Elsevier

Virus Research

Volume 158, Issues 1–2, June 2011, Pages 116-123
Virus Research

A novel member of the family Hepeviridae from cutthroat trout (Oncorhynchus clarkii)

https://doi.org/10.1016/j.virusres.2011.03.019Get rights and content

Abstract

Beginning in 1988, the Chinook salmon embryo (CHSE-214) cell line was used to isolate a novel virus from spawning adult trout in the state of California, USA. Termed the cutthroat trout (Oncorhynchus clarkii) virus (CTV), the small, round virus was not associated with disease, but was subsequently found to be present in an increasing number of trout populations in the western USA, likely by a combination of improved surveillance activities and the shipment of infected eggs to new locations. Here, we report that the full length genome of the 1988 Heenan Lake isolate of CTV consisted of 7269 nucleotides of positive-sense, single-stranded RNA beginning with a 5′ untranslated region (UTR), followed by three open reading frames (ORFs), a 3′ UTR and ending in a polyA tail. The genome of CTV was similar in size and organization to that of Hepatitis E virus (HEV) with which it shared the highest nucleotide and amino acid sequence identities. Similar to the genomes of human, rodent or avian hepeviruses, ORF 1 encoded a large, non-structural polyprotein that included conserved methyltransferase, protease, helicase and polymerase domains, while ORF 2 encoded the structural capsid protein and ORF 3 the phosphoprotein. Together, our data indicated that CTV was clearly a member of the family Hepeviridae, although the level of amino acid sequence identity with the ORFs of mammalian or avian hepeviruses (13–27%) may be sufficiently low to warrant the creation of a novel genus. We also performed a phylogenetic analysis using a 262 nt region within ORF 1 for 63 isolates of CTV obtained from seven species of trout reared in various geographic locations in the western USA. While the sequences fell into two genetic clades, the overall nucleotide diversity was low (less than 8.4%) and many isolates differed by only 1–2 nucleotides, suggesting an epidemiological link. Finally, we showed that CTV was able to form persistently infected cultures of the CHSE-214 cell line that may have use in research on the biology or treatment of hepevirus infections of humans or other animals.

Introduction

In 1991, a novel RNA virus that was not associated with disease was reported as widespread among trout broodstocks in California, USA (Hedrick et al., 1991). The virus could be isolated using the Chinook salmon embryo cell line, CHSE-214, in which it produced a slow, focal type of cytopathic effect (CPE) that did not result in destruction of the entire monolayer. This cell culture system was used to demonstrate the small, round virus had several characteristics in common with picornaviruses, and that detectable levels of the virus were present at spawning in the ovarian fluids of cutthroat trout (Oncorhynchus clarkii), rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis). Termed the cutthroat trout virus (CTV), increased surveillance efforts in subsequent years showed the virus was broadly distributed among trout populations in many areas of the western United States, likely by the widespread shipment of infected eggs to rearing facilities in new locations.

While waterborne exposures of juvenile rainbow trout, cutthroat trout or kokanee salmon (Oncorhynchus nerka) to relatively high doses of CTV did not produce mortality, the virus could be re-isolated from some of the animals for 4–6 weeks post-infection (Hedrick et al., 1991). Interestingly, prior exposures of juvenile rainbow trout to CTV provided significant reduction in mortality compared with unexposed controls following laboratory challenges with the fish rhabdovirus, Infectious hematopoietic necrosis virus. The increased resistance could be detected for up to 4 weeks post-CTV exposure and was potentially mediated by induction of an interferon response (Hedrick et al., 1994). More recently, unpublished epidemiological evidence suggests that CTV may be maintained in a life-long carrier state among some fraction of the population in which the virus is expressed at spawning, possibly due to the immunosuppressive effects associated with sexual maturation.

Hepatitis E virus (HEV) is the type species of the genus Hepevirus in the family Hepeviridae and has been the subject of several recent and comprehensive reviews (Emerson and Purcell, 2003, Lu et al., 2006, Okamoto, 2007, Panda et al., 2007, Vasickova et al., 2007, Mushahwar, 2008, Purcell and Emerson, 2008, Aggarwal and Naik, 2009, Meng, 2010). The virus is known as the causative agent of outbreaks of either self-limited or fulminant hepatitis in humans and a zoonotic agent that has been detected in a range of mammalian species from many areas of the world. Initially sequenced by Tam et al. (1991), HEV has a single-stranded, positive-sense, capped and poly-adenylated genome of approximately 7.2 kb in length containing three discontinuous and partially overlapping open reading frames coding for a set of structural and non-structural genes flanked by short 5′ and 3′ untranslated regions. Subsequent analysis of HEV sequences from humans and domestic animals revealed the presence of four genotypes that were associated with phylogeographic or epidemiologic differences (Lu et al., 2006, Okamoto, 2007). More recently, a novel HEV genotype was detected in rodents (Johne et al., 2010). The genome of avian hepeviruses shows a similar organization, but the shorter length (6.6 kb) and lower sequence identity (approximately 50% at the nucleotide level) appear sufficiently distinct to support the creation of a separate genus having at least three genotypes (Huang et al., 2004, Bilic et al., 2009, ICTV, 2010, Marek et al., 2010). Here, we report the sequence of the full genome of CTV and show it is a novel member of the family Hepeviridae.

Section snippets

Cell culture and propagation of virus

The 1988 isolate of CTV from adult cutthroat trout spawned at Heenan Lake in Northern California, USA was selected for whole genome sequencing. This isolate represents the index case and was used to provide the initial description of CTV by Hedrick et al. (1991). The Chinook salmon embryo (CHSE-214) cell line (ATCC CRL-1681) was propagated at 15 °C using minimum essential medium as described by Hedrick et al. (1994). To produce stocks of virus for sequence analysis, cultures of CHSE-214 cells

Characteristics of the virus

All isolates of CTV grew well in CHSE-214 cell cultures where they produced typical CPE after approximately 14 d of incubation (Fig. 1). Electron microscopy of pelleted cell culture fluid confirmed that CTV was a spherical virion of approximately 31 nm (30.94 ± 0.94) in diameter (Fig. 2), slightly smaller than the 37.5 nm initially reported by Hedrick et al. (1991) but in good agreement with size estimates for virions of other hepeviruses (Purcell and Emerson, 2008).

Sequencing of virus genome

The full length genome of the

Discussion

Our results indicate that the cutthroat trout virus is a candidate for inclusion in the family Hepeviridae, the first from an aquatic animal. Nucleotide sequence differences between CTV and hepeviruses from mammals or birds appeared to be sufficient to justify the creation of a novel genus, within the family that would include CTV as well as similar viruses isolated from fish or other aquatic animals in the future. We propose the genus name Cutrovirus to reflect the initial host, the cutthroat

Acknowledgements

The authors are grateful to the fish health experts in the Western USA who provided isolates of CTV for this study. Dr. Graham Young of the School of Aquatic and Fishery Sciences at the University of Washington and Dr. Penny Swanson of the Northwest Fisheries Science Center of the National Oceanographic and Atmospheric Administration provided helpful advice on the reproductive endocrinology of teleosts. We also thank two anonymous reviewers for suggestions that substantially improved the

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