Genotyping spring viraemia of carp virus and other piscine vesiculo-like viruses using reverse hybridisation

Abstract

A simple nylon membrane-based DNA macroarray was developed to genotype spring viraemia of carp virus (SVCV) and related viruses. Twenty-six viruses were genotyped using the array, and the results were confirmed by phylogenetic analysis of a 426 bp partial glycoprotein gene sequence. The array was not only capable of discriminating between the 4 main genogroups of cyprinid vesiculo-type viruses described previously, but also accurately sub-type the SVC viruses assigned to Genogroup I. The assay offers a practical solution for diagnostic laboratories that currently lack a sequencing capability to confirm the nature of PCR products generated in suspected SVCV cases.

First detection of pike fry-like rhabdovirus in barbel and spring viraemia of carp virus in sturgeon and pike in aquaculture in the Czech Republic

“…The sequences of the 550 bp region of the G gene of Czech isolates were identified as SVCV or pike fry-like rhabdovirus by the Basic Local Table 1). Phylogenetic analysis of the 401 nt sequences (corresponding to nucleotides 3545 to 3945 of GenBank sequence U18101) of newly and previously isolated vesiculo viruses (Björklund et al 1996, Stone et al 2003, Garver et al 2007, Miller et al 2007, Sheppard et al 2007, Teng et al 2007, Warg et al 2007, Yue et al 2008, Basic et al 2009) was done, as this allowed most isolates for which sequence data is available to be included. Phylo genetic analyses were also performed with subsets of the isolates for which 550 nt (nucleotides 3498 to 4047 of GenBank sequence U18101) and 520 nt (nucleotides 3498 to 4017 of GenBank se quence U18101) were available.…”

“…Phylogenetic analyses based on the 401 nt, as well as the 550 nt, region of the G gene showed that all Czech vesiculovirus isolates, except for the barbel isolate (V-630), were grouped with the representative SVCV isolates (Björklund et al 1996, Stone et al 2003, Garver et al 2007, Miller et al 2007, Sheppard et al 2007, Teng et al 2007, Warg et al 2007, Yue et al 2008, Basic et al 2009) in Genogroup I (Fig. 2) and therefore should be classified as SVCV.…”

“…The phylogenetic analyses further clustered the Czech isolates, including the isolate from sturgeon fry (V-637), which was linked with pathological changes and occurrence of mortality in its sturgeon host, into the Subgroup Id, supported by bootstrap values ≥990. The analyses divided the Czech isolates belonging to Subgroup Id, and the representative Id isolates (Björklund et al 1996, Stone et al 2003, Sheppard et al 2007, Basic et al 2009), into 2 distinct groups: Id1 and Id2 (Fig. 2).…”

“…glycoprotein G, the N and M proteins) are closely related, conventional serological methods such as the indirect fluorescent antibody test or ELISA can only detect the virus and do not allow further classification (Jorgensen et al 1989, Way 1991, Rodak et al 1993, Ahne et al 1998, Dixon & Longshaw 2005. For genogroup and subgroup classification, sequencing of 401 to 550 nucleotides (nt) of the G gene is most frequently used (Stone et al 2003, Garver et al 2007, Miller et al 2007, Sheppard et al 2007, Warg et al 2007, although an alternative target gene has been tested with similar results (Miller et al 2007). Molecular differentiation, specifically sequence analysis, of the G gene of aquatic vesiculoviruses is accepted by the World Organization for Animal Health (OIE) (OIE 2010) for the identification of SVCV in cell culture.…”

“…Members of the genus Vesi culo virus have a linear non-segmented singlestranded negative-sense RNA genome consisting of 5 genes in the order 3' N-P-M-G-L 5' coding for the viral nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase, respectively (Wunner & Peters 1991, Coll 1995, Ahne et al 2002. Phylogenetic analysis of the SVCV-related fish vesiculoviruses reveals 4 distinct genogroups (Stone et al 2003, Sheppard et al 2007. Genogroup I comprises all SVCV isolates, Genogroup III comprises a single isolate named pike fry rhabdovirus (PFRV), and Genogroups II and IV include infectious agents originally characterised as pike fry-like rhabdoviruses.…”