Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications; a systematic review

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing 42 recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009 to 2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 uM; incubation on ice or at room temperature (20 to 25C) for 5 to 120 min; and dye activation using LED or high light (500 to 800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever.

Consequently, viral infectivity measurements are included in guidelines of water reuse for potable 54 and non-potable purposes to demonstrate water reuse safety and evaluate water treatment 55 efficiencies through log-reduction value achievements (Farkas et

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The copyright holder for this preprint this version posted May 12, 2020. . https://doi.org/10.1101/2020.05.08.20095364 doi: medRxiv preprint One of the most established qPCR modifications to measure infectivity is capsid integrity qPCR, 76 an approach where samples are pre-treated with the intercalating azo dyes propidium monoazide 77 (PMA), ethidium monoazide (EMA) or their derivates PMAxx and PEMAX. First described 78 almost two decades ago by Nogva et al. (2003) to allow the identification of viable but non-79 cultivable bacteria, this technique has been successfully adapted to remove putatively false- viable measurement method has been especially useful for those viruses for which cell cultivation-

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based detection has been difficult, but has yet to be fully validated (Estes et al., 2019). One known 89 limitation of the azo intercalating dyes is their inability to differentiate viruses that have lost their 90 infectivity due to damaged nucleic acids but whose capsid remains intact, a condition often found 91 after UV-C treatment (Leifels et al., 2015). Moreover, there are numerous factors that affect the 92 efficacy of a method, including virus type, inactivation method, type of dye and its concentration.

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Incubation conditions and light source are also crucial in the applicability of capsid integrity qPCR 94 as reflected by the great range of capsid integrity pretreatment conditions in the literature.

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Consequently, the objectives of this review were to evaluate the efficiency of azo dye pretreatment 96 conditions as stated in current literature and to establish protocols and considerations of the capsid 97 integrity qPCR methods for virus infectivity monitoring.

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118 Figure 2: Schematic overview of a typical case study structure. One or more virus samples (taken from the 119 environment or a culture collection) are split in two and one portion is inactivated, the other not, before 120 filtration and concentration steps. Azo-dye pretreatment is then conducted under various incubation 121 conditions and concentrations of PMA, PMAxx, PEMAX or EMA, either in the presence or absence of 122 additives like surfactants and enzymes, before the tubes are exposed to light for photo activation. While 123 early studies used high-energy light sources (500 -800 Watt) to initiate the formation of the light induced 124 dye-genome complex, more recent articles have focused on low energy LED in the blue light spectrum. 125 Virus quantification is done with qPCR or ddPCR for quantitative, or endpoint PCR for qualitative, 126 detection after genome extraction.  CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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All known types of enteric viruses, whether they contain single-or double-stranded DNA or RNA, 135 with or without an envelope, have been studied with capsid-integrity qPCR methods ( Figure 3).

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The intercalating dye infectivity assay was successfully applied to most viruses tested, with few

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The copyright holder for this preprint this version posted May 12, 2020. . https://doi.org/10.1101/2020.05.08.20095364 doi: medRxiv preprint capsid disruption of MNV was more challenging than for other viruses in the same studies, i.e.,

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HAdV, poliovirus, rotavirus, and bacteriophages phiX174 and MS2, using heat treatment (Kim  is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
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(which was not certified by peer review)
The copyright holder for this preprint this version posted May 12, 2020.  is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted May 12, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted May 12, 2020.  CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted May 12, 2020. . https://doi.org/10.1101/2020.05.08.20095364 doi: medRxiv preprint conditions and photo activation can be modified and need to be optimized during assay 299 development.

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Starting with these initial experimental conditions, optimized protocols need to be generated based 302 on the virus under investigation and the sample origin.    CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 12, 2020.  The authors declare no conflict of interest.

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