Genome analysis of Ranavirus frog virus 3 isolated from American Bullfrog (Lithobates catesbeianus) in South America

Ranaviruses (family Iridoviridae) cause important diseases in cold-blooded vertebrates. In addition, some occurrences indicate that, in this genus, the same virus can infect animals from different taxonomic groups. A strain isolated from a Ranavirus outbreak (2012) in the state of Sao Paulo, Brazil, had its genome sequenced and presented 99.26% and 36.85% identity with samples of Frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV) ranaviruses, respectively. Eight potential recombination events among the analyzed sample and reference FV3 samples were identified, including a recombination with Bohle iridovirus (BIV) sample from Oceania. The analyzed sample presented several rearrangements compared to FV3 reference samples from North America and European continent. We report for the first time the complete genome of Ranavirus FV3 isolated from South America, these results contribute to a greater knowledge related to evolutionary events of potentially lethal infectious agent for cold-blooded animals.


Methods
This research project was approved by the ethics committee on the use of vertebrate animals of the Faculty of Animal Science and Food Engineering (Universidade de São Paulo), case number 1629310316. All experiments were performed in accordance with relevant guidelines and regulations.
In the present study, eight samples of liver, spleen and kidney collected from American Bullfrogs (Lithobates catesbeianus) in an Rv outbreak that occurred in 2012 in a frog farm located in the state of Sao Paulo, Brazil, confirmed by PCR and nucleotide sequencing using primers M151, M152, M153 and M154, were pooled and grinded to viral isolation in cell culture (BF-2 cells -bluegill fry ATCC ® CCL-91 TM ) following the protocol recommended by the World Organisation For Animal Health (OIE) 7 .
Extraction of viral DNA from cell culture was performed using the QIAamp DNA Mini Kit (Qiagen, Germany), following the manufacturer's instructions. The total DNA underwent quality analysis through 2% agarose gel electrophoresis and quantification by fluorescence in a Qubit ® 2.0 Fluorometer (ThermoFisher Scientific, USA). The DNA was subjected to purification steps using Agencourt AMPure XP magnetic bead (Beckman Coulter, USA) and was quantified again by fluorescence using the Qubit ® 2.0 Fluorometer (Thermo Fisher Scientific, USA).
DNA fragment libraries were prepared with 50 ng of purified DNA using a Nextera DNA sample preparation kit and sequenced using an Illumina ® MiSeq System (Illumina, USA). After quality control of the sequence reads using Trim Galore (https://github.com/FelixKrueger/TrimGalore) 8  For phylogenetic characterization, identity analysis, recombination detection and rearrangement analyzes of the genome under analysis, complete genome sequences of 19 members from Iridoviridae family representing all species of Rv that have the genome available were retrieved from GenBank ( Table 1).
The DNA sequences of Rv were aligned using the software MAFFT version 7 with default settings (https://mafft. cbrc.jp/alignment/server/) 9 . Phylogenetic reconstruction was generated by the Neighbor-Joining (NJ) method, Kimura 2-parameter model, with bootstrap nodal support for 1000 pseudoreplicates in MEGA software, version 6.0 10 .
Pairwise identity scores using the complete genome and in parallel the major capsid protein (MCP) of Rv were calculated using Sequence Demarcation Tool (SDT) 11 with default settings. The MCP gene is highly conserved among the different species of Rv, which makes it suitable for the presumptive diagnosis in infected animals through conventional PCR reaction 12 .
The recombination analysis among complete genomes of Rv FV3 was carried using RDP4 program version 4.95 in default settings, with p-value of <0.05. Genome rearrangement was assessed using Mauve 2.4.0 13

Results
phylogenetic analysis. The sample sequenced in this study (MH351268) was grouped in the Rv FV3-like clade, presenting a bootstrap value of 100% with sequences of the same viral species (Fig. 1).

Analysis of MCP gene identity among different species of Ranavirus.
Analyzing the MCP gene, the nucleotide identity among the samples under analysis was greater than 94% for all Rv species, except for Singapore grouper iridovirus (SGIV) isolates that showed identity below 69% (Fig. 2).
identity analysis among ranaviruses genomes. The sequenced Rv showed the highest nucleotide identity of the genome (99.26%) with North American FV3 isolates (AY548484 and KJ175144) and only 36.85% with SGIV isolates (AY521625 and NC_006549). The overall mean of identity among the genomes was 62.01% ( Supplementary Fig. S1).   Table 2). Several deletions of ORFs were identified in the sample under analysis compared to reference samples: deletion of an ORF between ORFs 31 and 32, two OFRs between ORFs 43 and 45, an ORF between ORFs 45 and 46, an ORF between ORFs 60 and 61, two ORFs between ORFs 64 and 65, one ORF between ORFs 83 and 84, one ORF between ORFs 88 and 89, and one ORF after ORF 94. As in the KJ175144 sample, the sample under analysis showed a combination of two ORFs in one (ORF 48) when compared to sample AY548484.

Rearrangement analysis among
The genome of the FV3 sample from the Netherlands (KJ175144) was also compared to the South American sample, due to the large recombinant region. This sample also showed several deletions and insertions within the genome compared to the sample under analysis. A high degree of rearrangement between Rv was evidenced (Fig. 3).

Analysis of rearrangement among different species of Ranavirus. When comparing the genomes
of different species of Rv, the only species that shows complete collinearity with the FV3 genome analyzed in this study was the genome of Bohle Iridovirus (BIV), all other species showed genomic inversions in some region of the genome compared to the analyzed FV3 sample (Fig. 4).

Discussion
The number of nucleotides, percentage of G + C and number of ORFs of the genome analyzed are in accordance with those described in the literature for the genotyped virus 14,15 .
The nucleotide phylogenetic reconstruction grouped the South American Rv genome in the FV3-like clade, presenting a 100% bootstrap value with genomic samples of the same viral species. In an intriguing way, KX185156 sample, derived from Rv of the BIV species, entered the clade of FV3-like presenting ≥98% of bootstrap value. BIV was the virus-causing epizootic outbreak with 100% mortality of captive tilapias of the specie Oreochromis mossambicus, within a period of 60 days in Australia 16 .
Similar to this work, other recent studies have also demonstrated the insertion of BIV-like into the clade of FV3-like 17 . The identity between the genome under analysis and the KX185156 (BIV) sample was 94.85%, a relatively high percentage because it is a different species within the same viral genus.
When the complete nucleotide sequences of the MCP gene (1392 bp) of different Rv species were analyzed, identities were greater than 94.00%, except for SGIV isolates, which showed identity below 69% compared to other species within the same genus. Similar results were described by Waltzek et al. 18 , comparing nucleotide sequences of the MCP gene between the SGIV and FV3 species isolated from sturgeon (Scaphirhynchus albus); the percentage of identity between samples was 69.4% and among FV3 and other species of Rv (FV3, BIV, CMTV, EHNV, ECV and ATV), the identity for the gene was >94.1%.  www.nature.com/scientificreports www.nature.com/scientificreports/ Through the acquisition of genes via recombination, members of viral families that present dsDNA adapt to their respective hosts 19 . Recombinant events are recognized as driving forces in the generation of pathogenic viruses and as a form of viral adaptation to new hosts [20][21][22][23] . All samples used for the recombination analysis (JQ654586, KX185156, AF389451, MF360246, KJ175144 and AY548484) showed recombination events (major parent or minor parent p < 0.05) with the South American sample of Rv FV3 in some region of the genome.
We also documented recombination (minor parent) between Rv FV3 and BIV. Events of recombination among Rv of different species were also reported by Claytor et al. 24 , who detected recombination between FV3 and CMTV viruses, generating a pathogenic chimeric Rv denominated RCVZ2 that infect amphibians. The authors isolated and analyzed strains from a commercial facility in different years (1998 and 2006), suggesting that the virus evolution occurred in the locality and that one or more CMTV virus genes have been rearranged through processes of recombination.  Some studies have identified naturally occurring recombination events among Rv isolates 25,26 . It is suggested that the inherent frequency of high recombination of Rv leads to a sharp rearrangement of the organization of the viral genome, presenting a diversified genomic organization as a reflex 27 . Rv that show greater sequence divergence can also show lower sequence collinearity over time derived from increased recombination of the viral genome 28 . International trade of live animals such as the American bullfrog (Lithobates catesbeianus) and cultured fish has been associated with the emergence of these viruses [29][30][31] .
Rearrangement and recombination were identified between FV3 samples collected in North America (AY548484 and KJ175144), Europa (MF360246) and South America (MH3512168), highlighting the role that the international trade in farmed animals may have played in the global dissemination of highly pathogenic Rv. Recombination in double-stranded DNA viruses (dsDNA) has been reported by some researchers, such as herpesvirus, poxvirus and bacteriophages [32][33][34] .
High rates of recombination of Rv in vitro have been suggested, however the mechanism and significance of these genomic changes remain unknown 27,28,35 . Thus, more investigations are needed to elucidate the mechanisms and meanings of Rv evolution, associating any relation with its ecology, host interaction and pathogenesis 28 .
Infectious diseases in insects, mammals, marsupials, amphibians, reptiles and fish are not yet fully understood. Globally, combating infectious diseases is a major goal of public and veterinary health efforts. Since Rv infects a wide variety of ecologically and economically important hosts, understanding the evolution of these viruses, including the importance of the genome rearrangements found among isolates in relation to host specificity and viral evolution will help predict and eventually prevent epizootics 36 .
This study adds and reinforces the potential recombination among Rv. Although this study provides information on recombination and rearrangements of Rv, future works should focus on understanding this genomic variability among these viruses and its relationship to viral ecology, host range, and pathogenesis.
In conclusion, we report here for the first time the genome of Rv FV3 isolated from South America. Our results contribute to a better understanding of the characteristics of viral agents potentially associated with severe outbreaks in cold-blooded vertebrates.

Data availability
The Ranavirus Frog Virus 3-like DNA sequence analysed in the present investigation was deposited in the GenBank database under the accession code MH351268.