Elsevier

Virus Research

Volume 118, Issues 1–2, June 2006, Pages 178-184
Virus Research

A novel deletion in the LTR region of a Greek small ruminant lentivirus may be associated with low pathogenicity

https://doi.org/10.1016/j.virusres.2005.12.010Get rights and content

Abstract

Greek small ruminant lentivirus (SRLV) strains remain relatively uncharacterized at the molecular level, despite the fact that lentiviral diseases of small ruminants are known to be widespread in the country. In the present study, we investigated the sequence diversity of the LTR region in Greek SRLV strains from sheep with and without disease symptoms, since sequence differences within this genomic area have been shown to lead to SRLVs with distinct replication rates. The AP-4 and AML (vis) motifs and the TATA-box were highly conserved among Greek strains, whereas the two AP-1 sites exhibited some substitutions. Pairwise comparisons with reference strains revealed that Greek LTR sequences were closer to the ovine strains (25.7% average divergence) rather than the caprine strain CAEV (59.1% average divergence). The most striking difference observed between the two groups of animals was a 13–14 nucleotide deletion in the strains obtained from the asymptomatic sheep. The deletion was located within the R region of LTR, which was also found to be much less homologous (39.6% average divergence) than the U3 and U5. Taken together, our data suggest that the R region of LTR may be involved in virus transcriptional activation. Furthermore, a specific deletion within this region may, at least in part, be associated with low pathogenicity of some SRLV strains.

Introduction

The small ruminant lentiviruses (SRLV) – which consist of maedi-visna virus of sheep (MVV) and caprine arthritis encephalitis virus of goats (CAEV) – constitute a divergent group of the lentivirus genus (Shah et al., 2004). They induce a lifelong infection, which can cause inflammatory and degenerative disease in various target organs including the mammary glands, lungs, synovia and brain (Pépin et al., 1998), often after a long latent period. The resulting lesions involve chronic inflammatory changes characterized by lymphoid hyperplasia and interstitial infiltration of mononuclear cells (Georgsson and Palsson, 1971, Pépin et al., 1998). Sheep and goats can become infected at an early age, but clinical signs are rarely evident before the second or third year of age (Berriatua et al., 2003). Following a long asymptomatic period, SRLV infections result in a multisystemic disease, the major manifestations of which are interstitial pneumonia, mastitis, encephalitis and arthritis (Houwers et al., 1988, Narayan and Clements, 1989, Van der Molen and Houwers, 1987).

SRLVs have a genetic organization that is typical of lentiviruses. Their genome is comprised of the gag, pol and env genes, and the tat, rev and vif open reading frames (Clements and Zink, 1996). Long terminal repeats (LTRs) – divided into the U3, R and U5 regions – flank the proviral DNA and provide the signals required for transcription, integration and polyadenylation of viral RNA (Pépin et al., 1998). They have also been shown to be responsible for the cellular tropism of the virus (Agnarsdottir et al., 2000). Lentiviral genomes are among the most rapidly evolving known. An elevated substitution rate is attributed to the low fidelity of the lentiviral reverse transcriptase, which has no proofreading exonuclease activity (Leroux et al., 1997).

SRLV isolates may be classified as rapid/high or slow/low according to their replication rate in vitro. The rapid/high strains replicate rapidly, inducing cell lysis and reaching high titers, whereas the slow/low grow slowly and to low titers (Barros et al., 2004, Lairmore et al., 1987, Querat et al., 1984, Woodward et al., 1995). Transcription of the proviral genome plays a crucial role in the virus life cycle, as it provides the template for synthesis of structural and regulatory proteins, and results in the generation of new copies of viral RNA. Transcription is regulated by the binding of various cellular proteins to DNA sequences, which reside within the LTR of the proviral genome. Several studies have demonstrated that AP-1, AP-4 and AML (vis) binding sites, which are located in the U3 promoter region of LTR, have a central role in the regulation of viral transcription (Barros et al., 2004, Barros et al., 2005, Campbell and Avery, 1996, Gabuzda et al., 1989, Gdovin and Clements, 1992, Hess et al., 1986, Sutton et al., 1997). Repeated sequences, which span the AP-1, AP-4 and AML (vis) motif-containing region, have been identified within the U3 of several virus isolates (Agnarsdottir et al., 2000, Barros et al., 2004, Barros et al., 2005, Campbell and Avery, 1996, Hess et al., 1989, Sargan et al., 1995). Due to the multiplication of these transcription factor binding sites, the repeats have been implicated in enhanced LTR-mediated transcription and subsequent increased virus replicative potential and accelerated disease progress. Proviral gene expression is also transactivated by the virus protein Tat, which mediates an increase in viral RNA synthesis (Hess et al., 1989) and RNA stability (Gdovin and Clements, 1992), through sequences located in U3. Therefore, sequence variations in SRLV LTRs may affect interactions with cellular transcription factors and may lead to an altered viral gene expression and replication. Indeed, it has been shown that sequence divergence between SRLV strains leads to LTRs with distinct transcriptional activities (Barros et al., 2004, Sargan et al., 1995).

In Greece, although lentiviral diseases of small ruminants have been described since 1967 (Exarchopoulos, 1967), SRLV strains remain relatively uncharacterized at the molecular level. Viral DNA sequences have only lately been detected by PCR in blood samples of sheep and goats from flocks with history of SRLV infections (Karanikolaou et al., 2005), and a partial sequence analysis of the gag gene of Greek strains has been recently published (Angelopoulou et al., 2005). Since sequences that control viral transcription are located in the LTR, and sequence differences (including sequence repeats) within this region have been shown to lead to SRLVs with distinct replication rates; the aim of the present study was to characterize this genomic area in Greek ovine lentivirus strains.

Section snippets

Animals and blood samples

Fifty sheep (2–12 years of age) from flocks in Northern Greece with history of MVV infections were used in this study. Forty-seven of them, which were from the same flock (flock I; Table 1), had no clinical signs of infection. The other three suffered from weight loss and dyspnoea, and each one of them belonged to a different flock (flocks II, III, IV; Table 1). Whole blood (10 mL) was collected in EDTA tubes by jugular venipuncture. Blood samples were centrifuged at 400 × g for 10 min, and buffy

Results

Forty-seven asymptomatic sheep from a flock with history of small ruminant lentiviral infections were analyzed for the presence of MVV in PBMC using a PCR protocol, which was based on the amplification of a portion of the LTR of the proviral genome. The area amplified spans part of the U3, the whole R and part of the U5 region. Forty-two animals were found to be positive. During agarose gel electrophoresis, however, it was observed that the bands of the PCR amplicons of all positive sheep moved

Discussion

Lentiviral diseases of small ruminants are widespread throughout the world. SRLV infections persist for life and carriers are considered a continuous potential source of virus for transmission. Although serological prevalence of SRLV within a flock can reach 90%, only a small proportion of animals develop clinical signs (Knowles, 1997). The mechanisms through which some lentiviruses kill their hosts, whereas others induce persistent infections are currently under investigation. It has been

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