Metavirome Survey of Eight Ray-Finned Fishes Revealed Domination of Giant Viral Members of Pandoravirus and Megaviricetes

are an essential source of food all around the world. The bulk of the microbial diversity in ecosystems is uncharacterized, with accounting for most of the This may be a major to therefore the of in the is critical. The to


Abstract Background
Fishes are an essential source of food all around the world. The bulk of the microbial diversity in marine ecosystems is uncharacterized, with viruses accounting for most of the remaining diversity. This may be a major danger to public health, therefore knowing the variety of viruses in the marine ecosystem is critical. The goal was to assess the virome of ray-nned shes in terms of variety and relation to human diseases.

Results
It was revealed that the viral kingdom was allocated 2,228,888 sequence reads. The pooled genome assay of the ray-nned shes indicated a signi cant abundance of viruses in three clades of the genus Pandoravirus (31%), the order of Herepesvirales (23%), and the kingdom of Bamfordvirae (10%). Furthermore, 432281/2031445 reads (21%) of mapped reads were unassigned viruses. According to the GiantVirusFinder ndings, 17931 hits (4.15$) were mapped to the genomes of known giantviruses.

Conclusion
Humans eat seafood regularly as one of their primary diets. Ray-nned shes have a long evolutionary history and, as demonstrated here, are hosts to a variety of large DNA viruses. Fish must be screened for viral infections that are linked to human illnesses.

Background
Fishes are one of the major sources of food worldwide. Aquatic animal species in marine ecosystems are exposed to a wide variety of microorganisms [1]. Viruses are the most abundant organisms on earth [1,2] and affect the nutrient and carbon cycle in aquatic environments [3]. According to the rst study conducted on two uncultured marine viral communities by Breitbart et al. in 2002, it was found that over 65% of microorganism diversity in marine communities is uncharacterized, and the majority of remaining diversity are viruses. On the other hand, the most abundant viral genome comprises 2-3% of the total population in marine communities, indicating high viral diversity [4].
Phylogenetic classi cation has shown approximately 72 orders (and 79 suborders), 514 families of bony sh have been identi ed [5]. According to calculations made on the population density of sh, it is determined that some sh shoal can occupy nearly three billion sh in a single school [6]. This rate represents a very dense population among vertebrates. On the other hand, this number of shes can harbor an increasing number of viruses compared to other vertebrates. Viruses are transmitted horizontally between shes, which may occur through contaminated feces [7]. A study with a meta-transcriptomics approach on four sh species conducted by Geoghegan et al. in 2018 showed densely shoaling sh had the highest number of viruses compared to the most unsocial sh species. Interestingly, identifying the number of potential novel viruses in shes as previously detected in mammals and birds may indicative cross-species transmission [8]. This can also occur by the horizontal transmission of viruses by birds or other sh predators.
Actinopterygii, known as ray-nned shes, with more than 32,000 species (9), contains nearly half of the living vertebrate species belonging to the Neopterygii (modern ray-ns) sub-class. Neopterygii is the most species-rich clade of bony shes (> 99.9%) [9,10]. Neopterygii comprises members, including Ginglymodi, Halecomorphi, and Teleostei. Teleostei is the dominant species of modern Actinopterygian with the highest number of shes. Also, non-teleost Actinopterygians are known as living fossils [9]. Ray-nned shes, which can live in different aquatic environments such as deep ocean trenches, high mountain streams, grow in extreme habitats with acidic, subzero, hypersaline, hypoxic, temporary, and fast-owing water environments, dating back to more than 400 million years ago [11]. It is necessary to say that in addition to their long history, ray shes are very important in providing human food. According to an estimate made in 2010, 80.6% of the catches were related to this sh species [12].
The vast diversity of ray-nned shes makes them a source of different viral species. The existence of billions of viral particles per milliliter of the ocean and as the most important cause of death in those waters make them capable of altering populations' structures in the ocean [13][14][15]. On the other hand, sh is the most important source of consumption in human food and in direct contact with ocean water that can carry various and even new emerging viruses [16][17][18][19]. A population of viruses can cause many infections occurring in marine environments. Therefore, it is necessary to study the viral presence in a sh [2]. A new approach that can be used to survey the diversity and richness of the environmental virus samples is metagenomics [2]. In a culture-independent analysis, metagenomics surveys the genetic content of all microbial organisms in an environmental sample using biotechnological and bioinformatics methods. Also, Metavirome evaluates the metagenome of the entire viral population in one sample [20,21].
The purpose of the current study was to investigate the genome ngerprints of the viral genome that could potentially pose a threat to public health. We also discuss the possibility of the evolutionary role of viral diversity in marine ecosystems and their impact on the emerging viral outbreak.
Assembly and quality control of raw FASTQ les Illumina sequencing les were polished for chimeric sequences, barcode errors, and duplication using the Trimmomatic tool [24]. Single-ended SRA entries containing FASTQ les were assembled by using Unicycler [25]. Assemblies are performed with the package's default settings. Contigs were checked for their quality through QUality ASsessment Tool (QUAST) [26].

Metavirome classi cation and taxonomic a liation
Reads from each sequencing run were mapped to the viral database's genomic sequences using the Kraken taxonomic assignment tool [27]. The number of reads mapped to reference was counted using the same Kraken tool. Furthermore, classi ed data were plotted by using Krona [28,29]. The root of the chart was set as viral families. The distribution of each viral taxa was estimated through the depth of sequence reads.
Mapping unidenti ed sequence reads to Giant viral database A pooled unassigned read of the genome of all eight ray-nned sh species was used to nd giant viral ngerprints. GiantVirusFinder package was used to identify the number of sequences reads to the genome of 288 known giantviruses [30]. The packaged supports a fast-greedy alignment algorithm to identify hits to the giantviruses [31]. However, the source code was modi ed to support blast version 2.8.1+. Furthermore, the expected threshold was changed to 0.1.

Metavirome survey
In this study, metagenomics data of eight ray-nned sh species were investigated for their virome.
Overall, 2228888 sequence reads were assigned to the virus kingdom. Pooled genome assay revealed a high abundance of Pandoravirus (31%), Herepesvirales (23%), and Bamfordvirae (10%) of all kingdom of viruses. In the following virome of each ray-nned sh will be presented. The presented study results are taxonomically assigned, and as some groups of viruses may not have some supper group classi cations like class, order, or family, we de ned them in their related sections. Accordingly, the genus of Pandoravirus, the order of Herpesvirales, the family of Polydnavirus, and the kingdom of Bamfordvirae are the roots of their clades, as shown in Fig. 1.
The order of Herpesvirales with 131,119 reads composed 28% of all the virome of A.schrenckii. In addition to Alloherpesviridae, the Herpesviridae family was contained in some members of human herpesviruses. Accordingly, the subfamily of Betaherpesvirinae (3% of all viruses) was comprised of Roseolovirus (41% of Betaherpesvirinae) and Cytomegalovirus (22% of Betaherpesvirinae), which was responsible for known human-associated viral pathogens. Accordingly, Human alphaherpesvirus 2 (1%) within the subfamily of Alphaherpesvirinae, Human betaherpesvirus 7 (5%), Human betaherpesvirus 6B (1%), and Human betaherpesvirus 6A (0.1%) within Betaherpesvirinae and Human gammaherpesvirus 8 (0.4%) belong to the Gammaherpesvirinae subfamily were constituted the human-associated viruses of the family of Herpesviridae.
The order of Orthornavirae with 6542 reads comprised two main sub-order of Kitrinoviricota (40%) and Pisuviricota (38%). The majority of viral species in these two sub-orders were those associated with vegetable and marine viral species. However, the Flaviviridae family (45%) within the Kitrinoviricota suborder consisted of major human viral pathogens, namely Hepacivrus (63%) and Tick-borne encephalitis virus (19%).

Diversity of virome of Hiodon tergisus
Virome analysis of H. tergisus showed an almost different distribution of viral taxonomic among the raynned shes. However, the sequence reads rate was signi cantly different from others as Pandoravirus with 142901 sequence reads comprised 41% of viruses in H. tergisus. Herpesvirales with 18%, Polydnaviridae with 11%, and Bamfordvirae with 4% were the other prevalent viral clades in the taxonomy assignments (Fig. 1E).

Metavirome assay of Polyodon spathula
The results revealed a similar distribution of viral populations in P. sapathula. Accordingly, genus Pandoravirus with 35% of all viruses was most prevalent, and the order of Herpesvirales followed it with 21% and Bamfordvirae with 6%. Other viral taxonomies are shown in Fig. 1F.

Analysis of viral species in Denticeps clupeoides
As shown in Fig. 1G, Pandoravirus with 32%, Herpesvirales with 29%, and Bamfordvirae with 8% were the most prevalent clades of the viral taxonomy of D. clupeoides. This result was relatively similar to those in other ray-nned shes.

Virome structure of Rutilus rutilus
As one member of ray-nned shes, the metagenomic data of R. rutilus were assessed 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 shown in Fig. 1H.

Prevalence of giantvirus signatures in Ray-nned shes' metagenomes
As a result, 432281/2031445 reads (21%) of mapped reads were unassigned viruses. To assess the distribution of giantviruses, the GiantVirusFinder package was employed. Accordingly, 17931 hits (4.148%) were mapped to the genome of 288 known giantviruses (e-value < 0.1). Interestingly, Fig. 2 shows the circulation of yet unidenti ed giantviruses in marine environments.

Discussion
There are so many different types of viruses in nature that we do not know them since we cannot cultivate them in laboratory conditions. Cultivation is not the only solution for viral typing. Due to day-today advances in scienti c elds and the discovery of various techniques and methods, it has become more accessible to achieve the desired information about microorganisms. Metagenomics utilizes some techniques using genetic information of any microorganism in its natural habitat, evolutionary history, and any other information required without the need for laboratory process [32].
In the present study, the metavirome of eight ray-nned sh species was investigated. According to the results, Pandoravirus with 31%, Herepesvirales with 23%, and Bamfordvirae with 10% had the highest abundance of viruses. It is noteworthy that these viruses were abundant in the ray-nned shes that were geographically belonged to the different parts of the world but mostly in freshwater and riverine. Where there is more connection with humans, we will give a brief review about these 8 species of sh in the following.
Acipenser schrenckii (Amur sturgeon), with a history of ~ 200 million years known as a living fossil [33,34]. Also, it is a large river species with a maximum length of 3 meters and a weight of 100 kilograms, strong adaptability, having mild temperament, tolerating in the range of 0-33°C but activating in the range of 13-26°C that inhabits the Amor River. This species is important in terms of genetic resource conservation and food source because it has high nutritional value in freshwater aquaculture [35][36][37].
The following ray-nned sh species was Odontobutis haifengensis, an endangered species native in China and inhabited by rivers [38,39]. The Odontobutis genus is distributed in freshwaters of Eastern-Asia and Yalu River in China [40,41].
Hiodon tergisus, is a North American freshwater sh and lives in the waters with 10-15 OC temperature during spawning. Its height and weight are about 130 mm and 24 gr, respectively. They feed on plankton, insects, mollusks, cray sh, and small shes [43][44][45][46].
Polyodon spathula (Paddle sh) is considered a living fossil (49) and native of the Northern Hemisphere. It homes in the riverine streams, broad rivers, and backwaters. It weighs more than 90 kg and can live up to 60 years. Also, it can act as a lter feeder into aquaculture [47][48][49]. The remarkable thing about this sh is its high nutritional value, provided through its caviar and meat [50].
Denticeps clupeoides is a small teleost species of sh that inhabits the freshwater of Western Africa. This species is a key taxon for nd relationships among lower teleosts [51][52][53].
Rutilus rutilus (common roach), as one of the studied ray-nned shes, live primarily in freshwater and has a length up to 500 mm, which is endemic in most parts of Europe and western Asia [54,55]. Common roach feeds on a variety of food sources in its natural environment [56,57]. It is also an important food source for humans.
The action of viruses in aquatic environments is pervasive and in uential. As a crucial factor in the mortality of microbial communities, Viruses can affect the food cycle of the marine environment. On the other hand, by causing infection, it can effectively control the composition of planktonic populations [58].
In addition to the effects of viruses on their environment, these intelligent entities can also acquire abilities from the same environment. An examination of the evolutionary history of viruses showed that they could receive genes from the hosts that they infect (61), especially it is a common mechanism in viruses like giantviruses [59]. Despite having the greatest diversity among vertebrates and having a variety of viruses that have played an important role in the evolution of vertebrates, there is little information about sh viruses and their association with human viruses. Most of the information is about viruses that infect humans [60]. Nevertheless, the information obtained suggests a possible link between marine caliciviruses and disease in humans. Also, the presence of avian in uenza in marine birds [58, 68] implicates a risk of aquatic virome posed to public health. Since shes are the most prosperous species among terrestrial and aquatic vertebrates, they can be a source for spreading new emerging viruses.
Furthermore, a small portion of genetically adapted viruses or quasispecies from the different environments can jump between species and cause infection [69]. Given that among viruses, DNA viruses have the highest recombination (genomic rearrangements) rate [69], and genetic changes in viruses can lead to cross-species transmission [70]. Furthermore, based on the present study results, which is also supported in a previous study [71], it can be implicated that viruses can jump from aquatic vertebrates to terrestrial vertebrates and pose a threat to the human population.

Conclusion
Population growth is a factor that leads people to use more food resources. One of these food sources is seafood, especially sh. The point is that food sources with the strong evolutionary background are supported by living many different viruses. As the ndings demonstrated a high abundance of different giant DNA viruses in some ray-nned shes, most of which are human seafood, it is necessary to investigate these aquatic species' virome further. This would help to enhance our knowledge of the human-associated illnesses these viruses may cause in the future.

Declarations
Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable.
Availability of data and materials: All data generated or analysed during this study are included in this published article.
Competing interests: The authors declare that they have no competing interests.
Funding: Not applicable.
Authors' contributions: FSA has been involved in data preparation, FASTQ les quality check and assembly, and she has written the rst draft of the manuscript. AM has been worked on viral toxonomy generation of the ray-nned sh, data visualization, detection of the giantvirus signatures of the unassigned viral taxa, and the research manager.