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. 2019 Dec 18;9:19398. doi: 10.1038/s41598-019-55741-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Design of the spiking study. (A) 100 mosquitoes were homogenised, centrifuged and the supernatant was subsampled five times, with a sixth subsample taken as a negative control. (B) The subsamples were spiked with differing dilutions of Ross River virus (RRV), a monopartite virus, and Umatilla virus (UMAV), a segmented virus. The 1:1 dilution represented the viral load of a single RRV-infected mosquito in a pool of 100. (C) Nucleic acid was extracted and split into three technical replicates. (D) Viral load was measured using metatranscriptomic sequencing, and reverse transcription droplet digital PCR (RT-ddPCR) and quantitative PCR (RT-qPCR). Created with BioRender.com.