A panoramic view of the virosphere in three wastewater treatment plants by integrating viral-like particle-concentrated and traditional non-concentrated metagenomic approaches

Abstract

Wastewater biotreatment systems harbor a rich diversity of microorganisms, and the effectiveness of biotreatment systems largely depends on the activity of these microorganisms. Specifically, viruses play a crucial role in altering microbial behavior and metabolic processes throughout their infection phases, an aspect that has recently attracted considerable interest. Two metagenomic approaches, viral-like particle-concentrated (VPC, representing free viral-like particles) and non-concentrated (NC, representing the cellular fraction), were employed to assess their efficacy in revealing virome characteristics, including taxonomy, diversity, host interactions, lifestyle, dynamics, and functional genes across processing units of three wastewater treatment plants (WWTPs). Our findings indicate that each approach offers unique insights into the viral community and functional composition. Their combined use proved effective in elucidating WWTP viromes. We identified nearly 50,000 viral contigs, with Cressdnaviricota and Uroviricota being the predominant phyla in the VPC and NC fractions, respectively. Notably, two pathogenic viral families, Asfarviridae and Adenoviridae, were commonly found in these WWTPs. We also observed significant differences in the viromes of WWTPs processing different types of wastewater. Additionally, various phage-derived auxiliary metabolic genes (AMGs) were active at the RNA level, contributing to the metabolism of the microbial community, particularly in carbon, sulfur, and phosphorus cycling. Moreover, we identified 29 virus-carried antibiotic resistance genes (ARGs) with potential for host transfer, highlighting the role of viruses in spreading ARGs in the environment. Overall, this study provides a detailed and integrated view of the virosphere in three WWTPs through the application of VPC and NC metagenomic approaches. Our findings enhance the understanding of viral communities, offering valuable insights for optimizing the operation and regulation of wastewater treatment systems.

Keywords: antibiotic resistance gene; auxiliary metabolic gene; metagenome; virome; virus; wastewater treatment system.

Figure 1

Metagenomic analysis pipeline for metagenome‐assembled genome (MAG) recovery, viral contig identification, and taxonomic classification. The primary analysis tools and the count of viral contigs or MAGs are delineated on the diagram. AMG, auxiliary metabolic gene; ARG, antibiotic resistance gene; NC, non‐concentrated; vOTU, viral operational taxonomic unit; VPC, viral‐like particle‐concentrated.

Figure 2

Quantity and quality of viral contigs derived from viral‐like particle‐concentrated (VPC) and non‐concentrated (NC) metagenomes. (A) and (B) Intersection of candidate viral contigs identified by four virus detection tools. (C) and (D) Contig quality of viruses identified from VPC (C) and NC (D) metagenomes. The number of viruses including proviruses (noted in brackets) at each quality level is annotated. Kernel density plots provide data distribution. Pie charts depict the proportion of viruses at each quality level.

Figure 3

Dynamics of viruses infecting different types of hosts across the wastewater treatment streams. (A) and (B) Relative abundance and diversity of viruses infecting archaea, bacteria, eukaryotes, and unknown hosts in NC and VPC metagenomes. The lines indicate vOTU numbers. (C) and (D) Relative abundance and diversity of vertebrate viruses. The lines indicate vOTU numbers. (E) Comparison of viral abundance and diversity revealed by NC and VPC metagenomic approaches. * indicates p < 0.01. ** indicates p < 0.005. *** indicates p < 0.001. NS. indicates no significant difference. NC, non‐concentrated; vOTU, viral operational taxonomic unit; VPC, viral‐like particle‐concentrated.

Figure 4

Diversity and relative abundance of viruses in each sample. (A) Alpha diversity of viruses. (B) Viral composition at the phylum level. (C) Viral composition at the family level. A1–A9 represent samples collected from WWTP A treating duckery wastewater. B1–B10 represent samples collected from WWTP B treating swine wastewater. C1–C3 represent samples collected from WWTP C treating municipal wastewater. WWTP, wastewater treatment plant.

Figure 5

Virus–prokaryote associations in WWTPs. (A) MAG numbers and virus–host events at the phylum level. The orange column indicates the number of virus–host linkages, while the blue column indicates the number of MAGs per phylum. The purple line indicates the average viral range of MAGs per prokaryotic phylum. The green line indicates the average host range of viruses infecting each prokaryotic phylum. (B) Virus–prokaryote association at the phylum level. (C) The top 30 virus–prokaryote associations at the family level. The chord width and bar length represent the number of virus–host linkages. (D) Linear and Spearman's correlation of Shannon index of vOTUs and MAGs. (E) Linear and Spearman's correlation of richness of vOTUs and MAGs. The gray shade indicates the confidence interval of the linear correlation. MAGs, metagenome‐assembled genomes; vOTUs, viral operational taxonomic units; WWTPs, wastewater treatment plants.

Figure 6

Representative auxiliary metabolic genes (AMGs) carried by viruses. (A) Expression of representative AMGs in viruses. Values are presented as scaled means of transcripts per million (TPM) values (n = 3). The cladogram represents clustering based on scaled TPM values of AMGs. (B) Arrangement of representative AMGs in viruses.

Figure 7

Antibiotic resistance genes (ARGs) harbored by viral contigs in NC metagenomes. (A) Numbers of ARGs harbored by NC viruses, VPC viruses, and MAGs. (B) Relative abundance of ARGs harbored by viruses. Relative abundance is presented as reads per kilobase per million mapped reads (RPKM) values. The green star symbol in the right histogram indicates an MAG of the host carrying a corresponding virus‐born ARG. (C) A network of ARGs co‐occurrence in viruses and prokaryotes. MAGs, metagenome‐assembled genomes; NC, non‐concentrated; vOTU, viral operational taxonomic unit; VPC, viral‐like particle‐concentrated.