Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus

Abstract

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107–101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107–101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0–100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28–95.6; 95% CI) and 89.8% (83.53–94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

Fish Iridoviridae: infection, vaccination and immune response

“…An SDDV-close European chub iridovirus (ECIV) and an unclassified three-spined stickleback (TSIV) were recently isolated and classified. They have been proposed as species within the MCVs genus [ 11 , 30 , 32 ]. Megalocytiviruses provoke systemic, often life-threatening diseases, characterised by enlarged cells called ‘inclusion body-bearing cells’ in infected organs and tissues such as the spleen, kidney, gastrointestinal tract, and gills.…”

“…This can all lead to mortality amongst wild and farmed marine and freshwater fish, including ornamental species [ 33 ]. This genus is gaining attention because it causes significant mortality that occasionally can reach up to 100% during epizootics in captive fish populations and experimental infections [ 29 , 32 , 34 , 35 ] (Table 1 ). Additional file 1 lists the main species of fish, both marine and freshwater, which have been described as susceptible to infection with megalocytiviruses.…”