Vector competence of selected North American Anopheles and Culex mosquitoes for Zika virus

Mosquito and Zika virus propagation

Four mosquito species were tested for their ability to become infected with and transmit ZIKV. Anopheles freeborni (F1 strain, MRA-130), An. quadrimaculatus (Orlando strain, MRA-139), and Culex tarsalis (Yolo strain, NR-43026, colony originated from Yolo County CA in 2003) were provided by BEI resources (National Institute of Allergy and Infectious Diseases, Manassas, VA). Aedes aegypti (Rockefeller strain) were used as a positive control.

All mosquito species were reared and maintained at the Penn State Millennium Sciences Complex insectary at 27 °C ± 1 °C, 12:12 hr light:dark cycle, and 80% relative humidity in 30 × 30 × 30-cm cages. Mosquitoes were provided with cotton pads soaked with 10% sugar for maintenance and fed anonymous expired human blood (Biological Specialty Corporation, Colmar, PA; anticoagulant = citrate phosphate dextrose adenine) using a glass feeder jacketed with 37 °C distilled water for colony propagation and experimental virus infections. Aedes aegypti larvae were fed on whole koi pellets (TetraPond, Koi Vibrance, Prestons, Australia), all Anopheles larvae were fed finely ground koi pellets, and Culex tarsalis larvae were fed a 1:1:1 mixture by weight of ground bovine liver powder (MP Biomedicals, Solon, OH), koi pellets, and rabbit pellets (Kaytee, Chilton, WI, USA).

ZIKV strain PRVABC59 (provided by BEI Resources, NR-50240; GenBank KU501215) was used for all mosquito infections. This virus was originally isolated in 2015 from infected human serum in Puerto Rico and passed once in Cercopithecus aethiops kidney epithelial cells (Vero 76, ATCC CRL-1586). Working stocks were made by passing the virus three times in Vero cells (grown in Dulbecco’s modified-essential media (10566-024; Thermo Fischer Scientific, Gibco, Waltham, MA, USA) with 10% fetal bovine serum, and 1 × penicillin–streptomycin), harvesting the supernatant, and storing the 300 µl aliquots at −70 °C until used for mosquito infections.

Mosquito vector competence for Zika virus

ZIKV aliquots were thawed briefly at 37 °C and were added to whole human blood in equal volumes. Aliquots were only thawed once to prevent any loss in virulence via multiple freeze–thaw cycles. We exposed adult female mosquitoes (3–7 days old) to ZIKV-infected blood via a glass feeder jacketed with 37 °C distilled water. Mosquitoes were allowed to feed to engorgement for up to 1 h. Mosquitoes were sorted by fed status under CO₂ anesthesia. All mosquitoes that took a blood meal were fully engorged and they were retained for transmission experiments; non-blood fed females were discarded. We stored a 200–300 ml aliquot of each blood meal pre-feeding to confirm ZIKV titer by plaque assay on Vero cells, as described below.

We assessed ZIKV infection, dissemination, and transmission at 7 days post-blood feeding (Aitken, 1977; Dodson, Kramer & Rasgon, 2011; Dodson, Kramer & Rasgon, 2012). We attempted to assess additional time points, but (except for Aedes aegypti) feeding rates on the artificial blood meal were poor and resulted in low numbers; additionally, sufficient numbers of mosquitoes did not survive past 10 days post-feeding. Assayed female mosquitoes were anesthetized with triethylamine (Sigma, St. Louis, MO, USA). Legs from each mosquito were removed with forceps and placed in a 2-ml tube filled with 1 ml mosquito diluent (MD: 20% heat-inactivated fetal bovine serum [FBS] in Dulbecco’s phosphate-buffered saline, 50 µg/ml penicillin/streptomycin, 50 ug/mL gentamicin, and 2.5 µg/mL fungizone) with a single sterile, zinc-plated, steel 4.5 mm ball (Daisy, Rogers, AR). To induce mosquitoes to salivate, the mouthparts of each mosquito were placed in a tapered capillary tube containing approximately 10 µl of 1:1 50% sucrose and FBS (Moudy et al., 2007; Dodson, Kramer & Rasgon, 2011). Saliva was collected for 30 min, after which the contents were expelled into 0.3 ml MD. Bodies were placed individually into 2-ml tubes filled with 1 ml MD and a single sterile, zinc-plated, steel 4.5 mm BB. All samples were stored at −70 °C until tested for ZIKV by Vero plaque assay. All mosquito experiments with infectious ZIKV were conducted in the Eva J. Pell ABSL3 Laboratory at Pennsylvania State University, according to established biosafety protocols (IRB #45458).

Zika virus plaque assay

We tested blood meals and mosquito samples for ZIKV infectious particles by Vero cell plaque assays (Payne et al., 2006). To identify infected mosquitoes, mosquito bodies were first assayed. If the body of that individual was positive, the legs and saliva were assayed to test for dissemination and transmission. Prior to the start of the assay, samples were thawed briefly at 37 °C. Bodies and legs were homogenized for 30 s with a TissueLyser (QIAGEN, Hilden, Germany) at 24 cycles/sec, and centrifuged for 1 min. Samples were kept on ice to prevent loss of viral infectivity during the assay, and after the initial infection concluded, samples were returned to −70 °C. Complete media (1 × Dulbecco’s modified-essential media (10566-024; Thermo Fischer Scientific, Waltham, MA, USA), 100 units/ml penicillin–streptomycin, and 10% fetal bovine serum) was removed from confluent monolayers of Vero cells in 6-well-plates, and monolayers inoculated with 100 µl of each clarified sample. Inoculated plates were placed in an incubator at 37 °C with 5% CO₂ for 2 h and were rocked every 30 min to prevent cell desiccation. After the 2 h incubation, a 3 ml overlay was applied to each well that comprised equal amounts of complete media and 1.2% agarose, and plates incubated for 3 days. A secondary 3 ml overlay was then applied that comprised equal amounts of complete media and 1.2% agarose, as well as 1.5% neutral red solution, and plates incubated for one additional day. After incubation, plates were inverted, placed on a white light transilluminator (Hall Productions, San Luis Obispo, CA, USA), and each well assessed as positive or negative for ZIKV by observation of plaques. Plaques were counted if they were present. Positive and negative controls were included with every assay to confirm that the assay worked as expected and to ensure cell viability (positive: diluted ZIKV-infected blood meal and ZIKV stocks; negative: complete cell culture media and MD). Infection rate was defined as the proportion of mosquitoes exposed to virus that had ZIKV-positive bodies. Dissemination and transmission rates were defined as the proportion of mosquitoes infected with ZIKV that had infectious viral particles present in their legs and saliva, respectively.