Seroneutralization of Porcine Reproductive and Respiratory Syndrome Virus Correlates with Antibody Response to the GP₅Major Envelope Glycoprotein

Abstract

Abstract. To determine the structural protein of the porcine reproductive and respiratory syndrome virus (PRRSV) involved in the production of neutralizing antibodies following clinical infection, correlation was studied between virus neutralization capability of convalescent pig sera and antibody response to the open reading frames (ORFs) 3-, 4-, 5-, and 7-encoded proteins GP 3 , GP 4 , GP 5 , and N, respectively. Individual virus genes were cloned into the pGEX-4T-1 vector, and the recombinant viral proteins were expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein. The resulting GST-ORF3, GST-ORF4, GST-ORF5, and GST-ORF7 recombinant fusion proteins were purified by electroelution and used as antigens for serologic testing by indirect enzyme-linked immunosorbent assay and western immunoblotting. The overall antibody (IgG and IgM) titers to PRRSV of pooled convalescent pig sera were first determined by indirect immunofluorescence, and then sera with specific IgG titers Ͼ1:1,024 were tested for their specific virus neutralization activity and reactivity to individual recombinant fusion proteins. Except for the early immune response (as revealed by the presence of specific IgM), neutralizing titers were correlated with anti-GP 5 titers but not with anti-GP 3 and anti-GP 4 titers. The correlation between virus neutralization and anti-GP 5 titers was significant (r ϭ 0.811, P Յ 0.001).Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pig pathogen structurally and genomically related to the equine arteritis virus (EAV), the murine lactate dehydrogenase-elevating virus, and the simian hemorrhagic fever virus. 20,28 These viruses have been recently classified in the genus Arterivirus, family Arteriviridae in the order Nidovirales, which also comprises members of the family Coronaviridae. 3 The genome of the PRRSV is a positive single-stranded polyadenylated RNA molecule approximately 15 kb in length containing 8 open reading frames (ORFs), which are transcribed in the infected cells as a nested set of subgenomic mRNAs. 8,20 The enveloped viral particles contain 3 major structural proteins, a glycosylated envelope protein of 25 kD (GP 5 ), an unglycosylated membrane (M) protein of 19 kD, and a nucleocapsid (N) protein of 14 kD, encoded by ORFs 5, 6, and 7, respectively. 16,17,21 The ORFs 2, 3, and 4 of the Lelystad virus, the European prototype of PRRSV, encode for putative membrane-associated glycoproteins, GP 2 , GP 3 , and GP 4 , respectively, with respective molecular masses of 30, 45, and 31 kD. 21 Kinetics of development of antiviral and neutralizing antibodies to PRRSV following clinical infection in pigs have been previously studied. 15,24 The deter- mination of the protein(s) responsible for the induction of neutralizing antibodies is a key element to consider for the development of an efficient recombinant vaccine. Several reports clearly established that the ectodomain of the G L glycoprotein of EAV, the counterpart of the GP 5 of PRRSV, co...

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP5 glycoprotein and improves immune response in challenged pigs

“…The VN antibodies response detected at 10 days post-challenge was the specific consequence of prior immunization with hAdV/synORF5 since normally, VN antibodies are detected after only 3 to 4 weeks in SPF pigs experimentally-or naturallyinfected with PRRSV [10,20]. The higher reactivity to GP5, as well as the higher VN activity of pigs sera that have been vaccinated either with hAdV/wtORF5 or hAdV/synORF5 is in agreement with previous findings indicating that GP5 is the primary structural glycoprotein of PRRSV involved in virus neutralization activity [6,7,25]. Even if high titers of VN antibodies appeared at day 10 post-challenge, the viremia could not be prevented (data not shown) as previously observed [10,26,27].…”

“…Circulating antibodies in PRRSV-infected pigs responsible for viral neutralization in cell cultures are mainly directed against GP5 [6]. Immunization of mice with Escherichia coli-expressed GST-ORF5 recombinant fusion protein, as well as with purified PRRSV, induced specific anti-GP5 neutralizing monoclonal antibodies [7].…”

The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain

“…PRRSV neutralization is correlated with antibodies directed against the GP 5 protein, both in vivo (Gonin et al, 1999;Kwang et al, 1999;Pirzadeh & Dea, 1998;Yoon et al, 1995) and in vitro (Pirzadeh & Dea, 1997;Weiland et al, 1999;Yang et al, 2000;Zhang et al, 1998). Monoclonal antibodies (mAbs) against the GP 4 protein have also been found to be IP: 52.247.194.177 On: Thu, 21 Mar 2019 22:53:41 neutralizing (Meulenberg et al, 1997), although mAbs against the GP 5 protein appeared to be much more effective (Weiland et al, 1999).…”

Antibody Repertoire Development in Fetal and Neonatal Piglets: XIX. Undiversified B Cells with Hydrophobic HCDR3s Preferentially Proliferate in the Porcine Reproductive and Respiratory Syndrome

“…IL-10 is up-regulated (37-39), but the appearance of virus-neutralizing (VN) Abs and CD4 ϩ antiviral responses is delayed until 28 dpi (40). When present, VN is often mediated by Abs to open reading frame (ORF)5 glycoprotein (41,42) and to a lesser extent epitopes on the M protein (membrane protein, ORF6; 43), even through robust responses to this highly immunogenic nucleocapsid protein (N protein, ORF7) are seen (44,45). Because PRRSV causes polyclonal B cell activation, the early non-VN Abs in PRRS may represent a Tindependent preadaptive response triggered through pathogen-associated molecular patterns.…”