B and T lymphocytes in rabbits change according to the sex and throughout the year.

The European rabbit (Oryctolagus cuniculus) is a good model in biomedicine used in research on several human diseases. The reference values of B and T cells and their subpopu- lations are very important to understand how the adaptive immune system is responding to infectious agents. The aim of this study was to determine values of B and T cells and their subpopulations in Polish mixed-breed rabbits, considering seasons of the year and sex. The study was performed on 200 Polish mixed-breed rabbits and the percentage of B and T lymphocytes was measured cytometrically using mouse anti-rabbit antibodies. The study revealed that the season of the year and sex of the animals affected the percentage of B- and T-cells and their subpopulations in peripheral blood. Statistically significant values of CD19+ B-cells in spring and autumn, of T CD5+ cells in spring and winter, of T CD4+ in spring, summer, autumn and winter, of T CD8+ in winter and of T CD25+ in spring were noted. Generally the highest values were found mainly in warm part of the year, while the lowest in colder months. A statistical significance was also observed between males and females - changes were found in T CD4+ and T CD25+ lymphocytes in spring, T CD8+ cells in winter and higher percentage was generally obtained in females than in males. The only exception was the T CD5+ subpopulation in which no differences were observed between the sexes and throughout the year. This is the first paper on adaptive immune system cell values in the European rabbit of domestic breeds.

Insights into the evolution of the new variant rabbit haemorrhagic disease virus (GI.2) and the identification of novel recombinant strains.

Rabbit haemorrhagic disease (RHD) is a viral disease that affects the European rabbit. RHD was detected in 1984 in China and rapidly disseminated worldwide causing a severe decline in wild rabbit populations. The aetiological agent, rabbit haemorrhagic disease virus (RHDV), is an RNA virus of the family Caliciviridae, genus Lagovirus. Pathogenic (G1-G6 or variants GI.1a-GI.1d) and non-pathogenic strains (GI.4) have been characterized. In 2010, a new variant of RHDV, RHDV2/RHDVb/GI.2, was detected in France. GI.2 arrived to the Iberian Peninsula in 2011, and several recombination events were reported. Here, we sequenced full genomes of 19 samples collected in Portugal between 2014 and 2016. New GI.2 recombinant strains were detected, including triple recombinants. These recombinants possess a non-structural protein p16 related to a non-pathogenic strain. Evolutionary analyses were conducted on GI.2 VP60 sequences. Estimated time to the most recent common ancestor (tMRCA) suggests an emergence of GI.2 in July 2008, not distant from its first detection in 2010. This is the first study on GI.2 evolution and highlights the need of continued monitoring and characterization of complete genome sequences when studying lagoviruses' evolution.

Epidemiology of RHDV2 (Lagovirus europaeus/GI.2) in free-living wild European rabbits in Portugal.

As the detection of the first outbreak of a novel aetiological agent of rabbit haemorrhagic disease commonly called RHDV2 or RHDVb (Lagovirus europaeus/GI.2, henceforth GI.2) in France in 2010, the virus rapidly spread throughout continental Europe and nearby islands such as Great Britain, Sardinia, Sicily, the Azores and the Canary Islands among others. The outbreaks of this new lagovirus cause important economic losses in rabbitries, and ecological disruptions by affecting the conservation of rabbit-sensitive top predators. We analysed 550 rabbit carcasses collected in the field between May 2013 and March 2016, to investigate the epidemiology of GI.2 in free-living populations and to perform a comparative analysis with the epidemiology of classical rabbit haemorrhagic disease virus forms (RHDV, henceforth GI.1) in Portugal. Rabbits were sexed, aged and liver and blood samples were collected for subsequent RHDV screening and serology. A total of 172 samples were PCR-positive to GI.2, whereas GI.1 strains were not detected in any of the samples. The outbreaks of GI.2 revealed a marked seasonality, with peaks during the breeding season (November-May). We also found that approximately, one-third of free-ranging European rabbits in Portugal have seroconverted to GI.2. We demonstrate that the GI.2 lagovirus is currently widespread in wild populations in Portugal and is affecting a high proportion of adults and juveniles. Therefore, ongoing monitoring and surveillance are required to assess the effects of GI.2 on wild rabbit populations, its evolution, and to guide management actions aimed at mitigating the impacts of rabbit declines in the ecosystem and in rural economies.

Survey of genetic diversity of IgG in wild and domestic rabbits.

We sequenced IgG from genomic DNA of 30 wild European rabbits of O. c. algirus and O. c. cuniculus subspecies from three regions and 15 domestic O. c. cuniculus. Genetic diversity was highest within Iberian wild populations. Only two new amino acid polymorphisms were found, both in O. c. algirus.

Complete genome sequence of two rabbit hemorrhagic disease virus variant b isolates detected on the Iberian Peninsula.

We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit hemorrhagic disease virus (RHDVb). Isolate N11 was detected in young domestic animals during a rabbit hemorrhagic disease (RHD) outbreak that occurred in 2011 on a rabbit farm in Navarra, Spain, while CBVal16 was isolated from a wild rabbit found dead in Valpaços, Northern Portugal, a year later. The viral sequences reported show 84.8-85.1 % and 78.3-78.5 % identity to RHDVAst/89 and RCV-A1 MIC-07, representative members of the pathogenic genogroup 1 RHDV and apathogenic rabbit calicivirus, respectively. In comparison with other RHDV isolates belonging to the previously known genogroups 1-6, RHDVb shows marked phenotypic differences, as it causes disease preferentially in young rabbits under 40 days of age and shows modified red blood cell agglutination profiles as well as antigenic differences that allow this variant to escape protection by the currently available vaccines.

Genetic characterization of CCL3, CCL4 and CCL5 in leporid genera Oryctolagus, Sylvilagus and Lepus.

The genetic diversity of C-C motif chemokine receptor 5 (CCR5) ligands CCL3, CCL4 and CCL5 in the leporid genera Oryctolagus, Sylvilagus and Lepus was studied. Our results demonstrate that the three CCR5 chemokine ligands are under strong purifying selection as a result of possible functional binding constraints.

Recurrent introgression of mitochondrial DNA among hares (Lepus spp.) revealed by species-tree inference and coalescent simulations.

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.

Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability.

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain ("MAV", Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863 bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain "Lausanne" and its attenuated field derivative strain "6918". The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to "Lausanne" and "6918". Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.

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.