RNA interference is essential to modulating the pathogenesis of mosquito-borne viruses in the yellow fever mosquito Aedes aegypti.

While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.

Antiviral Immunity and Virus-Mediated Antagonism in Disease Vector Mosquitoes.

More than 100 pathogens, spanning multiple virus families, broadly termed 'arthropod-borne viruses (arboviruses)' have been associated with human and/or animal diseases. These viruses persist in nature through transmission cycles that involve alternating replication in susceptible vertebrate and invertebrate hosts. Collectively, these viruses are among the greatest burdens to global health, due to their widespread prevalence, and the severe morbidity and mortality they cause in human and animal hosts. Specific examples of mosquito-borne pathogens include Zika virus (ZIKV), West Nile virus (WNV), dengue virus serotypes 1-4 (DENV 1-4), Japanese encephalitis virus (JEV), yellow fever virus (YFV), chikungunya virus (CHIKV), and Rift Valley fever virus (RVFV). Interactions between arboviruses and the immune pathways of vertebrate hosts have been extensively reviewed. In this review we focus on the antiviral immune pathways present in mosquitoes. We also discuss mechanisms by which mosquito-borne viruses may antagonize antiviral pathways in disease vectors. Finally, we elaborate on the possibility that mosquito-borne viruses may be engaged in an evolutionary arms race with their invertebrate vector hosts, and the possible implications of this for understanding the transmission of mosquito-borne viruses.

Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

Temperature-dependent effects on the replication and transmission of arthropod-borne viruses in their insect hosts.

The transmissibility of vector borne viruses can be affected by a combination of factors, both extrinsic (climatic changes, temperature, urbanization, among others) and intrinsic (genetics, life span, immunity, among others). Temperature is of particular importance since the insect vectors of arthropod-borne viruses (arboviruses) are ectothermic and acutely susceptible to temperature changes. Modeling suggests that with increasing global temperature and urbanization, arboviral diseases will continue to emerge or reemerge. This review highlights current literature regarding temperature-dependent effects on virus-vector interactions and their potential to influence the transmission dynamics and epidemiology of arboviral diseases.

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

Mosquito-borne viruses and suppressors of invertebrate antiviral RNA silencing.

The natural maintenance cycles of many mosquito-borne viruses require establishment of persistent non-lethal infections in the invertebrate host. While the mechanisms by which this occurs are not well understood, antiviral responses directed by small RNAs are important in modulating the pathogenesis of viral infections in disease vector mosquitoes. In yet another example of an evolutionary arms race between host and pathogen, some plant and insect viruses have evolved to encode suppressors of RNA silencing (VSRs). Whether or not mosquito-borne viral pathogens encode VSRs has been the subject of debate. While at first there would seem to be little evolutionary benefit to mosquito-borne viruses encoding proteins or sequences that strongly interfere with RNA silencing, we present here a model explaining how the expression of VSRs by these viruses in the vector might be compatible with the establishment of persistence. We also discuss the challenges associated with interrogating these viruses for the presence of suppressor proteins or sequences, as well as the candidates that have been identified in the genomes of mosquito-borne pathogens thus far.

Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

BACKGROUND: The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus), exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi) pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery. METHODOLOGY/PRINCIPAL FINDINGS: We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2) or Argonaute-2 (AGO-2). We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses. CONCLUSIONS/SIGNIFICANCE: This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

Acid sphingomyelinase deficiency contributes to resistance of scleroderma fibroblasts to Fas-mediated apoptosis.

BACKGROUND: Scleroderma (SSc) is characterized by excess production and deposition of extracellular matrix (ECM) proteins. Activated fibroblasts play a key role in fibrosis in SSc and are resistant to Fas-mediated apoptosis. Acid sphingomyelinase (ASMase), a major sphingolipid enzyme, plays an important role in the Fas-mediated apoptosis. OBJECTIVE: We investigated whether dysregulation of ASMase contributes to Fas-mediated apoptosis resistance in SSc fibroblasts. METHODS: Fibroblasts were isolated from SSc patients and healthy controls. Western blot was performed to analyze protein levels and quantitative real time RT-PCR was used to determine mRNA expression. Cells were transiently transfected with siRNA oligos against ASMase or transduced with adenoviruses overexpressing ASMase. Apoptosis was induced using anti-Fas antibody (1 µg/mL) and analyzed using caspase-3 antibody or Cell Death Detection ELISA. RESULTS: SSc fibroblasts showed increased resistance to Fas-mediated apoptosis. ASMase expression was decreased in SSc fibroblasts and Transforming Growth Factor beta (TGFß), the major fibrogenic cytokine involved in the pathogenesis of SSc, downregulated ASMase in normal fibroblasts. Forced expression of ASMase in SSc fibroblasts restored sensitivity of these cells to Fas-mediated apoptosis while blockade of ASMase was sufficient to induce partial resistance to Fas-induced apoptosis in normal fibroblasts. In addition, ASMase blockade decreased activity of protein phosphatase 2A (PP2A) through phosphorylation on Tyr(307) and resulted in activation of extracellular regulated kinase 1/2 (Erk1/2) and protein kinase B (Akt/PKB). CONCLUSION: In conclusion, this study suggests that ASMase deficiency promotes apoptosis resistance and contributes to activation of profibrotic signaling in SSc fibroblasts.