A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3)

Abstract

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3)

“…In light of the absence of a cell line that can support the replication of EEDV, detection of the virus for screening purposes and clinical diagnosis are currently limited to endpoint PCR (Kurobe et al, 2009) and real-time PCR (Glenney et al, 2016b). In this study, we developed a LAMP assay for EEDV detection in fish tissue with relatively high specificity and sensitivity, and therefore, represents a time-and cost-effective early diagnostic tool for the detection and quantification of this deadly virus.…”

“…All samples came from negative control (numbers 1-20) or an experimentally infected fish group (number 21 through 100) as described in Shavalier (2017). DNA was extracted from these tissue samples using the kit OMEGA (Bio-tek) as described above, after which the qLAMP was run in parallel with the SYBR Green qPCR assay as described by Glenney et al (2016b). Briefly, all quantitative PCR reactions were carried out in an Eppendorf Mastercycler® Realplex 2 with a total reaction volume of 20 μl.…”

“…The analytical sensitivity of the EEDV LAMP assessed by plasmid and infected tissue is considerably higher than those reported by Chen et al (2010) for the swine transmissible gastroenteritis coronavirus, Li and Ling (2014) for the tomato necrotic stunt virus, and Ma et al (2016) for the Eriocheir sinensis reovirus. The EEDV SYBR qPCR assay developed by Glenney et al (2016b) seems to be more sensitive with a detection limit of a single digit of plasmid copies (exact number of copies was not given). While this superior sensitivity of the SYBR qPCR may enable the detection of carrier fish, the developed LAMP assay was proven to be capable of detecting EEDV DNA in tissue samples after several orders of dilution.…”

“…Endpoint and quantitative PCR-based detection assays for EEDV have been developed that target stretches of the EEDV terminase gene (Glenney et al, 2016b;Kurobe et al, 2009). After uncovering of the molecular characterization of Salmonid Herpesvirus-4 and -5, it was determined that the current EEDV qPCR assay was unable to distinguish between Salmonid Herpesvirus-3, -4, and -5, as the viruses share high sequence similarity in the terminase gene.…”

“…After uncovering of the molecular characterization of Salmonid Herpesvirus-4 and -5, it was determined that the current EEDV qPCR assay was unable to distinguish between Salmonid Herpesvirus-3, -4, and -5, as the viruses share high sequence similarity in the terminase gene. This led Glenney et al (2016b) to design three primer sets based on the glycoprotein gene. Using these primer sets in a SYBR Green qPCR assay, the authors were able to amplify each virus individually.…”