The virus FD201807 was isolated from the kidney of a 3-month-old juvenile L. crocea exhibiting illness in 2018. Sequencing of the major capsid protein (MCP) gene has demonstrated that FD201807 belongs to the family Iridoviridae and has been classified as a member of the genus Megalocytivirus. FD201807 was maintained and propagated in the mandarin fish fry cell line-1 (MFF-1) (Dong et al., 2008). Specifically, MFF-1 cells, provided by Sun Yat-sen University, were inoculated into 75-cm² cell culture flasks and cultured in Dulbecco's modified Eagle's medium (DMEM), which contained 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, at 27°C in the presence of 5% CO₂. After establishing the confluent monolayer, 60 μL of the FD201807 viral suspension, with the viral loads of 2.5 × 10⁷ copies/μL, was added. And the viral culture was maintained until an 80% cytopathic effect was observed in the MFF-1 cells. For electron microscopy, the infected cells were digested with 0.25% trypsin for 1 min, then harvested and re-suspended in a fixative solution (0.1 M cacodylate buffer containing 2.5% glutaraldehyde, pH = 7.4), after a centrifugation at 1,800 rpm for 5 min, the cells were immersed in the fixative solution. Ultrathin sections were prepared and graphed with a transmission electron microscope (JEOL JSM-6380LV, Tokyo, Japan).

The virus-infected cells, stored at −80°C, were subjected to three freeze-thaw cycles. Following this, the cells were centrifuged at 8,000 rpm and 4°C for 40 min, and the supernatant was collected. Precipitation was achieved by centrifugation at 150,000 g and 4°C for 2 h, after which the pellet was resuspended in 0.01 M phosphate-buffered saline (PBS, pH 7.4). Subsequently, 2 mL of 60%, 2 mL of 50%, 2 mL of 40%, 2 mL of 35%, and 1 mL of 30% sucrose solutions were added stepwise from the bottom to the top of ultracentrifuge tubes. The viral suspension was then gently layered on top of the sucrose gradient solution. Further centrifugation was performed at 200,000 g and 4°C for 4 h, followed by the collection of the virus within the 50% sucrose gradient range. This procedure was repeated for two additional rounds of sucrose gradient centrifugation at 150,000 g for 1 h at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of PBS. Finally, the viral suspension underwent 10 min of ultrasonication in an ice bath (power: 200 W, ultrasound: 2 s, interval: 6 s) before being stored at −80°C.

Purified FD201807 virus proteins (40 μg) were analyzed using SDS-PAGE. A Mini-PROTEAN II cell system (Bio-Rad, Hercules, CA, U.S.A.) with a 5% stacking gel and a 12% separating gel was employed for discontinuous denaturing gel electrophoresis. Following staining with Coomassie Brilliant Blue R-250, the gel was thoroughly destained, scanned, and the image was saved. The SDS-PAGE band sizes were estimated using Quantity One software (Bio-Rad, U.S.A.), based on the sizes of the molecular weight markers.

A total of 200 μL of purified virus suspension was utilized for the extraction of viral genomic DNA using the viral genome DNA extraction kit (Tiangen Biotech Co. Ltd., Beijing), in accordance with the manufacturer's protocols. The extracted viral DNA was subsequently eluted with double-distilled water (ddH₂O) and stored at −20°C.

The extracted viral DNA was submitted to Sangon Biotech (Shanghai) Co., Ltd. for library construction and next-generation sequencing using the Illumina Hiseq 2000 (Illumina, San Diego, CA, USA). The initial sequencing data underwent quality assessment via FastQC 0.11.2 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality trimming of the Illumina sequencing data was performed using Trimmomatic 0.36 (Bolger et al., 2014), resulting in a refined dataset. For de novo assembly of the Illumina sequencing data, SPAdes 3.5.0 (Bankevich et al., 2012) was utilized. Following the completion of assembly, GapFiller 1.11 (Boetzer and Pirovano, 2012) was employed to fill gaps. Sequence correction was carried out using PrInSeS-G 1.0.0 (Massouras et al., 2010), which rectified errors introduced during assembly and addressed small fragment insertions or deletions. This process yielded the FD201807 virus genome. Based on the genome sequencing data, primers were designed using Oligo 6.44 (https://www.oligo.net/downloads.html) to amplify the terminal and contiguous N sequences of the genome for sequencing and cloning purposes (Table S1). The missing portions were supplemented according to the sequencing results.

Infectious spleen and kidney necrosis virus (ISKNV, GenBank accession no. AF371960) serves as a representative strain of the genus Megalocytivirus. Utilizing ISKNV as a reference, we aligned the FD201807 genome to this reference using Molecular Evolutionary Genetics Analysis (MEGA) software version 11 (Tamura et al., 2021). Significant differences were identified, and primers were designed for polymerase chain reaction (PCR) amplification and clone validation, culminating in the final FD201807 genome sequence. The circular genome schematic, which illustrates ORF distribution, GC content, and GC skew, was visualized using the BLAST Ring Image Generator (BRIG) version 0.95 (https://sourceforge.net/projects/brig/).

Gene annotation for the viral genome was performed using the Rapid Annotation using Subsystems Technology (RAST) server. Functional gene annotation was achieved through NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and comparisons with several databases, including the Nucleotide sequence database (NT), Non-Redundant protein sequence database (NR), Swiss-Prot protein knowledgebase (SWISSPROT), Translated EMBL Nucleotide Sequence Database (TREMBL), clusters of orthologous groups database (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG).

The genome sequence of FD201807 was aligned with complete gene sequences from various megalocytiviruses using the online software MAFFT (version 7.520, https://mafft.cbrc.jp/alignment/software/) (Katoh et al., 2019). A phylogenetic tree was constructed utilizing the neighbor-joining method in MEGA 11 software (Tamura et al., 2021), with a bootstrap test value of 1,000 and default parameters. The evolutionary relationships between the FD201807 genome and those of other megalocytiviruses were evaluated. Detailed information regarding the megalocytivirus genomes used in this study is presented in Table 1.

Viruses from infected cells were purified using sucrose density gradient centrifugation. Proteins from the supernatants of virus-infected cells, and purified viruses were sent to Novogene Biotech Co., Ltd. (Beijing, China) for label-free proteomics analysis. Total protein was extracted as follows: the sample was treated with an appropriate volume of protein lysis buffer (100 mM ammonium bicarbonate, 8 M urea, 0.2% SDS, pH 8.0) followed by 5 min of ultrasound treatment in an ice-water bath. After centrifugation at 12,000 g and 4°C for 15 min, the supernatant was collected, and 10 mM dithiothreitol (DTT) was added to react at 56°C for 1 h. Subsequently, an adequate amount of iodoacetamide was added, and the mixture was allowed to react at room temperature in the dark for 1 h. The sample was then precipitated with four volumes of pre-cooled acetone at −20°C for 3 h, followed by centrifugation at 12,000 g and 4°C for 15 min to collect the precipitate. The precipitate was washed with 1 mL of pre-cooled acetone and centrifuged at 12,000 g and 4°C for 15 min. Following this, the precipitate was collected, air-dried, and subsequently dissolved in an appropriate volume of protein solubilization buffer (6 M urea, 100 mM triethyl ammonium bicarbonate, pH 8.5). After assessing the protein quality, the sample underwent trypsin digestion and subsequent LC-MS/MS analysis. Mass spectrometry was performed using a data-dependent acquisition mode, with a full scan range from m/z 350 to 1,500 and a resolution of 60,000 (at m/z 200). The resolution for the second-level mass spectrum was set to 15,000 (at m/z 200). The resulting raw data from MS detection were designated as “.raw” files. These raw files were then imported into Proteome Discoverer 2.2 software for retrieval from the UniProt database and for peptide and protein quantification. Additional filtering was conducted using Proteome Discoverer 2.2 to enhance the quality of the analysis results and minimize the false-positive rate. Peptide spectrum matches (PSMs) with a credibility exceeding 99% were identified. Proteins that contained at least one unique peptide were classified as credible proteins. Only credible peptides and proteins were retained, and false discovery rate (FDR) validation was performed to exclude peptides and proteins with an FDR >1%.

Gene Ontology (GO) annotation was performed by conducting sequence alignment (BLAST) of the identified protein sequences against six widely recognized databases: protein family databases (Pfam), Protein fingerprint database (PRINTS), Protein domain database (ProDom), Simple Modular Architecture Research Tool (SMART), Protein site database (ProSite), and Protein Analysis Through Evolutionary Relationships (PANTHER). This was followed by analysis using the InterProScan software (http://www.ebi.ac.uk/interpro/search/sequence/).

Freeze-thaw infected cells were separated using 12% SDS-PAGE gels and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. Polyclonal antibodies raised in mice against the purified recombinant proteins (VP007L, VP058L, and VP121R) were employed as primary antibodies at a dilution of 1:1,000. Additionally, a monoclonal antibody targeting VP026R, generously provided by Sun Yat-sen University, was also utilized as a primary antibody at the same dilution of 1:1,000. The PVDF membrane was then subjected to immunodetection using a secondary antibody conjugated with horseradish peroxidase (HRP) at a dilution of 1:10,000 (Proteintech, Wuhan, China) and was visualized through enhanced chemiluminescence.

The names of viruses in this article are abbreviated as follows: ISKNV for Infectious spleen and kidney necrosis virus; PIV for Pompano iridovirus; RSIV for Red seabream iridovirus; LYCIV for Large yellow croaker iridovirus; LYCIV-ZS-2020 for Large yellow croaker iridovirus isolate Zhoushan; OSGIV for Orange-spotted grouper iridovirus; RBIV for Rock bream iridovirus; TRBIV for Turbot reddish body iridovirus; GSIV for Giant seaperch iridovirus; SACIV for South American cichlid iridovirus; SKIV for Spotted knifejaw iridovirus; TSGIV for Three spot gourami iridovirus; BCIV for Banggai cardinalfish iridovirus; AFIV for Angelfish iridovirus; and LMIV for Lateolabrax maculatus iridovirus.

Transmission electron microscopy was conducted on FD201807-infected cells, which exhibited a pathological rate of 80%. Compared to normal MFF-1 cells (Figure 1A), abundant viral particles were observed, scattered randomly within the cytoplasm of the infected cells (Figure 1B). FD201807-infected cells also appeared larger in size than the normal MFF-1 cell, as indicated by the differences in scale bar between Figures 1A, B, which aligns with a key symptom of Megalocytivirus infection. Upon magnification, viral particles and some empty capsids with a roughly hexagonal shape and bilayer envelope were visible, exhibiting a uniform size with a diameter of approximately 150 nm (Figures 1C, D).

Illumina HiSeq sequencing yielded 20,709,962 reads with a total base count of 3,106,494,300 bp. After Tirmmomatic clipping, 19,852,088 reads, or 2,753,330,175 bp were retained, with a Q20 base rate of 97.76%. A genome of 111,110 bp was assembled by processing these reads through SPAdes for assembly (> 24,000 × coverage), GapFiller for gap completion, and PrInSeS-G for sequence correction. The ends of the viral sequences were amplified using PCR, and the sequences were determined by Sanger sequencing. The final, complete genome of FD201807 is 112,214 bp in length with a G + C content of 53.53%, slightly lower than that of ISKNV (54.78%). NCBI BLAST genome comparison demonstrated that the FD201807 viral genome shared over 90% identity with 20 other genomes from the genus Megalocytivirus. Analysis of the genome termini indicated that the FD201807 genome DNA is both circularly permuted and terminally redundant, characteristics commonly shared among members of the Iridoviridae family (Chinchar et al., 2017).

We identified 130 putative ORFs encoding proteins with lengths ranging from 41 to 1,293 amino acids on both the sense (R) and antisense (L) DNA strands. Among these ORFs, 52 (40%) were transcribed in the forward orientation, whereas 78 (60%) were oriented in the reverse orientation (Figure 2). Each ORF's hypothetical amino acid sequence was BLAST-searched against the GenBank nr database. All ORFs matched homologous sequences within GenBank's Megalocytivirus entries, with 97 aligning fully with registered Megalocytivirus protein sequences. A substantial majority (65.4%, or 85) of FD201807 ORFs lacked predicted functions. Of the 130 ORFs, 44 encoded proteins with homology to functionally annotated proteins, and all 26 iridovirid core genes were identified. These core genes are believed to play essential roles in viral transcription, replication, and assembly (Eaton et al., 2007).

The putative gene products of FD201807 included at least 10 enzymes involved in transcription, nucleotide metabolism, and DNA replication. These included DNA-dependent RNA polymerase subunit H (ORF002L), ribonucleotide-diphosphate reductase subunit beta (ORF027R), the two largest subunits of DNA-dependent RNA polymerase (ORF031L and ORF036R), transcription elongation factor S-II (TFIIS, ORF032L), mRNA capping enzyme (ORF065L), deoxyribonucleoside kinase (ORF034R), D5 family NTPase (ORF111L), DNA polymerase (ORF023R), sucrose non-fermenting 2 (SNF2) family helicase (ORF064L), proliferating cell nuclear antigen (PCNA, ORF114R), and one RNase III enzyme (ORF088R). These FD201807 genes exhibited 94–97% amino acid sequence identity to their homologs in ISKNV (Dong et al., 2011). Detailed annotation information for each ORF is provided in Table S2.

Some inferred gene products of FD201807 contained highly conserved structural domains and enzymatic active site motifs involved in protein processing and modification. For instance, ORF015R featured a serine/threonine protein kinase domain, ORF026R contained three calcium-binding epidermal growth factor (EGF)-like domains, and ORF117L included a tyrosine-specific protein phosphatase active site.

Notably, FD201807 displayed a unique feature on the positive strand at positions 8,313–8,319, consisting of seven adenine residues, unlike other Megalocytivirus strains, which typically have eight consecutive adenine residues. This deletion was confirmed by a separate PCR followed by Sanger sequencing, indicating a one-adenosine deletion likely resulting in a truncated ORF011L and a unique ORF010R (Figure S1).

A phylogenetic tree was constructed using the complete genome sequences of megalocytiviruses within the family Iridoviridae that are available in GenBank (Figure 3). The results of the phylogenetic analysis indicated that FD201807 is classified within the RSIV genotype and is closely related to PIV and RSIV, exhibiting sequence identities of 98.98 and 98.92%, respectively. Other members of the RSIV genotype include RBIV, SKIV, OSGIV, GSIV, RSIV, LYCIV, and LYCIV-ZS-2020. Notably, two LYCIV strains are genetically distinct: one was isolated from Zhoushan in 2020 (LYCIV-ZS-2020), and the other from the Ningde area of Fujian Province in 2006 (LYCIV) (Ao and Chen, 2006). Our findings suggest that LYCIV-ZS-2020, FD201807, RSIV, and PIV form a sister clade to a group comprising four closely related megalocytiviruses: RBIV, SKIV, OSGIV, and GSIV (Figure 3). The earliest isolated strain of LYCIV (Ao and Chen, 2006) appears to be the earliest diverging member of the RSIV genotype. Additionally, AFIV, BCIV, and ISKNV constitute a monophyletic ISKNV genotype, while SACIV, TSGIV, and TRBIV are categorized under the TRBIV genotype.

The purified viral particles were subjected to SDS-PAGE analysis, which revealed approximately 22 distinct protein bands stained with Coomassie brilliant blue. Based on the protein sizes obtained by LC-MS/MS analysis, we indicate the regions where various viral proteins are located in Figure 4. Notably, primary viral proteins were concentrated in bands with molecular weights above 29 kDa, while proteins below 29 kDa showed lower abundance (Figure 4). Trypsin digestion followed by LC-MS/MS analysis identified 595 peptide segments from both the purified virus and cell culture supernatant, corresponding to 141 proteins (Table S3). Most peptide segments ranged from 8 to 18 amino acids in length, with a majority of proteins exhibiting molecular weights between 10 and 60 kDa (Figure S2). Amino acid sequences were searched against the Uniprot and GenBank databases using BLAST, leading to the identification of 54 viral proteins after excluding duplicates. Of these, 47 relatively abundant proteins contained multiple peptide fragments, while smaller or low-abundance proteins, such as VP004L, VP019L, and VP021R, contained only one identifiable peptide (Table 2).

Of the 54 viral proteins, 27 were found exclusively in the purified virus, while 8 proteins (VP004L, VP019L, VP026R, VP030L, VP060L, VP074R, VP102L, and VP114R) were present only in the cell culture supernatant (Figure 5). Additionally, two host-origin proteins, cytochrome c and ubiquitin-activating enzyme E1 were detected in both the MFF-1 cell culture supernatant and the purified virus.

Among the 54 viral proteins, 23 had functional and/or domain annotations, 6 proteins contained transmembrane domains, while another 6, including a SNF2 family helicase (VP064L), had an Arg-Gly-Asp (RGD) motif, which is linked to DNA repair (Table 2). Moreover, FD201807 ORFs 008L, 058L, and 122L are likely to encode envelope proteins, as their putative protein sequences share 97, 99, and 97% identity, respectively, with ISKNV envelope proteins 007L, 056L, and 118L (Dong et al., 2011). Notably, only three proteins (VP007L, VP078R, and VP081R) exhibited acetylation modifications.

To confirm the presence of the identified proteins, we utilized mouse antibodies as the primary antibody in Western blotting for VP007L (49.6 kDa), VP026R (100.8 kDa), VP058L (23.4 kDa), and VP121R (49.6 kDa). Samples containing the virus displayed distinct bands corresponding to the expected protein molecular weights (Figure 6).

The 54 identified viral proteins underwent Gene Ontology (GO) classification to help gain insights into functional enrichment across biological processes, cellular components, and molecular functions. The classification of biological processes indicated that viral proteins primarily participate in protein folding, oxidation–reduction, and translation processes. In terms of cellular component, the classification revealed that viral proteins predominantly originated from viral capsids, intracellular locales, and ribosomes. The molecular function classification highlighted roles in ATP binding, structural molecule activity, suggesting their involvement as structural components of ribosomes (Figure 7).