Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells

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 proliferation 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 publisherDis Aquat Org 73: [89][90][91][92][93][94][95][96][97][98][99][100][101] 2006 see Mackenzie 2005, Novoa et al. 2005). The host cell membranes function as the replication site for the synthesis of the nascent viral genomes. For instance, in many viruses such as mouse hepatitis virus, rubella virus, and Semliki Forest virus, these consist of generation and proliferation of endoplasmic reticulum (ER) and membrane vesicles that accumulate in the perinuclear region of infected cells (Magliano et al. 1998, Kujala et al. 2001, Gosert et al. 2002. Characterization of the viral replication complexes is an important aspect in virology and cell biology. In the present paper, the ultrastructure of the replication site in cells infected with TSV was visualized by TEM and ISH. MATERIALS AND METHODSShrimp specimens. Litopenaeus vannamei were collected from affected farms in Ecuador, Peru, and Colombia, fixed in 6% glutaraldehyde in phosphate buffer, and processed for TEM as previously described by Bonami et al. (1992). Histological examinations revealed that they exhibited characteristic lesions of TSV infection as previously reported by Lightner et al. (1995) (not shown). These shrimp were not tested for TSV by ISH.Specimens used for ISH were embedded in paraffin and hydrophilic Unicryl resin (British Bio-Cell International). For paraffin embedding, the specimen was a Litopenaeus vannamei (wt = 1 g) derived from a specific-pathogen-free (SPF) Kona stock (Moss et al. 2005), obtained from the Oceanic Institute...

Viral disease emergence in shrimp aquaculture: origins, impact and the effectiveness of health management strategies

“…Clinical signs and mortalities have been reported following natural or experimental infection of P. stylirostris, P. schmitti, P. setiferus, P. monodon and M. ensis. Susceptibility to disease may also vary in selected lines of shrimp and for different strains of TSV (Brock 1997;Overstreet et al 1997;Erickson et al 2002;Chang et al 2004;Nielsen et al 2005;Srisuvan et al 2005Srisuvan et al , 2006a. Selected lines of TSV-resistant P. vannamei have been developed (Argue et al 2002;White et al 2002;Xu et al 2003;Srisuvan et al 2006a).…”

“…Taura syndrome virus can be transmitted horizontally by injection, ingestion of infected tissue or exposure to infected shrimp or shrimp carcasses Hasson et al 1995;Lotz 1997;Lotz et al 2003;Srisuvan et al 2006a). Exposure to chronically infected shrimp can result in TSV transmission in the absence of disease (Lotz et al 2003).…”