DISEASES OF AQUATIC ORGANISMS Dis Aquat Org Vol. 73: 89–101, 2006 Published December 14 INTRODUCTION Taura syndrome (TS) is an economically important disease listed by the World Organization for Animal Health (OIE 2006). Since its first recognition in Ecuador in 1992, TS has rapidly spread to cultured penaeid shrimp-farming regions in many countries of the Americas, Asia, and Africa and has continued to devastate the shrimp industry for the last decade (Lightner et al. 1995, Nielsen et al. 2005, Srisuvan et al. 2005, Tang & Lightner 2005). The causative agent of TS is Taura syndrome virus (TSV), a member of the family Dicistroviridae, which is a non-enveloped icosahedral virus with a diameter of 32 nm and a single-stranded, positive-sense RNA genome of 10 205 nucleotides (Bonami et al. 1997, Mari et al. 2002, Mayo 2005). The principal host of TSV is Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei; Lightner et al. 1995, Pérez-Farfante & Kensley 1997). The intracellular biogenesis of TSV remains largely uninvestigated, although the virus has been an intense subject of research for almost 15 yr. Ultrastructural pathogenesis of many viruses, e.g. human parecho- virus type 1, dengue virus, hepatitis C virus, foot-and- mouth disease virus (FMDV), and severe acute respira- tory syndrome-associated coronavirus (SARS-CoV), has been characterized using transmission electron microscopy (TEM), in situ hybridization (ISH), and immunoelectron microscopy (IEM; Grief et al. 1997, Gosert et al. 2003, Krogerus et al. 2003, Goldsmith et al. 2004, Monaghan et al. 2004). Additionally, our laboratory has previously developed an ISH protocol using a specific cDNA probe to follow the intracellular translocation of hepatopancreatic parvovirus (HPV) in penaeid shrimp (Pantoja & Lightner 2001). Infection by all single-stranded, positive-sense RNA viruses is believed to involve the intracellular re- arrangement of membranes in the cytosol (for reviews, © Inter-Research 2006 · www.int-res.com *Email: thinnarat@hotmail.com Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells Thinnarat Srisuvan 1, 2, * , Carlos R. Pantoja 2 , Rita M. Redman 2 , Donald V. Lightner 2 1 Department of Livestock Development, 69/1 Phayathai Road, Bangkok 10400, Thailand 2 Department of Veterinary Science and Microbiology, University of Arizona, 1117 E. Lowell, Tucson, Arizona 85721, USA ABSTRACT: Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and pro- liferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV. KEY WORDS: Taura syndrome virus · TSV · In situ hybridization · Transmission electron microscopy · Litopenaeus vannamei Resale or republication not permitted without written consent of the publisher