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

Yanhai Wang,Xiaoguang Chen,Peiwen Liu,Jinbao Gu,Jing Li,Binbin Jin

doi:10.3390/v10040213

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.

Highlights

Small RNA regulatory pathways (SRRPs) control essential aspects of antiviral immune responses to arboviruses in mosquitoes
Results based on RNA-seq (Figure 10A) and reverse-transcription PCR (RT-PCR) (Figure 10B) analyses indicated that transcripts of PIWI1–7 and Argonaute 3 (Ago3)(1–2) are readily detected in Ae. albopitus adult females, whereas transcripts of PIWI1–7 and Ago3(1–2) are readily detected in Ae. albopitus adult females, whereas
PIWI8 and 9 transcripts were not detected in both the midgut and mosquito whole-body using RT-PCR and RNA-Seq analyses, so we continued to the midgut and mosquito whole-body using RT-PCR and RNA-Seq analyses, so we continued to explore the temporal expression patterns of P-element induced wimpy testis (PIWI) genes further using the RT-PCR method

Summary

Introduction

Small RNA (sRNA) regulatory pathways (SRRPs) control essential aspects of antiviral immune responses to arboviruses in mosquitoes. Viruses 2018, 10, 213; doi:10.3390/v10040213 www.mdpi.com/journal/viruses (siRNA), endogenous microRNA (miRNA), and P-element induced wimpy testis (PIWI)-mediated transcriptional silencing pathways, which present in mosquitoes [1,2]. 2 (Ago2)-dependent RNA-induced silencing complex (RISC), leading to complementary mRNA degradation. In contrast to siRNAs, miRNAs are processed from long endogenous RNA transcripts, namely, primary miRNA transcripts (pri-miRNA), which are recognized and cleaved into precursor miRNAs (pre-miRNAs) in the nucleus through a microprocessor-complex composed of the ribonuclease III (RNase III) enzymes, Drosha and Pasha (partner of Drosha) [3]. Mature miRNAs are loaded onto an effector Ago1-dependent RISC and guide the translational repression and the degradation of target transcripts [5]. PIWI-interacting RNA (piRNA)-mediated post-transcriptional regulation has been recently indicated to involve an antiviral defense mechanism [6]

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