Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae within the order Nidovirales which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus. The viral genome is a linear, positive-stranded RNA molecule of approximately 15.0 kb in length and possesses a cap structure at the 5Ј end and a poly(A) tail at the 3Ј end. Eight open reading frames (ORFs) are in the viral genome (9, 34). The first two open reading frames (ORF1a and ORF1ab) encode viral nonstructural (NS) polyproteins that are involved in polyprotein processing and genome transcription and replication (47). The viral structural proteins, encoded in ORFs 2 to 7, are expressed from six subgenomic capped and polyadenylated mRNAs that are synthesized as a 3Ј-coterminal nested set of mRNAs with a common leader sequence at the 5Ј end.The major viral envelope protein is glycoprotein 5 (GP5), which is encoded in ORF5 of the viral genome (29,35,36). GP5 is a glycosylated transmembrane protein of approximately 25 kDa (10,16,35). It has a putative N-terminal signal peptide and possesses three potential N-linked glycosylation sites which are located in a small ectodomain comprising the first 40 residues of the mature protein (28,35). In EAV and LDV, the major envelope glycoprotein forms a disulfide-l...
Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1␣ and NSP1 (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1 was found to inhibit both IFN regulatory factor 3 (IRF3)-and NF-B-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1. Moreover, when tested in a porcine myelomonocytic cell line, NSP1 inhibited Sendai virus-mediated activation of porcine IFN- promoter activity. We propose that this NSP1-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.
The field isolate of porcine epidemic diarrhea virus (PEDV) was serially passaged in Vero cells. The cell passaged PEDV, designated KPEDV-9, was tested for its pathogenicity in the neonatal pigs, immunogenicity and safety in the pregnant sows. The result indicated that KPEDV-9 at the 93rd passage revealed reduced pathogenicity in the neonatal pigs. Pregnant sows inoculated with the attenuated virus showed increased immune responses by ELISA. In addition, delivered piglets were protected from challenge of wild type PEDV. The safety test in pregnant sows indicated that all inoculated animals farrowed the average numbers of litters of piglets. The results of this study supported that the attenuated virus derived from serial passage could be applied as vaccine for protecting suckling piglets against PEDV infection.
Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, in two of its envelope glycoproteins, one in GP3 (position 131) and the other in GP5 (position 51). To determine the influence of these missing N-glycosylation sites on the distinct neutralization phenotype of PRRSV-01, a chimeric virus (FL01) was generated by replacing the structural genes of type II PRRSV strain FL12 cDNA infectious clone with those from PRRSV-01. N-glycosylation sites were reintroduced into GP3 and GP5 of FL01, separately or in combination, by site-directed mutagenesis. Reintroduction of the N-glycosylation site in either GP3 or GP5 allowed recovery of in vivo and in vitro glycan shielding capacity, with an additive effect when these sites were reintroduced into both glycoproteins simultaneously. Although the loss of these glycosylation sites has seemingly occurred naturally (presumably by passage through cell cultures), PRRSV-01 virus quickly regains these glycosylation sites through replication in vivo, suggesting that a strong selective pressure is exerted at these sites. Collectively, our data demonstrate the involvement of an N-glycan moiety located in GP3 in glycan shield interference.
The role of N-glycosylation of the three minor envelope glycoproteins (GP2, GP3, and GP4) of porcine reproductive and respiratory syndrome virus (PRRSV) on infectious virus production, interactions with the receptor CD163, and neutralizing antibody production in infected pigs was examined. By mutation of the glycosylation sites in these proteins, the studies show that glycan addition at N184 of GP2, N42, N50 and N131 of GP3 is necessary for infectious virus production. Although single-site mutants of GP4 led to infectious virus production, mutation of any two sites in GP4 was lethal. Furthermore, the glycosylation of GP2 and GP4 was important for efficient interaction with CD163. Unlike PRRSVs encoding hypoglycosylated form of GP5 that induced significantly higher levels of neutralizing antibodies in infected piglets, PRRSVs encoding hypoglycosylated forms of GP2, GP3 or GP4 did not. These studies reveal the importance of glycosylation of these minor GPs in the biology of PRRSV.
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