Does adaptation to vertebrate codon usage relate to flavivirus emergence potential?

<div><p>Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage reference. Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrate historically relevant trends of well-characterized viruses, in different viral species and genetic diversity within a single species. CAI can be a useful tool together with <i>in vivo</i> and <i>in vitro</i> kinetics, phylodynamics, and additional functional genomics studies to better understand species trafficking and viral emergence in a new host.</p></div

CAI and phylodynamics of West Nile virus lineage 2 sequences.

<p>A) Bayesian maximum clade credibility tree representing a time scaled phylogeny of a WNV lineage 2 polyprotein sequences. Bayesian posterior probabilities > 0.9 are marked with an asterisk at major nodes. Averages for the European epidemic lineage (yellow bar) and a 2^nd European lineage (highlighted in red) are shown. B) Malthusian fitness (<i>Wm</i>) was calculated from 2004–2014, and compared to LOESS trend lines generated from CAI values to the codon usages of C) human house-keeping genes, D) human immune/antiviral genes, E) pigeon (Columba livia) genes, and F) mosquito (<i>Culex pipiens</i>) genes. G) The Spearman’s rank correlation test was used to test if there were any correlations between the 2010 to 2012 CAI increase in mosquitoes and the CAI increase in vertebrate species from 2012–2014, as well as Wm. The ΔCAI, Δ<i>Wm</i> and <i>rho</i> are shown for clarity. # = for all correlations, <i>p</i>-values were < 0.05.</p

The CAI of Tobacco Mosaic virus to house-keeping genes.

<p>A violin dotplot of the CAI of complete Tobacco Mosaic virus (TMV) coding sequences to the codon usages of human house-keeping genes, antiviral/immune genes, and tobacco (<i>Nicotiana tabacum</i>) house-keeping genes. Average CAI values for each group are shown.</p

Flavivirus-CAI dynamics to associated species.

<p>(A) Bayesian-inferred phylogenetic tree of the complete ORF using nucleotide sequences. All nodes had a posterior probability > 0.9. Taxa were omitted for clarity. Associated viral vectors and vertebrate host groups are colored. The scale bar represents 0.06 mutations per site. (B) Box and whisker plot of CAI for each flavivirus species subgroup. For each group of sequences, the CAI was normalized by length, GC-percentage and amino-acid content. Black lines with asterisks signify CAI values that are significantly different.</p

Does adaptation to vertebrate codon usage relate to flavivirus emergence potential? - Fig 3

<p><b>CAI changes across time for (A) Yellow fever virus, (B) endemic strains of Dengue 2 virus, and (C) sylvatic strains of Dengue 2 virus.</b> For each codon usage table, the CAI was normalized by length, GC% and amino acid content for each dataset. Area of plot points reflects the density of sequences at a specific coordinate. A trend line was generated using LOESS, a non-parametric regression method, with 0.95 confidence interval shading. For B), CAI data to monkeys was removed for clarity, but was positioned in between the human table trend lines.</p

Biological and phylogenetic characteristics of West African lineages of West Nile virus

<div><p>The West Nile virus (WNV), isolated in 1937, is an arbovirus (arthropod-borne virus) that infects thousands of people each year. Despite its burden on global health, little is known about the virus’ biological and evolutionary dynamics. As several lineages are endemic in West Africa, we obtained the complete polyprotein sequence from three isolates from the early 1990s, each representing a different lineage. We then investigated differences in growth behavior and pathogenicity for four distinct West African lineages in arthropod (Ap61) and primate (Vero) cell lines, and in mice. We found that genetic differences, as well as viral-host interactions, could play a role in the biological properties in different WNV isolates <i>in vitro</i>, such as: (<i>i</i>) genome replication, (<i>ii</i>) protein translation, (<i>iii</i>) particle release, and (<i>iv</i>) virulence. Our findings demonstrate the endemic diversity of West African WNV strains and support future investigations into (<i>i</i>) the nature of WNV emergence, (<i>ii</i>) neurological tropism, and (<i>iii</i>) host adaptation.</p></div

Growth kinetics of West African West Nile virus strains.

<p>The strain lineage label is in reference to the strains in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006078#pntd.0006078.t001" target="_blank">Table 1</a>. Figs A-D show the amount of viral RNA equivalents isolated from cells (A and B) and supernatant (C and D) (log₁₀ of RNA copy number), the percent (% immunofluorescence) of cells infected (E and F) and the number of infectious viral particles (G and H) (log₁₀PFU/ml) over a 146-hour post-infection time period. The experiments were performed with Ap61 cells (left column) and Vero cells (right column). The error bars indicate the range in values of two independent experiments.</p

The genetic diversity of the West Nile virus lineages.

<p>A) Pairwise percent identity between nucleotide (blue) and amino acid (orange) sequences of the polyprotein. Sequences are labeled in the following format: accession number, 2-letter country code, and year of isolation. B) Genomic structure of West Nile virus with genes labeled. Alignments of known virulence motifs are shown. Codons of special interest are labeled by their position at each individual protein sequence and not by their position in the polyprotein. Sequences are labeled by country, year of isolation and phylogenetic lineage.</p

Mice mortality and virulence of West Nile virus <i>in vivo</i> of 5- to 6-week-old Swiss mice observed for 21 days.

<p>Mice mortality and virulence of West Nile virus <i>in vivo</i> of 5- to 6-week-old Swiss mice observed for 21 days.</p

Bayesian maximum clade credibility tree estimating the phylogenetic relationships of West Nile virus.

<p>Tree nodes with a posterior probability greater than 0.7 are displayed. Tree tip nodes are colored by proposed lineage and for visual clarity. For each sequence, the two-letter code representing a country of isolation is included in the sequence label. Branches are scaled in years before 2015. ^# NY99 strain.</p