bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Article 2 A Genome Segments Show Distinct Segregation and Codon Usage Patterns 3 4 Irene Hoxie 1,2* and John J. Dennehy1,2 5 1The Graduate Center of The City University of New York, New York City, NY, USA 6 2 Biology Department, Queens College of The City University of New York, Queens, NY, USA 7 *Correspondence: [email protected] 8 9 Abstract: Reassortment of the Rotavirus A (RVA) 11-segment dsRNA genome may generate new genome 10 constellations that allow RVA to expand its host range or evade immune responses. Reassortment may also 11 produce phylogenetic incongruities and weakly linked evolutionary histories across the 11 segments, 12 obscuring reassortant-specific epistasis and changes in substitution rates. To determine the co-segregation 13 patterns of RVA segments, we generated time-scaled phylogenetic trees for each of the 11 segments of 789 14 complete RVA genomes isolated from mammalian hosts and compared the segments’ geodesic distances. 15 We found that segments 4 (VP4) and 9 (VP7) occupied significantly different treespaces from each other 16 and from the rest of the genome. By contrast, segments 10 and 11 (NSP4 and NSP5/6) occupied nearly 17 indistinguishable treespaces, suggesting strong co-segregation. Host-species barriers appeared to vary by 18 segment, with segment 9 (VP7) presenting the least conservation by host species. Bayesian skyride plots 19 were generated for each segment to compare relative genetic diversity among segments over time. All 20 segments showed a dramatic decrease in diversity around 2007 coinciding with the introduction of RVA 21 vaccines. To assess selection pressures, codon adaptation indices and relative codon deoptimization indices 22 were calculated with respect to common host genomes. Codon usage varied by segment with segment 11 23 (NSP5) exhibiting significantly higher adaptation to host genomes. Furthermore, RVA codon usage patterns 24 appeared optimized for expression in humans and birds relative to the other hosts examined, suggesting that 25 translational efficiency is not a barrier in RVA zoonosis.

26 Keywords: Reassortment, Phylogeny, evolution, Genome constellations, Codon Bias, Genetic 27 diversity 28

29 1. Introduction 30 The high mutation rates and large population sizes of RNA allow them to rapidly explore 31 adaptive landscapes, expand host-ranges, and adapt to new environments. Segmented RNA viruses also 32 may undergo ‘reassortment’ whereby viruses swap entire genome segments during coinfection (1). 33 Reassortment may allow rapid evolution of specific viral traits such as, for example, the acquisition of novel 34 spike glycoproteins during the emergence of H1N1 A in 2009 (2). Similarly, reassortment among 35 segmented dsRNA may have significant implications for human health (3), but it is challenging 36 to determine the prevalence of rotavirus reassortment in nature. Our motivation here is to elucidate apparent 37 restrictions (or lack thereof) to RVA genetic exchange in nature by comparing the relative linkage between 38 each of the RVA segments as shown by phylogeny. In addition, we parse the evolutionary constraints that 39 may contribute to the distinct phylogenies of each segment. 40 The rotavirus genome consists of 11 segments of double-stranded RNA, each possessing a single 41 open reading frame, except for segment 11, which contains two genes. Rotaviruses are classified based on 42 the antigenicity of the VP6 protein into groups A through I (4). The consensus is that viruses from different 43 groups cannot reassort with one another (5), though some rare cross-group reassortment events appear to 44 have occurred (6). While mammalian RVA strains routinely reassort with other mammalian RVA strains, 45 reassortment between avian and mammalian RVA strains does not seem to occur outside of laboratories 46 (7). Nevertheless, there have been cases of avian strains infecting mammals (8) and causing encephalitis bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

47 (9), evidence of an avian RVA isolate with a mammalian VP4 gene (10), and some in vitro reassortment 48 assays (7, 11), most recently confirming that mono-reassortants with avian segments 3 and 4 can be 49 recovered using the SA11 reverse genetics system (12). Reassortment between RVA strains from different 50 mammalian species is also less common; however, it is not clear whether this lack of reassortment is due 51 to biological incompatibilities as opposed to genetic incompatibilities. Genotypes resulting from interhost- 52 species reassortment, while rare, have not only occurred, but have fixed in populations (13-21), indicating 53 that reassortment between even distantly related strains is potentially a significant driver of rotavirus 54 evolution (22-25). 55 56 1.1 Rotavirus A Genome and Proteins 57 While the RVA genome is double-stranded, RVA RNA is packaged into and exits as 58 positive-sense, single-stranded RNA (+ssRNA). The RNA-dependent-RNA-polymerase, VP1, and the 59 capping enzyme, VP3, are anchored to VP2 pentamers to form the replicase complex. This complex is 60 present at each of the rotavirus vertices through which +ssRNAs are released (26). The inner VP2 61 layer is enclosed by an intermediate layer composed of VP6 protein and outermost layer composed of the 62 glycoprotein (VP7) and the multimeric spike protein (VP4) (27). For reassortment to occur, +ssRNAs from 63 two or more parents must be packaged into the same virion. 64 In addition to the six structural proteins, the RVA genome also encodes five to six non-structural 65 proteins on five separate segments. The NTPase (NSP2) is a +ssRNA binding protein that forms a 66 doughnut-shaped octamer with a positively charged periphery, allowing +ssRNA to wrap around and bind 67 within its grooves during genome packaging (28, 29). NSP2 protein interactions with the +ssRNA are 68 therefore critical to stabilizing +ssRNA contacts (30). NSP2 also interacts with NSP5 to form viroplasms 69 (along with VP1, VP2, VP3, V6 and NSP4) (31) where genome packaging, replication, assembly of 70 progeny cores and double-layered particles (DLPs) occurs (32-34). To effect transmission, RVA encodes 71 an enterotoxin, NSP4, which elevates cytosolic Ca2+ in cells. This Ca2+ elevation causes diarrhea in hosts 72 (35-39). NSP4 also interacts with VP6 on DLPs during RVA production (40-43). 73 The non-structural protein encoded on segment 7, NSP3, hijacks cellular translation and is a 74 functional analog of the cellular poly(A)-binding protein (44-46). NSP3 binds the group-specific consensus 75 tetranucleotide *UGACC (44, 45, 47) located at the 3’ end of RVA ssRNA, which suggests inter-group 76 reassortment does not occur. The protein NSP1 disrupts cellular antiviral responses (48) and may also play 77 a role in host-range (49). Both segment 5 (NSP1), as well as segment 11 (NSP5/6) are especially prone to 78 genome insertions (50-52). 79 When reassortment occurs, the resulting reassortant virus must maintain the many protein and RNA 80 interactions required for efficient packaging and replication. Even if the reassortant virus is functional, it 81 may still be outcompeted by other genotypes, go extinct due to transmission bottlenecks, or evolve 82 compensatory mutations. These factors, along with the high genetic diversity of RVA strains, make 83 predicting reassortment facility or barriers to reassortment difficult. 84 85 1.2 Selective Pressures on Synonymous Sites 86 RVA is the most common cause of diarrheal disease in young children and is also an important 87 agricultural pathogen, particularly for cows and pigs. RVA vaccines RotaTeq and Rotarix have produced 88 substantial selective pressure on circulating RVA strains since they were first administered on a large scale 89 in 2006 and 2008 respectively. This selective pressure seems to have favored certain genome constellations. 90 Strains relevant to humans mostly consist of genogroup 1 (Wa-like) genes, genogroup 2 (DS-1-like) genes, 91 or genogroup 3 (AU-1-like) genes. Specific G and P types also associated more with specific genogroups 92 (53, 54). 93 Selective pressure on amino acid sequences to conserve protein interactions is a barrier to reassortment 94 compatibility, but segmented viruses are also under considerable selective pressure on synonymous sites as 95 RNA-protein and RNA-RNA interactions are critical in virus packaging (30, 39-42). RVA genome 96 assortment requires +ssRNA molecules from each segment to complex with one another before being 97 packaged, which relies on packaging signals on each segment. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

98 Synonymous sites may also be under selective pressure for certain codon usage patterns. Codon usage 99 bias results in varying levels of efficiency in translation, with highly expressed genes showing stronger bias 100 for codons abundantly available in the host tRNA pool (55, 56), and marginally expressed genes displaying 101 less codon bias (57). Codon usage adaptation to the host is a well-documented phenomenon in DNA viruses 102 (56, 58, 59) and is observed in RNA viruses as well (60-66), though the constraints of secondary RNA 103 structure, as well as the high rate of mutation in RNA viruses, may lower the relative effects of translational 104 selection in RNA viruses. Codon bias is also explained by drift and mutational pressure (i.e., bias towards 105 A/T/U or G/C mutations) as well as translational selection (67). Codon usage that is too similar to the host 106 can also lower efficiency if viral proteins cannot fold properly (68), and differential codon adaptation can 107 be a mechanism of controlling viral gene expression (69, 70). 108 Rotaviruses have been shown to exhibit codon bias (71, 72), but have especially divergent codon usage 109 patterns from humans relative to other RNA viruses (73). Codon usage can be a potential hindrance to 110 zoonosis if a virus infects a host that cannot efficiently translate the virus’ proteins. RVA’s broad host range 111 and ability to undergo genetic exchange makes RVA’s zoonotic potential a cause for some concern. 112 We show that RVA’s segments have distinctive evolutionary histories, demonstrating the impact 113 reassortment has had on mammalian RVA between the late 1950s to 2017. Because each RVA segment is 114 under different selective pressure, we also tested whether there was evidence for translational selection on 115 synonymous sites for each of the segments. To assess whether certain segments showed more codon 116 adaptation against different common host genomes, indicating translational efficiency differences, we 117 tested for neutrality in codon position 3, as well as variations in codon adaptive indices and relative codon 118 deoptimization indices for each segment. To test whether RVA showed signs of codon optimization to a 119 particular host, we compared strains isolated from specific hosts against their host genome and other RVA 120 host genomes. We found differences in codon usage patterns between segments, with segment 11 (NSP5) 121 having significantly higher codon adaptation to host genomes, however our study indicates codon usage is 122 not a barrier to rotavirus zoonosis. 123

124 2. Materials and Methods 125 2.1 Sequence Alignment and Phylogenetic Analysis 126 From all complete RVA genomes in NCBI’s Virus Variation Resource, 789 complete mammalian 127 rotavirus A genomes were chosen by excluding laboratory strains, avian strains, and to avoid sampling bias, 128 any genomes where three or more genomes shared the same sequence identity for NSP4. To ensure that the 129 analysis of each RVA segment employed the same set of strains, we filtered out the selected strains from 130 files containing all available genomes for each segment in Python v3.9.2. The pooled sequences of each of 131 the 11 RVA segments were independently aligned using MUSCLE v3.8.31 (74), and visually inspected for 132 obvious sequencing errors or low-quality sequences. Sequences that were unusually long, short, or 133 contained ‘N’ nucleotides were all removed. 134 We performed phylogenetic analyses using BEAST v1.10.4 (75). We used tip dating to calibrate 135 molecular clocks and generate time-scaled phylogenies. The analyses were run under an uncorrelated 136 relaxed clock model using a time-aware Gaussian Markov random field Bayesian skyride tree prior (76). 137 For VP1, VP2, VP3, VP6 and VP7, the alignments were run using a GTR + Γ + I substitution model and 138 partitioned by codon position. NSP1-NSP4 were further partitioned into non-translated and translated 139 regions. VP4 was further partitioned by the VP5* and VP8* protein domains. Due to the large insertions in 140 segment 11 (NSP5/6), this segment required three additional partitions based on insertion locations. Log 141 files in Tracer v1.7.1 (77) were analyzed to confirm sufficient effective sample size (ESS) values, and trees 142 were annotated using a 10% burn in. The alignments were run for three chains with a 500,000,000 Markov 143 chain Monte Carlo (MCMC) chain length, analyzed on Tracer v1.7.1, and combined using LogCombiner 144 v1.10.4 (78). Trees were annotated with a 10% burn-in and run in TreeAnnotator v1.10.4 (77). The best 145 trees were visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/), with the nodes 146 labeled with posterior probabilities and node bars representing 95% confidence intervals for the divergence bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

147 dates (SI Figure 1-11). Bayesian skyride plots were made in Tracer v1.7.1 to compare each segment’s 148 changes in effective population size (used as a proxy for relative diversity) since the root of the tree (~50 149 years prior to 2017 for most segments). 150 We used the R package ‘distory’ to calculate the geodesic distances between segments. Geodesic 151 distance uses topology and branch length to visualize the tree space of the 11 segments to determine which 152 segments share a close evolutionary history. To account for phylogenetic uncertainty, 350 post-burn-in, 153 randomly chosen trees were sampled from the BEAST v1.10.4 tree file for each segment. We applied multi- 154 dimensional scaling using the R package, ‘treespace’ to determine whether the ‘time to the most recent 155 common ancestor’ (TMRCA) was consistent between segments. The correlation coefficient of TMRCA 156 estimates from all pairwise comparisons of the 11 segment trees was used to estimate tree-distance and then 157 the matrix of tree distance was plotted. Variation in branch length between different segment trees was 158 visualized as a cloud of points where the center represents the mean of several hundred trees. Segments that 159 co-segregate overlapped in three-dimensional space, while segments that did not co-segregate were isolated 160 in space. 161 2.2 Codon Bias Analysis Comparison 162 The relative codon deoptimization index (RCDI) was used to assess if the codon usage of a gene 163 was similar to the codon usage of a reference genome (2,953 coding sequences for Sus scrofa (pig), 93,487 164 coding sequences for Homo sapiens (human), 6,017 coding sequences for Gallus gallus (chicken), 13,374 165 coding sequences from Bos taurus (cow), 1,194 coding sequences for Canis familiaris (dog), and 1,115 166 coding sequences for Oryctolagus cuniculus (rabbit)) (79). RCDI values range from 0.0 to 1.0 with 1.0 167 indicating maximum codon usage compatibility with a reference genome. Similarly, the codon adaptation 168 index (CAI) was used as a measure of codon usage adaptation to the most used synonymous codons of a 169 reference genome, and was used to predict the expression levels of genes (79). CAI values range between 170 0.0 and 1.0, where higher CAI values for a particular reference set indicates higher expression levels. To 171 determine if there were significant differences in CAI/RCDI values between the different segments, we 172 calculated CAI and RCDI values using the CAIcal server (http://genomes.urv.es/CAIcal/) for each segment 173 using a subset of RVA isolates containing multiple representatives of common mammalian RVA genotypes 174 (80). Statistical analyses were performed to assess whether RVA was genetically compatible in its codon 175 usage patterns with a set of RVA host reference genomes. To reduce bias that would result from analyzing 176 a small number of genes, the RVA host reference genomes were chosen based on the availability of a large 177 number of genes analyzed for their codon usage tables in the Codon Usage Database 178 (http://www.kazusa.or.jp/codon/). Reference sets for chicken, human, pig, dog, rabbit, and cow genomes 179 were used for the analysis. We performed Tukey’s Honest Significant Difference (HSD) test to compare 180 mean CAI and RCDI values between segments. We also used Tukey’s HSD tests to compare mean CAI 181 and RCDI values between different host genomes after combining all segments’ values. 182 To test for neutrality at the third codon position, a neutrality plot was made by comparing the GC 183 contents at the first, second and third codon positions. GC12 being the average of GC1 and GC2, was 184 plotted against GC3. If GC12 and GC3 are significantly correlated to one another, and the slope of the 185 regression line is close to 1, mutational bias is assumed to be primarily responsible for shaping codon usage 186 patterns rather than translational selection. Selection against mutation bias can lead to larger differences in 187 GC content between positions 1 and 2, and position 3 and little to no correlation between GC12 and GC3 188 (81). 189 RVA strains isolated from specific hosts (pig, human, cow and avian) were also compared to the 190 pig, human, cow and chicken genomes to test whether RVA genomes showed evidence of adapting to 191 specific hosts. If RVA genomes did not appear to differ in CAI and RCDI patterns based on host type, then 192 it would suggest mutational selection was the dominating force over translational selection, and that 193 selection at the translation level was weak or undetectable.

194 3. Results

195 3.1 Different Tree Space Occupied by the 11 Segments bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

196 Multi-dimensional scaling of the random, post-burn-in sampling of BEAST trees for each of the 11 197 segments revealed that segments 4 and 9 occupy distinct tree spaces from each other and from the rest of 198 the genome (Fig. 1). Segments 10 and 11 occupied indistinguishable treespace indicating close geodesic 199 distances and high levels of evolutionary linkage between them. Segments 3 (VP3), 5 (NSP1) and 6 (VP6) 200 also shared highly overlapping treespace with one another. Segment 2’s (VP2) evolutionary history was 201 most like segment 6 (VP6). None of the segments overlapped with segment 1 (VP1) except for segment 7 202 (NSP3). Segment 7 also had the most phylogenetic uncertainty of all 11 segments as shown by the larger 203 spread around the plot of the post-burn-in trees in Fig. 1. The best-supported trees for segments 1-3 (Fig. 204 2), segments 4-6, 9 (Fig. 3) and segments 7,8, 10, and 11 (Fig. 4) with the host species coded on the tree 205 and the branch lengths color coded by relative evolutionary rate (Fig. 2). While segment 4 had a more 206 independent evolutionary history from other segments, its tree suggested this segment has stricter host 207 boundaries than segment 9 (Fig. 3) indicating less opportunity for divergence due to selection responses to 208 host species change.

209 210 Figure 1. Multi-dimensional scaling plot using 350 post-burn-in BEAST trees from each of the 11 211 RVA segments. The clusters of points represent the geodesic distances between different trees. 350 212 randomly sampled post-burn-in trees were taken from each segment to account for phylogenetic 213 uncertainty. Points sharing the same color are from the same segment as shown in the legend. Points closer 214 to each other indicate close geodesic distances and high levels of evolutionary linkage. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

215 216 Figure 2. Time-scaled phylogenetic trees of Segments 1-3 for 789 mammalian RVA strains. The 217 phylogenies are time-scaled using tip-dating. Scale bars below each tree represent branch length time in 218 years. The branches are colored by rate. Cyan indicates the fastest evolutionary rate among all lineages, and 219 black represents the slowest rate of evolution. Colored asterisks specify the host species the strain was 220 isolated from as shown in the legend. Posterior probabilities, node bars for confidence intervals of the 221 divergence dates and tip labels for the strain names can be viewed in SI Figures 1-3. 222 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

223 224 Figure 3. Time-scaled phylogenetic trees of Segments 4, 6, 9, and 5 for 789 mammalian RVA strains. 225 Phylogenies are time-scaled using tip-dating. Scale bars below each tree represent branch length time in 226 years. The branches are colored by rate. Cyan indicates the fastest evolutionary rate among all lineages, and 227 black represents the slowest rate of evolution. Colored asterisks specify the host species the strain was 228 isolated from as shown in Figure 1. Posterior probabilities, node bars for confidence intervals of the 229 divergence dates and tip labels for the strain names can be viewed in SI Figures 4-7. 230 231 232 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

233 234 Figure 4. Time-scaled phylogenetic trees of Segments 8, 7, 10, and 11 for 789 mammalian RVA 235 strains. Phylogenies are time-scaled using tip-dating. Scale bars below each tree represent branch length 236 time in years. The branches are colored by rate. Cyan indicates the fastest evolutionary rate among all 237 lineages, and black represents the slowest rate of evolution. Colored asterisks specify the host species the 238 strain was isolated from as shown in Figure 1. Posterior probabilities, node bars for confidence intervals of 239 the divergence dates and tip labels for the strain names can be viewed in SI Figures 8-11. 240 241 3.2 Evolutionary rates 242 243 Segment 8 (NSP2) displayed the lowest mean substitution rate (1.48 x 10-3 substitutions per site per 244 year), while segment 4 (VP4) had the highest (3.77 x 10-3 substitutions per site per year). The mean rate for 245 segment 11 was likely skewed higher due to the frequency of large insertions into the segment. Segments bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

246 1-3 showed similar evolutionary rate changes at corresponding clades and time periods in their trees, with 247 the higher evolutionary rates occurring earlier in their evolutionary histories (Fig. 2) (for node confidence 248 intervals at divergence dates see SI Figures 1-11). Higher evolutionary rates tended to be observed along 249 branches leading towards non-human host isolates, particularly for VP7, which had more rate variation 250 across the tree than the other segments (Fig. 3). For example, the Ailuropoda melanoleuca (giant panda) 251 strain (represented by the green asterisk in Figures 2-4) possesses a genomic backbone that is clearly within 252 a cluster of pig and cow strains, except for segment 9. The giant panda’s RVA segment 9 occupies a more 253 divergent branch associated with a higher evolutionary rate than the rest of its segments. 254 Coefficients of variation (CoV) for each segment (Table 1) were consistently high, supporting the 255 assumption that a strict molecular clock is inappropriate for this analysis, and that a relaxed clock is a better 256 choice. All segments exhibited relatively similar TMRCA estimates with segments 1-3 possessing slightly 257 older TMRCA dates than the other segments (Table 1). Segment 1 had the oldest TMRCA estimate (~1957) 258 of all the segments, while segment 11 had the most recent TMRCA estimate (~1969). Node bars for 95% 259 confidence intervals of the node divergence dates are shown in SI Figures 1-11 for each segment. Since this 260 data set of 789 genomes includes strains collected from geographic areas across the world, it appears that 261 most of the mammalian RVA genetic diversity present today has evolved in the past ~60 years. 262 The Bayesian skyride plots indicate that RVA segments reached their peak diversity levels around the 263 year 2000, with a steep decline around the year 2007, which coincides with the introduction and broad- 264 scale use of RVA vaccination (Fig. 5).

265 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

266 267 Figure 5. Skyride plots of the 11 RVA segments. The X-axis represents years before 2017, with 0 268 indicating ~2017. The Y-axis represent effective population size and is a proxy for genetic diversity. All 269 segments show a sharp decrease in diversity roughly 10 years before 2017 (2006-2008). 270 271 Table 1. Mean rates, 95% highest posterior density (HPD), coefficient of variation, and date for the ‘time 272 to most recent common ancestor’ (TMRCA) for each segment’s phylogenetic tree. 273 Segment Mean Rate 95% HPD Coefficient of TMRCA (lower, upper) Variation Segment 1 (VP1) 1.724 x 10-3 1.610 x 10-3 1.835 x 10-3 2.410 1957 Segment 2 (VP2) 1.703 x 10-3 1.589 x 10-3 1.826 x 10-3 2.602 1963 Segment 3 (VP3) 1.948 x 10-3 1.814 x 10-3 2.087 x 10-3 2.436 1962 Segment 4 (VP4) 3.775 x 10-3 3.517 x 10-3 4.044 x 10-3 8.978 1964.5 Segment 5 (NSP1) 2.953 x 10-3 2.694 x 10-3 3.223 x 10-3 3.994 1964.5 Segment 6 (VP6) 1.694 x 10-3 1.541 x 10-3 1.857 x 10-3 3.412 1967.5 Segment 7 (NSP3) 1.615 x 10-3 1.454 x 10-3 1.778 x 10-3 5.061 1967 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Segment 8 (NSP2) 1.473 x 10-3 1.335 x 10-3 1.610 x 10-3 3.323 1966.5 Segment 9 (VP7) 2.660 x 10-3 2.400 x 10-3 2.940 x 10-3 3.375 1966.5 Segment 10 (NSP4) 1.583 x 10-3 1.420 x 10-3 1.743 x 10-3 2.599 1967.5 Segment 11 (NSP5/6) 2.281 x 10-3 1.935 x 10-3 2.635 x 10-3 3.285 1969 274 275 276 3.3 Differing codon usage patterns by segment 277 278 Compared to the other ORFs, NSP5 possessed significantly higher CAI scores and significantly 279 lower RCDI scores across all host genomes (see SI Figure 12 for Tukey-Kramer 95% pair-wise 280 confidence intervals). The wide range of values for each RVA segment suggests that, while there may 281 be differences in selective pressure on codon usage by segment, translational selection was relatively 282 weak compared to mutational selection for the rest of the genome. 283 Codon usage patterns for both mammalian and avian RVA appear more compatible with avian 284 genomes than mammalian genomes (Fig. 6, 7), however human genomes and avian genomes showed 285 similar compatibility between one another, with RVA genomes, and their CAI scores were not 286 significantly different. RVA had higher CAI values and RCDI scores closer to 1 for both human and 287 avian genomes relative to rabbit, cow, pig, and dog genomes. RVA compared against the pig genome 288 resulted in the lowest CAI scores for all segments and strains. In other words, based on CAI and RCDI 289 metrics, RVA is predicted to be least translationally efficient within pigs. 290 A neutrality plot revealed that NSP4 had the highest GC content in position 3 (GC3) relative to 291 the other segments. VP6 had the highest GC content in positions 1 and 2 (GC12) relative to the other 292 segments while NSP1 and VP3 had the lowest GC12 content. While VP6’s GC12 content was higher 293 than the rest of the RVA genome, VP6’s GC3 content was not. The slopes for all regression lines 294 deviated from 1 (Fig. 8), ranging from 0.043 (VP7) to 0.316 (NSP1), indicating that there was 295 significant selective pressure on position 3 for codon usage for all segments. The slopes indicate NSP1 296 is under more mutational pressure than the other segments, while VP7 is under more selective pressure 297 at position 3 compared with the other segments, however the lower GC3 values relative to GC12 in 298 VP7 actually indicates lower adaptation to the host genome. 299 The lower GC3 values relative to GC12 in VP1 and VP3 also showed low slopes (0.06 and 0.05 300 respectively), suggesting that they may also be under less mutation pressure and more translational 301 selection, however like VP7, the relatively low GC3 content in already high AU genome, suggests the 302 translational efficiency would be lower. The correlation coefficient overall for GC12 and GC3 was 303 0.261 (p < 0.001). While some segments/ORFs (VP1, VP2, VP4, NSP1, NSP2, NSP3 and NSP5) were 304 more significantly positively correlated between GC12 and GC3, segments 9 and 10 displayed no 305 significant correlation. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A 306

307 B 308 Figure 6. Boxplots of the Codon Adaptation Index and Relative Codon Deoptimization Index values 309 for RVA genes with respect to different RVA hosts. A. Sampled sequences representing common 310 genotypes for each of the 11 segments were measured for CAI with respect to the six hosts shown. The Y- 311 axis value ranges are different for each host as some hosts result in higher CAI values. Higher CAI values 312 indicate more efficient expression. B. RCDI values for the same sampled sequences from A plotted for each 313 segment with respect to each host. RCDI values closest to 1 indicate more similar codon usage patterns. 314 The Y-axis value ranges are different for each host. Tukey-Kramer 95% pair-wise confidence intervals are 315 shown in SI figure 12. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Tukey-cramer

Tukey HSD 95% confidence level

Differences in Means for RCDI Differences in Means for CAI 316 317 Figure 7. Tukey’s honest significance test for RCDI and CAI values between different host genomes. 318 Values used for this test are the combined values for all segments used in Figure 6. 319 320 321 3.4 Synonymous sites under selection, however evidence does not suggest translational selection to specific 322 hosts 323 324 When comparing strains isolated from different species, there was no evidence to support the 325 conclusion that RVAs adapt their codon usage to specific hosts (Fig. 9). While the bovine strains and avian 326 strains had RCDI values closer to 1.0 than the pig and human RVA strains, there were no significant 327 differences between RCDI values of avian isolates compared to avian genomes vs. RCDI values of bovine 328 isolates compared to avian genomes. Based on RCDI values, bovine strains were “most compatible” with 329 avian genomes, and human strains were “less compatible” to the human genome than to bovine strains. 330 Given that avian and mammalian RVAs do not exchange genetic information in nature, there is substantial 331 divergence between avian RVA isolates and mammalian RVA isolates. The fact that RVA strains show 332 more similar codon patterns between bovine and avian strains than between human and bovine strains 333 suggests that translational selection by host does not play a large role in codon bias. 334 Rotavirus genes overall had higher CAI scores and lower RDCI scores when contrasted with the avian 335 genome codon usage patterns. The lowest CAI and highest RDCI scores were observed when RVA genes 336 were compared with rabbit genomes. However, there was little variation dependent on the host the viral 337 strain was isolated from (i.e., avian RVA strains did not have significantly different scores when compared 338 to bovine RVA strains using the same reference codon usage patterns). There was no evidence for detectable 339 translational selection by host, however the observation that RVA was generally less optimized for the non- 340 human mammalian genomes suggests that RVA may have higher protein expression levels in humans. 341 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

342 343 344 Figure 8. Neutrality plot of RVA ORFs. GC percentages of codon position 3 plotted against GC 345 percentages of codon positions 1 and 2 (GC12) for the ORF for 11 RVA genes. Slopes significantly 346 deviating from 1 show evidence of natural selection, while slopes near 1 suggest neutrality wherein 347 mutational selection is the driving force of codon usage patterns.

Strains’ host

Host Genomes

Strains’ host

348 Host Genomes bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

349 Figure 9. Box plots of CAI and RCDI values for each ORF from strains isolated from specific hosts. 350 RCDI and CAI values were derived by sampling 7 complete genomes (77 segment genomes) for each host, 351 isolated from either avian (red), cow (green), human (blue), or pig (purple) hosts. These RVA genomes 352 were then separately compared to cow, chicken, human or pig codon usage charts.

353 4. Discussion 354 While segment exchange between different RVA genotypes is common, reassortment is not a random 355 process (82-86). However, the limits of segment exchange, whether ecological or mechanical, are poorly 356 understood. Some segment combinations work well together, whereas others are incompatible (87). 357 Numerous factors potentially could affect whether segments from different RVA genotypes are able to 358 reassort, including protein interactions, RNA-RNA and RNA-protein interactions, and the needs to maintain 359 host range and ensure RNA packaging efficiency. 360 The goal of this study was to better predict potential (or unlikely) genome constellations that may 361 emerge in nature and enhance our understanding of why some segments co-segregate and some do not. To 362 this end, we compared the evolutionary histories as well as some of the selective pressures acting on the 11 363 RVA segments, first by performing phylogenetic analyses on a large collection of complete RVA genomes, 364 and then by assessing the selective pressures acting on synonymous sites by segment and host type. We 365 estimated the diversity levels of each RVA segment over the last ~60 years and linked decreases in diversity 366 to the introduction of RVA vaccines between 2006 and 2008. 367 The segments that share highly similar tree space may share important interactions that rely on a higher 368 percentage of the sequence (e.g., selective pressure at synonymous sites), while the segments that inhabit 369 distinct tree space are likely more flexible on a variety of genetic backgrounds. RVA’s RNA segments are 370 under different evolutionary pressures, which is clear from the distinct evolutionary histories of the 371 segments (Fig. 1), and the significant difference in nucleotide composition between the segments, despite 372 coming from the same genome (Figs. 6 and 8). The zoonotic potential for rotaviruses makes understanding 373 restrictions on genetic exchange important, as outcrossing events can result in novel strains which may 374 cause more severe disease or be better able to evade a vaccine or immune response. Both codon usage and 375 reassortment potential can be important factors in viral host-range, and both are constrained by RNA 376 interactions, translational selection, and mutational bias. Understanding the relative constraints of the RVA 377 genome can help better assess the risk of zoonotic outbreaks and emerging strains. 378 Potential impact of rotavirus RNA and protein interactions on segment co-segregation 379 RVA protein and RNA molecules interact with each other in a variety of ways during infection and 380 assembly processes. Incompatibilities among different genotypes resulting from these interactions may 381 limit the diversity of genome constellations observed in nature. For example, RNA secondary structures 382 and segment-specific sequences found in the non-translated terminal regions (NTRs) may govern the 383 formation of the supramolecular RNA complex associated with segment packaging (30, 86, 88-91) 384 Sequence mismatches between segments from coinfecting RVAs may prevent segment interactions and co- 385 packaging, and hence, the generation of reassortant RVAs. One of the studies (88) also suggested that VP4 386 has less conserved terminal RNA structure, so the importance of these RNA interactions may vary 387 significantly by segment. 388 The order in which the segments associate to form the supramolecular RNA complex may be 389 sequential. In this scenario, the smallest segments interact first, then they recruit intermediate sized 390 segments before finally incorporating the largest segments (89). In addition, incompatibilities in the 3’- 391 NTRs of the smaller segments may have stronger effects on segment co-segregation than do the larger 392 segments. For instance, the smallest RVA segments, segments 10 and 11 must directly interact before they 393 can interact with larger segments. Evidence for this supposition that the smaller segments’ RNA structure 394 is under strong selective pressure along with its protein structure (i.e., synonymous mutations are often not 395 neutral) also comes from our observation that segments 10 and 11 co-segregated with one another more 396 strongly than other pairs of segments (Fig. 1), despite their many protein interactions with other segments. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

397 There is evidence for frequent inter-host-species reassortment of the segment 10, which encodes the 398 enterotoxin, NSP4, in nature (14, 92), so reassortment of segment 10 may be more dependent on the 399 sequence conservation of terminal +ssRNA between segments 10 and 11, than on protein interactions. In 400 addition to NSP5, segment 11 also encodes a second out of frame protein, NSP6 via leaky scanning. NSP6 401 is not required for virion function and sometimes not expressed in rotaviruses (93); however, it is 402 constrained due to overlapping with NSP5. Segment 11’s RNA structure is thought to have some functional 403 importance (88, 94) however segment 11 appears more tolerant of genome insertions than other segments 404 (50, 51), likely due to packaging signal duplication (51). The close evolutionary histories of segments 10 405 and 11 may also relate to NSP4-NSP5 interactions during viroplasm formation (33) 406 The treespaces of both segments 4 and 9 (Fig. 1) were notably distinct from the treespaces of rest 407 of the genome. RVA segment 4 encodes the spike protein, VP4, which is cleaved by trypsin into VP8* and 408 VP5*. VP4 interacts with different receptors depending on the strain, including sialoglycans and histo- 409 blood glycans (95-97) These different receptors partly explain why certain P types tend to dominate in 410 different populations, species, and age groups (97, 98). Our results showed that the gene tree of segment 4 411 was distinct from the rest of the genome, suggesting that segment 4 may reassort more readily than other 412 segments. However, segment 4 also appeared to have stricter host barriers than segment 9, which encodes 413 the other serotype protein, VP7 (Fig. 3). Other environmental studies have also found that segment 4 and 414 segment 9 are more likely to appear in different genetic backgrounds (99-101) Based on the high genetic 415 diversity of segments 4 and 9, and segment 4 seeming to have a less conserved role in RVA +ssRNA 416 assortment, reassortment into new genetic backgrounds may confer a selective advantage and a broader 417 host-range as strains can have an opportunity to evolve in a novel host they may otherwise be unable to 418 infect simply due to a barrier caused by another segment (e.g., poor receptor-binding). The fact that segment 419 4 had the greatest geodesic distance from the rest of the genome, and also possesses the most diversity in 420 RNA secondary structures, suggests that segment 4’s RNA secondary structures are less critical to the 421 segment’s function. Segment 9, on the other hand, is critical for the formation/stabilization of the 422 supramolecular RNA complex and for packaging the genome. Both segment 4 and 9 have been shown to 423 tolerate homologous recombination among highly divergent genotypes, including recombination events 424 resulting in the disruption of many amino acids, whilst still maintaining overall tertiary structure (102). 425 Due to the importance of VP1, VP2, and VP3 during the formation of the virion, synthesis of 426 dsRNA, and complexing with the 11 +ssRNA segments, one might expect these segments to be the least 427 likely to reassort independently. However, the critical VP1, VP2 and VP3 interactions are mostly protein- 428 protein interactions, so even a genetically distant strain could maintain a conserved amino acid sequence 429 allowing these segments some flexibility with their genetic background. Our results showed that, while 430 these three segments are generally associated with the larger “gene tree” of the rest of the genome, they 431 have a more independent evolutionary history than for example, segments 10 and 11, which almost entirely 432 overlap in tree space. 433 Interestingly, segment 7 (encoding NSP3) shared the closest evolutionary history with segment 1 (Fig. 434 1). We expected segment 1 to have the closest evolutionary history with segments 2 or 3 given their 435 proteins’ interactions, however segment 1’s high degree of structural conservation (5) and having less 436 functionally important RNA structure than the other segments may explain its tolerance for novel genetic 437 backgrounds. Segment 7/NSP3 may have less strain specific interactions with other segments resulting in 438 less fitness variation following a reassortment event. Segment 7 may have endured a significant 439 reassortment event around 1970 (Fig. 4, and SI Fig. 7) which also may explain its geodesic from the other 440 segments. NSP3’s primary function is to recognize a conserved group-specific sequence, *UGACC, present 441 on all group A RV segments and interact with host eIF4G, which is highly conserved among orthologs 442 (103). This suggests NSP3 genes should be flexible to many RVA genetic backgrounds and hosts. 443 Segment 5 (NSP1) was only particularly distinct in the treespace (Fig. 1) from segments 1, 4 and 9. 444 This was somewhat surprising as NSP1 has relatively low conservation, can tolerate insertions and 445 deletions, and is not required for rotavirus replication in vitro, although the RNA is still required for 446 packaging. NSP1 does however serve important roles in targeting the host’s antiviral response. It acts as an 447 interferon antagonist (104),and inhibits apoptosis (105, 106) and activation of NFkappaB (107). and may bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

448 play a role in host-range (49). A stricter host-range boundary could help explain NSP1’s more similar 449 evolutionary history with the rest of the genome, as NSP1 reassortment with different host strains may 450 confer a deleterious effect in vivo, despite the reassortant’s ability to compete in vitro (i.e., in the absence 451 of a significant immune response). 452 453 Rotavirus evolution following the introduction of rotavirus vaccines 454 The live-attenuated pentavalent vaccine, RotaTeq (Merck), was introduced in 2006, and the live- 455 attenuated monovalent vaccine, Rotarix (GlaxoSmithKline), was disseminated in 2008. RotaTeq contains 456 five human-bovine reassortant viruses with strain serotypes of G1P[5], G2P[5], G3P[5], G4P[5], and 457 G6P[8]), while Rotarix is comprised of the human G1P[8] strain, RIX4414. These vaccines provided 458 effective protection against contemporaneous globally dominant strains G1P[8], G2P[4], G3P[8], G4P[8], 459 and G9P[8]. Our results show a coincident decline in the relative diversities and effective population sizes 460 of all RVA segments after 2006. Supporting this analysis, several studies, including an analysis of G12 461 strains in Africa, G12 strains in Spain, and a lineage of P[8] strains also show a general decline in 462 diversity/effective population size after 2008 (108-110). 463 The introduction of the RVA vaccines resulted in a global reduction in RVA-associated mortality. 464 However, vaccine effectiveness varied substantially by region (111) and resulted in changes to the 465 circulating strain prevalence. For example, in post-vaccine era, the prevalence of G9P[8], G2P[4], and 466 G9P[4], G9P[8] increased, while the prevalence of G1P[8] and G3P[8] declined (112-115)The emergence 467 of rare genotypes or animal RVA reassortants in children also appears to be connected to the selective 468 pressure imposed by vaccination. For example, the increase in abundance of G12 and G11 genotypes (113, 469 114) and the appearance of atypical Wa-like and DS-1-like reassortants, such as the emergence of G1P[8] 470 with a DS-1-like backbone in Malawi (116), appear linked to increases in RVA vaccination. Given DS-1- 471 like and Wa-like segments are thought to have incompatibilities with one another, limiting their 472 reassortment potential (87), the vaccine-induced selection for mutations can be difficult to assess. That is, 473 it is difficult to determine whether emerging fixed mutations are the direct result of escape mutants or are 474 compensatory mutations resulting from novel reassortants. A study on G2P[8] evolution did not observe 475 evidence of vaccine-induced selection; however, another study focusing on P[6], P[4], and P[8] genes did 476 report substantial divergence from the vaccine strains. 477 Vaccine-induced selective pressure may partly explain the pattern observed when comparing the 478 geodesic distances of the segments. Like the present study, which found that segments 4 and 9 (encoding 479 serotype proteins VP4 and VP7) were especially amenable to reassorting into new genetic backgrounds, a 480 study of European Bluetongue virus (BTV) isolates also found that segments 2 and 6 (encoding the BTV 481 homologs of VP4 and VP7) were quite distant from the rest of the genome in treespace (117). However, 482 the BTV segments 2 and 6 were closer to one another in treespace, whereas in RVA segments 4 and 9 were 483 highly distinct from each other. Additionally, BTV segments 7 (encoding the inner capsid protein) and 10 484 (encoding NS3) shared close evolutionary histories with one another, and distinct from the rest of the 485 genome. Interestingly, in another BTV study on strains from India, segment 4 (encoding a protein 486 homologous to VP3 in RVA) was found to be the most isolated segment in treespace (118). While there are 487 BTV vaccines available, BTV does not infect humans, is not as globally distributed as RVA, and far fewer 488 serotypes circulate, so there may be more selective pressure on RVA serotype segments to reassort. 489 While RVA segments were unlinked and had different phylogenies and rate variations along their 490 trees, the segments’ patterns in relative diversity over time mostly matched each other. In Rift Valley fever 491 virus (119), which is a three-segmented -ssRNA arbovirus, different skyline plots were observed for each 492 segment, suggesting that the segments are evolving independently to some extent. In Rift Valley fever 493 study, the medium segment had a much larger effective population size than the small and large segments, 494 indicating that the medium segment experienced reassortment events more frequently than the small and 495 large segments, which tended to co-segregate. The differences in evolutionary patterns between RVA and 496 Rift Valley fever virus may be a consequence of their differing epidemiologies. Frequent outbreaks of Rift 497 Valley fever are limited to sub-Saharan Africa, and their transmission relies heavily on mosquitos and not 498 human-to-human transmission. Rotavirus is conversely, a globally present pathogen with many dominating bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

499 strains constantly circulating amongst humans, likely making it less sensitive to bottleneck effects. 500 A more thorough comparison of the geodesic distances between the spike and outer capsid proteins and 501 the rest of the genome in other dsRNA viruses is indicated. It would be interesting to ascertain whether the 502 patterns observed in RVA are also seen in other dsRNA viruses. While the G and P types are thought to 503 maintain host-barriers, this study suggests that is only the case with VP4. By contrast, VP7 may be more 504 flexible in host range and not evolutionarily linked to VP4. Rotavirus disease is typically discussed in terms 505 of ‘GXP[Y]’ strains, however segment 4 and segment 9 evolve independently both from each other and 506 from the rest of the genome. The prevalence of common G and P combinations in association with certain 507 backbones seems to have more to do with an ecological abundance of those strains and less to do with a 508 functional constraint on the virus. As this study analyzed strains from prior to 2017, the global declines in 509 RVA diversity in response to RVA vaccination may change. 510 511 Codon bias analysis shows codon usage differs between segments, but not between host strains 512 We contrasted the codon usage patterns of several common mammalian RVA genotypes with a set 513 of RVA host reference genomes. Our results showed that mammalian and avian RVA codon usage patterns 514 were most compatible with avian genomes (Fig. 6). For example, the highest CAI values and lowest RCDI 515 values in our comparative analysis suggested that RVA NSP5’s codon usage pattern was best adapted to 516 the chicken genome (Fig. 6). This finding suggests that RVA originated as an avian virus that subsequently 517 expanded its host range to include mammalian hosts. The fact that RVA appears better adapted to human 518 and avian genomes rather than the other mammalian genomes tested further suggests that RVA spread to 519 other mammalian hosts after adapting to humans, though more evidence is required to confirm this 520 hypothesis (Fig. 7). Additionally, NSP5’s relatively high CAI value and low RCDI value (Fig. 6; both 521 values approach 1) may indicate a strong selective advantage for NSP5’s codon optimization. Since 522 efficient NSP5 protein expression is critical for RVA viroplasm formation and replication (120-122), the 523 higher CAI value may indicate that NSP5’s codons are optimized to match avian hosts to maximize NSP5 524 expression in host cells. The variation in GC content between segments was also notable, as being from the 525 same genome, this suggests mutational bias towards AU or GC is not entirely responsible for RVA’s 526 observed codon bias. The low GC3 content and especially low GC3:GC12 ratio seen in VP3 (Fig. 8), in 527 contrast with the high GC3 content of NSP5 and NSP4 (relative to the rest of the genome), could suggest 528 it is beneficial for the rotavirus to have lower-efficiency VP3 expression relative to NSP4 and NSP5, during 529 infection. Varying the codon bias by gene, or maintaining suboptimal codon bias to the host, may sometimes 530 be beneficial (73, 123), a phenomena that has been observed for example, in hepatitis A virus (60). 531 Although the bovine and avian RVA strains sampled tended to have higher CAI scores and RCDI 532 scores closer to 1, there was no evidence of divergence in codon usage among bovine and avian strains. 533 The similarity in codon usage between host strains is in contrast with a study of influenza A which found 534 that avian and human influenza strains have distinctly different G/C vs. A/U contents from one another 535 (124). Our finding suggests that, while RVA strains may indeed experience an advantage from matching 536 the codon usage patterns of their hosts, it is unlikely that translational selection can counteract nucleic acid 537 selection (i.e., selection favoring synonymous substitutions improving virus survival and reproduction). 538 The wide range of CAI/RCDI values and GC3 content for some of the segments suggest that translational 539 selection is not an especially strong selective pressure for every gene. However, translational selection does 540 seem to be a stronger selective pressure at least in NSP5, based on its significant divergence from the rest 541 of the RVA genome’s codon usage (Fig. 8). The neutrality plot also shows that GC percentages at position 542 3 frequently are significantly different from the GC percentages at positions 1 and 2, which would indicate 543 that strong selection is occurring at position 3 (Fig. 8). The variation if GC content and codon usage by 544 segment could point to RVA using codon bias as a mechanism of controlling viral gene expression. While 545 there is no significant difference in codon usage between strains isolated from different hosts, notably RVA 546 in general is significantly more optimized to human and avian genomes, than pig and cow genomes. This 547 indicates translational efficiency is not a barrier for zoonosis between avian and mammalian strains. 548 There are some limitations in the assessment of codon usage patterns. Despite the potential 549 advantage of possessing a codon usage pattern that strongly resembles that of a host, codon usage patterns bioRxiv preprint doi: https://doi.org/10.1101/2021.03.20.436270; this version posted March 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

550 on their own are inadequate for making inferences or conclusions about the selective forces acting on virus 551 populations. Furthermore, a significant amount of codon usage pattern variation exists between genes the 552 same host, so forming conclusions regarding viral codon-level adaptation to the host is especially difficult. 553 That is, having high CAI values or RCDIs close to 1.0 does not necessarily mean that the virus is more 554 adapted to that host. It could, however, provide evidence that the viral genes are better expressed in a 555 particular host or that a specific gene of a virus may be more efficiently expressed. Additionally, we note 556 that rotaviruses have especially AU-rich genomes and their codon usage patterns diverge from human usage 557 patterns more than other human viruses (73). It would appear that the benefits of being AU-rich outweigh 558 any benefits conferred through codon optimization. 559 This study further supports caution when measuring for selection by comparing dN/dS ratios for 560 rotaviruses, as the selective pressure at synonymous sites varies significantly by segment. While the 561 evidence did not support RVA nucleotide composition or translation selection varying based on host strain, 562 certain segments were under stronger selectional (translational) rather than mutational pressure at codon 563 position 3. RVA’s indistinctive codon usage by host strain, suggests translational efficiency is not an 564 important host-range barrier for RVA. 565 Funding: We appreciate funding from the PSC-CUNY Research Award Program, the CUNY Advanced 566 Science Center Seed Program, and from the Queens College Biology Department’s Seymour Fogel 567 Endowment Fund for Genetic and Genomic Research. 568 569 Acknowledgments: We appreciate useful conversations from Nanami Kubota and Sarah McDonald 570 Esstman, relating to codon bias and rotavirus evolution. We are also grateful to Thomas Hoxie for 571 editing drafts of this article. 572 573 Conflicts of Interest: The authors declare no conflict of interest.

574 Author Contributions: Conceptualization, I.H.; Methodology, I.H.; Formal Analysis, I.H.; Investigation, 575 I.H.; Resources, I.H. and J.J.D.; Data Curation, I.H.; Writing – Original Draft Preparation, I.H.; Writing – 576 Review & Editing, I.H. and J.J.D.; Visualization, I.H.; Supervision, J.J.D.; Project Administration, J.J.D.; 577 Funding Acquisition, I.H. and J.J.D.

578 Data Availability Statement: All genomic data analyzed in this manuscript are publicly available 579 through NCBI’s Virus Variation Resource (https://www.ncbi.nlm.nih.gov/genome/viruses/variation/).

580 References 581

582

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