Diversity, Relationship, and Distribution of Virophages and Large Algal Viruses in Global Ocean Viromes.

Virophages are a group of small double-stranded DNA viruses that replicate and proliferate with the help of the viral factory of large host viruses. They are widely distributed in aquatic environments but are more abundant in freshwater ecosystems. Here, we mined the Global Ocean Viromes 2.0 (GOV 2.0) dataset for the diversity, distribution, and association of virophages and their potential host large viruses in marine environments. We identified 94 virophage sequences (>5 kbp in length), of which eight were complete genomes. The MCP phylogenetic tree showed that the GOV virophages were widely distributed on the global virophage tree but relatively clustered on three major branches. The gene-sharing network divided GOV virophages into 21 outliers, 2 overlaps, and 14 viral clusters, of which 4 consisted of only the GOV virophages. We also identified 45 large virus sequences, 8 of which were >100 kbp in length and possibly involved in cell-virus-virophage (C-V-v) trisome relationships. The potential eukaryotic hosts of these eight large viruses and the eight virophages with their complete genomes identified are likely to be algae, based on comparative genomic analysis. Both homologous gene and codon usage analyses support a possible interaction between a virophage (GOVv18) and a large algal virus (GOVLV1). These results indicate that diverse and novel virophages and large viruses are widespread in global marine environments, suggesting their important roles and the presence of complicated unknown C-V-v relationships in marine ecosystems.

Isolation and Identification of a Large Green Alga Virus (Chlorella Virus XW01) of Mimiviridae and Its Virophage (Chlorella Virus Virophage SW01) by Using Unicellular Green Algal Cultures.

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae (Chlorella sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage (Chlorella virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus (Chlorella virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes (n = 82) with Cafeteria roenbergensis virus (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting Cafeteriavirus of the family Mimiviridae. Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga Chlorella sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. IMPORTANCE Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, Cafeteria roenbergensis. In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga (Chlorella sp.), Mimiviridae large green alga virus, and virophage at the coculture level, with Chlorella sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the Mimiviridae giant virus Cafeteria roenbergensis virus, host virus of the virophage Mavirus, as well as a novel large virus of Mimiviridae that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.

Metagenomics combined with comprehensive validation as a public health risk assessment tool for urban and agricultural run-off.

Early detection of emerging and life-threatening pathogens circulating in complex environments is urgently required to combat infectious diseases. This study proposed a public health risk assessment workflow with three stages, pathogen screening, pathogen genotyping, and risk assessment. In stage one, pathogens were screened with metagenomic sequencing, microfluidic chip, and qPCR. In stage two, pathogens were isolated and genotyped with multi-locus sequence typing (MLST) or conventional PCR. Finally, virulence genes from metagenomic data were assessed for pathogenicity. Two regions (Donggang and Zhanjiang) with potential public health concerns were selected for evaluation, each of which comprised of one urban and one farming wastewater sampling location. Overall, metagenomic sequencing reflected the variation in the relative abundance of medically important bacteria. Over 90 bacterial pathogens were monitored in the metagenomic dataset, of which 56 species harbored virulence genes. In Donggang, a pathogenic Acinetobacter sp. reached high abundances in 2018 and 2020, whereas all pathogenic Vibrio spp. peaked in October 2019. In Zhanjiang, A. baumanni, and other Enterobacteriaceae species were abundantly present in 2019 and 2020, whereas Aeromonas and Vibrio spp. peaked in November-2017. Forty species were subsequently isolated and subtyped by MLST, half of which were prevalent genotypes in clinical data. Additionally, we identified the African Swine Fever Virus (ASFV) in water samples collected in 2017, ahead of the first reported ASFV outbreak in 2018 in China. RNA viruses like Hepatitis A virus (HAV) and Enterovirus 71 (EV71) were also detected, with concentrations peaking in April 2020 and April 2018, respectively. The dynamics of HAV and EV71 were consistent with local epidemic trends. Finally, based on the virulence gene profiles, our study identified the risk level in wastewater of two cities. This workflow illustrates the potential for an early warning of local epidemics, which helps to prioritize the preparedness for specific pathogens locally.