Diagnostic validation of three test methods for detection of cyprinid herpesvirus 3 (CyHV-3)

Abstract

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of koi herpesvirus disease in koi and common carp. The disease is notifiable to the World Organisation for Animal Health. Three tests -quantitative polymerase chain reaction (qPCR), conventional PCR (cPCR) and virus isolation by cell culture (VI) -were validated to assess their fitness as diagnostic tools for detection of CyHV-3. Test performance metrics of diagnostic accuracy were sensitivity (DSe) and specificity (DSp). Repeatability and reproducibility were measured to assess diagnostic precision. Estimates of test accuracy, in the absence of a gold standard reference test, were generated using latent class models. Test samples originated from wild common carp naturally exposed to CyHV-3 or domesticated koi either virus free or experimentally infected with the virus. Three laboratories in Canada participated in the precision study. Moderate to high repeatability (81 to 99%) and reproducibility (72 to 97%) were observed for the qPCR and cPCR tests. The lack of agreement observed between some of the PCR test pair results was attributed to cross-contamination of samples with CyHV-3 nucleic acid. Accuracy estimates for the PCR tests were 99% for DSe and 93% for DSp. Poor precision was observed for the VI test (4 to 95%). Accuracy estimates for VI/qPCR were 90% for DSe and 88% for DSp. Collectively, the results show that the CyHV-3 qPCR test is a suitable tool for surveillance, presumptive diagnosis and certification of individuals or populations as CyHV-3 free.KEY WORDS: CyHV-3 · Diagnostic validation · Precision · Accuracy · Quantitative PCR · Virus isolation Resale or republication not permitted without written consent of the publisherDis Aquat Org 123: 2017 ment on the Application of Sanitary and Phytosanitary Measures.An effective CyHV-3 diagnostic assay should detect the virus in diseased fish as well as in asymptomatic carriers of the virus with latent or persistent infections. Three diagnostic assays were evaluated for their performance characteristics in this study: a modified conventional polymerase chain reaction (cPCR) assay targeting the CyHV-3 thymidine kinase gene (cTK;Bercovier et al. 2005), a modified quantitative PCR (qPCR) assay targeting CyHV-3 open reading frame 89 (ORF89) (qORF89; Gilad et al. 2004) and virus isolation by culture on common carp brain (CCB) cells (VI;Neukirch et al. 1999). The unmodified cTK and qORF89 tests have been evaluated in inter-laboratory ring studies (Way 2008) and intralaboratory comparisons (Bergmann et al. 2010, Monaghan et al. 2015a). The qPCR test displayed high analytical specificity (ASp), detecting all known CyHV-3 isolates but not related virus species such as CyHV-1 or CyHV-2 (Gilad et al. 2004, Bergmann et al. 2010. It is currently the most commonly used diag nostic test for CyHV-3 and may prove to be suitable for use in surveillance programs to declare healthy populations of susceptible fish free of the virus (OIE 2015c). The cTK test is one of the PCR assays recommended by the OIE fo...

Analytical validation of two RT-qPCR tests and detection of spring viremia of carp virus (SVCV) in persistently infected koi Cyprinus carpio

“…Concordance correlation coefficient (Lin 1989(Lin , 2000 and Bland and Altman limits of agreement (Bland & Altman 1986, Barnhart et al 2007 were used as described previously (Clouthier et al 2017) to evaluate the statistical agreement in virus titer results obtained with the Q2N and PA tests. Virus titer agreement within and between the 2 tests is presented as point estimates as well as graphically to identify patterns within the data.…”

“…ASp) was extracted from traditional virus preparations of CyHV-3, SVCV, GrCRV, TenRV, VHSV, IHNV or IPNV. CyHV-3 DNA was prepared from infected whole-cell lysates as described by Clouthier et al (2017). Viral RNA for cDNA synthesis was extracted using the QiaAmp Viral RNA Extraction Kit (Qiagen) after clarification (2500 × g, 10 min, 4°C) and centrifugation (21100 × g, 90 min, 4°C) of in fected whole-cell lysates.…”

“…The semi-purified viral RNA or DNA was quantified using the Nanodrop 8000 (Nanodrop Technologies) and then stored at −80°C. Reverse transcription was performed as described by Clouthier et al (2017) except that 100 ng of RNA were used as the template.…”

“…pdf). Linearized plasmid DNA was prepared as described by Clouthier et al (2017), except that the restriction enzyme NcoI (New England Biolabs) was used to cut the DNA. The purified DNA was diluted and used as P3A and P3B positive control samples for the Q1G or Q2N tests.…”

“…The optimum quantity of RNA to use in the Q2N and Q1G tests was identified by adding a range of RNA (i.e. 100, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 ng) extracted from either naïve carp kidney (Clouthier et al 2017) or koi brain tissue (Clouthier et al 2020) seeded with 50 ng exogenous SVCV HHO-carp06 viral RNA from infected whole cell lysates (enriched).…”