Effects of Long Term Antibiotic Therapy on Human Oral and Fecal Viromes

Viruses are integral members of the human microbiome. Many of the viruses comprising the human virome have been identified as bacteriophage, and little is known about how they respond to perturbations within the human ecosystem. The intimate association of phage with their cellular hosts suggests their communities may change in response to shifts in bacterial community membership. Alterations to human bacterial biota can result in human disease including a reduction in the host''s resilience to pathogens. Here we report the ecology of oral and fecal viral communities and their responses to long-term antibiotic therapy in a cohort of human subjects. We found significant differences between the viral communities of each body site with a more heterogeneous fecal virus community compared with viruses in saliva. We measured the relative diversity of viruses, and found that the oral viromes were significantly more diverse than fecal viromes. There were characteristic changes in the membership of oral and fecal bacterial communities in response to antibiotics, but changes in fecal viral communities were less distinguishing. In the oral cavity, an abundance of papillomaviruses found in subjects on antibiotics suggests an association between antibiotics and papillomavirus production. Despite the abundance of papillomaviruses identified, in neither the oral nor the fecal viromes did antibiotic therapy have any significant impact upon overall viral diversity. There was, however, an apparent expansion of the reservoir of genes putatively involved in resistance to numerous classes of antibiotics in fecal viromes that was not paralleled in oral viromes. The emergence of antibiotic resistance in fecal viromes in response to long-term antibiotic therapy in humans suggests that viruses play an important role in the resilience of human microbial communities to antibiotic disturbances.     

Viral biogeography revealed by signatures in Sulfolobus islandicus genomes

Viruses are a driving force of microbial evolution. Despite their importance, the evolutionary dynamics that shape diversity in viral populations are not well understood. One of the primary factors that define viral population structure is coevolution with microbial hosts. Experimental models predict that the trajectory of coevolution will be determined by the relative migration rates of viruses and their hosts; however, there are no natural microbial systems in which both have been examined. The biogeographic distribution of viruses that infect Sulfolobus islandicus is investigated using genome comparisons among four newly identified, integrated, Sulfolobus spindle-shaped viruses and previously sequenced viral strains. Core gene sequences show a biogeographic distribution where viral genomes are specifically associated with each local population. In addition, signatures of host–virus interactions recorded in the sequence-specific CRISPR (clustered regularly interspaced short palindromic repeats) system show that hosts have interacted with viral communities that are more closely related to local viral strains than to foreign ones. Together, both proviral and CRISPR sequences show a clear biogeographic structure for Sulfolobus viral populations. Our findings demonstrate that virus–microbe coevolution must be examined in a spatially explicit framework. The combination of host and virus biogeography suggests a model for viral diversification driven by host immunity and local adaptation.     

Viral metagenomics

Viruses, most of which infect microorganisms, are the most abundant biological entities on the planet. Identifying and measuring the community dynamics of viruses in the environment is complicated because less than one percent of microbial hosts have been cultivated. Also, there is no single gene that is common to all viral genomes, so total uncultured viral diversity cannot be monitored using approaches analogous to ribosomal DNA profiling. Metagenomic analyses of uncultured viral communities circumvent these limitations and can provide insights into the composition and structure of environmental viral communities.     

CRISPR-Induced Distributed Immunity in Microbial Populations

In bacteria and archaea, viruses are the primary infectious agents, acting as virulent, often deadly pathogens. A form of adaptive immune defense known as CRISPR-Cas enables microbial cells to acquire immunity to viral pathogens by recognizing specific sequences encoded in viral genomes. The unique biology of this system results in evolutionary dynamics of host and viral diversity that cannot be fully explained by the traditional models used to describe microbe-virus coevolutionary dynamics. Here, we show how the CRISPR-mediated adaptive immune response of hosts to invading viruses facilitates the emergence of an evolutionary mode we call distributed immunity - the coexistence of multiple, equally-fit immune alleles among individuals in a microbial population. We use an eco-evolutionary modeling framework to quantify distributed immunity and demonstrate how it emerges and fluctuates in multi-strain communities of hosts and viruses as a consequence of CRISPR-induced coevolution under conditions of low viral mutation and high relative numbers of viral protospacers. We demonstrate that distributed immunity promotes sustained diversity and stability in host communities and decreased viral population density that can lead to viral extinction. We analyze sequence diversity of experimentally coevolving populations of Streptococcus thermophilus and their viruses where CRISPR-Cas is active, and find the rapid emergence of distributed immunity in the host population, demonstrating the importance of this emergent phenomenon in evolving microbial communities.     

Virioplankton: Viruses in Aquatic Ecosystems

The discovery that viruses may be the most abundant organisms in natural waters, surpassing the number of bacteria by an order of magnitude, has inspired a resurgence of interest in viruses in the aquatic environment. Surprisingly little was known of the interaction of viruses and their hosts in nature. In the decade since the reports of extraordinarily large virus populations were published, enumeration of viruses in aquatic environments has demonstrated that the virioplankton are dynamic components of the plankton, changing dramatically in number with geographical location and season. The evidence to date suggests that virioplankton communities are composed principally of bacteriophages and, to a lesser extent, eukaryotic algal viruses. The influence of viral infection and lysis on bacterial and phytoplankton host communities was measurable after new methods were developed and prior knowledge of bacteriophage biology was incorporated into concepts of parasite and host community interactions. The new methods have yielded data showing that viral infection can have a significant impact on bacteria and unicellular algae populations and supporting the hypothesis that viruses play a significant role in microbial food webs. Besides predation limiting bacteria and phytoplankton populations, the specific nature of virus-host interaction raises the intriguing possibility that viral infection influences the structure and diversity of aquatic microbial communities. Novel applications of molecular genetic techniques have provided good evidence that viral infection can significantly influence the composition and diversity of aquatic microbial communities.     

The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples

Viruses are the most abundant biological entities on our planet. Interactions between viruses and their hosts impact several important biological processes in the world's oceans such as horizontal gene transfer, microbial diversity and biogeochemical cycling. Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS) revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins. Cluster analyses of the viral sequences revealed hundreds to thousands of viral genes encoding various metabolic and cellular functions. Quantitative analyses of viral genes of host origin performed on the viral fraction of aquatic samples confirmed the viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such genetic material. Distributional and phylogenetic analyses of these host-derived viral sequences also suggested that viral acquisition of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated. The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying global distributions according to viral family. Recruitment of GOS viral sequence fragments against 27 complete aquatic viral genomes revealed that only one reference bacteriophage genome was highly abundant and was closely related, but not identical, to the cyanomyovirus P-SSM4. The co-distribution across all sampling sites of P-SSM4-like sequences with the dominant ecotype of its host, Prochlorococcus supports the classification of the viral sequences as P-SSM4-like and suggests that this virus may influence the abundance, distribution and diversity of one of the most dominant components of picophytoplankton in oligotrophic oceans. In summary, the abundance and broad geographical distribution of viral sequences within microbial fractions, the prevalence of genes among viral sequences that encode microbial physiological function and their distinct phylogenetic distribution lend strong support to the notion that viral-mediated gene acquisition is a common and ongoing mechanism for generating microbial diversity in the marine environment.     

Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome

Viruses are the most abundant known infectious agents on the planet and are significant drivers of diversity in a variety of ecosystems. Although there have been numerous studies of viral communities, few have focused on viruses within the indigenous human microbiota. We analyzed 2 267 695 virome reads from viral particles and compared them with 263 516 bacterial 16S rRNA gene sequences from the saliva of five healthy human subjects over a 2- to 3-month period, in order to improve our understanding of the role viruses have in the complex oral ecosystem. Our data reveal viral communities in human saliva dominated by bacteriophages whose constituents are temporally distinct. The preponderance of shared homologs between the salivary viral communities in two unrelated subjects in the same household suggests that environmental factors are determinants of community membership. When comparing salivary viromes to those from human stool and the respiratory tract, each group was distinct, further indicating that habitat is of substantial importance in shaping human viromes. Compared with coexisting bacteria, there was concordance among certain predicted host–virus pairings such as Veillonella and Streptococcus, whereas there was discordance among others such as Actinomyces. We identified 122 728 virulence factor homologs, suggesting that salivary viruses may serve as reservoirs for pathogenic gene function in the oral environment. That the vast majority of human oral viruses are bacteriophages whose putative gene function signifies some have a prominent role in lysogeny, suggests these viruses may have an important role in helping shape the microbial diversity in the human oral cavity.     

土壤病毒生态学研究方法

病毒是地球上最丰富的生物实体,每克土壤中可包含数以亿计的病毒,它不仅影响土壤中其它微生物的群落组成、土壤元素的生物地球化学循环,还会影响土壤微生物的物种进化,甚至影响植物、动物和人体健康。目前人们对土壤中病毒的种类及丰度、分布特征以及功能引起的生态环境效应还知之甚少。在概述病毒生态学研究方法的基础上,对土壤病毒的提取、纯化、定量及分子生态学方法等基本流程进行了比较分析,以期建立一套快速简便、高效稳定的适用于土壤病毒研究的方法,并用于研究土壤病毒的多样性及分布特征,探讨病毒在环境中的生存和传播机制,为土壤病毒的防控及开发利用提供支撑。     

Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses

Explorations of human microbiota have provided substantial insight into microbial community composition; however, little is known about interactions between various microbial components in human ecosystems. In response to the powerful impact of viral predation, bacteria have acquired potent defences, including an adaptive immune response based on the clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system. To improve our understanding of the interactions between bacteria and their viruses in humans, we analysed 13?977 streptococcal CRISPR sequences and compared them with 2?588?172 virome reads in the saliva of four human subjects over 17 months. We found a diverse array of viruses and CRISPR spacers, many of which were specific to each subject and time point. There were numerous viral sequences matching CRISPR spacers; these matches were highly specific for salivary viruses. We determined that spacers and viruses coexist at the same time, which suggests that streptococcal CRISPR/Cas systems are under constant pressure from salivary viruses. CRISPRs in some subjects were just as likely to match viral sequences from other subjects as they were to match viruses from the same subject. Because interactions between bacteria and viruses help to determine the structure of bacterial communities, CRISPR-virus analyses are likely to provide insight into the forces shaping the human microbiome.     

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.     

The world within: Quantifying the determinants and outcomes of a host's microbiome

The fungal, bacterial, and viral microbial communities embedded as endosymbionts within all free-living organisms are extremely diverse and encode the vast majority of genes in the biosphere. Microbes in a human, for example, account for 100 times more genes than their host; similar results are emerging for virtually all free-living organisms. Disease is the best studied host–microbe interaction, but endosymbiotic microbial populations and communities also are responsible for critical functions in their hosts including nutrient uptake (plants), reduction in inflammatory responses (animals), digestion (animals), anti-herbivore defenses (plants), and pathogen resistance. In spite of the tremendous diversity and functional importance of the microbial biome to free-living organisms, we have little predictive understanding of the biotic and abiotic factors controlling within-host microbial community composition or the spatial scales at which anthropogenic changes affect host and microbial community interactions and functions. Current research suggests that anthropogenic changes to nutrient supply and food web composition can affect biological systems at scales ranging from individuals to continents. However, while current studies are clarifying the effects of some of these drivers on the structure and functioning of ecosystems, we have far less knowledge of their effects on microbial communities residing within hosts. Given the accelerating progress in metagenome studies, we are poised to make rapid advances in understanding the determinants and effects of within-host microbial communities.     

Are low temperature habitats hot spots of microbial evolution driven by viruses?

There is an increasing body of evidence to show that viruses are important drivers of microbial evolution and that they can store a great deal of the Earth's microbial diversity in their genomes. Examination of microbial diversity in polar regions has revealed a higher than expected diversity of viruses, bacteria and eukaryotic microbes. Further, the few available studies in polar regions reveal that viral control of microbial mortality is important in these habitats. In this opinion article, we argue that strong relationships between viruses and their hosts in a range of polar habitats could be key in explaining why polar regions are in fact hot spots of microbial diversity and evolution. Further, we argue that periodic glaciations, and particularly the Neoproterozoic low-latitude glaciation, known as 'snowball Earth', could have been periods of intense diversification in aquatic refuges.     

The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches

The pool of mobile genetic elements (MGE) in microbial communities consists of plasmids, bacteriophages and other elements that are either self-transmissible or use mobile plasmids and phages as vehicles for their dissemination. By facilitating horizontal gene exchange, the horizontal gene pool (HGP) promotes the evolution and adaptation of microbial communities. Efforts to characterise MGE from bacterial populations resident in a variety of ecological habitats have revealed a surprisingly vast and seemingly untapped diversity. MGE, conferring such selectable traits as mercury or antibiotic resistance and degradative functions, have been readily acquired from diverse microbial communities. To circumvent the need to isolate microbial hosts, polymerase chain reaction (PCR)-based detection methods have frequently been used to assess the prevalence of MGE-specific sequences resident in the ‘microbial community’ HGP. As studies continue to reveal novel and distinct MGE, sequencing of newly isolated MGE from diverse habitats is essential for the continued development of DNA probes, PCR primers as well as for gene array and proteomics-based approaches. This minireview highlights insight gained from different methodological approaches, biased albeit largely toward plasmids in Gram-negative bacteria, used to study the HGP of naturally occurring microbial communities from various aquatic and terrestrial habitats.     

病毒编码的miRNA:基因表达新的调控因子

微小RNA(microRNA,miRNA)是一类长约22个核苷酸的RNA,在数量、序列、结构、表达和功能上具有多样性。目前,通过生物信息学手段和分子克隆方法,已发现了3518种miRNA,在控制细胞的生长发育、分化、凋亡等过程中发挥着十分重要的作用。最近研究发现疱疹病毒、多瘤病毒、逆转录病毒的某些病毒基因组也能够编码miRNA,这些miRNA在调控病毒基因自身表达以及病毒与宿主相互作用方面可能起重要的作用。某些病毒甚至能够利用宿主体内的miRNA调控其自身表达。找出病毒可能编码的miRNA,探索其对病毒感染、复制、表达的作用,有助于病毒分子生物学的研究,也会为研发防治病毒的新方法和新途径提供新的思路。     

Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis

H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G). Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.     

Genome-resolved viral ecology in a marine oxygen minimum zone

Oxygen minimum zones (OMZs) are critical to marine nitrogen cycling and global climate change. While OMZ microbial communities are relatively well-studied, little is known about their viruses. Here, we assess the viral community ecology of 22 deeply sequenced viral metagenomes along a gradient of oxygenated to anoxic waters (<0.02 μmol/l O₂) in the Eastern Tropical South Pacific (ETSP) OMZ. We identified 46 127 viral populations (≥5 kb), which augments the known viruses from ETSP by 10-fold. Viral communities clustered into six groups that correspond to oceanographic features. Oxygen concentration was the predominant environmental feature driving viral community structure. Alpha and beta diversity of viral communities in the anoxic zone were lower than in surface waters, which parallels the low microbial diversity seen in other studies. ETSP viruses were largely endemic, with the majority of shared viruses (87%) also present in other OMZ samples. We detected 543 putative viral-encoded auxiliary metabolic genes (AMGs), of which some have a distribution that reflects physico-chemical characteristics across depth. Together these findings provide an ecological baseline for viral community structure, drivers and population variability in OMZs that will help future studies assess the role of viruses in these climate-critical environments.     

Evolution and Horizontal Transfer of dUTPase-Encoding Genes in Viruses and Their Hosts

dUTPase is a ubiquitous and essential enzyme responsible for regulating cellular levels of dUTP. The dut gene exists as single, tandemly duplicated, and tandemly triplicated copies. Crystallized single-copy dUTPases have been shown to assemble as homotrimers. dUTPase is encoded as an auxiliary gene in a number of virus genomes. The origin of viral dut genes has remained unresolved since their initial discovery. A comprehensive analysis of dUTPase amino acid sequence relationships was performed to explore the evolutionary dynamics of dut in viruses and their hosts. Our data set, comprised of 24 host and 51 viral sequences, includes representative sequences from available eukaryotes, archaea, eubacteria cells, and viruses, including herpesviruses. These amino acid sequences were aligned by using a hidden Markov model approach developed to align divergent data. Known secondary structures from single-copy crystals were mapped onto the aligned duplicate and triplicate sequences. We show how duplicated dUTPases might fold into a monomer, and we hypothesize that triplicated dUTPases also assemble as monomers. Phylogenetic analysis revealed at least five viral dUTPase sequence lineages in well-supported monophyletic clusters with eukaryotic, eubacterial, and archaeal hosts. We have identified all five as strong examples of horizontal transfer as well as additional potential transfer of dut genes among eubacteria, between eubacteria and viruses, and between retroviruses. The evidence for horizontal transfers is particularly interesting since eukaryotic dut genes have introns, while DNA virus dut genes do not. This implies that an intermediary retroid agent facilitated the horizontal transfer process between host mRNA and DNA viruses.     

Metagenomic Analysis of Respiratory Tract DNA Viral Communities in Cystic Fibrosis and Non-Cystic Fibrosis Individuals

The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5′-triphosphate,3′-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota.