Ixodes scapularis salivary gland microRNAs are differentially expressed during Powassan virus transmission

Abstract

Successful tick feeding is facilitated by an assortment of pharmacologically-active factors in tick saliva that create an immunologically privileged micro-environment in the host's skin. Through a process known as saliva-assisted transmission, bioactive tick salivary factors modulate the host environment, promoting transmission and establishment of a tick-borne pathogen. This phenomenon was previously demonstrated for Powassan virus (POWV), a North American tick-borne flavivirus that is the causative agent of a severe neuroinvasive disease in humans. Here, we sought to characterize the Ixodes scapularis salivary gland microRNAs (miRNAs) expressed during the earliest period of POWV transmission to a mammalian host. POWV-infected and uninfected I. scapularis females were fed on naïve mice for 1, 3, and 6 hours, and Illumina next generation sequencing was used to characterize the salivary gland miRNA expression profiles of POWV-infected versus uninfected ticks. 379 salivary miRNAs were detected, of which 338 are reported here as putative novel I. scapularis miRNAs. 35 salivary gland miRNAs were significantly up-regulated and 17 miRNAs were significantly down-regulated in response to POWV infection. To investigate the potential role of salivary gland miRNAs in POWV replication in-vitro, we transfected miRNA inhibitors into VeroE6 cells to profile temporal POWV replication in mammalian cells. Together, the small RNA sequencing data and the in vitro miRNA inhibition assay suggest that the differentially expressed tick salivary miRNAs could act in regulating POWV replication in host tissues.

Figure 1

Summary of next generation sequencing of tick-derived small RNAs obtained from POWV-infected and uninfected Ixodes scapularis salivary glands at 1 hour, 3 hours, and 6 hours post tick attachment. Salivary glands were dissected from POWV-infected female I. scapularis ticks and uninfected female ticks that were fed on mice for 1, 3, or 6 hours. RNA was extracted from the salivary glands, and 21 small RNA libraries were generated then subjected to Illumina small RNA sequencing. (a–c) Size distribution of tick-derived small RNA reads with passing filters mapped to the I. scapularis genome. (d–f) Summary of tick-derived reads that match various small RNA categories from POWV-infected tick salivary glands at 1 hour, 3 hours, and 6 hours post tick attachment. (g–i) Summary of tick-derived reads that match various small RNA categories from uninfected tick salivary glands at 1 hour, 3 hours, and 6 hours post tick attachment.

Figure 2

Size distributions of virus-derived small RNAs. Size distribution of mappable reads to the POWV genome (POWV LB Strain, NCBI Reference Sequence: NC_003687.1) in small RNA libraries of POWV-infected female Ixodes scapularis tick salivary glands after (a) 1 hour, (b) 3 hours, and (c) 6 hours of tick attachment.

Figure 3

Predicted stem-loop structures of highly expressed microRNAs. The miRDeep2 software was used to identify potential miRNA precursors based on nucleotide length, star sequence, stem-loop folding, and homology to the I. scapularis reference genome. Figures (a) isc-miR-5307 (b) nDS978597_16878 and (c) nDS625977_65388 show the predicted stem-loop structures, star, and mature sequences of highly expressed miRNAs in I. scapularis salivary glands.

Figure 4

Overlap between the top 50 most abundant miRNAs expressed in POWV-infected 1 hour, 3 hour, and 6 hour fed Ixodes scapularis salivary glands, and uninfected 1 hour, 3 hour, and 6 hour fed I. scapularis salivary glands. In each experimental group, normalized read counts were totaled for every miRNA to generate a list of the top 50 most abundant miRNAs. The online tool, InteractiVenn, was used to generate an Edwards Venn diagram.

Figure 5

Ixodes scapularis salivary gland miRNAs with significant differential expression in response to POWV infection at a minimum of 1 time point. miRNAs with a Log₂ fold-change expression > |1| and FDR ≤ 0.1 were considered significantly differentially expressed. Values highlighted in red indicate significant up-regulation and values highlighted in green indicate significant down-regulation.

Figure 6

Overlap between the significantly up- and down-regulated Ixodes scapularis salivary gland miRNAs in response to POWV infection. The Venn diagrams show the overlap between the significantly up-regulated (a) or significantly down-regulated (b) salivary gland miRNAs after 1, 3, and 6 hours of POWV-infected tick feeding. miRNAs with a Log₂ fold-change expression > |1| and FDR ≤ 0.1 are considered significantly differentially expressed. Numbers in parenthesis indicate the total number of miRNAs with significant differential expression at a given time point.

Figure 7

qPCR validation of select mature miRNAs that are differentially expressed in response to POWV infection. Each graph depicts the differential expression (Log₂ fold change) of a select miRNA from POWV-infected salivary glands versus uninfected salivary glands based on NGS data (shown in orange) and qRT-PCR data (shown in blue). Significance for qRT-PCR-based differential expression is determined by the 2-tailed Student’s t test where *P < 0.05, **P < 0.01, ***P < 0.001. Significance for NGS-based differential expression is determined by a DESeq2-derived adjusted P-value (P-adj) where ^#P-adj < 0.1, ^##P-adj < 0.01, ^###P-adj < 0.001.

Figure 8

Ixodes scapularis saliva miRNAs regulate POWV replication in mammalian cells. VeroE6 cells were transfected with a miRNA inhibitor or mock-transfected with Lipofectamine only. 24 hours post-transfection, cells were infected with POWV (MOI = 0.05). Supernatant was collected daily for 96 hours and viral titers for each timepoint were determined via quantitative real-time PCR analysis. A 2-tailed Student’s T-test was used to analyze the significance of POWV titer differences between miRNA-transfected versus mock-transfected VeroE6 cells at each time point where *P < 0.05, **P < 0.01, ***P < 0.001.