# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 1 Introduction
### Selective Pressures on Synonymous Sites  
RVA is the most common cause of diarrheal disease in young children and is also an important agricultural pathogen, particularly for cows and pigs (Figure 1). RVA vaccines RotaTeq and Rotarix have produced substantial selective pressure on circulating RVA strains since they were first administered on a large scale in 2006 and 2008 respectively. This selective pressure seems to have favored certain genome constellations. Strains relevant to humans mostly consist of genogroup 1 (Wa-like) genes, genogroup 2 (DS-1-like) genes, or genogroup 3 (AU-1-like) genes. Specific G and P types are also associated more with specific genogroups [54; 55].  
Selective pressure on amino acid sequences to conserve protein interactions is a barrier to reassortment compatibility, but segmented viruses are also under considerable selective pressure on synonymous sites as RNA-protein and RNA-RNA interactions are critical in virus packaging [30; 39; 40; 41; 42]. RVA genome assortment requires +ssRNA molecules from each segment to form complexes with one another before being packaged, which relies on packaging signals on each segment.  
Synonymous sites may also be under selective pressure for certain codon usage patterns. Codon usage bias results in varying levels of efficiency in translation, with highly expressed genes showing stronger bias for codons abundantly available in the host tRNA pool [56; 57], and marginally expressed genes displaying less codon bias [58]. Codon usage adaptation to the host is a well-documented phenomenon in DNA viruses [57; 59; 60] and is observed in RNA viruses as well [61; 62; 63; 64; 65; 66; 67], though the constraints of a secondary RNA structure, as well as the high rate of mutation in RNA viruses, may lower the relative effects of translational selection in RNA viruses. Codon bias is also explained by drift and mutational pressure (i.e., bias towards A/T/U or G/C mutations) as well as translational selection [68]. Codon usage that is too similar to the host can also lower efficiency if viral proteins cannot fold properly [69], and differential codon adaptation can be a mechanism of controlling viral gene expression [70; 71].  
Rotaviruses have been shown to exhibit codon bias [72; 73], but have especially divergent codon usage patterns from humans relative to other RNA viruses [74]. Codon usage can be a potential hindrance to zoonosis if a virus infects a host that cannot efficiently translate the virus' proteins. RVA's broad host range and ability to undergo genetic exchange makes RVA's zoonotic potential a cause for some concern.  
We show that RVA's segments have distinct evolutionary histories, demonstrating the impact reassortment has had on mammalian RVA between the late 1950s to 2017. Because each RVA segment is under different selective pressure, we also tested whether there was evidence for translational selection on synonymous sites for each of the segments. To assess whether certain segments showed more codon adaptation against different common host genomes, indicating translational efficiency differences, we tested for neutrality in codon position 3, as well as variations in codon adaptive indices and relative codon deoptimization indices for each segment. To test whether RVA showed signs of codon optimization to a particular host, we compared strains isolated from specific hosts against their host genome and other RVA host genomes. We found differences in codon usage patterns between segments, with segment 11 (NSP5) having significantly higher codon adaptation to host genomes, however, our study indicates codon usage is not a barrier to rotavirus zoonosis.