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

Author:

Work Thierry M.1,Dagenais Julie1,Weatherby Tina M.2,Balazs George H.3,Ackermann Mathias4

Affiliation:

1. U.S. Geological Survey, National Wildlife Health Center, Honolulu Field Station, Honolulu, Hawaii, USA

2. Biological EM Facility, University of Hawaii, Honolulu, Hawaii, USA

3. NOAA, National Marine Fisheries Service, Pacific Islands Fisheries Science Center, Honolulu, Hawaii, USA

4. Institute of Virology, University of Zurich, Zurich, Switzerland

Abstract

ABSTRACT Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with chelonid herpesvirus 5 (ChHV5), which 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 of active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included (i) either in vitro cultures of ChHV5-positive tumor biopsy specimens (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and (ii) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies that revealed intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegument formation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign of active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures, in which 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 a model for culture of other viruses that are resistant to replication in conventional cell culture. IMPORTANCE A major challenge in virology is the study of viruses that cannot be grown in the laboratory. One example is chelonid herpesvirus 5 (ChHV5), which is associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of endangered marine turtles. Pathological examination 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, the 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 applications to other viruses, including those of humans.

Funder

DOI | U.S. Geological Survey

Publisher

American Society for Microbiology

Subject

Virology,Insect Science,Immunology,Microbiology

Reference82 articles.

1. Pritchard PCH . 1996. Evolution, phylogeny, and current status, p 1–29. InLutzPLMusickJA (ed), The biology of sea turtles, vol 1. CRC Press, Boca Raton, FL.

2. Using Expert Opinion Surveys to Rank Threats to Endangered Species: A Case Study with Sea Turtles

3. Hargrove S Work T Brunson S Foley AM Balazs G . 2016. Proceedings of the 2015 international summit on fibropapillomatosis: global status trends and population impacts p 1–85. U.S. Department of Commerce Washington DC.

4. Fibropapillomatosis of marine turtles

5. Retrospective pathology survey of green turtles Chelonia mydas with fibropapillomatosis in the Hawaiian Islands, 1993-2003

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3