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Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential

Nature Microbiology volume 5, pages 239–247 (2020)Cite this article

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Abstract

The recent Zika virus (ZIKV) and chikungunya virus epidemics highlight the explosive nature of arthropod-borne viruses (arboviruses) transmitted by Aedes spp. mosquitoes1,2. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses3. These are typically measured empirically by offering mosquitoes an infectious blood meal and temporally sampling mosquitoes to determine the infection and transmission status. This approach has been used for the better part of a century; however, it does not accurately capture the biology and behaviour of many mosquito vectors that refeed frequently (every 2–3 d)4. Here, we demonstrate that acquisition of a second non-infectious blood meal significantly shortens the EIP of ZIKV-infected Aedes aegypti by enhancing virus dissemination from the mosquito midgut. Similarly, a second blood meal increases the competence of this species for dengue virus and chikungunya virus as well as Aedes albopictus for ZIKV, suggesting that this phenomenon may be common among other virus–vector pairings and that A. albopictus might be a more important vector than once thought. Blood-meal-induced microperforations in the virus-impenetrable basal lamina that surrounds the midgut provide a mechanism for enhanced virus escape. Modelling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0, estimated from experimental data. This helps to explain how A. aegypti can sustain explosive epidemics such as ZIKV despite relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behaviour, EIP and vector competence.

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Fig. 1: Multiple feeding events increase dissemination and transmission rates of ZIKV, DENV and CHIKV in A. aegypti.
Fig. 2: Blood meal acquisition induces microperforations in the midgut basal lamina.
Fig. 3: Offering mosquitoes a second blood meal post-infection decreases the mean EIP and increases the transmission potential of ZIKV.
Fig. 4: Proposed model for double-feed-enhanced early dissemination and transmission.

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Data availability

Numerical source data underlying the graphs shown in Figs. 1 and 2c are associated with each figure. Additional data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank M. C. Thomas, M. Olson, T. Petruff and M. Correa who provided technical assistance with rearing, processing and virus testing of mosquitoes. This publication was supported by the Cooperative Agreement Number U01CK000509, funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services. This work was further supported by the US Department of Agriculture Hatch Funds and Multistate Research Project (CONH00773 and NE1443), the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA grant number UL1 TR001863 and KL2 TR001862) (J.L.W.) and the National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01 AI112970) (V.E.P.) and R21 AI129464 (B.D.F.).

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Authors and Affiliations

  1. Center for Vector-Borne and Zoonotic Diseases, Department of Environmental Sciences, The Connecticut Agricultural Experiment Station, New Haven, CT, USA

    Philip M. Armstrong, Patrick J. Conway, Angela Bransfield, Michael J. Misencik, Andrea Gloria-Soria, Theodore G. Andreadis, John J. Shepard & Doug E. Brackney

  2. Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, CT, USA

    Philip M. Armstrong, Hanna Y. Ehrlich, Theodore G. Andreadis, Virginia E. Pitzer & Doug E. Brackney

  3. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA

    Tereza Magalhaes, Megan R. Miller & Brian D. Foy

  4. Department of Biomedical Sciences, Quinnipiac University, Hamden, CT, USA

    Patrick J. Conway

  5. Department of Biostatistics, Yale School of Public Health, Yale University, New Haven, CT, USA

    Joshua L. Warren

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  1. Philip M. Armstrong
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Contributions

P.M.A., H.Y.E., V.E.P. and D.E.B. conceived and designed the experiments and wrote the manuscript. P.M.A., D.E.B., T.M., M.R.M., B.D.F., A.B., M.J.M. and A.G.-S. performed mosquito experiments including mosquito infections, dissections, transmission assays and the quantification of viral RNA and infectious virus particles. D.E.B. performed the retrograde infection assay and fluorescence microscopy. P.J.C. performed the CHP assay. J.J.S. and T.G.A. performed the scanning electron microscopy. P.M.A. and D.E.B. statistically analysed and interpreted the experimental data. H.Y.E., J.L.W. and V.E.P. performed the modelling and statistics associated with the modelling. P.M.A., V.E.P., B.D.F. and D.E.B. oversaw the project.

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Extended data

Extended Data Fig. 1 Non-infectious bloodmeals prior to infectious bloodmeals do not increase dissemination rates.

(a) Schematic of experimental design. At (b) 4, (c) 6, and (d) 9 dpi, midguts and legs were dissected from both cohorts and tested for the presence of ZIKV RNA by RT-qPCR. (●) single feed; (●) double feed. Data were analyzed by Two-sided Fisher’s exact test. Sample sizes (represented as a fraction of positive samples/ total samples) for each treatment/ timepoint are embedded in the figures above each experimental group. Center values represent the proportion and error bars represent the binomial SE of sample proportions.

Extended Data Fig. 2 Increased transmission of ZIKV RNA and infectious virions associated with acquisition of an additional non-infectious bloodmeal.

Aedes aegypti mosquitoes were offered a ZIKV infectious bloodmeal and at 3 dpi individuals in the double-feed groups were fed a second, non-infectious bloodmeal. 10 dpi mosquito saliva was collected and assayed for ZIKV RNA and infectious virions by RT-qPCR and plaque assay respectively. (●) single feed; (●) double feed. Data were analyzed by Two-sided Fisher’s exact test. (*) p<0.05. Sample sizes (represented as a fraction of positive samples/ total samples) for each treatment/ timepoint are embedded in the figures above each experimental group. Center values represent the proportion and error bars represent the binomial SE of sample proportions.

Extended Data Fig. 3 Multiple feeding events increase the potential of Aedes albopictus to transmit ZIKV.

(a) Schematic of experimental design. Paired bodies (MGI; midgut infection; % of mosquitoes with viral RNA in their bodies), legs (DI; disseminated infection; % of body positive mosquitoes with viral RNA in their legs) and salivary glands (SGI; salivary gland infection; % of leg positive mosquitoes with viral RNA in their salivary glands) were collected and assayed for the presence of viral RNA (b) 7 dpi and (c) 10 dpi. (d) SGI data from (b) and (c) analyzed as the % of ZIKV-exposed mosquitoes with a salivary gland infection. The data presented represents at least three experimental replicates. (●) single-feed; (●) double-feed. Data were analyzed by Two-sided Fisher’s exact test. (*) p<0.05, (**) p<0.01, (***) p<0.001. Sample sizes (represented as a fraction of positive samples/ total samples) for each treatment/ timepoint are embedded in the figures above each experimental group. Center values represent the proportion and error bars represent the binomial SE of sample proportions.

Extended Data Fig. 4 Increased dissemination rates associated with multiple feeding are not due to increased midgut replication.

Aedes aegypti mosquitoes were offered a ZIKV-infectious bloodmeal and 4 dpi individuals in the double feed groups were fed a second, non-infectious bloodmeal. At 7 dpi, mosquito midguts and legs were dissected and viral genomic equivalents were determined. (a) Midgut genome equivalents based on feeding status regardless if the infection was restricted to the midgut or disseminated (n=10/ group). (b) Midgut genome equivalents based on infection status (disseminated (n=14) vs. not disseminated (n=6) (that is midgut restricted)) regardless of feeding status. (●) single feed; (●) double feed. Data were analyzed by Two-sided T-test. Center values represent means and error bars represent SD.

Extended Data Fig. 5 Distributions of mean EIP estimates.

(●) experimental single-feed; (●) meta-analysis single-feed; (●) double feed, from a thinned subset (10,000 iterations) of each model’s respective posterior shape and rate estimates.

Extended Data Fig. 6 Comparison of R0 estimates from experimental data to published estimates from field studies.

Circles correspond to point estimates of R0 derived from an individual study and setting and/or methodology, while the lines show the corresponding 95% CI (n=41 estimates from 22 studies). Colors are used to show estimates from different regions (light blue: Oceania, dark blue: South America, dark green: Central America, gray: other) and our own experimental results (blue: single-feed model, red: double-feed model).

Extended Data Fig. 7 Results of a best/worst case scenario sensitivity analysis and a one-way random sampling sensitivity analysis.

(a) Scenario sensitivity analysis assessing the effect of varying each parameter according to its lowest and highest bounds on the difference in \(R_0\left( {{\it{R}}_{0_{DF}} - {\it{R}}_{0_{SF}}} \right)\) (blue=lowest value; red=highest value for each parameter). The initial difference of 0.71 was obtained by holding all parameters constant at their mean or given value (Supplementary Table 4). (b) Violin plots show the probably density of the difference in R0 \(\left( {{\it{R}}_{0_{DF}} - {\it{R}}_{0_{SF}}} \right)\) when we randomly sampled each parameter 10,000 times from its specified distribution (Supplementary Table 4), while holding all other parameters constant. Each violin spans the 98% quantile of the distribution of R0 differences with the width proportional to the probability of observing a particular value of difference. The horizontal line at 0.71 represents the difference in R0 when all parameters are held constant at their mean or given value.

Extended Data Fig. 8 Comparison of R0 and p.

(a) The mean value for 10,000 simulations of R0 is plotted for every value of p (blue= single-feed model, red= double-feed model, dashed black= reference line at R0=1), along with (b) the proportion of those simulations for which R0 >1.

Extended Data Fig. 9 Salivary Gland Infection Meta-Analysis.

Salivary Gland Infection (SGI) data aggregated from 8 published studies are plotted, with each observation (n=45) weighted by sample size and color-coded by study. The black line shows the best-fit gamma CDF model for salivary gland data and the blue line for combined meta-analysis single-feed dissemination data (data points not shown).

Extended Data Fig. 10 Results of an uncertainty analysis assessing the distribution of R0 under different feeding assumptions.

(a) We compared histograms of the single-feed R0 distribution specified by the uncertainty analysis (blue) and double-feed R0 distribution (red) and the overlap (purple) generated from 10,000 iterations of Latin Hypercube Sampling. The mean R0 for each respective distribution is also shown as a horizontal line (\({\it{R}}_{0_{SF}} = 6.68\), \({\it{R}}_{0_{DF}} = 7.55\)). (b) The distribution of the difference in \(R_0\left( {{\it{R}}_{0_{DF}} - {\it{R}}_{0_{SF}}} \right)\) is plotted for each random sample (n=10,000).

Supplementary information

Supplementary Information

Supplementary Tables 1–6 and Supplementary References.

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Source data

Source Data Fig. 1

Replicate data for Figure 1 with sample sizes and precise P values.

Source Data Fig. 2

Raw data for figure 2c with sample sizes and precise P values.

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Armstrong, P.M., Ehrlich, H.Y., Magalhaes, T. et al. Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential. Nat Microbiol 5, 239–247 (2020). https://doi.org/10.1038/s41564-019-0619-y

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