Capsid Integrity qPCR—An Azo-Dye Based and Culture-Independent Approach to Estimate Adenovirus Infectivity after Disinfection and in the Aquatic Environment

Abstract

Recreational, reclaimed and drinking source waters worldwide are under increasing anthropogenic pressure, and often contain waterborne enteric bacterial, protozoan, and viral pathogens originating from non-point source fecal contamination. Recently, the capsid integrity (ci)-qPCR, utilizing the azo-dyes propidium monoazide (PMA) or ethidium monoazide (EMA), has been shown to reduce false-positive signals under laboratory conditions as well as in food safety applications, thus improving the qPCR estimation of virions of public health significance. The compatibility of two widely used human adenovirus (HAdV) qPCR protocols was evaluated with the addition of a PMA/EMA pretreatment using a range of spiked and environmental samples. Stock suspensions of HAdV were inactivated using heat, UV, and chlorine before being quantified by cell culture, qPCR, and ci-qPCR. Apparent inactivation of virions was detected for heat and chlorine treated HAdV while there was no significant difference between ci-qPCR and qPCR protocols after disinfection by UV. In a follow-up comparative analysis under more complex matrix conditions, 51 surface and 24 wastewater samples pre/post UV treatment were assessed for enteric waterborne HAdV to evaluate the ability of ci-qPCR to reduce the number of false-positive results when compared to conventional qPCR and cell culture. Azo-dye pretreatment of non-UV inactivated samples was shown to improve the ability of molecular HAdV quantification by reducing signals from virions with an accessible genome, thereby increasing the relevance of qPCR results for public health purposes, particularly suited to resource-limited low and middle-income settings.

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications – A systematic review

“…Lack of efficacy of an azo dye method with avian influenza virus was suspected to be due to natural characteristics of an enveloped virus that make it difficult for EMA to penetrate the compromised capsid ( Graiver et al., 2010 ). However, the PMA dye assay was successfully applied to detect dengue virus, another enveloped, single-stranded RNA virus ( Huang et al., 2016 ), lending further support to the conclusion that PMA is more effective than in removing false positive signals in qPCR while not showing microbicidal effects ( Fittipaldi et al., 2012 ; Gedalanga and Olson, 2009 ; Leifels et al., 2019 ).…”

“…For other viruses, direct investigation for effects of strain origins, i.e., laboratory-grown strains and environmentally acquired strains, is limited, and comparisons of results among different studies would lead to biases due to diverse experimental protocols and conditions. Moreover, a limitation of azo dye studies on clinical, environmental and food samples is the absence of information regarding absolute quantification of infectious and non-infectious viruses to evaluate the assay performance, even though known concentrations of mengovirus ( Randazzo et al., 2018a , 2018b ) or murine norovirus ( Leifels et al., 2016 , 2019 ) have been added as internal controls in some studies investigating greywater used for irrigation or freshwater near a recreational bathing site. Other complicating factors include process recovery loss and inhibition effects in qPCR assays that appeared to affect RNA viruses more than DNA viruses regardless of azo dye pretreatment ( Leifels et al., 2016 ).…”

“…Seventy percent of the articles we researched used PMA or the derived PMAxx, and a third of the studies compared them to EMA and/or the derivate PEMAX ( Table 1 ). In general, PMA and PMAxx were found to be more suitable for capsid integrity qPCR, most likely due to their higher ability to enter thermally and chemically inactivated virions while not penetrating intact capsids or showing microbicidal effects ( Jeong et al., 2017 ; Kim et al., 2017 ; Moreno et al., 2015 ), when compared to infectious virus titers determined by cell culture ( Leifels et al., 2019 ).…”

Assessing the Occurrence of Waterborne Viruses in Reuse Systems: Analytical Limits and Needs

“…Intercalating dyes, such as propidium monoazide (PMA) and ethidium monoazide (EMA) in conjunction with qPCR (PMA-RT-qPCR and PMA-qPCR for RNA or DNA viruses, respectively), have been used to determine the potential infectivity of enteric viruses in water [11,70,71]. Treatment of virus suspensions with platinum (IV) chloride (PtCl4) has also been applied to discriminate between potentially infectious and thermally inactivated enteric hepatitis viruses in environmental samples [12,72,73].…”

“…Unfortunately, these methods cannot directly detect the infectivity of waterborne viruses. Various approaches have been developed to assess infectivity of waterborne enteric viruses using molecular methods, but they are specific to the virus and the mechanism of virus inactivation [10,11,12]. The mechanism of virus inactivation may vary by the type of virus, disinfectant, and other methods which may make the virus incapable of replication [13,14].…”