First Report on Natural Infection of Nodavirus in an Echinodermata, Sea Cucumber (Apostichopus japonicas).

Cross-species transmission of emerging viruses happens occasionally due to epidemiological, biological, and ecological factors, and it has caused more concern recently. Covert mortality nodavirus (CMNV) was revealed to be a unique shrimp virus that could cross species barrier to infect vertebrate fish. In the present study, CMNV reverse transcription-nested PCR (RT-nPCR)-positive samples were identified from farmed sea cucumber (Apostichopus japonicas) in the CMNV host range investigation. The amplicons of RT-nPCR from sea cucumber were sequenced, and its sequences showed 100% identity with the RNA-dependent RNA polymerase gene of the original CMNV isolate. Histopathological analysis revealed pathologic changes, including karyopyknosis and vacuolation of the epithelial cells, in the sea cucumber intestinal tissue. The extensive positive hybridization signals with CMNV probe were shown in the damaged epithelial cells in the in situ hybridization assay. Meanwhile, transmission electron microscopy analysis revealed CMNV-like virus particles in the intestine epithelium. All the results indicated that the sea cucumber, an Echinodermata, is a new host of CMNV. This study supplied further evidence of the wide host range of CMNV and also reminded us to pay close attention to its potential risk to threaten different aquaculture animal species.

An Unconventional Flavivirus and Other RNA Viruses in the Sea Cucumber (Holothuroidea; Echinodermata) Virome.

Sea cucumbers (Holothuroidea; Echinodermata) are ecologically significant constituents of benthic marine habitats. We surveilled RNA viruses inhabiting eight species (representing four families) of holothurian collected from four geographically distinct locations by viral metagenomics, including a single specimen of Apostichopus californicus affected by a hitherto undocumented wasting disease. The RNA virome comprised genome fragments of both single-stranded positive sense and double stranded RNA viruses, including those assigned to the Picornavirales, Ghabrivirales, and Amarillovirales. We discovered an unconventional flavivirus genome fragment which was most similar to a shark virus. Ghabivirales-like genome fragments were most similar to fungal totiviruses in both genome architecture and homology and had likely infected mycobiome constituents. Picornavirales, which are commonly retrieved in host-associated viral metagenomes, were similar to invertebrate transcriptome-derived picorna-like viruses. The greatest number of viral genome fragments was recovered from the wasting A. californicus library compared to the asymptomatic A. californicus library. However, reads from the asymptomatic library recruited to nearly all recovered wasting genome fragments, suggesting that they were present but not well represented in the grossly normal specimen. These results expand the known host range of flaviviruses and suggest that fungi and their viruses may play a role in holothurian ecology.

Enhancement of non-specific immune response in sea cucumber (Apostichopus japonicus) by Astragalus membranaceus and its polysaccharides.

In this study, the immunostimulatory effect of oral administration of different preparations (conventional fine powder [CP] and superfine powder [SP]) of Astragalus membranaceus root or its polysaccharides (APS) in sea cucumber (Apostichopus japonicus) was investigated. Sea cucumbers with an average initial weight of 49.3 +/- 5.65 g were fed with a diet containing 3% CP or SP or 0.3% APS over a period of 60 days. The non-specific humoral (phenoloxidase, lysozyme and agglutination titer) and cellular (phagocytic capacity and reactive oxygen species) responses were determined and compared with controls (no supplement) after 20, 40 and 60 days of feeding. Variation in the levels of responses was evident among different supplements. SP and APS significantly enhanced most of the immune parameters tested. Among the humoral responses, lysozyme activity significantly increased after feeding with SP-supplemented diet for 20, 40 or 60 days. Furthermore, lectin titer showed significant enhancement after 20 and 60 days of feeding with APS-supplemented diet. Significant increase in the production of reactive oxygen species was evident for all three supplements after 20 days of feeding, but no significant change in serum phenoloxidase activity was observed for any of the three supplements over the three different periods. Overall, significant modulation of the cellular responses was only noticed after 20 days of feeding with SP- or APS-supplemented diet. After 60 days, these two groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with Vibrio splendidus. These results indicated that dietary intake containing A. membranaceus root or its polysaccharides could enhance the immune responses of A. japonicus and improve its resistance to infection by V. splendidus.

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China.

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.