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

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 10(4) 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.