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. 2019 Apr 4;13(4):e0007281.
doi: 10.1371/journal.pntd.0007281. eCollection 2019 Apr.

Vector competence of Australian Aedes aegypti and Aedes albopictus for an epidemic strain of Zika virus

Leon E Hugo  1 , Liesel Stassen  2 , Jessica La  2 , Edward Gosden  2 , O'mezie Ekwudu  2 , Clay Winterford  3 , Elvina Viennet  4 , Helen M Faddy  4 , Gregor J Devine  1 , Francesca D Frentiu  2
Affiliations

Vector competence of Australian Aedes aegypti and Aedes albopictus for an epidemic strain of Zika virus

Leon E Hugo et al. PLoS Negl Trop Dis. .

Abstract

Background: Recent epidemics of Zika virus (ZIKV) in the Pacific and the Americas have highlighted its potential as an emerging pathogen of global importance. Both Aedes (Ae.) aegypti and Ae. albopictus are known to transmit ZIKV but variable vector competence has been observed between mosquito populations from different geographical regions and different virus strains. Since Australia remains at risk of ZIKV introduction, we evaluated the vector competence of local Ae. aegypti and Ae. albopictus for a Brazilian epidemic ZIKV strain. In addition, we evaluated the impact of daily temperature fluctuations around a mean of 28°C on ZIKV transmission and extrinsic incubation period.

Methodology/principal findings: Mosquitoes were orally challenged with a Brazilian ZIKV strain (8.8 log CCID50/ml) and maintained at either 28°C constant or fluctuating temperature conditions. At 3, 7 and 14 days post-infection (dpi), ZIKV RNA copies were quantified in mosquito bodies, as well as wings and legs, using qRT-PCR, while virus antigen in saliva (a proxy for transmission) was detected using a cell culture ELISA. Despite high body and disseminated infection rates in both vectors, the transmission rates of ZIKV in saliva of Ae. aegypti (50-60%) were significantly higher than in Ae. albopictus (10%) at 14 dpi. Both species supported a high viral load in bodies, with no significant differences between constant and fluctuating temperature conditions. However, a significant difference in viral load in wings and legs between species was observed, with higher titres in Ae. aegypti maintained at constant temperature conditions. For ZIKV transmission to occur in Ae. aegypti, a disseminated virus load threshold of 7.59 log10 copies had to be reached.

Conclusions/significance: Australian Ae. aegypti are better able to transmit a Brazilian ZIKV strain than Ae. albopictus. The results are in agreement with the global consensus that Ae. aegypti is the major vector of ZIKV.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Constant and fluctuating temperature regimes.
Temperature set points for the maintenance of mosquitoes in the constant and fluctuating temperature regimes. Relative humidity was set at 75% and a 12:12 h day:night light cycle was used.
Fig 2
Fig 2. ZIKV RNA copies in Ae. aegypti and Ae. albopictus bodies.
Comparison of ZIKV RNA copies in the bodies of Ae. aegypti and Ae. albopictus maintained at 28°C constant or fluctuating temperature conditions. The amount of ZIKV RNA copies in mosquito bodies were quantified by qRT-PCR at 3, 7 and 14 dpi. Each point on the plot represents an individual mosquito. All plots show the median value ± interquartile range (IQR).
Fig 3
Fig 3. ZIKV RNA copies in Ae. aegypti and Ae. albopictus wings and legs.
Comparison of ZIKV RNA copies in the wings and legs of Ae. aegypti and Ae. albopictus maintained at 28°C constant or fluctuating temperature conditions. The amount of ZIKV RNA copies in mosquito wings and legs were quantified by qRT-PCR at 3, 7 and 14 dpi. Each point on the plot represents an individual mosquito. All plots show the median value ± interquartile range (IQR).
Fig 4
Fig 4. In vivo distribution of ZIKV infection in Ae. aegypti mosquitoes using immunofluorescence assay (IFA) with whole mosquito microscopy.
Mosquitoes were examined by IFA for ZIKV by staining with an anti-Flavivirus NS1 protein monoclonal antibody (green) and DAPI staining for DNA (blue). (A) An example of a whole mosquito body section showing ZIKV infection in the midgut (m), head (h) and salivary glands (s). (B) Quantification of anti-ZIKV staining density. Staining areas were quantified by image analysis and were expressed as the area of ZIKV staining divided by the area of DAPI staining for each organ/tissue. Data are presented for midguts, bodies (whole mosquitoes minus the area covered by midgut tissue), heads and salivary glands for mosquitoes that had infection in at least one tissue/organ. Post hoc comparisons of the main effect of days post infection on ZIKV staining density was carried out for each organ by Sidak’s method. Lines join comparisons where significant increases in ZIKV staining density had occurred for the main effect of days post infection. (C-E) High resolution images of ZIKV in infected mosquito midgut, head and salivary glands, respectively.
Fig 5
Fig 5. Immunofluorescence of whole Ae. aegypti mosquito sections.
Whole Ae. aegypti mosquito sections showing infection and dissemination of ZIKV throughout mosquito tissues over a 14 day incubation period. Representative sections from different mosquitoes were selected at various time points. (A) 3 dpi (B) 7 dpi (C) 10 dpi and (D) 14 dpi. Immunofluorescence staining was performed as described for Fig 4. Green, ZIKV infection. Blue, DNA.
Fig 6
Fig 6. ZIKV infection in Ae. aegypti ovaries.
(A) Quantification of ZIKV staining density relative to DNA over time, calculated as described for Fig 4. (B) High resolution image of ZIKV staining in the follicular epithelium of mature oocytes within Ae. aegypti ovaries. Green, ZIKV infection. Blue, DNA. Post hoc comparisons of the main effect of days post infection on ZIKV staining density was carried out for each organ by Sidak’s method. Lines join comparisons where significant increases in ZIKV staining density had occurred for the main effect of days post infection.
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Grants and funding

Funding was provided by the Australia National Health and Medical Research Council (NHMRC) grant number APP1125317 to FDF and GJD, an Australian Infectious Diseases seed grant “Zika virus vector biology, diagnostics and vaccines” (Pls Young, Hall and Devine), and a donation from John and Elizabeth Hunter. Australian governments fund the Australian Red Cross Blood Service to provide blood, blood products and services to the Australian community. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.