# Rotavirus A Genome Segments Show Distinct Segregation and Codon Usage Patterns
## 1 Introduction
### Rotavirus A Genome and Proteins  
While the RVA genome is double-stranded, RVA RNA is packaged into and exits capsids as positive-sense, single-stranded RNA (+ssRNA). The RNA-dependent-RNA-polymerase, VP1, and the capping enzyme, VP3, are anchored to VP2 pentamers to form the  
Figure 1: Phylogeny of segment 9 (VP7) of Rotavirus A colored by host species. Stars indicate avian-mammalian RVA spillover events (avian RVA to fox, avian RVA to raccoon, bovine RVA to turkeys). The alignment for this tree was made using complete VP7 sequences representing all known, available G types and hosts, to display the known-host range and host-boundary patterns for RVA. The VP7 (G) genotypes are labeled by clade. The tree file is available (SI file S1) with accession numbers for all sequences used. The phylogeny was run in BEAST v1.10.4 using a birth-death tree prior under an uncorrelated relaxed clock and a GTR+1+G substitution model, and the alignment was partitioned by translated and non-translated regions. Branch lengths correspond to substitutions.  
replicate complex. This complex is present at each of the rotavirus capsid vertices through which +ssRNAs are released [27]. The inner VP2 layer is enclosed by an intermediate layer composed of VP6 protein, and the outermost layer is composed of the glycoprotein (VP7) and the multimeric spike protein (VP4) [28].  
In addition to the six structural proteins, the RVA genome also encodes five to six non-structural proteins on five separate segments. The NTPase (NSP2) is a +ssRNA binding protein that forms a doughnut-shaped octamer with a positively charged periphery, allowing +ssRNA to wrap around and bind within its grooves during genome packaging [29; 30]. NSP2 protein interactions with the +ssRNA are therefore critical to stabilizing +ssRNA contacts [31]. NSP2 also interacts with NSP5 to form viroplasms (along with other viral proteins) [32], where genome packaging, replication, assembly of progeny cores, and double-layered particles (DLPs) occurs [33; 34; 35]. To effect transmission, RVA encodes NSP4, an endoplasmic reticulum associated viroporin and enterotoxin, which elevates cytosolic Ca\({}^{2+}\) in cells. This Ca\({}^{2+}\) elevation ultimately results in diarrhea/vomiting in hosts [36; 37; 38; 39; 40]. NSP4 also interacts with VP6 on DLPs during RVA production [41; 42; 43; 44]. The non-structural protein encoded on segment 7, NSP3, hijacks cellular translation and is a functional analog of the cellular poly (A)-binding protein [45; 46; 47]. NSP3 binds the group-specific consensus tetranucleotide *UGACC [45; 46; 48] located at the 3' end of RVA ssRNA, which suggests inter-group reassortment does not occur. Lastly, NSP1 disrupts cellular antiviral responses [49], so may also play a role in the host-range [50]. Both segment 5 (NSP1), as well as segment 11 (NSP5/6) are especially prone to genome insertions [51; 52; 53].  
For reassortment to occur, +ssRNAs from two or more parents must be packaged into the same virion. When reassortment occurs, the resulting reassortant virus must maintain the many protein and RNA interactions required for efficient packaging and replication. Even if the reassortment virus is functional, it may still be outcompeted by other genotypes, go extinct due to transmission bottlenecks, or evolve compensatory mutations. These factors, along with the high genetic diversity of RVA strains, make predicting reassortment facility or barriers to reassortment difficult.