An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein

Herpesvirus Infection Induces both Specific and Heterologous Antiviral Antibodies in Carp

“…CyHV-3 was replicated in cells from common carp brain (CCB), kindly provided by the Collection of Cell Lines at the Veterinary Medicine and German Reference Laboratory (Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany). The novirhabdovirus VHSV-07.71 ( 28 ) was replicated in Epithelioma papulosum cyprini cells from the fathead minnow Pimephales promelas (ATCC, CRL-2872) as previously described ( 11 , 29 ). Briefly, the abovementioned cell lines were grown at 25°C in a 5% CO 2 atmosphere with RPMI-Dutch modified cell culture medium that was buffered with 20 mM HEPES and supplemented with 10% fetal calf serum (FCS), 1 mM pyruvate, 2 mM glutamine, 50 µg/ml gentamicin, and 2.5 µg/ml fungizone.…”

“…The design, construction, expression, and purification of fragment frg11 (amino acid residues 56–110) of the glycoprotein G of VHSV-07.71 strain (AJ233396) were performed as previously described ( 11 , 30 ). The sequence of the CyHV-3-U reference strain (NC009127) was used as the source of the ORF149 cDNA sequence to design fragment II (amino acid residues 42–159) as described before ( 6 ).…”

“…The sequence of the CyHV-3-U reference strain (NC009127) was used as the source of the ORF149 cDNA sequence to design fragment II (amino acid residues 42–159) as described before ( 6 ). The fragments frg11 VHSV and frgII CyHV-3 were selected because of their immunodominance among the VHSV ( 11 , 12 ) and CyHV-3 ( 6 ) epitopes, respectively. Alignment of the 119 amino acids of frgII CyHV-3 and the 56 amino acids of frg11 VHSV resulted in only a 12% match (15 conserved positions randomly distributed throughout both sequences) (Clone Manager vs9, Denver, CO, USA), minimizing possible cross-reactivities among them.…”

“…The nucleotide frg sequences were chemically synthesized (GeneArt, Regensburg, Germany) and subcloned into pRSETa expression vectors that added poly-histidine tag tails to their amino-terminal ends. The plasmids were then transfected into E. coli BL21, cultured, and induced with IPTG followed by the purification of extracts by Ni 2+ affinity chromatography as previously described ( 6 , 11 , 30 , 31 ). Purified frgs were kept frozen in 20 mM sodium acetate pH 4.5 to increase their solubility.…”

“…Natural non-specific IgM-binding levels were also reported when using CyHV-3 recombinant proteins for indirect ELISA ( 6 ). Furthermore, similar difficulties were found when analyzing sera from fish surviving novirhabdovirus infections, such as those caused by viral hemorrhagic septicemia virus (VHSV) or infectious hematopoietic necrosis virus (IHNV) using indirect ELISAs ( 7 – 10 ), including those employing recombinant fragments ( 11 , 12 ). Such difficulties were generally justified by the “sticky” nature of the IgM molecules to different surfaces and in different fish species ( 13 , 14 ), despite the addition of background reducing agents ( 6 , 11 – 13 , 15 , 16 ).…”