Aedes aegypti uses RNA interference in defense against Sindbis virus infection

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an important anti-viral defense mechanism. The <it>Aedes aegypti </it>genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use <it>Ae. aegypti </it>as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family <it>Togaviridae</it>, genus <it>Alphavirus</it>).</p> <p>Results</p> <p>SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate <it>Ago2</it>, <it>Dcr2</it>, or <it>TSN </it>mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut <it>Ago2</it>, <it>Dcr2</it>, and <it>TSN </it>transcript levels during infection revealed that only <it>TSN </it>transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.</p> <p>Conclusion</p> <p>We show that silencing RNAi components in <it>Ae. aegypti </it>results in transient increases in SINV replication. Furthermore, <it>Ae. aegypti </it>RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in <it>Ae. aegypti</it>.</p

A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is due in part to mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated Mosquito Small RNA Genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) composed of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of crosstalk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses

Human cellular restriction factors that target HIV-1 replication

Recent findings have highlighted roles played by innate cellular factors in restricting intracellular viral replication. In this review, we discuss in brief the activities of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G), bone marrow stromal cell antigen 2 (BST-2), cyclophilin A, tripartite motif protein 5 alpha (Trim5α), and cellular microRNAs as examples of host restriction factors that target HIV-1. We point to countermeasures encoded by HIV-1 for moderating the potency of these cellular restriction functions

Generation of Multiple Arbovirus-like Particles Using a Rapid Recombinant Vaccinia Virus Expression Platform

As demonstrated by the 2015 Zika virus outbreak in the Americas, emerging and re-emerging arboviruses are public health threats that warrant research investment for the development of effective prophylactics and therapeutics. Many arboviral diseases are underreported, neglected, or of low prevalence, yet they all have the potential to cause outbreaks of local and international concern. Here, we show the production of virus-like particles (VLPs) using a rapid and efficient recombinant vaccinia virus (VACV) expression system for five tick- and mosquito-borne arboviruses: Powassan virus (POWV), Heartland virus (HRTV), severe fever with thrombocytopenia syndrome virus (SFTSV), Bourbon virus (BRBV) and Mayaro virus (MAYV). We detected the expression of arbovirus genes of interest by Western blot and observed the expression of VLPs that resemble native virions under transmission electron microscopy. We were also able to improve the secretion of POWV VLPs by modifying the signal sequence within the capsid gene. This study describes the use of a rapid VACV platform for the production and purification of arbovirus VLPs that can be used as subunit or vectored vaccines, and provides insights into the selection of arbovirus genes for VLP formation and genetic modifications to improve VLP secretion and yield

Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Arboviruses can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important arboviruses, including dengue virus (DENV), Zika virus (ZIKV), Chikungunya (CHIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of DENV (as a model system for mosquito-borne viruses more generally) that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP)-the time taken for DENV virus to be transmissible after infection-is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicate that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field