The Ancient Evolutionary History of Polyomaviruses.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses.

UNLABELLED: Hepadnaviruses (hepatitis B viruses [HBVs]) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the known host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs, which allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motifs I, II, and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topologies, characterized by three transmembrane domains. Most striking was the fact that BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host codivergence over millions of years and host species jumping. IMPORTANCE: Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full-length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegills, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that, although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.

Myxobolus axelrodi n. sp. (Myxosporea: Myxobolidae) a parasite infecting the brain and retinas of the cardinal tetra Paracheirodon axelrodi (Teleostei: Characidae).

An investigation of mortalities in a group of cardinal tetras Paracheirodon axelrodi Meyers, 1936, a popular ornamental fish, revealed myxozoan parasites in ventricles of the brains in 3/10 fish and the ocular retina of a fourth. Parasite impacts were unclear, as additional histopathological findings were present, including bacterial dermatitis and meningitis. Ethanol-preserved specimens pooled from multiple fish were used for morphological characterization of myxospores. Elongate, teardrop myxospores were 20.5 ± 0.7-µm (mean ± SD; range = 19.0-21.8 µm) long, 6.6 ± 0.5-µm (5.7-7.9 µm) wide, and 5.1 ± 0.4-µm (4.8-5.9 µm) thick (valvular width). Two, unequally sized, apical, pyriform polar capsules were in the same plane as the sutural ridge. The larger measured 9.9 ± 0.8-µm (8.0-11.2 µm) long and 3.8 ± 0.3-µm (3.2-4.8 µm) wide. The smaller was 4.1 ± 0.3-µm (3.5-4.5 µm) long and 2.0 ± 0.1-µm (1.8-2.3 µm) wide. Identical 1912 bp 18S rRNA sequences were obtained from two pooled spore samples from tetra brains, which did not match any sequences in the NCBI nr/nt database. Phylogenetically, these parasites grouped loosely within a clade containing Myxobolus spp. from other South American characins and Unicauda spp. from siluriform catfish. Myxospores shared some morphological similarities with Myxobolus inaequus from the unrelated glass knifefish (Order: Gymnotiformes), but were genetically divergent (<85 % similarity) from other myxozoan parasites of South American characins and shared few morphological features or tissue predilection sites. Based on host and tissue tropism, spore morphology, and 18S rRNA sequencing, we report this isolate as a previously unknown species, Myxobolus axelrodi n. sp.

Microscopic and Molecular Evidence of the First Elasmobranch Adomavirus, the Cause of Skin Disease in a Giant Guitarfish, Rhynchobatus djiddensis.

Only eight families of double-stranded DNA (dsDNA) viruses are known to infect vertebrate animals. During an investigation of papillomatous skin disease in an elasmobranch species, the giant guitarfish (Rhynchobatus djiddensis), a novel virus, distinct from all known viral families in regard to particle size, morphology, genome organization, and helicase phylogeny was discovered. Large inclusion bodies containing 75-nm icosahedral viral particles were present within epithelial cell nuclei in the proliferative skin lesions. Deep metagenomic sequencing revealed a 22-kb circular dsDNA viral genome, tentatively named guitarfish "adomavirus" (GAdoV), with only distant homology to two other fish viruses, Japanese eel endothelial cell-infecting virus (JEECV) and a recently reported marbled eel virus. Phylogenetic analysis of the helicase domain places the guitarfish virus in a novel clade that is equidistant between members of the Papillomaviridae and Polyomaviridae families. Specific PCR, quantitative PCR, and in situ hybridization were used to detect, quantify, and confirm that GAdoV DNA was localized to affected epithelial cell nuclei. Changes in the viral titer, as well as the presence of a hybridization signal, coincided with the progression and then final resolution of gross and microscopic lesions. The results indicate that GAdoV is the causative agent of the proliferative skin lesions.IMPORTANCE Cartilaginous fish, including the sharks and rays, evolved from ancestral fish species at least 400 million years ago. Even though they are the descendants of one of the most ancient vertebrate lineages, reports of viral diseases in these species are rare and poorly documented. Deep sequencing revealed a highly divergent virus, tentatively named guitarfish adomavirus, that is distantly related to known papillomaviruses and polyomaviruses. Out of the eight predicted viral genes, only the helicase could be identified as viral by sequence homology searches (BLAST), exemplifying the difficulties of discovering novel viruses within seas of unidentifiable "dark matter" associated with deep sequencing data. The novel adomavirus represents the first viral genome shown to cause clinical disease in a cartilaginous fish species, the giant guitarfish. Our findings demonstrate that emerging fish viruses are fertile ground to expand our understanding of viral evolution in vertebrates.

Two new species of betatorqueviruses identified in a human melanoma that metastasized to the brain.

The role of viral infections in the etiology of brain cancer remains uncertain. Prior studies mostly focused on transcriptome or viral DNA integrated in tumor cells. To investigate for the presence of viral particles, we performed metagenomics sequencing on viral capsid-protected nucleic acids from 12 primary and 8 metastatic human brain tumors. One brain tumor metastasized from a skin melanoma harbored two new human anellovirus species, Torque teno mini virus Emory1 (TTMV Emory1) and Emory2 (TTMV Emory2), while the remaining 19 samples did not reveal any exogenous viral sequences. Their genomes share 63-67% identity with other TTMVs, and phylogenetic clustering supports their classification within the Betatorquevirus genus. This is the first identification of betatorqueviruses in brain tumors. The viral DNA was in its expected non-integrated circular form, and it is unclear if the viruses contributed to tumor formation. Whether the viruses originated from blood, or the primary skin tumor could not be ascertained. Overall, our results demonstrate the usefulness of viral metagenomics to detect previously unknown exogenous virus in human brain tumors. They further suggest that active viral infections are rare events in brain tumors, but support a follow-up larger scale study to quantify their frequency in different brain tumor subtypes.

Complete Sequence of the Smallest Polyomavirus Genome, Giant Guitarfish (Rhynchobatus djiddensis) Polyomavirus 1.

Polyomaviruses are known to infect mammals and birds. Deep sequencing and metagenomic analysis identified the first polyomavirus from a cartilaginous fish, the giant guitarfish (Rhynchobatus djiddensis). Giant guitarfish polyomavirus 1 (GfPyV1) has typical polyomavirus genome organization, but is the smallest polyomavirus genome (3.96 kb) described to date.