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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Available online at www.sciencedirect.com Journal of Virological Methods 147 (2008) 226–234 Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling Robert M. Bowers a, Scott E. Lapatra b, Arun K. Dhar a,∗ a Advanced BioNutrition Corporation, 7155-H Columbia Gateway Drive, Columbia, MD 21046, United States b Clear Springs Foods, Inc., Research Division, P.O. Box 712, Buhl, ID 83316, United States Received 22 May 2007; received in revised form 28 August 2007; accepted 5 September 2007 Abstract Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabir- navirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR® Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout ␤-actin and elongation factor 1␣ (EF-1␣) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 ◦C for IPNV NS, trout ␤-actin, and EF-1␣ genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 107 RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24 h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin could be used for the detection and quantification of IPNV. The development of a non-lethal detection method will be useful for the detection of IPNV and potentially other viruses of finfish in farmed and wild fish. © 2007 Elsevier B.V. All rights reserved. Keywords: IPNV; Real-time RT-PCR; SYBR Green; Non-lethal detection; Rainbow trout 1. Introduction America to Europe, Asia, and South Africa (Hill, 2000). IPNV is highly contagious and transmitted through a variety of routes Infectious pancreatic necrosis virus (IPNV), the etiological including contaminated water (Magyar and Dobos, 1994), inges- agent of infectious pancreatic necrosis disease (IPN), is a major tion of infected material, direct contact with secretions from viral pathogen of wild and cultured salmonids (Wolf, 1988). In infected fish (e.g., feces, sexual fluids), and contact with any salmonid farming, the disease can cause high mortality in both contaminated surface. Adult fish can also serve as asymptomatic fry and juveniles in freshwater as well as in smolts during their carriers of the virus, eventually shedding the virus particles into first month after transfer to seawater (Jarp et al., 1994). This virus the environment where other fish can become infected (Reno et is prevalent in salmonid hatcheries around the world, from the al., 1978). These carriers may hamper the successful detection of IPNV preceding an outbreak. For this reason, it is critical that highly sensitive detection methods be developed for rapid ∗ Corresponding author. Tel.: +1 410 730 8600; fax: +1 410 730 9311. management of infected stock. E-mail addresses: [email protected], arun [email protected] IPNV is a bisegmented double-stranded RNA virus belong- (A.K. Dhar). ing to the family Birnaviridae, genus Aquabirnavirus. The two 0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.09.003 Author's personal copy R.M. Bowers et al. / Journal of Virological Methods 147 (2008) 226–234 227 genome segments are designated Segment A and Segment B. Recent advances in infectious disease diagnosis in human, The virions are non-enveloped icosahedrons measuring 60 nm plant, and terrestrial agricultural species are being applied grad- in size. Segment A of the IPNV genome is 3.1 kb and contains ually to the health and management of economically important a large open reading frame encoding a 106 kDa polyprotein aquatic species. One such example is the application of real-time arranged in the order NH2-preVP2-VP4 (NS-protease)-VP3- PCR in gene expression studies and viral disease diagnosis in fish COOH. The polyprotein is co-translationally cleaved by the and shellfish aquaculture. Real-time PCR was developed in 1992 NS-protease to produce preVP2 and VP3. The preVP2 is cleaved (Higuchi et al., 1992, 1993) and quickly became a major tool for further to yield mature VP2, which makes up at least 62% of the gene expression studies, and more recently, for disease diagnosis capsid protein (Dobos et al., 1979). The Segment A encoded (Bustin, 2000; Mackay et al., 2002; Niesters, 2002). The sim- polyprotein also contains a 15-kDa non-structural VP5 protein, plicity, sensitivity, dynamic range of detection, reproducibility, which is found only in infected cells (Magyar and Dobos, 1994). and amenability to high throughput screening makes real-time Segment B of the IPNV genome is 2.8 kb and presumed to PCR an attractive tool for viral detection (Mackay et al., 2002; encode the RNA-dependent RNA polymerase, designated VP1. Niesters, 2002). A variety of fluorescently based methods are VP1 exists as a free polypeptide and as a genome linked protein currently available for the detection of real-time PCR prod- in the virion, termed VPg (Dobos, 1995). ucts. These include the non-specific DNA-binding dye SYBR® Currently, IPNV detection relies on the isolation of Green I, target-specific fluorescently labeled linear oligoprobes, virus from suspect fish using established fish cell lines, 5-nuclease oligoprobes, molecular becons, and self-fluorescing then confirmed using a variety of immunological assays, amplicons (Mackay et al., 2002). Among these, detection by such as serum neutralization (Hill, 2000), flow cytometry, SYBR® Green I is the simplest and least expensive method immunofluorescense, immunoperoxidase, immunodot-blotting, because it does not require the design of fluorescently labeled immunostaphylococcus-protein A (Saint-Jean et al., 2001 #4), oligoprobes. Additionally, the specificity of the amplified prod- and enzyme-linked immunosorbent (ELISA) assays (Nicholson uct can be determined by examining the melting curve of the and Caswell, 1982). Virus isolation in cell culture continues to amplicon (Ririe and Rasmussen, 1997). Real-time PCR assays be the gold standard for IPNV detection (OIE, 2003). The major employing either SYBR® Green I (Dhar et al., 2001, 2002)or drawback to this approach is the time required to isolate and con- TaqMan probes (Overturf et al., 2001; Tang and Lightner, 2001; firm the replicating agent as IPNV, which may be up to 10–14 Durand and Lightner, 2002; de la Vega et al., 2004; Gilad et al., days after virus inoculation. Reverse transcriptase-polymerase 2004; Munir and Kibenge, 2004; Tang et al., 2004; Lockhart et chain reaction (RT-PCR) (Blake et al., 1995; Saint-Jean et al., al., 2007; Purcell et al., 2006; Rajendran et al., 2006) have been 2001; Kerr and Cunningham, 2006) and in situ hybridization developed to detect and quantify several viral pathogens of fish (ISH) (Alonso et al., 2004) were developed for the detection of and shrimp. These tests have potential as routine virus screening IPNV; both are rapid and more sensitive than cell culture assay. A tools that would improve the management of viral diseases in multiplex RT-PCR assay to detect IPNV, infectious hematopoi- commercial hatcheries. etic necrosis virus and viral hemorrhagic septicemia virus was Here, we report the development of a real-time RT-PCR developed by Williams et al. (1999). In a study by Saint-Jean assay using SYBR® Green I chemistry for the detection and et al. (2001), the sensitivity of RT-PCR was compared to the quantitation of IPNV. Using this method, IPNV was detected immunological assays mentioned above. The RT-PCR and flow and quantified in spleen, head kidney, and pectoral fin tissues cytometry methods were demonstrated to be the most sensitive of laboratory-challenged rainbow trout (Oncorhynchus mykiss). of the detection methods assessed, detecting virus as early as The ability to detect IPNV in pectoral fin samples could be 4 h post-challenge in CHSE-214 cells.