Development of a monoclonal antibody against viral haemorrhagic septicaemia virus (VHSV) genotype IVa

Abstract

The viral haemorrhagic septicaemia virus (VHSV) comprises 4 major genotypes and a number of subtypes with, in most cases, distinct geographical distribution. A quick and simple detection method that can discriminate the different genotypes is desirable for a quick and more efficient prevention of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected with each of the viral protein genes, it was shown that the MAb VHS-10 recognizes a nonlinear genotype IVa-specific epitope on the VHSV N-protein.

Typing of viral hemorrhagic septicemia virus by monoclonal antibodies

“…In general, it seems that the epitopes are concentrated at two sites on the N-protein with putatively higher antigenicity [aa 38-48 and aa 103-121 in the N-terminal-core domain of the N-protein (Albertini et al, 2006)]. We also deduced in a previous study (Ito et al, 2010) that aa 38-46 of the N-protein forms a variable region in VHSV, due to the presence of many substitutions in this region. The epitopic domains on the N-protein of the mAbs are concentrated in two regions with high antigenicity for a mouse.…”

“…Immunization of mice. Immunization of BALB/c mice with purified VHSV DK-F1, DK-5151, KRRV9601, 1p52, 4p168, JF00Ehi1 and Goby 1-5 was performed as described previously (Ito et al, 2010). Immunization of mice with the VHSV genotype Ib isolate 1p40 was performed as described by Lorenzen et al (1988).…”

“…Cell fusion. Cell fusion was performed as described previously (Ito et al, 2010). Cell fusion of spleen cells from mice immunized with 1p40 was performed as described by Lorenzen et al (1988).…”

“…Access to other tests for VHSV typing, such as a reliable immunoassay that is more convenient than molecular techniques, would be an improvement. mAbs against VHSV have been established in several laboratories (Lorenzen et al, 1988(Lorenzen et al, , 1990Mourton et al, 1990Mourton et al, , 1992Sanz & Coll, 1992;Sanz et al, 1993;Fernandez-Alonso et al, 1998;Ito et al, 2010). Some are widely used as diagnostic reagents, such as IP5B11, which reacts with all known VHSV isolates (Lorenzen et al, 1988); others are neutralizing and are used for serological discrimination into serotypes by seroneutralization test (Olesen et al, 1993).…”

“…Some are widely used as diagnostic reagents, such as IP5B11, which reacts with all known VHSV isolates (Lorenzen et al, 1988); others are neutralizing and are used for serological discrimination into serotypes by seroneutralization test (Olesen et al, 1993). Recently, a mAb reacting specifically against VHSV genotype IVa, mAb VHS-10, was reported (Ito et al, 2010). This work prompted us to immunize BALB/c mice with various genotypes of VHSV and to develop a range of mAbs, enabling us to develop a quick immunochemical test for discriminating between the genotypes and subtypes of VHSV.…”