Figure 1.
(A) ORFs as they are referred to in Lauck et al., 2011 [9], labeled sequentially 5′-3′: ORF1a-ORF9. Asterisks denote ORFs identified in SHFV-krc1 and SHFV-krc2 not reported in Lauck et al., 2011 [9]. (B) ORFs as they are named in Snijder et al., 2013 [17], labeled 5′-3′: ORF1a-ORF7, with duplicated ORFs designated by a “prime” (e.g. ORF2a’). Expression products are given in bold.
Figure 2.
Infection frequency of SHFV-krc1 and SHFV-krc2 in the Kibale red colobus.
SHFV-krc1 (green) and SHFV-krc2 (purple) infections were identified by “unbiased” deep sequencing and confirmed by strain-specific qRT-PCR.
Figure 3.
Viral loads of SHFV-krc1 and SHFV-krc2 in the Kibale red colobus.
Comparison of SHFV-krc1 (green) and SHFV-krc2 (purple) viral loads from all animals positive for either virus (A) and viral loads from mono-infections vs. co-infections of SHFV-krc1 (B) and SHFV-krc2 (C). RNA was isolated from blood plasma and quantitative RT-PCR was performed using strain-specific primers and probes designed from deep sequencing data. Statistical significance was assessed using a two-tailed t-test performed on log-transformed values (CI = 95%).
Figure 4.
Pairwise comparison of nucleotide identity among variants of SHFV-krc1 and SHFV-krc2 from Kibale red colobus (RC).
Full coding sequences for each isolate were aligned using CLC Genomics Workbench. Numbers show percent nucleotide identity between two variants within (A) SHFV-krc1 or (B) SHFV-krc2. Colors highlight similarity, with red representing the most similar sequences and yellow representing sequences with the lowest degree of nucleotide identity. The same color scale was used for (A) and (B).
Figure 5.
Overall nucleotide diversity of SHFV-krc1 and SHFV-krc2.
Mean (± S.E.) πS (A), πN (B), and πN/πS (C) in monkeys infected with SHFV-krc1 (green) and SHFV-krc2 (purple). Paired t-tests were performed to compare mean values between SHFV-krc1 and SHFV-krc2.
Figure 6.
Nucleotide diversity of SHFV-krc1 and SHFV-krc2 by ORF.
Interaction graphs comparing mean πS (A) and πN (B) in ORFs from SHFV-krc1 (green) and SHFV-krc2 (purple). In the case of πN there was a significant ORF-by-virus interaction (F13, 459 = 4.39; p<0.001). Comparison of mean πs (blue) to πN (red) within ORFs of SHFV-krc1 (C) and SHFV-krc2 (D) revealed substantial differences among ORFs within each virus.
Figure 7.
Nucleotide diversity across ORF3 and ORF5 of SHFV-krc1 and SHFV-krc2.
Mean πS (blue) and πN (red) in sliding windows of 9 codons across the coding region of ORF5 (A,B) and ORF3 (C,D). Overlapping ORFs are shown at the bottom. Grey boxes represent predicted transmembrane domains, with striped grey boxes representing a hydrophobic region unique to the SHFVs. Green lines depict putative sites of N-glycosylation, with dashed green lines showing sites that are variably glycosylated. Yellow boxes show predicted signal peptide cleavage sites that vary in location in GP5 of SHFV-krc1 and SHFV-krc2 and were not found in GP3 of SHFV-krc1. The purple box corresponds to the unique region of highly variable acidic residues found only in ORF3 of SHFV-krc2.
Figure 8.
Relationship between viral load and nucleotide diversity.
(A) Synonymous nucleotide diversity (πS) and (B) nonsynonymous nucleotide diversity (πN) were plotted against log-transformed viral loads for both SHFV-krc1 (green) and SHFV-krc2 (purple) infections. A significant correlation between nucleotide diversity and viral load was found for both πS (r2 = 0.2465, p = 0.0015) and πN (r2 = 0.1749, p = 0.0090).