Transmission of Single HIV-1 Genomes and Dynamics of Early Immune Escape Revealed by Ultra-Deep Sequencing

We used ultra-deep sequencing to obtain tens of thousands of HIV-1 sequences from regions targeted by CD8+ T lymphocytes from longitudinal samples from three acutely infected subjects, and modeled viral evolution during the critical first weeks of infection. Previous studies suggested that a single virus established productive infection, but these conclusions were tempered because of limited sampling; now, we have greatly increased our confidence in this observation through modeling the observed earliest sample diversity based on vastly more extensive sampling. Conventional sequencing of HIV-1 from acute/early infection has shown different patterns of escape at different epitopes; we investigated the earliest escapes in exquisite detail. Over 3–6 weeks, ultradeep sequencing revealed that the virus explored an extraordinary array of potential escape routes in the process of evading the earliest CD8 T-lymphocyte responses – using 454 sequencing, we identified over 50 variant forms of each targeted epitope during early immune escape, while only 2–7 variants were detected in the same samples via conventional sequencing. In contrast to the diversity seen within epitopes, non-epitope regions, including the Envelope V3 region, which was sequenced as a control in each subject, displayed very low levels of variation. In early infection, in the regions sequenced, the consensus forms did not have a fitness advantage large enough to trigger reversion to consensus amino acids in the absence of immune pressure. In one subject, a genetic bottleneck was observed, with extensive diversity at the second time point narrowing to two dominant escape forms by the third time point, all within two months of infection. Traces of immune escape were observed in the earliest samples, suggesting that immune pressure is present and effective earlier than previously reported; quantifying the loss rate of the founder virus suggests a direct role for CD8 T-lymphocyte responses in viral containment after peak viremia. Dramatic shifts in the frequencies of epitope variants during the first weeks of infection revealed a complex interplay between viral fitness and immune escape

Capturing Single Cell Genomes of Active Polysaccharide Degraders: An Unexpected Contribution of Verrucomicrobia

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Genomic sequencing of five cells, representing the most predominant, polysaccharide-active Verrucomicrobia phylotype, revealed significant enrichment in genes encoding a wide spectrum of glycoside hydrolases, sulfatases, peptidases, carbohydrate lyases and esterases, confirming that these organisms were well equipped for the hydrolysis of diverse polysaccharides. Remarkably, this enrichment was on average higher than in the sequenced representatives of Bacteroidetes, which are frequently regarded as highly efficient biopolymer degraders. These findings shed light on the ecological roles of uncultured Verrucomicrobia and suggest specific taxa as promising bioprospecting targets. The employed method offers a powerful tool to rapidly identify and recover discrete genomes of active players in polysaccharide degradation, without the need for cultivation

Forest floor community metatranscriptomes identify fungal and bacterial responses to N deposition in two maple forests

Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 75,000 carbohydrate active enzyme transcript sequences (CAZymes) in each metatranscriptome. Parallel ribosomal RNA surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa

Species within the Neoromicia bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, Neoromicia capensis have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae, Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the Adenoviridae, Coronaviridae, and Herpesviridae families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout Neoromicia populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard.S1 File. Virome sequence data information from the Parvoviridae and Papillomaviridae families (with references and figures).S1 Table. Neoromicia samples collected to investigate the South African Neoromicia virome.S3 Table. Neoromicia samples pooled for molecular detection of selected viruses.S4 Table. Alpha- and Betacoronavirus genera hemi-nested RT-PCR primers.S5 Table. Pairwise similarities inferred from distance estimations of full length genomes of the Circoviridae family. Sequence similarities of viruses in the Circoviridae family inferred from estimated evolutionary divergence calculated from pairwise distances. Full genomes were aligned and trimmed to 1075 overlapping positions. All ambiguous positions were removed for each sequence pair. Analyses were conducted in MEGA7 [41].S6 Table. Pairwise similarities inferred from distance estimations of an L gene region between selected Bunyavirales. The table shows pairwise sequence similarities inferred from evolutionary divergence estimates of 249 positions of compared Bunyavirales. The number of base differences per site from between sequences were converted to percentage of similarities. Standard errors for distance estimates are shown above the diagonal. Codon positions included were 1±3 as well as noncoding. Ambiguous positions were removed for each sequence pair as per pairwise deletion. Estimates were analysed in MEGA7 [41].S7 Table. Pairwise similarities inferred from distance estimations of a 605bp conserved segment of the coronavirus RNA dependent RNA polymerase gene. The table shows pairwise sequence similarities inferred from evolutionary divergence estimates of 605 positions of compared coronaviruses. The number of base differences per site from between sequences were converted to percentage of similarities. The sequences from this study are highlighted in grey and closest similarities to sequences from other studies are indicated in bold. Standard errors for distance estimates are shown above the diagonal in grey text. Codon positions included were 1±3 as well as noncoding and ambiguous positions were removed for each sequence pair as per pairwise deletion. Estimates were analysed in MEGA 7 [41].S8 Table. Genome annotation of BtCoVNeo5038 with similarities to compared lineage C betacoronaviruses. The percentage similarities were inferred from pairwise distance estimates of the base pair and amino acid differences per site for each gene and of the overall genome. The estimates were calculated in MEGA7 [41] using pairwise deletion to treat gaps. All percentage similarities are given in comparison to BtCoVNeoV5038. Accession number of compared betacoronaviruses are listed in order: KC869678.4, KX574227, EF065505.1, EF065509.1, JX869059.2, KF958702.1, KF917527.1, and KJ477102.1.S9 Table. Pairwise similarities inferred from distance estimations between betacoronavirus full genomes. The table shows pairwise similarities inferred from evolutionary divergence estimates of betacoronavirus full genomes. The number of base differences per site between sequences were converted to percentage similarities. Within lineage similarities are indicated in shaded blocks. Standard errors for distance estimates are shown above the diagonal in grey text. Codon positions included were 1±3 as well as noncoding and ambiguous positions were removed for each sequence pair as per pairwise deletion. Estimates were analysed in MEGA 7 [41].S10 Table. Coronavirus strains and Genbank accession numbers of sequences used in the full genome phylogeny.This work was financially supported in part by the National Research Foundation (NRF) of South Africa: the South African Research Chair held by WMgrant no. 98339, as well as grant numbers 92524, 85756, and 91496. The opinions, findings and conclusions expressed are those of the authors alone, and the NRF accepts no liability in this regard for research supported. Additional support was obtained byWMfrom the Poliomyelitis Research Foundation (grant number: 12/14). The Research Trust of the National Health Laboratory Service and the Medical Research Council was awarded to JW. This research was partially supported by the Grant or Cooperative Agreement Number [5 NU2GGH001874-02-00], funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services. MG was supported by funding from the NRF's Innovation bursary award (grant UID: 79409), the Poliomyelitis Research Foundation (grant no. 13/48), and the postgraduate study abroad bursary program of the University of Pretoria, which funded a research visit to the Los Alamos National Laboratory in New Mexico.http://www.plosone.orgam2018Mammal Research InstituteMedical Virolog

California condor microbiomes: Bacterial variety and functional properties in captive-bred individuals.

Around the world, scavenging birds such as vultures and condors have been experiencing drastic population declines. Scavenging birds have a distinct digestive process to deal with higher amounts of bacteria in their primary diet of carcasses in varying levels of decay. These observations motivate us to present an analysis of captive and healthy California condor (Gymnogyps californianus) microbiomes to characterize a population raised together under similar conditions. Shotgun metagenomic DNA sequences were analyzed from fecal and cloacal samples of captive birds. Classification of shotgun DNA sequence data with peptide signatures using the Sequedex package provided both phylogenetic and functional profiles, as well as individually annotated reads for targeted confirmatory analysis. We observed bacterial species previously associated with birds and gut microbiomes, including both virulent and opportunistic pathogens such as Clostridium perfringens, Propionibacterium acnes, Shigella flexneri, and Fusobacterium mortiferum, common flora such as Lactobacillus johnsonii, Lactobacillus ruminus, and Bacteroides vulgatus, and mucosal microbes such as Delftia acidovorans, Stenotrophomonas maltophilia, and Corynebacterium falsnii. Classification using shotgun metagenomic reads from phylogenetic marker genes was consistent with, and more specific than, analysis based on 16S rDNA data. Classification of samples based on either phylogenetic or functional profiles of genomic fragments differentiated three types of samples: fecal, mature cloacal and immature cloacal, with immature birds having approximately 40% higher diversity of microbes

Correction: California condor microbiomes: Bacterial variety and functional properties in captive-bred individuals.

[This corrects the article DOI: 10.1371/journal.pone.0225858.]

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in <i>Neoromicia</i> bats within South Africa

<div><p>Species within the <i>Neoromicia</i> bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, <i>Neoromicia capensis</i> have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the <i>Circoviridae</i> family, and also identified sequence data from the <i>Adenoviridae</i>, <i>Coronaviridae</i>, <i>Herpesviridae</i>, <i>Parvoviridae</i>, <i>Papillomaviridae</i>, <i>Phenuiviridae</i>, and <i>Picornaviridae</i> families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the <i>Adenoviridae</i>, <i>Coronaviridae</i>, and <i>Herpesviridae</i> families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous <i>Neoromicia</i>-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout <i>Neoromicia</i> populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard.</p></div