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First Report on Natural Infection of Nodavirus in an Echinodermata, Sea Cucumber (Apostichopus japonicas).
Wang C
,
Yao L
,
Wang W
,
Sang S
,
Hao J
,
Li C
,
Zhang Q
.
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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.
Figure 1. The topography and schematic diagram of the intestine of sea cucumber (Apostichopus japonicas) in this study. (a) Topography of sea cucumber collected from the polyculture ponds infected with covert mortality nodavirus (CMNV). (b) Schematic diagram of sea cucumber. (c) Schematic diagram of the longitudinal section of sea cucumber. Intestinal tissue is pointed by the black arrow.
Figure 2. Electrophoretogram of amplicons from the CMNV RT-nPCR assay and phylogenetic analysis. (a) Electrophoretogram of amplicons from the second-step PCR of the CMNV RT-nPCR assay. M: DL2000 molecular weight marker. Lanes 1–8: the PCR products of four RNA samples from sea cucumbers’ intestines (each sample was done with two replicates; samples 1, 2, 3, and 4 were shown in lanes 1 and 2, 3 and 4, 5 and 6, 7 and 8, respectively). P: positive control; N: negative control. (b) Analysis of the phylogenetic tree based on the deduced amino acid sequences of the RdRp gene from the CMNV-positive sea cucumber sample and other nodaviruses (abbreviations of other viruses shown in Table 1). The CMNV isolate of the sea cucumber sampled from farming ponds is highlighted in red. Viral species of Alphanodavirus genus and Betanodavirus genus are shown in pink and lavender background, respectively. The phylogeny tree was derived using the neighbor-joining method by the MEGA 6.0 program. The scale bar is 0.01.
Figure 3. Micrographs of hematoxylin and eosin-phloxine (H&E) staining and in situ hybridization (ISH) of the intestine of sea cucumber naturally infected with CMNV. (a) Micrographs of H&E staining of the intestine. (b) Magnified micrographs from the red-framed areas of (a). Karyopyknosis (red arrows) and extensive vacuolation (yellow arrows) were observed in the intestinal epithelial cells. (c) Micrographs of ISH of the intestine. (d) Magnified micrographs from the red-framed areas of (c). Intense CMNV positive hybridization signals (colored deep-purple) were detected in the intestinal epithelial cells. Scale bars: (a,c) 200 µm, (b,d) 50 µm.
Figure 4. Micrographs of H&E staining and ISH of the intestinal tissue of sea cucumber of negative control. (a) Micrographs of H&E staining of negative control intestinal tissue. (b) Magnified micrographs from the red-framed areas of (a). No obvious histopathological change was observed in the intestinal tissue. (c) Micrographs of ISH of negative control intestinal tissue. (d) Magnified micrographs from the red-framed areas of (c). No CMNV positive hybridization signal was detected in the intestinal tissue. Scale bars: (a,c) 200 µm, (b,d) 50 µm.
Figure 5. TEM micrographs of an ultrathin section of the intestinal epithelial cells of sea cucumber from the farm infected with CMNV. (b–d) show magnified micrographs in the black-framed areas of (a–c), respectively. Note the scattering of CMNV-like virus particles (red arrows) at the intestinal villus epithelium cells. Scale bars: (a) 10 µm, (b) 2 µm, (c) 500 nm, (d) 100 nm.
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