Figure 1.
Schematic of the synthesis of rabbit HBGA ligands.
(A) Schematic of the synthesis of H type 2, A type 2, B type 2 and Ley. (B) Structural representation of H type 1, H type 2 and H type 3. (C) Structural representation of core structures of O-glycans. The symbol presentation of O-glycans is based on the nomenclature used in Essentials of Glycobiology textbook (http://www.ncbi.nlm.nih.gov/books/NBK1908) and adopted by the Consortium for Functional Glycomics (http://www.functionalglycomics.org).
Figure 2.
Evolutionary tree of RHDV strains and non-pathogenic rabbit calicivirus strains (RCV, RCV-A1 and MRCV) according to nucleotide sequences of the gene encoding the capsid protein VP60.
The 6 RHDV strains used in this study are marked with a dot (GenBank accession numbers: G1 JF438967, G2 FR823355, G3 FR823354, G4 AJ535094, G5 AM085133, and G6 AJ969628). The genetic group of each strain (G1 to G6 according to Le Gall-Reculé et al.[17] and Group 1 to 4 according to Kerr et al. [20]) is annotated. For the G1 strain, the genetic group is noted as defined in Muller et al. [19] (IB2a). Capsid sequences where recombination was previously detected were excluded from the analyses [76], [77]. The newly obtained sequences were checked for recombination using RDP3 [78]. Evolutionary distances were computed using the p-distance method. Alignment gaps and missing data were eliminated only in pairwise sequence comparisons (Pairwise deletion option). The European brown hare syndrome virus strain (GenBank accession number X98002) was used as an outgroup to root the tree. The tree was constructed using the neighbor-joining method [79] with the pairwise deletion option. Reliability of the tree was assessed by bootstrap with 1000 replicates and is indicated in the nodes for relevant clusters. Several genetic distance methods were used and similar results were obtained, but only p-distance is shown. The phylogenetic analyses were conducted in MEGA 4 [80]. Similar trees were obtained using the Maximum Likelihood (ML) method as implemented in Garli v1.0 [81].
Figure 3.
RHDV strains binding to synthetic oligosaccharides.
RHDV liver extract binding used in a range of dilutions for detection of binding to human serum albumin (HSA) and polyacrylamide (PAA) conjugated oligosaccharides. Vertical grey line symbolizes the same amount of viral RNA, whereas the dotted line symbolizes the same amount of viral RNA as the solid lines, though detected with a different antibody system since the G6 strain was less well recognized than the other strains by the Lp4 polyclonal antibody.
Table 1.
RHDV agglutination titres of human red blood cells.
Figure 4.
Immunohistochemistry of A, B and H expression and RHDV binding to duodenum and trachea.
Staining of the duodenum, trachea and liver of A+B+ rabbits by an anti-A, an anti-B and the H type 2 specific UEA-I lectin. Binding of RHDV (shown a G3 strain) was performed by incubating liver extracts on tissue sections followed by detection using an anti-RHDV monoclonal antibody as described in the “materials and methods” section. Negative controls correspond to section where the liver extracts were omitted. The simple bar corresponds to 100 µm and the double bar to 200 µm. A 4x higher magnification is inserted into the RHDV/duodenum picture, showing the labeling of the crypt epithelial cells. The white star is the Lieberkühn crypts (surface layer of the duodenal mucosa). The black star is the Brünners' glands (deep layer of the duodenal mucosa). The arrow indicates the epithelial layer of the trachea. The triangle indicates the liver parenchyma.
Figure 5.
Expression of H type 2, A and B in rabbit duodenum.
Relative expression of H type 2, A and B where the individual rabbits are sorted according to H type 2, A and B. A- rabbits are shown with stars in graph of H type 2 expression.
Figure 6.
MALDI-TOF MS of O-glycans released from Rabbit Duodenum.
Glycans were permethylated prior to MALDI-TOF analysis. Structures were assigned taking into account the molecular weight value and the fragment ions obtained upon MS/MS analysis. Non annotated structures correspond to non fucosylated O-glycans and permethylation adducts. A: Profile of fucosylated permethylated O-glycans released from rabbit duodenum (Sample 2). B: Profile of fucosylated permethylated O-glycans released from rabbit duodenum (Sample 4). C: Profile of fucosylated permethylated O-glycans released from rabbit duodenum (Sample 6).
Table 2.
Detection of A and B with mass spectrometry and antibodies.
Figure 7.
Binding of 6 RHDV strains to rabbit duodenum.
Relative binding of G1–G6 to 84 individual wild and non-wild rabbit duodenums. Rabbits are ordered according to G4 binding in all graphs. The rabbits below the detection threshold of G4 and therefore in no particular order are displayed with gray bars.
Table 3.
Correlation between weak, medium and strong RHDV binding and A, B and H type 2 expression or no expression.
Figure 8.
Enzymatic removal of A, B and H from three different rabbit duodenums.
The H epitope was cleaved from an A−B− rabbit duodenum and G1–G6 binding was tested against H removal and a mock treated control (A). A and H were cleaved from an A+B− rabbit duodenum and RHDV binding was tested against A removal, A followed by H removal and a mock treated control (B). A, B and H were removed from an A+B+ rabbit duodenum where A removal, A and H removal, B removal, B and H removal and a mock treated control were tested for G1–G6 binding (C). Control of the enzymatic removal of A, B and H are shown (D).
Figure 9.
RHDV challenge of A+B+ and A−B− rabbits.
31 rabbits were infected with an A and B binding G4 strain at 105, 107 or 109 virus copies. Rabbits either succumbed to infection or were sacrificed after 11 days. Duodenum and liver samples were collected at time of death. Duodenum was phenotyped for A, B and H expression and G4 binding. Duodenum and liver were also assayed for virus RNA via real time RT-PCR. (A) G4 binding plotted against B expression levels. (B) Viral load of liver and duodenum of all dead and surviving rabbits. Duodenum RNA is plotted as arbitrary units since the RNA was normalized against β actin to account for tissue preparation variations. Liver RNA is plotted as copy numbers since the tissue state due to infection did not allow for reliable quantification of rabbit RNA. (C–D) Virus RNA levels of liver or duodenum for 105 infectious dose, where A+B+ (circle), A−B− (triangle), dead rabbits (black symbol) and surviving rabbits (white symbol).
Table 4.
A+B+ and A−B− phenotypes and survival and death of rabbits from G4 challenge experiment.
Table 5.
Correlation of B expression and RHDV binding of rabbits from Claira, not RHDV infected, and Canohès, RHDV survivors, percentage in parenthesis.
Table 6.
Correlation of A and B phenotypes of rabbits from three populations in Australia with G2 strain binding (percentages in parenthesis).
Figure 10.
Summary of the RHDV strains specificities for ABH antigens.
The size of the A, B and H letters represents the level of expression in the rabbit duodenum and the volume and color intensities of the ovals correspond to the ability of each strain to recognize the epitopes. The strains shown above the ABH antigens (G2–G5) serially replaced each other in France (G2: 1988–1990; G3: 1990–1997; G4: 1993–1999; G5: 1994–2009), whereas those shown below evolved independently.