A total of 28 and 35 cultured L. crocea (20–30 cm in length) were collected from marine cages in Ningbo, Zhejiang, and Ningde, Fujian, respectively, during the period from 2019 to 2021 (Figure 1). Six wild L. crocea (10–14 cm in length) were collected from the southern Yellow Sea (34°N, 121.5°E) of China by bottom trawl in 2019 (Figure 1). The collected cultured L. crocea individuals showed clinical symptoms such as anorexia and abnormal swimming. The heart, liver, kidney, spleen, eye, and gill tissues were dissected from each individual, part of which was stored in 4% paraformaldehyde (4% PFA) solution (Sinopharm, Beijing, China) for in situ RNA hybridization and histopathological analysis, or in the RNAstore solution (Tiangen, Beijing, China) for molecular biological detection and 2.5% glutaraldehyde solution (Sinopharm, Beijing, China) for transmission electron microscopy (TEM) analysis, respectively. The remaining part was then stored in dry ice for virus purification. The collected wild L. crocea samples were stored in liquid nitrogen and brought back to the laboratory for further analysis.

The frozen tissues of L. crocea were first homogenized thoroughly in TN buffer (20 mM Tris/HCl, 400 mM NaCl, pH 7.4) and then subjected to the purification of viral particles according to a previously reported protocol (Xu et al., 2020). The purified virus particles were filtered through a 0.22-μm membrane prior to further use.

The fish cell line [epithelioma papilloma cyprini (EPC)] was used for virus proliferation (Xiao et al., 2012). EPC cells were cultured in Eagle’s minimal essential medium (EMEM, Sigma-Aldrich, United States) in a 25-cm² flask (Corning, United States) with 10% FBS at 25°C. One hundred milliliter of purified virus suspension was inoculated into a confluent monolayer of EPC cells. The same volume of EMEM was used as a mock infection in EPC cells as control. After adsorption for 1 h at 25°C, 5 mL of medium supplemented with 2% FBS was added into the flasks, and the infected cells were incubated at 25°C in the incubator (SANYO, Japan). The infected cells were examined daily under an inverted phase-contrast microscope (Nikon, Japan). When 70–80% of cultured cells showed cytopathic effect (CPE), the cultures were collected in 2.5% glutaraldehyde for TEM analysis or in an RNAstore solution for RNA extraction.

Approximately 40 mg tissues or cell cultures fixed in RNAstore solution and liquid nitrogen were used for total RNA extraction by TIANamp Marine Animal RNAprep pure Tissue Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer’s instructions. The concentration and purity of the extracted RNA was determined by Nanodrop 2000c (Thermo Scientific, Waltham, MA, United States).

By using the total RNA as template, the first-step PCR of the RT-nPCR was conducted by using the PrimeScript One Step RT-PCR Kit (Version 2.0) (TaKaRa, Dalian, China) in total of 25 μL reaction mixture, including 12.5 μL first-step buffer, 1 μL PrimeScript first-step enzyme mix, 1 μL forward primer (Noda-F1: 5′-AAATACGGCGATGACG-3′, 10 μM), 1 μL reverse primer (Noda-R1: 5′-ACGAAGTGCCCACAGAC-3′, 10 μM), 1 μL RNA template, and 8.5 μL ddH₂O. The programmed reaction was then performed for the reverse transcription at 50°C for 30 min, predenaturation at 94°C for 3 min followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 50 s, and the final extension at 72°C for 5 min. The second-step PCR of the RT-nPCR was carried out by using the TaKaRa Premix Taq™ (Ex Taq™ Version 2.0) in a 25 μL reaction mixture, which included 12.5 μL Premix Taq, 1 μL forward primer (CMNV-D-F1: 5′-TCGCGTATTCGTGGAT-3′, 10 μM), 1 μL reverse primer (CMNV-D-R1: 5′-TAGGGTCAAAAGGTGTAGT-3′, 10 μM), 1 μL product of the first-step RT-PCR, and 9.5 μL ddH₂O. The second-step PCR was performed in a similar way as the first-step PCR except for the incubation for the reverse transcription.

Two target CMNV-specific RNA-dependent RNA polymerase (RdRp) gene fragments of 619 and 413 bp (GenBank accession number KM112247) were amplified by the first- and second-step PCRs, respectively. The PCR products were sequenced by using the Sanger sequencing method.

All the samples for TaqMan probe-based reverse transcription quantitative PCR (TaqMan RT-qPCR) were analyzed and quantified in three duplicates for targeting the same gene as previously described (Li et al., 2018).

The samples that were positive for CMNV from above TaqMan RT-qPCR assay were subjected to histopathological analysis with hematoxylin and eosin-phloxine (H&E) staining by using regular histological methods as previously described (Lightner, 1996).

The samples positive for CMNV in TaqMan RT-qPCR assay were subjected to in situ hybridization (ISH) analysis according to the previous procedures (Piette et al., 2008; Chen et al., 2014; Zhang et al., 2017). Nuclear Fast Red solution (Sigma-Aldrich, St. Louis, MO, United States) was added dropwise on the surface of the sections and dyed the nucleus for 2 min for counterstain postcolor development of BCIP/NBT in the ISH. The sections were visualized and photographed by using the Nikon Eclipse E80i microscope (Nikon Co., Tokyo, Japan).

The samples preserved in 2.5% glutaraldehyde with the corresponding number were taken out and treated with 1% osmic acid for 2 h, and then dehydrated and embedded in Spurr’s resin and stained with uranyl acetate and lead citrate following the protocols of Panphut et al. (2011). All specimens were visualized under JEOL JEM-1200 transmission electron microscopy at 80 kV.

Five and two pairs of primers (Table 1) were designed based on the original sequences of CMNV RdRp and capsid protein genes, respectively (GenBank accession numbers MT270124 and MT270123). cDNAs were synthesized by using the extracted total RNA of L. crocea samples which were positive for CMNV with SMART^® MLV-Reverse Transcriptase (TaKaRa) according to the procedure described by Xu et al. (2020). The reaction mixture and procedures for CMNV genes cloning PCR were referred to the previous report (Xu et al., 2020) with modifications of annealing temperatures and extension time (Table 1). The PCR product was subjected to electrophoresis on a 2% agarose gel and sequenced by the Sanger method (Sangon Biotech, Shanghai, China). Sequence analysis was conducted by using BioEdit (Ver. 7.1.3) and BLAST.¹ The phylogenetic tree based on the RdRp and capsid protein genes was generated using the neighbor-joining method of MEGA 7.0 (Tamura et al., 2011).

The results of RT-nPCR assay indicated that CMNV-positive rates of 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. The RT-qPCR test yielded 25.00% (7/28), 22.86% (8/35), and 16.67% (1/6) CMNV-positive rates in L. crocea collected from Ningbo, Ningde, and the Yellow Sea, respectively, with a range of 12.73–3,108.33 copy numbers/μg of total RNA (Table 2). The standard curve showed that the RT-qPCR had a high correlation coefficient (R² = 0.999) within the range of 2.21 × 10⁹ – 2.21 × 10⁰ RNA copies/reaction (Supplementary Figure 1).

For further confirmation of the CMNV infection in the CMNV-positive L. crocea samples, 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). No positive hybridization signals appeared on the sections from the same samples without CMNV-specific RNA probes, or from the CMNV negative samples determined by RT-nPCR (Supplementary Figures 2, 3).

The impact of CMNV infection on the heart, eye, liver, spleen, and gill tissues of L. crocea was investigated by histopathology of H&E-stained sections as shown in Figure 2. Moderate dissolution-like necrotic muscle fibers and hemocyte infiltration in the fibromuscular stroma were observed in cardiac muscle (Figures 2C,D). Severe vacuolation and karyopyknosis were found in hepatocytes (Figures 2G,H) and bipolar cells layer of the retina (Figures 2O,P). Moderate vacuolation and degenerated erythrocytes were observed in the spleen (Figures 2K,L). Disorders of the secondary lamella were also viewed in the gills (Figures 2S,T).

Under the TEM, massive CMNV-like particles with approximate 28–32 nm in diameter and spherical to icosahedral shapes were observed in the blood cells in the kidney (Figures 3A–C) and cardiac muscle cells of the heart of the L. crocea (Figures 3D–F).

Five amplicons of the nucleotide sequence of RdRp, with the sizes of 783, 649, 1,683, 413, and 881 bp were obtained by PCR (Figure 4A, lanes 1–5). Two amplicons of the nucleotide sequence of capsid protein gene, with the sizes of 662 and 906 bp, were generated by PCR (Figure 4A, lanes 6 and 7). Then, by sequencing and assembling the overlapping amplicons, the nucleotide sequences of RdRp and capsid protein genes of CMNV isolated from L. crocea were determined, which were 3,132 and 1,314 bp in length, respectively.

Searching results of BLASTx showed that the obtained sequences of RdRp and capsid protein genes of CMNV isolated from L. crocea had 99% similarities to the corresponding gene sequences of CMNV isolates from P. vannamei (MT270124 and MT270123) and L. polyactis (MW625911 and MW625912). The phylogenetic analysis indicated that both the RdRp and capsid protein gene sequences of CMNV isolated from L. crocea were clustered tightly with the corresponding sequences of other CMNV isolates. However, it showed a high variation rate with the other members of Alpha Nodavirus, including Drosophila melanogaster American nodavirus (DmANV), flock house virus (FHV), black beetle virus (BBC), P. vannamei nodavirus (PvNV), and M. rosenbergii nodavirus (MrNV), etc. (Figure 4B).

The CPE was first observed by light microscopy at 4 days postinoculation (dpi) of tissue filtrates. The cells shrank, their boundaries became invisible, and a degree of cell fusion appeared. The cell monolayer was destroyed on day four (Figure 5A). After three consecutive passages, the CPE became more consistent. Cell cultures were positive for CMNV by PCR assay (Figure 5B). The expected fragment with 619 and 413 bp in size of the CMNV partial RdRp gene was cloned and sequenced from the inoculated cells. The phylogenetic analysis showed that CMNV isolated from EPC cell cultures was clustered into the same branch with the known CMNV isolates (Figure 5C). Typical CMNV-like virus inclusion bodies could be observed around the lysed cells under the TEM (Figure 5D).