Cross-reactive antibody responses to nsp1 and nsp2 of Porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural proteins (nsps) play a key role in processing and maturation of the repertoire of structural and nsps of the virion, but little is known about the anti-nsp immune response. Here, it was hypothesized that pronounced antibody responses are generated to PRRSV nsp1 and nsp2, as they are present in infected cells and cytolytic infection releases viral proteins into interstitial spaces. Accordingly, nsp1 and nsp2 were cloned and expressed, and antibody responses in the sera of infected and vaccinated pigs were determined. Pigs mounted significant cross-reactive antibody responses that appeared equivalent to or greater than the response to nucleocapsid (N). Antibody reactivity to nsp1 and N was highly dependent on refolding of denatured proteins, suggesting that the porcine antibody response is directed primarily to conformational epitopes. The proteins reacted with sera from pigs infected with other PRRSV strains, indicating that multiple epitopes are conserved. Antibody responses to nsp1 and nsp2 were much higher than those to nsp4, which is encoded on the same RNA molecule and is equivalent in predicted antigenicity. These findings suggest either that nsp1 and nsp2 are highly immunogenic or that they are expressed at higher levels than nsp4 in PRRSV-infected cells, or both. Strong antibody responses to nsp1 and nsp2 may benefit the host by limiting potentially pathological consequences of viral protease activities encoded in these proteins that are released from dying cells. The identification of strain-specific antibody responses to a highly variable region of nsp2 may also provide the basis for immunoassays that differentiate serological responses of vaccines from field isolates. INTRODUCTIONA new viral disease of pigs, typified by late-term abortions and stillbirths in sows and interstitial pneumonia in nursery pigs, was detected in North America in 1987(Hill, 1990Keffaber, 1989) and in Europe in 1990(Paton et al., 1991, and then characterized as porcine reproductive and respiratory syndrome (PRRS) caused by Porcine reproductive and respiratory syndrome virus (PRRSV) Wensvoort et al., 1991). The causative agent is a small, enveloped, positive-stranded RNA virus. It is a member of the family Arteriviridae, which includes Equine arteritis virus (den Boon et al., 1991;Meulenberg et al., 1993), Lactate dehydrogenase-elevating virus of mice (Plagemann & Moennig, 1992) and Simian hemorrhagic fever virus (Zeng et al., 1995), in the order Nidovirales (Cavanagh, 1997). PRRSV predominantly infects macrophages and establishes a persistent infection in resident macrophages of numerous lymphoid tissues (Lawson et al., 1997).A complex immunological interaction exists between PRRSV and pigs that involves both induction and subversion of host defences . Exposure to PRRSV induces an immune response that protects pigs against re-exposure to the same virus. However, pigs exposed to PRRSV also demonstrate prolonged viraemia and persistent infection, may conti...

Antigen-Specific B-Cell Responses to Porcine Reproductive and Respiratory Syndrome Virus Infection

“…The GP5 endodomain contains the 72 carboxyl-terminal amino acids within the viral envelope, is highly conserved, and is recognized by serum from pigs infected with a wide variety of PRRSV isolates (unpublished data). DNA sequences were subcloned into expression plasmid pET24b as fusion proteins containing an amino-terminal myc tag and a carboxyl-terminal 6ϫ histidine tag and were expressed in BL21(DE3)-RP cells (Stratagene, La Jolla, CA) (4,31). Proteins were expressed and purified using a modification of the Qiagen Ni-nitrilotriacetic acid agarose affinity column purification method for denatured protein isolation.…”

“…Recombinant PRRSV nucleocapsid (N), envelope glycoprotein 5 ectodomain (GP5 5Ј total), envelope glycoprotein 5 endodomain (GP5 3Ј), and nonstructural protein 2 (nsp2) proteins and polypeptides were prepared by PCR amplification and cloning from cDNA of PRRSV strain VR2332 (4,31). The GP5 ectodomain contains 52 amino acids external to the viral envelope, is highly polymorphic, and includes the putative primary neutralization epitope (4,21,53).…”

Presence of free haptoglobin alpha 1S-subunit in acute porcine reproductive and respiratory syndrome virus infection

“…Inclusion bodies were resuspended and homogenized in 50 ml solution containing 0.1 % Triton X-100, 10 mM b-mercaptoethanol and 1 mM PMSF. Expressed rHp-a1S protein was purified by using a Qiagen Ni-NTA agarose-affinity resin as described previously, except that urea was replaced with 6 M guanidine (Johnson et al, 2007). Purity and molecular mass of purified protein was confirmed by SDS-PAGE and MALDI-TOF.…”

“…Amplification was performed in a GeneAmp PCR system 2400 (Perkin Elmer) with one cycle of 95 uC for 10 min, 35 cycles at 94 uC for 45 s, 55 uC for 45 s and 72 uC for 45 s and then 72 uC for 10 min. Gel-purified 270 bp products were sequenced (Advanced Genetics Analysis Center, University of Minnesota, USA) and cloned into a modified pET 24b (Novagen) vector (Johnson et al, 2007), which was restriction ). Colonies were initially tested by small-scale test expression by using Ni-NTA spin column (Qiagen) according to the manufacturer's directions.…”

“…Colonies were initially tested by small-scale test expression by using Ni-NTA spin column (Qiagen) according to the manufacturer's directions. Large-scale protein purification was performed as described previously (Johnson et al, 2007) with the following modifications. IPTG-(1 mM) induced bacterial cells were pelleted by centrifugation at 9000 r.p.m.…”