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Article

Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass

by
Xue-Dong Yu
1,
Fei Ke
2,*,
Qi-Ya Zhang
2 and
Jian-Fang Gui
1,2,*
1
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
2
The Innovative Academy of Seed Design, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
*
Authors to whom correspondence should be addressed.
Pathogens 2023, 12(5), 730; https://doi.org/10.3390/pathogens12050730
Submission received: 12 April 2023 / Revised: 10 May 2023 / Accepted: 16 May 2023 / Published: 17 May 2023
(This article belongs to the Special Issue Aquatic Animal Pathogens: Epidemiology, Pathogenesis, Diagnosis, Preventive and Therapeutic Measures and Host–Pathogen Relationships)
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Abstract

:
Ranaviruses are promiscuous pathogens that threaten lower vertebrates globally. In the present study, two ranaviruses (SCRaV and MSRaV) were isolated from two fishes of the order Perciformes: mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides). The two ranaviruses both induced cytopathic effects in cultured cells from fish and amphibians and have the typical morphologic characteristics of ranaviruses. Complete genomes of the two ranaviruses were then sequenced and analyzed. Genomes of SCRaV and MSRaV have a length of 99, 405, and 99, 171 bp, respectively, and both contain 105 predicted open reading frames (ORFs). Eleven of the predicted proteins have differences between SCRaV and MSRaV, in which only one (79L) possessed a relatively large difference. A comparison of the sequenced six ranaviruses from the two fish species worldwide revealed that sequence identities of the six proteins (11R, 19R, 34L, 68L, 77L, and 103R) were related to the place where the virus was isolated. However, there were obvious differences in protein sequence identities between the two viruses and iridoviruses from other hosts, with more than half lower than 55%. Especially, 12 proteins of the two isolates had no homologs in viruses from other hosts. Phylogenetic analysis revealed that ranaviruses from the two fishes clustered in one clade. Further genome alignment showed five groups of genome arrangements of ranaviruses based on the locally collinear blocks, in which the ranaviruses, including SCRaV and MSRaV, constitute the fifth group. These results provide new information on the ranaviruses infecting fishes of Perciformes and also are useful for further research of functional genomics of the type of ranaviruses.

1. Introduction

Ranaviruses are members of the genus Ranavirus (family Iridoviridae) [1], which are nucleocytoplasmic large DNA viruses (NCLDVs). Ranaviruses have been isolated from several poikilotherms, including fishes [2,3,4], amphibians [5,6,7], and reptiles [8]. Several of the poikilotherms are important farmed animals. Thus, ranaviruses represent a great threat to these animals and the related culture industry. The complete genomes of more than 100 ranavirus isolates have been sequenced, including two isolated in our lab, the Rana grylio virus (RGV) and Andrias davidianus ranavirus (ADRV) [6,8,9,10,11,12,13,14,15], which promoted the understanding of virus infection and virus–host interactions. According to the report of the International Committee of Taxonomy of Viruses (ICTV), four genomic phenotypes, frog virus 3 (FV3)-like, Ambystoma tigrinum virus (ATV)-like, common midwife toad virus (CMTV)-like, and Singapore grouper iridovirus (SGIV)-like, has been reported in ranaviruses based on whole genome dot plot comparisons [1], in which RGV was grouped in FV3-like and ADRV was grouped in CMTV-like.
It has been reported that aquaculture has become the fastest-growing agricultural production industry in the world, and a major contributor is China [16,17,18]. Mandarin fish (Siniperca chuatsi, also known as Chinese perch) and largemouth bass (Micropterus salmoides) are two fishes belonging to the Order Perciformes, which have a delicious taste and high nutrition as food. Thus, the culture of the two fishes has been rapidly developing in recent years in China. It has been reported that the annual production of mandarin fish and largemouth bass in China has been more than between 330 and 600 kilotons in recent years [19]. However, economic losses caused by diseases in these fishes are becoming a serious challenge. One of the important viral pathogens in the aquaculture of the two fishes is the ranavirus, which has been isolated from the two fishes in recent years [20,21,22]. Although there are genome sequences of ranaviruses isolated from the two fishes in the GenBank database, a detailed analysis of the genome architecture and comparison with other ranaviruses are not reported.
In the present study, we isolated a ranavirus from diseased mandarin fish and a ranavirus from diseased largemouth bass. The complete genome of the two ranaviruses was determined. Further genome comparison and analysis revealed the characteristics of the two viruses.

2. Materials and Methods

2.1. Sample Collection

Diseased largemouth bass and mandarin fish were collected from aquafarms in Hubei province of China from June 2021 to July 2022. Tissues of liver, spleen, and kidney of the diseased fishes were collected for virus isolation.

2.2. Virus Isolation

Collected tissues were homogenized in phosphate-buffered saline (PBS) and clarified by centrifugation at 10,000× g for 5 min. The supernatants were filtered through a 0.45 μm sterile filter (Millipore, Burlington, MA, USA) and used to infect cell lines.
Different aquatic animal cell lines, Chinese giant salamander thymus cell (GSTC), Epithelioma Papulosum Cyprini (EPC), and Siniperca chuatsi skin cell (SCSC), which were preserved in our lab, were used in virus isolation and infection. The cells were cultured in M199 medium supplemented with 10% fetal bovine serum (FBS) at 25 °C, except the SCSC cells were cultured in L15 medium with 10% FBS. For virus isolation, monolayers of these cells were inoculated with the above tissue homogenates and incubated at 25 °C. The cells were harvested when advanced cytopathic effects (CPE) were observed, and the supernatant was used for the next round of infection until a stable CPE was obtained. Finally, the infected cells were collected and used as virus stocks after being frozen and thawed. The virus titers were measured by using a 50% tissue culture infectious dose (TCID50) assay as described previously [5].

2.3. Electron Microscopy

Cells were collected at 48 h post-infection (hpi) by centrifugation at 1000× g for 5 min. Cell pellets were pre-fixed with 2.5% glutaraldehyde, followed by post-fixed with 1% osmium tetroxide (OsO4), then dehydrated stepwise and embedded in Epon-812. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a Hitachi HT-7700 transmission electron microscope (TEM) at 80 KV.

2.4. Genomic DNA Extraction and Sequencing

Virus particles were purified from collected infected cells by ultracentrifugation, as described previously [6]. Genomic DNA was extracted from the purified virus particles by using the phenol-chloroform method. Briefly, virus suspensions were mixed with Proteinase K and RNase A (Takara, Dalian, China) and digested in a 56 °C water bath for 30 min. Then, the phenol chloroform isoamyl alcohol solution (25:24:1) was added. After shaking and centrifugation, the top water phase was transferred to a clean EP tube. The DNA was precipitated by 3 M sodium acetate and ethanol and stored at −80 °C for further use.
For genomic DNA sequencing, the insertion libraries were constructed with SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer’s instructions and sequenced using a PacBio Sequel II instrument (CCS; The Beijing Genomics Institute, Beijing, China).

2.5. Genome Annotation and Analysis

The DNA composition, structure, nucleotide, and amino acid sequences were analyzed with the DNASTAR program (Lasergene, Madison, WI, USA) as described previously [23]. The open reading frames (ORFs) were predicted using SnapGene software (version 6.1.1) and NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 12 December 2022). The following criteria were considered during ORF prediction: (1) the length was at least 120 bp, (2) the predicted ORF was not located in another larger ORF, (3) overlapping ORFs should have homologs in other sequenced iridoviruses [6]. Comparisons of homologous sequences among different viruses were performed by using BLAST programs (blastn for DNA sequence and blastp for protein sequence). All coding protein sequences of ranavirus were collected from GenBank. Multiple sequence alignments were conducted with ClustalX 1.83, and sequence identities were calculated with the MegAlign program. For a detailed comparison of the ORFs between SCRaV, MSRaV, and other ranaviruses, nine strains of ranaviruses were selected, including the four isolated from mandarin fish and largemouth bass previously and five others representing different genomic types of ranaviruses.
For phylogenetic analysis, the 26 iridovirus core proteins from SCRaV, MSRaV, and other completely sequenced iridoviruses were collected, identified based on homology comparison, and concatenated separately, and a reminder is needed that the Shrimp hemocyte iridescent virus and Cherax quadricarinatus iridovirus just have 24 core proteins. The MUSCLE program in Mega software (version 11.0.11) was used to make alignment, and a phylogenetic tree was constructed by the Neighbor-Joining method with default parameters. The Multiple genome alignment, including all 6 isolates from mandarin fish and largemouth bass (SCRaV, MSRaV, mandarin fish ranavirus strain NH-1609, largemouth bass virus strain Alleghany, largemouth bass virus strain GDOU, and largemouth bass virus strain Pine), RGV, FV3, ADRV, CMTV, ATV, epizootic hematopoietic necrosis virus (EHNV), SGIV, and grouper iridovirus (GIV), was performed with the progressive Mauve plugin in Geneious software (version 2023.0.2) [24].

3. Results

3.1. Virus Isolation and Identification

Tissue extracts from the diseased largemouth bass and mandarin fish both induced cytopathic effect (CPE) in several cultured cells, including SCSC, EPC, and GSTC. Infection of the cells with supernatants from the infected cells still caused typical CPE. A representative CPE in the three cells is shown in Figure 1. The two viruses’ infections both induced the lysis or detachment of cells. In the fibroblast-like SCSC cells, the infected cells lysed or detached rapidly, and only about half of the cells retained at the culture surface at 24 hpi, which formed a discrete distribution. At 48 hpi, most of the SCSC cells have lysed, and the remaining cells became round, indicating their death. For the epithelioid EPC and GSTC cells, a few plaques formed at 24 hpi, and plaques enlarged with infection time due to the lysis and detachment of infected cells. The CPE in SCSC cells seemed more serious than in the other two cells. Infection of GSTC with ADRV, a previously identified ranavirus, was used as a control, which showed similar CPE with the two viruses.
Ultrastructural observations were performed with SCRaV-infected SCSC cells and MSRaV-infected GSTC cells, respectively. As shown in Figure 2, serious cytoplasmic vacuolation was observed in SCRaV-infected SCSC cells, which caused difficulties in finding cellular organelles (Figure 2A). Cell shrinkage was observed in MSRaV-infected GSTC cells with a compacted and deformed nucleus (Figure 2B). Several regions that were full of mature or immature viral particles can be found in the cells (cytoplasm of GSTC). Intact virions in the ultrathin section are hexagonal or approximately circular, with a diameter of about 160 nm. Paracrystalline arrays that were formed by virion accumulation can be observed in a small number of cells (Figure 2C).

3.2. Architecture and General Features of the Two Virus Genomes

The complete genome sequence of the two viruses was determined. The genome of SCRaV consists of 99,405 bp with 105 potential ORFs, and the genome of MSRaV consists of 99, 171 bp with 105 potential ORFs. Detailed information about the predicted ORFs and comparisons with their homologs of other ranaviruses, including the four other ranaviruses (MFRV, LMBV-G, LMBV-A, LMBV-P) isolates from mandarin fish and largemouth bass worldwide were shown in Table 1 and Table S2. The length of the predicted proteins of the two viruses (SCRaV and MSRaV) both ranged from 49 to 1354 aa. There are very high sequence identities between the proteins of the two viruses. Most of their proteins (94/105) have sequence identities of 100% with the homolog. Ten proteins have sequence identities ranging from 92.5% to 99.9% with their homolog. Sequence identity lower than 90% was only obtained in one protein (79L) between the two viruses, which encodes a predicted neurofilament triplet H1-like protein.
Genome and encoding proteins of SCRaV and MSRaV were then compared with the previously sequenced four ranaviruses from the mandarin fish and largemouth bass worldwide. The results showed that the genome sequence identity between SCRaV and MSRaV was 99.92%, and a range of 98.68–99.88% was obtained between SCRaV and the other four isolates (Table 1). Most of the coding proteins of the six ranaviruses isolated from the two fishes possessed high identities, more than 96% among their homologs. It could be observed that the four isolates from China had higher similarity in genome sequences and coding proteins than the two from the USA (Table S2), especially the six proteins (11R, 19R, 34L, 68L, 77L, and 103R), in which 11R and 68L contain domains of LPXTG-anchored collagen-like adhesin and 77L contains a domain of DNA polymerase III subunit.
However, the sequence identity between the two viruses and ranaviruses from other hosts is not high. Although the sequence identity of the major capsid protein (MCP) between the two viruses and other ranaviruses could reach more than 83%, more than half of the proteins of the two viruses share sequence identity of less than 55% with homologs of ranaviruses from other hosts. There are still several proteins possessing sequence identity lower than 30% (the lowest was 22.3%) with its homolog, and 12 proteins cannot find homologs in iridoviruses from other hosts.
The schematic diagrams of the genome organization of SCRaV and MSRaV are shown in Figure 3. The two viruses have the same genome organization and gene composition. Combined with function analysis, the predicted genes were clustered as genes encoding structural proteins, nucleotide metabolism-related genes, DNA replication- and transcription-related genes, virus–host interaction-related genes, and unknown genes. Detailed information about the genes are described below. Because of the high sequence identity between the two viruses, gene and protein descriptions were mainly performed based on SCRaV.

3.3. Structural Proteins

SCRaV 104R was predicted to encode the major capsid protein (MCP), which contains 463 aa. Among the viral proteins, the MCP of SCRaV and MSRaV has the highest sequence identity with their homologs of ranaviruses infecting other animals. For example, they had a sequence identity of 84% with ADRV MCP and 83.6% with RGV MCP. SCRaV 1L and 16R encode two myristylated membrane proteins corresponding to ADRV 2L/RGV 2L and ADRV 58L/RGV 53R, respectively, which belong to core genes of iridoviruses and have been identified as envelope proteins of ranaviruses [25,26]. SCRaV 1L and 16R have sequence identities ranging from 70.5% to 75.2% and 55.4% to 63.7% with their homologs of the last five ranaviruses in Table 1, respectively. There are several other predicted proteins containing transmembrane domain (SCRaV 5R/8R/9R/56L/86R/98R), which could contain envelope proteins.

3.4. Nucleotide Metabolism Related Genes

There are 4 predicted proteins that could involve in nucleotide metabolism. SCRaV 71L encodes a protein of 141 aa, which contains domains of the deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase) family. SCRaV 69L (387 aa) and 94L (562 aa) are two homologs of ribonucleotide reductase (RNR) subunit that could catalyze the synthesis of deoxyribonucleotides that was used as precursors of DNA synthesis. SCRaV 99L (189 aa) contains the domain of deoxyribonucleoside kinase (dNK) or thymidine kinase (TK), which is a key enzyme in the salvage of deoxyribonucleosides. The four proteins all have homologs in other ranaviruses.

3.5. DNA Replication- and Transcription-Related Genes

For the proteins that could be involved in DNA strand replication, SCRaV 66R encodes a homolog of DNA polymerase, which has a length of 1004 aa and contains a 3′–5′ exonuclease domain and a B-family DNA polymerase domain. SCRaV 37R encodes a protein of 955 aa, which contains a domain of primase and the D5_N family. SCRaV 12L (261 aa) is a homolog of the p31K protein of ranaviruses, which has been identified as the virus single-stranded DNA binding (SSB) protein [27]. SCRaV 100L encodes a protein of 242 aa, whose homologs in other ranaviruses have been considered a homolog of proliferating cell nuclear antigens (PCNA) [28]. In addition, SCRaV 77L (284 aa) contains a domain of DNA polymerase III subunits gamma/tau. SCRaV 82L (566 aa) contains a DNA polymerase III subunit gamma/tau and an SAP domain. SCRaV 75L (356 aa) encodes a putative RAD2 family DNA repair protein, which could be involved in ranavirus DNA recombination and repair [29]. SCRaV 31L (173 aa) contains a domain of Holliday junction resolvases.
For the proteins that could be involved in genome transcription, there are 3 putative subunits of DNA-directed RNA polymerase (RNAP) II. SCRaV 45R encodes a protein of 1354 aa, which is the putative largest subunit of RNAP (Rpb1). SCRaV 74R has a length of 1094 aa and could be the β subunit of RNAP (Rpb2). SCRaV 28R encodes a protein of 159 aa and contains an RNAP Rpb5 domain. Besides the RNAP subunits, there are possible transcription factors. SCRaV 22L (91 aa) is a transcription elongation factor SII-like protein. SCRaV 40L (253 aa) contains a domain of the poxvirus late transcription factor VLTF3 superfamily. SCRaV 7R (634 aa) contains a domain of transcription termination factor.
In addition, other viral proteins may be involved in genome replication and transcription. For example, SCRaV 48L (400 aa) contains a domain of superfamily II DNA or RNA helicase. SCRaV 97L (949 aa) contains a domain of DEAD-like helicases superfamily and a C-terminal helicase domain of the SNF family helicases.

3.6. Virus–Host Interaction Related Genes

Several SCRaV or MSRaV proteins possess domain/motif that has been identified in host proteins, which indicates that these viral proteins could have functions in virus–host interactions. SCRaV 23R encodes a protein of 385 aa, which contains a domain of the ribonuclease III family. SCRaV 26R (270 aa) is a putative eukaryotic translation initiation factor 2α (eIF-2α)-like protein. SCRaV 41L encodes a protein of 171 aa containing a domain of the apoptosis regulator proteins of the Bcl-2 family. SCRaV 61L (85 aa) contains a domain of lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF). SCRaV 70L (91 aa) contains caspase activation and recruitment domain. SCRaV 72L (237 aa) is a putative tumor necrosis factor receptor (TNFR). SCRaV 95L (79 aa) contains a domain of insulin-like growth factor.

3.7. SCRaV- and MSRaV-Specific Genes

Sequence analysis also revealed 12 putative genes (4R, 15L, 17L, 25R, 34L, 36R, 43L, 46R, 53R, 54L, 81L, and 93L) that no homologs were found for their encoding proteins in viruses of other hosts, which could be considered as specific genes for SCRaV and MSRaV (or SCRaV/MSRaV-like viruses) (Figure 3 and Table 1). It should be noticed that there are 16 genes of SCRaV/MSRaV, including the 12 genes that cannot be found homologs in the compared viruses (ADRV, RGV, FV3, EHNV, and SGIV) in Table 1, but 4 of them (19R, 20R, 44L, and 49L) had homologs in other ranaviruses that were not listed in the table. Most of the specific genes encode hypothetical proteins that no conserved domains/motifs can be found. Only two proteins contain known domains. The 4R protein contains an N-terminal immunoglobulin (Ig)-like domain, and the 17L protein contains a domain of tumor necrosis factor receptor (TNFR), which could be involved in virus–host interactions.
In addition, ORF prediction and analysis also showed that SCRaV/MSRaV encodes five putative proteins (11R, 32L, 33L, 67L, and 68L) that contain domains of LPXTG-anchored collagen-like adhesins. The amino acid length of the 5 predicated proteins is 245, 240, 257, 243, and 288 aa, respectively. Sequence alignment and motif search showed that they all contain variable-length regions full of Gly-X-X repeats, which is a character of LPXTG-anchored collagen-like adhesin. Although homologs of the five proteins could be found in some ranaviruses, the sequence identity between the five proteins and their homologs is low, which made most of their homologs do not contain the LPXTG-anchored collagen-like adhesins domain. So, the five proteins can also be considered SCRaV/MSRaV-specific proteins.

3.8. Phylogenetic Analysis

A phylogenetic tree was constructed based on the proteins of core genes from 56 iridoviruses, including 35 ranavirus isolates (Figure 4). All the ranavirus isolates clustered in a big branch, which could be divided into small branches, including FV3/RGV-like, CMTV/ADRV-like, EHNV/ATV-like, largemouth bass virus (LMBV)/SCRaV-like, and SGIV-like viruses. The two viruses, MSRaV and SCRaV, were clustered with the other largemouth bass virus and mandarin fish ranavirus isolates, which indicated that they belonged to LMBV-like viruses.

3.9. Genome Comparison

We tried to perform a dot plot analysis to determine the genome similarity degrees between the two viruses and other ranaviruses, but no obvious collinearity can be found, possibly because of the low sequence identity between the two virus genomes and other ranaviruses. Then, a genome-wide alignment was carried out and revealed the genomic arrangement of the aligned ranaviruses (Figure 5). The genome of the 14 ranaviruses can be divided into more than 20 locally collinear blocks (LCBs), which were indicated by different colors in the figure. It can be observed that there were 5 types of genomic arrangement in the aligned ranavirus genomes based on the arrangement of LCBs. All the ranaviruses isolated from mandarin fish and largemouth bass, including SCRaV and MSRaV, have the same genomic arrangement and belong to the first type named SCRaV/MSRaV/LMBV-like or Santee-Cooper ranavirus (SCRV), and RGV and FV3 have the same second type of genomic arrangement. ADRV and CMTV possess the third type of genomic arrangement. ATV and EHNV have the fourth type of genomic arrangement. SGIV and GIV have the fifth type of genomic arrangement. LCBs arrangement of SCRaV/MSRaV/LMBV-like viruses was obviously different from the other four types. For example, the LCB at genome regions of about 75–80 kbp in SCRaV/MSRaV/LMBV-like viruses were located at regions of about 16–23 kbp in RGV and FV3, at regions about 93–101 kbp in ADRV and CMTV, and regions of about 103–111 kbp in ATV. The 3′-end of the genome of the FV3-, CMTV-, and ATV-like viruses all correspond to a central region located at 35–37 kbp of genomes from SCRaV and MSRaV. Arrangement of these LCBs revealed the genomic insertion, inversion, and rearrangement among the ranaviruses and also indicated that SCRaV and MSRaV-like viruses have unique genome arrangements in ranaviruses. Thus, combined with the genome type represented by SGIV and GIV, there are 5 genome types in the sequenced ranaviruses.

4. Discussion

Fish ranaviruses are getting more and more attention for the development of the aquaculture industry, such as these infecting fishes of the order Perciformes. However, a detailed analysis of the genome architecture of ranaviruses from Perciformes fish and a comparison with other ranaviruses was lacking. In the present study, based on two newly isolated ranaviruses from mandarin fish and largemouth bass, genome characters of the types of ranaviruses were analyzed.
Sequence comparison showed that there was highly sequence identity between SCRaV and MSRaV, which indicated that the two viruses should belong to one species. Among the eleven proteins that possessed differences between the two viruses, the 79L (predicted neurofilament triplet H1-like protein) of the two viruses had identities lower than 90%, which hinted that the proteins, especially the 79L, could determine the characteristics of the two viruses. We also observed that the proteins among the SCRaV/MSRaV-like viruses isolated in China possessed more sequence identity than that of virus isolates of the USA, and vice versa, especially for six proteins, including a DNA polymerase subunit, which indicated that these proteins may be associated with the regional divergence and replication efficacy of the viruses.
Sequence divergence between the type of ranavirus and other ranaviruses (e.g., FV3/RGV-like, ATV/EHNV-like, CMTV/ADRV-like, and SGIV-like) is relatively high, which indicated that the ranaviruses isolated from mandarin fish and largemouth bass have their own characters. Up to now, reports on gene functions of the type of ranaviruses are few. It could be observed that the MCP of SCRaV and MSRaV have the highest sequence identity with its homolog of other ranaviruses, which indicated the high homology of MCPs among ranaviruses. On the contrary, several proteins possessing low homology with other ranaviruses were found. The viral proteins that could be involved in virus–host interactions all belonged to the low homology proteins, which indicated the adaptation to a specific host.
Genome-wide recombination, deletion, insertion, and inversion have been reported in ranaviruses [6,10,14,30]. Our genome alignment showed the sequence inversion and insertion among different types of ranaviruses. The inversion and insertion may be an adaption of viruses to different hosts or environments, which can be used as the basis to classify different types of ranaviruses and also would help in the identification or prediction of emerging and re-emerging ranaviruses. Combined with the results from sequence identity comparison, genome-wide alignment, and phylogenetic analysis, the SCRaV and MSRaV or SCRV-like viruses constitute a unique type/group in ranaviruses.
NCLDVs usually encode their own proteins to conduct DNA replication and transcription. Our previous study with ADRV and RGV has revealed the replication and transcription machinery of ranaviruses [27]. For DNA replication, the viral DNA polymerase, helicase/primase, PCNA, and SSB should be key components of the replisome. The four proteins were identified in SCRaV and MSRaV encoded proteins (SCRaV 66R, 37R, 100L, and 12L), which indicated that the core components of the replisome of SCRaV and MSRaV were similar with ranaviruses infecting amphibians. Interestingly, domain/motif search showed that two proteins of SCRaV (77L and 82L) contain domains of DNA polymerase III subunits. DNA polymerase III is the main enzyme in bacterial DNA replication [31]. Whether the two proteins participated in ranavirus DNA replication needs to be researched in the future. For DNA transcription, there are 3 predicted RNAP subunits (45R, 74R, and 28R) and 3 possible transcription factors (22L, 40L, and 7R) in SCRaV-encoded proteins, but the number is lower than the need for a complete RNAP in eukaryotes [32,33]. There should be host factors involved in the genome transcription of SCRaV-like viruses, as occurred in ADRV and RGV [27].
To facilitate virus infection, viruses usually encode multiple proteins to regulate cellular processes [34]. Immune responses are important strategies to resist virus infection. It has been reported that two proteins of ranaviruses, the homolog of RNase III and eIF2α, have the ability to regulate the activation of host interferon responses [35,36,37,38]. The two proteins were both identified in SCRaV encoded proteins (23R and 26R), although the eIF2α homolog of SCRaV only has a sequence identity of about 30% with corresponding homologs of other ranaviruses. Other cellular processes include inflammation and apoptosis. SCRaV encodes homologs of LITAF, TNFR, and apoptosis regulator (61L, 72L, 41L, and 70L), which could have functions in the regulation of cell death and inflammation and prompt virus infection, as reported in other ranaviruses [39,40,41]. Interestingly, SCRaV-like ranavirus was found to encode a homolog of insulin-like growth factor (SCRaV 95L). Its homolog in ranaviruses was only found in SGIV, which could modulate cell proliferation and apoptosis [42]. In vitro synthesized viral insulin-like peptides have activities in mammalian cells [43]. However, its function in SCRaV-like viruses in vivo need to be investigated in the future.
It should be noted that there are 5 predicted proteins containing characters of LPXTG-anchored collagen-like adhesins that are mainly found in Enterococci and function as a virulence factor [44]. Whether they also have a function in viral virulence in SCRaV and MSRaV infection remains unknown up to now.
In conclusion, the present study provided a complete genome analysis for SCRaV/MSRaV/LMBV-like ranaviruses, especially the genome architecture and variations compared with other ranaviruses. These results provided new information for understanding the genetic evolution of ranaviruses from fish species and other animals and also facilitated the early warning of fish ranavirus epidemics.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens12050730/s1, Table S1: Virus name and accession number, Table S2: Information of six ranavirus isolates from Siniperca chuatsi and Micropterus salmoides.

Author Contributions

Conceptualization, F.K. and J.-F.G.; methodology, F.K. and X.-D.Y.; writing—original draft preparation, F.K. and X.-D.Y.; writing—review and editing, F.K. and Q.-Y.Z.; supervision, Q.-Y.Z. and J.-F.G.; funding acquisition, Q.-Y.Z. and F.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31972839), and the National Key R & D Plan of the Ministry of Science and Technology of China (2022YFF1000302).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The complete genome sequence of SCRaV and MSRaV have been submitted into NCBI GenBank. The accession number of SCRaV is OQ267588, and MSRaV is OQ267587. Data is contained within the article or supplementary material. The raw data is available upon reasonable request from the corresponding author.

Acknowledgments

We would like to thank Yuan Xiao and Zhen-Fei Xing at the Analysis and Testing Center of Institute of Hydrobiology, Chinese Academy of Sciences for their assistance with transmission electron microscopy analysis.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Cytopathic effect caused by SCRaV and MSRaV in SCSC, EPC, and GSTC cells, and ADRV in GSTC cells (ADRV/GSTC) in different time point. Bar = 100 μm.
Figure 1. Cytopathic effect caused by SCRaV and MSRaV in SCSC, EPC, and GSTC cells, and ADRV in GSTC cells (ADRV/GSTC) in different time point. Bar = 100 μm.
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Figure 2. Ultrastructure observation of (A,C) SCRaV-infected SCSC (48 hpi) and (B,D) MSRaV-infected GSTC cells (48 hpi). N, nucleus. PA, paracrystalline array. CS, Cell shrinkage.
Figure 2. Ultrastructure observation of (A,C) SCRaV-infected SCSC (48 hpi) and (B,D) MSRaV-infected GSTC cells (48 hpi). N, nucleus. PA, paracrystalline array. CS, Cell shrinkage.
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Figure 3. Schematic diagram of the genome organization of SCRaV (A) and MSRaV (B). The SCRaV and MSRaV genome are 99,405 bp and 99,171 bp in size, respectively, and both contain 105 predicted ORFs. The scale is in kilobase pairs. Arrows indicate the size, location, and orientation of the ORFs. The iridovirus core genes and SCRaV/MSRaV specific genes were shown in black and blue color, respectively. There are 12 SCRaV/MSRaV-specific genes that have no homologs in viruses infecting other animals, including a TNFR-like protein encoded by 17L.
Figure 3. Schematic diagram of the genome organization of SCRaV (A) and MSRaV (B). The SCRaV and MSRaV genome are 99,405 bp and 99,171 bp in size, respectively, and both contain 105 predicted ORFs. The scale is in kilobase pairs. Arrows indicate the size, location, and orientation of the ORFs. The iridovirus core genes and SCRaV/MSRaV specific genes were shown in black and blue color, respectively. There are 12 SCRaV/MSRaV-specific genes that have no homologs in viruses infecting other animals, including a TNFR-like protein encoded by 17L.
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Figure 4. Phylogenetic analysis of the evolutionary relationship among the two ranaviruses and other iridovirus strains based on 26 iridoviral core protein sequences. The two viruses in the present study are indicated by yellow triangles. The ranavirus isolates from mandarin fish and largemouth bass clustered in a clade. The sequences used in the analysis are collected in Table S1.
Figure 4. Phylogenetic analysis of the evolutionary relationship among the two ranaviruses and other iridovirus strains based on 26 iridoviral core protein sequences. The two viruses in the present study are indicated by yellow triangles. The ranavirus isolates from mandarin fish and largemouth bass clustered in a clade. The sequences used in the analysis are collected in Table S1.
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Figure 5. Whole genome alignment of SCRaV, MSRaV, and ranaviruses from other animals. Each genome displays several locally collinear blocks showing in different colored blocks. Related blocks with similar colors and patterns were connected by lines with different colors. There are the following five groups of genomic arrangements: SCRaV and MSRaV-like, RGV and FV3, ADRV and CMTV, ATV and EHNV, SGIV and GIV. The two viruses in the present study are indicated by yellow triangles.
Figure 5. Whole genome alignment of SCRaV, MSRaV, and ranaviruses from other animals. Each genome displays several locally collinear blocks showing in different colored blocks. Related blocks with similar colors and patterns were connected by lines with different colors. There are the following five groups of genomic arrangements: SCRaV and MSRaV-like, RGV and FV3, ADRV and CMTV, ATV and EHNV, SGIV and GIV. The two viruses in the present study are indicated by yellow triangles.
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Table
Table 1. Characterization of predicted open reading frames (ORFs) of SCRaV and MSRaV a. ORFs of SCRaV were used as reference (the first column).
Table 1. Characterization of predicted open reading frames (ORFs) of SCRaV and MSRaV a. ORFs of SCRaV were used as reference (the first column).
ORF/aaNucleotide PositionPredicted Function/Conserved DomainkDaMSRaV c
(OQ267587)		MFRV c
(MG941005)		LMBV-G c
(MW630113)		LMBV-A c
(MK681855)		LMBV-P c
(MK681856)		ADRV c
(KC865735)		RGV c
(JQ654586)		FV3 c
(AY548484)		EHNV c
(MT510742)		SGIV c
(NC_006549)
ORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID dORF/AA%ID d
1L/345 b1–1038myristylated membrane protein37.21L/345100123L/35497.54L/3451001L/35496.11L/35496.12L/32575.22L/32375.22L/32075.21L/35074.419R/34270.5
2L/2901044–1916hypothetical protein 33.22L/290100124L/2901005L/2901002L/29098.62L/29098.63L/29145.13L/292443L/279442L/27944.718R/28536.7
3R/4041943–3157hypothetical protein44.73R/404100NA/NANA6R/4041003R/40499.53R/40499.54R/40451.44R/40451.23R/43851.23R/40450.816L/41339
4R/2533184–3945N-terminal immunoglobulin (Ig)-like domain27.34R/2531001R/2531007R/2531004R/25398.44R/16598.2NA/NANANA/NANANA/NANANA/NANANA/NANA
5R/543982–4146TM6.15R/541002R/54100NA/NANANA/NANANA/NANA5R/6045.55R/6047.74R/6047.74R/6048.815L/5945.7
6R/1414187–4612hypothetical protein 15.56R/1411003R/1411008R/1411005R/14197.95R/14197.980L/13937.633R/10435.431R/13937.667R/13937.614L/14133.1
7R/6344627–6531transcription termination factor Rho67.77R/62498.44R/59894.29R/39689.46R/63396.36R/62194.479L/6405134R/6445132R/6295068R/6584912L/102431.1
8R/626581–6769TM6.88R/621006R/62100NA/NANANA/NANANA/NANA78L/6362.135R/6362.133R/6363.869R/6363.811L/6253.2
9R/1196872–7231L protein-like protein, TM12.89R/1191008R/11910011R/991007R/99997R/999977L/10655.236R/10653.134R/10653.170R/10752.59L/15442.5
10L/1637277–7768hypothetical protein16.210L/1631009L/16698.212R/24426.354/28842.9NA/NANANA/NANA72R/11547.736L/20744.8NA/NANA7L/30729.5
11R/2457500–8237LPXTG-anchored collagen-like adhesin24.411R/24510010R/24898.812R/24498.89R/10246.69R/10246.675L/14454.638R/9139.265L/545447L/11251.48L/23040.5
12L/2618285–9070p31K protein 29.312L/26110011L/26110013L/26110010L/26299.210L/26299.285L/2617727R/2617725R/2627760R/304776R/25964.4
13R/3749190–10,314hypothetical protein42.313R/37410012R/37410014R/37410011R/37498.911R/37498.9NA/NANANA/NANANA/NANANA/NANA4L/36522.3
14L/35410,373–11,437hypothetical protein39.214L/35410014L/35410015L/35410012L/35499.412L/35499.460R/23765.952L/35564.552L/35564.854R/35564.83R/38155.8
15L/7111,525–11,740hypothetical protein7.615L/71100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
16R/503 b11,706–13,217myristylated membrane protein53.116R/50310017R/50310016R/50399.813R/50399.213R/50399.258L/52263.753R/52263.353R/52263.553L/52363.988L/50655.4
17L/19113,299–13,874Tumor necrosis factor receptor20.517L/19110019L/19110017L/19110014L/19196.314L/19196.3NA/NANANA/NANANA/NANANA/NANANA/NANA
18R/22013,940–14,602DNA methyltransferase25.418R/22010020R/22010018R/22010015R/22097.715R/22097.724L/21465.390R/21464.883R/21465.320L/21465.3NA/NANA
19R/19214,587–15,165Methylase of polypeptide chain release factors20.519R/19210021R/17710019R/17710016R/17794.916R/17794.9NA/NANANA/NANANA/NANANA/NANANA/NANA
20R/19715,271–15,864hypothetical protein2220R/19710022R/19710020R/19710017R/1969717R/19697NA/NANANA/NANANA/NANANA/NANANA/NANA
21L/14715,925–16,368immediate early protein ICP-1816.421L/14710023L/14710021L/14710018L/14798.618L/14798.626L/15739.989R/15740.582R/15740.522L/15739.286R/15449
22L/91 b16,431–16,706transcription elongation factor S-II10.322L/9110024L/9198.9NA/NANANA/NANANA/NANA27L/9256.388R/9256.381R/9256.323L/9255.285R/9251.7
23R/385 b16,736–17,893RNAseIII42.223R/38510025R/38510022R/38510019R/37099.219R/38599.228R/37260.787L/37161.780L/37161.724R/37261.484L/37552.3
24L/46417,939–19,333ATPase-dependent protease52.424L/46410026L/46410023L/46499.820L/46499.620L/46499.629L/55852.386R/57252.679R/57252.825L/64552.883R/44541.6
25R/10119,415–19,720hypothetical protein11.325R/10110027R/10110024R/10110021R/10110021R/101100NA/NANANA/NANANA/NANANA/NANANA/NANA
26R/27019,764–20,576putative eIF-2 alpha-like protein30.626R/27010028R/27010025R/27010022R/27098.222R/27097.884L/23330.728R/6931.126R/7631.461R/25930.7NA/NANA
27R/957 b20,666–23,539tyrosine kinase107.227R/95710029R/95710026R/95710023R/95799.723R/95799.783L/83754.629R/97053.627R/9705362R/97054.178L/79045.9
28R/15923,550–24,029DNA-directed RNA polymerase II subunit1828R/15910030R/15910027R/15910024R/15910024R/15910082L/17545.130R/16243.828R/16243.863R/16944.1160L/16239.7
29R/24824,149–24,895capsid maturation protease27.829R/24810032R/24810028R/24810025R/24899.225R/24899.2NA/NANANA/NANANA/NANA65R/27427156L/27023.9
30R/15924,994–25,473hypothetical protein17.930R/15998.733R/15910029R/15910026R/15896.626R/15896.681R/9832.131L/9832.729L/9833.766R/16136.3158L/13838.2
31L/17325,518–26,039hypothetical protein19.131L/17310034L/17310030L/17310027L/11489.327L/17399.450L/18467.461R/18467.4NA/NANANA/NANA157R/17459.2
32L/24026,072–26,794LPXTG-anchored collagen-like adhesin Scl2/SclB22.532L/24010035L/24099.631L/24099.628L/24097.528L/24097.175L/14436.7NA/NANA65L/5451.140R/24040.556R/24635.8
33L/25726,802–27,575LPXTG-anchored collagen-like adhesin Scl2/SclB24.833L/25710036L/25710032L/25799.629L/25798.829L/25798.875L/14435.138R/914565L/5455.139R/18340.845L/24236.1
34L/17727,575–28,108hypothetical protein18.734L/17710037L/17710033L/17710030L/17793.830L/17793.2NA/NANANA/NANANA/NANANA/NANANA/NANA
35L/223 b28,163–28,834hypothetical protein25.735L/22310038L/22310034L/22310031L/22310031L/22310089R/21972.923L/21972.921L/21972.986R/21972.454R/21568.9
36R/56328,859–30,550hypothetical protein63.836R/56310039R/56310035R/53910032R/53998.732R/53998.7NA/NANANA/NANANA/NANANA/NANANA/NANA
37R/955 b30,673–33,540hypothetical protein107.537R/95599.940R/95599.836R/95599.633R/95599.533R/95599.588L/9757524R/97575.222R/97376.185L/9737652L/96868
38R/7033,573–33,785TM7.738R/7010042R/70100NA/NANANA/NANANA/NANA99L/7052.512R/7052.511R/7054.296L/7052.5103R/9738.8
39L/297 b33,832–34,725hypothetical protein3339L/29710044L/29710037L/29710034L/29799.734L/29799.798R/29765.313L/2976612L/29765.795R/29766.7118R/31959.7
40L/253 b34,770–35,531replicating factor29.640L/25310045L/25399.638L/25399.235L/25399.635L/25399.61R/25661.51R/25661.51R/25661.9100R/25662.4116R/25853.5
41L/17135,589–36,104myeloid cell leukemia protein18.741L/17110046L/17110039L/15010036L/15099.336L/15099.3101R/13730.6105R/13730.897R/13730.899R/13730.5115R/15224.5
42L/22636,107–36,787hypothetical protein24.742L/22610047L/22610040L/21410037L/21499.137L/21499.1100R/22839.6104R/22340.596R/22339.698R/22840.5111R/25541
43L/7836,866–37,102TM8.743L/7810048L/78100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
44L/10337,104–37,415TM1144L/10310049L/10310038L/25310038L/10310038L/103100NA/NANANA/NANANA/NANANA/NANANA/NANA
45R/1354b37,201–41,265DNA-dependent RNA polymerase a subunit147.145R/135410050R/135499.941R/135399.939R/126399.739R/126399.79R/129464.99R/129464.78R/129364.87R/130364.9104L/126862.4
46R/6941,298–41,507hypothetical protein6.246R/55100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
47R/13641,525–41,935hypothetical protein14.947R/13610051R/136100NA/NANANA/NANANA/NANA55L/37952.657R/37951.655R/37952.4NA/NANANA/NANA
48L/40141,582–42,787helicase-like protein44.648L/40010052L/40110042L/40110040L/40198.540L/40198.554R/4315556L/43155.255L/4315551R/43154.7152R/41248
49L/4942,794–42,943TM5.249L/49100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
50R/13642,982–43,392hypothetical protein15.550R/13610053R/13610043R/13610041R/13698.541R/13698.552L/13437.859R/13438.6NA/NANA49L/13437.8151L/19539.3
51R/492 b43,401–44,879Serine/threonine protein kinases53.851R/49210054R/49299.844R/49299.842R/49299.242R/49299.251L/49845.260R/49845.657R/49845.648L/49846.3150L/50835.4
52L/17344,939–45,460hypothetical protein19.652L/17310056L/17310045L/17210043L/17299.443L/17299.4NA/NANANA/NANANA/NANA33L/16049148R/15940.8
53R/6045,433–45,615hypothetical protein6.553R/6010057R/60100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
54L/7145,634–45,849hypothetical protein7.954L/7110058L/71100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
55L/12345,885–46,256hypothetical protein13.755L/12310059L/12310046L/12310044L/12399.244L/12399.238L/1244077R/12439.270R/1244035L/12241.1NA/NANA
56L/7946,264–46,503TM8.556L/7910060L/79100NA/NANANA/NANANA/NANA39L/8864.876R/8863.469R/8863.436L/8863.4143L/7951.9
57R/21646,591–47,241hypothetical protein2457R/21610061R/21610047R/21699.545R/21610045R/21610040R/10741.175L/8843.1NA/NANA37R/23435.9145R/16520.5
58L/32447,338–48,312NTPase helicase-like protein36.158L/32410063L/32410048L/32410046L/32499.746L/32499.735R/35044.380L/32444.673L/32444.631R/32444.9146L/32447.2
59L/40548,433–49,650hypothetical protein44.659L/40510065L/40510049L/40510047L/40510047L/405100NA/NANANA/NANANA/NANANA/NANA147L/34424.5
60L/37749,677–50,810hypothetical protein41.360L/37710067L/37710050L/37710048L/37798.448L/37798.434R/44741.881L/36442.774L/37042.330R/39364.6137R/46141.1
61L/8550,859–51,116lipopolysaccharide-induced TNF-alpha factor-like protein9.561L/8510068L/85100NA/NANANA/NANANA/NANA33R/8467.182L/8466.375L/8465.9NA/NANA136R/10466.7
62R/7351,175–51,396hypothetical protein8.262R/7310069R/73100NA/NANANA/NANANA/NANA32L/7360.383R/7358.976R/7358.928L/7360.3119R/8330.4
63L/20851,446–52,072hypothetical protein23.763L/20810070L/20810051L/20810049L/20898.649L/20898.630R/21239.185L/22439.578L/21239.526R/25538.6122L/21029.1
64L/37152,139–53,254hypothetical protein42.664L/37110071L/37110052L/37110050L/37199.550L/37199.248R/35230.962L/3523259L/3523245R/35232.3123L/36230.5
65R/18253,321–53,869hypothetical protein20.465R/18210072R/18210053R/18210051R/18298.951R/18298.9NA/NANANA/NANANA/NANANA/NANA126R/18544.4
66R/1004 b53,929–56,943DNA polymerase113.766R/100410074R/100410054R/100410052R/100499.852R/100499.847L/101375.863R/101375.960R/101375.844L/101375.6128R/10968.8
67L/24357,000–57,731LPXTG-anchored collagen-like adhesin Scl2/SclB23.467L/24310075L/24310055L/23810053L/23897.553L/23897.575L/14442.938R/9155.6NA/NANA47L/11246.455R/24059.6
68L/28857,737–58,603LPXTG-anchored collagen-like adhesin Scl2/SclB28.968L/28899.776L/28810056L/28810054L/28896.554L/17389.575L/1444038R/9156.3NA/NANA39R/18358.8112R/35553.1
69L/387 b58,652–59,815ribonucleotide reductase beta subunit44.169L/38710077L/38710057L/38710055L/38710055L/38710042R/38777.973L/38777.767L/38777.738R/38777.747L/38474.9
70L/9159,932–60,207caspase recruitment domain protein10.270L/9110078L/91100NA/NANANA/NANANA/NANA43L/9539.868R/9542.164R/9542.141L/9542.148L/9137.7
71L/14160,269–60,694deoxyuridine 5’-triphosphate nucleotidohydrolase15.171L/14199.380L/14110058L/14110056L/14110056L/14110044L/16457.367R/16457.363R/16457.342L/16457.349L/15557.9
72L/23760,795–61,508tumor necrosis factor receptor, TM25.472L/23710081L/23710059L/23710057L/23799.657L/23799.6NA/NANANA/NANANA/NANANA/NANA51L/23136.7
73L/17861,588–62,124hypothetical protein2073L/17810083L/17810060L/17810058L/17899.458L/17899.445L/17841.566R/17840.6NA/NANANA/NANA75R/17838.9
74R/1094 b62,188–65,472DNA-dependent RNA polymerase II120.874R/109410084R/109410061R/109410059R/109499.659R/109499.646R/122170.465L/122169.962L/122169.943R/12277073L/110365.8
75L/356 b65,516–66,586DNA repair enzyme RAD24075L/35610085L/35610062L/35610060L/35698.960L/35698.912L/36359.9102R/36359.995R/36359.910L/36459.597L/38259.4
76R/154 b66,655–67,119hypothetical protein17.776R/15410086R/15410063R/15499.461R/15410061R/15410013R/15576.6101L/15576.694L/15576.611R/15576.698R/26767.6
77L/28467,653–68,507DNA polymerase III subunits gamma and tau29.177L/28410089L/28410064L/28499.762L/26391.162L/26391.467R/2903345L/38337.342L/8534.778L/2853320L/32235.9
78L/13668,563–68,973hypothetical protein15.678L/1361009L/13610065L/13610063L/13698.563L/13698.566R/13662.946L/13662.145L/13662.1NA/NANA21L/13960.3
79L/28769,019–69,882neurofilament triplet H1-like protein30.879L/1978692L/1918666L/1368664L/3119764L/23982.665R/16953.447L/14452.946L/8137.180L/20355.322L/16644.3
80L/13669,916–70,326hypothetical protein1680L/13699.393L/13698.567L/13698.565L/13699.365L/13699.364R/1383648L/13838.147L/13838.181L/13834.524L/15127.9
81L/7370,366–70,587hypothetical protein8.681L/7310094L/73100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA
82L/56670,628–72,328SAP domain-containing protein61.782L/60292.595L/60493.268L/32090.466L/56088.667L/38395.662R/50848.850L/49947.749L/24949.283L/54150.825L/51054.5
83R/56672,401–74,101hypothetical protein63.283R/56610096R/56610070R/56610067R/56699.368R/56699.161L/56146.251R/56146.651R/56146.684R/56146.626R/56639.9
84L/14474,152–74,586hypothetical protein15.884L/14410097L/14410071L/14410068L/14410069L/14410090L/14860.122R/17260.820R/14860.188L/14961.838L/17044.7
85L/879 b74,614–77,253TM96.386R/11399.999R/113100NA/NANANA/NANANA/NANA91L/96041.621R/85241.819R/85140.489L/90741.639L/105138.7
86R/11376,955–77,2962-cysteine adaptor domain protein1285L/87910098L/87999.972L/87999.969L/87999.270L/87999.392R/7842.420L/7946.218L/7848.5NA/NANANA/NANA
87R/50377,329–78,840hypothetical protein54.387R/503100100R/50310073R/50310070R/50399.871R/50399.893R/50254.519L/50254.917L/50255.190R/50254.743R/66739.3
88R/28178,924–79,769hypothetical protein3188R/281100101R/28110074R/28110071R/28110072R/28110095L/21645.717R/27546.716R/27546.191R/29147.4132R/27539.7
89L/315 b79,982–80,929ABC-ATPase35.889L/315100103L/29310075L/29310072L/30099.373L/30099.396L/31580.916R/31580.915R/32280.992L/30880.9134L/32371.2
90L/11980,896–81,255hypothetical protein13.790L/119100104L/11910076L/11910073L/11999.274L/11999.297L/11952.115R/8460.814R/11952.193L/11852.1135L/11246.7
91L/1162 b81,325–84,813TM129.791L/1162100105L/116210077L/116299.974L/116299.875L/116299.868L/11656044R/116560.141R/116559.877R/11656057L/116852
92L/9184,867–85,142TM10.292L/91100107L/91100NA/NANANA/NANANA/NANANA/NANANA/NANA40R/1284475R/26844NA/NANA
93L/10685,201–85,521hypothetical protein12.393L/106100108L/10610078L/10110075L/10610076L/106100NA/NANANA/NANANA/NANANA/NANANA/NANA
94L/56285,585–87,273ribonucleotide reductase alpha subunit62.994L/562100109L/56299.879L/56210076L/56299.677L/56299.671L/56578.841R/56578.538R/56578.173R/25478.964R/57270.3
95L/7987,364–87,603insulin-like growth factor8.495L/79100110L/79100NA/NANANA/NANANA/NANANA/NANANA/NANANA/NANANA/NANA62R/25638.7
96L/205 b87,643–88,260NIF/NLI interacting factor23.496L/205100111L/20510080L/20510077L/20599.578L/20599.572L/21359.240R/21359.237R/20959.272R/21159.261R/20450.5
97L/949 b88,278–91,127NTPase106.497L/949100112L/94910081L/94910078L/94999.479L/94999.410L/94868.210L/94868.29L/94868.28L/94868.160R/97060.6
98R/13291,149–91,547TM14.698R/132100113R/13210082R/13210079R/13210080R/13210011R/1374611R/13743.110R/13743.19R/13747.559L/14632.4
99L/189 b91,617–92,186deoxyribonucleoside kinase20.899L/189100115L/18910083L/18910080L/18999.581L/18999.522L/19552.792R/19552.285R/19552.218L/19555.267L/19153.6
100L/242 b92,238–92,966hypothetical protein25.8100L/242100116L/24210084L/24210081L/24299.282L/24299.223L/24560.591R/24561.384R/24560.919L/26060.968L/27250
101L/59493,049–94,833hypothetical protein66.4101L/59499.8117L/59410085L/59410082L/59499.583L/59499.520R/60534.994L/59333.687L/60534.917R/61734.669L/54832
102R/147 b94,867–95,310thiol oxidoreductase 16.6102R/147100118R/14710086R/14710083R/14799.384R/14799.319L/15062.395R/15061.688R/15061.616L/1506170R/15254.6
103R/41395,316–96,557hypothetical protein48.5103R/39896.4119R/36888.91R/38793.784R/39081.885R/39083.818L/4143696R/38137.489R/38838.115L/36837.171R/27438.8
104R/463 b96,670–98,061major capsid protein50.1104R/463100120R/4631002R/46310085R/46399.486R/46399.417L/4638497R/46383.690R/46383.214L/46383.472R/46373.7
105R/382 b98,174–99,322immediate early protein ICP-46 43.9105R/382100121R/3821003R/38210086R/38299.787R/38299.716L/39557.198R/39557.691R/39557.913L/39557.6162L/38250.7
a TM, transmembrane domain; aa, number of amino acids of each protein; kDa, molecular mass of each protein as predicted by Detaibio website tools; ID, identity; NA, not annotated (denotes no corresponding homologous ORF in the genome). MFRV, mandarin fish ranavirus; LMBV-G, largemouth bass virus strain GDOU; LMBV-A, largemouth bass virus strain Alleghany; LMBV-P, largemouth bass virus strain Pine; ADRV, Andrias davidianus ranavirus; RGV, Rana grylio virus; FV3, frog virus 3; EHNV, epizootic hematopoietic necrosis virus; SGIV, Singapore grouper iridovirus. b Core genes of iridoviruses. c Corresponding homologous ORFs in the indicated virus genomes based on BLASTP analysis. d Amino acid identities were calculated using the ClustaW method in the MegAlign program.
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MDPI and ACS Style

Yu, X.-D.; Ke, F.; Zhang, Q.-Y.; Gui, J.-F. Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass. Pathogens 2023, 12, 730. https://doi.org/10.3390/pathogens12050730

AMA Style

Yu X-D, Ke F, Zhang Q-Y, Gui J-F. Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass. Pathogens. 2023; 12(5):730. https://doi.org/10.3390/pathogens12050730

Chicago/Turabian Style

Yu, Xue-Dong, Fei Ke, Qi-Ya Zhang, and Jian-Fang Gui. 2023. "Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass" Pathogens 12, no. 5: 730. https://doi.org/10.3390/pathogens12050730

APA Style

Yu, X. -D., Ke, F., Zhang, Q. -Y., & Gui, J. -F. (2023). Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass. Pathogens, 12(5), 730. https://doi.org/10.3390/pathogens12050730

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