Complete genome sequence of a novel papillomavirus in Siamese fighting fish (Betta splendens) from Trinidad and Tobago

ABSTRACT

Here, we announce the complete genome of a previously undescribed papillomavirus from a betta fish, Betta splendens. The genome is 5,671 bp with a GC content of 38.2%. Variants were detected in public databases. This genome is most similar to papillomaviruses that infect sea bass (52.9 % nucleotide identity).

ANNOUNCEMENT

Papillomaviruses (PVs) are double-stranded circular DNA viruses that belong to the consortium of small DNA tumor viruses (1). Papillomavirus genomes range in size from 5 to 8.6 kb. Virions are non-enveloped, icosahedral particles approximately 55 nm in diameter and have been identified in most classes of vertebrates (1–3). To date, 16 PVs that infect fishes or have a putative fish host have been identified. The genomes of these viruses are notably smaller (5–6 kb) than mammalian PVs, possess a subset of the open reading frames (ORFs) named in that lineage, and constitute the exclusive members of the subfamily, Secondpapillomavirinae (family, Papillomaviridae; genus, Alefpapillomavirus) (4). The core ORFs in this group of viruses that are shared with mammalian PVs include E1, E2, L1, and L2. An E4 homolog has been identified in some genomes. Notably, homologs of the E6/E7 proteins associated with neoplasia are encoded in some of these fish PV genomes. In cases where PVs are associated with clinical disease in fishes, often multiple small to large, raised nodules that appear gray or black in color are observed. They are typically benign (1, 3, 5).

Betta fish (Betta splendens; Siamese fighting fish) are freshwater fish native to southern Asia and commercially important in the ornamental fish trade (6–8). Although bacterial and viral diseases have been described in this species, the extent of putative microbial pathogens is unknown (9–11). Here, a moribund betta fish that presented with lethargy and later died (October 2018) at The University of the West Indies, St. Augustine, School of Veterinary Medicine, was examined. No raised nodules were observed. DNA was extracted from pooled liver and splenic tissues using a DNeasy Blood & Tissue DNA extraction kit (Qiagen). DNA was subjected to Repli-G (Qiagen) preamplification and submitted to GENEWIZ, Inc. A DNA library was created using a NEB NextUltra DNA Library Preparation kit, and sequencing was performed using a 2 × 150 paired-end configuration on an Illumina HiSeq instrument. Reads were quality trimmed and assembled using Megahit (v1.2.9) and screened for viral sequences in Cenote Taker 2 (12, 13). The genome of a novel papillomavirus consisting of 5,671-bp genome with a GC content of 38.2% was recovered. Reads were mapped to the draft genome using CLC Genomics Workbench (v21.0.2) and represented 2.84% (3,116,785) of the total reads (109,600,338). Average coverage was 81,380×.

Six partially overlapping ORFs including E6/E7 homologs (OncN and OncC), replicative helicase (E1), E2 protein, and the capsid proteins L1 and L2 were found. The inferred intron-containing L1 sequence is reminiscent of fish papillomaviruses MZ570863 and KX643372. Highest amino acid identity was observed for seabass PV (QYW06029) E1 protein (54.3%). Lower sequence identity (23.9%–54.3%) was observed for the E1 proteins of other fish-associated PVs (Fig. 1).

Fig 1.

Genome map and phylogenetic analyses of fish papillomavirus E1 proteins. (A) Genome map depicting predicted coding sequences (CDS). Black arrows illustrate splice donor/ acceptor sites. MacVector (v18.6.1) was used to circularize the genome and annotate genome features. Gene names were assigned based on BLASTP search results. (B) Midpoint rooted maximum likelihood phylogeny of E1 proteins of fish papillomaviruses. E1 proteins from papillomaviruses with fish hosts were recovered from the NCBI database via BLASTP query (9/2023). Sequences were aligned with the Geneious Prime (v2023.2.1) plugin MUSCLE v.5.1 using the PPP algorithm (14). The alignment was used to infer a maximum likelihood phylogenetic tree in IQ-Tree 1.6.12 using model GTR + F + I + G4 (15). The phylogenetic tree was visualized using iTOL (V6) (16). Branch tips are labeled with sample accession number, and the betta papillomavirus is highlighted in bold. (C) Pairwise comparisons of fish-associated PV E1 amino acid sequences using the sequence demarcation tool (v1.3) (17). The betta papillomavirus is highlighted in bold.

While this virus was identified in a moribund fish, the association with morbidity is unclear. The complete genome presented here supports future development of molecular tools to further our understanding of the prevalence of this virus in ornamental fish and its role as a potential infectious agent.

DATA AVAILABILITY

The genome sequence has been deposited in GenBank under accession number OQ633419. The raw reads were deposited in BioProject, PRJNA880623; BioSample, SAMN30874153; SRA, SRR21609248. Variant genomes were assembled from SRA DRR320248 and SRR18231402.