Quantification of virus genes provides evidence for seed-bank populations of phycodnaviruses in Lake Ontario, Canada.
ABSTRACT: Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000?copies?ml(-1) and were considered 'high abundance', whereas the other two Prasinovirus-related genes peaked at abundances <1000?copies?ml(-1) and were considered 'low abundance'. Of the genes related to chloroviruses, one peaked at ca 1600?copies?ml(-1), whereas the other reached only ca 300?copies?ml(-1). Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100-1000?copies?ml(-1) while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100?copies?ml(-1). Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.
Project description:Viruses strongly influence the ecology and evolution of their eukaryotic hosts in the marine environment, but little is known about their diversity and distribution. Prasinoviruses infect an abundant and widespread class of phytoplankton, the Mamiellophyceae, and thereby exert a specific and important role in microbial ecosystems. However, molecular tools to specifically identify this viral genus in environmental samples are still lacking. We developed two primer sets, designed for use with polymerase chain reactions and 454 pyrosequencing technologies, to target two conserved genes, encoding the DNA polymerase (PolB gene) and the major capsid protein (MCP gene). While only one copy of the PolB gene is present in Prasinovirus genomes, there are at least seven paralogs for MCP, the copy we named number 6 being shared with other eukaryotic alga-infecting viruses. Primer sets for PolB and MCP6 were thus designed and tested on 6 samples from the Tara Oceans project. The results suggest that the MCP6 amplicons show greater richness but that PolB gave a wider coverage of Prasinovirus diversity. As a consequence, we recommend use of the PolB primer set, which will certainly reveal exciting new insights about the diversity and distribution of prasinoviruses at the community scale.
Project description:The Agrocybe aegerita mitochondrial genome contains a truncated family B DNA polymerase gene (Aa-polB P1) whose nucleotide sequence is 86% identical to the previously described and potentially functional Aa-polB gene. A tRNA(Met) gene occurs at the 3' end of the Aa-polB P1 gene. The Aa-polB P1 gene could result from reverse transcription of an Aa-polB mRNA primed by a tRNA(Met) followed by the integration of the cDNA after recombination at the mitochondrial tRNA locus. Two naturally occurring alleles of Aa-polB P1 carry one or two copies of the disrupted sequence. In strains with two copies of Aa-polB P1, these copies are inverted relative to one another and separated by a short sequence carrying the tRNA(Met) gene. Both A. aegerita mitochondrial family B DNA polymerases were found to be related to other family B DNA polymerases (36 to 53% amino acid similarity), including the three enzymes of the archaebacterium Sulfolobus solfataricus. If mitochondria originated from a fusion between a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium, then the A. aegerita family B DNA polymerase genes could be remnants of the archaebacterial genes.
Project description:Marine dinitrogen (N2) fixation studies have focused nearly exclusively on cyanobacterial diazotrophs; however ?-proteobacteria are an abundant component of the marine community and have been largely overlooked until recently. Here we present a phylogenetic analysis of all nifH ?-proteobacterial sequences available in public databases and qPCR data of a ?-proteobacterial phylotype, Gamma A (UMB), obtained during several research cruises. Our analysis revealed a complex diversity of diazotrophic ?-proteobacteria. One phylotype in particular, Gamma A, was described in several traditional and quantitative PCR studies. Though several ?-proteobacterial nifH sequences have been described as laboratory contaminants, Gamma A is part of a large cluster of sequences isolated from marine environments and distantly related to the clade of contaminants. Using a TaqMan probe and primer set, Gamma A nifH DNA abundance and expression were analyzed in nearly 1000 samples collected during 15 cruises to the Atlantic and Pacific Oceans. The data showed that Gamma A is an active, cosmopolitan diazotroph found throughout oxygenated, oligotrophic waters reaching maximum abundances of 8 x 104 nifH DNA copies l-1 and 5 x 105 nifH transcript copies l-1. Gamma A nifH transcript abundances were on average 3 fold higher than nifH DNA abundances. The widespread distribution and activity of Gamma A indicate that it has potential to be a globally important N2 fixing organism.
Project description:Phycodnaviruses are widespread algae-infecting large dsDNA viruses and presently contain six genera: Chlorovirus, Prasinovirus, Prymnesiovirus, Phaeovirus, Coccolithovirus and Raphidovirus. The members in Prasinovirus are identified as marine viruses due to their marine algal hosts, while prasinovirus freshwater relatives remain rarely reported.Here we present the complete genomic sequence of a novel phycodnavirus, Dishui Lake Phycodnavirus 1 (DSLPV1), which was assembled from Dishui Lake metagenomic datasets. DSLPV1 harbors a linear genome of 181,035 bp in length (G?+?C content: 52.7%), with 227 predicted genes and 2 tRNA encoding regions. Both comparative genomic and phylogenetic analyses indicate that the freshwater algal virus DSLPV1 is closely related to the members in Prasinovirus, a group of marine algae infecting viruses. In addition, a complete eukaryotic histone H3 variant was identified in the genome of DSLPV1, which is firstly detected in phycodnaviruses and contributes to understand the interaction between algal virus and its eukaryotic hosts.It is in a freshwater ecosystem that a novel Prasinovirus-related viral complete genomic sequence is discovered, which sheds new light on the evolution and diversity of the algae infecting Phycodnaviridae.
Project description:BACKGROUND:The PENPACT-1 trial compared virologic thresholds to determine when to switch to second-line antiretroviral therapy (ART). Using PENPACT-1 data, we aimed to describe HIV-1 drug resistance accumulation on first-line ART by virologic threshold. METHODS:PENPACT-1 had a 2 × 2 factorial design, randomizing HIV-infected children to start protease inhibitor (PI) versus nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART, and switch at a 1000 copies/mL versus 30,000 copies/mL threshold. Switch criteria were not achieving the threshold by week 24, confirmed rebound above the threshold thereafter, or Center for Disease Control and Prevention stage C event. Resistance tests were performed on samples ?1000 copies/mL before switch, resuppression, and at 4-years/trial end. RESULTS:Sixty-seven children started PI-based ART and were randomized to switch at 1000 copies/mL (PI-1000), 64 PIs and 30,000 copies/mL (PI-30,000), 67 NNRTIs and 1000 copies/mL (NNRTI-1000), and 65 NNRTI and 30,000 copies/mL (NNRTI-30,000). Ninety-four (36%) children reached the 1000 copies/mL switch criteria during 5-year follow-up. In 30,000 copies/mL threshold arms, median time from 1000 to 30,000 copies/mL switch criteria was 58 (PI) versus 80 (NNRTI) weeks (P = 0.81). In NNRTI-30,000, more nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations accumulated than other groups. NNRTI mutations were selected before switching at 1000 copies/mL (23% NNRTI-1000, 27% NNRTI-30,000). Sixty-two children started abacavir + lamivudine, 166 lamivudine + zidovudine or stavudine, and 35 other NRTIs. The abacavir + lamivudine group acquired fewest NRTI mutations. Of 60 switched to second-line, 79% PI-1000, 63% PI-30,000, 64% NNRTI-1000, and 100% NNRTI-30,000 were <400 copies/mL 24 weeks later. CONCLUSIONS:Children on first-line NNRTI-based ART who were randomized to switch at a higher virologic threshold developed the most resistance, yet resuppressed on second-line. An abacavir + lamivudine NRTI combination seemed protective against development of NRTI resistance.
Project description:Quantitative plasma viral load (VL) at 1000 copies /mL was recommended as the threshold to confirm antiretroviral therapy (ART) failure by the World Health Organization (WHO). Because of ongoing challenges of using plasma for VL testing in resource-limited settings (RLS), especially for children, this study collected 717 DBS and paired plasma samples from children receiving ART ≥1 year in Mozambique and compared the performance of DBS using Abbott's VL test with a paired plasma sample using Roche's VL test. At a cut-off of 1000 copies/mL, sensitivity of DBS using Abbott DBS VL test was 79.9%, better than 71.0% and 63.9% at 3000 and 5000 copies/mL, respectively. Specificities were 97.6%, 98.8%, 99.3% at 1000, 3000, and 5000 copies/mL, respectively. The Kappa value at 1000 copies/mL, 0.80 (95% CI: 0.73, 0.87), was higher than 0.73 (95% CI: 0.66, 0.80) and 0.66 (95% CI: 0.59, 0.73) at 3000, 5000 copies/mL, respectively, also indicating better agreement. The mean difference between the DBS and plasma VL tests with 95% limits of agreement by Bland-Altman was 0.311 (-0.908, 1.530). Among 73 children with plasma VL between 1000 to 5000 copies/mL, the DBS results were undetectable in 53 at the 1000 copies/mL threshold. While one DBS sample in the Abbott DBS VL test may be an alternative method to confirm ART failure at 1000 copies/mL threshold when a plasma sample is not an option for treatment monitoring, because of sensitivity concerns between 1,000 and 5,000 copies/ml, two DBS samples may be preferred accompanied by careful patient monitoring and repeat testing.
Project description:To investigate the responses to switching to second-line regimens among patients who had received a long-term first-line antiretroviral therapy.Patients switching to second-line regimens from June 2008 to June 2015 were enrolled from an observational cohort. In addition, patients continuing first-line therapy and had a viral load <1000?copies/mL were included as controls in July 2012. All these patients were followed-up for 36 months or until June 2016. The virological, immunological outcomes, and drug resistance were evaluated. Virological failure was defined as viral load ?1000?copies/mL after 6 months of treatment since the start of the study.There were 304 patients switching to second-line regimens and 46 patients remaining on first-line therapy enrolled while having received first-line therapy for a median of 7.6 years. Patients with plasma viral load (VL) ?1000?copies/mL before switching to second-line regimens had a sharp decline in the proportion of virological failure with 26.7%, 20.4%, and 17.0% at 12, 24, and 36 months after regimen switch, respectively (trend test, P?<?.001). Among these patients, individuals with drug resistance (DR) had a better virological responses as compared with those without DR after regimen switching. While patients with VL <1000?copies/mL at inclusion remained a high rate of viral suppression after switching to second-line regimens. So did patients continuing first-line therapy. Among patients with VL ?1000?copies/mL before switching to second-line regimens, the rates of drug resistance were decreased from 79.4% at inclusion to 7.5% at 36 months of regimen switch, with the proportion of NRTI- and NNRTI-related drug resistance from 67.2% and 79.4% to 5.4% and 7.5%, respectively. No PI-related resistance was found. Having self-reported missing doses within a month at follow-ups were independently associated with virological failure at 36 months of switching.HIV-infected patients had viral load ?1000?copies/mL at regimen switch after a long duration of first-line therapy had good virological responses to second-line regimens, especially those harbored drug resistant variants at regimen switch. However, patients with suppressive first-line therapy did not appear to benefit virologically from switching to second-line regimens.
Project description:The World Health Organization (WHO) guidelines on antiretroviral therapy (ART) define treatment failure as 2 consecutive viral loads (VLs) ?1000?copies/mL. There is, however, little evidence supporting 1000 copies as an optimal threshold to define treatment failure. Objective of this study was to assess the correlation of the WHO definition with the presence of drug-resistance mutations in patients who present with 2 consecutive unsuppressed VL in a resource-limited setting.In 10 nurse-led clinics in rural Lesotho children and adults on first-line ART for ?6 months received a first routine VL. Those with plasma VL ?80?copies/mL were enrolled in a prospective study, receiving enhanced adherence counseling (EAC) and a follow-up VL after 3 months. After a second unsuppressed VL genotypic resistance testing was performed. Viruses with major mutations against ?2 drugs of the current regimen were classified as "resistant".A total of 1563 adults and 191 children received a first routine VL. Of the 138 adults and 53 children with unsuppressed VL (?80?copies/mL), 165 (116 adults; 49 children) had a follow-up VL after EAC; 108 (74 adults; 34 children) remained unsuppressed and resistance testing was successful. Ninety of them fulfilled the WHO definition of treatment failure (both VL ?1000?copies/mL); for another 18 both VL were unsuppressed but with <1000?copies/mL. The positive predictive value (PPV) for the WHO failure definition was 81.1% (73/90) for the presence of resistant virus. Among the 18 with VL levels between 80 and 1000?copies/mL, thereby classified as "non-failures", 17 (94.4%) harbored resistant viruses. Lowering the VL threshold from 1000?copies/mL to 80?copies/mL at both determinations had no negative influence on the PPV (83.3%; 90/108).The current WHO-definition misclassifies patients who harbor resistant virus at VL below 1000?c/mL as "nonfailing." Lowering the threshold to VL ?80?copies/mL identifies a significantly higher number of patients with treatment-resistant virus and should be considered.
Project description:Giant viruses in the genus Chlorovirus (family Phycodnaviridae) infect eukaryotic green microalgae. The prototype member of the genus, Paramecium bursaria chlorella virus 1, was sequenced more than 15 years ago, and to date there are only 6 fully sequenced chloroviruses in public databases. Presented here are the draft genome sequences of 35 additional chloroviruses (287 - 348 Kb/319 - 381 predicted protein encoding genes) collected across the globe; they infect one of three different green algal species. These new data allowed us to analyze the genomic landscape of 41 chloroviruses, which revealed some remarkable features about these viruses.Genome colinearity, nucleotide conservation and phylogenetic affinity were limited to chloroviruses infecting the same host, confirming the validity of the three previously known subgenera. Clues for the existence of a fourth new subgenus indicate that the boundaries of chlorovirus diversity are not completely determined. Comparison of the chlorovirus phylogeny with that of the algal hosts indicates that chloroviruses have changed hosts in their evolutionary history. Reconstruction of the ancestral genome suggests that the last common chlorovirus ancestor had a slightly more diverse protein repertoire than modern chloroviruses. However, more than half of the defined chlorovirus gene families have a potential recent origin (after Chlorovirus divergence), among which a portion shows compositional evidence for horizontal gene transfer. Only a few of the putative acquired proteins had close homologs in databases raising the question of the true donor organism(s). Phylogenomic analysis identified only seven proteins whose genes were potentially exchanged between the algal host and the chloroviruses.The present evaluation of the genomic evolution pattern suggests that chloroviruses differ from that described in the related Poxviridae and Mimiviridae. Our study shows that the fixation of algal host genes has been anecdotal in the evolutionary history of chloroviruses. We finally discuss the incongruence between compositional evidence of horizontal gene transfer and lack of close relative sequences in the databases, which suggests that the recently acquired genes originate from a still largely un-sequenced reservoir of genomes, possibly other unknown viruses that infect the same hosts.
Project description:BACKGROUND:Many individuals failing first-line antiretroviral therapy (ART) in sub-Saharan Africa never initiate second-line ART or do so after significant delay. For people on ART with a viral load more than 1000 copies/ml, the WHO recommends a second viral load measurement 3 months after the first viral load and enhanced adherence support. Switch to a second-line regimen is contingent upon a persistently elevated viral load more than 1000 copies/ml. Delayed second-line switch places patients at increased risk for opportunistic infections and mortality. METHODS:To assess the potential benefits of a simplified second-line ART switch strategy, we use an individual-based model of HIV transmission, progression and the effect of ART which incorporates consideration of adherence and drug resistance, to compare predicted outcomes of two policies, defining first-line regimen failure for patients on efavirenz-based ART as either two consecutive viral load values more than 1000 copies/ml, with the second after an enhanced adherence intervention (implemented as per current WHO guidelines) or a single viral load value more than 1000 copies/ml. We simulated a range of setting-scenarios reflecting the breadth of the sub-Saharan African HIV epidemic, taking into account potential delays in defining failure and switch to second-line ART. FINDINGS:The use of a single viral load more than 1000 copies/ml to define ART failure would lead to a higher proportion of persons with nonnucleoside reverse-transcriptase inhibitor resistance switched to second-line ART [65 vs. 48%; difference 17% (90% range 14-20%)], resulting in a median 18% reduction in the rate of AIDS-related death over setting scenarios (90% range 6-30%; from a median of 3.1 to 2.5 per 100 person-years) over 3 years. The simplified strategy also is predicted to reduce the rate of AIDS conditions by a median of 31% (90% range 8-49%) among people on first-line ART with a viral load more than 1000 copies/ml in the past 6 months. For a country of 10 million adults (and a median of 880?000 people with HIV), we estimate that this approach would lead to a median of 1322 (90% range 67-3513) AIDS deaths averted per year over 3 years. For South Africa this would represent around 10?215 deaths averted annually. INTERPRETATION:As a step towards reducing unnecessary mortality associated with delayed second-line ART switch, defining failure of first-line efavirenz-based regimens as a single viral load more than 1000 copies/ml should be considered.