Chelonid herpesvirus 5 virus isolation data

Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with Chelonid herpesvirus 5 (ChHV5) that has historically been refractory to growth in tissue culture. Here, we show for the first time de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative for active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included 1) either in-vitro culturing of ChHV5-positive tumor biopsies or organotypic cultures consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with ChHV5-negative or -positive turtle keratinocytes and 2) keratinocyte maturation, which occurred naturally in explanted biopsies or was induced by bringing the raft cultures to the air-media interface. These findings were confirmed by detailed electron microscopic studies revealing intranuclear capsid factories, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegumentation, and envelopment to complete de novo virus production. Ongoing membrane synthesis was also observed as a sign for active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayered cell cultures where most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch’s postulates for FP. Moreover, the principles described here may serve as model to culture other viruses that are resistant to replication in cell culture.