Agua Salud Alphavirus Infection, Dissemination and Transmission in Aedes aegypti Mosquitoes

Abstract

Mosquitoes are competent vectors for many important arthropod-borne viruses (arboviruses). In addition to arboviruses, insect-specific viruses (ISV) have also been discovered in mosquitoes. ISVs are viruses that replicate in insect hosts but are unable to infect and replicate in vertebrates. They have been shown to interfere with arbovirus replication in some cases. Despite the increase in studies on ISV-arbovirus interactions, ISV interactions with their hosts and how they are maintained in nature are still not well understood. In the present study, we investigated the infection and dissemination of the Agua Salud alphavirus (ASALV) in the important mosquito vector Aedes aegypti through different infection routes (per oral infection, intrathoracic injection) and its transmission. We show here that ASALV infects the female Ae. aegypti and replicates when mosquitoes are infected intrathoracically or orally. ASALV disseminated to different tissues, including the midgut, salivary glands and ovaries. However, we observed a higher virus load in the brain than in the salivary glands and carcasses, suggesting a tropism towards brain tissues. Our results show that ASALV is transmitted horizontally during adult and larval stages, although we did not observe vertical transmission. Understanding ISV infection and dissemination dynamics in Ae. aegypti and their transmission routes could help the use of ISVs as an arbovirus control strategy in the future.

Keywords: Aedes aegypti; Agua Salud alphavirus; horizontal transmission; insect-specific viruses.

Figure 1

ASALV infection rate (A) and load (B) in female Ae aegypti mosquitoes following intrathoracic injection and oral infection. Adult female mosquitoes (7–13 days old) were fed or injected with ASALV and RNA was isolated from the collected mosquitoes at 0, 7 and 14 dpi. ASALV was quantified using the qRT-PCR infection rate (A), or genome copy numbers (B) were calculated using corresponding standard curves. Each data point represents individual mosquitoes (whole bodies). Error bars indicate 95% confidence intervals for the infection rate, n = sample size.

Figure 2

ASALV-specific small RNA production in the Ae. aegypti female midgut. Adult female Ae. aegypti mosquitoes were fed with ASALV, and the midgut of 15 female mosquitoes was pooled at 14 dpi. Size distribution of small RNAs mapping to the ASALV genome (white) or antigenome (grey) (A). Distribution of the 21 nt small RNAs along the ASALV genome (white) and antigenome (grey) (B).

Figure 3

ASALV dissemination to different tissues in ASALV-fed or injected adult Ae. aegypti females. The RNA of different tissue pools (15 mosquitoes/pool), dissected at 7 and 14 dpi, was isolated and the ASALV load was determined by qPCR (A). RNA of brain tissue, salivary glands (sg) and carcass pools (5 mosquitoes/pool) were isolated from ASALV-injected mosquitoes at 7 dpi (B). Six pools per tissue were analysed for ASALV load in the carcass, salivary gland and brain in ASALV-injected Ae. aegypti females. ASALV load was determined by qPCR and relative ASALV quantification using the ribosomal S7 gene as a housekeeping gene. ***: p < 0.001. Each data point represents dissected tissue pools consisting of 15 (A) or 5 (B) mosquitoes.

Figure 4

ASALV can be horizontally transmitted from female to male Ae. aegypti mosquitoes. ASALV-injected females were kept with uninfected males for 7 days. ASALV genome copy numbers were quantified in female and male mosquitoes using qRT-PCR. The results represent the combined data of two independent repeats. Each data point represents an individual mosquito (whole body sample).