Abstract
Covert mortality nodavirus (CMNV) recently became more prevalent, severely affecting farmed shrimps, and also other invertebrates and teleost fish, in the coastal areas of China. For better understanding of its prevalence and susceptibility of large yellow croaker Larimichthys crocea to CMNV, an epidemiological survey was carried out in the main producing areas (Ningbo, Zhejiang, China, and Ningde, Fujian, China) and its offshore feeding grounds in the Southern Yellow Sea. The collected cultured L. crocea individuals showed clinical symptoms such as anorexia and abnormal swimming and diagnosed for CMNV infection by using RT-nPCR assay. The positive rates for CMNV in the collected L. crocea samples from Ningbo, Ningde, and the Yellow Sea were 14.29% (4/28), 20.00% (7/35), and 16.67% (1/6), respectively. Furthermore, CMNV diagnosis using TaqMan probe-based reverse transcription quantitative PCR (TaqMan RT-qPCR) yielded positive rates of 25.00% (7/28), 22.86% (8/35), and 16.67% (1/6) respectively in the above-mentioned samples, and with a range of 12.73–3,108.33 of CMNV copy numbers/μg total RNA. Phylogenetic tree analysis based on the RNA-dependent RNA polymerase (RdRp) and capsid protein genes showed that CMNV isolates from L. crocea samples were clustered tightly with the original isolate of CMNV from Penaeus vannamei. The histopathological studies indicated that severe cellular necrosis occurred in the heart, liver, spleen, eye, and gill of naturally infected fish. Stronger positive signals for CMNV-specific probes appeared in the necrotic cells and tissues in the in situ hybridization (ISH) analysis. Transmission electron microscopy (TEM) revealed the presence of numerous 30 nm diameter CMNV-like viral particles. The purified CMNV suspension could cause a significant cytopathic effect (CPE) in epithelioma papilloma cyprini (EPC) cells and resulted in the formation of typical inclusion bodies, and also visible CMNV particles around the lysed EPC cells under the TEM. Taken together, all results of this study demonstrated that L. crocea is susceptible to CMNV. The prevalence of CMNV and its potential impacts on the wild population of L. crocea see the need for further investigations.
Highlights
Covert mortality nodavirus (CMNV), an emerging alphanodavirus, severely impacted the shrimp aquaculture industries in China and Southeast Asian countries in the past decade (Zhang et al, 2014; Flegel, 2015; Thitamadee et al, 2016)
For further confirmation of the CMNV infection in the CMNV-positive L. crocea samples, in situ hybridization (ISH) was conducted by using a CMNV-specific RNA probe
The results showed that positive purple hybridization signals of CMNV probe could be observed in the cardiac muscle (Figures 2A,B), hepatocytes (Figures 2E,F), spleen (Figures 2I,J), bipolar cell of the retina (Figures 2M,N), and chloride cells of gill filament (Figures 2Q,R)
Summary
Covert mortality nodavirus (CMNV), an emerging alphanodavirus, severely impacted the shrimp aquaculture industries in China and Southeast Asian countries in the past decade (Zhang et al, 2014; Flegel, 2015; Thitamadee et al, 2016). Epizootic investigations recorded CMNV prevalence in farmed shrimp at the annual rates of 45.93%, 27.91%, 20.85%, 26.8%, and 16.3% respectively during the period of 2013–2017 in the major shrimp producing provinces in China (Zhang et al, 2017). Except for wide geographical prevalence, CMNV has shown much broader host ranges. Varying prevalence (from 7 to 18%) of CMNV infection was reported in wild small yellow croaker Larimichthys polyactis sampled from coastal water of China in 2018–2019 (Xu et al, 2021). The above studies revealed that CMNV has been widely distributed in the farmed aquatic animals of coastal areas, and the wild hosts from the offshore waters. The appearance of certain negative effects on teleost fishes has raised concerns about the prevalence of CMNV in both cultured and wild fish from the coastal areas
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