Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater.

Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater.

Publication date: Dec 26, 2023

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e. g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 μM and 100 μM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 μM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed -0. 52-1. 15, 0. 9-1. 51, and 0. 31-1. 69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater.

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Concepts Keywords
Adenovirus Adenoviridae
Efficiency adenovirus
Epidemiology Antigens, Viral
Inactivated Antigens, Viral
Laboratories Capsid
capsid integrity
Capsid Proteins
Capsid Proteins
Coloring Agents
Coloring Agents
Enterovirus Infections
Hepatitis A
human pathogenic viruses
influenza viruses
One Health
viability qPCR
viral inactivation
waterborne viruses


Type Source Name
disease VO Viruses
disease MESH infection
disease IDO cell
drug DRUGBANK Propidium
disease IDO assay
disease VO efficiency
disease MESH hepatitis A
drug DRUGBANK Influenza A virus
disease VO Enterovirus
drug DRUGBANK Influenza B virus
disease VO GII
disease MESH hepatitis
disease MESH influenza
disease VO inactivation
disease VO viability
disease MESH polio
disease VO Respiratory syncytial virus
disease MESH COVID 19 pandemic
disease VO population
drug DRUGBANK Coenzyme M
disease VO time
disease MESH genetic markers
disease IDO infectivity
drug DRUGBANK Water
drug DRUGBANK Gold
disease VO storage
drug DRUGBANK Etoperidone
disease VO viable
disease IDO host
disease VO Rotavirus
disease VO vaccine strain
drug DRUGBANK Phosphate ion
disease VO volume
disease VO USA
disease VO device
drug DRUGBANK Polyethylene glycol
disease VO protocol
drug DRUGBANK Magnesium sulfate
drug DRUGBANK Human Serum Albumin
disease MESH Enterovirus Infections

Original Article

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