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Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool

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posted on 07.01.2021, 05:22 by Jana Batovska, PT Mee, SE Lynch, Tim Sawbridge, Brendan Rodoni
© 2019, The Author(s). The ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses. Practical application of this technique, however, requires a clear understanding of its analytical sensitivity and specificity. To assess this, five dilutions (1:1, 1:20, 1:400, 1:8,000 and 1:160,000) of Ross River virus (RRV) and Umatilla virus (UMAV) isolates were spiked into subsamples of a pool of 100 Culex australicus mosquitoes. The 1:1 dilution represented the viral load of one RRV-infected mosquito in a pool of 100 mosquitoes. The subsamples underwent nucleic acid extraction, mosquito-specific ribosomal RNA depletion, and Illumina HiSeq sequencing. The viral load of the subsamples was also measured using reverse transcription droplet digital PCR (RT-ddPCR) and quantitative PCR (RT-qPCR). Metatranscriptomic sequencing detected both RRV and UMAV in the 1:1, 1:20 and 1:400 subsamples. A high specificity was achieved, with 100% of RRV and 99.6% of UMAV assembled contigs correctly identified. Metatranscriptomic sequencing was not as sensitive as RT-qPCR or RT-ddPCR; however, it recovered whole genome information and detected 19 other viruses, including four first detections for Australia. These findings will assist arbovirus surveillance programs in utilising metatranscriptomics in routine surveillance activities to enhance arbovirus detection.

Funding

The authors thank Russell Barnes for collecting the mosquitoes and Karen Brown for morphologically identifying them; Jonathan Darbro and Elise Kho for providing the Ross River virus-infected Aedes notoscriptus specimens; and Tracie Webster for her assistance in sequencing the samples and re-demultiplexing the reads. The authors also thank the two anonymous reviewers whose important feedback helped to improve this manuscript. This work was funded by the Biosciences Research Innovation Fund Program provided by the Victorian Department of Jobs, Precincts and Regions. The mosquito trapping was performed as part of the Victorian Arbovirus Disease Control Program, which is funded by the Department of Health and Human Services. JB is supported by an Australian Government Research Training Program Scholarship.

Biosciences Research Innovation Fund Program by the Victorian Department of Jobs, Precincts and Regions

Department of Health and Human Services

Australian Government Research Training Program Scholarship

History

Publication Date

01/01/2019

Journal

Scientific Reports

Volume

9

Issue

1

Article Number

19398

Pagination

13p.

Publisher

Springer Nature

ISSN

2045-2322

Rights Statement

The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.