In Vitro Replication of Chelonid Herpesvirus 5 in Organotypic Skin Cultures from Hawaiian Green Turtles (Chelonia mydas)

Thierry M. Work, Julie Dagenais, Tina M. Weatherby, George H. Balazs, Mathias Ackermann

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 (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and 2) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies revealing intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegumentation, and envelopment to complete de novo virus production. 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 monolayer 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 conventional cell culture.Importance: A major challenge in virology is viruses that cannot be grown in the laboratory. One example is Chelonid herpesvirus 5 (ChHV5) associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of endangered marine turtles. Pathology shows that ChHV5 is shed in skin. Here, we show that ChHV5 will grow in vitro if we replicate the complex three- dimensional structure of turtle skin. Moreover, lytic virus growth requires a close interplay between fibroblasts and keratinocytes. Finally, morphogenesis of herpesviral growth in three dimensional cultures reveals a far richer, and likely more realistic, array of capsid morphologies than that encountered in traditional monolayer cell cultures. Our findings have application to other viruses including those of humans.