Sustained Wolbachia-mediated blocking of dengue virus isolates following serial passage in Aedes aegypti cell culture

Abstract

Wolbachia is an intracellular endosymbiont of insects that inhibits the replication of a range of pathogens in its arthropod hosts. The release of Wolbachia into wild populations of mosquitoes is an innovative biocontrol effort to suppress the transmission of arthropod-borne viruses (arboviruses) to humans, most notably dengue virus. The success of the Wolbachia-based approach hinges upon the stable persistence of the 'pathogen blocking' effect, whose mechanistic basis is poorly understood. Evidence suggests that Wolbachia may affect viral replication via a combination of competition for host resources and activation of host immunity. The evolution of resistance against Wolbachia and pathogen blocking in the mosquito or the virus could reduce the public health impact of the symbiont releases. Here, we investigate if dengue 3 virus (DENV-3) is capable of accumulating adaptive mutations that improve its replicative capacity during serial passage in Wolbachia wMel-infected cells. During the passaging regime, viral isolates in Wolbachia-infected cells exhibited greater variation in viral loads compared to controls. The viral loads of these isolates declined rapidly during passaging due to the blocking effects of Wolbachia carriage, with several being lost all together and the remainder recovering to low but stable levels. We attempted to sequence the genomes of the surviving passaged isolates but, given their low abundance, were unable to obtain sufficient depth of coverage for evolutionary analysis. In contrast, viral loads in Wolbachia-free control cells were consistently high during passaging. The surviving isolates passaged in the presence of Wolbachia exhibited a reduced ability to replicate even in Wolbachia-free cells. These experiments demonstrate the challenge for dengue in evolving resistance to Wolbachia-mediated blocking.

Keywords: Aedes aegypti; Wolbachia; dengue virus; evolution.

Figure 1.

Sequential passaging of twelve DENV-3 isolates in Aag-2, Aag-2Tet, and Aag-2wMel cells. (A) Log₁₀ transformed viral genome copies of the twelve isolates at every passage, as measured via qRT-PCR. Blue-themed circles denote Aag-2-passaged isolates (1–3). Green-themed squares denote Aag-2Tet-passaged isolates (4–6). Red-themed triangles denote Aag-2wMel-passaged isolates (7–12). Data points at 0 log₁₀ copies/µl indicate that viral loads were below detection limits, which are at ten genome copies/µl (dotted line). (B) Log₁₀ transformed viral genome copies in three cell lines inoculated with P₀ to verify Wolbachia-induced blocking phenotype at five days post-inoculation. Mean and SEM are shown in graphs (n = 5 per cell line). (C) Wolbachia densities in Aag-2wMel cells at every passage measured as the ratio of Wolbachia gene WD0513 to mosquito gene rps17.

Figure 2.

Replicative fitness assay of the nine remaining isolates following sequential passaging. Log₁₀ transformed viral genome copy numbers in the supernatants of (A) Aag-2, (B) Aag-2Tet, and (C) Aag-2wMel cells were quantified via qRT-PCR at six days post-inoculation by each isolate. Each data point represents one biological replicate. Data points at 0 log₁₀ copies/µl indicate that viral copies were below detection limits at ten genome copies/µl (dotted line) and were excluded from data analysis. Mean and SEM are shown in graphs (n = 5 per isolate).