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Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80-100nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found.
Fig. 1. Histopathologic changes in intestine tissue of sea cucumbers with APED. (a) Histological examination of the intestine lesions in epithelium layers. Intestinal villi arranged irregularly and the epithelium layer obviously damaged or even disappeared (arrowhead) accompanied with empty areas for histolysis (arrow). Bar, 50 μm. (b) Viral inclusion bodies were observed in epithelium layer. The size of these obviously spherical inclusion bodies ranges between 0.6 and 1 μm, stained purple (arrow). Bar, 3 μm.
Fig. 2. Pathologic changes in cells of intestine epithelium of sea cucumbers with APED. (a) A cytopathic cell with two vesicles full of virions (arrow). Bar, 200Â nm. (b) A nucleus of virus-infected cell was of homogenous materials and hyper-stained, with no nucleoli observed, and the endoplasmic reticulum obviously swelled and formed a separation cisternae (arrow) between the nucleus and the cytoplasm. Bar, 200Â nm. (c) Empty vesicles dispersed in granular areas of cytopathic cell of intestine epithelium, some with clear membrane (arrow), and some with blurry margins (arrowhead). Bar, 200Â nm. (d) Virus-containing vesicles in cytopathic cells of intestine, in which some virus are light-colored in the center (arrow) as if they are hollow and some are over-colored with petal-like pattern (arrowhead). Orderly tubular structures can also be found around the vesicles. Bar, 50Â nm. Inset: a typical virus particle, with an envelope and regular petal-like surface projections (arrow) surrounding the periphery of the particle. Bar, 100Â nm.
Fig. 3. Ultrastructural characteristics of virus assembly in cytopathic cells of APED. (a) A cytopathic cell of intestine epithelium, with a large area of granular material (arrowhead) and empty vesicles (arrow), devoid of organelles, and only mitochondria sometimes seen (open arrow). Bar, 200Â nm. (b) Nucleocapsid inclusion (arrow), hyper-stained, in association with virus-containing vesicles or double-membrane vesicles. Bar, 100Â nm. (c) Tubular structures, averaging 25Â nm in diameter, within some virus-containing vesicles; some ends of the tubular structures swell and are similar to the spherical virions (arrow). Bar, 100Â nm. (d) Tubular structures within an enclosed vesicle in cytopathic cell of intestine. Bar, 100Â nm.
Fig. 4. Tubular structures outside the virus-containing vesicles. (a) Tubular structures (open arrow) arrayed with large space but in the same direction; nucleocapsid inclusions (arrow), empty vesicle (arrowhead) and virus-containing vesicles dispersed among them. Bar, 100Â nm. (b) Tubular structures arranged orderly around the virus-containing vesicles in cytopathic cells. Bar, 100Â nm.
Fig. 5. Double-membrane vesicles and the assembly of virus particles. (a) Double-membrane vesicles in cytopthic cells of intestine, typically composed of accumulations of multiple single-membrane (arrow) vesicles enclosed within an outer membrane (arrowhead). Bar, 100Â nm. (b) Double-membranes vesicles (arrowhead), probably coming from rough endoplasmic reticula, and a double-membrane vesicle with a large space between the inner (arrow) and outer (open arrow) membranes of the vesicle, virus particles located between the membranes. Bar, 200Â nm. (c) Some vesicles with dark blurry edge areas around clear membrane (arrow) are also found in the cytopathic cells. Bar, 200Â nm. (d) The virions (arrowheads) always exist in the dark blurry margins around the membrane, if the vesicles are not full of virions; virions acquired an envelope by budding into the cisternae (arrows) and formed mostly spherical, sometimes pleomorphic, particles, which suggest these vesicles seem to be the site of the viral assembly. Bar, 200Â nm.
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