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HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. Tracking clonal behavior by proviral “zip codes” indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP- cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in the sense orientation of genes were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP- population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics. [ABSTRACT FROM AUTHOR]

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process. [ABSTRACT FROM AUTHOR]

Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes. [ABSTRACT FROM AUTHOR]

It has been proposed that in wild ecosystems viruses are often plant mutualists, whereas agroecosystems favour pathogenicity. We seek evidence for virus pathogenicity in wild ecosystems through the analysis of plant-virus coevolution, which requires a negative effect of infection on the host fitness. We focus on the interaction between Arabidopsis thaliana and Cucumber mosaic virus (CMV), which is significant in nature. We studied the genetic diversity of A. thaliana for two defence traits, resistance and tolerance, to CMV. A set of 185 individuals collected in 76 A. thaliana Iberian wild populations were inoculated with different CMV strains. Resistance was estimated from the level of virus multiplication in infected plants, and tolerance from the effect of infection on host progeny production. Resistance and tolerance to CMV showed substantial genetic variation within and between host populations, and depended on the virus x host genotype interaction, two conditions for coevolution. Resistance and tolerance were co-occurring independent traits that have evolved independently from related life-history traits involved in adaptation to climate. The comparison of the genetic structure for resistance and tolerance with that for neutral traits (QST/FST analyses) indicated that both defence traits are likely under uniform selection. These results strongly suggest that CMV infection selects for defence on A. thaliana populations, and support plant-virus coevolution. Thus, we propose that CMV infection reduces host fitness under the field conditions of the wild A. thaliana populations studied. [ABSTRACT FROM AUTHOR]

Resistance against different antibiotics appears on the same bacterial strains more often than expected by chance, leading to high frequencies of multidrug resistance. There are multiple explanations for this observation, but these tend to be specific to subsets of antibiotics and/or bacterial species, whereas the trend is pervasive. Here, we consider the question in terms of strain ecology: explaining why resistance to different antibiotics is often seen on the same strain requires an understanding of the competition between strains with different resistance profiles. This work builds on models originally proposed to explain another aspect of strain competition: the stable coexistence of antibiotic sensitivity and resistance observed in a number of bacterial species. We first identify a partial structural similarity in these models: either strain or host population structure stratifies the pathogen population into evolutionarily independent sub-populations and introduces variation in the fitness effect of resistance between these sub-populations, thus creating niches for sensitivity and resistance. We then generalise this unified underlying model to multidrug resistance and show that models with this structure predict high levels of association between resistance to different drugs and high multidrug resistance frequencies. We test predictions from this model in six bacterial datasets and find them to be qualitatively consistent with observed trends. The higher than expected frequencies of multidrug resistance are often interpreted as evidence that these strains are out-competing strains with lower resistance multiplicity. Our work provides an alternative explanation that is compatible with long-term stability in resistance frequencies. [ABSTRACT FROM AUTHOR]

Knowledge of the within-host frequencies of resistance-associated amino acid variants (RAVs) is important to the identification of optimal drug combinations for the treatment of hepatitis C virus (HCV) infection. Multiple RAVs may exist in infected individuals, often below detection limits, at any resistance locus, defining the diversity of accessible resistance pathways. We developed a multiscale mathematical model to estimate the pre-treatment frequencies of the entire spectrum of mutants at chosen loci. Using a codon-level description of amino acids, we performed stochastic simulations of intracellular dynamics with every possible nucleotide variant as the infecting strain and estimated the relative infectivity of each variant and the resulting distribution of variants produced. We employed these quantities in a deterministic multi-strain model of extracellular dynamics and estimated mutant frequencies. Our predictions captured database frequencies of the RAV R155K, resistant to NS3/4A protease inhibitors, presenting a successful test of our formalism. We found that mutational pathway maps, interconnecting all viable mutants, and strong founder effects determined the mutant spectrum. The spectra were vastly different for HCV genotypes 1a and 1b, underlying their differential responses to drugs. Using a fitness landscape determined recently, we estimated that 13 amino acid variants, encoded by 44 codons, exist at the residue 93 of the NS5A protein, illustrating the massive diversity of accessible resistance pathways at specific loci. Accounting for this diversity, which our model enables, would help optimize drug combinations. Our model may be applied to describe the within-host evolution of other flaviviruses and inform vaccine design strategies. [ABSTRACT FROM AUTHOR]

Virus ecology and evolution play a central role in disease emergence. However, their relative roles will vary depending on the viruses and ecosystems involved. We combined field studies, phylogenetics and experimental infections to document with unprecedented detail the stages that precede initial outbreaks during viral emergence in nature. Using serological surveys we showed that in the absence of large-scale outbreaks, horses in Mongolia are routinely exposed to and infected by avian influenza viruses (AIVs) circulating among wild birds. Some of those AIVs are genetically related to an avian-origin virus that caused an epizootic in horses in 1989. Experimental infections showed that most AIVs replicate in the equine respiratory tract without causing lesions, explaining the absence of outbreaks of disease. Our results show that AIVs infect horses but do not spread, or they infect and spread but do not cause disease. Thus, the failure of AIVs to evolve greater transmissibility and to cause disease in horses is in this case the main barrier preventing disease emergence. [ABSTRACT FROM AUTHOR]

Bats are newly identified reservoirs of hantaviruses (HVs) among which very divergent HVs have been discovered in recent years. However, their significance for public health remains unclear since their seroprevalence as well as antigenic relationship with human-infecting HVs have not been investigated. In the present study archived tissues of 1,419 bats of 22 species from 6 families collected in 5 south and southwest provinces in China were screened by pan-HV RT-PCR following viral metagenomic analysis. As a result nine HVs have been identified in two bat species in two provinces and phylogenetically classified into two species, Laibin virus (LAIV, ICTV approved species, 1 strain) and Xuan son virus (XSV, proposed species, 8 strains). Additionally, 709 serum samples of these bats were also analyzed by ELISA to investigate the seroprevalence and cross-reactivity between different HVs using expressed recombinant nucleocapsid proteins (rNPs) of LAIV, XSV and Seoul virus (SEOV). The cross-reactivity of some bat sera were further confirmed by western blot (WB) using three rNPs followed by fluorescent antibody virus neutralization test (FAVNT) against live SEOV. Results showed that the total HV seropositive rate of bat sera was 18.5% (131/709) with many cross reacting with two or all three rNPs and several able to neutralize SEOV. WB analysis using the three rNPs and their specific hyperimmune sera demonstrated cross-reactivity between XSV/SEOV and LAIV/XSV, but not LAIV/SEOV, indicating that XSV is antigenically closer to human-infecting HVs. In addition a study of the distribution of the viruses identified an area covering the region between Chinese Guangxi and North Vietnam, in which XSV and LAIV circulate within different bat colonies with a high seroprevalence. A circulation sphere of bat-borne HVs has therefore been proposed. [ABSTRACT FROM AUTHOR]

Despite ongoing efforts to control transmission, rabies prevention remains a challenge in many developing countries, especially in rural areas of China where re-emerging rabies is under-reported due to a lack of sustained animal surveillance. By taking advantage of detailed genomic and epidemiological data for the re-emerging rabies outbreak in Yunnan Province, China, collected between 1999 and 2015, we reconstruct the demographic and dispersal history of domestic dog rabies virus (RABV) as well as the dynamics of dog-to-dog and dog-to-human transmission. Phylogeographic analyses reveal a lower diffusion coefficient than previously estimated for dog RABV dissemination in northern Africa. Furthermore, epidemiological analyses reveal transmission rates between dogs, as well as between dogs and humans, lower than estimates for Africa. Finally, we show that reconstructed epidemic history of RABV among dogs and the dynamics of rabid dogs are consistent with the recorded human rabies cases. This work illustrates the benefits of combining phylogeographic and epidemic modelling approaches for uncovering the spatiotemporal dynamics of zoonotic diseases, with both approaches providing estimates of key epidemiological parameters. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomics study of 111 B. multivorans sputum isolates from one CF patient through three stages of infection including an early incident isolate, deep sampling of a one-year period of chronic infection occurring weeks before a lung transplant, and deep sampling of a post-transplant infection. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found the incident isolate was basally related to the rest of the strains and more susceptible to antibiotics from three classes (β-lactams, aminoglycosides, quinolones). The chronic infection isolates diversified into multiple, distinct genetic lineages and showed reduced antimicrobial susceptibility to the same antibiotics. The post-transplant reinfection isolates derived from the same source as the incident isolate and were genetically distinct from the chronic isolates. They also had a level of susceptibility in between that of the incident and chronic isolates. We identified numerous examples of potential parallel pathoadaptation, in which multiple mutations were found in the same locus or even codon. The set of parallel pathoadaptive loci was enriched for functions associated with virulence and resistance. Our GWAS analysis identified statistical associations between a polymorphism in the ampD locus with resistance to β-lactams, and polymorphisms in an araC transcriptional regulator and an outer membrane porin with resistance to both aminoglycosides and quinolones. Additionally, these three loci were independently mutated four, three and two times, respectively, providing further support for parallel pathoadaptation. Finally, we identified a minimum of 14 recombination events, and observed that loci carrying putative parallel pathoadaptations and polymorphisms statistically associated with β-lactam resistance were over-represented in these recombinogenic regions. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development. [ABSTRACT FROM AUTHOR]

Recent discoveries on the origins of modern humans from multiple archaic hominin populations and the diversity of human papillomaviruses (HPVs) suggest a complex scenario of virus-host evolution. To evaluate the origin of HPV pathogenesis, we estimated the phylogeny, timing, and dispersal of HPV16 variants using a Bayesian Markov Chain Monte Carlo framework. To increase precision, we identified and characterized non-human primate papillomaviruses from New and Old World monkeys to set molecular clock models. We demonstrate specific host niche adaptation of primate papillomaviruses with subsequent coevolution with their primate hosts for at least 40 million years. Analyses of 212 HPV16 complete genomes and 3582 partial sequences estimated ancient divergence of HPV16 variants (between A and BCD lineages) from their most recent common ancestors around half a million years ago, roughly coinciding with the timing of the split between archaic Neanderthals and modern Homo sapiens, and nearly three times longer than divergence times of modern Homo sapiens. HPV16 A lineage variants were significantly underrepresented in present African populations, whereas the A sublineages were highly prevalent in European (A1-3) and Asian (A4) populations, indicative of viral sexual transmission from Neanderthals to modern non-African humans through multiple interbreeding events in the past 80 thousand years. Remarkably, the human leukocyte antigen B*07:02 and C*07:02 alleles associated with increased risk in cervix cancer represent introgressed regions from Neanderthals in present-day Eurasians. The archaic hominin-host-switch model was also supported by other HPV variants. Niche adaptation and virus-host codivergence appear to influence the pathogenesis of papillomaviruses. [ABSTRACT FROM AUTHOR]

RNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication (albeit within a permissive host environment). A 33.5 kilobase (kb) nidovirus has been considered close to the upper size limit for such entities; conversely, the minimal cellular DNA genome is in the 100–300 kb range. This large difference presents a daunting gap for the transition from primordial RNP to contemporary DNA-RNP-based life. Whether or not RNA viruses represent transitional steps towards DNA-based life, studies of larger RNA viruses advance our understanding of the size constraints on RNP entities and the role of genome size in virus adaptation. For example, emergence of the largest previously known RNA genomes (20–34 kb in positive-stranded nidoviruses, including coronaviruses) is associated with the acquisition of a proofreading exoribonuclease (ExoN) encoded in the open reading frame 1b (ORF1b) in a monophyletic subset of nidoviruses. However, apparent constraints on the size of ORF1b, which encodes this and other key replicative enzymes, have been hypothesized to limit further expansion of these viral RNA genomes. Here, we characterize a novel nidovirus (planarian secretory cell nidovirus; PSCNV) whose disproportionately large ORF1b-like region including unannotated domains, and overall 41.1-kb genome, substantially extend the presumed limits on RNA genome size. This genome encodes a predicted 13,556-aa polyprotein in an unconventional single ORF, yet retains canonical nidoviral genome organization and expression, as well as key replicative domains. These domains may include functionally relevant substitutions rarely or never before observed in highly conserved sites of RdRp, NiRAN, ExoN and 3CLpro. Our evolutionary analysis suggests that PSCNV diverged early from multi-ORF nidoviruses, and acquired additional genes, including those typical of large DNA viruses or hosts, e.g. Ankyrin and Fibronectin type II, which might modulate virus-host interactions. PSCNV's greatly expanded genome, proteomic complexity, and unique features–impressive in themselves–attest to the likelihood of still-larger RNA genomes awaiting discovery. [ABSTRACT FROM AUTHOR]

The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains. [ABSTRACT FROM AUTHOR]

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among multiple nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on the genotyping of three variant sites in the orthologs of the β-tubulin gene found to confer resistance in the free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites to identify additional mechanisms that might contribute to BZ resistance in the field. Here, we took an unbiased genome-wide mapping approach in the free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we find that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in the β-tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and segregates within the C. elegans population, suggesting that selection in natural niches could enrich for resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the C. elegans population, including many novel molecular variants. Population genetic analyses indicate that ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence that these alleles likely occurred recently because of local selective pressures. Additionally, we find purifying selection at all five β-tubulin genes, despite predicted loss-of-function variants in ben-1, indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one β-tubulin gene. Furthermore, we use genome-editing to show that the most common parasitic nematode β-tubulin allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent of ben-1 and the other β-tubulin loci, suggesting that there are multiple mechanisms underlying BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for the study of mechanisms that give rise to BZ resistance. [ABSTRACT FROM AUTHOR]

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5′ mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin. [ABSTRACT FROM AUTHOR]

Infection with human BK polyomavirus, a small double-stranded DNA virus, potentially results in severe complications in immunocompromised patients. Here, we describe the in vivo variability and evolution of the BK polyomavirus by deep sequencing. Our data reveal the highest genomic evolutionary rate described in double-stranded DNA viruses, i.e., 10−3–10−5 substitutions per nucleotide site per year. High mutation rates in viruses allow their escape from immune surveillance and adaptation to new hosts. By combining mutational landscapes across viral genomes with in silico prediction of viral peptides, we demonstrate the presence of significantly more coding substitutions within predicted cognate HLA-C-bound viral peptides than outside. This finding suggests a role for HLA-C in antiviral immunity, perhaps through the action of killer cell immunoglobulin-like receptors. The present study provides a comprehensive view of viral evolution and immune escape in a DNA virus. [ABSTRACT FROM AUTHOR]

Candida albicans is an opportunistic fungal pathogen that can infect oral mucosal surfaces while being under continuous flow from saliva. Under specific conditions, C. albicans will form microcolonies that more closely resemble the biofilms formed in vivo than standard in vitro biofilm models. However, very little is known about these microcolonies, particularly genomic differences between these specialized biofilm structures and the traditional in vitro biofilms. In this study, we used a novel flow system, in which C. albicans spontaneously forms microcolonies, to further characterize the architecture of fungal microcolonies and their genomics compared to non-microcolony conditions. Fungal microcolonies arose from radially branching filamentous hyphae that increasingly intertwined with one another to form extremely dense biofilms, and closely resembled the architecture of in vivo oropharyngeal candidiasis. We identified 20 core microcolony genes that were differentially regulated in flow-induced microcolonies using RNA-seq. These genes included HWP1, ECE1, IHD1, PLB1, HYR1, PGA10, and SAP5. A predictive algorithm was utilized to identify ten transcriptional regulators potentially involved in microcolony formation. Of these transcription factors, we found that Rob1, Ndt80, Sfl1 and Sfl2, played a key role in microcolony formation under both flow and static conditions and to epithelial surfaces. Expression of core microcolony genes were highly up-regulated in Δsfl1 cells and down-regulated in both Δsfl2 and Δrob1 strains. Microcolonies formed on oral epithelium using C. albicans Δsfl1, Δsfl2 and Δrob1 deletion strains all had altered adhesion, invasion and cytotoxicity. Furthermore, epithelial cells infected with deletion mutants had reduced (SFL2, NDT80, and ROB1) or enhanced (SFL2) immune responses, evidenced by phosphorylation of MKP1 and c-Fos activation, key signal transducers in the hyphal invasion response. This profile of microcolony transcriptional regulators more closely reflects Sfl1 and Sfl2 hyphal regulatory networks than static biofilm regulatory networks, suggesting that microcolonies are a specialized pathogenic form of biofilm. [ABSTRACT FROM AUTHOR]

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species. [ABSTRACT FROM AUTHOR]

The deep history and early diversification of retroviruses remains elusive, largely because few retroviruses have been characterized in vertebrates other than mammals and birds. Endogenous retroviruses (ERVs) documented past retroviral infections and thus provide ‘molecular fossils’ for studying the deep history of retroviruses. Here we perform a comprehensive phylogenomic analysis of ERVs within the genomes of 92 non-avian/mammalian vertebrates, including 72 fishes, 4 amphibians, and 16 reptiles. We find that ERVs are present in all the genomes of jawed vertebrates, revealing the ubiquitous presence of ERVs in jawed vertebrates. We identify a total of >8,000 ERVs and reconstruct ~450 complete or partial ERV genomes, which dramatically expands the phylogenetic diversity of retroviruses and suggests that the diversity of exogenous retroviruses might be much underestimated in non-avian/mammalian vertebrates. Phylogenetic analyses show that retroviruses cluster into five major groups with different host distributions, providing important insights into the classification and diversification of retroviruses. Moreover, we find retroviruses mainly underwent frequent host switches in non-avian/mammalian vertebrates, with exception of spumavirus-related viruses that codiverged with their ray-finned fish hosts. Interestingly, ray-finned fishes and turtles appear to serve as unappreciated hubs for the transmission of retroviruses. Finally, we find retroviruses underwent many independent water-land transmissions, indicating the water-land interface is not a strict barrier for retrovirus transmission. Our analyses provide unprecedented insights into and valuable resources for studying the diversification, key evolutionary transitions, and macroevolution of retroviruses. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster has played a key role in our understanding of invertebrate immunity. However, both functional and evolutionary studies of host-virus interaction in Drosophila have been limited by a dearth of native virus isolates. In particular, despite a long history of virus research, DNA viruses of D. melanogaster have only recently been described, and none have been available for experimental study. Here we report the isolation and comprehensive characterisation of Kallithea virus, a large double-stranded DNA virus, and the first DNA virus to have been reported from wild populations of D. melanogaster. We find that Kallithea virus infection is costly for adult flies, reaching high titres in both sexes and disproportionately reducing survival in males, and movement and late fecundity in females. Using the Drosophila Genetic Reference Panel, we quantify host genetic variance for virus-induced mortality and viral titre and identify candidate host genes that may underlie this variation, including Cdc42-interacting protein 4. Using full transcriptome sequencing of infected males and females, we examine the transcriptional response of flies to Kallithea virus infection and describe differential regulation of virus-responsive genes. This work establishes Kallithea virus as a new tractable model to study the natural interaction between D. melanogaster and DNA viruses, and we hope it will serve as a basis for future studies of immune responses to DNA viruses in insects. [ABSTRACT FROM AUTHOR]

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype. [ABSTRACT FROM AUTHOR]

The bromodomain protein Brd4 promotes HIV-1 latency by competitively inhibiting P-TEFb-mediated transcription induced by the virus-encoded Tat protein. Brd4 is recruited to the HIV LTR by interactions with acetyl-histones3 (AcH3) and AcH4. However, the precise modification pattern that it reads and the writer for generating this pattern are unknown. By examining a pool of latently infected proviruses with diverse integration sites, we found that the LTR characteristically has low AcH3 but high AcH4 content. This unusual acetylation profile attracts Brd4 to suppress the interaction of Tat with the host super elongation complex (SEC) that is essential for productive HIV transcription and latency reversal. KAT5 (lysine acetyltransferase 5), but not its paralogs KAT7 and KAT8, is found to promote HIV latency through acetylating H4 on the provirus. Antagonizing KAT5 removes AcH4 and Brd4 from the LTR, enhances the SEC loading, and reverses as well as delays, the establishment of latency. The pro-latency effect of KAT5 is confirmed in a primary CD4+ T cell latency model as well as cells from ART-treated patients. Our data thus indicate the KAT5-AcH4-Brd4 axis as a key regulator of latency and a potential therapeutic target to reactivate latent HIV reservoirs for eradication. [ABSTRACT FROM AUTHOR]

Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi. [ABSTRACT FROM AUTHOR]

Host shifts, where a pathogen invades and establishes in a new host species, are a major source of emerging infectious diseases. They frequently occur between related host species and often rely on the pathogen evolving adaptations that increase their fitness in the novel host species. To investigate genetic changes in novel hosts, we experimentally evolved replicate lineages of an RNA virus (Drosophila C Virus) in 19 different species of Drosophilidae and deep sequenced the viral genomes. We found a strong pattern of parallel evolution, where viral lineages from the same host were genetically more similar to each other than to lineages from other host species. When we compared viruses that had evolved in different host species, we found that parallel genetic changes were more likely to occur if the two host species were closely related. This suggests that when a virus adapts to one host it might also become better adapted to closely related host species. This may explain in part why host shifts tend to occur between related species, and may mean that when a new pathogen appears in a given species, closely related species may become vulnerable to the new disease. [ABSTRACT FROM AUTHOR]

In the United States, the introduction of the heptavalent pneumococcal conjugate vaccine (PCV) largely eliminated vaccine serotypes (VT); non-vaccine serotypes (NVT) subsequently increased in carriage and disease. Vaccination also disrupts the composition of the pneumococcal pangenome, which includes mobile genetic elements and polymorphic non-capsular antigens important for virulence, transmission, and pneumococcal ecology. Antigenic proteins are of interest for future vaccines; yet, little is known about how the they are affected by PCV use. To investigate the evolutionary impact of vaccination, we assessed recombination, evolution, and pathogen demographic history of 937 pneumococci collected from 1998–2012 among Navajo and White Mountain Apache Native American communities. We analyzed changes in the pneumococcal pangenome, focusing on metabolic loci and 19 polymorphic protein antigens. We found the impact of PCV on the pneumococcal population could be observed in reduced diversity, a smaller pangenome, and changing frequencies of accessory clusters of orthologous groups (COGs). Post-PCV7, diversity rebounded through clonal expansion of NVT lineages and inferred in-migration of two previously unobserved lineages. Accessory COGs frequencies trended toward pre-PCV7 values with increasing time since vaccine introduction. Contemporary frequencies of protein antigen variants are better predicted by pre-PCV7 values (1998–2000) than the preceding period (2006–2008), suggesting balancing selection may have acted in maintaining variant frequencies in this population. Overall, we present the largest genomic analysis of pneumococcal carriage in the United States to date, which includes a snapshot of a true vaccine-naïve community prior to the introduction of PCV7. These data improve our understanding of pneumococcal evolution and emphasize the need to consider pangenome composition when inferring the impact of vaccination and developing future protein-based pneumococcal vaccines. [ABSTRACT FROM AUTHOR]

Deep sequencing was used to determine complete nucleotide sequences of echovirus 11 (EV11) strains isolated from a chronically infected patient with CVID as well as from cases of acute enterovirus infection. Phylogenetic analysis showed that EV11 strains that circulated in Israel in 1980-90s could be divided into four clades. EV11 strains isolated from a chronically infected individual belonged to one of the four clades and over a period of 4 years accumulated mutations at a relatively constant rate. Extrapolation of mutations accumulation curve into the past suggested that the individual was infected with circulating EV11 in the first half of 1990s. Genomic regions coding for individual viral proteins did not appear to be under strong selective pressure except for protease 3C that was remarkably conserved. This may suggest its important role in maintaining persistent infection. [ABSTRACT FROM AUTHOR]

The disease state of amebiasis, caused by Entamoeba histolytica, varies from asymptomatic to severe manifestations that include dysentery and extraintestinal abscesses. The virulence factors of the pathogen, and host defense mechanisms, contribute to the outcomes of infection; however, the underlying genetic factors, which affect clinical outcomes, remain to be fully elucidated. To identify these genetic factors in E. histolytica, we used Illumina next-generation sequencing to conduct a comparative genomic analysis of two clinical isolates obtained from diarrheal and asymptomatic patients (strains KU50 and KU27, respectively). By mapping KU50 and KU27 reads to the genome of a reference HM-1:IMSS strain, we identified two genes (EHI_089440 and EHI_176590) that were absent in strain KU27. In KU27, a single AIG1 (avrRpt2-nduced ene 1) family gene (EHI_176590) was found to be deleted, from a tandem array of three AIG1 genes, by homologous recombination between the two flanking genes. Overexpression of the EHI_176590 gene, in strain HM-1:IMSS cl6, resulted in increased formation of cell-surface protrusions and enhanced adhesion to human erythrocytes. The EHI_176590 gene was detected by PCR in 56% of stool samples from symptomatic patients infected with E. histolytica, but only in 15% of stool samples from asymptomatic individuals. This suggests that the presence of the EHI_176590 gene is correlated with the outcomes of infection. Taken together, these data strongly indicate that the AIG1 family protein plays a pivotal role in E. histolytica virulence via regulation of host cell adhesion. Our in-vivo experiments, using a hamster liver abscess model, showed that overexpression or gene silencing of EHI_176590 reduced and increased liver abscess formation, respectively. This suggests that the AIG1 genes may have contrasting roles in virulence depending on the genetic background of the parasite and host environment. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii picoplast hioedoins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1’s involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites. [ABSTRACT FROM AUTHOR]

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms. [ABSTRACT FROM AUTHOR]

The factors that determine the pattern and rate of spread of influenza virus at a continental-scale are uncertain. Although recent work suggests that influenza epidemics in the United States exhibit a strong geographical correlation, the spatiotemporal dynamics of influenza in Australia, a country and continent of approximately similar size and climate complexity but with a far smaller population, are not known. Using a unique combination of large-scale laboratory-confirmed influenza surveillance comprising >450,000 entries and genomic sequence data we determined the local-level spatial diffusion of this important human pathogen nationwide in Australia. We used laboratory-confirmed influenza data to characterize the spread of influenza virus across Australia during 2007–2016. The onset of established epidemics varied across seasons, with highly synchronized epidemics coinciding with the emergence of antigenically distinct viruses, particularly during the 2009 A/H1N1 pandemic. The onset of epidemics was largely synchronized between the most populous cities, even those separated by distances of >3000 km and those that experience vastly diverse climates. In addition, by analyzing global phylogeographic patterns we show that the synchronized dissemination of influenza across Australian cities involved multiple introductions from the global influenza population, coupled with strong domestic connectivity, rather than through the distinct radial patterns of geographic dispersal that are driven by work-flow transmission as observed in the United States. In addition, by comparing the spatial structure of influenza A and B, we found that these viruses tended to occupy different geographic regions, and peak in different seasons, perhaps indicative of moderate cross-protective immunity or viral interference effects. The highly synchronized outbreaks of influenza virus at a continental-scale revealed here highlight the importance of coordinated public health responses in the event of the emergence of a novel, human-to-human transmissible, virus. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen. [ABSTRACT FROM AUTHOR]

The global-scale epidemiology and genome-wide evolutionary dynamics of influenza B remain poorly understood compared with influenza A viruses. We compiled a spatio-temporally comprehensive dataset of influenza B viruses, comprising over 2,500 genomes sampled worldwide between 1987 and 2015, including 382 newly-sequenced genomes that fill substantial gaps in previous molecular surveillance studies. Our contributed data increase the number of available influenza B virus genomes in Europe, Africa and Central Asia, improving the global context to study influenza B viruses. We reveal Yamagata-lineage diversity results from co-circulation of two antigenically-distinct groups that also segregate genetically across the entire genome, without evidence of intra-lineage reassortment. In contrast, Victoria-lineage diversity stems from geographic segregation of different genetic clades, with variability in the degree of geographic spread among clades. Differences between the lineages are reflected in their antigenic dynamics, as Yamagata-lineage viruses show alternating dominance between antigenic groups, while Victoria-lineage viruses show antigenic drift of a single lineage. Structural mapping of amino acid substitutions on trunk branches of influenza B gene phylogenies further supports these antigenic differences and highlights two potential mechanisms of adaptation for polymerase activity. Our study provides new insights into the epidemiological and molecular processes shaping influenza B virus evolution globally. [ABSTRACT FROM AUTHOR]

Recognizing the life cycle of an organism is key to understanding its biology and ecological impact. Emiliania huxleyi is a cosmopolitan marine microalga, which displays a poorly understood biphasic sexual life cycle comprised of a calcified diploid phase and a morphologically distinct biflagellate haploid phase. Diploid cells (2N) form large-scale blooms in the oceans, which are routinely terminated by specific lytic viruses (EhV). In contrast, haploid cells (1N) are resistant to EhV. Further evidence indicates that 1N cells may be produced during viral infection. A shift in morphology, driven by meiosis, could therefore constitute a mechanism for E. huxleyi cells to escape from EhV during blooms. This process has been metaphorically coined the ‘Cheshire Cat’ (CC) strategy. We tested this model in two E. huxleyi strains using a detailed assessment of morphological and ploidy-level variations as well as expression of gene markers for meiosis and the flagellate phenotype. We showed that following the CC model, production of resistant cells was triggered during infection. This led to the rise of a new subpopulation of cells in the two strains that morphologically resembled haploid cells and were resistant to EhV. However, ploidy-level analyses indicated that the new resistant cells were diploid or aneuploid. Thus, the CC strategy in E. huxleyi appears to be a life-phase switch mechanism involving morphological remodeling that is decoupled from meiosis. Our results highlight the adaptive significance of morphological plasticity mediating complex host–virus interactions in marine phytoplankton. [ABSTRACT FROM AUTHOR]

Burkholderia cenocepacia causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic B. cenocepacia infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone B. cenocepacia ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum B. cenocepacia ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in katG and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS. [ABSTRACT FROM AUTHOR]

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen. [ABSTRACT FROM AUTHOR]

By combining high-throughput sequencing (HTS) with experimental evolution, we can observe the within-host dynamics of pathogen variants of biomedical or ecological interest. We studied the evolutionary dynamics of five variants of Potato virus Y (PVY) in 15 doubled-haploid lines of pepper. All plants were inoculated with the same mixture of virus variants and, variant frequencies were determined by HTS in eight plants of each pepper line, at each of six sampling dates. We developed a method for estimating the intensities of selection and genetic drift in a multi-allelic Wright-Fisher model, applicable whether these forces are strong or weak, and in the absence of neutral markers. This method requires variant frequency determination at several time points, in independent hosts. The parameters are the selection coefficients for each PVY variant and four effective population sizes Ne at different time-points of the experiment. Numerical simulations of asexual haploid Wright-Fisher populations subjected to contrasting genetic drift (Ne ∈ [10, 2000]) and selection (|s| ∈ [0, 0.15]) regimes were used to validate the method proposed. The experiment in closely related pepper host genotypes revealed that viruses experienced a considerable diversity of selection and genetic drift regimes. The resulting variant dynamics were accurately described by Wright-Fisher models. The fitness ranks of the variants were almost identical between host genotypes. By contrast, the dynamics of Ne were highly variable, although a bottleneck was often identified during the systemic movement of the virus. We demonstrated that, for a fixed initial PVY population, virus effective population size is a heritable trait in plants. These findings pave the way for the breeding of plant varieties exposing viruses to stronger genetic drift, thereby slowing virus adaptation. [ABSTRACT FROM AUTHOR]

Reassortment, which is the exchange of genome sequence between viruses co-infecting a host cell, plays an important role in the evolution of segmented viruses. In the human influenza virus, reassortment happens most frequently between co-existing variants within the same lineage. This process breaks genetic linkage and fitness correlations between viral genome segments, but the resulting net effect on viral fitness has remained unclear. In this paper, we determine rate and average selective effect of reassortment processes in the human influenza lineage A/H3N2. For the surface proteins hemagglutinin and neuraminidase, reassortant variants with a mean distance of at least 3 nucleotides to their parent strains get established at a rate of about 10−2 in units of the neutral point mutation rate. Our inference is based on a new method to map reassortment events from joint genealogies of multiple genome segments, which is tested by extensive simulations. We show that intra-lineage reassortment processes are, on average, under substantial negative selection that increases in strength with increasing sequence distance between the parent strains. The deleterious effects of reassortment manifest themselves in two ways: there are fewer reassortment events than expected from a null model of neutral reassortment, and reassortant strains have fewer descendants than their non-reassortant counterparts. Our results suggest that influenza evolves under ubiquitous epistasis across proteins, which produces fitness barriers against reassortment even between co-circulating strains within one lineage. [ABSTRACT FROM AUTHOR]

Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, H. contortus, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36’A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36’A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the H. contortus GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted. [ABSTRACT FROM AUTHOR]

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal. [ABSTRACT FROM AUTHOR]

The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T0) mutant pools were calculated for each gene. We identified a total of 273 ubutaneous itness (scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments. [ABSTRACT FROM AUTHOR]

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them—including Archaea—encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system—often from different groups—and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body location. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by-product of environmental adaptation. Understanding the role of capsules in natural environments might enlighten their function in pathogenesis. [ABSTRACT FROM AUTHOR]

Cross-species transmission (CST) has led to many devastating epidemics, but is still a poorly understood phenomenon. HIV-1 and HIV-2 (human immunodeficiency virus 1 and 2), which have collectively caused over 35 million deaths, are the result of multiple CSTs from chimpanzees, gorillas, and sooty mangabeys. While the immediate history of HIV is known, there are over 45 lentiviruses that infect specific species of primates, and patterns of host switching are not well characterized. We thus took a phylogenetic approach to better understand the natural history of SIV recombination and CST. We modeled host species as a discrete character trait on the viral phylogeny and inferred historical host switches and the pairwise transmission rates between each pair of 24 primate hosts. We identify 14 novel, well-supported, ancient cross-species transmission events. We also find that lentiviral lineages vary widely in their ability to infect new host species: SIVcol (from colobus monkeys) is evolutionarily isolated, while SIVagms (from African green monkeys) frequently move between host subspecies. We also examine the origins of SIVcpz (the predecessor of HIV-1) in greater detail than previous studies, and find that there are still large portions of the genome with unknown origins. Observed patterns of CST are likely driven by a combination of ecological circumstance and innate immune factors. [ABSTRACT FROM AUTHOR]

The interplay of different virus species in a host cell after infection can affect the adaptation of each virus. Endogenous viral elements, such as endogenous pararetroviruses (PRVs), have arisen from vertical inheritance of viral sequences integrated into host germline genomes. As viral genomic fossils, these sequences can thus serve as valuable paleogenomic data to study the long-term evolutionary dynamics of virus–virus interactions, but they have rarely been applied for this purpose. All extant PRVs have been considered autonomous species in their parasitic life cycle in host cells. Here, we provide evidence for multiple non-autonomous PRV species with structural defects in viral activity that have frequently infected ancient grass hosts and adapted through interplay between viruses. Our paleogenomic analyses using endogenous PRVs in grass genomes revealed that these non-autonomous PRV species have participated in interplay with autonomous PRVs in a possible commensal partnership, or, alternatively, with one another in a possible mutualistic partnership. These partnerships, which have been established by the sharing of noncoding regulatory sequences (NRSs) in intergenic regions between two partner viruses, have been further maintained and altered by the sequence homogenization of NRSs between partners. Strikingly, we found that frequent region-specific recombination, rather than mutation selection, is the main causative mechanism of NRS homogenization. Our results, obtained from ancient DNA records of viruses, suggest that adaptation of PRVs has occurred by concerted evolution of NRSs between different virus species in the same host. Our findings further imply that evaluation of within-host NRS interactions within and between populations of viral pathogens may be important. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity. [ABSTRACT FROM AUTHOR]

APOBEC3 (A3) family proteins are DNA cytosine deaminases recognized for contributing to HIV-1 restriction and mutation. Prior studies have demonstrated that A3D, A3F, and A3G enzymes elicit a robust anti-HIV-1 effect in cell cultures and in humanized mouse models. Human A3H is polymorphic and can be categorized into three phenotypes: stable, intermediate, and unstable. However, the anti-viral effect of endogenous A3H in vivo has yet to be examined. Here we utilize a hematopoietic stem cell-transplanted humanized mouse model and demonstrate that stable A3H robustly affects HIV-1 fitness in vivo. In contrast, the selection pressure mediated by intermediate A3H is relaxed. Intriguingly, viral genomic RNA sequencing reveled that HIV-1 frequently adapts to better counteract stable A3H during replication in humanized mice. Molecular phylogenetic analyses and mathematical modeling suggest that stable A3H may be a critical factor in human-to-human viral transmission. Taken together, this study provides evidence that stable variants of A3H impose selective pressure on HIV-1. [ABSTRACT FROM AUTHOR]

Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed ‘ClickSeq’ due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies’s (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these ‘intermediate’ species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs. [ABSTRACT FROM AUTHOR]

Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a high-throughput approach to quantify the selection that monoclonal antibodies exert on all single amino-acid mutations to a viral protein. This approach, mutational antigenic profiling, involves creating all replication-competent protein variants of a virus, selecting with antibody, and using deep sequencing to identify enriched mutations. We use mutational antigenic profiling to comprehensively identify mutations that enable influenza virus to escape four monoclonal antibodies targeting hemagglutinin, and validate key findings with neutralization assays. We find remarkable mutation-level idiosyncrasy in antibody escape: for instance, at a single residue targeted by two antibodies, some mutations escape both antibodies while other mutations escape only one or the other. Because mutational antigenic profiling rapidly maps all mutations selected by an antibody, it is useful for elucidating immune specificities and interpreting the antigenic consequences of viral genetic variation. [ABSTRACT FROM AUTHOR]

Individuals vary extensively in the way they respond to disease but the genetic basis of this variation is not fully understood. We found substantial individual variation in resistance and tolerance to the fungal pathogen Metarhizium anisopliae Ma549 using the Drosophila melanogaster Genetic Reference Panel (DGRP). In addition, we found that host defense to Ma549 was correlated with defense to the bacterium Pseudomonas aeruginosa Pa14, and several previously published DGRP phenotypes including oxidative stress sensitivity, starvation stress resistance, hemolymph glucose levels, and sleep indices. We identified polymorphisms associated with differences between lines in both their mean survival times and microenvironmental plasticity, suggesting that lines differ in their ability to adapt to variable pathogen exposures. The majority of polymorphisms increasing resistance to Ma549 were sex biased, located in non-coding regions, had moderately large effect and were rare, suggesting that there is a general cost to defense. Nevertheless, host defense was not negatively correlated with overall longevity and fecundity. In contrast to Ma549, minor alleles were concentrated in the most Pa14-susceptible as well as the most Pa14-resistant lines. A pathway based analysis revealed a network of Pa14 and Ma549-resistance genes that are functionally connected through processes that encompass phagocytosis and engulfment, cell mobility, intermediary metabolism, protein phosphorylation, axon guidance, response to DNA damage, and drug metabolism. Functional testing with insertional mutagenesis lines indicates that 12/13 candidate genes tested influence susceptibility to Ma549. Many candidate genes have homologs identified in studies of human disease, suggesting that genes affecting variation in susceptibility are conserved across species. [ABSTRACT FROM AUTHOR]