specific RT-PCR assay for detection of replicating CyHV-3 in infected fish tissues and cell cultures has been described [86]. ELISA tests have been developed to detect specific anti-CyHV-3 antibodies in carp serum [22].…”