RNA-Seq of untreated wastewater to assess COVID-19 and emerging and endemic viruses for public health surveillance.

Background: The COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored.
Methods: A retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing.
Findings: This is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments.
Interpretation: RNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time.
Funding: UK Research and Innovation (UKRI) Global Challenges Research Fund (GCRF) grant number H54810, as supported by Research England.
Competing Interests: This study was principally funded by the 10.13039/100016270Global Challenges Research Fund (GCRF) awarded to chief investigator TM and GCRF grant co-investigators RSK, SA, PM, AT, AS, RG, EA, TB, UV, AA, AH as supported by Research England under UKRI. SS and CH received financial support from the 10.13039/501100001602Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273, a Science Foundation Ireland's Spokes Programme which is co-funded under the 10.13039/501100008530European Regional Development Fund under Grant Number SFI/14/SP APC/B3032, and a research grant from 10.13039/100005565Janssen Biotech, Inc. The authors declare no conflict of interest, financial or otherwise.
(© 2023 The Author(s).)