# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 4 Discussion
### Rotavirus Evolution Following the Introduction of Rotavirus Vaccines  
The live-attenuated pentavalent vaccine, RotaTeq (Merck, West Point, U.S.), was introduced in 2006, and the live-attenuated monovalent vaccine, Rotarix (GlaxoSmithKline, Rixensart, Belgium, U.S.), was disseminated in 2008. RotaTeq contains five human-bovinereassortment viruses with strain serotypes of G1P [5], G2P [5], G3P [5], G4P [5], and G6P [8]), while Rotarix is comprised of the human G1P [8] strain, RIX4414. These vaccines provided effective protection against contemporaneous globally dominant strains G1P [8], G2P [4], G3P [8], G4P [8], and G9P [8]. Our results show a coincident decline in the relative diversities and effective population sizes of all RVA segments after 2006. Supporting this analysis, several studies, including an analysis of G12 strains in Africa, G12 strains in Spain, and a lineage of P [8] strains also show a general decline in diversity/effective population size after 2008 [116; 117; 118]. The introduction of the RVA vaccines resulted in a global reduction in RVA-associated mortality. However, vaccine effectiveness varied substantially by region [119] and resulted in changes to the circulating strain prevalence. For example, in a post-vaccine era, the prevalence of G9P [8], G2P [4], and G9P [4], G9P [8] increased, while the prevalence of G1P [8] and G3P [8] declined [120; 121; 122; 123]. The emergence of rare genotypes or animal RVA reassortments in children also appears to be connected to the selective pressure imposed by vaccination. For example, the increase in abundance of G12 and G11 genotypes [121; 122] and the appearance of atypical Wa-like and DS-1-like reassortments, such as the emergence of G1P [8] with a DS-1-like backbone in Malawi [124], appear linked to increases in RVA vaccination. Given DS-1-like and Wa-like segments are thought to have incompatibilities with one another, limiting their reassortment potential [95], the vaccine-induced selection for mutations can be difficult to assess. That is, it is difficult to determine whether emerging fixed mutations are the direct result of escape mutants or are compensatory mutations resulting from novel reassortments. A study on G2P [8] evolution did not observe evidence of vaccine-induced selection; however, another study focusing on P [6], P [4], and P [8] genes did report substantial divergence from the vaccine strains. Vaccine-induced selective pressure may partly explain the pattern observed when comparing the geodesic distances of the segments. Like the present study, which found that segments 4 and 9 (encoding serotype proteins VP4 and VP7) were especially amenable to reassorting into new genetic backgrounds, a study of European Bluetongue virus (BTV) isolates also found that segments 2 and 6 (encoding the BTV homologs of VP4 and VP7) were quite distant from the rest of the genome in tree space [125]. However, the BTV segments 2 and 6 were closer to one another in tree space, whereas in RVA segments 4 and 9 were highly distinct from each other. Additionally, BTV segments 7 (encoding the inner capsid protein) and 10 (encoding NS3) shared close evolutionary histories with one another, and distinct from the rest of the genome. Interestingly, in another BTV study on strains from India, segment 4 (encoding a protein homologous to VP3 in RVA) was found to be the most isolated segment in tree space [126]. While there are BTV vaccines available, BTV does not infect humans, is not as globally distributed as RVA, and far fewer serotypes circulate, so there may be more selective pressure on RVA serotype segments to reassort. While RVA segments were unlinked and had different phylogenies and rate variations along with their trees, the segments' patterns in relative diversity over time mostly matched each other. In the Rift Valley fever virus [127], which is a three-segmented -ssRNA arbovirus, different skyline plots were observed for each segment, suggesting that the segments are evolving independently to some extent.