Anopheles gambiae mosquitoes (Keele strain) were originally obtained from The National Institutes of Health (Bethesda, MD, USA). Mosquitoes were reared at the PSU Millennium Sciences Complex insectary in 30 × 30 × 30 cm cages under the following environmental conditions: 27 ± 1 °C; 12:12 hours light:dark diurnal cycle; 80% relative humidity. For reproduction, mosquitoes were maintained on expired anonymous human blood using a membrane feeder as previously described . Larvae were fed on ground fish flakes (TetraMin). Adult mosquitoes were provided with 10% sucrose solution as a carbohydrate source.
Anopheles gambiae Mos55 and Sua5B cells were grown in Schneider’s media (Gibco/Thermo Fisher Scientific, Waltham MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), 100 µg/ml of streptomycin (Gibco/Thermo Fisher Scientific) and 100 units/ml of penicillin (Gibco/Thermo Fisher Scientific) at 28 °C. African green monkey kidney (Vero, ATCC CCL-81) cells (ATCC, Manassas, Virginia, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/Thermo Fisher Scientific) supplemented with 10% FBS (Gibco/Thermo Fisher Scientific), 100 µg/ml of streptomycin (Gibco/Thermo Fisher Scientific) and 100 units/ml of penicillin (Gibco/Thermo Fisher Scientific) at 37 °C in 5% CO2.
AgDNV (GenBank: EU233812.1) stocks were prepared from Sua5B cells which are persistently infected with this virus . Cells were lysed by vortexing with sterile 3 mm borosilicate glass beads for 5 min and centrifuged for 10 min at 12,000×g at 4 °C in a benchtop microcentrifuge to remove cell debris. Cleared lysates were used for infections and injections or stored at − 80 °C.
MAYV strains BE AN343102 and BE AR505411 were obtained from BEI Resources (Manassas, VA, USA). BE AN343102 is a genotype D strain originally isolated from a monkey in Para (Brazil) in May 1978. BE AR505411 is a genotype L strain originally isolated from Haemagogus janthinomys mosquitoes in Para (Brazil) in March 1991. To produce viral stocks, viruses were propagated on Vero cells and stored at − 80 °C. MAYV stocks were quantified using focus-forming assay (see below).
qPCR for AgDNV quantification
Total DNA was extracted from 100 µl of AgDNV stocks or mosquito homogenates using E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, NorCross, GA, USA) kit according to the manufacturer’s instructions. qPCR was performed using PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, USA) on a Rotor-Gene Q qPCR machine (Qiagen, Hilden, Germany) using the following primers: 5′-GGC ATC AAT GTG GGA CCA AG-3′ (forward) and 5′-CCG TTA GCA AGC GTT GTC TG-3′ (reverse), and thermocycling conditions: 95 °C for 2 min for initial denaturation; 40 cycles at 95 °C for 10 s, 60 °C for 40 s, 72 °C for 30 s for DNA amplification and data acquisition; 55–99 °C (5 s per increment) for the melt curve analysis. A standard curve was generated using a dilution series of a plasmid encoding AgDNV genome as previously described .
Mouse monoclonal anti-chikungunya virus E2 envelope glycoprotein antibodies, clone CHK-48 (produced in vitro) (NR-44002) were obtained from BEI Resources, NIAID, NIH. These antibodies were shown to be cross-reactive with MAYV antigens of both BE AN343102 and BE AR505411 strains. Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibodies, Alexa Fluor 488 (A-11029) were purchased from Invitrogen. Both antibodies were used in focus-forming assays at a dilution of 1:1000.
Focus-forming assay for MAYV quantification
Vero cells were seeded in 96-well plates at a density of 3 × 104 cells/well. Ten-fold serial dilutions of virus samples in serum-free DMEM were made in 96-well plates and 30 µl of prepared solutions per well were used for infections. Cells were infected at 37 °C for 2 hours, infectious solutions removed, and cells covered with 100 µl of DMEM containing 1% methylcellulose (Sigma-Aldrich, St. Louis, MO, USA). After 24 hours, overlay medium was removed, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline (PBS) (Gibco/Thermo Fisher Scientific) for 15 min and permeabilized and blocked using a blocking solution containing 3% bovine serum albumin (Sigma-Aldrich) and 0.125% TritonX in PBS for 30 min. Primary and secondary antibodies were diluted in blocking solution and incubated for 2 hours and 1 hour, respectively. All incubations and washing steps using PBS were carried out at room temperature. After the final wash, cells were dried briefly to remove the excess of liquid, and MAYV foci immediately counted using an Olympus BX41 inverted microscope equipped with an UPlanFI 4X objective and a FITC filter.
Mixed AgDNV and Mayaro virus infections in cell culture
Mos55 cells were seeded in 6-well plates, infected with AgDNV using 100 µl of AgDNV stock obtained from Sua5B cells (~1011 genome equivalents (GE)/ml) in serum-free Schneider’s medium or mock-infected, incubated for 4 days to establish AgDNV infection and superinfected with MAYV at an MOI ≤ 0.01. Medium samples were collected at 0, 4, 8, 12, 24, 48, 72 and 120 hours post-superinfection and MAYV titers were evaluated using focus-forming assays. Cells were infected in triplicates using two different MAYV strains (BE AN and BE AR), and the experiment was performed twice.
Mosquito injections with AgDNV
Two to three-day-old An. gambiae female mosquitoes were infected with AgDNV via intrathoracic injection. Mosquitoes were anesthetized on ice and injected with 69 nl of AgDNV stock (1011 GE/ml) or complete Schneider’s medium as a control, using a Nanoject II injector (Drummond Scientific, Broomall, PA, USA).
Vector competence assays
Four days after injection with AgDNV, An. gambiae female mosquitoes were fed for 30–45 min on infected human blood spiked with 107 infectious MAYV (BE AN343102 strain) particles per ml. After feeding, mosquitoes were anesthetized on ice, fully engorged females selected and placed in cardboard cages. Ten days after blood-feeding, survived mosquitoes were anesthetized with triethylamine (Sigma-Aldrich) and processed for vector competence assays. To collect saliva samples, mosquitoes were forced to salivate in glass capillaries filled with a mix of 50% sucrose solution and FBS (1:1) for 30 min. Body, legs and saliva samples were collected in mosquito diluent containing 20% of FBS, 100 µg/ml of streptomycin, 100 units/ml of penicillin, 50 µg/ml gentamicin, and 2.5 µg/ml Amphotericin B in PBS. Body and legs samples were homogenized by a single zinc-plated, steel, 4.5 mm bead using TissueLyser II (Qiagen) at 30 Hz for 2 min and centrifuged at 4000× rpm at 4 °C for 5 min in a bench top centrifuge to clear the homogenates. Samples were stored at − 80 °C. 20 µl of body and legs samples were used to prepare 10-fold serial dilutions as described above, and 30 µl of undiluted saliva samples in mosquito diluent were used in focus-forming assay.
Infection frequency data between treatments were analyzed using Fisher’s exact text. To determine relationships between AgDNV and MAYV body titer, the Spearman’s rank correlation test was used, as assumptions for Pearson’s correlation were violated. All statistical analyses were performed in GraphPad Prism version 7 for Windows (GraphPad Software, San Diego, CA).