title: ORF1 but not ORF2 dependent differences are important for in vitro replication of PCV2 in porcine alveolar macrophages singularly or coinfected with PRRSV.
abstract: The objective of this study was to investigate cytokine expression and in vitro replication of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in pulmonary alveolar macrophages (PAMs) emphasizing PCV2 open-reading frame (ORF) origin (PCV2a or PCV2b) and PRRSV strain. Chimeric PCV2 viruses composed of different combinations of ORF1 and ORF2 of PCV2a or PCV2b (chimera PCV2a-2b and chimera PCV2b-2a) were constructed and five different PRRSV isolates were utilized: Type 1 (SD 01-08) or type 2 (NC16845b, VR-2332, MN-184, JA-142). PAMs were infected singularly or with combinations of PCV2b, PCV2a, chimera PCV2a-2b, and chimera PCV2b-2a, and one of the five PRRSV isolates. Real-time PCR was used to test PAMs (PCV2 mRNA) and supernatants (PRRSV RNA, PCV2 DNA, PCV2 mRNA) harvested at 24, 48, 72 and 96h post inoculation (hpi). Levels of IFN-γ, TNF-α and IL-10 were determined by quantitative ELISAs. PCV2 replication in PAMs was limited to groups inoculated with PCV2 strains containing ORF1 of PCV2a (PCV2a, chimera PCV2a-2b). Furthermore, in supernatants, PCV2 mRNA was only detected in groups coinfected with PRRSV regardless of strain at 48hpi supporting an enhancing effect of PRRSV on PCV2 infection. Changes in cytokine levels were minimal and associated with PRRSV strain for TNF-α. In summary, in vitro differences in PCV2 replication in PAMs inoculated with different PCV2-PRRSV combinations were independent of PCV2 ORF2 origin with minimal effects of concurrent PRRSV infection perhaps indicating that PCV2-specific changes in ORF1 may be more important than those in ORF2.
authors.affiliation: Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250, USA.
authors.forename: Avanti
authors.initials: A
authors.lastname: Sinha
authors.forename: Kathy
authors.initials: K
authors.lastname: Lin
authors.forename: Michelle
authors.initials: M
authors.lastname: Hemann
authors.forename: Huigang
authors.initials: H
authors.lastname: Shen
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Beach
authors.forename: Carmen
authors.initials: C
authors.lastname: Ledesma
authors.forename: Xiang-Jin
authors.initials: XJ
authors.lastname: Meng
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Halbur
authors.forename: Tanja
authors.initials: T
authors.lastname: Opriessnig
mesh_terms: D000818:Animals; D002470:Cell Survival; D018173:Circoviridae Infections / Q000821:virology*; D017925:Circovirus / Q000502:physiology*; D016207:Cytokines / Q000032:analysis / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D016676:Macrophages, Alveolar / Q000166:cytology / Q000276:immunology / Q000821:virology*; D016366:Open Reading Frames; D019316:Porcine respiratory and reproductive syndrome virus / Q000502:physiology*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D034421:Sus scrofa; D013552:Swine
chemical_list: D016207:Cytokines; D012367:RNA, Viral