Genetic variations are instrumental for unraveling phage evolution and deciphering their functional implications. Here, we explore the underlying fine-scale genetic variations in the gut phageome, especially structural variations (SVs). By using virome-enriched long-read metagenomic sequencing across 91 individuals, we identified a total of 14,438 nonredundant phage SVs and revealed their prevalence within the human gut phageome. These SVs are mainly enriched in genes involved in recombination, DNA methylation, and antibiotic resistance. Notably, a substantial fraction of phage SV sequences share close homology with bacterial fragments, with most SVs enriched for horizontal gene transfer (HGT) mechanism. Further investigations showed that these SV sequences were genetic exchanged between specific phage-bacteria pairs, particularly between phages and their respective bacterial hosts. Temperate phages exhibit a higher frequency of genetic exchange with bacterial chromosomes and then virulent phages. Collectively, our findings provide insights into the genetic landscape of the human gut phageome.

BACKGROUND: Pneumonia is typically caused by a variety of pathogenic microorganisms. Traditional research often focuses on the infection of a few microorganisms, whereas metagenomic studies focus on the impact of the bacteriome and mycobiome on respiratory diseases. Reports on the virome characteristics of pediatric pneumonia remain relatively scarce.
METHODS: We employed de novo assembly and combined homology- and feature-based methods to characterize the respiratory virome in whole-genome DNA sequencing samples from oropharynx (OP) swabs, nasopharynx (NP) swabs, and bronchoalveolar lavage fluids (BALF) of children with pneumonia.
RESULTS: Significant differences were observed in the alpha and beta diversity indexes, as well as in the composition of the oropharyngeal virome, between pneumonia cases and controls. We identified 1137 viral operational taxonomic units (vOTUs) with significant differences, indicating a preference of pneumonia-reduced vOTUs for infecting Prevotella, Neisseria, and Veillonella, whereas pneumonia-enriched vOTUs included polyomavirus, human adenovirus, and phages targeting Staphylococcus, Streptococcus, Granulicatella, and Actinomyces. Comparative analysis revealed higher relative abundances and prevalence rates of pneumonia-enriched OP vOTUs in NP and BALF samples compared to pneumonia-reduced vOTUs. Additionally, virome analysis identified six pediatric patients with severe human adenovirus or polyomavirus infections, five of whom might have been undetected by targeted polymerase chain reaction (PCR)-based testing.
CONCLUSIONS: This study offers insights into pediatric pneumonia respiratory viromes, highlighting frequent transmission of potentially pathogenic viruses and demonstrating virome analysis as a valuable adjunct for pathogen detection.

Shrews being insectivores, serve as natural reservoirs for a wide array of zoonotic viruses, including the recently discovered Langya henipavirus (LayV) in China in 2018. It is crucial to understand the shrew-associated virome, viral diversity, and new viruses. In the current study, we conducted high-throughput sequencing on lung samples obtained from 398 shrews captured along the eastern coast of China, and characterized the high-depth virome of 6 common shrew species (Anourosorex squamipes, Crocidura lasiura, Crocidura shantungensis, Crocidura tanakae, Sorex caecutiens, and Suncus murinus). Our analysis revealed numerous shrew-associated viruses comprising 54 known viruses and 72 new viruses that significantly enhance our understanding of mammalian viruses. Notably, 34 identified viruses possess spillover-risk potential and six were human pathogenic viruses: LayV, influenza A virus (H5N6), rotavirus A, rabies virus, avian paramyxovirus 1, and rat hepatitis E virus. Moreover, ten previously unreported viruses in China were discovered, six among them have spillover-risk potential. Additionally, all 54 known viruses and 12 new viruses had the ability to cross species boundaries. Our data underscore the diversity of shrew-associated viruses and provide a foundation for further studies into tracing and predicting emerging infectious diseases originated from shrews.

Viruses as the prevailing biological entities are poorly understood in underground realms. Here, we establish the first metagenomic Groundwater Virome Catalogue (GWVC) comprising 280,420 viral species ( ≥ 5 kb) detected from 607 monitored wells in seven geo-environmental zones throughout China. In expanding ~10-fold the global portfolio of known groundwater viruses, we uncover over 99% novel viruses and about 95% novel viral clusters. By linking viruses to hosts from 119 prokaryotic phyla, we double the number of microbial phyla known to be virus-infected in groundwater. As keystone ultrasmall symbionts in aquifers, CPR bacteria and DPANN archaea are susceptible to virulent viruses. Certain complete CPR viruses even likely infect non-CPR bacteria, while partial CPR/DPANN viruses harbor cell-surface modification genes that assist symbiont cell adhesion to free-living microbes. This study reveals the unknown viral world and auxiliary metabolism associated with methane, nitrogen, sulfur, and phosphorus cycling in groundwater, and highlights the importance of subsurface virosphere in viral ecology.

Co-composting with exogenous microbial inoculant, presents an effective approach for the harmless utilization of livestock manure and agroforestry wastes. However, the impact of inoculant application on the variations of viral and antibiotic resistance genes (ARGs) remains poorly understood, particularly under varying manure quantity (low 10 % vs. high 20 % w/w). Thus, employing virome and metagenomic sequencing, we examined the influence of Streptomyces-Bacillus Inoculants (SBI) on viral communities, phytopathogen, ARGs, mobile genetic elements, and their interrelations. Our results indicate that SBI shifted dominant bacterial species from Phenylobacterium to thermotropic Bordetella, and the quantity of manure mediates the effect of SBI on whole bacterial community. Major ARGs and genetic elements experienced substantial changes with SBI addition. There was a higher ARGs elimination rate in the composts with low (∼76 %) than those with high manure (∼70 %) application. Virus emerged as a critical factor influencing ARG dynamics. We observed a significant variation in virus community, transitioning from Gemycircularvirus- (∼95 %) to Chlamydiamicrovirus-dominance. RDA analysis revealed that Gemycircularvirus was the most influential taxon in shaping ARGs, with its abundance decreased approximately 80 % after composting. Collectively, these findings underscore the role of microbial inoculants in modulating virus communities and ARGs during biowaste co-composting.

Foodborne diseases are major public health problems globally. Metagenomics has emerged as a widely used tool for pathogen screening. In this study, we conducted an updated Tn5 transposase-assisted RNA/DNA hybrid co-tagmentation (TRACE) library construction approach. To address the detection of prevalent known foodborne viruses and the discovery of unknown pathogens, we employed both specific primers and oligo-T primers during reverse transcription. The method was validated using clinical samples confirmed by RT-qPCR and compared with standard RNA-seq library construction methods. The mapping-based approach enabled the retrieval of nearly complete genomes (>95%) for the majority of virus genome segments (86 out of 88, 97.73%), with a mean coverage depth of 21,494.53× (ranging from 77.94× to 55,688.58×). Co-infection phenomena involving prevalent genotypes of Norovirus with Astrovirus and Human betaherpesvirus 6B were observed in two samples. The updated TRACE-seq exhibited superior performance in viral reads percentages compared to standard RNA-seq library preparation methods. This updated method has expanded its target pathogens beyond solely Norovirus to include other prevalent foodborne viruses. The feasibility and potential effectiveness of this approach were then evaluated as an alternative method for surveilling foodborne viruses, thus paving the way for further exploration into whole-genome sequencing of viruses.

The gut microbiota is involved in the pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D), but few studies have focused on the role of the gut virome in IBS-D. We aimed to explore the characteristics of the gut virome in patients with IBS-D, its interactions with bacteria and metabolites, and the associations between gut multiomics profiles and symptoms. This study enrolled twelve patients with IBS-D and eight healthy controls (HCs). The stool samples were subjected to metavirome sequencing, 16S rRNA gene sequencing, and untargeted metabolomic analysis. The participants completed relevant scales to assess the severity of their gastrointestinal symptoms, depression, and anxiety. The results revealed unique DNA and RNA virome profiles in patients with IBS-D with significant alterations in the abundance of contigs from Siphoviridae, Podoviridae, Microviridae, Picobirnaviridae, and Tombusviridae. Single-omics co-occurrence network analyses demonstrated distinct differences in the gut virus, bacteria, and metabolite network patterns between patients with IBS-D and HCs. Multiomics networks revealed that short-chain fatty acid-producing bacteria occupied more core positions in IBS-D networks, but had fewer links to viruses. Amino acids and their derivatives exhibit unique connectivity patterns and centrality features within the IBS-D network. The gastrointestinal and psychological symptom factors of patients with IBS-D were highly clustered in the symptom-multiomics network compared with those of HCs. Machine learning models based on multiomics data can distinguish IBS-D patients from HCs and predict the scores of gastrointestinal and psychological symptoms. This study provides insights into the interactions among gut viruses, bacteria, metabolites, and clinical symptoms in patients with IBS-D, indicating further classification and personalized treatment for IBS-D.

BACKGROUND: Haematological patients exhibit immune system abnormalities that make them susceptible to viral infections. Understanding the relationship between the virome in the blood plasma of haematological patients and their clinical characteristic is crucial for disease management. We aimed to explore the presence of viral pathogens and identify close associations between viral infections and various clinical features.
RESULTS: A total of 21 DNA viruses and 6 RNA viruses from 12 virus families were identified from 1383 patients. Patients with haematological diseases exhibited significantly higher diversity, prevalence, and co-detection rates of viral pathogens. During fever episodes, pathogen detection was notably higher, with Epstein-Barr virus (EBV) and Mucorales infections being the most probable culprits for fever symptoms in non-haematological patients. The detection rate of torque teno virus (TTV) significantly increases in haematological patients after transplantation and during primary lung infections. Additionally, TTV-positive patients demonstrate significantly higher absolute neutrophil counts, while C-reactive protein and procalcitonin levels are notably lower. Furthermore, TTV, cytomegalovirus, and parvovirus B19 (B19V) were found to be more prevalent in non-neutropenic patients, while non-viral pathogenic infections, such as Gram-negative bacteria and Mucorales, were more common in neutropenic patients. Pegivirus C (HPgV-C) infection often occurred post-transplantation, regardless of neutropenia. Additionally, some viruses such as TTV, B19V, EBV, and HPgV-C showed preferences for age and seasonal infections.
CONCLUSIONS: Analysis of the plasma virome revealed the susceptibility of haematological patients to plasma viral infections at specific disease stages, along with the occurrence of mixed infections with non-viral pathogens. Close associations were observed between the plasma virome and various clinical characteristics, as well as clinical detection parameters. Understanding plasma virome aids in auxiliary clinical diagnosis and treatment, enabling early prevention to reduce infection rates in patients and improve their quality of life. Video Abstract.

The insect order Blattodea (cockroaches and termites) has drawn substantial research attention for their dietary habits and lifestyle of living with or around humans. In the present study, we focused on the discovery of RNA viruses hidden in Blattodea insects using the publicly available RNA sequencing datasets. Overall, 136 distinctive RNA viruses were identified from 36 Blattodea species, of which more than 70 % were most closely related to the invertebrate-associated viral groups within Picornavirales, Sobelivirales, Bunyaviricetes, Jingchuvirales, Durnavirales, Lispiviridae, Orthomyxoviridae, Permutotetraviridae, Flaviviridae and Muvirales. Several viruses were associated with pathogens of vertebrates (Paramyxoviridae), plants (Tymovirales), protozoa (Totiviridae), fungi (Narnaviridae) and bacteria (Norzivirales). Collectively, 93 complete or near-complete viral genomes were retrieved from the datasets, and several viruses appeared to have remarkable temporal and spatial distributions. Interestingly, the newly identified Periplaneta americana dicistrovirus displayed a remarkable distinct bicistronic genome arrangement from the well-recognized dicistroviruses with the translocated structural and non-structural polyprotein encoding open reading frames over the genome. These results significantly enhance our knowledge of RNA virosphere in Blattodea insects, and the novel genome architectures in dicistroviruses and other RNA viruses may break our stereotypes in the understanding of the genomic evolution and the emergence of potential novel viral species.

Mangrove forests, a critical coastal ecosystem, face numerous anthropogenic threats, particularly from aquaculture activities. Despite the acknowledged significance of viruses in local and global biogeochemical cycles, there is limited knowledge regarding the community structure, genomic diversity, and ecological roles of viruses in mangrove forests ecosystems, especially regarding their responses to aquaculture. In this study, we identified 17,755 viral operational taxonomic units (vOTUs) from nine sediments viromes across three distinct ecological regions of the mangrove forests ecosystem: mangrove, bare flat, and aquaculture regions. Viral assemblages varied among three regions, and the pathogenic viruses associated with marine animals, such as the white spot syndrome virus (WSSV) from Nimaviridae, were identified in this study. The relative abundance of Nimaviridae in the bare flat region was higher than in other regions. Furthermore, viruses in distinct mangrove forests sediments regions have adapted to their environments by adopting distinct survival strategies and encoding various auxiliary metabolic genes involved in carbon metabolism and antibiotic resistance. These adaptations may have profound impacts on biogeochemical cycles. This study provides the first insights into the effects of vegetation cover and aquaculture on the community structure and ecological roles of viruses in mangrove forests sediments. These findings are crucial for understanding the risks posed by anthropogenic threats to mangrove forests ecosystems and informing effective management strategies.