title: Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay.
abstract: Pseudorabies virus (PrV) causes Aujeszky's disease (AD), which affects mainly swine, but also cattle, sheep, and wild animals, resulting in substantial economic losses due to animal mortality and lost productivity worldwide. To combat PrV, eradication programs using PrV strains lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable, easy-to-use, and sensitive tests that can detect PrV infection in pigs infected with either wild-type virus or vaccine strain (gE-deleted) virus. To meet this demand, we used the baculovirus-insect cell system to produce recombinant glycoprotein B (gB) as antigen for an immune assay. The high GC-content (70% average) of the gB gene from the Argentinian PrV CL15 strain necessitated the use of betaine as a PCR enhancer to amplify the extracellular domain. Recombinant gB was expressed at high levels and reacted strongly with sera from PrV infected pigs. We used the recombinant gB to develop an agar gel immunodiffusion (AGID) test for detection of PrV antibodies. Compared to the gold standard virus neutralization (VN) assay, the AGID sensitivity and specificity were 95% and 96.6% respectively. Thus, recombinant gB produced in the baculovirus-insect cell system is a viable source of antigen for the detection of PrV antibodies in AGID tests. Considering its relatively lower cost, simplicity of use and result interpretation, our AGID is a valuable alternative tool to the VN assay.
authors.affiliation: Department of Virology, Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina; CONICET (Scientific Research Council), Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina.
authors.forename: María Soledad
authors.initials: MS
authors.lastname: Serena
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Geisler
authors.affiliation: Department of Virology, Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina; CONICET (Scientific Research Council), Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina.
authors.forename: Germán Ernesto
authors.initials: GE
authors.lastname: Metz
authors.affiliation: Department of Immunology, Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina.
authors.forename: Eduardo Carlos
authors.initials: EC
authors.lastname: Mórtola
authors.affiliation: Department of Virology, Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina; CONICET (Scientific Research Council), Faculty of Veterinary Sciences, National University of La Plata, La Plata, Argentina. Electronic address: mariagabrielaecheverria@yahoo.com.ar.
authors.forename: María Gabriela
authors.initials: MG
authors.lastname: Echeverría
mesh_terms: D000818:Animals; D000956:Antigens, Viral; D016367:Baculoviridae; D005779:Immunodiffusion / Q000379:methods; D009500:Neutralization Tests / Q000379:methods; D011557:Pseudorabies / Q000175:diagnosis / Q000821:virology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins
keywords: Agar Gel Immunodiffusion; Aujeszky’s disease; Baculovirus insect cell system; Glycoprotein B; Pseudorabies