Novel Insights on Hantavirus Evolution: The Dichotomy in Evolutionary Pressures Acting on Different Hantavirus Segments

<div><p>Background</p><p>Hantaviruses are important emerging zoonotic pathogens. The current understanding of hantavirus evolution is complicated by the lack of consensus on co-divergence of hantaviruses with their animal hosts. In addition, hantaviruses have long-term associations with their reservoir hosts. Analyzing the relative abundance of dinucleotides may shed new light on hantavirus evolution. We studied the relative abundance of dinucleotides and the evolutionary pressures shaping different hantavirus segments.</p><p>Methods</p><p>A total of 118 sequences were analyzed; this includes 51 sequences of the S segment, 43 sequences of the M segment and 23 sequences of the L segment. The relative abundance of dinucleotides, effective codon number (ENC), codon usage biases were analyzed. Standard methods were used to investigate the relative roles of mutational pressure and translational selection on the three hantavirus segments.</p><p>Results</p><p>All three segments of hantaviruses are CpG depleted. Mutational pressure is the predominant evolutionary force leading to CpG depletion among hantaviruses. Interestingly, the S segment of hantaviruses is GpU depleted and in contrast to CpG depletion, the depletion of GpU dinucleotides from the S segment is driven by translational selection. Our findings also suggest that mutational pressure is the primary evolutionary pressure acting on the S and the M segments of hantaviruses. While translational selection plays a key role in shaping the evolution of the L segment. Our findings highlight how different evolutionary pressures may contribute disproportionally to the evolution of the three hantavirus segments. These findings provide new insights on the current understanding of hantavirus evolution.</p><p>Conclusions</p><p>There is a dichotomy among evolutionary pressures shaping a) the relative abundance of different dinucleotides in hantavirus genomes b) the evolution of the three hantavirus segments.</p></div

ENC-GC₃ plot.

<p>Correlation between GC₃ and the effective codon usage statistic (ENC) among (a) S segment, (b) M segment and (c) L segment. The red line represents the ENC expected values (ENC*) and the ENC values are shown in blue. ENC values for the L segment are significantly lower than that for the S segment (45.49±1.52 vs 50.11±2.52; P<0.0001) or the M segment (45.49±1.52 vs 47.93±3.11; P = 0.0003), suggesting that mutational pressure is the predominant evolutionary force acting on the S and M segments; while translational selection predominates in the evolution of the L segment.</p

GpU dinucleotide depletion in the S segment is linked to translational selection.

<p>The intracodon GpU O/E ratio for the S segment was significantly lower than that for the non-coding region of this segment (0.70±0.07 vs 0.95±0.14; P< 0.0001); clearly supporting translational selection as the major driver of GpU depletion in the S segment of hantaviruses.</p

Relative abundance of dinucleotides in hantavirus genomes.

<p>(a) The mean ±standard deviation of dinucleotide O/E ratios for hantaviruses S segment is 1.0±0.25. The O/E ratios for most of the dinucleotides lie inside the confidence interval of 0.75–1.25 with the exceptions of CpG and GpU dinucleotides (underrepresented) and CpA dinucleotides (overrepresented). (b) The mean±standard deviation of dinucleotide O/E ratios for hantaviruses M segment is 1.0±0.25. CpG dinucleotides were underrepresented and CpA and UpG dinucleotides were overrepresented as the O/E ratios for these dinucleotides were located outside the confidence interval of 0.75–1.25. (c) The mean ±standard deviation of dinucleotide O/E ratios for hantaviruses L segment is 1.0±0.22. CpG and CpA dinucleotides were the most deviant dinucleotides as their O/E ratios were located outside the confidence interval of 0.78–1.22. (d) Comparison of the average dinucleotide O/E ratios for all three segments of hantaviruses. The depletion of CpG dinucleotides was common across all the three segments of hantaviruses.</p

Hantavirus genomes are depleted for CpG dinucleotides and underlying evolutionary pressure is specific to CpG (not GpC) dinucleotides.

<p>(a) Among the 3 segments of hantaviruses the CpG dinucleotide O/E ratios for the M segment were significantly lower than that for the S segment (0.22±0.04 vs 0.28±0.06; P<0.0001) and that for the L segment (0.22±0.04 vs 0.29±0.05; P<0.0001). The differences in CpG O/E ratios of the S segment and the L segment were not significant (0.28±0.06 vs 0.29±0.05; P = 0.18). (b) The CpG/GpC O/E ratios were significantly lower for the M segment of hantaviruses as compared to those for the S segment (0.22±0.04 vs 0.29±0.07; P<0.0001) and for the L segment (0.22±0.04 vs 0.28±0.04; P<0.0001); clearly demonstrating that CpG dinucleotides but not GpC dinucleotides are susceptible to the underlying evolutionary pressures.</p

Avoidance of GpU-containing codons in the S segment of hantaviruses.

<p>Box plots showing the RSCU values of synonymous GpU-containing codons. The RSCU values of GpU-containing codons are shown in yellow boxes. GpU-containing codons were avoided in the S segment of hantaviruses as evidenced by median RSCU values of less than one. The average RSCU values for GpT-containing codons in the S segment were lower as compared those in the M segment (0.91±0.32 vs 1.46±0.25; P<0.0001) or the L segment (0.91±0.32 vs 1.7±0.27; P<0.0001); this is a reflection of GpU dinucleotides depletion from the S segment.</p

CpG dinucleotide depletion in the M segment is linked to evolutionary lineage of host.

<p>(a) In M segment the CpG O/E ratios were significantly lower in insectivore borne viruses than that in rodent borne viruses (0.19±0.03 vs 0.22±0.03; P = 0.0077). The average CpG O/E ratios were lower in insectivore borne hantaviruses as compared to rodent borne hantaviruses for S segment (0.25±0.08 vs 0.29±0.06; P = 0.1344) and L segment (0.26±0.03 vs 0.30±0.05; P = 0.1481); however the difference was not statistically significant. (b) The GpU O/E ratios was not statistically different between insectivore borne and rodent borne hantaviruses in S segment (0.75±0.06 vs 0.72±0.06; P = 0.2289), M segment (0.97±0.02 vs 0.97±0.03;P = 0.9355) and L segment (0.89±0.05 vs 0.87±0.03).</p

Neutrality plot.

<p>Scatter plot demonstrating significant correlation between GC₃ (X-axis) and GC_1,2 (Y-axis) among (a) S segment (<i>r</i>² = 0.203, P<0.0001) and (b) M segment (<i>r</i>² = 0.274, P<0.0001), implying a major role for mutational pressure (and not translational selection) in the evolution of the S and the M segments. (c) There was no correlation between GC₃ and GC_1,2 in the L segment (<i>r</i>² = 0.039, P = 0.234) suggesting that translational selection may play an important role in the evolution of this segment.</p

Avoidance of CpG-containing codons in all three segments of hantaviruses.

<p>Box plots with RSCU values of synonymous CpG-containing codons. Data for CpG-containing codons are shown in red boxes. The single letter code for amino acids is indicated within parenthesis adjacent to the codons. The median RSCU values of all CpG-containing codons were less than 1 in all the 3 segments, clearly suggesting that CpG-containing codons were avoided in hantavirus genomes.</p