Mutability dynamics of an emergent single stranded DNA virus in a naïve host

Abstract

Quasispecies variants and recombination were studied longitudinally in an emergent outbreak of beak and feather disease virus (BFDV) infection in the orange-bellied parrot (Neophema chrysogaster). Detailed health monitoring and the small population size (<300 individuals) of this critically endangered bird provided an opportunity to longitudinally track viral replication and mutation events occurring in a circular, single-stranded DNA virus over a period of four years within a novel bottleneck population. Optimized PCR was used with different combinations of primers, primer walking, direct amplicon sequencing and sequencing of cloned amplicons to analyze BFDV genome variants. Analysis of complete viral genomes (n = 16) and Rep gene sequences (n = 35) revealed that the outbreak was associated with mutations in functionally important regions of the normally conserved Rep gene and immunogenic capsid (Cap) gene with a high evolutionary rate (3.41×10(-3) subs/site/year) approaching that for RNA viruses; simultaneously we observed significant evidence of recombination hotspots between two distinct progenitor genotypes within orange-bellied parrots indicating early cross-transmission of BFDV in the population. Multiple quasispecies variants were also demonstrated with at least 13 genotypic variants identified in four different individual birds, with one containing up to seven genetic variants. Preferential PCR amplification of variants was also detected. Our findings suggest that the high degree of genetic variation within the BFDV species as a whole is reflected in evolutionary dynamics within individually infected birds as quasispecies variation, particularly when BFDV jumps from one host species to another.

Figure 1. Bayesian phylogenetic tree inferred evolutionary relationships among BFDV full genome sequences from orange-bellied parrots.

Maximum clade credibility tree automatically rooted by using relaxed molecular clock model in Beast v1.7.5. Labels at branch tips refer to GenBank accession number, and with country code, original sample ID, collection site and year of isolation in parentheses. Nodes with posterior probability of ≥0.95 are indicated with asterisks and with a hash for P≥0.7. Inferred nonsynonymous substitutions at codons are indicated at the appropriate lineages where the majority of genomes in a clade possess a particular substitution then the ancestral node has been labeled.

Figure 2. Bayesian phylogenetic inference of evolutionary relationship among Rep gene sequences from orange-bellied parrots.

Maximum clade credibility tree automatically rooted by using relaxed molecular clock model in Beast v1.7.5. Labels at branch tips refer to GenBank accession number, and with country code, original sample ID, collection site and year of isolation in parentheses. Nodes with posterior probability of ≥0.9 are indicated with asterisks and with a hash for P≥0.6. Inferred nonsynonymous substitutions (blue colour) at codons are indicated at the appropriate lineages where the majority of genomes in a clade possess a particular substitution then the ancestral node has been labeled.

Figure 3. DNA folds analysis for demonstrating loop-like DNA structure within recombination breakpoint locations.

Predicted DNA fold analysis showing recombination breakpoint locations within loop structures in the BFDV genome (12-0827-201213, GenBank accession: KC693651) using tools available in Geneious 6.1.6. The first recombination breakpoint location at thymidine nt location 1311 (P≤0.001) as circled was a consistently predicted loop structure in all recombinant genomes (n = 15), a second recombination breakpoint location at 1238 (P≤0.05) is also shown (arrow) in a smaller loop structure. Colors of nucleotides represent base-pair probabilities (red = high, green = mid, blue = low).

Figure 4. Alignment of 13 Rep sequences showing BFDV quasispecies variants in orange-bellied parrots.

The variants 12-364QA1 and 12-364QA2 (GenBank accession numbers: KF188694 and KF188695 respectively) originated from a single bird, 10-1018-QB1 to 10-1018-QB7 (GenBank accession numbers: KF188692, KF188696-KF188701 respectively) originated from another orange-bellied parrot (10-1018), 08-423CQ1 and 08-423CQ2 (GenBank accession numbers: KF188683 and KF188702 respectively) represented orange-bellied parrot 08-423, while 08-448-QD1 and 08-448-QD2 (GenBank accession numbers: KF188686 and KF188703 respectively) originated from another orange-bellied parrot. For each individual BFDV sequence, asterisks indicate those where full genome (1993 bp) sequences were performed as well and supported by at least 8 clones. Variants from these (suffixes 2–7) came from multiple sequencing of PCR amplicons directly as well as cloned products. From Table 1 the primer 2 forward (5′-AACCCTACAGACGGCGAG-3′) and 4 reverse (5′-GTCACAGTCCTCCTTGTACC-3′) are indicated by arrows.