The genome of the human immunodeficiency virus is highly prone to recombination, although it is not obvious whether recombinants arise infrequently or whether they are constantly being spawned but escape identification because of the massive and rapid turnover of virus particles. Here we use fluorescence in situ hybridization to estimate the number of proviruses harboured by individual splenocytes from two HIV patients, and determine the extent of recombination by sequencing amplified DNA from these cells. We find an average of three or four proviruses per cell and evidence for huge numbers of recombinants and extensive genetic variation. Although this creates problems for phylogenetic analyses, which ignore recombination effects, the intracellular variation may help to broaden immune recognition.

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.

1. Viruses are the most abundant biological entities on the planet, and sediments provide a highly suitable environment for them. This review presents the first comparative synthesis of information on the fresh water and marine viriobenthos and explores differences and similarities to the better known virioplankton. We present methods for studying life cycles of the viriobenthos, data on viral distribution and diversity, interactions with host microbes, and information on the role of viruses in benthic food webs and biogeochemical cycles. 2. Most approaches developed for the virioplankton are also applicable to viriobenthos, although methods for analysing benthic viruses may differ in important details. 3. Benthic viruses are very abundant in both marine and freshwater sediments, where 10 7 -10 10 can occur in 1 g of dry sediment. Although information on viral production (VP) and decay rates in freshwater sediments is very limited, the data suggest that VP and decay could also be high. These data highlight the potential ecological importance of benthic viruses, suggesting that they could play a key role in prokaryotic mortality and in biogeochemical cycles. 4. There is clear indirect evidence for the importance of viriobenthos in marine and freshwater ecosystems. However, large numbers of visibly infected cells have not been observed, suggesting limited effects on prokaryote population and community dynamics. The apparent paradox between high viral abundance and low impact is currently unresolved, while several aspects of viral life cycles in sediments (e.g. chronic infection) are almost completely unknown. 5. Studies on viriobenthic diversity and community structure are at a pioneering stage. First results from a few studies using pulsed-field gel electrophoresis and especially from metagenomic analyses indicate, however, that viriobenthic assemblages are both highly diverse and distinct from the virioplankton.6. Estimates of global viral abundance in the top 1 m of fresh water and marine sediments are 0.5 and 28.7 · 10 28 viruses respectively. Similar rough estimates of production are 0.6 and 34.4 · 10 28 viruses day )1 , suggesting an average turnover time of 20 h.

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Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 ؋ 10 9 to 7.2 ؋ 10 9 particles ml of wet sediment ؊1 and from undetectable to 22.2 ؋ 10 7 particles ml ؊1 h ؊1 , respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.

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