Full-length genome sequence analysis of enzootic nasal tumor virus reveals an unusually high degree of genetic stability

Abstract

Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1_NA) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1_EU). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1_EU genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1_NA and ENTV-1_EU reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K_a/K_s ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.

Introduction

Enzootic nasal adenocarcinoma (ENA) and ovine pulmonary adenocarcinoma (OPA) are contagious neoplasms of sheep and goats affecting the epithelial cells of the nose and distal lung, respectively (De las Heras et al., 2003, Palmarini and Fan, 2003). Jaagsiekte sheep retrovirus (JSRV), an ovine betaretrovirus, is the causative agent of OPA (Palmarini et al., 1999a). A similar betaretrovirus etiology has long been assumed for ENA due to the consistent detection of enzootic nasal tumor virus (ENTV) in nasal tumors and exudate of animals with ENA (De las Heras et al., 1995, De las Heras et al., 1993, De las Heras et al., 1991). Two distinct viruses are implicated in ENA, one in sheep (ENTV-1) (Cousens et al., 1999) and one in goats (ENTV-2) (Ortín et al., 2003) and successful transmission of ENA from goat to goat using clarified or concentrated nasal fluid with immunological evidence of the presence of ENTV-2 is described (De las Heras et al., 1995).

The ovine genome is colonized by more than 27 endogenous retroviral sequences (ERVs) (Arnaud et al., 2007). Ovine ERVs share a high degree of sequence homology with JSRV and ENTV, and are designated endogenous JSRV (enJSRV). Past studies noted significant expression of enJSRV proteins in many tissues including the thymus, Peyer's patches, spleen, bone marrow, peripheral lymph nodes, leukocytes, lung, kidney and uterine endometrial epithelium of the fetal lamb (Palmarini et al., 1996, Palmarini et al., 2000), but extracellular release of endogenous particles was not observed (Spencer et al., 2003). Due to the similarity of enJSRV sequences with that of exogenous JSRV and ENTV it is believed that expression of enJSRV proteins in the fetal thymus causes an immune tolerance to exogenous virus via a mechanism of clonal deletion of self reactive T cells (Palmarini et al., 2004, Spencer et al., 2003). Moreover, no evidence for circulating antibodies has been reported for either ENA or OPA when using antigen from natural sources (Ortín et al., 1998, Sharp and Herring, 1983, Summers et al., 2002). The prevalence and geographical distribution of ENA is unclear as serological screening for the virus is not possible and reports of ENA in the USA and Canada are limited.

To date, only one full-length genome sequence of ENTV-1 has been determined (accession number NC_007015, hereafter referred to as ENTV-1_EU) (Cousens et al., 1999). This sequence was derived from a cDNA phage library of genomic RNA extracted from the purified nasal exudate of a spontaneous case of ENA in Europe. Currently, there is little epidemiological and no sequence data pertaining to ENTV-1 or -2 in North America despite the fact that intranasal tumors occur in sheep throughout Canada and the United States (Cutlip and Young, 1982, Duncan et al., 1967, Madewell et al., 1976, McKinnon et al., 1982, Rings and Rojko, 1985, Young et al., 1961). There are no known cell culture systems that support the growth of ENTV-1 (or JSRV), nor is there an infectious molecular clone of ENTV-1. For this reason, there is very little known about the pathogenesis or the prevalence of ENA. The purpose of the present study was to elucidate the full-length genome sequence of ENTV-1 from naturally occurring cases of ENA in Canada and the United States in order to examine the genetic heterogeneity of ENTV-1 in naturally infected sheep and to expand the limited sequence information that is currently available. To this end, ten tumor samples from spontaneous cases of ENA in Canada and the United States were obtained and the complete nucleotide sequence of the ENTV-1 provirus determined. Phylogenetic analysis revealed that these North American isolates are genetically stable and closely related to ENTV-1 from Europe. Despite this marked sequence conservation, four distinct regions of variability were identified.