Clearance of Human-Pathogenic Viruses from Sludge: Study of Four Stabilization Processes by Real-Time Reverse Transcription-PCR and Cell Culture

Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed

Development of an Effective Method for Recovery of Viral Genomic RNA from Environmental Silty Sediments for Quantitative Molecular Detection ▿

Nine approaches to recover viral RNA from environmental silty sediments were newly developed and compared to quantify RNA viruses in sediments using molecular methods. Four of the nine approaches employed direct procedures for extracting RNA from sediments (direct methods), and the remaining five approaches used indirect methods wherein viral particles were recovered before RNA extraction. A direct method using an SDS buffer with EDTA to lyse viral capsids in sediments, phenol-chloroform-isoamyl alcohol to extract RNA, isopropanol to concentrate RNA, and magnetic beads to purify RNA resulted in the highest rate of recovery (geometric mean of 11%, with a geometric standard deviation of 0.02; n = 7) of poliovirus 1 (PV1) inoculated in an environmental sediment sample. The direct method exhibiting the highest rate of PV1 recovery was applied to environmental sediment samples. One hundred eight sediment samples were collected from the Takagi River, Miyagi, Japan, and its estuary from November 2007 to April 2009, and the genomic RNAs of enterovirus and human norovirus in these samples were quantified by reverse transcription (RT)-quantitative PCR (qPCR). The human norovirus genome was detected in one sample collected at the bay, although its concentration was below the quantification limit. Meanwhile, the enterovirus genome was detected in two samples at the river mouth and river at concentrations of 8.6 × 102 and 2.4 × 102 copies/g (wet weight), respectively. This is the first report to obtain quantitative data for a human pathogenic virus in a river and in estuarine sediments using RT-qPCR