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. 2015 Jan 22;9(1):e0003386.
doi: 10.1371/journal.pntd.0003386. eCollection 2015 Jan.

Discovery of mosquito saliva microRNAs during CHIKV infection

Payal D Maharaj  1 , Steven G Widen  2 , Jing Huang  1 , Thomas G Wood  2 , Saravanan Thangamani  3
Affiliations

Discovery of mosquito saliva microRNAs during CHIKV infection

Payal D Maharaj et al. PLoS Negl Trop Dis. .

Abstract

Mosquito borne pathogens are transmitted to humans via saliva during blood feeding. Mosquito saliva is a complex concoction of many secretory factors that modulate the feeding foci to enhance pathogen infection and establishment. Multiple salivary proteins/factors have been identified/characterized that enhance pathogen infection. Here, we describe, for the first time, the identification of exogenous microRNAs from mosquito saliva. MicroRNAs are short, 18-24 nucleotide, non-coding RNAs that regulate gene expression, and are generally intracellular. However, circulating miRNAs have been described from serum and saliva of humans. Exogenous miRNAs have not been reported from hematophagous arthropod saliva. We sought to identify miRNAs in the mosquito saliva and their role in Chikungunya virus (CHIKV) infection. Next generation sequencing was utilized to identify 103 exogenous miRNAs in mosquito saliva of which 31 miRNAs were previously unidentified and were designated novel. Several miRNAs that we have identified are expressed only in the CHIKV infected mosquitoes. Five of the saliva miRNAs were tested for their potential to regulated CHIKV infection, and our results demonstrate their functional role in the transmission and establishment of infection during blood feeding on the host.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Identification of microRNAs in Aedes spp. saliva.
Aedes aegypti and Aedes albopictus were intra-thoracically infected with Chikungunya virus. At 10 days post infection, saliva was collected from both infected and uninfected mosquitoes. Small RNAs were extracted from the saliva and subjected to deep sequencing, small RNA libraries were created and mapped to Ae.aegypti and An.gambiae miRNA databases. Figure a) size distribution and percentages of small RNAs in Ae.aegypti saliva, b) percentages of 18–24 nucleotide microRNAs in Ae.aegypti saliva library, c) size distribution and percentages of small RNAs in Ae. albopictus saliva, d) percentages of 18–24 nucleotide microRNAs in Ae. albopictus saliva library.
Figure 2
Figure 2. Saliva microRNAs regulate CHIKV replication in mosquito and mammalian cells.
Mosquito (AAG-2 and C6/36) and mammalian (BHK-21) cells were transfected with miRNA inhibitors, a) MIR-12, b) MIR-125, c) MIR-184, d) MIR-375 and e) MIR-2940, and then infected with CHIKV at 72 hours post-transfection. Supernatant was collected daily for 72 hours and viral titers for each timepoint were determined via standard plaque assay on Vero cells.
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