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.2018 Aug 31;12(8):e0006754.
doi: 10.1371/journal.pntd.0006754. eCollection 2018 Aug.

Demonstration of efficient vertical and venereal transmission of dengue virus type-2 in a genetically diverse laboratory strain of Aedes aegypti

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Demonstration of efficient vertical and venereal transmission of dengue virus type-2 in a genetically diverse laboratory strain of Aedes aegypti

Irma Sánchez-Vargas et al. PLoS Negl Trop Dis..

Abstract

Aedes aegypti is the primary mosquito vector of dengue viruses (DENV; serotypes 1-4). Human-mosquito transmission cycles maintain DENV during epidemics but questions remain regarding how these viruses survive when human infections and vector abundance are minimal. Aedes mosquitoes can transmit DENV within the vector population through two alternate routes: vertical and venereal transmission (VT and VNT, respectively). We tested the efficiency of VT and VNT in a genetically diverse laboratory (GDLS) strain of Ae. aegypti orally infected with DENV2 (Jamaica 1409). We examined F1 larvae from infected females generated during the first and second gonotrophic cycles (E1 and E2) for viral envelope (E) antigen by amplifying virus in C6/36 cells and then performing an indirect immunofluorescence assay (IFA). RT-PCR/nested PCR analyses confirmed DENV2 RNA in samples positive by IFA. We observed VT of virus to larvae and adult male progeny and VNT of virus to uninfected virgin females after mating with males that had acquired virus by the VT route. We detected no DENV2 in 30 pools (20 larvae/pool) of F1 larvae following the first gonotrophic cycle, suggesting limited virus dissemination at 7 days post-infection. DENV2 was detected by IFA in 27 of 49 (55%) and 35 of 51 (68.6%) F1 larval pools (20 larvae/pool) from infected E2 females that received a second blood meal without virus at 10 or 21 days post-infection (E2-10d-F1 and E2-21-F1), respectively. The minimum filial infection rates by IFA for E2-10d-F1 and E2-21d-F1 mosquitoes were 1:36 and 1:29, respectively. The VNT rate from E2-10d-F1 males to virgin (uninfected) GDLS females was 31.6% (118 of 374) at 8 days post mating. Twenty one percent of VNT-infected females receiving a blood meal prior to mating had disseminated virus in their heads, suggesting a potential pathway for virus to re-enter the human-mosquito transmission cycle. This is the first report of VNT of DENV by male Ae. aegypti and the first demonstration of sexual transmission in Aedes by naturally infected males. Our results demonstrate the potential for VT and VNT of DENV in nature as mechanisms for virus maintenance during inter-epidemic periods.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. DENV2 JAM1409 infection of GDLSAe.aegypti.
GDLS mosquitoes were blood fed with DENV2 Jamaica1409 and virus titers of individual mosquitoes were determined at different day’s post-infection (dpi) by plaque assay. The number of mosquitoes assayed from each group, percentage of infection, means titers and blood meal titers are shown. Bars indicate mean values of titers ±SEM, ** P<0.0002. BMnV = blood meal no virus.
Fig 2
Fig 2. IFA of ovaries and oocytes dissected from females infected with DENV2 after second gonotrophic cycle.
A. Microscopy images of ovaries of Groups I and II from control (left) and DENV2 infected females (right), showing infected ovaries and oviducts.B. Microscopy images of single oocytes from Group II females infected with DENV2. Arrows denote IFA positive germarium and follicles. DAPI signal (blue) and DENV2 antigen (FITC/green).
Fig 3
Fig 3. Vertical transmission of DENV2 to 4th instar larvae.
A. Schematic of steps followed to obtain F1 progeny 4th instar larvae samples from the first (E1) and second (E2) gonotrophic cycle of GDLS mosquitoes infected orally with DENV2. After exposure to a blood meal containing virus, female mosquitoes were divided into four groups. Each group was offered no further blood meals (E1) a BMnV 7, 10 and 21 days later (E2-7d, E2-10d and E2-21d respectively). Eggs were collected for each group. After hatching, all larva pools were screened for the presence of DENV2.B. RT-N-PCR and IFA of infected C6/36 cells determined DENV2 minimum filial infection (MFIR) and vertical transmission rates (VTR).
Fig 4
Fig 4. DENV2 infection in the reproductive tract of male mosquito progeny (E2-F1) of infected GDLS female.
DENV2 E antigen was detected in testis and peripheral tissue of accessory glands by IFA using DENV2 specific 3H5 monoclonal antibody.A. Reproductive tract of male mosquito progeny (E2-10d-F1) from DENV2 infected GDLS female.B. Reproductive tract of male mosquito progeny from GDLS non-infected female.
Fig 5
Fig 5. FIR in male progeny showing lack of correlation between DENV2 titer in the female parent and proportion of infected male progeny.
DENV2 titers for each infected parental GDLS female within each replicate were measured separately to determine the MFIR in male progeny. Female mosquitoes were orally infected with DENV2 and received a second BMnV at 10 dpi. E2 eggs were collected from each female placed in numbered vials. Only E2 eggs from females positive for DENV2 were hatched to rear to F1 adults to determine MFIR. No significant correlation between female parent titer and proportion progeny infected was detected (Pearson correlation, r = 0.049, P = 0.804; r = 0.267, P = 0.176; r = 0.013, P = 0.933, respectively). Non-infected female mosquitoes with a non-infected male progeny (negative control) were clustered at the origin (arrow); 1st replicate (red circle); 2nd replicate (dark blue triangle); 3rd replicate (green square).
Fig 6
Fig 6. Venereal infection rates of GDLS females mated to males vertically infected with DENV2.
A. Schematic of steps used in VNT assay. Male progeny (E2-10d-F1; designated 1.1,1.2, etc.) of DENV2 orally infected females were allowed to mate either with virgin non-infected females fed with non-infectious blood meal (3 days before) or non-blood fed at a ratio of 1:2. After mating, IFA determined infection status of reproductive tissues of males. Live females were maintained until 8 dpi at 28°C. Thereafter, infection rates were determined in female reproductive tracts by IFA following virus amplification in C6/36 cells. AG = male accessory gland; RT = female reproductive tract.B. Infection rates of non-infected females (fed with non-infectious blood meal or non-blood fed) mated with males vertically infected with DENV2.
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