piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues

Abstract

The Asian tiger mosquito, Aedes albopictus, is a competent vector for the majority of arboviruses. The mosquito innate immune response is a primary determinant for arthropod-borne virus transmission, and the midgut is the first barrier to pathogen transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. However, the roles that the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway play in antiviral immunity in Ae. albopictus and its midgut still need further exploration. This study aimed to explore the profiles of both viral-derived and host-originated piRNAs in the whole body and midgut infected with Dengue virus 2 (DENV-2) in Ae. albopictus, and to elucidate gene expression profile differences of the PIWI protein family between adult females and their midguts. A deep sequencing-based method was used to identify and analyze small non-coding RNAs, especially the piRNA profiles in DENV-2-infected Ae. albopictus and its midgut. The top-ranked, differentially-expressed piRNAs were further validated using Stem-loop qRT-PCR. Bioinformatics analyses and reverse-transcription PCR (RT-PCR) methods were used to detect PIWI protein family members, and their expression profiles. DENV-2 derived piRNAs (vpiRNA, 24⁻30 nts) were observed in both infected Ae. albopictus and its midgut; however, only vpiRNA in the whole-body library had a weak preference for adenine at position 10 (10A) in the sense molecules as a feature of secondary piRNA. These vpiRNAs were not equally distributed, instead they were derived from a few specific regions of the genome, especially several hot spots, and displayed an obvious positive strand bias. We refer to the differentially expressed host piRNAs after DENV infection as virus-induced host endogenous piRNAs (vepiRNAs). However, we found that vepiRNAs were abundant in mosquito whole-body tissue, but deficient in the midgut. A total of eleven PIWI family genes were identified in Ae. albopictus; however, only AalPiwi5⁻7 and AalAgo3(1⁻2) were readily detected in the midgut. The characteristics of piRNAs in DENV-2-infected Ae. albopictus adult females were similar to those previously described for flavivirus infections but were not observed in the midgut. The reduced levels of vepiRNAs and incomplete expression of PIWI pathway genes in midgut samples from DENV-2-infected Ae. albopictus suggests that viral regulation of host piRNAs may not be an important factor in the midgut.

Keywords: Aedes albopictus; Dengue virus; antiviral immunity; midgut; piRNA.

Figure 1

The nucleotide length distribution of sequence tags obtained from the four Aedes albopictus small RNA (sRNA) libraries. Size distribution and relative frequency in each sample are shown for the small RNAs derived from (A) uninfected adult females and dengue virus type-2 (DENV-2)-infected adult females; and (B) midguts from uninfected and DENV-2-infected adult females. Abbreviations: nt, length of small RNA read in nucleotides.

Figure 2

Virus-derived small RNAs (vsRNAs) size distribution varies among dengue virus 2 (DENV-2)-infected adult females and midguts from DENV-2-infected adult females. Size distribution and relative frequency in each sample are shown for the DENV-2-specific small RNAs (vsRNA) derived from (A) DENV-2-infected adult females and (B) midguts from DENV-2-infected adult females. Abbreviations: nt, length of small RNA read in nucleotides. Note the logarithmic scale on the Y-axes.

Figure 3

Characterization of virus-derived small RNAs (vsRNAs). (A) vsRNA strand preference of DENV-2-infected adult females and midguts from DENV-2-infected adult females; (B) Abundances of three classes of DENV-2 derived vsRNA from DENV-2-infected adult females and midguts from DENV-2-infected adult females. Mean vsRNA distribution by sRNA size group.

Figure 4

Frequency map of the distance between 13–30 nt virus-derived small RNAs (vsRNAs) that mapped to strands of the dengue virus 2 (DENV-2) genome. (A) DENV-2-infected adult females; (B) midguts from DENV-2-infected adult females. vsRNAs that mapped to the positive and negative strands of the DENV-2 genome are shown in blue and red, respectively. The organization of the DENV-2 genome is shown in the lower panel.

Figure 5

The virus derived piRNAs (vpiRNA) genome coverage distribution in Dengue virus 2 (DENV-2)-infected Aedes albopictus and midgut. (A) DENV-2-infected adult females; (B) midguts from DENV-2-infected adult females. vsRNAs that mapped to the positive and negative strand of the DENV-2genome are shown in blue and red, respectively. Organization of the DENV-2 genome is shown in the lower panel.

Figure 6

Characterization of virus derived P-element induced wimpy testis (PIWI)-interacting RNA (piRNAs) (vpiRNA). The upper panel (A,B) shows the base composition of repeat-derived piRNAs of DENV-2-infected adult females and DENV-2-infected female midguts. The X-axis represents the nucleotide positions relative to the 5′ ends of the piRNAs. The Y-axis represents the percentage of base bias. The lower panel (C,D) shows ping-pong pair analyses of DENV-2-infected adult females and DENV-2-infected female midguts. The length of overlap is shown on the horizontal axes. Indicated above each axis is the number of possible overlapping pairs of small RNAs with the specified overlap size. Indicated below each axis is the relative frequency of the 5′ base identity for overlapping sequences. The color code for the bases is indicated in the centre box.

Figure 7

Quantification of DENV-2 infection induced host endogenous differentially expressed piRNAs (vepiRNAs) by stem–loop RT–PCR. (A) quantification of DENV-2 infection induced upregulated vepiRNA expression level in DENV-2 infected adult females; (B) quantification of DENV-2 infection induced downregulated vepiRNA expression level in DENV-2 infected adult females; (C) quantification of DENV-2 infection induced downregulated vepiRNA expression level in midguts from DENV-2 infected adult females; (D) The PCR products were run on 3% agarose gel in 1X TBE stained with ethidium bromide. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 8

Quantification of DENV-2 derived piRNAs (vpiRNA) by stem-loop RT-PCR. (A) quantification of vpiRNA expression level in DENV-2 infected adult females; (B) quantification of vpiRNA expression level in midguts from DENV-2 infected adult females; (C) The PCR products were run on 3% agarose gel in 1X TBE stained with ethidium bromide. Note: vpiRNA2191731, vpiRNA2191732, vpiRNA0705792, vpiRNA0705793, vpiRNA3130226 and vpiRNA0705787 differed by only a few nucleotide base lengths at the 5′ and 3′ ends. A similar pattern was also found in vpiRNA0676933 and vpiRNA1747127. **: p < 0.01; ***: p < 0.001.

Figure 9

Phylogenetic and molecular evolutionary analyses of PIWI protein family members of mosquitoes. Bootstrap values for 1000 replicate analyses are shown at the branching points. The bar at the bottom indicates the branch length, corresponding to the mean number of differences (0.05) per residue along each branch. Gene ID refers to Table S7.

Figure 10

Expression profiles of PIWI protein family members of Aedes albopictus. (A) RNA-seq expression profile. tBLASTx was performed against Ae. albopictus and midgut RNA-seq data spanning serial time points post-DENV-2 infection (SRP077936). Counts were normalized to the number of million reads per sample; (B) RT-PCR spatial expression analysis of PIWI protein family member genes. Ae. albopictus Rps7 was used as an internal control. The analyses were performed on the following samples: 0C: uninfected carcass; 0Mg: uninfected midgut; 1MgC: control midgut at 1 dpi; 1MgD: DENV-infected midgut at 1 dpi; 1CC: control carcass at 1 dpi; 1CD: DENV-infected carcass at 1 dpi; 5MgC: control midgut at 5 dpi; 5MgD: DENV-infected midgut at 5 dpi; 5CC: control carcass at 5 dpi; 5CD: DENV-infected carcass at 5 dpi.