POLARIZED ENTRY AND RELEASE IN EPITHELIAL-CELLS OF BLACK CREEK CANAL VIRUS, A NEW-WORLD HANTAVIRUS

Black Creek Canal (BCC) virus is a newly identified hantavirus from Fl orida which is carried by the cotton rat (Sigmodon hispidus) and is as sociated with hantavirus pulmonary syndrome (HPS). We have investigate d the interaction of BCC virus with polarized epithelial cells to exam ine whether entry and release of this virus occur at specific plasma m embrane domains. The polarized Vero C1008 monkey kidney cell line was grown on permeable filters and infected with BCC virus either through the apical or basolateral surface. As shown by indirect immunofluoresc ence and radioimmunoprecipitation analysis, cells infected through the apical surface demonstrated a high level of susceptibility to BCC vir us infection. In contrast, Vero C1008 cells infected basolaterally exh ibited a barely detectable level of BCC virus-synthesized proteins. Ti tration of virus from apical and basolateral media of infected cells h as demonstrated that virus titers released from the apical surface are about 1,200-fold greater than the titer of virus released into the ba solateral media. The site of BCC virus release from polarized cells is , therefore, different from that previously described for release of o ther members of the family Bunyaviridae and may reflect one of the det erminants of hantavirus pathogenesis, In addition, we have shown that BCC viral glycoproteins are expressed at the plasma membrane on the ap ical surface of polarized cells, Electron microscopy studies of the in fected cells revealed evidence of BCC virus budding at the plasma memb rane. This strongly indicates that, in contrast to most other members of the Bunyaviridae, BCC virus is assembled at the plasma membrane. Si nce the same site of virus assembly was recently described for Sin Nom bre virus, it is likely that all of the new American hantaviruses asso ciated with HPS utilize this same type of virus maturation.