DOI: 10.5061/dryad.1vhhmgqzb
title: Mechanical transmission of Dengue Virus by Aedes aegypti may influence disease transmission dynamics during outbreaks data

description: 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.
description: Virus and cell
 maintenance The clinical isolate 2015 DENV-2
 (TW2015; GenBank: KU365901)(19) was propagated in <em>Ae.
 aegypti</em> 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%
 CO<sub>2</sub> 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% CO<sub>2</sub>.
 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 <em>Ae. aegypti
 </em>(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 <em>Ifnar</em><sup>-/-</sup> mice and
 <em>Ifngr</em><sup>-/-</sup> 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.<strong> </strong> 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 <em>Ae. aegypti </em>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. <br> 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 <em>p</em> &lt; 0.05 was used throughout (*<em>p</em> &lt; 0.05; **<em>p</em> &lt; 0.01; ***<em>p</em> &lt; 0.001; ****<em>p</em> &lt; 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.