Electronic versions

DOI

  • Rachel C Williams
    School of Environmental and Natural Sciences, Bangor University
  • William B Perry
    University of NottinghamCardiff University
  • Kathryn Lambert-Slosarska
    School of Environmental and Natural Sciences, Bangor University
  • Ben Futcher
    School of Environmental and Natural Sciences, Bangor UniversityBangor University
  • Cameron Pellett
    School of Environmental and Natural Sciences, Bangor UniversityBangor University
  • India Richardson-O'Neill
    School of Environmental and Natural Sciences, Bangor UniversityBangor University
  • Steve Paterson
    University of Liverpool
  • Jasmine M S Grimsley
    UK Health Security Agency
  • Matthew J Wade
    UK Health Security Agency
  • Andrew J Weightman
    Cardiff University
  • Kata Farkas
    School of Environmental and Natural Sciences, Bangor UniversityBangor University
  • Davey L Jones
    School of Environmental and Natural Sciences, Bangor UniversityBangor University

Wastewater-based epidemiology (WBE) has been demonstrably successful as a relatively unbiased tool for monitoring levels of SARS-CoV-2 virus circulating in communities during the COVID-19 pandemic. Accumulated biobanks of wastewater samples allow retrospective exploration of spatial and temporal trends for public health indicators such as chemicals, viruses, antimicrobial resistance genes, and the possible emergence of novel human or zoonotic pathogens. We investigated virus resilience to time, temperature, and freeze-thaw cycles, plus the optimal storage conditions to maintain the stability of genetic material (RNA/DNA) of viral +ssRNA (Envelope - E, Nucleocapsid - N and Spike protein - S genes of SARS-CoV-2), dsRNA (Phi6 phage) and circular dsDNA (crAssphage) in wastewater. Samples consisted of (i) processed and extracted wastewater samples, (ii) processed and extracted distilled water samples, and (iii) raw, unprocessed wastewater samples. Samples were stored at -80 °C, -20 °C, 4 °C, or 20 °C for 10 days, going through up to 10 freeze-thaw cycles (once per day). Sample stability was measured using reverse transcription quantitative PCR, quantitative PCR, automated electrophoresis, and short-read whole genome sequencing. Exploring different areas of the SARS-CoV-2 genome demonstrated that the S gene in processed and extracted samples showed greater sensitivity to freeze-thaw cycles than the E or N genes. Investigating surrogate and normalisation viruses showed that Phi6 remains a stable comparison for SARS-CoV-2 in a laboratory setting and crAssphage was relatively resilient to temperature variation. Recovery of SARS-CoV-2 in raw unprocessed samples was significantly greater when stored at 4 °C, which was supported by the sequencing data for all viruses - both time and freeze-thaw cycles negatively impacted sequencing metrics. Historical extracts stored at -80 °C that were re-quantified 12, 14 and 16 months after original quantification showed no major changes. This study highlights the importance of the fast processing and extraction of wastewater samples, following which viruses are relatively robust to storage at a range of temperatures.

Keywords

  • Wastewater/virology, RNA, Viral, SARS-CoV-2, Freezing, Temperature, DNA, Viral, COVID-19/virology
Original languageEnglish
Pages (from-to)121879
JournalWater research
Volume259
Early online date3 Jun 2024
DOIs
Publication statusPublished - 1 Aug 2024
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