Evolutionary mechanisms of persistence and diversification of a calicivirus within endemically infected natural host populations

Abstract

In order to understand the evolutionary mechanisms of persistence and diversification within the Caliciviridae, we have been exploiting endemic infection of feline calicivirus within five geographically distinct household groups of cats. By sequencing immunodominant and variable regions of the capsid gene, we identified the relative contribution of the different evolutionary processes employed by the virus to ensure its long-term survival in the host population. Such strategies included progressive evolution of a given variant of a strain through mutation accumulation within an individual, sequential reinfection with either a variant of the same strain or with a different strain, and mixed infection. Recombination between different strains in this study has been reported in detail elsewhere (K. P. Coyne et al., J. Gen. Virol. 87:921-926, 2006). Here, we provide evidence to suggest that true long-term persistent infection in individuals is relatively rare, with the majority of apparent viral carriers undergoing a combination of progressive evolution and cyclical reinfection. Progressive evolution at the individual level and variant reinfection at both the individual and population levels were associated with positive selection. Two measures of evolution rate were determined; for a virus progressively evolving within an individual (1.32 x 10(-2) to 2.64 x 10(-2) substitutions per nucleotide per year, i.e., no transmission) and for a strain circulating within a population (3.84 x 10(-2) to 4.56 x 10(-2) substitutions per nucleotide per year, i.e., including transmission). Reiteration of both progressive evolution and variant reinfection appeared to lead to a gradual increase in the diversity of a given strain of virus, both in the individual and in the population, until eventually new strains emerged.

FIG. 1.

(A) Unrooted neighbor-joining tree of 143 FCV consensus sequences (including the published sequence FCV F9, GenBank accession number M86379), for a 420-nucleotide region of the FCV capsid gene. One consensus sequence was excluded, as it contained two strains. Shaded areas represent the 10 distinct virus strains identified in this study. Strain names, percent diversity, and the number of variants within each strain are indicated next to the corresponding cluster. Evolutionary distances were calculated using the Jukes-Cantor model, with the scale bar indicating percent divergence. Numbers at major nodes are the bootstrap values of ≥80% out of 1,000 replicates (MEGA 3.0). (B) Changes in viral diversity over time for the three strains (A1, D1, and D2) for which the most data were available. Shaded areas represent the ranges of viral strain diversity (Jukes-Cantor distance analysis) present in the cat groups at the time of sampling, with putative bottlenecks at 19 months (strain A) and 6 months (strain D2).

FIG. 2.

(A) Unrooted neighbor-joining tree of 91 FCV consensus sequences from samples isolated from colony D. Phylogenetic relationships are highlighted for three representative cats, numbers 1, 4, and 6. Colored circles represent viruses isolated from each cat, with the time at which each isolate was collected shown in months. Evolutionary distances were calculated using the Jukes-Cantor model, with the scale bar indicating percent divergence. (B) All possible pair-wise comparisons of genetic distances against time for all consensus sequences for cats 1, 4, and 6. Symbols below the line represent comparisons made within a strain and were used to calculate lines of best fit. Symbols above the line for cat 1 represent comparisons made between strains. (C) Unrooted neighbor-joining trees of cloned sequences derived for viruses isolated from cats 1, 4, and 6. Colored circles represent times at which each FCV sample was obtained. The mean interisolate variability is shown in bold, and the pair-wise ranking score is in italics. Mean intraisolate variability and E values are shown in brackets. Numbers at major nodes are the bootstrap values of ≥80% out of 1,000 replicates (MEGA 3.0). The scale bar indicates percent divergence.

FIG. 3.

(A) Bar chart showing nucleotide distance comparisons for 65 pairs of FCV isolates that were isolated on consecutive samplings from 24 cats (refer to the text for an explanation of the ranking system). (B) Box and whisker plot comparing the distributions of nucleotide distances for sequential sequences ranked 1 (progressive evolution), 2 (within-strain transmissions), and 3 (between-strain transmissions). The number of paired sequences that were placed within each ranking system is indicated. Median upper quartile (Q3, where 75% of data are less than or equal to this value) and lower quartile (Q1, where 25% of data are less than or equal to this value) distance values are shown for each rank. *, distance values outside the whiskers are classified as outliers.

FIG. 4.

Codon-based analysis of selection pressures on regions C and E of the FCV capsid gene (Datamonkey). The main figure shows a plot of dN-dS values for each predicted codon of the consensus sequences. The red line represents all variants of strain D1. The blue line represents previously published sequences from epidemiologically unrelated strains of FCV. For each data set, the codons predicted to be evolving under positive selection by SLAC (P = 0.1) are indicated by red and blue asterisks above the plot. Codon mutations known to disrupt neutralizing B-cell epitopes are indicated by downward-pointing arrows above the plot. The blue shaded areas correspond to FCV capsid variable regions (C, 5′ and 3′ HVRs). Codon 1 is equivalent to codon 383 of the FCV capsid protein (53). Bars above and below this main plot represent the results of integrated analyses (SLAC, FEL, and REL) for three other strains (D2, A1, and A2) and for three individual cats (progressive evolvers, cat 4 and cat 2 [8], and sequential reinfection with a variant of a strain [cat 6]), respectively. Gray areas correspond to those regions predicted to be evolving under positive selection in each data set (P = 0.25 for SLAC and FEL; Bayes factor of 10 for REL).

FIG. 5.

Predicted structure of the FCV F9 capsid P2 surface domain (6), highlighting regions identified in this study that are predicted to be under positive selection. The yellow region corresponds to the 5′ HVR, known to contain linear epitopes. The magenta and green regions correspond to the left and right boundaries of the 3′ HVR, known to contain conformational epitopes. The cyan region contains variable region C and maps close to the 3′ HVR. The FCV P2 domain as modeled contains six β-strands, labeled A, B, C, D-E, E, and F.