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Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra‐strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non‐Japanese isolates of the PPV‐Dideron (D) strain, we identified five positively selected sites in the PPV‐D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV‐D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra‐strain biological variation ascribed to positive selection. This is the first evidence of intra‐strain variation in plum pox virus ascribed to positive selection, especially reversible evolution that contributes to overcoming bottleneck by plant quarantine.

Significance Recent surveys of the microbial communities living on and in the human body—the human microbiome—have revealed strong variation in community membership between individuals. Some of this variation is stable over time, leading to speculation that individuals might possess unique microbial “fingerprints” that distinguish them from the population. We rigorously evaluated this idea by combining concepts from microbial ecology and computer science. Our results demonstrated that individuals could be uniquely identified among populations of 100s based on their microbiomes alone. In the case of the gut microbiome, >80% of individuals could still be uniquely identified up to a year later—a result that raises potential privacy concerns for subjects enrolled in human microbiome research projects. Community composition within the human microbiome varies across individuals, but it remains unknown if this variation is sufficient to uniquely identify individuals within large populations or stable enough to identify them over time. We investigated this by developing a hitting set-based coding algorithm and applying it to the Human Microbiome Project population. Our approach defined body site-specific metagenomic codes: sets of microbial taxa or genes prioritized to uniquely and stably identify individuals. Codes capturing strain variation in clade-specific marker genes were able to distinguish among 100s of individuals at an initial sampling time point. In comparisons with follow-up samples collected 30–300 d later, ∼30% of individuals could still be uniquely pinpointed using metagenomic codes from a typical body site; coincidental (false positive) matches were rare. Codes based on the gut microbiome were exceptionally stable and pinpointed >80% of individuals. The failure of a code to match its owner at a later time point was largely explained by the loss of specific microbial strains (at current limits of detection) and was only weakly associated with the length of the sampling interval. In addition to highlighting patterns of temporal variation in the ecology of the human microbiome, this work demonstrates the feasibility of microbiome-based identifiability—a result with important ethical implications for microbiome study design. The datasets and code used in this work are available for download from huttenhower.sph.harvard.edu/idability .

Epstein‐Barr virus (EBV) is a human tumor virus and is etiologically linked to various malignancies. Certain EBV‐associated diseases, such as Burkitt lymphomas and nasopharyngeal carcinomas, are endemic and exhibit biased geographic distribution worldwide. Recent advances in deep sequencing technology enabled high‐throughput sequencing of the EBV genome from clinical samples. Rapid cloning and sequencing of cancer‐derived EBV genomes, followed by reconstitution of infectious virus, have also become possible. These developments have revealed that various EBV strains are differentially distributed throughout the world, and that the behavior of cancer‐derived EBV strains is different from that of the prototype EBV strain of non‐cancerous origin. In this review, we summarize recent progress and future perspectives regarding the association between EBV strain variation and cancer. Recent advances in deep sequencing technology enabled high‐throughput sequencing of the EBV genome from clinical samples. We summarize recent progress and future perspectives regarding the association between EBV strain variation and cancer.

Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota. We generated complete genomes of the type strains G. apicola wkB1 ᵀ and S. alvi wkB2 ᵀ (isolated from Apis), as well as draft genomes for four other strains from Bombus . G. apicola and S. alvi were found to occupy very different metabolic niches: The former is a saccharolytic fermenter, whereas the latter is an oxidizer of carboxylic acids. Together, they may form a syntrophic network for partitioning of metabolic resources. Both species possessed numerous genes [type 6 secretion systems, repeats in toxin (RTX) toxins, RHS proteins, adhesins, and type IV pili] that likely mediate cell–cell interactions and gut colonization. Variation in these genes could account for the host fidelity of strains observed in previous phylogenetic studies. Here, we also show the first experimental evidence, to our knowledge, for this specificity in vivo: Strains of S. alvi were able to colonize their native bee host but not bees of another genus. Consistent with specific, long-term host association, comparative genomic analysis revealed a deep divergence and little or no gene flow between Apis and Bombus gut symbionts. However, within a host type (Apis or Bombus), we detected signs of horizontal gene transfer between G. apicola and S. alvi , demonstrating the importance of the broader gut community in shaping the evolution of any one member. Our results show that host specificity is likely driven by multiple factors, including direct host–microbe interactions, microbe–microbe interactions, and social transmission.

Objective Rhodococcus equi is an opportunistic pathogen that causes disease worldwide in young foals and immunocompromised humans. The interactions of R. equi with the host immune system have been described; however, most studies have been conducted using a few well-characterized strains. Because biological differences between R. equi strains are not well characterized, it is unknown if experimental results will replicate when different strains are used. Therefore, our objective was to compare the growth and biofilm formation of low-passage-rate clinical isolates of R. equi to higher-passage-rate, commonly studied isolates to determine whether strain-to-strain variation exists. Results Twelve strains were used: 103+, ATCC 33701, UKVDL206 103S harboring a GFP-expressing plasmid, a plasmid-cured 33701 (higher-passage-rate) and seven low-passage clinical isolates. Generation time in liquid revealed fast, moderate-fast, moderate-slow, and slow-growing isolates. The higher-passage-rate isolates were among the moderate-slow growing strains. A strain’s rate of growth did not correspond to its ability to form biofilm nor to its colony size on solid media. Based on our results, care should be taken not to extrapolate in vitro work that may be conducted using different R. equi strains. Further work is needed to evaluate the effect that the observed differences may have on experimental results.

Genomes computationally inferred from large metagenomic data sets are often incomplete and may be missing functionally important content and strain variation. We introduce an information retrieval system for large metagenomic data sets that exploits the sparsity of DNA assembly graphs to efficiently extract subgraphs surrounding an inferred genome. We apply this system to recover missing content from genome bins and show that substantial genomic sequence variation is present in a real metagenome. Our software implementation is available at https://github.com/spacegraphcats/spacegraphcats under the 3-Clause BSD License.

Background Toxoplasma gondii is an important pathogen and model organism for studying mechanisms of immune evasion and defense. Within the same strain, model organisms are typically assumed to be isogenic; for T. gondii , within-strain genetic divergence has been detected based on phenotypic changes and older molecular techniques but not characterized at the genomic level. We therefore used Oxford Nanopore long-read sequencing to characterize three independently maintained T. gondii ME49 isolates: 2015T and 2020T (obtained from ATCC and propagated in cell culture), and 2000B (propagated in mice). Results We de novo assembled a new T. gondii ME49 reference genome and, using state-of-the-art variant calling combined with pangenomic genotyping, detected variants between the sequenced isolates. Our new reference genome exceeded existing reference genomes in continuity (NG50 = 6.68 Mb versus 1.2 Mb in RefSeq) and structural accuracy, resolving all chromosomes except for a single break in the ribosomal DNA region. For isolates 2000B and 2020T, we identified 106 and 128 variants, respectively, across a final call set of 79 SNVs, 93 INDELs, and five structural variants; 18 small non-synonymous variants included genes associated with T. gondii life cycle (AP2X-8) and virulence in vivo (6-phosphogluconate dehydrogenase). A 13 kb expansion in the ROP8-ROP2A virulence locus increased the copy number of ROP2A-ROP8 genes in isolates 2000B and 2020T from three to six. Conclusions We provide an improved T. gondii ME49 reference genome and demonstrate the potentially confounding effect of intra-strain genetic heterogeneity, highlighting the need for continuous genomic monitoring for long-term genetic identity.

Background Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus , Lactobacillus helveticus and Lactobacillus delbrueckii . Results Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats. Conclusions Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.

Gut symbionts can augment resistance to pathogens by stimulating host-immune responses, competing for space and nutrients, or producing antimicrobial metabolites. Gut microbiota of social bees, which pollinate many crops and wildflowers, protect hosts against diverse infections and might counteract pathogen-related bee declines. Bumble bee gut microbiota, and specifically abundance of Lactobacillus ‘Firm-5’ bacteria, can enhance resistance to the trypanosomatid parasite Crithidia bombi. However, the mechanism underlying this effect remains unknown. We hypothesized that the Firm-5 bacterium Lactobacillus bombicola, which produces lactic acid, inhibits C. bombi via pH-mediated effects. Consistent with our hypothesis, L. bombicola spent medium inhibited C. bombi growth via reduction in pH that was both necessary and sufficient for inhibition. Inhibition of all parasite strains occurred within the pH range documented in honey bees, though sensitivity to acidity varied among strains. Spent medium was slightly more potent than HCl, d- and l-lactic acids for a given pH, suggesting that other metabolites also contribute to inhibition. Results implicate symbiont-mediated reduction in gut pH as a key determinant of trypanosomatid infection in bees. Future investigation into in vivo effects of gut microbiota on pH and infection intensity would test the relevance of these findings for bees threatened by trypanosomatids.

Many regions of the Epstein–Barr virus (EBV) genome, repeated and unique sequences, contribute to the geographical variation observed between strains. Here we use a large alignment of curated EBV genome sequences to identify major sites of variation in the genome of type 1 EBV strains; the CAO deletion in latent membrane protein 1 (LMP1) is the most frequent major indel present in the unique regions of EBV strains from various parts of the world. Principal component analysis was used to identify patterns of sequence variation and nucleotide positions in the sequences that can distinguish EBV from some different geographical regions. Viral genome sequence variation also affects interpretation of genetic content; known genes, origins of replication and gene expression control regions explain most of the viral genome but there are still a few sections of unknown function. One of these EBV genome regions contains a large inverted repeat sequence (invR) within the IR-1 major internal repeat array. We deleted this invR sequence and showed that this abolished the ability of the virus to transform human B cells into lymphoblastoid cell lines. This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.