Diagnosis and Identification of IPNV in Salmonids by Molecular Methods

Isolation in cell culture and detection by PCR‐based technology of IPNV‐like virus from leucocytes of carrier turbot, Scophthalmus maximus (L.)

“…In recent years, RT-PCR has become a commonly used tool for the detection of IPNV, in both cell culture (Rimstad, Hornes, Olsvik & Hyllseth 1990;López-Lastra, Gonzalez, Jashes & Sandino 1994;Wang, Wi & Lee 1997) and infected salmonid fish (Blake, Schill, McAllister, Lee, Singer & Nicholson 1995;Novoa, Blake, Nicholson & Figueras 1995;Rodriguez et al 2001;Taksdal, Dannevig & Rimstad 2001), even in the carrier state (Rodriguez et al 2001;Taksdal et al 2001), providing significant improvement in speed, sensitivity and repetitiveness compared with the traditional detection methods (Dopazo & Barja 2002). The use of nested PCR increases significantly the sensitivity achieved by RT-PCR (Rimstad et al 1990;López-Lastra et al 1994) and also facilitates sequencing and other approaches to RT-PCR amplicon characterization.…”

Restriction Fragment Length Polymorphisms and Sequence Analysis: an Approach for Genotyping Infectious Pancreatic Necrosis Virus Reference Strains and Other Aquabirnaviruses Isolated from Northwestern Spain

“…Unincorporated nucleotides were removed from the solution by using a prewashed Spin column (Sepharose 100; Centricon). The sample was vacuum dried and resuspended with 4 l of loading buffer (deionized formamide and 25 mM EDTA [pH 8.0] at a ratio of 5:1 containing 50 mg of blue dextran per ml). The sample was denatured at 90°C for 2 min, immediately transferred into an ice bath, and loaded onto a 7% polyacrylamide gel in an ABI model 373A DNA sequencer.…”

“…Antigenically unrelated aquatic birnaviruses constitute serogroup B, which includes a single serotype, serotype B1 (reference strain, TV-1). However, this classification is controversial because serotyping may yield ambiguous or even nonrepeatable results due to the lack of standardized antisera (8,14). Therefore, at present, an increasing number of researchers are employing molecular techniques for typing and classification of these viruses (3,8).…”

Detection of infectious pancreatic necrosis virus from rainbow trout, Oncorhynchus mykiss (Walbaum), using the macrophage lysis method

“…Infectious pancreatic necrosis virus (IPNV), belonging to the family Birnaviridae , genus Aquabirnavirus (Faquet, Mayo, Maniloff, Desselberger & Ball 2005), infects salmonid species, such as members of the genera Salmo , Salvelinus and Oncorhynchus (Reno 1998). It can cause high mortalities in young salmonids (Reno 1998; Dopazo & Barja 2002) although resistance against the disease increases with age (Dopazo & Barja 2002). Asymptomatic carriers can transmit the virus both horizontally and vertically (Dopazo & Barja 2002).…”

“…It can cause high mortalities in young salmonids (Reno 1998; Dopazo & Barja 2002) although resistance against the disease increases with age (Dopazo & Barja 2002). Asymptomatic carriers can transmit the virus both horizontally and vertically (Dopazo & Barja 2002). To reduce the risk of such transmission a more sensitive method for detecting IPNV has been developed where macrophages from head kidney are lysed and inoculated onto CHSE‐214 cells (Gahlawat, Munro & Ellis 2004) as an option to standard methods using RT‐PCR (Taksdal, Dannevig & Rimstad 2001) or inoculation of sonicated whole kidney homogenates on fish cell cultures (standard cell culture test) (Smail, Burnside, Watt & Munro 2003).…”