Transcriptomic and small RNA response to Mayaro virus infection in Anopheles stephensi mosquitoes

Abstract

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naïve regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint shows a number of proteases regulated at 2 and 7 dpi, potentially representative of Toll or melanization pathway activation, and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.

Fig 1. Principal Components Analysis (PCA) on filtered read counts mapping to annotated genes from the AsteI2 build of the An. stephensi genome in Vectorbase.

A., B., and C. are read counts from samples in the 2, 7, and 14 dpi groupings respectively. In all PCAs, blue is Mayaro infected, and red are control.

Fig 2. Volcano plots visualizing differential expression of An. stephensi transcripts in response to Mayaro infection.

The Y-axis shows -log10 transformed P-values, and the X-axis shows log2 transformed fold change values. Red points represent transcripts depleted by more than -1 log2FC in response to infection with a FDR < 0.05, while blue points are transcripts enriched by more than 1 log2FC in response to infection with a P value < 0.05. A.—C. are transcripts regulated in the 2 dpi, 7 dpi, and 14 dpi groupings respectively, while D. and E. are transcripts regulated in the infected treatment between 2–7 dpi and 7–14 dpi respectively.

Fig 3. Venn diagrams representing the number of shared transcripts differentially expressed at 2, 7, and 14 dpi.

A. represents the repressed transcripts and B. represents the enriched transcripts.

Fig 4. Results of g:Profiler analysis on differentially expressed transcripts in each contrast considered.

Y-axis is -log10 transformed P values, and X-axis are GO categories for molecular function (MF), biological process (BP), and cellular component (CC) with the number of significant categories with FDR < 0.05 in parenthesis next to each category. Points that are transparent are those that are not significant with FDR > 0.05. Displayed are 2 dpi (A.), 7 dpi (B.), 14 dpi (C.), 2 to 7 dpi (D.), and 7 to 14 dpi (E.).

Fig 5. The top histogram represents the number of miRNAs shared between treatments (intersection size), and each row below the histogram represents a treatment.

The lines connecting treatments below the top histogram represent treatments which share that number of miRNAs, and the histogram to the side of the treatments represents the number of miRNAs contained within each treatment. V denotes a viral sample, C denotes a control, D2, D7, and D14 represent 2, 7, and 14 dpi respectively.

Fig 6. Principal Components Analysis (PCA) on read counts mapping to miRNAs identified in the AsteI2 build of the An. stephensi genome in Vectorbase.

A.—C. are the 2 dpi, 7 dpi, and 14 dpi groupings respectively. In all PCAs, blue is Mayaro infected, and red is control.

Fig 7. Volcano plots visualizing differential expression of identified An. stephensi miRNAs in response to Mayaro infection.

The Y-axis shows -log10 transformed P-values, and the X-axis shows log2 transformed fold change values. Red points represent transcripts depleted by more than -1 log2FC in response to infection with a FDR < 0.05, while blue points are transcripts enriched by more than 1 log2FC in response to infection with a FDR < 0.05. A.—C. are the 2 dpi, 7 dpi, and 14 dpi groupings respectively, while D. and E. are miRNAs regulated in the infected treatment between 2–7 dpi and 7–14 dpi respectively.

Fig 8. Histograms demonstrating read depth across the Mayaro virus (MAYV) genome for reads with a siRNA size profile (18-23nt) and a piRNA size profile (24–35 nt).

Y-axis is read depth in reads per million, and X-axis is position in viral genome. Blue color reads map to the negative strand while red color reads map to the positive strand. A.—C.: 2, 7 and 14 dpi respectively of control (left) and infected (right) time points. At the bottom is a map of the MAYV genome. D. Length distributions of the total small RNAs in An. stephensi days after the blood meal with control versus MAYV-infected blood. The asterisks mark an artificial RNA size marker present in these libraries. Bottom row are the length distributions of MAYV-specific small RNAs, with the inset showing the progressive decrease of piRNA-long reads and the arrows noting the increase in the siRNA proportion. E. Sequence logos showing the nucleotide base compositions of the MAYV-specific small RNAs, which are mostly siRNAs, over the different days post blood meal.