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Short Communication |
1 Université de Lyon, F-69007 Lyon, France
2 Université Lyon 1, F-69007 Lyon, France
3 INRA, UMR754 ‘Rétrovirus et Pathologie Comparée’, Lyon, France
4 Ecole Nationale Vétérinaire de Lyon, Lyon, France
5 Ecole Pratique des Hautes Etudes, Lyon, France
6 IFR128 BioSciences Gerland Lyon Sud, Lyon, France
7 Laboratoire Départemental Vétérinaire, Gap, France
8 Hospices Civils de Lyon, Lyon, France
Correspondence
François Guiguen
guiguen{at}univ-lyon1.fr
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Present address: MMD Lab of Viral Pathogenesis, University of Kansas Medical Center, 5000 WHE, 3901 Rainbow Blvd, Kansas City, KS 66160, USA. 
Present address: UR66, Pharmacologie et Toxicologie, INRA-180 Chemin de Tournefeuille, BP 3, 31931 Toulouse Cedex 9, France.
The GenBank/EMBL/DDBJ accession numbers for the gag and LTR sequences, respectively, reported in this paper are EU375926–EU375932 and EU375968–EU375971 for ibex #1; EU375933–EU375937 and EU375972–EU375973 for ibex #2; EU375938–EU375941 and EU375974–EU375976 for ibex #3; EU375942–EU375945 and EU375977–EU375979 for hybrid #1; EU375946–EU375951 and EU375980–EU375982 for hybrid #2; EU375952–EU375953 and EU375983–EU375985 for hybrid #3; EU375954–EU375958 and EU375986–EU375988 for goat #1; EU375959–EU375961, EU526865 and EU375989–EU375991 for goat #2; and EU375962–EU375967 for goat #3.
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; Peterhans et al., 2004). SRLV origins remain obscure (Katzourakis et al., 2007; Querat et al., 1990) and similar viruses have not been found in wild small ruminants despite the frequent capacity of lentiviruses to cross species barriers, perhaps related to their high within-species genetic variation and propensity for recombination (VandeWoude & Apetrei, 2006). SRLVs are usually transmitted from dam to offspring by infected monocytes in milk or colostrum, but contact or venereal transmission are also possible, as in the classical epizootic of VMV in Iceland following the introduction of contagious Karakul rams (Palsson, 1972); contagion between goats is greatly reduced if barriers prevent physical contact (Peterhans et al., 2004). The possibility of transmission of SRLV to endangered wild small ruminants at a site in the French Alps where a small herd of heavily infected goats share grazing grounds with a small group of wild ibexes (Capra ibex) was investigated. Close contact between the goats and ibexes studied here has been confirmed by routine and specific observations (Richomme et al., 2006) and the birth of several goat/ibex hybrids. Proviral gag [matrix (MA) region] and long terminal repeat (LTR) sequences were obtained by PCR from goat, ibex and hybrid white blood cells. Blood was withdrawn from the jugular vein by Vacutainer into dry tubes for serology by ELISA as described previously (Guiguen et al., 2000) and onto EDTA for cellular studies and DNA extraction. The small group of ibexes, initially derived from a single pregnant female and isolated from other ibex groups, has been observed by the French wildlife survey over 12 years on high pastures shared with the domestic goat flock. The first offspring was a male (ibex #1) that remained dominant in the group until his death when his son (ibex #3) replaced him. Animals in the group (up to seven in total) were trapped at intervals for veterinary and serological surveillance. The founder female died shortly before our study. The domestic goat flock had a high turnover, as animals were frequently culled and replaced, and typically consisted of five to seven adult nannies and their kids. They were in regular summer contact with the ibexes and hybrid offspring often resulted. At the time of sampling, five of six adult nannies were seropositive for CAEV. Four hybrid females born to different dams in the same year at an age of approximately 8 months were purchased before they had been released to their summer pastures where they could contact the ibex population. They were maintained in stables at the Lyon Veterinary School for 2 years (authorization for animal testing no. 5759; experimental protocol approved by the Ethics Committee of the Ecole Nationale Vétérinaire de Lyon, on 17 April 2002). Three animals were seropositive for CAEV on arrival and were housed together; the fourth was, and remained, seronegative for CAEV and was kept in a separate but adjacent pen. Peripheral blood samples taken after 1 year (age approximately 20 months) provided DNA for investigation of SRLV proviral sequences. A third ibex (ibex #2), which was positive for SRLV by PCR, was sampled at a site >100 km away.
Peripheral blood mononuclear cells (PBMCs) were concentrated from 10–50 ml blood by Ficoll centrifugation (Narayan et al., 1983) and, when viable, monitored for the development of syncytia indicative of SRLV infection (Narayan et al., 1980) in simple culture or co-culture with susceptible goat synovial membrane cells. Genomic DNA was extracted from 5x106 uncultured PBMCs using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer's instructions. DNA concentration and quality were determined spectrophotometrically and stocks were kept at –70 °C before amplification of proviral elements.
A 512 bp canonical fragment of the gag gene comprising the MA region and the first 24 nt of the capsid region was amplified using Pfu polymerase (Promega) by nested PCR with primers and conditions described previously (Chebloune et al., 1996). Primers, numbered according to the CAEV reference strain Co (CAEV-Co) (Saltarelli et al., 1990), were: GEX5 (5'-GAAGTGTTGCTGCGAGAGGTCTTG-3', nt 393–416) and GEX3 (5'-TGCCTGATCCATGTTAGCTTGTGC-3', nt 1291–1268) for the first amplification; and GIN5 (5'-GATAGAGACATGGCGAGGCAAGT-3', nt 524–546) and GIN3 (5'-GAGGCCATGCTGCATTGCTACTGT-3', nt 1036–1013) for the second amplification.
Proviral LTRs were amplified by single-round PCR using primers U35 (5'-CTGTGAGACATGGGCTAAAGAGGAC-3', nt 8819–8843) and U53 (5'-GCTGCGAGAGCCGCTCTGGTATTGC-3', nt 163–139).
The PCR mix in a final volume of 50 µl consisted of 1x Pfu DNA polymerase buffer [20 mM Tris/HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1 mg nuclease-free BSA ml–1], 200 µM dNTP, 1.25 U Pfu DNA polymerase, 0.5 µM each primer and 500 ng DNA. Each amplification included a positive control (CAEV-Co-infected GSM cells) and negative control (ultrapure water) run in parallel. All PCR products were observed by electrophoresis through 1 % agarose gel containing 1 µg ethidium bromide ml–1 in 1x TAE buffer.
Amplicons from three independent PCRs from each animal were purified using the Montage PCR kit (Millipore), then A-tailed by incubation for 10 min at 72 °C with Taq polymerase and dATP for cloning into the pGEM-T Easy Vector System 1 (Promega). Ligation products were used to transform MAX Efficiency DH5-
chemically competent cells (Invitrogen) and plasmid DNA was extracted using the Plasmid Mini kit (Qiagen) and observed by EcoRI digestion to identify inserts with the expected size. With the exception of gag sequences from hybrid #3, where only two clones could be obtained, three to seven clones per locus were sequenced for each animal using an ABI Prism 3100 Genetic Analyzer at the ‘Sequencing Technical Platform’, IFR128, Lyon, France, using GIN5' and GIN3' primers for the gag gene and U35 and U53 primers for the LTR region. GenBank accession numbers are given in the legend of Fig. 1.
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), with manual adjustment where necessary. Different sequences from individual animals generally varied by only 0.2–1.4 % (Table 1). Goat #3 provides an interesting exception, with a single clone of gag (clone #4) that diverged by 8 % from the mean of the other five clones, leading to an intra-animal divergence of 3.5 %. In contrast, the LTR of goat #1 was virtually identical in all three clones. The ibex and hybrid proviral sequences varied between different animals by <2 %, whereas those from goats formed a diverse group with >7.5 % variation between animals. Finally, ibex and hybrid proviruses were similar in their gag and LTR sequences (<1.9 % variation) and both differed more considerably from the goat sequences in both gag (>6.5 %) and LTR (approx. 4 %) sequences; see Table 1.
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) constructed using the neighbour-joining method (Saitou & Nei, 1987), with bootstrap values determined over 1000 iterations (Felsenstein, 2002). Trees were formatted using the program NJPLOT (Perriere & Gouy, 1996). The gag sequences (Fig. 1a) show that proviral sequences from goats #2 and #3 clustered together, with the sequences of clone #4 from goat #3 and CAEV-Co being more distantly related. All sequences from ibexes and hybrids bunched together with those from goat #1 on a separate branch, well supported by a bootstrap value of 998. This might suggest a common origin for these proviruses, but the LTR sequences (Fig. 1b) of goat #1 clustered with those from goat #2 and CAEV-Co, whereas the ibex and hybrid proviruses formed a distinct group, strongly supported by the bootstrap values. Trees constructed using different algorithms produced very similar results (not shown) and homologous sequences from reference VMV fell far outside either grouping (not shown).
The gag proviral sequences from ibexes, hybrids and goat #1 showed a 6 nt deletion compared with CAEV-Co and the other goats in this study, leading to a 2 aa deletion in the MA protein (Fig. 2). This deletion is not of itself responsible for the division into two groups on the tree, because when the deletion was artificially replaced in the sequences, the resulting trees were not significantly altered (not shown). Surprisingly, this marker was present in all the hybrids sequenced, despite their having different mothers, and in all the ibex sequences, including those originating from a female (ibex #2) from a distant site. Analysis of synonymous and non-synonymous nucleotide changes using the Datamonkey software (www.datamonkey.org) indicated positive selection at three sites and negative selection at 12 sites, suggesting a trend toward conservation of the amino acid sequence deduced from the gag gene.
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. Unfortunately, no DNA remained from goat #3 and she had been culled. The amplified sequences varied between 316 and 318 nt in length and sequences varied little in individual goats, but somewhat more in the ibexes and hybrids (Table 1; see LTR intra-animal divergence); animals of the same group (goat, ibex and hybrid) varied by <2 % (Table 1; see intra-group divergence). Ibex proviral LTRs differed from those of goats by 3.9 %, but only by 1.9 % from those from hybrids. Examination of the LTR sequences shows that, whereas all sequences preserved a canonical TATA box, there were sporadic changes to the AP1, AP4 and AML sites postulated to be involved in proviral gene expression. Interestingly, two clones, one from an ibex and the other from a hybrid, showed single nucleotide substitutions in the polyadenylation signal, suggesting that these proviruses may be inactive. Other clones from the same animals showed a canonical polyadenylation signal.
These results indicate that wild-living ibexes in the French Alps can carry SRLV proviral sequences related to CAEV. These sequences may derive from domestic goats herded on high pastures where close contact with the mountain goats occurs, as attested by the birth of goat/ibex hybrids. It was found that the ibexes and the hybrids carry a subset of proviral sequences that distinguish them from most sequences from domesticated animals. The 5' region of the gag gene was studied in this report. This region is known to be both genetically and antigenically variable (Grego et al., 2005; Pisoni et al., 2006), but has unfortunately not been exploited for taxonomic studies of SRLV that use the CA region of the gag gene and/or pol sequences (Leroux et al., 1995, 1996; Shah et al., 2004a; Zanoni, 1998). All sequences from ibexes and hybrids shared a striking marker deletion of 6 nt corresponding to the absence of a glutamine–glutamic acid pair, which has not been described in CAEV type sequences, although it has been documented in one otherwise quite distinct VMV-type sequence from a Spanish sheep (Reina et al., 2006). The sequences were more closely related to typical published CAEV sequences than to VMV sequences (not shown), but a more precise taxonomic situation of our sequences in the SRLV group is not yet possible.
The marker deletion in gag MA was shared by one domestic goat and, even when the deletion was artificially restored in the sequences, the gag from goat #1 grouped with the ibex and hybrid sequences, not with the other goats (not shown). This strongly suggests a common origin, but the LTR sequences from goat #1 differed from those of the ibexes and hybrids and resembled those from other domestic goats (Fig. 1b). The hybrids must have acquired their infection from their dams or by horizontal transmission within the small flock, as they had not encountered wild ibexes at the time of purchase. The ibex-type LTR has never been reported from goats or sheep, but must have been present either in a culled animal or at low frequency within the flock. It is, however, the major sequence in wild goats and the hybrids. This might represent selection of a variant sequence in a different host context; however, passage of CAEV and VMV between sheep and goats is not accompanied by major genetic changes in the virus (Pisoni et al., 2005; Shah et al., 2004a, b), although sheep and goats are further apart genetically than goats and ibexes. Alternatively, a variant of SRLV may be present in some wild ibexes, as suggested by the very similar, though distinct, sequences from ibex #2 from a distant site. The rarity of ibexes that are seropositive for SRLV suggests that any such virus must be poorly cross-reactive with CAEV antigens used for testing; indeed, two of our ibexes carrying provirus detectable by PCR were seronegative by ELISA. The infectious virus isolated from samples from ibex #1 may prove to be a source of specific ibex SRLV sequences for more accurate testing of wild populations.
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ACKNOWLEDGEMENTS |
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Received 14 January 2008;
accepted 19 February 2008.
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