# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 4 Discussion
### Codon Bias Analysis Shows Codon Usage Differs between Segments, but Not between Host Strains  
We contrasted the codon usage patterns of several common mammalian RVA genotypes with a set of RVA host reference genomes. Our results showed that mammalian and avian RVA codon usage patterns were most compatible with avian genomes (Figure 7). For example, the highest CAI values and lowest RCDI values in our comparative analysis suggested that RVA NSP5's codon usage pattern was best adapted to the chicken genome (Figure 7). This finding suggests that RVA originated as an avian virus that subsequently expanded its host range to include mammalian hosts. The fact that RVA appears better adapted to human and avian genomes rather than the other mammalian genomes tested further suggests that RVA spread to other mammalian hosts after adapting to humans, though more evidence is required to confirm this hypothesis (Figure 8). Additionally, NSP5's relatively high CAI value and low RCDI value (Figure 7; both values approach 1) may indicate a strong selective advantage for NSP5's codon optimization. Since efficient NSP5 protein expression is critical for RVA viroplasm formation and replication [128; 129; 130], the higher CAI value may indicate that NSP5's codons are optimized to match avian hosts to maximize NSP5 expression in host cells. The variation in GC content between segments was also notable, as being from the same genome, this suggests mutational bias towards AU or GC is not entirely responsible for RVA's observed codon bias. The low GC3 content and especially low GC3 GC12 ratio seen in VP3 (Figure 9), in contrast with the high GC3 content of NSP5 and NSP4 (relative to the rest of the genome), could suggest it is beneficial for the rotavirus to have lower-efficiency VP3 expression relative to NSP4 and NSP5, during infection. Varying the codon bias by gene, or maintaining suboptimal codon bias to the host, may sometimes be beneficial [69; 74], a phenomenon which has been observed for example, in hepatitis A virus [61]. Although the bovine and avian RVA strains sampled tended to have higher CAI scores and RCDI scores closer to 1, there was no evidence of divergence in codon usage among bovine and avian strains. The similarity in codon usage between host strains is in contrast with a study of influenza A which found that avian and human influenza strains have distinctly different G/C vs. A/U contents from one another [131]. Our finding suggests that, while RVA strains may indeed experience an advantage from matching the codon usage patterns of their hosts, it is unlikely that translational selection can counteract nucleic acid selection (i.e., selection favoring synonymous substitutions improving virus survival and reproduction). The wide range of CAI/RCDI values and GC3 content for some of the segments suggest that translational selection is not an especially strong selective pressure for every gene. However, translational selection does seem to be a stronger selective pressure at least in NSP5, based on its significant divergence from the rest of the RVA genome's codon usage (Figure 9). The neutrality plot also shows that GC percentages at position 3 frequently are significantly different from the GC percentages at positions 1 and 2, which would indicate that strong selection is occurring at position 3 (Figure 9). The variation in GC content and codon usage by segment could point to RVA using codon bias as a mechanism of controlling viral gene expression. While there is no significant difference in codon usage between strains isolated from different hosts (Figure 10), notably RVA, in general, is significantly more optimized to human and avian genomes, than pig and cow genomes. This indicates translational efficiency is not a barrier for zoonosis between avian and mammalian strains. There are some limitations in the assessment of codon usage patterns.