Virus evolution in Wolbachia- infected Drosophila

Abstract

Wolbachia, a common vertically transmitted symbiont, can protect insects against viral infection and prevent mosquitoes from transmitting viral pathogens. For this reason, Wolbachia-infected mosquitoes are being released to prevent the transmission of dengue and other arboviruses. An important question for the long-term success of these programmes is whether viruses can evolve to escape the antiviral effects of Wolbachia. We have found that Wolbachia altered the outcome of competition between strains of the DCV virus in Drosophila. However, Wolbachia still effectively blocked the virus genotypes that were favoured in the presence of the symbiont. We conclude that Wolbachia did cause an evolutionary response in viruses, but this has little or no impact on the effectiveness of virus blocking.

Keywords: Drosophila melanogaster; Wolbachia; antiviral resistance; experimental evolution.

Figure 1.

Effect of Wolbachia on virus allele frequencies when selecting on a genetically diverse viral population. A genetically diverse population of DCV was passaged through Wolbachia-infected or Wolbachia-free flies and then sequenced. (a) Principal components analysis on allele frequencies of SNPs, where each point is an independent virus population (biological replicate). (b) Differences in the frequency of SNPs along the viral genome. The sequence reads were mapped to the DCV-C reference genome, and the heatmap shows the difference in the frequency of the allele carried by a given DCV strain between Wolbachia treatments (frequency in Wolbachia-infected flies minus frequency in Wolbachia-free flies; red is a higher allele frequency in Wolbachia-infected flies). The tree was computed from the Euclidian distance computed from these differences in allele frequencies. (c) DCV phylogeny with bootstrap support for the nodes. (d) Differences in the frequency of isolate- or clade-specific variants between viral populations that had evolved in Wolbachia-infected and Wolbachia-free flies (positive numbers are more common in the presence of Wolbachia). The number of SNPs is shown in parentheses, and the number in bold is the mean difference in frequency across all those SNPs. NA stands for cases where no isolate- or clade-specific SNPs could be found. (Online version in colour.)

Figure 2.

Wolbachia's effect on viral titres and virus-induced mortality in single virus infections. (a) Growth curves of three DCV isolates in Wolbachia-free and Wolbachia-infected flies. The lines are asymptotic exponential curves and the shaded areas are 95% confidence intervals. (b) Survival curves following infection with DCV. (c) Wolbachia effect on virus-induced mortality expressed as –ln(hazard ratio) where the hazard ratio is the probability of flies dying in Wolbachia-infected flies relative to their Wolbachia-free counterpart. Error bars are standard errors and ***p < 0.001. (Online version in colour.)

Figure 3.

Presence of DCV and Wolbachia effect on viral titres in selection experiment 2. (a) Ct values obtained from qPCR reactions targeting DCV RNA in virus populations at different passages during selection. Populations passaged in Wolbachia-free flies were only checked for DCV infection at passage 10. Fractions at the top of the plot indicate the number of biological replicates for which DCV was detected relative to the total number of replicates (n = 25 in each selection treatment). (b) Virus titre of the DCV populations 3 days post-infection in Wolbachia-free (black) and -infected (red) flies. Horizontal bold lines and dots indicate mean titres and values per biological replicate, respectively. Error bars are standard errors. (Online version in colour.)