Addition of palmitate restores RVFV infection in the presence of A769662.

<p><b>A.</b> U2OS cells were pretreated with 100 µM palmitate overnight and 100 µM A769662 or PBS was added 1 hour prior to infection with RVFV (MOI 1). Cells were incubated for 10 hours, and processed for immunofluorescence. (RVFV-N, green; nuclei, blue) <b>B.</b> Quantification of <b>A.</b> Data are displayed as the normalized percent infection relative to the untreated control at MOI 1.25±SD in triplicate experiments; * indicates p<0.05 compared to untreated vehicle control.</p

Additional arboviruses are restricted by AMPK.

<p>WT or AMPKα1/AMPKα2^−/− MEFs were infected with serial dilutions of KUNV (<b>A</b>), SINV (<b>E</b>), or VSV (<b>I</b>) and processed for immunofluorescence. (Virus, green; nuclei, blue). Quantifications of the percent infection for KUNV (<b>B</b>), SINV (<b>F</b>) and VSV (<b>J</b>) are shown as representatives of triplicate experiments. LKB1^−/−;LKB1 and LKB1^−/−;Vec MEFs were infected with serial dilutions of KUNV (<b>C</b>), SINV (<b>G</b>), and VSV (<b>K</b>) and processed for immunofluorescence. (Virus, green; nuclei, blue). Quantifications of the percent infection are shown for KUNV (<b>D</b>), SINV (<b>H</b>) and VSV (<b>L</b>) are shown as representatives of triplicate experiments.</p

AMPK restricts RVFV infection.

<p><b>A.</b> Plaque assays were performed on wild type (WT) and AMPKα1/AMPKα2^−/− MEFs. Representative data from triplicate experiments is shown. <b>B.</b> Quantification of plaques from <b>A.</b> presented as the normalized mean±SD relative to the number of wild type plaques from three experiments. <b>C.</b> The diameter of 30 representative plaques in each duplicate well from <b>A.</b> was used to calculate the average plaque area, displayed as the normalized mean+SD in triplicate experiments. <b>D.</b> WT or AMPKα1/AMPKα2^−/− MEFs were infected with serial dilutions of RVFV, incubated for 16 hours, and processed for immunofluorescence. (RVFV-N, green; nuclei, blue). <b>E.</b> Quantification of <b>D.</b> presented as percent of infected cells. A representative of three experiments is shown. <b>F.</b> One-step growth curve of RVFV in WT or AMPKα1/AMPKα2^−/− MEFs. RVFV grown in WT or AMPKα1/AMPKα2^−/− MEFs for 4, 8, or 12 hours was tittered on BHK cells and is presented as the normalized mean of triplicate experiments ±SD. * indicates p<0.05.</p

AMPK restricts RVFV RNA replication.

<p><b>A.</b> Northern blot of genomic S segment and N mRNA from RVFV (MOI 1) grown in WT or AMPKα1/AMPKα2^−/− MEFs for 4, 8, or 12 hours. A representative of triplicate experiments is shown. <b>B–C.</b> Quantification of RVFV mRNA (<b>B</b>) or genomic RNA (<b>C</b>) in WT or AMPKα1/AMPKα2^−/− MEFs displayed as the normalized fold change from WT 4 hours. A representative of triplicate experiments is shown. <b>D.</b> RVFV binding assay. RVFV (MOI 10) was bound to WT or AMPKα1/AMPKα2^−/− MEFs at 4°C for 1 hour, then washed, and treated with PBS or trypsin to remove bound virus. qRTPCR was performed on isolated RNA to detect RVFV S genome. Data are displayed as the average ΔΔCT of triplicate experiments normalized to GAPDH control. * indicates p<0.05. <b>E.</b> 2DG (12 mM), A769662 (100 uM) or Ammonium Chloride (NH₄Cl, 12 mM) was added either 1 hour prior to infection with RVFV (MOI 1), with infection, or 1, 2, or 4 hours post infection. After 10 hours of infection cells were fixed and processed for immunofluorescence. Data are displayed as the average percent infection relative to the post entry level of infection (NH₄Cl added at 4 hpi) ± SD from triplicate experiments. * indicates p<0.05.</p

Acetyl-CoA Carboxylase Activity is Tightly Regulated by AMPK during RVFV Infection.

<p><b>A.</b> Phosphorylation of AMPK and downstream effectors upon RVFV infection. WT MEFs were infected with RVFV (MOI 1) for 4 or 8 hours. Lysates were collected and assayed by immunoblot for phospho-AMPK, phospho-ACC, and phospho-eEF2. Total protein was assayed for each and Tubulin was measured as a loading control. Representative blot of triplicate experiments is shown. <b>B.</b> Phosphorylation of AMPK and downstream effectors in WT and AMPKα1/AMPKα2^−/− MEFs. Cells were treated with AMPK activators 2DG (12 mM), oligomycin (OM, 10 µM), and A769662 (100 µM) for 4 hours. Lysates were collected and assayed by immunoblot as above. Representative blot of triplicate experiments shown. <b>C.</b> Phosphorylation of AMPK and ACC upon treatment with UV-inactivated RVFV. WT MEFs were infected with live or UV-inactivated RVFV (MOI 1) for 4 or 8 hours. Lysates were collected and assayed by immunoblot as above. Representative blot of triplicate experiments is shown. <b>D.</b> Blocking fatty acid synthesis inhibits RVFV infection. MEFs were treated with the fatty acid synthase inhibitors Cerulenin (45 pM) and C75 (12.5 µM) or the AMPK activator A769662 (100 µM), infected with RVFV (MOI 1), and processed for immunofluorescence. Data are displayed as the normalized average percent infection relative to the untreated control ± SD in triplicate experiments. * indicates p<0.05. <b>E.</b> WT MEFs were treated with 100 µM A769662 for 10 hours and stained for cellular lipids with BODIPY lipophilic fluorescent dye. (BODIPY, red; nuclei, blue). Representative images from triplicate experiments are shown. <b>F.</b> Quantification of <b>E.</b> presented as integrated BODIPY intensity per cell relative to untreated control ± SD in triplicate experiments. * indicates p<0.05. <b>G.</b> WT and AMPKα1/AMPKα2^−/− MEFs were grown overnight and stained for cellular lipids with BODIPY lipophilic fluorescent dye. (BODIPY, red; nuclei, blue). Representative images from triplicate experiments are shown. <b>H.</b> Quantification of <b>G.</b> presented as integrated BODIPY intensity per cell relative to WT ± SD in triplicate experiments. * indicates p<0.05.</p

Ultrastructural Analysis Reveals Virus-Dependent Vesicular Compartment

<div><p>(A) Uninfected cells with intact Golgi.</p><p>(B) Vesicles were generated at 10 h postinfection throughout the cytoplasm of cells pretreated with dsRNA against GFP and infected with DCV.</p><p>(C–E) Cells were pretreated with dsRNA against COPI <i>(bCOP)</i> (C), COPII <i>(sec23)</i> (D), or <i>SREBP</i> (E), infected with DCV, and prepared for electron microscopy.</p><p>(F) Immunoelectron microscopy of <i>Drosophila</i> cells infected with DCV and the RNA replication machinery was visualized using anti-DCV helicase and a secondary antibody coupled to 10-nm gold particles. The surfaces of cytoplasmic vesicles (arrows) are stained.</p><p>(G) Higher-magnification view of DCV helicase–labeled vesicle.</p><p>(H) Immunoelectron microscopy of <i>Drosophila</i> cells infected with DCV and the RNA replication machinery was visualized using anti-DCV helicase and a secondary antibody coupled to 5-nm gold particles. The Golgi was visualized using an anti-Golgi antibody <i>(DG13)</i> and a secondary antibody coupled to 12-nm gold particles.</p></div

COPI-Dependent Golgi Disassembly in DCV Infected Cells

<div><p>Confocal analysis of cells pretreated with the indicated dsRNA and infected with DCV.</p><p>(A) Golgi morphology of DCV-infected control cells (GFP) reveals that the normal punctate staining in uninfected cells is dispersed during viral replication.</p><p>(B–F) Loss of COPI <i>(bCOP)</i> (B), <i>SREBP</i> (E), or <i>CG3523</i> (F) but not COPII <i>(sec23)</i> (C) or <i>Syx5</i> (D) results in a decrease in viral infection. Note that the Golgi stain is reduced in uninfected COPI, COPII, <i>SREBP, CG2523,</i> and <i>Syx5,</i> but only the loss in COPI, <i>SREBP,</i> or <i>CG3523</i> results in a decrease in DCV replication. Green, anti-Golgi <i>(DG13);</i> red, anti-DCV; blue, Hoescht 33342.</p></div

Instrument-Free Point-of-Care Molecular Detection of Zika Virus

The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors’ offices, clinics, and at home

Smartphone-Based Mobile Detection Platform for Molecular Diagnostics and Spatiotemporal Disease Mapping

Rapid and quantitative molecular diagnostics in the field, at home, and at remote clinics is essential for evidence-based disease management, control, and prevention. Conventional molecular diagnostics requires extensive sample preparation, relatively sophisticated instruments, and trained personnel, restricting its use to centralized laboratories. To overcome these limitations, we designed a simple, inexpensive, hand-held, smartphone-based mobile detection platform, dubbed “smart-connected cup” (SCC), for rapid, connected, and quantitative molecular diagnostics. Our platform combines bioluminescent assay in real-time and loop-mediated isothermal amplification (BART-LAMP) technology with smartphone-based detection, eliminating the need for an excitation source and optical filters that are essential in fluorescent-based detection. The incubation heating for the isothermal amplification is provided, electricity-free, with an exothermic chemical reaction, and incubation temperature is regulated with a phase change material. A custom Android App was developed for bioluminescent signal monitoring and analysis, target quantification, data sharing, and spatiotemporal mapping of disease. SCC’s utility is demonstrated by quantitative detection of Zika virus (ZIKV) in urine and saliva and HIV in blood within 45 min. We demonstrate SCC’s connectivity for disease spatiotemporal mapping with a custom-designed website. Such a smart- and connected-diagnostic system does not require any lab facilities and is suitable for use at home, in the field, in the clinic, and particularly in resource-limited settings in the context of Internet of Medical Things (IoMT)

Smartphone-Based Mobile Detection Platform for Molecular Diagnostics and Spatiotemporal Disease Mapping

Rapid and quantitative molecular diagnostics in the field, at home, and at remote clinics is essential for evidence-based disease management, control, and prevention. Conventional molecular diagnostics requires extensive sample preparation, relatively sophisticated instruments, and trained personnel, restricting its use to centralized laboratories. To overcome these limitations, we designed a simple, inexpensive, hand-held, smartphone-based mobile detection platform, dubbed “smart-connected cup” (SCC), for rapid, connected, and quantitative molecular diagnostics. Our platform combines bioluminescent assay in real-time and loop-mediated isothermal amplification (BART-LAMP) technology with smartphone-based detection, eliminating the need for an excitation source and optical filters that are essential in fluorescent-based detection. The incubation heating for the isothermal amplification is provided, electricity-free, with an exothermic chemical reaction, and incubation temperature is regulated with a phase change material. A custom Android App was developed for bioluminescent signal monitoring and analysis, target quantification, data sharing, and spatiotemporal mapping of disease. SCC’s utility is demonstrated by quantitative detection of Zika virus (ZIKV) in urine and saliva and HIV in blood within 45 min. We demonstrate SCC’s connectivity for disease spatiotemporal mapping with a custom-designed website. Such a smart- and connected-diagnostic system does not require any lab facilities and is suitable for use at home, in the field, in the clinic, and particularly in resource-limited settings in the context of Internet of Medical Things (IoMT)