UV inactivation of Adenovirus Type 4 measured by integrated cell culture qPCR

Abstract

Recent changes to water quality regulations may increase the prevalence of ultraviolet (UV) disinfection in water treatment applications. Adenoviruses currently pose a tremendous challenge to UV disinfection due to the high dose requirements for inactivation. This study validates a strategy combining cell culture and quantitative polymerase chain reaction (qPCR) for direct quantification of infectious adenoviruses in disinfection studies. Using primary liver carcinoma cell monolayers grown in well trays or flasks, post-infection washing, and a 24-hr incubation period, the time and material requirements for the infectivity assays were reduced significantly in comparison to traditional assays based on cytopathogenic effects. With this integrated cell culture quantitative PCR (ICC-qPCR) strategy, a standard curve was used to quantify infectious adenoviruses and ultimately determine relative inactivation for a disinfection study. Using ICC-qPCR, UV doses of approximately 10, 34, 69, and 116 mJ/cm² corresponded to 1, 2, 3, and 4-log inactivation of adenovirus 4 in water, respectively. The results indicate that the new ICC-qPCR strategy represents a practical alternative for the quantification of adenoviruses in disinfection studies.

Keywords:

• Adenovirus infectivity
• ICC-qPCR
• UV
• disinfection

Acknowledgments

This research was supported by the National Science Foundation (NSF) Water Quality Center at Arizona State University. This work was performed while Daniel Gerrity was on appointment as a United States Department of Homeland Security (DHS) Fellow under the DHS Scholarship and Fellowship Program. All opinions expressed in this paper are the authors' and do not necessarily reflect the policies and views of NSF and DHS.

Notes

^a Position based on GenBank accession numbers: AY458656 (hexon gene of Adenovirus Type 4).

^b FAM, 6-carboxyfluorescein, fluorescence reporter dye; TAMRA, 6-carboxytetramethylrhodamine, fluorescence quencher dye.

^a Ad4 UV inactivation measured using ICC-qPCR and incubation for 1 day; calculated as the average of the 3 replicate experiments using regression equations.

^b Ad2 UV inactivation measured using CPE-based method for unspecified incubation period.^{[ 22 ]}

^c Ad40 UV inactivation measured using CPE-based method and incubation for 12–24 days.^{[ 17 ]}

^d Ad41 UV inactivation measured using cell culture mRNA reverse-transcriptase PCR and incubation for 5–7 days.^{[ 21 ]}

^e LT2ESTWR dose requirements for viral UV inactivation credits.^{[ 3 ]}

^f Phosphate-buffered saline.

^g Inactivation data was reported based on dose rather than inactivation level.