Effects of sunlight on bacteriophage viability and structure

Current estimates of viral abundance in natural waters rely on direct counts of virus-like particles (VLPs), using either transmission or epifluorescence microscopy. Direct counts of VLPs, while useful in studies of viral ecology, do not indicate whether the observed VLPs are capable of infection and/or replication. Rapid decay in bacteriophage viability under environmental conditions has been observed. However, it has not been firmly established whether there is a corresponding degradation of the virus particles, To address this question, viable and direct counts were carried out employing two Chesapeake Bay bacteriophages in experimental microcosms incubated for 56 h at two depths in the York River estuary, Viruses incubated in situ in microcosms at the surface yielded decay rates in full sunlight of 0.11 and 0.06 h(-1) for CB 38 Phi, and CB 7 Phi, respectively, The number of infective particles in microcosms in the dark and at a depth of 1 m was not significantly different from laboratory controls, with decay rates averaging 0.052 h(-1) for CB 38 Phi and 0.037 h(-1) for CB 7 Phi. Direct counts of bacteriophages decreased in the estuarine microcosms, albeit only at a rate of 0.028 h(-1), and were independent of treatment, Destruction of virus particles is concluded to be a process separate from loss of infectivity, It is also concluded that strong sunlight affects the viability of bacteriophages in surface waters, with the result that direct counts of VLPs overestimate the number of bacteriophage capable of both infection and replication, However, in deeper waters, where solar radiation is not a significant factor, direct counts should more accurately estimate numbers of viable bacteriophage

Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e≤0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9×107, and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L−1 respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L−1. Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L−1. In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny

Analysis of a viral metagenomic library from 200 m depth in Monterey Bay, California constructed by direct shotgun cloning

<p>Abstract</p> <p>Background</p> <p>Viruses have a profound influence on both the ecology and evolution of marine plankton, but the genetic diversity of viral assemblages, particularly those in deeper ocean waters, remains poorly described. Here we report on the construction and analysis of a viral metagenome prepared from below the euphotic zone in a temperate, eutrophic bay of coastal California.</p> <p>Methods</p> <p>We purified viruses from approximately one cubic meter of seawater collected from 200m depth in Monterey Bay, CA. DNA was extracted from the virus fraction, sheared, and cloned with no prior amplification into a plasmid vector and propagated in <it>E. coli </it>to produce the MBv200m library. Random clones were sequenced by the Sanger method. Sequences were assembled then compared to sequences in GenBank and to other viral metagenomic libraries using BLAST analyses.</p> <p>Results</p> <p>Only 26% of the 881 sequences remaining after assembly had significant (E ≤ 0.001) BLAST hits to sequences in the GenBank nr database, with most being matches to bacteria (15%) and viruses (8%). When BLAST analysis included environmental sequences, 74% of sequences in the MBv200m library had a significant match. Most of these hits (70%) were to microbial metagenome sequences and only 0.7% were to sequences from viral metagenomes. Of the 121 sequences with a significant hit to a known virus, 94% matched bacteriophages (Families <it>Podo</it>-, <it>Sipho</it>-, and <it>Myoviridae</it>) and 6% matched viruses of eukaryotes in the Family <it>Phycodnaviridae </it>(5 sequences) or the Mimivirus (2 sequences). The largest percentages of hits to viral genes of known function were to those involved in DNA modification (25%) or structural genes (17%). Based on reciprocal BLAST analyses, the MBv200m library appeared to be most similar to viral metagenomes from two other bays and least similar to a viral metagenome from the Arctic Ocean.</p> <p>Conclusions</p> <p>Direct cloning of DNA from diverse marine viruses was feasible and resulted in a distribution of virus types and functional genes at depth that differed in detail, but were broadly similar to those found in surface marine waters. Targeted viral analyses are useful for identifying those components of the greater marine metagenome that circulate in the subcellular size fraction.</p

A Minimal Model for Multiple Epidemics and Immunity Spreading

Pathogens and parasites are ubiquitous in the living world, being limited only by availability of suitable hosts. The ability to transmit a particular disease depends on competing infections as well as on the status of host immunity. Multiple diseases compete for the same resource and their fate is coupled to each other. Such couplings have many facets, for example cross-immunization between related influenza strains, mutual inhibition by killing the host, or possible even a mutual catalytic effect if host immunity is impaired. We here introduce a minimal model for an unlimited number of unrelated pathogens whose interaction is simplified to simple mutual exclusion. The model incorporates an ongoing development of host immunity to past diseases, while leaving the system open for emergence of new diseases. The model exhibits a rich dynamical behavior with interacting infection waves, leaving broad trails of immunization in the host population. This obtained immunization pattern depends only on the system size and on the mutation rate that initiates new diseases

Assessing the Diversity and Specificity of Two Freshwater Viral Communities through Metagenomics

Transitions between saline and fresh waters have been shown to be infrequent for microorganisms. Based on host-specific interactions, the presence of specific clades among hosts suggests the existence of freshwater-specific viral clades. Yet, little is known about the composition and diversity of the temperate freshwater viral communities, and even if freshwater lakes and marine waters harbor distinct clades for particular viral sub-families, this distinction remains to be demonstrated on a community scale

Impacts of Poultry House Environment on Poultry Litter Bacterial Community Composition

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly Gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens

Population Dynamics and Diversity of Viruses, Bacteria and Phytoplankton in a Shallow Eutrophic Lake

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 × 107 and 1.3 × 108 virus-like particles ml−1 and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus–host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht

Microviridae Goes Temperate: Microvirus-Related Proviruses Reside in the Genomes of Bacteroidetes

The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated

The GAAS Metagenomic Tool and Its Estimations of Viral and Microbial Average Genome Size in Four Major Biomes

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions