Volume 8, Issue 3 (Journal of Clinical and Basic Research (JCBR) 2024)
jcbr 2024, 8(3): 14-22 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Askari F S, Mohebbi A. Metavirome survey of eight ray-finned fishes: Domination of giant viral members from pandoravirus and megaviricetes. jcbr 2024; 8 (3) :14-22
URL: http://jcbr.goums.ac.ir/article-1-452-en.html
URL: http://jcbr.goums.ac.ir/article-1-452-en.html
1- Vista Aria Rena Gene Corporation, Gorgan, Golestan Province, Iran
2- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran ,alirezaa2s@gmail.com
2- Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran ,
Full-Text [PDF 4535 kb]
(308 Downloads)
| Abstract (HTML) (1026 Views)
Discussion
Numerous viruses remain unidentified by nature due to our inability to cultivate them in laboratory conditions. However, viral typing does not solely rely on cultivation. Thanks to continuous advancements in scientific fields and the discovery of various techniques and methods, obtaining essential information about microorganisms has become more feasible. Metagenomics employs techniques that utilize genetic information from microorganisms in their natural habitats, providing insights into their evolutionary history and other pertinent information without the need for laboratory cultivation (31). In this study, the metavirome of eight ray-finned fish species was investigated. The results revealed that Pandoravirus accounted for 31%, Herpesvirales for 23%, and Bamfordvirae for 10% of the total viral sequence fingerprints. Notably, these viruses were prevalent in ray-finned fish populations distributed across the world, primarily in freshwater and riverine environments. Below is a brief overview of the eight fish species under investigation.
Acipenser schrenckii (Amur sturgeon) is known as a living fossil with a history dating back approximately 200 million years (32,33). The Amur sturgeon is a large river species, capable of reaching up to 3 meters in length and weighing up to 100 kilograms. It displays strong adaptability and can thrive in temperatures ranging from 0°C to 33°C, becoming active within the range of 13°C to 26°C. The Amur sturgeon holds significance for genetic resource conservation and is a valuable food source due to its high nutritional value in freshwater aquaculture (34-36).
Odontobutis haifengensis is an endangered species native to China and inhabits river environments (37,38). The Odontobutis genus is distributed in freshwater regions of Eastern Asia and the Yalu River in China (39). Rhinogobius giurinus is an amphidromous goby species inhabiting East Asia's marine, brackish, and freshwater environments. This ornamental fish feeds on aquatic insects, invertebrates, small fishes, zooplankton, phytoplankton, and plant detritus (40).
Hiodon tergisus is a North American freshwater fish that prefers spawning in temperatures between 10-15°C. This species typically grows to about 130 mm and weighs approximately 24 grams. Its diet includes plankton, insects, mollusks, crayfish, and small fishes (41–44). Furthermore, Polyodon spathula (Paddlefish) is considered a living fossil, native to the Northern Hemisphere, and inhabits riverine streams, broad rivers, and backwaters. It can grow to weigh over 90 kg and live up to 60 years. Paddlefish is also known for its filter-feeding behavior and has applications in aquaculture (45-47). Notably, this fish is valued for its high nutritional content, particularly its caviar and meat (48).
Denticeps clupeoides is a small teleost species of fish found in the freshwater regions of Western Africa. It holds importance for understanding relationships among lower teleosts (49,50). Additionally, Rutilus rutilus (Common roach) primarily inhabits freshwater environments and can grow up to 500 mm long. These fish are endemic to various regions in Europe and western Asia (51,52). Common roaches have a diverse diet that includes various food sources in their natural environment (53,54), and they are also a significant food source for humans.
Viruses are pervasive and influential in aquatic environments, significantly affecting microbial community mortality and the marine food cycle. Moreover, viruses can control planktonic population composition through infection (55), and they can acquire genes from their hosts, which is a common mechanism observed in viruses, particularly giant viruses (56). Host-switching, a phenomenon identified in the evolutionary history of viruses, further demonstrates the intricate links between viruses and their hosts (57).
Accordingly, one fascinating outcome of this meta-virome study was the high prevalence of Pandoravirus and Megaviricetes, both of which belong to the group of viruses known as giant viruses. These double-stranded DNA viruses were first identified in Acanthamoeba in 2003 (58). Giant viruses are found in terrestrial and aquatic environments and often interact with humans. Some giant viruses have been linked to human-associated diseases (59-61). The results of this study diverge from previously published articles, as they indicate a higher abundance of RNA viruses in aquatic environments (57).
In a study by Costa et al. on the virome of ten freshwater ray-finned fish species across seven taxonomic orders and thirteen locations within the Murray-Darling Basin in Australia, the most abundant vertebrate-associated viruses in ray-finned fishes belonged to the Arenaviridae and Astroviridae families (62). Additionally, studies by Geoghegan et al. demonstrated that the Astroviridae family had the highest abundance compared to other viral families in fish viromes (7,63,64). Despite variations in results, the viruses identified in fish were related to vertebrates and, in some cases, human-associated diseases. The presence of RNA viruses, DNA viruses, and giant viruses in fish populations and their connection to humans suggest potential evolutionary links between these viruses.
While information about viruses that infect fish remains limited, the high diversity among fish species and their significant role in vertebrate evolution implies that they might be a source of emerging viruses. Moreover, the exchange of genetically adapted viruses or quasispecies between different environments can lead to cross-species transmission, particularly in DNA viruses that exhibit high recombination rates (65). Virus genetic changes can facilitate such transmissions, making it crucial to consider the potential threats posed by aquatic viromes to public health. Although this study did not assess the completeness and integrity of human-associated viral genomes and giant viruses, mapping reference genomes will address this in future studies.
Conclusion
Population growth drives the need for more food resources, with seafood, especially fish, being one of the primary sources. The key point is that various viruses bolster food sources with a robust evolutionary history. As the findings have shown, there is a high abundance of giant DNA viruses in some ray-finned fish, many of which are consumed by humans. Therefore, further investigation into the virome of these aquatic species is crucial. This research would enhance our understanding of the potential human-associated illnesses these viruses might cause.
Acknowledgement
Not applicable.
Funding sources
Not applicable.
Ethical statement
Not applicable.
Conflicts of interest
The authors declare that they have no competing interests.
Author contributions
FSA has been involved in data preparation, FASTQ file quality checks and assembly, and has written the first draft of the manuscript. AM has been working on viral taxonomy generation for ray-finned fish, data visualization, detection of antivirus signatures in unassigned viral taxa, and has served as the research manager.
Full-Text: (277 Views)
Introduction
Fish represent one of the major sources of food worldwide. Aquatic animal species in marine ecosystems are exposed to a wide variety of microorganisms (1). Viruses, as the most abundant organisms on Earth (1,2), significantly influence the nutrient and carbon cycles in aquatic environments (3). A study conducted by Breitbart et al. in 2002 on two uncultured marine viral communities revealed that over 65% of the microbial diversity in aquatic communities is uncharacterized, with most of the remaining diversity comprising viruses. Furthermore, the most abundant viral genome constitutes only 2-3% of the total population in marine communities, indicating high viral diversity (4).
Phylogenetic classification has identified approximately 72 orders (And 79 suborders) and 514 families of bony fish (5). Calculations based on fish population density have shown that some fish shoals can consist of nearly three billion fish in a single school, making this an exceptionally dense population among vertebrates. Consequently, this large number of fish can harbor an increasing number of viruses compared to other vertebrates. Viruses are horizontally transmitted between fish, often through contaminated feces (6). A meta-transcriptomics study conducted by Geoghegan et al. in 2018 on four fish species demonstrated that densely shoaling fish had the highest number of viruses compared to less social fish species. Interestingly, identifying the number of potential novel viruses in fish, as previously detected in mammals and birds, may indicate cross-species transmission (7), which can occur through the horizontal transmission of viruses by birds or other fish predators.
Actinopterygii, known as ray-finned fishes, encompasses more than 32,000 species (5) and represents nearly half of the living vertebrate species within the Neopterygii (Modern ray-fins) sub-class. Neopterygii is the most species-rich clade of bony fishes (>99.9%) (8,9) and includes members such as Ginglymodi, Halecomorphi, and Teleostei. Teleostei, with the most species, is the dominant group of modern Actinopterygians. Non-teleost Actinopterygians are often called living fossils (8). Ray-finned fishes exhibit a remarkable ability to adapt to different aquatic environments, including deep ocean trenches, high mountain streams, and extreme habitats with conditions like acidity, subzero temperatures, hypersalinity, hypoxia, temporariness, and fast-flowing water, with a history dating back over 400 million years (10). It is worth noting that, in addition to their lengthy evolutionary history, ray-finned fishes are crucial for human food consumption. In 2010, it was estimated that 80.6% of fish catches were attributed to this group (11).
The extensive diversity of ray-finned fishes makes them a potential source of viral species. The presence of billions of viral particles per milliliter of ocean water, along with the significant role of viruses in causing mortality in aquatic environments, highlights their capacity to influence population structures in the ocean (12,13). Fish, being a primary source of human food, are in direct contact with ocean water and can carry various potentially new and emerging viruses (14-17). Viral populations can lead to numerous infections in marine environments, emphasizing the importance of studying the presence of viruses in fish (Suttle, 2007). A valuable approach for assessing the diversity and richness of environmental virus samples is metagenomics (2), which conducts a culture-independent analysis to survey the genetic content of all microbial organisms in an environmental sample using biotechnological and bioinformatics methods. Additionally, metavirome evaluation assesses the metagenome of the entire viral population in a single sample (18,19).
The primary aim of this study was to investigate the genome fingerprints of viral genomes that could potentially pose a threat to public health. Furthermore, we discuss the possible evolutionary role of viral diversity in marine ecosystems and its impact on the emergence of viral outbreaks.
Methods
Recovery of metagenomics resource
Metadata for the metagenome were retrieved from the Sequence Read Archive (SRA) (20) using the Galaxy server (21), as previously described (22). The data were obtained from a BioProject focused on ray-finned fishes with the accession number PRJNA493014, which included eight SRA experiments. The SRA accession numbers were as follows: SRR7903826 for Acipenser schrenckii (Amur sturgeon), SRR7903827 for Odontobutis haifengensis, SRR7903828 for Odontobutis yaluensis (Dark sleeper), SRR7903829 for Rhinogobius giurinus, SRR7903830 for Hiodon tergisus (Mooneye), SRR7903831 for Polyodon spathula (Paddlefish), SRR7903832 for Denticeps clupeoides (Denticle herring), and SRR7903833 for Rutilus rutilus (Common roach).
Assembly and quality control of raw FASTQ files
Illumina sequencing files were processed to remove chimeric sequences, correct barcode errors, and eliminate duplications using the Trimmomatic tool (23). Single-ended SRA entries containing FASTQ files were assembled using Unicycler (24) with the default package settings. Contig quality was assessed using the Quality Assessment Tool (25).
Metavirome classification and taxonomic affiliation
Reads from each sequencing run were aligned to genomic sequences in the viral database using the Kraken taxonomic assignment tool (26). The number of reads mapped to the reference was counted using the same Kraken tool. Additionally, classified data were visualized using Krona (27,28). The root of the chart was set as viral families, and the distribution of each viral taxon was estimated based on the depth of sequence reads.
Mapping unidentified sequence reads to the giant viral database
A pooled set of unassigned reads from the genomes of all eight ray-finned fish species was used to identify giant viral fingerprints. The GiantVirusFinder package was employed to identify the number of sequence reads that matched the genomes of 288 known giant viruses (29). The package supports a fast-greedy alignment algorithm for identifying hits to giant viruses (30). The source code was modified to support blast version 2.8.1+, and the expected threshold was adjusted to 0.1.
Results
Metavirome survey
This study investigated metagenomic data from eight ray-finned fish species for their virome. Overall, 2,228,888 sequence reads were assigned to the viral kingdom. A pooled genome assay revealed a high abundance of Pandoravirus (31%), Herpesvirales (23%), and Bamfordvirae (10%) among all virus kingdoms. The following sections will present the virome of each ray-finned fish. The study results presented here are taxonomically assigned. For some groups of viruses that lack supergroup classifications like class, order, or family, we have defined them in their respective sections. Consequently, the genus Pandoravirus, the order Herpesvirales, the family Polydnavirus, and the kingdom Bamfordvirae are the root classifications for their respective clades, as shown in Figure 1.
Diversity of viral species in Acipenser Schrenckii
According to viral taxonomy, the metagenomics data of Acipenser schrenckii comprised 464,973 classified reads and 91,977 unclassified reads. A. schrenckii included three main clades: Pandoravirus, Herpesvirales, and Bamfordvirae, accounting for 5% of the total virome. As illustrated in Figure 1, the genus Pandoravirus made up 34% of all viruses, followed by Herpesvirales at 28%. The most prevalent Pandoraviral species were P. malecodensis (28%), P. dulcis (24%), P. neocaledonia (10%), P. salinus (9%), P. quercus (4%), and P. inopinatum (2%). Unclassified Pandoravirus species constituted 24% of this lineage (Figure 1).
The order Herpesvirales, with 131,119 reads, accounted for 28% of A. schrenckii's virome. In addition to Alloherpesviridae, the Herpesviridae family was represented by some members of human herpesviruses. Specifically, the subfamily Betaherpesvirinae (3% of all viruses) included Roseolovirus (41% of Betaherpesvirinae) and Cytomegalovirus (22% of Betaherpesvirinae), both responsible for known human-associated viral pathogens. Other human-associated viruses from the family Herpesviridae included Human alphaherpesvirus 2 (1%) within the subfamily Alphaherpesvirinae, Human betaherpesvirus 7 (5%), Human betaherpesvirus 6B (1%), and Human betaherpesvirus 6A (0.1%) within Betaherpesvirinae, and Human gammaherpesvirus 8 (0.4%) belonging to the Gammaherpesvirinae subfamily.
The kingdom Bamfordvirae and its order Nucleocytoviricota comprised two major clades of large viral families: Megaviricetes (65%) and Poxviridae (26%). The class Megaviricetes included three families, namely Phycodnaviridae (71%), Mimiviridae (20%), and Pimascovirales (9%). The family Mimiviridae encompassed two genera, Mimivirus (24%) and Cafeteriavirus (11%), along with five different species: Moumouvirus (58%), Megavirus chiliensis (22%), Acanthamoeba polyphaga mimivirus (2%), Cafeteria roenbergensis virus (11%), and Yellowstone lake mimivirus (5%).
Viral population of Odontobutis haifengensis
In the virome of O. haifengnis, the most abundant viral orders were Herpesvirales (19%), Pandoravirus (17%), Orthornavirae (10%), and Bamfordvirae (10%). The family Herpesviridae (14%) constituted 74% of the order Herpesvirales. Within the Alphaherpesvirinae subfamily, Human alphaherpesvirus 2 accounted for a mere 0.05% of the viral population. The Betaherpesvirinae subfamily was represented by Human betaherpesvirus 6B (0.6% of all viral sequence reads) and Human betaherpesvirus 7 (0.1%).
As depicted in Figure 2, the giant Pandoravirus was predominantly composed of P. salinus (37%), P. macleodensis (22%), P. dulcis (12%), P. inopinatum (6%), P. neocaledonia (5%), and P. quercus (3%). Additionally, 1,773 unassigned reads were attributed to other Pandoravirus species, accounting for 16% of this category.
The order Orthornavirae, with 6,542 reads, consisted of two main sub-orders: Kitrinoviricota (40%) and Pisuviricota (38%). The majority of viral species in these sub-orders were associated with plant and marine viral species. Notably, the Flaviviridae family (45%) within the Kitrinoviricota sub-order included major human viral pathogens, namely Hepacivirus (63%) and Tick-borne encephalitis virus (19%).
Fish represent one of the major sources of food worldwide. Aquatic animal species in marine ecosystems are exposed to a wide variety of microorganisms (1). Viruses, as the most abundant organisms on Earth (1,2), significantly influence the nutrient and carbon cycles in aquatic environments (3). A study conducted by Breitbart et al. in 2002 on two uncultured marine viral communities revealed that over 65% of the microbial diversity in aquatic communities is uncharacterized, with most of the remaining diversity comprising viruses. Furthermore, the most abundant viral genome constitutes only 2-3% of the total population in marine communities, indicating high viral diversity (4).
Phylogenetic classification has identified approximately 72 orders (And 79 suborders) and 514 families of bony fish (5). Calculations based on fish population density have shown that some fish shoals can consist of nearly three billion fish in a single school, making this an exceptionally dense population among vertebrates. Consequently, this large number of fish can harbor an increasing number of viruses compared to other vertebrates. Viruses are horizontally transmitted between fish, often through contaminated feces (6). A meta-transcriptomics study conducted by Geoghegan et al. in 2018 on four fish species demonstrated that densely shoaling fish had the highest number of viruses compared to less social fish species. Interestingly, identifying the number of potential novel viruses in fish, as previously detected in mammals and birds, may indicate cross-species transmission (7), which can occur through the horizontal transmission of viruses by birds or other fish predators.
Actinopterygii, known as ray-finned fishes, encompasses more than 32,000 species (5) and represents nearly half of the living vertebrate species within the Neopterygii (Modern ray-fins) sub-class. Neopterygii is the most species-rich clade of bony fishes (>99.9%) (8,9) and includes members such as Ginglymodi, Halecomorphi, and Teleostei. Teleostei, with the most species, is the dominant group of modern Actinopterygians. Non-teleost Actinopterygians are often called living fossils (8). Ray-finned fishes exhibit a remarkable ability to adapt to different aquatic environments, including deep ocean trenches, high mountain streams, and extreme habitats with conditions like acidity, subzero temperatures, hypersalinity, hypoxia, temporariness, and fast-flowing water, with a history dating back over 400 million years (10). It is worth noting that, in addition to their lengthy evolutionary history, ray-finned fishes are crucial for human food consumption. In 2010, it was estimated that 80.6% of fish catches were attributed to this group (11).
The extensive diversity of ray-finned fishes makes them a potential source of viral species. The presence of billions of viral particles per milliliter of ocean water, along with the significant role of viruses in causing mortality in aquatic environments, highlights their capacity to influence population structures in the ocean (12,13). Fish, being a primary source of human food, are in direct contact with ocean water and can carry various potentially new and emerging viruses (14-17). Viral populations can lead to numerous infections in marine environments, emphasizing the importance of studying the presence of viruses in fish (Suttle, 2007). A valuable approach for assessing the diversity and richness of environmental virus samples is metagenomics (2), which conducts a culture-independent analysis to survey the genetic content of all microbial organisms in an environmental sample using biotechnological and bioinformatics methods. Additionally, metavirome evaluation assesses the metagenome of the entire viral population in a single sample (18,19).
The primary aim of this study was to investigate the genome fingerprints of viral genomes that could potentially pose a threat to public health. Furthermore, we discuss the possible evolutionary role of viral diversity in marine ecosystems and its impact on the emergence of viral outbreaks.
Methods
Recovery of metagenomics resource
Metadata for the metagenome were retrieved from the Sequence Read Archive (SRA) (20) using the Galaxy server (21), as previously described (22). The data were obtained from a BioProject focused on ray-finned fishes with the accession number PRJNA493014, which included eight SRA experiments. The SRA accession numbers were as follows: SRR7903826 for Acipenser schrenckii (Amur sturgeon), SRR7903827 for Odontobutis haifengensis, SRR7903828 for Odontobutis yaluensis (Dark sleeper), SRR7903829 for Rhinogobius giurinus, SRR7903830 for Hiodon tergisus (Mooneye), SRR7903831 for Polyodon spathula (Paddlefish), SRR7903832 for Denticeps clupeoides (Denticle herring), and SRR7903833 for Rutilus rutilus (Common roach).
Assembly and quality control of raw FASTQ files
Illumina sequencing files were processed to remove chimeric sequences, correct barcode errors, and eliminate duplications using the Trimmomatic tool (23). Single-ended SRA entries containing FASTQ files were assembled using Unicycler (24) with the default package settings. Contig quality was assessed using the Quality Assessment Tool (25).
Metavirome classification and taxonomic affiliation
Reads from each sequencing run were aligned to genomic sequences in the viral database using the Kraken taxonomic assignment tool (26). The number of reads mapped to the reference was counted using the same Kraken tool. Additionally, classified data were visualized using Krona (27,28). The root of the chart was set as viral families, and the distribution of each viral taxon was estimated based on the depth of sequence reads.
Mapping unidentified sequence reads to the giant viral database
A pooled set of unassigned reads from the genomes of all eight ray-finned fish species was used to identify giant viral fingerprints. The GiantVirusFinder package was employed to identify the number of sequence reads that matched the genomes of 288 known giant viruses (29). The package supports a fast-greedy alignment algorithm for identifying hits to giant viruses (30). The source code was modified to support blast version 2.8.1+, and the expected threshold was adjusted to 0.1.
Results
Metavirome survey
This study investigated metagenomic data from eight ray-finned fish species for their virome. Overall, 2,228,888 sequence reads were assigned to the viral kingdom. A pooled genome assay revealed a high abundance of Pandoravirus (31%), Herpesvirales (23%), and Bamfordvirae (10%) among all virus kingdoms. The following sections will present the virome of each ray-finned fish. The study results presented here are taxonomically assigned. For some groups of viruses that lack supergroup classifications like class, order, or family, we have defined them in their respective sections. Consequently, the genus Pandoravirus, the order Herpesvirales, the family Polydnavirus, and the kingdom Bamfordvirae are the root classifications for their respective clades, as shown in Figure 1.
Diversity of viral species in Acipenser Schrenckii
According to viral taxonomy, the metagenomics data of Acipenser schrenckii comprised 464,973 classified reads and 91,977 unclassified reads. A. schrenckii included three main clades: Pandoravirus, Herpesvirales, and Bamfordvirae, accounting for 5% of the total virome. As illustrated in Figure 1, the genus Pandoravirus made up 34% of all viruses, followed by Herpesvirales at 28%. The most prevalent Pandoraviral species were P. malecodensis (28%), P. dulcis (24%), P. neocaledonia (10%), P. salinus (9%), P. quercus (4%), and P. inopinatum (2%). Unclassified Pandoravirus species constituted 24% of this lineage (Figure 1).
The order Herpesvirales, with 131,119 reads, accounted for 28% of A. schrenckii's virome. In addition to Alloherpesviridae, the Herpesviridae family was represented by some members of human herpesviruses. Specifically, the subfamily Betaherpesvirinae (3% of all viruses) included Roseolovirus (41% of Betaherpesvirinae) and Cytomegalovirus (22% of Betaherpesvirinae), both responsible for known human-associated viral pathogens. Other human-associated viruses from the family Herpesviridae included Human alphaherpesvirus 2 (1%) within the subfamily Alphaherpesvirinae, Human betaherpesvirus 7 (5%), Human betaherpesvirus 6B (1%), and Human betaherpesvirus 6A (0.1%) within Betaherpesvirinae, and Human gammaherpesvirus 8 (0.4%) belonging to the Gammaherpesvirinae subfamily.
The kingdom Bamfordvirae and its order Nucleocytoviricota comprised two major clades of large viral families: Megaviricetes (65%) and Poxviridae (26%). The class Megaviricetes included three families, namely Phycodnaviridae (71%), Mimiviridae (20%), and Pimascovirales (9%). The family Mimiviridae encompassed two genera, Mimivirus (24%) and Cafeteriavirus (11%), along with five different species: Moumouvirus (58%), Megavirus chiliensis (22%), Acanthamoeba polyphaga mimivirus (2%), Cafeteria roenbergensis virus (11%), and Yellowstone lake mimivirus (5%).
Viral population of Odontobutis haifengensis
In the virome of O. haifengnis, the most abundant viral orders were Herpesvirales (19%), Pandoravirus (17%), Orthornavirae (10%), and Bamfordvirae (10%). The family Herpesviridae (14%) constituted 74% of the order Herpesvirales. Within the Alphaherpesvirinae subfamily, Human alphaherpesvirus 2 accounted for a mere 0.05% of the viral population. The Betaherpesvirinae subfamily was represented by Human betaherpesvirus 6B (0.6% of all viral sequence reads) and Human betaherpesvirus 7 (0.1%).
As depicted in Figure 2, the giant Pandoravirus was predominantly composed of P. salinus (37%), P. macleodensis (22%), P. dulcis (12%), P. inopinatum (6%), P. neocaledonia (5%), and P. quercus (3%). Additionally, 1,773 unassigned reads were attributed to other Pandoravirus species, accounting for 16% of this category.
The order Orthornavirae, with 6,542 reads, consisted of two main sub-orders: Kitrinoviricota (40%) and Pisuviricota (38%). The majority of viral species in these sub-orders were associated with plant and marine viral species. Notably, the Flaviviridae family (45%) within the Kitrinoviricota sub-order included major human viral pathogens, namely Hepacivirus (63%) and Tick-borne encephalitis virus (19%).
|
Figure 1. The taxonomic plot of acipenser schrenckii virome
.PNG) |
|
Figure 2. The taxonomic plot of odontobutis haifengensis virome
.PNG) |
Virome of Odontobutis yaluensis
The virome of O. yaluensis included three main viral orders: Herpesvirales (24%), Bamfordvirae (15%), and Pandoravirus (8%). Additional orders comprised Baculoviridae (6%) and Orthornavirae (5%). The Herpesvirales encompassed three major families: Herpesviridae (79%), Alloherpesviridae (15%), and Malacoherpesviridae (5%). Within the Herpesviridae family, the viruses Human alphaherpesvirus 3 and Human betaherpesvirus 6B were found in the Alphaherpesvirinae and Betaherpesvirinae sub-families, respectively (Figure 3).
The sub-order Nucleocytoviricota within Bamfordvirae included two prominent families: avian Poxviridae and giant viruses of Megaviricetes. The Mimiviridae family accounted for 5% of Bamfordvirae and consisted of Mimivirus (59%), Moumouvirus (35%), and Cafeteriavirus (5%). The genus Mimivirus was represented solely by Megavirus chiliensis.
Further investigation into giant viruses was conducted within the Pandoravirus taxonomy. Recognized species included P. macleodensis (30%), P. salinus (27%), P. dulcis (14%), P. inopinatum (5%), P. neocaledonia (5%), and P. quercus (3%), while the remaining 16% of the taxonomy represented other Pandoraviruses (Figure 3).
The virome of O. yaluensis included three main viral orders: Herpesvirales (24%), Bamfordvirae (15%), and Pandoravirus (8%). Additional orders comprised Baculoviridae (6%) and Orthornavirae (5%). The Herpesvirales encompassed three major families: Herpesviridae (79%), Alloherpesviridae (15%), and Malacoherpesviridae (5%). Within the Herpesviridae family, the viruses Human alphaherpesvirus 3 and Human betaherpesvirus 6B were found in the Alphaherpesvirinae and Betaherpesvirinae sub-families, respectively (Figure 3).
The sub-order Nucleocytoviricota within Bamfordvirae included two prominent families: avian Poxviridae and giant viruses of Megaviricetes. The Mimiviridae family accounted for 5% of Bamfordvirae and consisted of Mimivirus (59%), Moumouvirus (35%), and Cafeteriavirus (5%). The genus Mimivirus was represented solely by Megavirus chiliensis.
Further investigation into giant viruses was conducted within the Pandoravirus taxonomy. Recognized species included P. macleodensis (30%), P. salinus (27%), P. dulcis (14%), P. inopinatum (5%), P. neocaledonia (5%), and P. quercus (3%), while the remaining 16% of the taxonomy represented other Pandoraviruses (Figure 3).
|
Figure 3. The taxonomic plot of odontobutis yaluensis virome
.PNG) |
Distribution of the viral population in Rhinogobius giurinus
In the virome of R. giurinus, 59% of the metagenomics data comprised three major orders: Pandoravirus (24%), Herpesvirales (20%), and Bamfordvirae (15%). Other viral taxa included Polydnaviridae (5%), Baculoviridae (3%), and Caudovirales (3%). Additional virus-associated sequence reads can be found in Figure 4.
The genus Pandoravirus consisted of P. dulcis (38%), P. macleodensis (20%), P. salinus (9%), P. quercus (9%), P. neocaledonia (7%), and P. inopinatum (3%). Furthermore, Pandoraviruses made up 15% of this giant virus category. Herpesviridae comprised 79% of the Herpesvirales order, with Alphaherpesvirinae containing Human alphaherpesvirus two and Human alphaherpesvirus 3. The Betaherpesvirinae subfamily included Roseolovirus, Human betaherpesvirus 6B, Human betaherpesvirus 6A, and Human betaherpesvirus 7. Reads associated with Human gammaherpesvirus 4 and Human gammaherpesvirus 8 were observed in the Gammaherpesvirinae subfamily (Figure 4).
Bamfordvirae contained two main superorders, Nucleocytoviricota (85%) and Preplasmiviricota (15%). Similar to Preplasmiviricota, Poxviridae within Nucleocytoviricota consisted of avian viruses. Megaviricetes within Nucleocytoviricota included three major groups of giant viruses, namely Phycodnaviridae (89%), Mimiviridae (5%), and Pimascovirales (5%). Phycodnaviridae consisted of several species, including Phaeovirus (23%), Prymnesiovirus (19%), Prasinovirus (9%), Raphidovirus (7%), Chlorovirus (1%), Chrysochromulina ericina virus (0.7%), Aureococcus anophagefferens virus (0.7%), and Orpheovirus IHUMI-LCC2 (0.3%). The species in the Mimiviridae family were as follows: Moumouvirus (53%), M. chiliensis (24%), Cafeteria roenbergensis virus (21%), and Yellowstone lake mimivirus (1%). In addition, the order of Pimascovirales included the Iridoviridae (65%) and Ascovirus (35%) families.
Diversity of the virome of Hiodon tergisus
The virome analysis of H. tergisus showed a distinct distribution of viral taxonomy compared to other ray-finned fishes. The sequence reads rate was significantly different, with Pandoravirus accounting for 41% of the viruses in H. tergisus. Herpesvirales constituted 18%, Polydnaviridae 11%, and Bamfordvirae 4% of the prevalent viral clades in the taxonomy assignments (Figure 5).
In the virome of R. giurinus, 59% of the metagenomics data comprised three major orders: Pandoravirus (24%), Herpesvirales (20%), and Bamfordvirae (15%). Other viral taxa included Polydnaviridae (5%), Baculoviridae (3%), and Caudovirales (3%). Additional virus-associated sequence reads can be found in Figure 4.
The genus Pandoravirus consisted of P. dulcis (38%), P. macleodensis (20%), P. salinus (9%), P. quercus (9%), P. neocaledonia (7%), and P. inopinatum (3%). Furthermore, Pandoraviruses made up 15% of this giant virus category. Herpesviridae comprised 79% of the Herpesvirales order, with Alphaherpesvirinae containing Human alphaherpesvirus two and Human alphaherpesvirus 3. The Betaherpesvirinae subfamily included Roseolovirus, Human betaherpesvirus 6B, Human betaherpesvirus 6A, and Human betaherpesvirus 7. Reads associated with Human gammaherpesvirus 4 and Human gammaherpesvirus 8 were observed in the Gammaherpesvirinae subfamily (Figure 4).
Bamfordvirae contained two main superorders, Nucleocytoviricota (85%) and Preplasmiviricota (15%). Similar to Preplasmiviricota, Poxviridae within Nucleocytoviricota consisted of avian viruses. Megaviricetes within Nucleocytoviricota included three major groups of giant viruses, namely Phycodnaviridae (89%), Mimiviridae (5%), and Pimascovirales (5%). Phycodnaviridae consisted of several species, including Phaeovirus (23%), Prymnesiovirus (19%), Prasinovirus (9%), Raphidovirus (7%), Chlorovirus (1%), Chrysochromulina ericina virus (0.7%), Aureococcus anophagefferens virus (0.7%), and Orpheovirus IHUMI-LCC2 (0.3%). The species in the Mimiviridae family were as follows: Moumouvirus (53%), M. chiliensis (24%), Cafeteria roenbergensis virus (21%), and Yellowstone lake mimivirus (1%). In addition, the order of Pimascovirales included the Iridoviridae (65%) and Ascovirus (35%) families.
Diversity of the virome of Hiodon tergisus
The virome analysis of H. tergisus showed a distinct distribution of viral taxonomy compared to other ray-finned fishes. The sequence reads rate was significantly different, with Pandoravirus accounting for 41% of the viruses in H. tergisus. Herpesvirales constituted 18%, Polydnaviridae 11%, and Bamfordvirae 4% of the prevalent viral clades in the taxonomy assignments (Figure 5).
.PNG)
.PNG)
Pandoravirus was composed of P. macleodensis (30%), P. salinus (18%), P. dulcis (16%), P. neocaledonia (13%), P. quercus (2%), and P. inopinatum (2%). The remaining 20% of this taxonomy consisted of other Pandoraviruses. Herpesvirales included three prominent families: Herpesviridae (48%), Alloherpesviridae (48%), and Malacoherpesviridae (2%). Alphaherpesvirinae contained half of the Herpesvirales, while Beta- and Gammaherpesvirinae accounted for 20% and 28% of the other viruses in this family, respectively. Human-associated viruses within the Herpesviridae family included Human alphaherpesvirus 2 (3%), Human betaherpesvirus 6B (4%), Human betaherpesvirus 7 (2%), Human betaherpesvirus 6A (0.3%), and Human gammaherpesvirus 8 (0.4%).
Polydnaviridae only contained insect viruses. The kingdom Bamfordvirae also included two main phyla: Nucleocytoviricota (89%) and Preplasmiviricota (11%). The class Megaviricetes included two families and one order of giant viruses, namely Phycodnaviridae (84%), Mimiviridae (4%), and Pimascovirales (12%). Phaeocystis globosa virus (56%), Ectocarpus siliculosus virus 1 (21%), Ostreococcus lucimarinus virus 2 (10%), Bathycoccus sp. RCC1105 virus BpV (0.8%), Micromonas sp. RCC1109 virus MpV1 (0.6%), Paramecium bursaria Chlorella virus A1 (1%), Heterosigma akashiwo virus 01 (2%), Aureococcus anophagefferens virus (0.9%), Orpheovirus IHUMI-LCC2 (0.7%), and Chrysochromulina ericina virus (0.7%) were the species within the Phycodnaviridae family. Four species, including Moumouvirus (51%), M. chiliensis (24%), Cafeteria roenbergensis virus (22%), and Yellowstone lake mimivirus (1%), were found in the Mimiviridae family. Pimascovirales was composed of seven species: Ranavirus (40%), Chloriridovirus (34%), Iridovirus (8%), Trichoplusia ni ascovirus 2c (7%), Spodoptera frugiperda ascovirus 1a (5%), Heliothis virescens ascovirus 3a (5%), and Lymphocystivirus (1%).
Metavirome assay of Polyodon spathula
The results revealed a similar distribution of viral populations in P. sapathula. The genus Pandoravirus was the most prevalent, comprising 35% of all viruses, followed by Herpesvirales with 21% and Bamfordvirae with 6%. Other viral taxonomies are displayed in Figure 6. Pandoravirus comprised six species, including P. macleodensis (29%), P. dulcis (21%), P. salinus (13%), P. neocaledonia (9%), P. quercus (3%), and P. inopinatum (2%). Additionally, 25% of this genus consisted of other unassigned Pandoravirus species (19393 reads).
The taxonomy of Herpesvirales consisted of two families, including Herpesviridae (63%) and Alloherpesviridae (31%), and one genus, Aurivirus (5%). Similar to other fish, Alphaherpesvirinae contained Human alphaherpesvirus 2 (4%) and Human alphaherpesvirus 3 (2%). Human betaherpesvirus 6B (17%) and Human betaherpesvirus 7 (11%) represented the human-associated Betaherpesvirinae. No human viruses were observed in the Gammaherpesvirinae subfamily.
The class Megaviricetes constituted 67% of the phylum Nucleocytoviricota and 53% of the kingdom Bamfordvirae. In P. spathula, the viral taxonomy of Megaviricetes consisted of two families, Phycodnaviridae (59%) and Mimiviridae (29%), along with one order, Pimascovirales (11%). The family Phycodnaviridae comprised several genera, including Prymnesiovirus (51%), Prasinovirus (20%), Chlorovirus (10%), Phaeovirus (5%), and Raphidovirus (3%). The Mimiviridae family included four species: Moumouvirus (77%), M. chiliensis (14%), Cafeteria roenbergensis virus (5%), and Yellowstone lake mimivirus (1%). Pimascovirales was composed of four genera, namely Chloriridovirus (65%), Ascovirus (16%), Ranavirus (14%), and Lymphocystivirus (5%).
Analysis of viral species in Denticeps clupeoides
As shown in Figure 7, Pandoravirus (32%), Herpesvirales (29%), and Bamfordvirae (8%) were the most prevalent clades in the viral taxonomy of D. clupeoides. This result was relatively similar to that observed in other ray-finned fishes. The six prevalent Pandoravirus species were P. macleodensis (34%), P. dulcis (16%), P. salinus (15%), P. neocaledonia (12%), P. quercus (%), and P. inopinatum (2%).
Similarly to other ray-finned fishes, Herpesviridae constituted the majority (82%) of the Herpesvirales order, followed by Alloherpesviridae (18%) and Aurivirus (1%). As reported previously, only Herpesviridae contained human-associated herpes viruses. This included Human alphaherpesvirus 2 (4%) in the Alphaherpesvirinae subfamily, Human betaherpesvirus 6B (28%) in Betaherpesvirinae, and Human gammaherpesvirus 8 (1%) in Gammaherpesvirinae.
The third prevalent viral taxonomy, the kingdom Bamfordvirae, comprised two phyla: Nucleocytoviricota (90%) and Preplasmiviricota (10%). Megaviricetes was the main class of Nucleocytoviricota, consisting of two prominent families, Phycodnaviridae (90%) and Mimiviridae (4%), along with one order, Pimascovirales (6%). The family Phycodnaviridae contained six genera, including Prymnesiovirus (88%), Chlorovirus (4%), Phaeovirus (0.8%), Prasinovirus (0.8%), Raphidovirus (0.5%), and Coccolithovirus (0.08%). Pimascovirales included four genera, Ranavirus (73%), Chloriridovirus (16%), Ascovirus (10%), and Marseillevirus (0.1%), and three species, Heliothis virescens ascovirus 3a (4%), Trichoplusia ni ascovirus 2c (4%), and Spodoptera frugiperda ascovirus 1a (1%). Mimiviridae comprised two genera, Mimivirus (26%) and Cafeteriavirus (18%), and three species, Moumouvirus (55%), Megavirus chiliensis (26%), and Cafeteria roenbergensis virus (18%).
Polydnaviridae only contained insect viruses. The kingdom Bamfordvirae also included two main phyla: Nucleocytoviricota (89%) and Preplasmiviricota (11%). The class Megaviricetes included two families and one order of giant viruses, namely Phycodnaviridae (84%), Mimiviridae (4%), and Pimascovirales (12%). Phaeocystis globosa virus (56%), Ectocarpus siliculosus virus 1 (21%), Ostreococcus lucimarinus virus 2 (10%), Bathycoccus sp. RCC1105 virus BpV (0.8%), Micromonas sp. RCC1109 virus MpV1 (0.6%), Paramecium bursaria Chlorella virus A1 (1%), Heterosigma akashiwo virus 01 (2%), Aureococcus anophagefferens virus (0.9%), Orpheovirus IHUMI-LCC2 (0.7%), and Chrysochromulina ericina virus (0.7%) were the species within the Phycodnaviridae family. Four species, including Moumouvirus (51%), M. chiliensis (24%), Cafeteria roenbergensis virus (22%), and Yellowstone lake mimivirus (1%), were found in the Mimiviridae family. Pimascovirales was composed of seven species: Ranavirus (40%), Chloriridovirus (34%), Iridovirus (8%), Trichoplusia ni ascovirus 2c (7%), Spodoptera frugiperda ascovirus 1a (5%), Heliothis virescens ascovirus 3a (5%), and Lymphocystivirus (1%).
Metavirome assay of Polyodon spathula
The results revealed a similar distribution of viral populations in P. sapathula. The genus Pandoravirus was the most prevalent, comprising 35% of all viruses, followed by Herpesvirales with 21% and Bamfordvirae with 6%. Other viral taxonomies are displayed in Figure 6. Pandoravirus comprised six species, including P. macleodensis (29%), P. dulcis (21%), P. salinus (13%), P. neocaledonia (9%), P. quercus (3%), and P. inopinatum (2%). Additionally, 25% of this genus consisted of other unassigned Pandoravirus species (19393 reads).
The taxonomy of Herpesvirales consisted of two families, including Herpesviridae (63%) and Alloherpesviridae (31%), and one genus, Aurivirus (5%). Similar to other fish, Alphaherpesvirinae contained Human alphaherpesvirus 2 (4%) and Human alphaherpesvirus 3 (2%). Human betaherpesvirus 6B (17%) and Human betaherpesvirus 7 (11%) represented the human-associated Betaherpesvirinae. No human viruses were observed in the Gammaherpesvirinae subfamily.
The class Megaviricetes constituted 67% of the phylum Nucleocytoviricota and 53% of the kingdom Bamfordvirae. In P. spathula, the viral taxonomy of Megaviricetes consisted of two families, Phycodnaviridae (59%) and Mimiviridae (29%), along with one order, Pimascovirales (11%). The family Phycodnaviridae comprised several genera, including Prymnesiovirus (51%), Prasinovirus (20%), Chlorovirus (10%), Phaeovirus (5%), and Raphidovirus (3%). The Mimiviridae family included four species: Moumouvirus (77%), M. chiliensis (14%), Cafeteria roenbergensis virus (5%), and Yellowstone lake mimivirus (1%). Pimascovirales was composed of four genera, namely Chloriridovirus (65%), Ascovirus (16%), Ranavirus (14%), and Lymphocystivirus (5%).
Analysis of viral species in Denticeps clupeoides
As shown in Figure 7, Pandoravirus (32%), Herpesvirales (29%), and Bamfordvirae (8%) were the most prevalent clades in the viral taxonomy of D. clupeoides. This result was relatively similar to that observed in other ray-finned fishes. The six prevalent Pandoravirus species were P. macleodensis (34%), P. dulcis (16%), P. salinus (15%), P. neocaledonia (12%), P. quercus (%), and P. inopinatum (2%).
Similarly to other ray-finned fishes, Herpesviridae constituted the majority (82%) of the Herpesvirales order, followed by Alloherpesviridae (18%) and Aurivirus (1%). As reported previously, only Herpesviridae contained human-associated herpes viruses. This included Human alphaherpesvirus 2 (4%) in the Alphaherpesvirinae subfamily, Human betaherpesvirus 6B (28%) in Betaherpesvirinae, and Human gammaherpesvirus 8 (1%) in Gammaherpesvirinae.
The third prevalent viral taxonomy, the kingdom Bamfordvirae, comprised two phyla: Nucleocytoviricota (90%) and Preplasmiviricota (10%). Megaviricetes was the main class of Nucleocytoviricota, consisting of two prominent families, Phycodnaviridae (90%) and Mimiviridae (4%), along with one order, Pimascovirales (6%). The family Phycodnaviridae contained six genera, including Prymnesiovirus (88%), Chlorovirus (4%), Phaeovirus (0.8%), Prasinovirus (0.8%), Raphidovirus (0.5%), and Coccolithovirus (0.08%). Pimascovirales included four genera, Ranavirus (73%), Chloriridovirus (16%), Ascovirus (10%), and Marseillevirus (0.1%), and three species, Heliothis virescens ascovirus 3a (4%), Trichoplusia ni ascovirus 2c (4%), and Spodoptera frugiperda ascovirus 1a (1%). Mimiviridae comprised two genera, Mimivirus (26%) and Cafeteriavirus (18%), and three species, Moumouvirus (55%), Megavirus chiliensis (26%), and Cafeteria roenbergensis virus (18%).
|
Figure 6. The taxonomic plot of polyodon spathula virome
.PNG) |
Virome structure of Rutilus rutilus
As a member of the ray-finned fishes, the metagenomic data of R. rutilus were analyzed for viral sequence reads. Pandoravirus (35%), Herpesvirales (25%), and Bamfordvirae (18%) were the dominant taxonomical groups of viruses in R. rutilus. Other viral members are depicted in Figure 8. Members of Pandoravirus were similar to those found in other ray-finned fishes mentioned earlier. In this regard, P. macleodensis (34%), P. dulcis (18%), P. salinus (11%), P. neocaledonia (10%), P. quercus (7%), and P. inopinatum (2%) were the Pandoravirus species observed in the virome of R. rutilus.
Furthermore, Herpesviridae (63%), Alloherpesviridae (33%), and Malacoherpesviridae (2%) constituted the Herpesvirales taxonomy in R. rutilus. Human alphaherpesvirus 3 (0.7%) in the genus Varicellovirus and Human alphaherpesvirus 2 (4%) in the genus Simplexvirus were observed in Alphaherpesvirinae. Human betaherpesvirus 6B (23%) was the only human-associated herpesvirus in Betaherpesvirinae. Moreover, Human gammaherpesvirus 8 (5%) in the genus Rhadinovirus was observed solely in the Gammaherpesvirinae subfamily.
The Kingdom Bamfordvirae was taxonomically composed of two main phyla, Nucleocytoviricota (94%) and Preplasmiviricota (6%). Megaviricetes made up 36% of Nucleocytoviricota. Phycodnaviridae was the major family (93%) within the lineage of Megaviricetes, followed by Pimascovirales (4%) and Mimiviridae (3%). Six genera found in the virome of R. rutilus included Prymnesiovirus (71%), Prasinovirus (11%), Chlorovirus (5%), Phaeovirus (5%), Raphidovirus (0.9%), and Coccolithovirus (0.007%). The giant viral species within the family included Phaeocystis globosa virus (71%), Ostreococcus lucimarinus virus 2 (11%), Aureococcus anophagefferens virus (6%), Ectocarpus siliculosus virus 1 (5%), Paramecium bursaria Chlorella virus A1 (4%), Heterosigma akashiwo virus 01 (0.9%), Chrysochromulina ericina virus (0.4%), and Orpheovirus IHUMI-LCC2 (0.1%). The order of Pimascovirales contained five genera, including Chloriridovirus (43%), Ranavirus (35%), Ascovirus (11%), Iridovirus (10%), and Lymphocystivirus (0.5%). Additionally, the family of Mimiviridae comprised two genera, namely Cafeteriavirus (43%) and Mimivirus (28%). It also included five species within Mimiviridea, including Cafeteria roenbergensis virus (43%), Moumouvirus (28%), Acanthamoeba polyphaga mimivirus (15%), Megavirus chiliensis (14%), and Yellowstone lake mimivirus (0.9%).
Prevalence of giant virus signatures in ray-finned fishes' metagenomes
As a result, 432,281 out of 2,031,445 of the viral reads (21%) were unassigned to any known viruses. The GiantVirusFinder package was employed to assess the distribution of giant viruses. Accordingly, 17,931 hits (4.148%) were mapped to the genomes of 288 known giant viruses (e-value < 0.1). Interestingly, Figure 9 depicts the presence of unidentified giant viruses in marine environments.
As a member of the ray-finned fishes, the metagenomic data of R. rutilus were analyzed for viral sequence reads. Pandoravirus (35%), Herpesvirales (25%), and Bamfordvirae (18%) were the dominant taxonomical groups of viruses in R. rutilus. Other viral members are depicted in Figure 8. Members of Pandoravirus were similar to those found in other ray-finned fishes mentioned earlier. In this regard, P. macleodensis (34%), P. dulcis (18%), P. salinus (11%), P. neocaledonia (10%), P. quercus (7%), and P. inopinatum (2%) were the Pandoravirus species observed in the virome of R. rutilus.
Furthermore, Herpesviridae (63%), Alloherpesviridae (33%), and Malacoherpesviridae (2%) constituted the Herpesvirales taxonomy in R. rutilus. Human alphaherpesvirus 3 (0.7%) in the genus Varicellovirus and Human alphaherpesvirus 2 (4%) in the genus Simplexvirus were observed in Alphaherpesvirinae. Human betaherpesvirus 6B (23%) was the only human-associated herpesvirus in Betaherpesvirinae. Moreover, Human gammaherpesvirus 8 (5%) in the genus Rhadinovirus was observed solely in the Gammaherpesvirinae subfamily.
The Kingdom Bamfordvirae was taxonomically composed of two main phyla, Nucleocytoviricota (94%) and Preplasmiviricota (6%). Megaviricetes made up 36% of Nucleocytoviricota. Phycodnaviridae was the major family (93%) within the lineage of Megaviricetes, followed by Pimascovirales (4%) and Mimiviridae (3%). Six genera found in the virome of R. rutilus included Prymnesiovirus (71%), Prasinovirus (11%), Chlorovirus (5%), Phaeovirus (5%), Raphidovirus (0.9%), and Coccolithovirus (0.007%). The giant viral species within the family included Phaeocystis globosa virus (71%), Ostreococcus lucimarinus virus 2 (11%), Aureococcus anophagefferens virus (6%), Ectocarpus siliculosus virus 1 (5%), Paramecium bursaria Chlorella virus A1 (4%), Heterosigma akashiwo virus 01 (0.9%), Chrysochromulina ericina virus (0.4%), and Orpheovirus IHUMI-LCC2 (0.1%). The order of Pimascovirales contained five genera, including Chloriridovirus (43%), Ranavirus (35%), Ascovirus (11%), Iridovirus (10%), and Lymphocystivirus (0.5%). Additionally, the family of Mimiviridae comprised two genera, namely Cafeteriavirus (43%) and Mimivirus (28%). It also included five species within Mimiviridea, including Cafeteria roenbergensis virus (43%), Moumouvirus (28%), Acanthamoeba polyphaga mimivirus (15%), Megavirus chiliensis (14%), and Yellowstone lake mimivirus (0.9%).
Prevalence of giant virus signatures in ray-finned fishes' metagenomes
As a result, 432,281 out of 2,031,445 of the viral reads (21%) were unassigned to any known viruses. The GiantVirusFinder package was employed to assess the distribution of giant viruses. Accordingly, 17,931 hits (4.148%) were mapped to the genomes of 288 known giant viruses (e-value < 0.1). Interestingly, Figure 9 depicts the presence of unidentified giant viruses in marine environments.
.PNG)
.PNG)
|
Figure 9. A bar chart representing the number of mapped hits to known giant viruses. Reads with fewer than 20 hits were categorized as "Others." The data comprise 17,931 total reads of unassigned viruses with significant alignment scores.
.PNG) |
Discussion
Numerous viruses remain unidentified by nature due to our inability to cultivate them in laboratory conditions. However, viral typing does not solely rely on cultivation. Thanks to continuous advancements in scientific fields and the discovery of various techniques and methods, obtaining essential information about microorganisms has become more feasible. Metagenomics employs techniques that utilize genetic information from microorganisms in their natural habitats, providing insights into their evolutionary history and other pertinent information without the need for laboratory cultivation (31). In this study, the metavirome of eight ray-finned fish species was investigated. The results revealed that Pandoravirus accounted for 31%, Herpesvirales for 23%, and Bamfordvirae for 10% of the total viral sequence fingerprints. Notably, these viruses were prevalent in ray-finned fish populations distributed across the world, primarily in freshwater and riverine environments. Below is a brief overview of the eight fish species under investigation.
Acipenser schrenckii (Amur sturgeon) is known as a living fossil with a history dating back approximately 200 million years (32,33). The Amur sturgeon is a large river species, capable of reaching up to 3 meters in length and weighing up to 100 kilograms. It displays strong adaptability and can thrive in temperatures ranging from 0°C to 33°C, becoming active within the range of 13°C to 26°C. The Amur sturgeon holds significance for genetic resource conservation and is a valuable food source due to its high nutritional value in freshwater aquaculture (34-36).
Odontobutis haifengensis is an endangered species native to China and inhabits river environments (37,38). The Odontobutis genus is distributed in freshwater regions of Eastern Asia and the Yalu River in China (39). Rhinogobius giurinus is an amphidromous goby species inhabiting East Asia's marine, brackish, and freshwater environments. This ornamental fish feeds on aquatic insects, invertebrates, small fishes, zooplankton, phytoplankton, and plant detritus (40).
Hiodon tergisus is a North American freshwater fish that prefers spawning in temperatures between 10-15°C. This species typically grows to about 130 mm and weighs approximately 24 grams. Its diet includes plankton, insects, mollusks, crayfish, and small fishes (41–44). Furthermore, Polyodon spathula (Paddlefish) is considered a living fossil, native to the Northern Hemisphere, and inhabits riverine streams, broad rivers, and backwaters. It can grow to weigh over 90 kg and live up to 60 years. Paddlefish is also known for its filter-feeding behavior and has applications in aquaculture (45-47). Notably, this fish is valued for its high nutritional content, particularly its caviar and meat (48).
Denticeps clupeoides is a small teleost species of fish found in the freshwater regions of Western Africa. It holds importance for understanding relationships among lower teleosts (49,50). Additionally, Rutilus rutilus (Common roach) primarily inhabits freshwater environments and can grow up to 500 mm long. These fish are endemic to various regions in Europe and western Asia (51,52). Common roaches have a diverse diet that includes various food sources in their natural environment (53,54), and they are also a significant food source for humans.
Viruses are pervasive and influential in aquatic environments, significantly affecting microbial community mortality and the marine food cycle. Moreover, viruses can control planktonic population composition through infection (55), and they can acquire genes from their hosts, which is a common mechanism observed in viruses, particularly giant viruses (56). Host-switching, a phenomenon identified in the evolutionary history of viruses, further demonstrates the intricate links between viruses and their hosts (57).
Accordingly, one fascinating outcome of this meta-virome study was the high prevalence of Pandoravirus and Megaviricetes, both of which belong to the group of viruses known as giant viruses. These double-stranded DNA viruses were first identified in Acanthamoeba in 2003 (58). Giant viruses are found in terrestrial and aquatic environments and often interact with humans. Some giant viruses have been linked to human-associated diseases (59-61). The results of this study diverge from previously published articles, as they indicate a higher abundance of RNA viruses in aquatic environments (57).
In a study by Costa et al. on the virome of ten freshwater ray-finned fish species across seven taxonomic orders and thirteen locations within the Murray-Darling Basin in Australia, the most abundant vertebrate-associated viruses in ray-finned fishes belonged to the Arenaviridae and Astroviridae families (62). Additionally, studies by Geoghegan et al. demonstrated that the Astroviridae family had the highest abundance compared to other viral families in fish viromes (7,63,64). Despite variations in results, the viruses identified in fish were related to vertebrates and, in some cases, human-associated diseases. The presence of RNA viruses, DNA viruses, and giant viruses in fish populations and their connection to humans suggest potential evolutionary links between these viruses.
While information about viruses that infect fish remains limited, the high diversity among fish species and their significant role in vertebrate evolution implies that they might be a source of emerging viruses. Moreover, the exchange of genetically adapted viruses or quasispecies between different environments can lead to cross-species transmission, particularly in DNA viruses that exhibit high recombination rates (65). Virus genetic changes can facilitate such transmissions, making it crucial to consider the potential threats posed by aquatic viromes to public health. Although this study did not assess the completeness and integrity of human-associated viral genomes and giant viruses, mapping reference genomes will address this in future studies.
Conclusion
Population growth drives the need for more food resources, with seafood, especially fish, being one of the primary sources. The key point is that various viruses bolster food sources with a robust evolutionary history. As the findings have shown, there is a high abundance of giant DNA viruses in some ray-finned fish, many of which are consumed by humans. Therefore, further investigation into the virome of these aquatic species is crucial. This research would enhance our understanding of the potential human-associated illnesses these viruses might cause.
Acknowledgement
Not applicable.
Funding sources
Not applicable.
Ethical statement
Not applicable.
Conflicts of interest
The authors declare that they have no competing interests.
Author contributions
FSA has been involved in data preparation, FASTQ file quality checks and assembly, and has written the first draft of the manuscript. AM has been working on viral taxonomy generation for ray-finned fish, data visualization, detection of antivirus signatures in unassigned viral taxa, and has served as the research manager.
Article Type: Research |
Subject:
Microbiology
References
1. Hatfull GF. Dark Matter of the Biosphere: the Amazing World of Bacteriophage Diversity. J Virol. 2015;89(16):8107-10. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Suttle CA. Marine viruses - major players in the global ecosystem. Nat Rev Microbiol. 2007;5(10):801-12. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Roux S, Enault F, Robin A, Ravet V, Personnic S, Theil S, et al. Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics. PLoS One. 2012;7(3):e33641. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Breitbart M, Salamon P, Andresen B, Mahaffy JM, Segall AM, Mead D, et al. Genomic analysis of uncultured marine viral communities. Proc Natl Acad Sci U S A. 2002;99(22):14250-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
5. Betancur-R R, Wiley EO, Arratia G, Acero A, Bailly N, Miya M, et al. Phylogenetic classification of bony fishes. BMC Evol Biol. 2017;17(1):162. [View at Publisher] [DOI] [PMID] [Google Scholar]
6. Kurath G, Winton J. Complex dynamics at the interface between wild and domestic viruses of finfish. Curr Opin Virol. 2011;1(1):73-80. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Geoghegan JL, Di Giallonardo F, Cousins K, Shi M, Williamson JE, Holmes EC. Hidden diversity and evolution of viruses in market fish. Virus Evol. 2018;4(2):vey031. . [View at Publisher] [DOI] [PMID] [Google Scholar]
8. Friedman M. The early evolution of ray-finned fishes. Palaeontology. 2015;58(2):213-28. [View at Publisher] [DOI] [Google Scholar]
9. Romano C. A Hiatus Obscures the Early Evolution of Modern Lineages of Bony Fishes. Front Earth Sci (Lausanne). 2021;8:618853. [View at Publisher] [DOI] [Google Scholar]
10. Hughes LC, Ortí G, Huang Y, Sun Y, Baldwin CC, Thompson AW, et al. Comprehensive phylogeny of ray-finned fishes (Actinopterygii) based on transcriptomic and genomic data.Proc Natl Acad Sci U S A. 2018;115(24):6249-54. [View at Publisher] [DOI] [PMID] [Google Scholar]
11. Vaitla B, Collar D, Smith MR, Myers SS, Rice BL, Golden CD. Predicting nutrient content of ray-finned fishes using phylogenetic information. Nat Commun. 2018;9(1):3742. [View at Publisher] [DOI] [PMID] [Google Scholar]
12. Suttle CA, Chan AM, Cottrell MT. Infection of phytoplankton by viruses and reduction of primary productivity. Nature. 1990;347(6292):467-9. [View at Publisher] [DOI] [Google Scholar]
13. Proctor LM, Fuhrman JA. Viral mortality of marine bacteria and cyanobacteria. Nature. 1990;343(6253):60-2. [View at Publisher] [DOI] [Google Scholar]
14. Aswad A, Katzourakis A. A novel viral lineage distantly related to herpesviruses discovered within fish genome sequence data. Virus Evol. 2017;3(2):vex016. [View at Publisher] [DOI] [Google Scholar]
15. Ruboyianes R, Worobey M. Foamy-like endogenous retroviruses are extensive and abundant in teleosts. Virus Evol. 2016;2(2):vew032.. [View at Publisher] [DOI] [PMID] [Google Scholar]
16. Parry R, Wille M, Turnbull OMH, Geoghegan JL, Holmes EC. Divergent Influenza-Like Viruses of Amphibians and Fish Support an Ancient Evolutionary Association. Viruses. 2020;12(9):1042. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. López-Bueno A, Mavian C, Labella AM, Castro D, Borrego JJ, Alcami A, et al. Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream. J Virol. 2016;90(19):8768-79. [View at Publisher] [DOI] [PMID] [Google Scholar]
18. Garza DR, Dutilh BE. From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems. Cell Mol Life Sci. 2015;72(22):4287-308. [View at Publisher] [DOI] [PMID] [Google Scholar]
19. Herath D, Jayasundara D, Ackland D, Saeed I, Tang SL, Halgamuge S. Assessing Species Diversity Using Metavirome Data: Methods and Challenges. Comput Struct Biotechnol J. 2017;15:447-55. [View at Publisher] [DOI] [PMID] [Google Scholar]
20. Leinonen R, Sugawara H, Shumway M. The sequence read archive. Nucleic Acids Res. 2011;39(Database issue):D19-21. [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Afgan E, Baker D, Batut B, Van Den Beek M, Bouvier D, Cech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018;46(W1):W537-44. [View at Publisher] [DOI] [PMID] [Google Scholar]
22. Mohebbi A. Analysis of the RNA-Seq Data of Solanum tuberosum Revealed Viral Sequence Reads of a Severe Laboratory-Developed Strain of SARS-CoV-2 Containing Novel Substitutions. Journal of Clinical and Basic Research (JCBR). 2022;6(4):13-8. [View at Publisher] [DOI] [Google Scholar]
23. Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-20. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics. 2013;29(8):1072-5. [View at Publisher] [DOI] [PMID] [Google Scholar]
26. Wood DE, Salzberg SL. Kraken: Ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014;15(3):R46. [View at Publisher] [DOI] [PMID] [Google Scholar]
27. Cuccuru G, Orsini M, Pinna A, Sbardellati A, Soranzo N, Travaglione A, et al. Orione, a web-based framework for NGS analysis in microbiology. Bioinformatics. 2014;30(13):1928-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
28. Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC Bioinformatics. 2011;12:385. [View at Publisher] [DOI] [PMID] [Google Scholar]
29. Kerepesi C, Grolmusz V. The "Giant Virus Finder" discovers an abundance of giant viruses in the Antarctic dry valleys. Arch Virol. 2017;162(6):1671-6. [View at Publisher] [DOI] [PMID] [Google Scholar]
30. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7(1-2):203-14. [View at Publisher] [DOI] [PMID] [Google Scholar]
31. Seshadri R, Kravitz SA, Smarr L, Gilna P, Frazier M. CAMERA: A Community Resource for Metagenomics. PLoS Biol. 2007;5(3):e75. [View at Publisher] [DOI] [PMID] [Google Scholar]
32. Jin SB, Zhang Y, Dong X, Xi QK, Song D, Fu HT, et al. Comparative transcriptome analysis of testes and ovaries for the discovery of novel genes from Amur sturgeon (Acipenser schrenckii). Genet Mol Res. 2015;14(4):18913-27. [View at Publisher] [DOI] [PMID] [Google Scholar]
33. Zhang X, Zhou J, Li L, Huang W, Ahmad H, Li H, et al. Full-length transcriptome sequencing and comparative transcriptomic analysis to uncover genes involved in early gametogenesis in the gonads of Amur sturgeon (Acipenser schrenckii). Front Zool. 2020;17:11. [View at Publisher] [DOI] [PMID] [Google Scholar]
34. Zhuang P, Kynard B, Zhang L, Zhang T, Zhang Z, Li D. Overview of biology and aquaculture of Amur sturgeon (Acipenser schrenckii) in China. Journal of Applied Ichthyology. 2002;18(4-6):659-64. [View at Publisher] [DOI] [Google Scholar]
35. Zhang Y, Fan Z, Wu D, Li J, Xu Q, Liu H, et al. Dietary magnesium requirement on dietary minerals and physiological function of juvenile hybrid sturgeon (Acipenser schrenckii♀ × Acipenser baerii♂). Aquaculture International. 2021;29(4):1697-709. [View at Publisher] [DOI] [Google Scholar]
36. Cai L, Taupier R, Johnson D, Tu Z, Liu G, Huang Y. Swimming Capability and Swimming Behavior of Juvenile Acipenser schrenckii. J Exp Zool A Ecol Genet Physiol. 2013;319(3):149-55. [View at Publisher] [DOI] [PMID] [Google Scholar]
37. Zhong L, Wang M, Li D, Tang S, Zhang T, Bian W, et al. Complete mitochondrial genome of Odontobutis haifengensis (Perciformes, Odontobutiae): A unique rearrangement of tRNAs and additional non-coding regions identified in the genus Odontobutis. Genomics. 2018;110(6):382-8. [View at Publisher] [DOI] [PMID] [Google Scholar]
38. WU HL. A revision of the genus Odontobutis from China with description of a new species. J Shanghai Fish Univ. 1993;2:52-61. [View at Publisher] [Google Scholar]
39. Zang X, Wang X, Zhang G, Wang Y, Ding Y, Yin S. Complete mitochondrial genome and phylogenic analysis of Odontobutis yaluensis, Perciformes, Odontobutidae. Mitochondrial DNA A DNA Mapp Seq Anal. 2016;27(3):1965-7. [View at Publisher] [DOI] [PMID] [Google Scholar]
40. Ju YM, Wu JH, Kuo PH, Hsu KC, Wang WK, Lin FJ, et al. Mitochondrial genetic diversity of Rhinogobius giurinus (Teleostei: Gobiidae) in East Asia. Biochem Syst Ecol. 2016;69:60-6. [View at Publisher] [DOI] [Google Scholar]
41. Snyder DE, Douglas SC. Description and identification of Mooneye, Hiodon tergisus, protolarvae. Trans Am Fish Soc. 1978;107(4):590-4. [View at Publisher] [DOI] [Google Scholar]
42. Hilton EJ, Lavoué S. A review of the systematic biology of fossil and living bony-tongue fishes, Osteoglossomorpha (Actinopterygii: Teleostei). Neotropical Ichthyology. 2018;16:e180031. [View at Publisher] [DOI] [Google Scholar]
43. Glenn CL. Seasonal growth and diets of young-of-the-year Mooneye (Hiodon tergisus) from the Assiniboine River, Manitoba. Trans Am Fish Soc. 1978;107(4):587-9. [View at Publisher] [DOI] [Google Scholar]
44. Mclnerny MC, Held JW. First-Year Growth of Seven Co-Occurring Fish Species of Navigation Pool 9 of the Mississippi River. J Freshw Ecol. 1995;10(1):33-41. [View at Publisher] [DOI] [Google Scholar]
45. Naiel M, Elnakeeb M, Vasilyeva L, Sudakova N, Anokhina A, Gewida ahmed GA. Paddlefish, Polyodon spathula: Historical, current status and future aquaculture prospects in Russia. Int Aquat Res. 2021;13(2):89-107. [View at Publisher] [DOI] [Google Scholar]
46. Thurmer CR, Patel RR, Riveros GA, Alexander QG, Ray JD, Netchaev A, et al. Instrumenting Polyodon spathula (Paddlefish) Rostra in Flowing Water with Strain Gages and Accelerometers. Biosensors. 2020;10(4):37. [View at Publisher] [DOI] [PMID] [Google Scholar]
47. Kramer N, Phelps Q, Tripp S, Herzog D. Exploitation of paddlefish Polyodon spathula (Walbaum, 1792) in the Mississippi River. J Appl Ichthyol. 2019;35(1):355-9. [View at Publisher] [DOI] [Google Scholar]
48. Elnakeeb MA, Vasilyeva LM, Sudakova NV, Aokhina AZ, Gewida AGA, Alagawany M, et al. Evaluate the metabolism responses of
cultured paddlefish, polyodon spathula (walbaum, 1792), towards some ecological stressors in the volga-caspian basin using fuzzy modeling control. Adv Anim Vet Sci. 2021;9(6):773-86. [View at Publisher] [DOI] [Google Scholar]
49. Di Dario F, de Pinna MC. The supratemporal system and the pattern of ramification of cephalic sensory canals in Denticeps clupeoides (Denticipitoidei, Teleostei): additional evidence for monophyly of Clupeiformes and Clupeoidei. Pap Avulsos Zool. 2006;46:107-23. [View at Publisher] [DOI] [Google Scholar]
50. Picolo F, Grandchamp A, Piégu B, Rolland AD, Veitia RA, Monget P. Genes Encoding Teleost Orthologs of Human Haploinsufficient and Monoallelically Expressed Genes Remain in Duplicate More Frequently Than the Whole Genome. Int J Genomics. 2021;2021:9028667. [View at Publisher] [DOI] [PMID] [Google Scholar]
51. Bowes RE, Näslund J, Greenberg L, Bergman E. Cobble substrate in a surface bypass reduces bypass acceptance by common roach Rutilus rutilus. Ecol Eng. 2021;172:106402. [View at Publisher] [DOI] [Google Scholar]
52. Hamilton PB, Lockyer AE, Webster TMU, Studholme DJ, Paris JR, Baynes A, et al. Investigation into Adaptation in Genes Associated with Response to Estrogenic Pollution in Populations of Roach (Rutilus rutilus) Living in English Rivers. Environ Sci Technol. 2020;54(24):15935-45. [View at Publisher] [DOI] [PMID] [Google Scholar]
53. Nahon S, Roussel JM, Jaeger C, Menniti C, Kerhervé P, Mortillaro JM, et al. Characterization of trophic niche partitioning between carp (Cyprinus carpio) and roach (Rutilus rutilus) in experimental polyculture ponds using carbon (δ13C) and nitrogen (δ15N) stable isotopes. Aquaculture. 2020;522:735162. [View at Publisher] [DOI] [Google Scholar]
54. Łuczyńska J, Paszczyk B, Łuczyński MJ, Kowalska-Góralska M, Nowosad J, Kucharczyk D. Using Rutilus rutilus (L.) and Perca fluviatilis (L.) as Bioindicators of the Environmental Condition and Human Health: Lake Łańskie, Poland. Int J Environ Res Public Health. 2020;17(20):7595. [View at Publisher] [DOI] [PMID] [Google Scholar]
55. Suttle CA. Viruses in the sea. Nature. 2005;437(7057):356-61. [View at Publisher] [DOI] [Google Scholar]
56. Schulz F, Roux S, Paez-Espino D, Jungbluth S, Walsh DA, Denef VJ, et al. Giant virus diversity and host interactions through global metagenomics. Nature. 2020;578(7795):432-6. [View at Publisher] [DOI] [PMID] [Google Scholar]
57. Shi M, Lin XD, Chen X, Tian JH, Chen LJ, Li K, et al. The evolutionary history of vertebrate RNA viruses. Nature. 2018;556(7700):197-202. [View at Publisher] [DOI] [PMID] [Google Scholar]
58. dos Santos Oliveira J, Lavell AA, Essus VA, Souza G, Nunes GHP, Benício E, et al. Structure and physiology of giant DNA viruses. Curr Opin Virol. 2021;49:58-67. [View at Publisher] [DOI] [PMID] [Google Scholar]
59. Colson P, Scola B La, Raoult D. Giant Viruses of Amoebae: A Journey Through Innovative Research and Paradigm Changes. Annu Rev Virol. 2017;4(1):61-85. [View at Publisher] [DOI] [PMID] [Google Scholar]
60. Colson P, Aherfi S, La Scola B, Raoult D. The role of giant viruses of amoebas in humans. Curr Opin Microbiol. 2016;31:199-208. [View at Publisher] [DOI] [PMID] [Google Scholar]
61. Scheid P. A strange endocytobiont revealed as largest virus. Curr Opin Microbiol. 2016;31:58-62. [View at Publisher] [DOI] [PMID] [Google Scholar]
62. Costa VA, Mifsud JCO, Gilligan D, Williamson JE, Holmes EC, Geoghegan JL. Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia. Virus Evol. 2021;7(1):veab034. [View at Publisher] [DOI] [PMID] [Google Scholar]
63. Geoghegan JL, Di Giallonardo F, Wille M, Ortiz-Baez AS, Costa VA, Ghaly T, et al. Virome composition in marine fish revealed by meta-transcriptomics. Virus Evol. 2021;7(1):veab035. [View at Publisher] [DOI] [PMID] [Google Scholar]
64. Geoghegan JL, Giallonardo F Di, Wille M, Ortiz-Baez AS, Costa VA, Ghaly T, et al. Host evolutionary history and ecology shape virome composition in fishes. bioRxiv. [Preprint] [View at Publisher] [DOI] [Google Scholar]
65. Dennehy JJ. Evolutionary ecology of virus emergence. Ann N Y Acad Sci. 2017;1389(1):124-46. [View at Publisher] [DOI] [PMID] [Google Scholar]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0).