A real-time
assay coupled with a non-invasive
tissue sampling was developed for the detection and quantification of fish viruses. As a proof of principles, data were presented for the detection and quantification of infectious hypodermal
necrosis virus (IHNV) in
trout. The primers were designed for IHNV nucleocapsid (N), and surface
glycoprotein (G) genes, and
trout &bgr;-
actin and
elongation factor-l&agr; (EF-I &agr;) were used as internal control for the
assay. The
reaction conditions for the real-time RT-PCR were optimized using cDNA derived from IHNV-infected Epithelioma papulosum cyprinid (EPC) cells. Using both N- and G-
gene primers, IHNV was successfully detected in liver,
kidney,
spleen,
adipose tissue and pectoral fin samples of laboratory-challenged and wild samples. The dissociation curves with a single melting peak at expected temperature (85° C. for the N-
gene and 86.5° C. for the G-
gene) confirmed the specificity of the N- and G-gene amplicons. The IHNV N- and the G-
gene expression levels in different tissues of laboratory challenged samples were in the order of
spleen, liver,
kidney,
adipose tissue and pectoral fin, however in the field-collected samples the order of
gene expression was liver,
kidney, pectoral fin,
adipose tissue, and
spleen. The N- and G-gene expressions in spleen were found to be dramatically lower in the field-collected samples compared to the laboratory-challenged samples indicating a
potential difference in the IHNV replication in the laboratory as opposed to
field conditions. The real-time
PCR assay was found to be rapid,
highly sensitive, and reproducible. Based upon the ability to detect the
virus in pectoral fins a non-invasive detection method for IHNV and other fish viruses is developed. Such a non-invasive
tissue sampling coupled with real-time
PCR assay is very valuable for large-scale
virus screening of fish in
aquaculture facilities as well as for epidemiological studies.