For many enveloped viruses, entry into a host cell is primarily through the binding of cellular receptors and subsequent endocytosis of the viral particle into the cells. The fusion of envelope with the endosomal membrane releases viral capsid into the cytosol of the infected cell (reviewed in reference 1). However, for some enveloped viruses, alternative pathways for cell-to-cell transmission have been described (reviewed in references 2 to 4). One emerging model proposes that some viruses can use long, filamentous intercellular connections (nanotubes) as a means to transport infectious viral materials to neighboring naive cells. Previously, intercellular nanotubes have been described as nanotubules, tunneling nanotubes, and bridging conduits (5-8; reviewed in reference 9). The fundamental feature of the intercellular nanotube is a long membrane-bound extension that connects two neighboring cells and can also link multiple cells together to form complex cellular networks (6). Nanotubes are 50 to 200 nm in diameter and can span several cell distances. These structures are primarily composed of filamentous actin (F-actin) and also contain myosin as a motor to drive the movement of organelles or other cargo into neighboring cells (6, 9). Intercellular nanotubes offer cellular communication over long distances, particularly for transporting relatively large cellular materials (10).In this study, we investigated whether porcine reproductive and respiratory syndrome virus (PRRSV) utilizes intercellular nanotubes as an alternative pathway to spread infection. PRRSV is an enveloped, positive-sense, single-stranded RNA virus. The viral genome is about 15 kb in length. The 5= two-thirds of the viral genome encodes two large replicase polyproteins, pp1a and pp1ab, which are proteolytically processed into at least 14 functional nonstructural proteins (nsp1 to nsp12, with nsp1 autocleaved into nsp1␣/nsp1 and nsp7 autocleaved into nsp7␣/ nsp7) (reviewed in reference 11). Recently, two novel proteins,