Production of Infectious Dengue Virus in <i>Aedes aegypti</i> Is Dependent on the Ubiquitin Proteasome Pathway

<div><p>Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of <i>Aedes aegypti</i> and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show <i>in vivo</i> that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.</p></div

Proteasome inhibition of β2 and β5 subunits decouples infectious DENV-2 production from viral RNA replication in mosquito midguts.

<p>(A) Silencing efficiencies of β subunits of the proteasome were determined by gene specific qPCR, and expression values were normalized against dsRNA control targeting random sequences from pGEM T easy vector 10 days after dsRNA inoculation. N = 10. (B) No statistically significant difference was observed in virus titer per midgut at 6 dpbm after knockdown of β1, β2 and β5 subunits. Mean ± SEM, N = 7–16. (C) No statistically significant differences were observed in DENV2 viral RNA levels per midgut 6 dpbm after β1, β2 and β5 subunits knockdown. Mean ± SEM, N = 7–16. (D) log(PFU/Copy Number) was significantly lower after β2 and β5 knockdown. Mean ± SEM, N = 20–22. Student’s t test, **p<0.01.</p

Percentage of DENV2-infected mosquitoes after knockdown of proteasome subunits (p value; Fischer’s Exact Test).

<p>Percentage of DENV2-infected mosquitoes after knockdown of proteasome subunits (p value; Fischer’s Exact Test).</p

Knockdown of UBE2A and DDB1 decouples infectious DENV2 production from viral RNA replication in mosquitoes.

<p>(A) In the infected midguts, virus titers declined significantly after knockdown of UBE2A and DDB1 at 6 dpbm. N = 12–16. Student’s t test, *p < 0.05. (B) In the infected midguts, no statistically significant differences were observed in DENV2 viral RNA levels 6 dpbm after gene knockdown. N = 12–16. (C) Ratio of midgut infectious titers to viral RNA levels 6 dpbm after gene knockdown. N = 12–16. Student’s t test, *p < 0.05. (D) In infected heads/thoraces (HT), virus titers at 8 days post intra-thoracic inoculation declined significantly after knockdown of UBE2A and DDB1. N = 8–10. Student’s t test, **p < 0.01, ***p<0.001. (E) In infected heads/thoraces (HT), no statistically significant differences were observed in DENV2 viral RNA levels after gene knockdown. N = 8–10. (F) Ratio of head/thorax (HT) infectious titers to viral RNA levels after gene knockdown 8 dpi. N = 12–16. Student’s t test, **p<0.01.</p

Characterization of DENV-2 replication in the midguts and heads/thoraces of <i>Ae</i>. <i>aegypti</i> following ingestion of an infectious blood meal.

<p>(A) In the midgut, viral titers increased linearly until 8 dpbm and declined thereafter. In contrast, viral RNA remained stable between 8 to 21 dpbm. Mean ± SEM, N = 8–10. (B) In the heads/thoraces (HT), the increase in both infectious particles and viral RNA are positively correlated over time. Viral RNA copy number increases with increasing viral titers. Mean ± SEM, N = 8–10. (C-D) A corresponding decrease in PFU/Copy number was observed in the midgut over time, with no significant change in the head/thorax (HT).</p

Ingestion of blood meal does not modulate gene expression of <i>UBE2A</i> and <i>DDB1</i>.

<p>Sugar fed and blood fed mosquitoes were harvested at different time-points and individual midguts were dissected for analyses. Gene expression levels for (A) <i>UBE2A</i> and (B) <i>DDB1</i> were measured using qRT-PCR and normalized to GAPDH. Expression levels of <i>UBE2A</i> and <i>DDB1</i> in blood fed mosquitoes remained consistently unchanged over the course of 14 days, whereas ingestion of a sugar meal increases expression levels of <i>UBE2A</i> and <i>DDB1</i> significantly until 8 days relative to the blood fed mosquitoes. Mean ± SEM. N = 12. Student’s t test, **p < 0.01, ***p<0.001, ****p<0.0001.</p

Flowchart showing numbers of participants in both intervention and control groups at all stages of the trial.

<p>Flowchart showing numbers of participants in both intervention and control groups at all stages of the trial.</p

Proportions of dengue infections in 1,655 Thai school children with paired serum samples at 10 schools randomised to insecticide-treated school uniforms (intervention) or untreated school uniforms (control).

<p>Proportions of dengue infections in 1,655 Thai school children with paired serum samples at 10 schools randomised to insecticide-treated school uniforms (intervention) or untreated school uniforms (control).</p

Existing and developing control methods.

<p>Existing methods (upper green region) and methods under development (lower yellow region) are enumerated and separated by those that affect larval mosquito stages (left) and those that affect adult mosquito stages (right). Methods that target a particular sub-stage within a mosquito’s life cycle are oriented vertically with those sub-stages.</p

Approaches to Refining Estimates of Global Burden and Economics of Dengue

<div><p>Dengue presents a formidable and growing global economic and disease burden, with around half the world's population estimated to be at risk of infection. There is wide variation and substantial uncertainty in current estimates of dengue disease burden and, consequently, on economic burden estimates. Dengue disease varies across time, geography and persons affected. Variations in the transmission of four different viruses and interactions among vector density and host's immune status, age, pre-existing medical conditions, all contribute to the disease's complexity. This systematic review aims to identify and examine estimates of dengue disease burden and costs, discuss major sources of uncertainty, and suggest next steps to improve estimates. Economic analysis of dengue is mainly concerned with costs of illness, particularly in estimating total episodes of symptomatic dengue. However, national dengue disease reporting systems show a great diversity in design and implementation, hindering accurate global estimates of dengue episodes and country comparisons. A combination of immediate, short-, and long-term strategies could substantially improve estimates of disease and, consequently, of economic burden of dengue. Suggestions for immediate implementation include refining analysis of currently available data to adjust reported episodes and expanding data collection in empirical studies, such as documenting the number of ambulatory visits before and after hospitalization and including breakdowns by age. Short-term recommendations include merging multiple data sources, such as cohort and surveillance data to evaluate the accuracy of reporting rates (by health sector, treatment, severity, etc.), and using covariates to extrapolate dengue incidence to locations with no or limited reporting. Long-term efforts aim at strengthening capacity to document dengue transmission using serological methods to systematically analyze and relate to epidemiologic data. As promising tools for diagnosis, vaccination, vector control, and treatment are being developed, these recommended steps should improve objective, systematic measures of dengue burden to strengthen health policy decisions.</p></div