title: Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection.
abstract: BACKGROUND
  Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.
  
  
  RESULTS
  Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20-22 h p.i.), but the logarithmic infection phase (days 2-3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-gamma (IFN-gamma), as well as the more-potent experimental antiviral agent AK-2.
  
  
  CONCLUSIONS
  The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.
authors.affiliation: Division of Basic Biomedical Science, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA. bcafruny@usd.edu
authors.forename: William A
authors.initials: WA
authors.lastname: Cafruny
authors.forename: Richard G
authors.initials: RG
authors.lastname: Duman
authors.forename: Grace H W
authors.initials: GH
authors.lastname: Wong
authors.forename: Suleman
authors.initials: S
authors.lastname: Said
authors.forename: Pam
authors.initials: P
authors.lastname: Ward-Demo
authors.forename: Raymond R R
authors.initials: RR
authors.lastname: Rowland
authors.forename: Eric A
authors.initials: EA
authors.lastname: Nelson
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007371:Interferon-gamma / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000821:virology*; D018613:Microscopy, Confocal; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019316:Porcine respiratory and reproductive syndrome virus / Q000187:drug effects / Q000472:pathogenicity* / Q000502:physiology*; D013552:Swine / Q000821:virology*; D050257:Tubulin Modulators / Q000494:pharmacology; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D019384:Nucleic Acid Synthesis Inhibitors; D050257:Tubulin Modulators; D015638:Cytochalasin D; D007371:Interferon-gamma; D003078:Colchicine