Identification of viral pathogen diversity in sewage sludge by metagenome analysis

Abstract

The large diversity of viruses that exist in human populations are potentially excreted into sewage collection systems and concentrated in sewage sludge. In the U.S., the primary fate of processed sewage sludge (class B biosolids) is application to agricultural land as a soil amendment. To characterize and understand infectious risks associated with land application, and to describe the diversity of viruses in human populations, shotgun viral metagenomics was applied to 10 sewage sludge samples from 5 wastewater treatment plants throughout the continental U.S, each serving between 100,000 and 1,000,000 people. Nearly 330 million DNA sequences were produced and assembled, and annotation resulted in identifying 43 (26 DNA, 17 RNA) different types of human viruses in sewage sludge. Novel insights include the high abundance of newly emerging viruses (e.g., Coronavirus HKU1, Klassevirus, and Cosavirus) the strong representation of respiratory viruses, and the relatively minor abundance and occurrence of Enteroviruses. Viral metagenome sequence annotations were reproducible and independent PCR-based identification of selected viruses suggests that viral metagenomes were a conservative estimate of the true viral occurrence and diversity. These results represent the most complete description of human virus diversity in any wastewater sample to date, provide engineers and environmental scientists with critical information on important viral agents and routes of infection from exposure to wastewater and sewage sludge, and represent a significant leap forward in understanding the pathogen content of class B biosolids.

Figure 1

(A) Pie chart showing the MG-RAST subsystem annotation distribution for all contiguous sequences longer than 200 bp. (B) Pie chart demonstrating the distribution of tBLASTx virus contig annotations. Eukaryote pathogens do not include human viral pathogens. Human viral pathogen annotations represented ~0.1% of total contigs.

Figure 2

Heat map demonstrating the relative abundance and occurrence for human viral pathogens. Relative abundance is defined as the log₁₀[reads mapped to a virus contig divided by the total reads in the sample]. The dashed box represents replicated samples. Tables S3 and S4 provide virus identification to the highest taxonomic level.

Figure 3

Principle component analysis of human viral pathogen population occurrence values. PCA plots were produced using the Sorensen similarity index.

Figure 4

Comparison of the relative abundances of contigs, from replicate samples. Technical replicates refer to replicated sequencing of the same DNA/cDNA, while biological replicates refers to sequencing of DNA/cDNA extracted separately from the same sewage sludge sample. Slope and r² values refer to data fitting to a straight line.