ORIGINAL RESEARCH ARTICLE published: 01 December 2014 doi: 10.3389/fmicb.2014.00655 Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma Marta Canuti 1 *†, Cathy V. Williams 2, Sashi R. Gadi 3, Maarten F. Jebbink 1, Bas B. Oude Munnink 1, Seyed Mohammad Jazaeri Farsani 1,4, John M. Cullen 3 and Lia van der Hoek 1* 1 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, Netherlands 2 Duke Lemur Center, Duke University, Durham, NC, USA 3 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA 4 Department of Virology, Tehran University of Medical Sciences, Tehran, Iran Edited by: Cancer is one of the leading health concerns for human and animal health. Since Hirofumi Akari, Kyoto University, the tumorigenesis process is not completely understood and it is known that some Japan viruses can induce carcinogenesis, it is highly important to identify novel oncoviruses Reviewed by: and extensively study underlying oncogenic mechanisms. Here, we investigated a case Kevin Coombs, University of Manitoba, Canada of diffuse histiocytic sarcoma in a 22 year old slow loris (Nycticebus coucang), using a Peter Tijssen, Université du Québec, broad spectrum virus discovery technique. A novel parvovirus was discovered and the Canada phylogenetic analysis performed on its fully sequenced genome demonstrated that it *Correspondence: represents the first member of a novel genus. The possible causative correlation between Marta Canuti, Department of this virus and the malignancy was further investigated and 20 serum and 61 organ samples Biology, Memorial University of Newfoundland, 232 Elizabeth Ave, from 25 animals (N. coucang and N. pygmaeus) were screened for the novel virus but St John’s, NL A1B 3X9, Canada only samples collected from the originally infected animal were positive. The virus was e-mail: [email protected]; present in all tested organs (intestine, liver, spleen, kidneys, and lungs) and in all banked [email protected]; serum samples collected up to 8 years before death. All attempts to identify a latent viral Lia van der Hoek, Laboratory of Experimental Virology, Department form (integrated or episomal) were unsuccessful and the increase of variation in the viral of Medical Microbiology, Academic sequences during the years was consistent with absence of latency. Since it is well known Medical Centre, University of that parvoviruses are dependent on cell division to successfully replicate, we hypothesized Amsterdam, Meibergdreef 15, that the virus could have benefitted from the constantly dividing cancer cells and may not 1105 AZ Amsterdam, Netherlands e-mail: [email protected] have been the cause of the histiocytic sarcoma. It is also possible to conjecture that the †Present address: virus had a role in delaying the tumor progression and this report might bring new exciting Marta Canuti, Department of opportunities in recognizing viruses to be used in cancer virotherapy. Biology, Memorial University of Newfoundland, St. John’s, Canada Keywords: parvovirus, virus discovery, slow loris, Nycticebus coucang, histiocytic sarcoma, VIDISCA, hematopoietic tumor, oncovirus INTRODUCTION study of diseases in non-human primates, which in turn might be The Sunda slow loris (Nycticebus coucang), a Strepsirrhine pri- beneficial for human health. mate which belongs to the family Lorisidae, is a nocturnal, arbo- We investigated a case of histiocytic sarcoma (HS) in a N. cou- real prosimian species native to Indonesia (Sumatra), western cang identified at the Duke Lemur Center (DLC) in Durham, Malaysia (Peninsular Malaysia), Singapore and southern Thailand North Carolina. HS is a highly aggressive hematopoietic tumor (Nekaris and Streicher, 2008). Slow loris, listed as a vulnerable defined as a malignant proliferation of cells showing morpholog- species by the International Union for Conservation and Nature ical and immunophenotypic features of mature tissue histiocytes, (IUCN) (IUCN Red List of Threatened Species, available at: www. cells of the innate immune system derived from bone marrow iucnredlist.org), are protected by law in Malaysia, Thailand, and monocytes which differentiate into dendritic cells and tissue Indonesia since their conservation status is a matter of concern. localized macrophages (Fulmer and Mauldin, 2007; Takahashi The biggest threat endangering those animals is the pet trade— and Nakamura, 2013). Tumors can be localized or disseminated they are the most commonly protected primates species sold as with lymph nodes being the most common site of proliferation, exotic pets in southeast Asia (Nekaris and Nijman, 2007), but followed by organs of the gastrointestinal tract, spleen, soft tis- habitat loss and the fact they are killed as crop pests also jeop- sues, and skin (Takahashi and Nakamura, 2013). It is a rare type ardize their survival (Nekaris and Streicher, 2008). Because of of cancer in humans and cases have been reported in other species these reasons a better understanding of the causes and the dynam- of animals, including chickens, dogs, cats, camels, macaques, and ics of diseases in N. coucang is advantageous for conservation lemurs (Fulmer and Mauldin, 2007; Friedrichs and Young, 2008; attempts and captive management of this species (Remick et al., Soshin et al., 2008; Molenaar et al., 2009; Remick et al., 2009; 2009). In addition, valuable information can be gathered by the Takahashi and Nakamura, 2013). Although the cause of HS is www.frontiersin.org December 2014 | Volume 5 | Article 655 | 1 Canuti et al. Novel slow loris parvovirus largely unknown, a viral etiology can be postulated as the exis- MATERIALS AND METHODS tence of viruses involved in the development of hematopoietic CLINICAL CASE cancers have been recognized, as in the case of Epstein Barr The virus was identified in a 22 year old, male Nycticebus coucang and human T-lymphotropic virus-induced human lymphomas (slow loris) named Buddha, held in captivity for 22 years at the and similar viruses in non-human primates (Miller et al., 1972; Duke University Primate Center and which had no prior signif- Donahue et al., 1992; Feichtinger et al., 1992; Vereide and Sugden, icant health issues. The individual was diagnosed with neoplasia 2009; Qayyum and Choi, 2014). Additionally it has been proven and euthanized due to poor condition, although a routine physi- that the subgroup J avian leukosis virus is associated with the cal inspection 8 months before death revealed an enlarged spleen. development of histiocytic sarcomatosis in chickens (Pandiri A complete postmortem examination was performed. et al., 2009) and an association between persistent Epstein Barr virus infection and human HS has been reported (Kramer et al., CLINICAL SAMPLES 1985). In this study we applied a sequence independent virus Representative tissues of Buddha were collected and fixed in 10% discovery technique combined with high throughput sequenc- neutral formalin. Fixed tissue was processed routinely into paraf- ing, VIDISCA-454 (De Vries et al., 2011), to investigate the fin blocks and sections were stained with hematoxylin and eosin possible involvement of a previously unknown virus in the devel- and reviewed by a board of certified veterinary pathologist. opment of HS in a N. coucang. Several sequences with homol- Twenty serum samples (belonging to 16 individuals: 11 N. cou- ogy to parvoviral genes were identified indicating the presence cang and 5 Nycticebus pygmaeus) and 61 organs collected at of a novel parvovirus, whose genome was subsequently fully necropsy (belonging to 17 individuals: 11 N. coucang—including sequenced. Buddha—and 6 N. pygmaeus) were screened for the presence of Parvoviruses (viral family Parvoviridae) are small non- thevirus.Organsamplesincluded17livers,6spleens,15kid- enveloped single stranded DNA viruses which are able to infect neys, 10 lungs, 6 hearts, 7 intestines (5 small intestines and 2 large a wide range of species of vertebrates (subfamily Parvovirinae) intestines). Altogether these samples belonged to 25 individuals and arthropods (subfamily Densovirinae). According to the lat- (18 N. coucang and 7 N. pygmaeus) with various types of disease. est ICTV classification (2013) 8 genera are recognized within the Liver, spleen, kidney, lung, and intestine samples from Buddha subfamily Parvovirinae and 5 of them include viruses infecting were available as well as serum or whole blood collected on 4 dif- primates (Cotmore et al., 2013; Cotmore and Tattersall, 2014). ferent time points: year 2000 (serum), year 2005 (serum), year Some parvoviruses classified within the genus Dependoparvovirus 2007 (whole blood), and year 2008 (serum). need the presence of a helper virus for a productive infection Serum samples and organs were stored at −80◦C until virolog- while viruses within other genera—the so called autonomous ical examinations were performed. parvoviruses—are S phase–dependent: cells must undergo the Housing management and sample collection from the ani- S phase of growth for viral replication to occur (Berns, mals in this report were approved by the appropriate federal and 1990). institutional regulatory authorities. The spectrum of parvovirus induced diseases, which mainly involve young individuals, is very wide and varies from more VIRUS DISCOVERY severe forms [like severe enteritis