UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR

doi:10.1016/j.watres.2005.06.013

Water Research

Volume 39, Issue 15, September 2005, Pages 3643-3649

https://doi.org/10.1016/j.watres.2005.06.013 Get rights and content

Abstract

Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation. Adenovirus is a double-stranded DNA virus and produces messenger RNA (mRNA) during replication in host cells. The presence of viral mRNA in host cells is definitive evidence of infection. We recently developed a rapid and reliable cell culture-mRNA RT-PCR assay to detect and quantify adenovirus infectivity. Viral mRNA recovered from cell cultures 5–7 days after infection was purified on oligo-dT latex, treated with DNase, and amplified by RT-PCR using the primers specific for a conserved region of the hexon late mRNA transcript. Treatment of approximately 10⁴ Ad41 with different doses of 254 nm germicidal UV radiation resulted in a dose-dependent loss of infectivity. As UV doses were increased from 75 to 200 mJ/cm², virus survival decreased and no virus infectivity (measured by detectable mRNA) was found at a dose of 225 mJ/cm² or higher. Our results using the cell culture mRNA RT-PCR assay indicate that Ad41 is more resistant to UV radiation than in a previous study using a conventional cell culture infectivity assay. Results were more similar to those found for Ad 40 using CPE as a measure of infectivity in another previous study.

Introduction

Germicidal 254 nm UV irradiation has been receiving increased attention as an alternative to chemical disinfection of drinking water and wastewater because UV effectively inactivates cysts and oocysts of protozoan intestinal parasites that are highly resistant to chlorination (Clancy et al., 1998; Linden et al., 2002) and it produces limited toxic disinfection by-products (Haider et al., 2002). Germicidal UV is primarily absorbed by nucleic acids of microorganisms, causing photoproducts such as thymine dimers on the same nucleic acid strand (Harm, 1980). If the damage is not repaired, DNA replication is blocked, leading to inactivation of microorganisms. Numerous studies have demonstrated that UV disinfection can effectively inactivate most microorganisms including waterborne pathogens (Linden et al., 2002; Zimmer and Slawson, 2002).

Human enteric adenoviruses, which include the enteric adenovirus serotypes 40 (Ad40) and 41 (Ad41), are important waterborne pathogens. They are the second most commonly identified agent of pediatric gastroenteritis next to rotavirus in many studies (Brandt et al., 1985; McIver et al., 2001) and they have been placed on the United States (US) Environmental Protection Agency (EPA) contaminant candidate list (CCL) (USEPA, 1998). Despite the importance of controlling the enteric adenoviruses in drinking water, only a few studies have been conducted on UV inactivation of enteric adenoviruses (Meng and Gerba, 1996; Thurston-Enriquez et al., 2003). The past studies were performed by observation of infected cells for cytopathogenic effects (CPE) in order to quantify virus infectivity and UV inactivation rates. However, human enteric adenoviruses are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain CPE. This makes it difficult to obtain reliable and reproducible data on response to UV disinfection because inoculated cell cultures often deteriorate before the appearance of distinctive CPE as evidence of infectivity. We recently developed a new analytical method using cell culture mRNA RT-PCR to reliably measure the infectivity of Ad41(Ko et al., 2003). This method is not dependent upon visual inspection for CPE but rather a definitive molecular test for presence or absence of mRNA in infected cells. The objective of this study was to determine 254 nm UV inactivation kinetics of enteric Ad41 using the recently developed cell culture-mRNA RT-PCR for virus infectivity assay. UV inactivation of Ad41 was compared to that of the widely used virus biodosimeter of UV disinfection, male-specific (F+) RNA coliphage MS2.

Section snippets

MS2 assay and stock

Bacteriophage MS2 (ATCC # 15597-B1) was grown and assayed by single agar layer method using Escherichia coli C3000 (ATCC # 15597) (USEPA, 2001). The virus was purified from infected cell lysates of double agar layer plaque assay plates with confluent lysis by extracting it with an equal volume of chloroform, centrifuging it at 4000g for 30 min, and recovering the supernatant fluid. The resulting supernatant was recovered as virus stock and stored frozen in aliquots at −80 °C for use in

Results

Table 1 and Fig. 1 show the inactivation kinetics of Ad41 and MS2 as a function of UV dose. Unlike MS2, whose maximum log₁₀ reduction is >6, the maximum detectable log₁₀ reduction of Ad 41 is only 3.8 due to the lower titer of Ad41 viral stock. Ad41 was very resistant to UV, with a small fraction of the initial virus surviving even at a UV dose of 200 mJ/cm² (3.6 log₁₀ reduction), and the detection limit (>3.8 log₁₀ reduction) was reached at the UV dose of 225 mJ/cm² or higher. The reduction of

Discussion

There have been only a few studies on UV inactivation of enteric adenoviruses in spite of the importance of these viruses as waterborne pathogens (Meng and Gerba, 1996; Thurston-Enriquez et al., 2003). Furthermore, the results from these two studies are not in agreement. A likely reason for the limited number of studies and the inconsistent results in the past is the lack of a reliable method to quantify the infectivity of enteric adenoviruses. To overcome the difficulties of estimating enteric

Conclusion

UV resistance of enteric adenovirus serotype 41 was characterized by using a recently developed mRNA RT-PCR method. Our study indicated that enteric adenovirus Ad41 was very resistant to 254 nm germicidal UV. Approximately 222 mJ/cm² of UV dose would be required for 4 log₁₀ inactivation with our experimental conditions, which was significantly higher than previously reported using a conventional CPE-based cell culture infectivity assay method. This is a considerably higher dose than currently

Acknowledgments

This study was supported by grants from the American Water Works Association Research Foundation (AWWARF RFP# 2591), the Water Environment Research Federation (98-HHE-2), and the US Environmental Protection Agency (Agreement # R829012).

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UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR

Abstract

Introduction

Section snippets

MS2 assay and stock

Results

Discussion

Conclusion

Acknowledgments

Water Res.

Pathology

Water Res.

Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments

J. Environ. Engr.

Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine

Appl. Environ. Microbiol.

Adenoviruses and pediatric gastroenteritis

J. Infect. Dis.

UV light inactivation of Cryptosporidium oocysts

J. Am. Water Works Assoc. J.

Comparative inactivation of enteroviruses and adenovirus 2 by UV light

Appl. Environ. Microbiol.

Biological Effects of Ultraviolet Radiation