Quantification of human astroviruses in sewage using real-time RT-PCR

Abstract

Human astroviruses constitute a significant cause of acute diarrhea in children. Viral transmission occurs via the fecal–oral route, predominantly person-to-person, but the consumption of fecally contaminated water and shellfish has also been implicated. Viral pathogen detection in water, especially, in wastewater, is difficult and PCR is widely used to detect these viruses. Despite the recent development of real-time quantitative PCR, quantification of astroviruses in sewage had not been available up to now. We have developed a method to quantify astroviruses in sewage. For this purpose, we designed a set of primers and a fluorogenic probe located at the 3′-end of the genome of human astroviruses. The amplified region was cloned and the plasmid was transcribed to generate calibration standards for quantification. After validation of the standards, the method was evaluated in artificially contaminated samples. To validate the method on naturally contaminated samples, raw and treated wastewater samples were collected monthly for one year in a sewage treatment plant. Astrovirus genomes were detected in all samples collected at the entrance to the sewage treatment plant, with a mean value of 4.1×10 6 astrovirus genomes for 100 ml. Effluents were less strongly contaminated, with a mean value of 1.01×10 4 astrovirus genomes. The high prevalence of astroviruses in sewage treatment plant effluents indicates that these plants are not efficiently eliminating the virus. This is a major public health concern and new techniques of depuration are needed. Our method could be effectively used in evaluating new treatment processes to reduce the viral load in the effluent of treatment plants.