Development of real‐time PCR assays for detection of megalocytiviruses in imported ornamental fish

Abstract

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.

Detection of infectious spleen and kidney necrosis virus (ISKNV) and turbot reddish body iridovirus (TRBIV) from archival ornamental fish samples

“…2 & 3). Partial MCP gene sequence obtained from an outbreak of megalocytivirus disease involving threespot gouramis in Florida in 1991 and 1992 (Fraser et al 1993, Gias et al 2011) was identical with the sequence of all 3 newly characterised megalocytiviruses as well as RBIV/Tp/45/08 and suggests that this isolate also belongs to this TRBIV Clade 2.…”

“…4) suggests that the TRBIV genotype may have been the major genotype affecting ornamental fish from the time when megalocytiviruses appeared to emerge in ornamental fish in the late 1980s until the early 1990s. This assessment is based on Case 1 (angelfish), Case 2 (dwarf gourami) and Cases 3 and 4 (oscars and keyhole cichlids) that were submitted in 1986, 1988 and 1991, respectively, and partial sequence data from the MCP gene of an isolate originally obtained from threespot gouramis suffering mortalities during 1991 and 1992 that were reported by Gias et al (2011) (Fig. 4).…”

“…Gene sequences for the MCP and ATPase genes annotated in GenBank as megalocytiviruses were used to generate initial alignments. Additionally, a partial fragment of the MCP gene sequence obtained by Gias et al (2011) from cell culture material that originated from threespot gourami showing microscopic megalocytivirus-like lesions (Fraser et al 1993) was manually transcribed from Fig. 1 in Gias et al (2011), since it was not available in GenBank, and included in the alignment (after editing to remove primer sequences) of megalocytiviral MCP gene sequences.…”

Naturally concurrent infections of bacterial and viral pathogens in disease outbreaks in cultured Nile tilapia (Oreochromis niloticus) farms

“…Semi-nested PCR primers were designed to target the major capsid protein gene of megalocytivirus (GenBank JQ253374.1), which is a genus within the family Iridoviridae. MegaI and MegaII primers for the first step PCR were used to amplify 214-bp according to the method described by Gias et al (2011). To increase the method's sensitivity, MegaIII primer was newly designed in this study and was used together with MegaI primer in the secondary PCR reaction to amplify a 108-bp …”

Molecular confirmation of infectious spleen and kidney necrosis virus (ISKNV) in farmed and imported ornamental fish in Australia

“…TaqMan real-time PCR assays are commonly utilised in diagnostic laboratories to provide rapid, highly sensitive and sequence-specific results. Universal megalocytivirus SYBR Green qPCRs have previously been developed by Caipang et al (2003), Gias et al (2011) and Rimmer et al (2012), but, while more expensive, TaqMan qPCRs are generally more specific and reproducible than SYBR Green assays (Gunson et al 2006, Beld et al 2007, Purcell et al 2011.…”