W. van der Loo

Sharing of Endogenous Lentiviral Gene Fragments among Leporid Lineages Separated for More than 12 Million Years

ABSTRACT Lentiviruses are causal agents of severe pathologies of a variety of mammals, including cattle and humans (e.g., AIDS and different types of lymphoma). While endogenous forms of lentivirus do not occur in these species, A. Katzourakis and coworkers (A. Katzourakis, M. Tristem, O. G. Pybus, and R. J. Gifford, Proc. Natl. Acad. Sci. USA 104:6261-6265, 2007) recently reported the presence in the genome of the European rabbit (Oryctolagus cuniculus) of multiple sequences defining a lentiviral subgroup elegantly referred to as RELIK (rabbit endogenous lentivirus type K). Sequence comparisons indicated that the RELIK ancestor may have integrated into the rabbit lineage more than 7 million years ago. We have substantiated this by producing sequence data certifying the sharing of RELIK sequences among leporid lineages that diverged some 12 million years ago.

Evidence for recombination in the major capsid gene VP60 of the rabbit haemorrhagic disease virus (RHDV)

SummaryRabbit haemorrhagic disease (RHD) is a highly fatal disease caused by a virus of the family Caliciviridae. Whereas recombination is well documented in other members of this family, the extent of recombination has so far not been studied in RHDV. To reach a better evaluation of the possible role of recombination in the evolution of RHDV virulence, we have searched for recombination events in RHDV by analysing 43 complete sequences of the major capsid gene VP60. Phylogenetic analyses revealed two well separated groups. Clear evidence for recombination was found for the Hartmannsdorf strain which shows different phylogenetic profiles depending on the region of the capsid examined.

The evolution of the immunoglobulin heavy chain variable region (IgVH) in Leporids: an unusual case of transspecies polymorphism

In domestic rabbit (Oryctolagus cuniculus), three serological types have been distinguished at the variable domain of the antibody H chain, the so-called VHa allotypes a1, a2, and a3. They correspond to highly divergent allelic lineages of the VH1 gene, which is the gene rabbit utilizes in more than 80% of VDJ rearrangements. The sharing of serological VHa markers between rabbit and snowshoe hare (Lepus americanus) has suggested that the large genetic distances between rabbit VH1 alleles (9–14% nucleotide differences) can be explained by unusually long lineage persistence times (transspecies polymorphism). Because this interpretation of the serological data is uncertain, we have determined the nucleotide sequences of VH genes expressed in specimens of Lepus species. Two sequence groups were distinguished, one of which occurred only in hare specimen displaying serological motifs of the rabbit VHa-a2 allotype. Sequences of this group are part of a monophyletic cluster containing the VH1 sequences of the rabbit a2 allotype. The fact that this “transspecies a2 cluster” did not include genes of other rabbit VHa allotypes (a1, a3, and a4) is incompatible with the existence of a common VHa ancestor gene within the species, and suggests that the divergence of the VHa lineages preceded the Lepus vs Oryctolagus split. The sequence data are furthermore compatible with the hypothesis that the VHa polymorphism can be two times older than the divergence time between the Lepus and Oryctolagus lineages, which was estimated at 16–24 million years.

Detection of positive selection in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV)

Mutations were analysed in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV), a calicivirus responsible for high mortality rates in both wild and domestic European rabbits (Oryctolagus cuniculus). Likelihood of positive selection was estimated using the PAML software applied to 43 non-identical complete sequences of the major capsid protein. Three codons showed signs of positive selection (with posterior probabilities over 95%), one of them is located in the region containing the major antigenic determinants (region E). The presence of positively selected codons (PSCs) in other regions may suggest the existence of other antigenic regions on the major capsid protein that stimulate protective immune responses. At all the 3 PSCs, variation contributes to putative N-glycosylation sites of the protein. An N-glycosylation site is deleted in the non-pathogenic strain RCV. Some of the substitutions at PSCs may alter the polarity and the charge of the protein with possible implications in the protein structure and host interaction. The detection of PSCs should allow a better understanding of the interaction between RHDV and the rabbit immune system.

Extensive gene conversion between CCR2 and CCR5 in domestic cat (Felis catus)

Homogenization of the CC‐motif chemokine receptors CCR2 and CCR5 of cat (Felis catus) is documented and shown to be the outcome of gene conversion within the feline lineage. All regions were concerned, except the three extracellular protein domains (N‐ and C‐tails, and ECL2), suggesting that structural differentiation at these domains could be related to pathogen susceptibility.

Genetic diversity at the hinge region of the unique immunoglobulin heavy gamma (IGHG) gene in leporids (Oryctolagus, Sylvilagus and Lepus).

Unlike other species, European rabbit (Oryctolagus cuniculus) possesses only one immunoglobulin gamma class. Allelic diversity at the Ig (immunoglobulin) gamma constant region encoded by the unique IGHG (immunoglobulin heavy gamma) gene is moreover much reduced. In the European rabbit, the genetic variation at IGGH hinge region is limited to a single nucleotide substitution, which causes a Met–Thr interchange at amino acid position 9 (IMGT hinge numbering). We have analysed the diversity at this region more in‐depth by, (1) analysing the allelic variation in 11 breeds of domestic European rabbit (Oryctolagus cuniculus cuniculus), and (2) sequencing the gamma hinge exon in wild specimens of six species of rabbit (Oryctolagus and Sylvilagus) and hares (Lepus), including the two Oryctolagus subspecies (O. cuniculus cuniculus and O. cuniculus algirus). It appeared that among leporid species, amino acid changes occur exclusively at positions 8 and 9. However, while position 8 is occupied by either Pro or Ser residues, four different residues can occur at position 9 (Met, Thr, Pro and Leu). This variation concerns sites of potential O‐glycosylation and/or proteolytic cleavage, suggesting that the underlying genetic diversity could be the outcome of selection. Preservation of the gamma hinge polymorphism in domestic stocks could therefore be important. We report here a polymerase chain reaction/restriction fragment length polymorphism protocol that has allowed the monitoring of the heterozygosity levels at the gamma hinge in 11 breeds of domestic European rabbit.

The allotypic patchwork pattern of the rabbit IGKC1 allele b5wf: genic exchange or common ancestry?

Abstract The protein sequences of different alleles of the rabbit immunoglobulin IGKC1 gene can differ at more than 40% of the amino acid positions. This exceptional degree of allelic divergence raises questions concerning the causal underlying mechanisms. We report the DNA sequence of the coding region of an allotype which is associated with the mitochondrial lineage A (Southwestern Spain). At the serological level, this b5wf allotype presents a patchwork of antigenic determinants which in domestic breeds are characteristic of the b4, b5, and b6 allotypes. The inferred protein sequence of the b5wf allotype was found to differ from that of the b4, b5, and b6 allotypes at 25, 10, and 15% of the amino acid positions, respectively. Sequence comparisons show that the b4-specific epitopes of the b5wf allotype are probably due to a shared ThrThrGlnThr motif at Kabat positions 153–156. Similarly, the shared b5-specific determinants should relate to the motifs 161ThrSerLys163 and/or 182LysSerAspGlu185. A monoclonal antibody binding epitope shared among the b5wf, b5, and b6 sequences appeared to be correlated with the presence of Asp190. Although there is evidence of interallelic genic exchange, sequence comparisons suggest that the apparent mosaic structure of the b5wf allotype is better explained by common ancestry and point mutation.

Genetic characterization of the chemokine receptor CXCR4 gene in lagomorphs: comparison between the families Ochotonidae and Leporidae

Chemokines receptors are transmembrane proteins that bind chemokines. Chemokines and their receptors are known to play a crucial role in the immune system and in pathogen entry. There is evidence that myxoma virus, the causative agent of myxomatosis, can use the chemokine receptor CXCR4 to infect cells. This virus causes a benign disease in its natural host, Sylvilagus, but in the European rabbit (Oryctolagus cuniculus) it causes a highly fatal and infectious disease known as myxomatosis. We have characterized the chemokine receptor CXCR4 gene in five genera of the order Lagomorpha, Ochotona (Ochotonidae), and Oryctolagus, Lepus, Bunolagus and Sylvilagus (Leporidae). In lagomorphs, the CXCR4 is highly conserved, with most of the protein diversity found at surface regions. Five amino acid replacements were observed, two in the intracellular loops, one in the transmembrane domain and two in the extracellular loops. Oryctolagus features unique amino acid changes at the intracellular domains, putting this genus apart of all other lagomorphs. Furthermore, in the 37 European rabbits analysed, which included healthy rabbits and rabbits with clinical symptoms of myxomatosis, 14 nucleotide substitutions were obtained but no amino acid differences were observed.

The trans-species nature of rabbit b locus polymorphism is supported by studies on the snow-shoe hare

In the European rabbit ( Oryctolagus cuniculus ) there are two isotypes of the immunoglobulin (Ig) kappa light (L) chain: K1 and K2 (Benammar and Cazenave 1982; Heidmann and Rougeon 1983). The predominantly expressed K1 chain differs from all other known Ig L chains due to the presence of an extra disulfide bond between the variable and the constant domain. This constant domain (CK1) is encoded by theb locus (Oudin 1960; reviewed in Mage 1986). With allelic variation at nearly 50% of the amino acid positions, this locus is among the most polymorphic of protein loci known in vertebrates. Patterns of b locus gene diversity are very similar to those described for loci of the major histocompatibility complex ( Mhc; reviewed in Klein und O’hUigin 1995) and suggest that the rabbit CK1 chain is exposed to balancing selection (van der Loo and Verdoodt 1992; van der Loo 1993). The acquisition of numerous differences between alleles could theoretically be explained either by very fast substitution rates or long persistence time of allelic lineages (Klein et al. 1993; Graser et al. 1996). For Mhc loci, the available evidence fails to support the former but strongly supports the latter explanation (Klein 1980, 1987). This issue remains largely unsettled as far as theb locus alleles are concerned (Mariaméet al. 1987; van der Loo et al. 1995). Sequence determination of rabbitCK1 homologous loci in related species may verify whether allelic lineages can be shared between species, as was shown to be the case for Mhc polymorphism (‘trans-species polymorphism’; Figueroa and Klein 1987; Klein et al. 1993; Graser et al. 1996). Sequences are reported here of CK1 homologous polymerase chain reaction (PCR) products obtained with germline DNA of the snow-shoe hare ( Lepus americanus ). DNA was extracted from a blood clot of a specimen collected near Fairbanks (Alaska) with the help of John Bryant of the University of Alaska. Primer pair (OCCKP4 and OCCKM3) and PCR conditions were recently reported in this journal (van der Loo et al. 1995). The PCR products were cloned in pCR II with the TA cloning Kit (Invitrogen, San Diego, CA). Inserted sequences of nine clones were determined using the M13 primers of the plasmid. The consensus sequence of the CK1 exon homologous region is shown in Figure 1. The inferred amino acid sequences (Fig. 2) display the most salient characteristics of the rabbit CK1 domain. These are the supplementary cysteine at position 171 and the potential glycosylation site Asn174-Ser177. Consequently, they have been considered to reflect a gene locus homologous to the rabbit locus in L. americanus(bla locus).

PHYLOGENY OF THE RABBIT γ-CHAIN DETERMINANTS: A d12-LIKE ANTIGENIC DETERMINANT IN PRONOLAGUS RUPESTRIS

A survey for constant region γ‐chain allotypes (de locus) was undertaken in different lagomorph genera. As yet only Pronolagus rupestris, a paleolaginae, showed the presence of a determinant similar to rabbit d12 although it lacked the widespread e15 determinant.