Antigenic and Biological Characterization of ORF2–6 Variants at Early Times Following PRRSV Infection

Abstract

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) challenges efforts to develop effective and broadly acting vaccines. Although genetic variation in PRRSV has been extensively documented, the effects of this variation on virus phenotype are less well understood. In the present study, PRRSV open reading frame (ORF)2–6 variants predominant during the first six weeks following experimental infection were characterized for antigenic and replication phenotype. There was limited genetic variation during these early times after infection; however, distinct ORF2–6 haplotypes that differed from the NVSL97-7895 inoculum were identified in each of the five pigs examined. Chimeric viruses containing all or part of predominant ORF2–6 haplotypes were constructed and tested in virus neutralization and in vitro replication assays. In two pigs, genetic variation in ORF2–6 resulted in increased resistance to neutralization by autologous sera. Mapping studies indicated that variation in either ORF2–4 or ORF5–6 could confer increased neutralization resistance, but there was no single amino acid substitution that was predictive of neutralization phenotype. Detailed analyses of the early steps in PRRSV replication in the presence and absence of neutralizing antibody revealed both significant inhibition of virion attachment and, independently, a significant delay in the appearance of newly synthesized viral RNA. In all pigs, genetic variation in ORF2–6 also resulted in significant reduction in infectivity on MARC-145 cells, suggesting variation in ORF2–6 may also be important for virus replication in vivo. Together, these data reveal that variation appearing early after infection, though limited, alters important virus phenotypes and contributes to antigenic and biologic diversity of PRRSV.

Identification and characterization of small molecule inhibitors of porcine reproductive and respiratory syndrome virus

“…Compounds were assayed for anti-PRRSV activity in MARC-145 cells using a focusreduction assay as previously described (Wu et al, 2011;Evans et al, 2017). For most assays, 10 g of each compound, or DMSO diluent control, were incubated with varying amounts of PRRSV NVSL97-7895 for 1 hr at 37°C and inoculated in duplicate onto MARC-145 cells seeded the previous day at 3x10 5 cells/well in a 12-well plate.…”

Glycoprotein 3 of porcine reproductive and respiratory syndrome virus exhibits an unusual hairpin-like membrane topology

“…A multitude of linear antigenic regions were identified in GP3 proteins from both PRRSV-1 and PRSSV-2 strains, but antibodies directed against most epitopes do not neutralize viral infectivity (19–25). However, it was recently reported that PRRSV viruses which escaped from the antibody response in experimentally infected pigs contain mutations not only in its main glycoprotein GP5, but also in GP3 (26, 27). In transfected and infected cells GP3 is retained in the endoplasmic reticulum (ER), the viral budding site.…”

Leveraging genetic time series data to improve detection of natural selection

“…Nevertheless, there is indirect evidence of selection, particularly in the ORF5-encoded envelope protein GP5, which experiences rapid evolution in the ectodomain (Meng et al, 1995). A good review of recent evidence for selection in PRRSV can be found in Evans et al (2017).…”

“…2.1), but we analyze two small sequencing datasets obtained from the blood samples of a small number of Large White and Landrace commercial crossbred pigs. One study, the "virus study", isolated viral RNA from seven pigs in order to understand evolution of the virus during early infection (Evans et al, 2017). The other study, the "host study", isolated mRNA from 16 pigs in order to understand host gene expression changes in response to infection (Schroyen et al, 2016).…”

“…Two maintained high levels of viremia throughout 35 dpi (prolonged), and three initially cleared the virus only to experience rebound viremia by 41 dpi (rebound). Viral RNA was isolated from the NVSL97-7895-derived inoculum, day 7 sera from all pigs, and late day sera from prolonged and rebound pigs as described in Evans et al (2017). Briefly, viral RNA was isolated from sera, reverse transcribed to cDNA, and then ORF2-6 and nsp2 were amplified using PRRSV-specific primers.…”