Jennifer A. Dill

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.

Francisella noatunensis subsp. orientalis infection in Indo-Pacific reef fish entering the United States through the ornamental fish trade

Infections caused by Piscirickettsia-like organisms (PLO) appeared in Taiwanese tilapia, Oreochromis spp., culture in the early 1990s (Chen et al. 1994; Chern & Chao 1994). Additional reports quickly appeared making it evident that PLO posed an emerging threat to a range of fish species in both fresh and salt water (Mauel & Miller 2002). Early reports were based largely on gross and histologic features of the disease and failure of the organisms to react serologically or in PCR assays for Piscirickettsia salmonis. Improved culture and molecular detection methods determined later PLO cases to be caused by Francisella spp. bacteria capable of survival and growth within macrophages (Ottem et al. 2009; Birkbeck, Feist & Verner-Jeffreys 2011; Colquhoun & Duodu 2011). Taxonomic clarification of the pleomorphic 0.5to 1.7-lm bacteria indicates a single species, Francisella noatunensis, for all fish isolates. Two subspecies are recognized, F. noatunensis subsp. noatunensis (Fnn) and F. noatunensis subsp. orientalis (Fno), with apparent predilections for cold and warmwater fish, respectively (Ottem et al. 2009). Regardless of the bacterial subspecies involved, infections share clinical and pathologic features, including acute to chronic mortalities accompanied by non-specific signs. Most notable grossly is splenomegaly and renomegaly, with whitish-tan nodules corresponding histologically to granulomas typified by vacuolated macrophages containing the bacteria (Birkbeck et al. 2011; Colquhoun & Duodu 2011). Some granulomas develop cuffs of lymphocytes (Mauel et al. 2007) and with time may undergo central necrosis and fibrous encapsulation (Hsieh et al. 2006; Birkbeck et al. 2011). The spleen and kidney are most severely affected, although lesions have been described in all organ systems (Birkbeck et al. 2011; Colquhoun & Duodu 2011). Evidence for a PLO induced disease, suggestive of francisellosis, in the continental United States was reported from California in 1998 involving white seabass, Atractoscion nobilis (Ayres) (Chen et al. 2000). To the best of our knowledge, however, francisellosis has not emerged in the marine tropical fish trade in this country, although movements of live fish are likely involved in dissemination of the agent, and it has been predicted that the number of affected species will continue to rise (Birkbeck et al. 2011). This report describes Fno infections diagnosed in Indo-Pacific reef fish following international shipment to and distribution in the United States from Asian waters. Correspondence E Soto, Ross University, School of Veterinary Medicine, Department of Pathobiology, P.O. Box 334, Basseterre, St. Kitts, West Indies (e-mail: esoto@rossvet.edu.kn)

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

ABSTRACT 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.

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

ABSTRACT 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.

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.

Adomaviruses: an emerging virus family provides insights into DNA virus evolution

Adenoviruses, papillomaviruses, and polyomaviruses are collectively known as small DNA tumor viruses. Although it has long been recognized that small DNA tumor virus oncoproteins and capsid proteins show a variety of structural and functional similarities, it is unclear whether these similarities reflect descent from a common ancestor, convergent evolution, horizontal gene transfer among virus lineages, or acquisition of genes from host cells. Here, we report the discovery of a dozen new members of an emerging virus family, the Adomaviridae, that unite a papillomavirus/polyomavirus-like replicase gene with an adenovirus-like virion maturational protease. Adomaviruses were initially discovered in a lethal disease outbreak among endangered Japanese eels. New adomavirus genomes were found in additional commercially important fish species, such as tilapia, as well as in reptiles. The search for adomavirus sequences also revealed an additional candidate virus family, which we refer to as xenomaviruses, in mollusk datasets. Analysis of native adomavirus virions and expression of recombinant proteins showed that the virion structural proteins of adomaviruses are homologous to those of both adenoviruses and another emerging animal virus family called adintoviruses. The results pave the way toward development of vaccines against adomaviruses and suggest a framework that ties small DNA tumor viruses into a shared evolutionary history. Author Summary In contrast to cellular organisms, viruses do not encode any universally conserved genes. Even within a given family of viruses, the amino acid sequences encoded by homologous genes can diverge to the point of unrecognizability. Although members of an emerging virus family, the Adomaviridae, encode replicative DNA helicase proteins that are recognizably similar to those of polyomaviruses and papillomaviruses, the functions of other adomavirus genes have been difficult to identify. Using a combination of laboratory and bioinformatic approaches, we identify the adomavirus virion structural proteins. The results link adomavirus virion protein operons to those of other midsize non-enveloped DNA viruses, including adenoviruses and adintoviruses.

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

ABSTRACT 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. 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.

Investigation of epizootic papillomatosis in bluegill Lepomis macrochirus (Rafinesque 1810) using next‐generation sequencing

Orocutaneous neoplasms in fish have been recognized for over a century (Mawdesley-Thomas 1975). Most are benign epidermal hyperplasias and papillomas, with rare squamous cell carcinomas (Groff 2004; Roberts 2012). In the brown bullhead Ameiurus nebulosus (Lesueur), evidence suggests a possible chemical aetiology (Grizzle, Melius & Strength 1984; Pinkney, Harshbarger & Rutter 2014), while virus or virus-like particles have been demonstrated in other species (Coffee, Casey & Bowser 2013). Some sources relate tumour development to potential interactions between environmental contaminants and viruses (Smith, Ferguson & Hayes 1989). Several tumour-associated virus particles have been tentatively identified based on morphology only, including herpesviruses, retroviruses, adenoviruses and others (Anders & Yoshimizu 1994; Coffee et al. 2013). Examples of herpesviral-induced neoplastic conditions include salmonid herpesvirus II and cyprinid herpesvirus I, both of which cause mortalities in young fish and papillomas in some older survivors (Kimura & Yoshimizu 1989; Sano et al. 1991). The genus Epsilonretrovirus contains the complex retroviruses that infect fish (Edien et al. 2010; Goff 2013), several of which have been partially or completely sequenced (Bowser & Casey 1993; Holzschu et al. 1995; Basta et al. 2009; Coffee et al. 2013). The pathology of retroviralassociated tumours in fish has been reviewed by Coffee et al. 2013. While some associations are well supported by sequence data and transmission trials, others are based entirely on electron microscopy and the detection of reverse transcriptase activity. Epithelial hyperplasia, papillomas and squamous cell carcinomas on the skin, fins and lips of bluegill displayed at a public aquarium and from a private pond prompted investigation of a suspected viral aetiology using next-generation sequencing (NGS). Advanced molecular techniques, such as NGS and in situ hybridization, have made it possible to establish more definitive relationships between viruses and neoplasia. Potentially confounding investigations of suspected tumorigenic retroviruses are the presence of fragmented, partial or complete retroviral genomes, representing previous, often ancient, integrations into host germ lines. Endogenous viral element (EVE) is a term used for such endogenous viruses and fragments (Holmes 2011). Retroviruses are common sources of EVEs, as host genome integration is essential to their replication Correspondence A C Camus, Department of Pathology, College of Veterinary Medicine, University of Georgia, 501 DW Brooks, Drive, Athens, GA 30602, USA (e-mail: camus@uga.edu)

Disseminated nocardiosis associated with the isolation of Nocardia nova in a longsnout seahorse Hippocampus reidi (Ginsburg)

Of the more than 50 species of Nocardia, only N. asteroides, N. seriolae and N. salmonicida are recognized fish pathogens. However, many reports only include identification to the genus level, suggesting greater species diversity could be involved. Nocardiosis affects many fish species and summaries can be found in multiple sources (Cornwell et al. 2011; Lewis & Chinabut 2011; Plumb & Hanson 2011). N. asteroides was first reported from neon tetra Paracheirodon innesi (Meyers) in 1962 and has caused epizootics primarily in freshwater (Valdez & Conroy 1963; Chen, Tung & Tsai 1989; Chen & Tung 1991). N. seriolae causes disease in marine fish worldwide, particularly in Asia, and is a major pathogen of cultured yellowtail and amberjack (Chen et al. 2000; Huang et al. 2004; Wang, Yuan & Jin 2005; Shimahara et al. 2008). Since its isolation from sockeye salmon Onchorhynchus nerka (Walbaum) and recognition as a species, N. salmonicida (originally Streptomyces salmonis) has received little attention and information is limited (Isik et al. 1999). Nocardiosis is a chronic, systemic granulomatous disease characterized by progressive lethargy and emaciation. Infections can be confused both clinically and grossly with those of more common mycobacteriosis. Gross lesions can include skin ulcers, muscle necrosis and organomegaly, with small nodules, often numerous, in parenchymal organs and the gills. Microscopically, nodules are variously described as granulomas and abscesses, frequently with necrotic centres, peripheral macrophages, lymphocytes, possibly giant cells, and variable fibrous encapsulation (Lewis & Chinabut 2011). The filamentous branching bacteria, normal inhabitants of soil, fresh and salt water, stain weakly and irregularly Gram-positive, poorly or not at all with the Ziehl–Neelsen acid-fast stain and are best visualized with modified acid-fast stains, such as Fite’s Chandler et al. 1980. While reports of epizootics are increasing, particularly in Asian mariculture, little information is available on economic impacts of the disease. However, losses of 15–17% have been reported in cultured sea bass, Lateolabrax japonicus (Cuvier), and yellow croaker, Larimichthys crocea (Richardson), respectively (Chen et al. 2000; Wang et al. 2005). Although contaminated feed has been suggested as a source of infection, the pathogenesis of natural disease is poorly understood (Sinderman 1990). Transmission has been demonstrated experimentally by injection, dermal abrasion, immersion, feeding and cohabitation (Itano et al. 2006). Infections progress slowly at warmer temperatures, with Correspondence A Camus, Department of Pathology, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, USA (e-mail: camus@uga.edu)

Lipoid liver disease, atherosclerosis and glomerular lipidosis in a Gulf flounder Paralichthys albigutta (Jordan & Gilbert 1882): a case report

Nutritional disease is uncommon in fish from unspoiled native habitats, where, if food items are available in sufficient quantify, natural feeds provide a balance of essential nutrients. However, under artificial conditions, provision of natural diets may be impractical or impossible. In public aquaria, significant efforts are often made to replicate natural diets, but specific nutrient requirements for many ornamental species are unknown and must be extrapolated from similar fish types (Lovell 2000; Roberts 2002). Providing proper nutrition can be further complicated in displays housing diverse fish species that, in addition to having differing dietary requirements, may also require feed of a certain size or feed presented in a specific manner to be acceptable (Lovell 2000; Sales & Janssens 2003). Even among fish of the same species, requirements can vary with age and differences in body size can result in smaller fish being outcompeted, while larger more aggressive individuals can consume feed in excess. Energy requirements vary among fish species, based on behaviour, environmental temperature, life stage, and body size, but are lower than those of terrestrial vertebrates, as energy is not required to regulate body temperature and less is needed to maintain posture or excrete nitrogenous wastes (Pannevis & Earle 1994; Lovell 2000; Sales & Janssens 2003). Dietary lipids are a major energy source for fish, especially carnivores, and certain fatty acids are essential to different species. For example, carnivorous marine species require highly peroxidizable n-3 series polyunsaturated fatty acids (PUFAs), while freshwater fish require both n-3 and n-6 series PUFAs (Lovell 2000; Sargent, Tocher & Bell 2002). Obesity is unexpected in wild fish and often minimized in cultured food fish, where feeding rates are closely managed to maximize profitability (Hoole et al. 2001). Although it has been suggested that fish eat to meet their energy requirements, obesity can result from chronic overfeeding or in fish allowed to feed ad libitum (Hoole et al. 2001; Wilson 2002). Lipid levels in a carcass are strongly influenced by dietary intake, and while associated degenerative changes are most common in the liver, multiple tissues can be affected (Earle 1995; Hoole et al. 2001; Sargent et al. 2002). This report describes multi-organ pathologic lesions in a Gulf flounder (Paralichthys albigutta) that are similar to those associated with excessive lipid consumption and disturbances of lipid metabolism in other vertebrates. In addition to lipoid liver disease, atherosclerosis and glomerular lipidosis were observed in the bulbus arteriosus and kidney, respectively. Correspondence A Camus, Department of Pathology, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, USA (e-mail: camus@uga.edu)