Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula.

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.

Sharing of endogenous lentiviral gene fragments among leporid lineages separated for more than 12 million years.

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.

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.

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

Rabbit 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.

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.

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.

Genetic variation at chemokine receptor CCR5 in leporids: alteration at the 2nd extracellular domain by gene conversion with CCR2 in Oryctolagus, but not in Sylvilagus and Lepus species.

Whereas in its natural host (Sylvilagus sps.) the effects of myxoma virus infections are benign, in European rabbit (Oryctolagus cuniculus), it causes a highly infectious disease with very high mortality rate, known as myxomatosis. There is evidence that, as with HIV-1 virus in human, myxoma virus may use chemokine receptors such as CCR5 of the host target cell for entry and activation of pathways of immune avoidance. We have characterized and compared CCR5 genes of leporid species with different susceptibility levels to myxomatosis. The CCR5 protein of O. cuniculus differs markedly from all those known from other species. The most striking was the replacement of a specific peptide motif of the second extracellular loop (ECL2) by a motif, which in other species characterizes the CCR2 molecules. While absent in Sylvilagus and Lepus species, this CCR2 imposed CCR5-ECL2 alteration was observed in all genomes of 25 European rabbits, representing the subspecies O. cuniculus algirus and O. cuniculus cuniculus. Allelic variation at the rabbit CCR5 locus confirmed that the gene conversion predates the subspecies split (1-2 Ma).

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 V(H) a allotypes a1, a2, and a3. They correspond to highly divergent allelic lineages of the V(H) 1 gene, which is the gene rabbit utilizes in more than 80% of VDJ rearrangements. The sharing of serological V(H) a markers between rabbit and snowshoe hare (Lepus americanus) has suggested that the large genetic distances between rabbit V(H) 1 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 V(H) 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 V(H) a-a2 allotype. Sequences of this group are part of a monophyletic cluster containing the V(H) 1 sequences of the rabbit a2 allotype. The fact that this "transspecies a2 cluster" did not include genes of other rabbit V(H) a allotypes (a1, a3, and a4) is incompatible with the existence of a common V(H) a ancestor gene within the species, and suggests that the divergence of the V(H) a lineages preceded the Lepus vs Oryctolagus split. The sequence data are furthermore compatible with the hypothesis that the V(H)a polymorphism can be two times older than the divergence time between the Lepus and Oryctolagus lineages, which was estimated at 16-24 million years.

Hotspot variation at the CH2-CH3 interface of leporid IgG antibodies (Oryctolagus, Sylvilagus and Lepus).

The European rabbit (Oryctolagus cuniculus) is the only species known to express only one subclass of gamma class immunoglobulin (IgG) antibodies. The rabbit IGHGCH2 (second domain of the gene region encoding the IgG heavy chain constant region) or e locus presents two serologically defined alleles, the e14 and e15 allotypes. These are correlated with amino acid variation at position 309, which is located within the target region of the neonatal FcRn receptor. The e14 and e15 markers were also observed in other lagomorph species. Population genetic research has indicated that polymorphism at this locus is sustained by selection. We present here the IGHGCH2 exon sequences for 12 species of rabbit and hare (genera Oryctolagus, Sylvilagus and Lepus). The inferred amino acid sequences reveal that, despite an overall sequence identity of 97%, five different residues can occur at position 309. As for Oryctolagus, the e15 allotype was always associated with the presence of an Ala309 codon. In all but one case, this codon defined an allotype-specific ThaI restriction site. The potential of PCR/ThaI restriction fragment length polymorphism (RFLP) analyses for studying IGHGCH2 variation within and between populations is emphasized.

Restriction fragment alleles of the rabbit IGHG genes with reference to the rabbit IGHGCH2 or e locus polymorphism.

Among domesticated mammals, rabbit (Oryctolagus cuniculus) is the only species possessing not more than one subclass of immunoglobulin (IgG) antibodies. The rabbit IGHGCH2 or e locus presents two serologically defined alleles, the e14 and e15 allotypes, which are correlated with amino acid variation at the IgG CH2-CH3 interface. Genetic studies, while revealing the adaptive value of this polymorphism, have relied so far entirely upon allo-antisera. Here we show how these alleles can be distinguished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The proposed PCR-RFLP approach allows the monitoring of IGHG locus diversity in rabbit.

Genetic analysis and mapping of biochemical markers in an F2 intercross of two inbred strains of the rabbit (Oryctolagus cuniculus).

A total of 40 biochemical and four immunological markers found to be polymorphic in the rabbit in previous studies were screened in the AX/JU and IIIVO/JU inbred strains. Although the strains are considered unrelated, only eight (biochemical) markers werefound to be polymorphic between the two strains. These eight markers were analyzed in an F2 intercross population. Linkage was found for Est-5 and C on chromosome 1 and for Es-1, Est-2, Est-4, Est-6 and HP on linkage group VI. Two polymorphic markers, Es-3 and Mhr-1 could not be linked to any of the other markers.

Clinimetric evaluation of the pain observation scale for young children in children aged between 1 and 4 years after ear, nose, and throat surgery.

This study assessed the reliability, validity, and responsiveness of a new pain measure for children aged 1 to 4 years that was developed from the Children's Hospital of Ontario Pain Scale and its Neonatal Infant Pain Scale. Pain in 311 children, aged 1 to 4 years, was measured by two observers at fixed intervals after adenotonsillectomy (n = 114), adenotomy (n = 109), or insertion of ventilation tubes (grommets) (n = 88) until discharge using a dichotomous pain scale of 9 behavioral and physiological categories. The scale proved to be strongly homogeneous. The interobserver agreement was substantial for 7 items. On these final 7 items, the ability to distinguish between patients with differing degrees of pain and the sensitivity to detect changes over time within each patient were substantial. The resulting Pain Observation Scale for Young Children is reliable and easy to use for assessment of short- and longer-lasting pain after ear, nose, and throat surgery and may be used for assessing pain with other conditions.

Cytonuclear disequilibria in wild populations of rabbit (Oryctolagus cuniculus L.) suggest unequal allele turnover rates at the b locus (IGKC1).

DNA sequence comparisons suggest that evolutionary rates at the rabbit IGKC1 locus can differ among allelic lineages. Here we address the question of whether population turnover rates can vary among IGKC1 alleles. We studied the distribution of sixteen IGKC1 (or b-locus) allotypes in areas comprising the aboriginal species range (Iberian peninsula). Rabbits in this area belong to one of two distantly related mitochondrial lineages (mtDNA types) A and B. In the more recent distribution area of the species, all rabbits belong to the mtDNA type B lineage, and IGKC1 alleles b4 and b5 comprise over 90% of the gene pool. These two alleles are also predominant in areas of mtDNA type B prevalence within the Iberian range. However, in areas of mtDNA type A prevalence, the b4 and b5 allotypes are rare or absent; they apparently have been replaced by serologically related, but distinct, 'endemic' variants. The cytonuclear disequilibria were highly significant, also within the subsample consisting of populations from Spain. These observations suggest that allelic persistence times for the predominant IGKC1 lineages could be shorter than the divergence time of the major mtDNA lineages A and B. In contrast, the relative gene frequencies of the IGKC1 allele b9 were similar among the type A and type B rabbits; it was present in most populations at low frequency. In consequence, persistence times of the b9 allele appear to be longer than the divergence time of lineages A and B. The data reported here are in agreement with the DNA sequence data, providing further proof that the molecular clock can run at different rates among allelic lineages at the rabbit IGKC1 locus.

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

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.

Compensatory aspects of allele diversity at immunoglobulin loci: gene correlations in rabbit populations devoid of light chain diversity (Oryctolagus cuniculus L.; Kerguelen Islands).

Is there a selective advantage of increased diversity at one immunoglobulin locus when diversity at another locus is low? A previous paper demonstrated excess heterozygosity at the rabbit light chain b locus when heterozygosity was low at the heavy chain constant region e locus. Here we consider the reverse situation by analyzing allele distributions at heavy chain loci in populations fixed for the light chain b locus. We analyzed the a locus that encodes the predominantly expressed heavy chain variable region, and the d and e loci that control different parts of the Ig gamma class constant region. While there was excess heterozygosity, genetic differentiation between localities was extensive and was most pronounced for females. This was in marked contrast with observations in areas where b-locus diversity was important and confirms a negative correlation between e- and b-locus heterozygosity. Trigenic disequilibria corresponded to a significant negative correlation between e- and a-locus heterozygosity due mainly to strong variation among localities within the context of pronounced (digenic) linkage disequilibria. Although substantial, the average increase in a/e-locus single heterozygosity implemented by higher order disequilibria within localities was not significant.

Characterization and DNA sequence of the b6w2 allotype of the rabbit immunoglobulin kappa 1 light chain (b locus).

The b6w2 allotype of the constant region of the rabbit immunoglobulin kappa 1 (K1) light chain (b locus) was discovered in wild populations from northern Spain. At the serological level, the b6w2 allotype is characterized by the presentation of all b6-specific epitopes, while an allotypic determinant which is shared between the nominal b5 and b6 allotypes is lacking. The DNA fragment encoding the b6w2 allotype was amplified by means of the polymerase chain reaction, and sequenced directly by dideoxy-DNA-sequencing. When compared with the sequence of the nominal b6 allele, the b6w2 sequence differs at eleven nucleotide positions (96.5% similarity). This variation corresponds to amino acid replacements at 1) the three positions C-terminal to the peptidyl junction with the variable region (amino acid positions 109-111); 2) the four positions N-terminal to the interdomain disulfide bond (167-170); and 3) two positions in the vicinity of the interchain disulfide bond (190 and 210). The nature and distribution of the observed nucleotide substitutions strongly suggest a possible role of the extra interdomain disulfide bond in the unusual evolutionary dynamics of the rabbit K1 light chain.