Development of an AGID based on baculovirus expressed Pseudorabies virus glycoprotein B

Abstract

Event Abstract Back to Event Development of an AGID based on baculovirus expressed Pseudorabies virus glycoprotein B MARIA S. SERENA1, 2, 3, GERMAN E. METZ2, 3, CARLOS J. PANEI1, 2, 3, ALEJANDRA E. LARSEN1, CAROLINA ASPITIA4, MARTIN PALACIOS4, CECILIA M. GALOSI2, 5, EDUARDO C. MORTOLA1* and MARIA G. ECHEVERRIA2, 3 1 LABORATORIO DE INMUNOLOGIA VETERINARIA, FACULTAD DE VETERINARIA - UNLP, Argentina 2 LABORATORIO DE VIROLOGIA, FACULTAD DE CIENCIAS VETERINARIAS - UNLP, Argentina 3 CONICET, Argentina 4 BECARIO PIITAP - UNIVERSIDAD NACIONAL DE LA PLATA, Argentina 5 CIC, Argentina Introduction The pseudorabies virus (PrV) is a member of the genus Varicellovirus, subfamily Alphaherpesvirinae and family Herpesviridae, characterized as the etiologic agent of Aujeszky’s disease in swine. Aujeszky’s disease is a one of the most infectious diseases affecting the swine industry which causes central nervous system symptoms and is characterized by an acute and often fatal infection in piglets, while in older pigs it causes disorders such as encephalitis, pneumonia and abortion. Economically, the greatest cost of the disease is associated with the acute phase of the infection, in which the abortion rate and neonatal mortality are quite high. The glycoprotein B, a major constituent of the viral envelope, exists has a disulphide-linked complex consisting of three glycoproteins: gBa, gBb and gBc. The primary translation product consists of 913aa, while the mature form of the glycoprotein after remove the signal sequence has 855aa. The fully glycosylated (gBa) is a 120kDa polypeptide that is cleaved between Arg444 and Ser445 by a cellular protease to yield two subunits, gBb (68kDa) and gBc (55kDa). The gB is indispensable for virus replication, plays an essential role in membrane fusion events during virus infection and cell-to-cell spread of virus and belongs to the class of glycoproteins that are most conserved among herpesviruses. A variety of ELISA tests have been developed for diagnosis of PrV using viral glycoproteins as antigen but any of this proteins were use in agar gel immunodiffusion test (AGID). Serological tests such as virus neutralization (VN) and ELISA require special facilities or commercial equipment, in contrast, the AGID which was designed for other virus animal’s as an effective tool for the detection of specific antibodies, is a fast technique that does not require special conditions and can be performed at any veterinary diagnostic laboratory. The baculovirus expression system has been used to produce large quantities of antigenically active and processed freeing glycoproteins. The BaculoDirect Expression System facilitates direct transfer of the gene of interest into the baculovirus genome in vitro without the need for additional cloning or recombination in bacterial or insect cells. The resulting recombinant baculovirus DNA is transfected directly into insect cells to generate recombinant virus and to screen for expression. The backbone contains strong polyhedrin promoter for high protein expression and a C-terminal or N-terminal 6xHis and V5 tag for detection and purification. This report describes the development of a simple AGID test based on the gB of PrV produced in BaculoDirect Expression System to examine a porcine serum samples with a sensitivity and specificity comparable with VN test (gold standard test). Materials and methods For recombinant gB production, Continuous Porcine Kidney cell line (CPK) was infected with the CL-15 PrV strain. Infected cells were collected, treated with proteinase K and total DNA was extracted with commercial kit (Promega). The gB gene was amplified by polymerase chain reaction (PCR) using a designed pair of primers. Engineered modifications were made to the gene protein that included deletion of two different regions, the signal peptide and the transmembrane domain. A recombinant baculovirus encoding CL-15 PrV gB was isolated in Sf9 cells using a consensus pENTR™/D-TOPO clone and the BaculoDirect™ C-Term Baculovirus Expression System (Life Technologies) according to the manufacturer’s instructions. Recombinant baculovirus clones were plaque-purified once in Sf9 cells, tested for gB expression by immunoblotting, and a clone expressing the full-length membrane bound version of the gB protein with C-terminal 6xHis and V5 epitope tags was identified. SDS-PAGE and immunoblotting analysis were performed using infected High Five™ cell lysates and supernatants at three days post-infection with a mouse anti-HisTag monoclonal antibody (GEHealthtcare) and a swine polyclonal PrV antibody as the primary antibody and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse antibody or rabbit anti-swine antibody (Sigma) as the secondary antibody. The supernatant culture contained gB recombinant protein (gBa-rec) was concentrated by 0.2µ dialysis membrane and its concentration was determinate by the standard method of Bradford. The AGID test was performed using 1% agar granulated in Tris buffer 0.2M and NaCl 0.85%, pH 7.2. The test was carried out in Petri dish using a punch of six wells of 4mm in diameter and incubated at room temperature in a humid chamber for 48hs. Different concentration of protein and dilutions of PrV positive serum samples were analyzed for determine the optimal condition of assay. A panel of 297 swine serum samples previously analyzed by VN test was obtained from the serum bank of the Virology Laboratory of the Veterinary Faculty of La Plata City (Buenos Aires, Argentina) and used for AGID test with the gBa-rec protein as the antigen. The Kappa index, sensitivity (SS) and specificity (SP) values were determined. Results The expression of recombinant protein (gBa-rec) in the supernatant of the insect cells culture was confirmed by SDS-PAGE. The analysis revealed the presence of a unique band of approximately 120 kD, that belongs to the predicted molecular weight of the gB protein. The antigenicity was confirmed by immunoblotting with swine polyclonal PrV and mouse anti-HisTag monoclonal antibodies. The concentration of protein in the stock solution was 1000g/ml. In the AGID test, clean precipitation lines were obtained with the recombinant protein antigen at 500g/ml, and undiluted PrV positive serum samples. No positive reactions were observed with PrV negative control sera. When we compared the results obtained using our in house AGID test to those obtained with a VN test, 170 out of 297 pig serum samples were positive in both assays, nine serum samples that were positive by neutralization scored negative with AGID, and four samples that were negative by neutralization scored positive with AGID. The Kappa coefficient (0.90, CI 95% 0.86–0.95), SS (94.97%) and SP (96.61%) values estimated were satisfactory and indicate substantial agreement between the two methods compared. Discussion Diagnosis of PrV is based on virus isolation, histopathology, immunohistochemical staining and antibody detection by VN and ELISA. All of them require special laboratory facilities or expensive commercial equipment. In contrast, the AGID test is very easy to perform, does not need expensive equipment and it is read with the naked eye, could be performed in any laboratory with minimal equipment and low cost. A variety of ELISA tests have been developed using recombinant viral glycoproteins as antigen but the use of AGID technique in the diagnosis of PrV has not been reported and there are not commercial kits available. The gB protein is one important protein that induce a high immunity response against PrV infection and belongs to the class of glycoproteins that are most conserved among herpesviruses. The eradication programs are based on the use of marked vaccines that lack the nonessential glycoprotein E (gE). It enables the detection of wild type PrV-infected pigs in vaccinated populations by using serological assays that detected antibodies to gE. When vaccination is stopped the pig population will be fully susceptible for new PrV infections, not only gE-positive PrV strains will threaten the PrV free status but also gE-negative derivatives from vaccine strains that are still circulating in the region. Thus, is necessary a sensitive and specific test in order to detect vaccinated as well as wild type virus infected pigs. In this research the expression of PrV recombinant gB and its use as antigen to develop a AGID test was achieved. The advantage of recombinant viral antigens is that they are available in unlimited quantities, the production and quality control processes is simple, no nucleic acids or other viral or external proteins are included, therefore is less toxic and feasible even if virus cannot be cultivated. The engineered modifications that were made to the gB gene protein allows recovering the recombinant protein from the supernatant cell culture with the subsequent advantages in production and purification of the protein. We showed here that the AGID is a simple and reliable serological assay based on a homogeneous and permanent source of antigen that guarantees its use and availability for veterinary PrV diagnosis. Virus neutralization has been recognized as the reference method for serology and is considered as gold standard test for PrV infection, although in general it has been widely replaced by ELISA assay because of its suitability for large-scale testing. The VN is not only very expensive but also time consuming and both require expensive equipment and trained technicians. These conditions prevent to carry out this technique in routine veterinary laboratory. With our preliminary results showed here, we can conclude that the AGID test it seems to be a valuable alternative tool because it identifies positive animals with a specificity and sensitivity similar to those of VN. However, further immunoserologic analysis are necessary to evaluate the accuracy of our test in order to complete the validation process. Keywords: pseudorabies virus, AGID, recombinant antigens, baculovirus, Serology test Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Veterinary and Comparative Immunology Citation: SERENA MS, METZ GE, PANEI CJ, LARSEN AE, ASPITIA C, PALACIOS M, GALOSI CM, MORTOLA EC and ECHEVERRIA MG (2015). Development of an AGID based on baculovirus expressed Pseudorabies virus glycoprotein B. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00236 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 Apr 2015; Published Online: 14 Sep 2015. * Correspondence: Dr. EDUARDO C MORTOLA, LABORATORIO DE INMUNOLOGIA VETERINARIA, FACULTAD DE VETERINARIA - UNLP, LA PLATA, Argentina, ecmortol@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers MARIA S SERENA GERMAN E METZ CARLOS J PANEI ALEJANDRA E LARSEN CAROLINA ASPITIA MARTIN PALACIOS CECILIA M GALOSI EDUARDO C MORTOLA MARIA G ECHEVERRIA Google MARIA S SERENA GERMAN E METZ CARLOS J PANEI ALEJANDRA E LARSEN CAROLINA ASPITIA MARTIN PALACIOS CECILIA M GALOSI EDUARDO C MORTOLA MARIA G ECHEVERRIA Google Scholar MARIA S SERENA GERMAN E METZ CARLOS J PANEI ALEJANDRA E LARSEN CAROLINA ASPITIA MARTIN PALACIOS CECILIA M GALOSI EDUARDO C MORTOLA MARIA G ECHEVERRIA PubMed MARIA S SERENA GERMAN E METZ CARLOS J PANEI ALEJANDRA E LARSEN CAROLINA ASPITIA MARTIN PALACIOS CECILIA M GALOSI EDUARDO C MORTOLA MARIA G ECHEVERRIA Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.