Ethics Statement

This study was carried out in strict accordance with recommendations set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animals and animal facilities were under the control of the School of Veterinary Medicine with oversight from the University of Wisconsin Research Animal Resource Center. The protocol was approved by the University of Wisconsin Animal Care and Use Committee (Approval #V01327).

Cells and viruses

African Green Monkey kidney cells (Vero; ATCC #CCL-81) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 100 U/ml of penicillin, 100 μg/ml of streptomycin and incubated at 37 °C in 5% CO 2 . Aedes albopictus mosquito cells, (C6/36; ATCC #CRL-1660) were maintained in MEM supplemented with 10% FBS, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin and incubated at 28 °C in 5% CO 2 . ZIKV strain PRVABC59 (GenBank:KU501215), originally isolated from a traveler to Puerto Rico with three rounds of amplification on Vero cells, was obtained from Brandy Russell (CDC, Ft. Collins, CO). Virus stocks were prepared by inoculation onto a confluent monolayer of C6/36 mosquito cells.

Mosquito strains and colony maintenance

Ae. aegypti used in this study were maintained at the University of Wisconsin-Madison as previously described42. Two lines of mosquitoes were used in this study. Wild type (WT) mosquitoes (not infected with Wolbachia) were established from several hundred eggs collected from 41 ovitraps placed around the municipality of Bello (commune 1), a northwest suburb of Medellin, Colombia. The Wolbachia-infected ((COL)wMel; infected with the wMel strain of Wolbachia pipientis) mosquito line was generated by crossing wild-caught Aedes aegypti with a wMel-infected laboratory strain of Ae. aegypti using a scheme of mating to field-collected males as developed by Yeap et al.43 to generate a Colombian genetic background Wolbachia-infected mosquito. The wMel-infected laboratory population of Ae. aegypti originated from eggs provided by Scott O’Neill (Monash University, Victoria Australia). Briefly, females from the wMel-infected laboratory population were backcrossed to wild-caught males until generation five at which time the lines were closed (restricting matings to mosquitoes from within the line) with the population size maintained at several thousand adults. An outcrossed line then was established by continuous backcrossing laboratory mosquitoes to the progeny of wild-caught Ae. aegypti from the Bello region (same location as eventual release zones). The WT line was derived from material collected from the same 41 ovitraps distributed throughout the suburb of Bello, Colombia. This line has never had any previous contact with Wolbachia-infected mosquitoes. Wild-caught males from the Bello region were routinely collected and introduced into the (COL)wMel and WT colony cages after each generation to prevent inbreeding effects and to ensure the genetic homogeneity of these two lines. Wolbachia infection status was routinely verified using PCR with primers specific to the IS5 repeat element20.

Exposure to infective bloodmeal

Mosquitoes were exposed to ZIKV by feeding on isoflurane anesthetized ZIKV-infected Ifnar−/− mice. These mice have abrogated type I interferon signaling and as a result develop lethal infection and a high viremia. Ifnar−/− mice on the C57BL/6 background were obtained from Eva Harris (University California-Berkeley, Berkeley, CA) and were bred in the pathogen-free animal facilities of the University of Wisconsin-Madison School of Veterinary Medicine. Groups of three-week-old mixed sex mice were used for mosquito exposures. Mice were infected in the left, hind foot pad with 106 plaque forming units (PFU) of ZIKV in 50 μl of animal diluent (AD: 1% heat-inactivated FBS in Dulbecco’s PBS). Uninfected mosquitoes (both WT and (COL)wMel) were allowed to feed on mice two or three days post infection at which time sub-mandibular blood draws were performed and serum was collected to verify viremia. Mice fed upon two days post infection (biological replicate one) yielded an infectious bloodmeal concentration of 6.02 log 10 PFU/ml ± 0.67 (mean ± standard deviation; n = 4) and mice fed upon three days post infection (biological replicate two) yielded an infectious bloodmeal concentration of 4.74 log 10 PFU/ml ± 0.06. A third biological replicate was performed, whereby mosquitoes were exposed to a ZIKV-infected bloodmeal via water-jacketed membrane feeder maintained at 36.5 °C30. Bloodmeals consisted of defibrinated sheep blood (HemoStat Laboratories) and fresh virus supernatant, yielding an infectious bloodmeal concentration of 8.0 log 10 PFU/ml (bloodmeal titer was determined after feeding).

Vector Competence

Infection, dissemination and transmission rates were determined for individual mosquitoes and sample sizes were chosen using long established procedures25,44,45. Briefly, three- to six-day-old female mosquitoes were sucrose starved for 14 to 16 hours prior to exposure to mice or membrane feeder. Mosquitoes that fed to repletion were randomized, separated into cartons in groups of 20–30 and maintained on 0.3 M sucrose in an environmental chamber at 26.5 °C ± 1 °C, 75% ± 5% relative humidity and with a 12 hour photoperiod within the Department of Pathobiological Sciences BSL3 Insectary facility at the University of Wisconsin-Madison. All samples were screened by plaque assay on Vero cells. Dissemination was indicated by virus-positive legs. Transmission was defined as release of infectious virus with salivary secretions, i.e., the potential to infect another host and was indicated by virus positive-salivary secretions.

Viral Quantification

All ZIKV screens from mosquito tissues and titrations for virus quantification from mouse serum or virus stocks were completed by plaque assay on Vero cell cultures. Duplicate wells were infected with 0.1 ml aliquots from serial 10-fold dilutions in growth media and virus was adsorbed for one hour. Following incubation, the inoculum was removed and monolayers were overlaid with 3 ml containing a 1:1 mixture of 1.2% oxoid agar and 2X DMEM (Gibco, Carlsbad, CA) with 10% (vol/vol) FBS and 2% (vol/vol) penicillin/streptomycin. Cells were incubated at 37 °C in 5% CO 2 for four days for plaque development. Cell monolayers then were stained with 3 ml of overlay containing a 1:1 mixture of 1.2% oxoid agar and 2X DMEM with 2% (vol/vol) FBS, 2% (vol/vol) penicillin/streptomycin and 0.33% neutral red (Gibco). Cells were incubated overnight at 37 °C and plaques were counted. Viral RNA from human serum samples from Colombia was quantified by qRT-PCR using the primers and probe designed by Lanciotti et al.46. The RT-PCR was performed using the iTaqTM Universal One-Step RT-qPCR kit (BioRad, Hercules, CA) on an iCycler® instrument (BioRad, Hercules, CA). Primers and probe were used at final concentrations of 600 nm and 100 nm respectively. Cycling conditions were as follows: 37 °C for 15 min, 50 °C for 30 min and 95 °C for 2 min, followed by 50 cycles of 95 °C for 15 sec and 60 °C for 1 min. Virus concentration was determined by interpolation onto an internal standard curve made up of a 7-point dilution series of in vitro transcribed RNA.

Statistical Analysis

Infection, dissemination and transmission rates were analyzed using an Exact unconditional test47. This test replaces Fisher’s exact test. It also is exact but has the advantage of being more sensitive in detecting differences (i.e., its statistical power is higher) in the case of sample sizes less than 10047. Each biological replicate consisted of mosquitoes from distinct generations to take into account stochastic variations.