Summary

Background: Dengue virus outbreaks are increasing in number and severity worldwide. Viral transmission is assumed to require a minimum time period of viral replication within the mosquito midgut. It is unknown if alternative transmission periods not requiring replication are possible.

Methods: We used a mouse model of dengue virus transmission to investigate the potential of mechanical transmission of dengue virus. We investigated minimal viral titres necessary for development of symptoms in bitten mice and used resulting parameters to inform a new model of dengue virus transmission within a susceptible population.

Findings: Naïve mice bitten by mosquitoes immediately after they took partial blood meals from dengue infected mice showed symptoms of dengue virus, followed by mortality. Incorporation of mechanical transmission into mathematical models of dengue virus transmission suggests that this supplemental transmission route could result in larger outbreaks which peak sooner.

Interpretation: The potential of dengue transmission routes independent of midgut viral replication has implications for vector control strategies that target mosquito lifespan and suggest the possibility of similar mechanical transmission routes in other disease-carrying mosquitoes.

Virus and cell maintenance

The clinical isolate 2015 DENV-2 (TW2015; GenBank: KU365901)(19) was propagated in Ae. aegypti mosquitoes and Vero 76 cells (RRID: CVCL_0603). The viral titre was determined via plaque-formation assay using BHK-21 clone 13 cells (RRID: CVCL_1915). Vero cells were grown at 37°C, 5% CO₂ in 1× Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% foetal bovine serum (FBS), 1× MEM nonessential amino acid solution, and 1× antibiotic-antimycotic solution. BHK-21 clone 13 cells were grown in 1× DMEM supplemented with 2% FBS and 1× antibiotic-antimycotic solution at 37°C, 5% CO₂. All reagents were purchased from Thermo Fisher Scientific. All experiments were conducted using viral samples obtained following four passages of the original virus. All cell lines were purchased from the Food Industry Research and Development Institute, Taiwan. For mycoplasma testing: after thawing cells and culturing for at least 48 hours, 1 ml of medium was centrifuged at high speed at 13,200 rpm for 10 minutes. The supernatant was then removed, and 20 μL of water was added for reconstitution, after which the sample was boiled at 95°C for 10 minutes. Treated samples were subjected to PCR, followed by gel electrophoresis to test for the presence of mycoplasma contamination. This test was conducted approximately once per month. Mycoplasma testing was detected using a PCR with a thermal profile of step one: 94°C for 2 min; 30 cycles of step two: 94°C for 30 sec, 60°C for 2 min, 72°C for 1 min; and step three: 72°C for 5 min. The following primers were used for PCR in mycoplasma testing:

Mycoplasma forward primer: 5’-GGGAGCAAACAGGATTAGATACCCT-3’

Mycoplasma reverse primer: 5’-TGCACCATCTGTCACTCTGTTAACCTC-3’. 

Plaque-formation assay

The viral titre of mouse serum stored at -80°C was determined by a previously described plaque-formation assay(30). In brief, virus-containing serum samples from mice were serially diluted in serum-free DMEM and added to a BHK-21 clone 13 cell monolayer for virus adsorption at 37°C for 2 hours. After the diluted samples were removed, the BHK-21 clone 13 cells were overlaid with DMEM containing 1% methylcellulose (4000 cps, Sigma-Aldrich), 5% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 2.5 mM HEPES, and 1× antibiotic-antimycotic solution and cultured for 6 days at 37°C. The overlaid medium was then gently removed, and the cells were stained with Rapid Gram Stain solution (Tonyar Biotech) for 2 hours to stain plaques before being washed with water. Plaques were counted, with viral titres expressed as plaque-forming units (PFU)/mL.

Mosquito husbandry

Ae. aegypti (Higgs strain; Akbari lab) mosquito eggs were hatched in deionized water under deoxygenated conditions. Hatched larvae were fed powdered yeast and goose liver (NTN Fishing Bait). Newly emerged mosquitoes were collected in rearing cages and fed a 10% sucrose solution for maintenance at 28°C in 70% relative humidity with a 12-hour light/dark cycle. Five-day-old female mosquitoes were starved for 16 hours prior to mechanical DENV transmission experiments.

Ethics

All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals published by the National Research Council, Taiwan (1996). The animal protocol was approved by the Institutional Animal Care and Use Committee of the National Health Research Institutes (NHRI-IACUC-107054-A). The Laboratory Animal Center at the NHRI where mouse husbandry was conducted received full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International in 2015. 

Mouse husbandry

AGB6 mice, which are deficient in both type I and II interferon signalling, used for all experiments were a cross between Ifnar^-/- mice and Ifngr^-/- mice (both with a C57BL/6 background; Jackson Laboratory, Bar Harbor, ME, USA)(19,31). Mice were bred and maintained in the ABSL-2 animal feeding room of the Laboratory Animal Center at the NHRI at 22 ± 2°C in 55 ± 10% relative humidity with a 12-hour light/dark cycle. All food, water, aspen chips, and cages were sterilized before use. Mouse health monitoring was performed every 3, 6, or 12 months by Serology, Microbiology and Parasites, using the standard operating protocol of the NHRI Animal Center (https://lac.nhri.org.tw/08-2/).

Kaohsiung city household infection data analysis

Household-level infection data were obtained from the Department of Health, Kaohsiung City Government. In this dataset, the time between DENV infections reported from single households containing multiple individuals (as determined via Dengue NS1 Rapid Test (SD BIOLINE Inc., Korea; sensitivity and specificity estimated at 92 and 98%, respectively) assays) was calculated. 

Infection of mice with DENV

Eight- to ten-week-old, 18–24 g female and/or 22–25 g male AGB6 mice were challenged intravenously with either 1000 PFU of DENV(32) in 100 μL of serum-free DMEM or with 1× PBS (control) three days before the start of all experiments. For DENV infection via injection of mosquito proboscis extracts, female Ae. aegypti were first allowed to feed from DENV-infected mice until either half or fully engorged. Mosquito proboscises were collected and dissected in groups of one, four, or ten; these were then ground in 100 µL of 1× PBS prior to inoculating naïve AGB6 mice via intravenous injection.

Infection of AGB6 mice with DENV via mechanical transmission

Mice were intraperitoneally injected with the anaesthetics Rompun (16 mg/kg, Bayer Animal Health) and Ketalar (100 mg/kg, Pfizer). Anesthetized mice were individually placed on top of the mesh covering a mosquito cage and exposed to three to five mosquitoes that had been starved for 16 hours. These mosquitoes were allowed to feed from DENV-infected AGB6 mice until they were half engorged, as confirmed via visual observation. The mice were then immediately removed and naïve AGB6 mice were placed on the top of the cage. The mosquitoes were allowed to resume feeding until fully engorged. The number of successful mosquito bites was determined by counting the number of blood-engorged mosquitoes. Mouse serum was collected by retro-orbital bleeding on days 0, 2, 4, and 6 after mosquito biting, and body weight and survival were monitored for 10 days. 

Sample sizes were based on the practical experimental considerations over mice handling. No selection criteria were applied when selecting mice for testing, and no animal data were excluded from analyses. Researchers were not blinded to the infection status of the mice during experiments.

Quantification of DENV genomic RNA 

Mosquitoes were dissected either two, four or six days after biting non-infected/DENV-infected mice. Proboscis tissues were collected, and RNA was extracted using a standard Trizol-based protocol. Extracted RNA was reverse transcribed to cDNA using a SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific) prior to real-time quantitative PCR [RT-qPCR] analysis. RNA fragments encompassing the qPCR target were produced and used to generate an absolute standard curve as described previously(33). DENV genomic RNA was quantified using a RT-qPCR detection system (ABI) with a thermal profile of 95°C for 3 min, 40 cycles of 95°C for 2 sec, and 60°C for 20 sec. The following primers were used for RT-qPCR analysis in this study:

DENV2 forward primer: 5’-TCG CTG CCC AAC ACA AG-3’

DENV2 reverse primer: 5’-CAT GTT CTT TTT GCA TGT GAA C-3’.  

Statistics

Mosquitoes were randomly selected from cages, and mice were assigned to different groups according to average weight. A significance level of p < 0.05 was used throughout (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Mosquito and mouse serum titres were tested using non-parametric Mann–Whitney tests. Mouse body weights were tested using parametric unpaired t-tests. A log-rank (Mantel-Cox) test was used to compare survival distributions. Four biological replicates were conducted for all experiments. Statistical analyses were conducted using the GraphPad Prism 6 statistical software and R software.  

Role of funders

Study Funders had no role in study design, data collection, data analyses, interpretation, or writing.