Cooperative interactions in the West Nile virus mutant swarm

Abstract

Background: RNA viruses including arthropod-borne viruses (arboviruses) exist as highly genetically diverse mutant swarms within individual hosts. A more complete understanding of the phenotypic correlates of these diverse swarms is needed in order to equate RNA swarm breadth and composition to specific adaptive and evolutionary outcomes.

Results: Here, we determined clonal fitness landscapes of mosquito cell-adapted West Nile virus (WNV) and assessed how altering the capacity for interactions among variants affects mutant swarm dynamics and swarm fitness. Our results demonstrate that although there is significant mutational robustness in the WNV swarm, genetic diversity also corresponds to substantial phenotypic diversity in terms of relative fitness in vitro. In addition, our data demonstrate that increasing levels of co-infection can lead to widespread strain complementation, which acts to maintain high levels of phenotypic and genetic diversity and potentially slow selection for individual variants. Lastly, we show that cooperative interactions may lead to swarm fitness levels which exceed the relative fitness levels of any individual genotype.

Conclusions: These studies demonstrate the profound effects variant interactions can have on arbovirus evolution and adaptation, and provide a baseline by which to study the impact of this phenomenon in natural systems.

Figure 1

Haplotype accumulation ofWest Nile virusduring passage in mosquito cell culture. Unique shades and/or patterns represent unique haplotypes identified by high-fidelity molecular cloning and sequencing of 15-20 clones of nt 1311-3248 following passage on C6/36 mosquito cells (some data modified from [22]).

Figure 2

Distribution of relative fitness values among 40 biological clones of WNV following passage on mosquito cell culture (WNV CP40). Relative fitness refers to the proportion of individual WNV CP40 clones relative to the control virus (WNV MARM) following 72 h of competition on mosquito cell culture. The solid line represents the mean +/- standard deviation and the dotted line represents the fitness of the combined population. The circled data point represents the highest fitness clone isolated, WNV CP40-12.

Figure 3

Relationship between levels of co-infection and relative fitness of WNV CP40-12 on mosquito cell culture. a. Individual points represent relative fitness values +/- standard error of duplicate competition assays carried out for 72 h at indicated MOIs. A significant negative correlation between MOI and relative fitness was measured (linear regression analysis, r² = 0.68, p = 0.0035). b. Relationship between WNV titer and relative fitness during competition following infection at an MOI of 10. Individual points represent duplicate measurements of output virus following 24 (circles), 48(squares), or 72 (triangles) hours of competition. Results demonstrate a negative correlation between WNV titer and relative fitness (Pearson correlation, r = -0.88, p = 0.0195).

Figure 4

Relationship between levels of co-infection and genetic diversity of WNV. Bars depict normalized Shannon entropy (S_n) on both the nucleotide (nt) and amino acid (aa) levels and circles depict relative differences in mean hamming distance for populations following a single passage of WNV CP40 at indicated MOIs. All measures of genetic diversity are for nt 1311-3248 and 17-22 clones were sequenced and analyzed for each measure. A significant positive correlation between co-infection (MOI) and S_n for both nt and aa was measured (linear regression analyses, r² = 0.99, p < 0.05).

Figure 5

Fitness distributions of WNV CP40 with or without co-infection on mosquito cell culture. Individual data points represent relative fitness values of 40 clones isolated following 24 h growth at MOI 10 (with co-infection) or 0.01 (without co-infection). Solid lines represent means +/- standard deviations and dashed lines represent relative fitness values of combined populations. The inset graph depicts individual viral titers of clones following 72 h growth on mosquito cell culture. Significantly higher relative fitness values and significantly lower viral titers were measured in the MOI 10 group (t-tests, p < 0.05), as indicated by the *.