Assessment of commercial test kits for identification of spring viraemia of carp virus

Abstract

Two test kits for the identification of spring viraemia of carp virus (SVCV), one an enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antiserum, and the other an indirect fluorescent antibody test (IFAT) using a mouse monoclonal antibody, were assessed for specificity using a range of virus isolates. The test viruses were selected from 4 recently described genogroups of piscine rhabdoviruses: Genogroup I (SVCV), Genogroup II (grass carp rhabdovirus), Genogroup III (pike fry rhabdovirus) and Genogroup IV ('tench rhabdovirus'). The test viruses included SVCV isolates from all 4 subgroups of Genogroup I. The ELISA was non-specific for these viruses and did not distinguish between SVCV and isolates from the other 3 Genogroups. However, the IFAT was too specific and detected SVCV isolates from only 1 of the 4 SVCV subgroups. Reliance on these test kits alone could result in misidentification of this OIE notifiable disease. KEY WORDS: SVCV · IFAT · ELISA · Serological comparison · Identification Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [25][26][27][28][29] 2005 MATERIALS AND METHODSViruses and propagation. Viruses were selected from distinct branches of the neighbour-joining distance tree (Table 1) within a Genogroup or subgroup (Stone et al. 2003, Dikkeboom et al. 2004 and grown at 20°C in Epithelioma papulosum cyprini cells (Fijan et al. 1983) or fathead minnow cells (ATCC CCL 42) (isolate WI02-131 only) as described previously (Hill et al. 1975, Way 1991, and harvested when complete cytopathic effect (CPE) was observed. The viruses were titrated in 96-well cell cultivation plates and TCID 50 values were calculated according to Kärber (1931). For the IFAT, virusinfected cell culture harvests were diluted to 1:100 and 1:1000 in cell culture medium and used to inoculate the appropriate cell cultures on 24-well IWAKI plates (Scientific Laboratory Supplies). Following incubation for 16 to 20 h at 20°C, the medium was removed, and the cells were fixed with the solution provided with the test kit, or with 80% acetone for the in-house IFAT (see below). For the ELISA, virus-infected cell culture harvests were diluted in diluent supplied with the kit. The remaining steps of the ELISA and IFAT were conducted according to the procedures supplied with the kits; cells inoculated with both the 1:100 and 1:1000 dilutions of virus were tested in the IFAT. An in-house rabbit polyclonal antiserum (produced against SVCV isolate S30) was used as a positive control for the IFAT.Test kits. Kit SVC048, which is an ELISA using a rabbit polyclonal antiserum was obtained from TestLine, (Brno, Czech Republic) and Kit BIO K12, which is an IFAT using a mouse monoclonal antibody (MAb) came from Bio-X Diagnostics SPRL (Jemelle, Belgium). Tests were conducted according to the procedures specified by the manufacturers with the following exceptions. TestLine specified the use of doubling dilutions of sample from 1:2 to 1:256, but doubling dilutions from 1:20 to 1...

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

“…Consequently, ELISA (Rodak at al. 1993, Dixon & Longshaw 2005 detected a high portion of positive samples from the overall tested samples (27 of 178) during the study period 1995 to 2008. Virus-infected samples giving a positive result with a polyclonal antibody detecting all genogroups of piscine vesiculoviruses (Rodak at al.…”

“…Virus-infected samples giving a positive result with a polyclonal antibody detecting all genogroups of piscine vesiculoviruses (Rodak at al. 1993, Dixon & Longshaw 2005 were subsequently used for PCR and sequencing of a G gene region, apart from 4 presumed vesiculovirus isolates detected by ELISA which were not further investigated in the present study. Using primers specific to SVCV (Koutná et al 2003) 1 of these 4 isolates (PL01) was previously detected in a carp host (Cyprinus carpio) diagnosed with spring viraemia of carp disease (M. Koutná et al unpubl.…”

“…To identify potential vesiculovirus inducing CPE in cell cultures, a test kit from TestLine, Brno, based on the sandwich ELISA antigen detection method (Rodak at al. 1993, Dixon & Longshaw 2005 was applied. Virus identification was based on vesi culovirus specific polyclonal antibodies: swine antibody as the microplate-coating agent and a primary rabbit antibody.…”

“…Since piscine vesiculovirus antigenic determinants (e.g. 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).…”

Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library

“…The produced 29 kDa specific scFv antibodies were confirmed by Western blot assay. Monoclonal antibodies have been used in protocols developed for the rapid and sensitive detection and diagnosis of SVCV, for example, antigen capture ELISA, sandwich ELISA and IFA (Dixon and Longshaw, 2005;Reschova et al, 2007). In order to further characterization of scFvs in this study, they were applied to assess specificity by ELISA and immunofluorescent.…”