Liang Yao

Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R²) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

Covert mortality nodavirus (CMNV), from the Nodaviridae family, is characterized by its unique cross-species transmission and wide epidemic distribution features. In this study, Macrobrachium rosenbergii was proved to be infected naturally by CMNV, which further expand the known host range of CMNV. Here, 61.9% (70/113) of the M. rosenbergii samples collected from Jiangsu Province were CMNV positive in the TaqMan RT-qPCR assay, which indicated the high prevalence of CMNV in M. rosenbergii. Meanwhile, the sequences of CMNV RdRp gene cloned from M. rosenbergii were highly identical to that of the original CMNV isolate from Penaeus vannamei. In situ hybridization (ISH) and histology analysis indicated that the intestine, gill, hepatopancreas and ovary were the targeted organs of CMNV infection in M. rosenbergii, and obvious histopathological damage including vacuolation and karyopyknosis were occurred in the above organs. Notably, the presence of CMNV in gonad alerted its potential risk of vertical transmission in M. rosenbergii. Additionally, numerous CMNV-like particles could be observed in tissues of hepatopancreas and gill under transmission electron microscopy. Collectively, our results call for concern of the potential negative impact of the spread and prevalence of CMNV in M. rosenbergii on its aquaculture, as well as providing a renewed orientation for further investigation and exploration of the diverse pathogenic factors causing M. rosenbergii diseases.

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.