Production and characterization of monoclonal antibodies to a Brazilian bovine herpesvirus type 5

Abstract

Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein Cnegative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes.

Ruminant alphaherpesviruses related to bovine herpesvirus 1

“…A quantitative real time PCR is available for the detection and quantitation of CpHV-1 DNA in goats 5 . There also exists monoclonal antibodies that can distinguish between BoHV-1 and BoHV-5 by immunofluorescence, immunoprecipitation or western blot [45,76,89]. More recently, an immunofluorescence assay able to differentiate the five related herpesviruses has been developed.…”

Identificação e diferenciação de herpesvírus bovino tipos 1 e 5 isolados de amostras clínicas no Centro-Sul do Brasil, Argentina e Uruguai (1987-2006)

“…Entretanto, as técnicas utilizadas no presente estudo -PCR e seqüenciamento do amplicon -identificaram esse isolado inequivocamente como BoHV-1. O AcM utilizado por Oldoni et al (2004) possivelmente é direcionado para um epitopo da gC que é conservado entre isolados de BoHV-5 e que apresenta alguma similaridade em determinadas amostras de BoHV-1, podendo assim apresentar reatividade com esses vírus.…”

Histopathological, immunohistochemical, and molecular study of BHV-5 infection in the central nervous system of experimentally infected calves

“…Non-specific binding was blocked with 3% skim milk in phosphate-buffered saline for 1 h, and endogenous biotin was blocked as recommended by the manufacturer 6 . The 2F9 monoclonal antibody against BHV-5gC protein (Oldoni et al 2004) (diluted 1:200) was added to the tissue sections, which were incubated for 2 h at 37°C. A biotinylated goat anti-mouse antibody 7 was used as the secondary antibody.…”

“…IHC using the gC-specific 2F9 monoclonal antibody (Vogel et al 2003, Oldoni et al 2004, Hübner et al 2005) is highly specific as the antibody reacts only to the targeted protein and demonstrates no immunostaining in uninfected calf brains. In a previous study (Meyer et al 2001) 7 of 8 experimentally infected calves presented with neurological signs and similar histopathological changes, but only 5 calves were IHC positive.…”

“…A definitive diagnosis can be made via virus isolation from cell cultures combined with other techniques such as immunoperoxidase staining (Oldoni et al 2004), restriction endonuclease digestion (Meyer et al 2001), or polymerase chain reaction (PCR) (Sá E Silva et al 2007). However, virus isolation is not always possible due to confounding factors, such as the distance from the farm to the laboratories, the time of death, and the sample collection and isolation procedures (Franco e Roehe 2007).…”