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. 2024 May 21;16(6):816.
doi: 10.3390/v16060816.

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters

Pablo Puchades-Colera  1 , Azahara Díaz-Reolid  1 , Inés Girón-Guzmán  1 , Enric Cuevas-Ferrando  1 , Alba Pérez-Cataluña  1 , Gloria Sánchez  1
Affiliations

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters

Pablo Puchades-Colera et al. Viruses. .

Abstract

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

Keywords: capsid integrity (RT)-qPCR; enteric viruses; reclaimed water; virus contamination indicator; wastewater.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Presence of human enteric viruses, crAssphage, and somatic coliphages in influent and effluent wastewater samples over 10 months. Each colored square represents mean Log10 genome copies (gc)/L or Log10 plaque-forming units (pfu)/L values. Abbreviations: human norovirus genotype I (HuNoV GI), human norovirus genotype II (HuNoV GII), rotavirus (RV), limit of detection (LoD).
Figure 2
Figure 2
Temporary distribution of enteric viruses, crAssphage and somatic coliphages on influent samples. Bars represent mean Log10 genome copies (gc)/L or Log10 plaque-forming units (pfu)/L values. * p < 0.05 between seasons for HuNoV GII. Abbreviations: human norovirus genotype I (HuNoV GI), human norovirus genotype II (HuNoV GII), rotavirus (RV), limit of detection (LoD).
Figure 3
Figure 3
Mean reduction in human enteric viruses, crAssphage, and somatic coliphages after wastewater treatment. Abbreviations: human norovirus genotype I (HuNoV GI), human norovirus genotype II (HuNoV GII), rotavirus (RV), genome copies (gc), pfu (plaque-forming units).
Figure 4
Figure 4
Spearman’s correlation heatmap on the presence of enteric viruses ((RT)-qPCR) and fecal contamination indicators in effluent samples. Abbreviations: human norovirus genotype I (HuNoV GI), human norovirus genotype II (HuNoV GII), rotavirus (RV).
Figure 5
Figure 5
Optimization of PMAxx-RT-qPCR assay for influent wastewater samples. Abbreviations: human norovirus genotype I (HuNoV GI), human norovirus genotype II (HuNoV GII), rotavirus (RV), cycle threshold (Ct), limit of detection (LoD).
Figure 6
Figure 6
Monitoring of human enteric viruses on raw samples (black dots) and with optimized PMAxx 100 µM treatment (pink dots) in influent wastewater and reclaimed water for (A) human norovirus genotype I (HuNoV GI), (B) human norovirus genotype II (HuNoV GII), and (C) rotavirus (RV). Black crosses mean negative samples. Abbreviations: cycle threshold (Ct), limit of detection (LoD).
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