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Even during extended periods of effective immunological control, a substantial dynamic of the viral genome can be observed in different cellular compartments in HIV-1 positive individuals, indicating the persistence of active viral reservoirs. To obtain further insights, we studied changes in the proviral as well as in the viral HIV-1 envelope (Env) sequence along with transcriptional, translational and viral outgrowth activity as indicators for viral dynamics and genomic intactness. Our study identified distinct reservoir patterns that either represented highly sequence-diverse HIV-1 populations or only a single / few persisting virus variants. The single dominating variants were more often found in individuals starting ART during early infection phases, indicating that early treatment might limit reservoir diversification. At the same time, more sequence-diverse HIV reservoirs correlated with a poorer immune status, indicated by lower CD4 count, a higher number of regimen changes and more co-morbidities. Furthermore, we noted that in T-cell populations in the peripheral blood, replication-competent HIV-1 is predominantly present in Lymph node homing TN (naïve) and TCM (central memory) T cells. Provirus genomes archived in TTM (transitional memory) and TEM (effector memory) T cells more frequently tended to carry inactivating mutations and, population-wise, possess changes in the genetic diversity. These discriminating properties of the viral reservoir in T-cell subsets may have important implications for new early therapy strategies, underscoring the critical role of early therapy in preserving robust immune surveillance and constraining the viral reservoir. Author summary: Our study demonstrates that a poor immune status associates with and may form a basis for HIV reservoir diversification. Reservoirs for replication-competent, infectious viruses are mainly found in TN and TCM, which harbor a small number of intact viral variants. A distinct population of archived HIV sequences, residing in TTM and TEM, is characterized by a shifted genetic diversity. While the former cells are likely to harbor a promptly re-activatable latent reservoir, the latter with often defective but more variable viral genomes could thus represent a source for retroviral recombination and reactivation. [ABSTRACT FROM AUTHOR]

Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations. Author summary: Most plant-pathogenic bacteria are specialists and have a narrow host range. However, the true host range of plant-pathogenic species is usually not well defined. Here, we demonstrate a variation in the host range of different clones of the specialist pathogen Clavibacter michiganensis (Cm), which is mainly reported to cause disease in tomato. We characterized a Cm clone library by combining dual-host virulence phenotyping, using tomato and eggplant, and genomic approaches. Our analysis showed that individual Cm clones harbor a differential host range. While most clones were solely pathogenic on tomato, a few were pathogenic on eggplant, and some were non-pathogenic in either host. Comparative genomic analyses identified that non-pathogenic clones lacked the chp/tomA pathogenicity island, a major virulence determinant of Cm. In addition, we found that eggplant-pathogenic clones harbored a distinct allelic variant of the putative secreted serine hydrolase chpG, a known immune elicitor in eggplant. We demonstrated that the presence of this allelic variant in eggplant-pathogenic Cm clones allows them to evade detection and cause disease. Our study provides novel insights into the phenotypic complexity within the population of bacterial plant pathogens and establishes a link between phenotypic variations and distinct genetic features. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy. Author summary: Adeno-associated virus (AAV) is a viral vector that is universally applied in human gene therapy. Despite their versatile applications in gene therapy, the origin and evolution of AAV serotypes have not been extensively studied. In this study, positive selection upon diversification of AAV capsid genes was carefully investigated by evolutionary analysis. Numerous diversifying selected sites were dispersed among the AAV lineages, and evolutionary pathways were inferred for two selected sites of the AAV2 capsid gene. Furthermore, the putative immunological drives underlying the selection of AAV2 capsid genes were experimentally dissected. This work provides a novel interpretation of the evolution and diversity of AAV serotypes and might be helpful for their engineering and application in gene therapy. [ABSTRACT FROM AUTHOR]

Genetic variants in Epstein-Barr virus (EBV) have been strongly associated with nasopharyngeal carcinoma (NPC) in South China. However, different results regarding the most significant viral variants, with polymorphisms in EBER2 and BALF2 loci, have been reported in separate studies. In this study, we newly sequenced 100 EBV genomes derived from 61 NPC cases and 39 population controls. Comprehensive genomic analyses of EBV sequences from both NPC patients and healthy carriers in South China were conducted, totaling 279 cases and 227 controls. Meta-analysis of genome-wide association study revealed a 4-bp deletion downstream of EBER2 (coordinates, 7188–7191; EBER-del) as the most significant variant associated with NPC. Furthermore, multiple viral variants were found to be genetically linked to EBER-del forming a risk haplotype, suggesting that multiple viral variants might be associated with NPC pathogenesis. Population structure and phylogenetic analyses further characterized a high risk EBV lineage for NPC revealing a panel of 38 single nucleotide polymorphisms (SNPs), including those in the EBER2 and BALF2 loci. With linkage disequilibrium clumping and feature selection algorithm, the 38 SNPs could be narrowed down to 9 SNPs which can be used to accurately detect the high risk EBV lineage. In summary, our study provides novel insight into the role of EBV genetic variation in NPC pathogenesis by defining a risk haplotype of EBV for downstream functional studies and identifying a single high risk EBV lineage characterized by 9 SNPs for potential application in population screening of NPC. Author summary: Two genome-wide association studies (GWAS) of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) in South China have revealed different risk loci in EBER region and BALF2 gene of EBV, respectively. More EBV genomes isolated from healthy donors and NPC biopsies are included in this study. The meta-analysis of EBV genomes derived from 279 NPC cases and 227 healthy controls identifies a risk haplotype of EBV comprising of multiple genetic variants having strong linkage disequilibrium with each other. Furthermore, a high risk lineage of EBV characterized by 9 single nucleotide polymorphisms (SNPs) is found to be enriched in NPC of South China. The risk haplotype of EBV will be useful for downstream studies of disease mechanisms and the high risk EBV lineage may be included in screening protocol for early detection of NPC in the population. [ABSTRACT FROM AUTHOR]

This article discusses the importance of genomic surveillance in studying pathogen invasions, using the example of the ash dieback invasion of Europe caused by Hymenoscyphus fraxineus. The ash dieback invasion has had significant economic and ecological impacts, and understanding the genetic variation within the pathogen is crucial for effective management strategies. The article highlights the significance of different types of genomic variation, such as nucleotide substitutions, haplotype diversity, and structural genomic variation, in invasion biology. The authors also discuss various genomic surveillance strategies, including targeted resequencing, whole genome resequencing, and pangenomics, and suggest the potential of air sequencing as a pathogen surveillance method. [Extracted from the article]

Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses. Author summary: Research in virology is primarily motivated by human pathogens, such as SARS-CoV-2 in the case of the family Coronaviridae in the order Nidovirales. Studies of these and few model viruses describe virus-host interactions on the molecular level and are essential for developing virus control measures, but they must accommodate a vast range of viral natural diversity to allow generalizations. Here, we redefine our understanding of the genetic and genomic diversity in corona- and other nidoviruses of poorly sampled hosts. We mine more than 269,000 publicly accessible raw sequencing datasets for the presence of viral sequences using high-performance computing and discover 40 nidoviruses including 13 coronaviruses from a wide range of vertebrates. Some of the novel viruses from aquatic hosts have extraordinary features such as segmented genomes and recombinant genes coding for structural proteins. Our study suggests that gene exchange between diverse nidovirus species from different virus families might be more frequent than previously thought and can result in abrupt genomic innovations that in turn might facilitate host jumps even across vertebrate class borders. The growing list of newly discovered (corona)viruses enables an evolutionary perspective across virus divergency scales in different hosts on the wet lab-acquired knowledge about few viruses. [ABSTRACT FROM AUTHOR]

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments. Author summary: Two species of arteriviruses, a family of RNA viruses, can cause severe reproductive disorders and respiratory diseases in infected piglets that result in significant economic losses in the swine industry. The respective viruses conventionally known as PRRSV-1 and PRRSV-2 exhibit high genetic variability and immune evasion capabilities, which complicate the development of effective vaccines and control measures. In this study, we have described a unique mechanism by which PRRSV-2 infection stimulates RAB18 expression through RIG-I/MAVS-mediated canonical NF-κB activation. RAB18 then recruits HSPA8 and PLIN2 for CMA-mediated degradation and subsequent lipolysis, facilitating viral replication and assembly. Our study provides new insights into the interplay between innate immunity, RAB18, and the CMA-mediated lipophagy pathway during RNA virus infection. [ABSTRACT FROM AUTHOR]

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections. Author summary: Our study investigates the world of RNA viruses and viroids, which are constantly evolving due to error-prone replication. These pathogens exist as quasispecies, featuring dominant master sequences and numerous variants. Understanding how new infectious strains emerge from this genetic diversity remains challenging. Focusing on potato spindle tuber viroid (PSTVd), targeted mutagenesis of specific stem and loop regions was performed to generate mutant pools. Plants were infected with mutant pools, and progeny were analyzed following cell-to-cell movement and long-distance trafficking. Our results reveal that mutants with 3D structures similar to the wild type viroid are more likely to accumulate within quasispecies. This accumulation is associated with the ability to move between cells and travel long distances within the plant host. Even mutants with impaired mobility can be spread by functional counterparts within the quasispecies. This study highlights the role of RNA 3D structures in shaping viroid quasispecies evolution. Mutants resembling the wild type viroid have a greater chance of persisting within quasispecies and may give rise to new infectious variants. These findings offer insight into viroid quasispecies dynamics and identify RNA 3D structure as a promising avenue for resistance strategies. [ABSTRACT FROM AUTHOR]

Microbial pathogens often harbor substantial functional diversity driven by structural genetic variation. Rapid adaptation from such standing variation threatens global food security and human health. Genome-wide association studies (GWAS) provide a powerful approach to identify genetic variants underlying recent pathogen adaptation. However, the reliance on single reference genomes and single nucleotide polymorphisms (SNPs) obscures the true extent of adaptive genetic variation. Here, we show quantitatively how a combination of multiple reference genomes and reference-free approaches captures substantially more relevant genetic variation compared to single reference mapping. We performed reference-genome based association mapping across 19 reference-quality genomes covering the diversity of the species. We contrasted the results with a reference-free (i.e., k-mer) approach using raw whole-genome sequencing data in a panel of 145 strains collected across the global distribution range of the fungal wheat pathogen Zymoseptoria tritici. We mapped the genetic architecture of 49 life history traits including virulence, reproduction and growth in multiple stressful environments. The inclusion of additional reference genome SNP datasets provides a nearly linear increase in additional loci mapped through GWAS. Variants detected through the k-mer approach explained a higher proportion of phenotypic variation than a reference genome-based approach and revealed functionally confirmed loci that classic GWAS approaches failed to map. The power of GWAS in microbial pathogens can be significantly enhanced by comprehensively capturing structural genetic variation. Our approach is generalizable to a large number of species and will uncover novel mechanisms driving rapid adaptation of pathogens. Author summary: Mapping trait variation to polymorphism within species has become a cornerstone of modern biology. Applications in microbial pathogens (both bacteria and fungi) have produced major insights into their ecology, emergence of resistance, gains in virulence and climatic adaptation. However, microbial populations collected from the environment often express major adaptive traits governed by complex genetic variation. Standard genome-wide association studies (GWAS) based on single nucleotide polymorphisms have typically failed to reveal the full extent of loci contributing to such traits. We provide the first quantitative assessment of GWAS in a fungal pathogen comprehensively accounting for complex sequence variation. We used an environmental collection of the global fungal pathogen of wheat, Zymoseptoria tritici, as a case study. We analyzed a panel of 145 strains collected across the global distribution range and gathered trait variation data on 49 distinct traits. We integrated complex sequence variation among strains systematically into the association mapping and found that multiple approaches are needed to cover satisfactorily causal loci for trait variation. Our approach is generalizable to many microbial species and will uncover novel mechanisms driving rapid host adaptation in microbial populations. [ABSTRACT FROM AUTHOR]

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection. Author summary: Different genotypes of B. burgdorferi have been associated with different rates of dissemination, but the genetic basis of these differences is not known. We report the genomes of 299 B. burgdorferi isolates from patients with Lyme disease. We find that whole genome sequence (WGS) type A isolates are a genetically divergent group of isolates characterized by an enlarged pan-genome, an expanded surface lipoproteome encoded on a unique set of plasmids, including lp28-1 and lp56, and increased rates of dissemination. Using genome-wide association methods applied to the B. burgdorferi pan-genome, we identify loci associated with dissemination. The near-clonal nature of B. burgdorferi populations means that relationships of individual loci to dissemination are relatively weak after adjusting for the lineage structure among the isolates, implying that experimental studies and larger cohorts are needed to identify the causal alleles within a lineage mediating these effects. Across the isolates studied, an increasing number of surface-expressed lipoproteins was associated with an increased probability of dissemination in humans. The results underscore how strain-specific genetic variation—particularly among surface lipoproteins located on plasmids—is linked to the phenotype of human dissemination. More broadly, this approach provides a foundation for future studies linking spirochete genotype to the diverse clinical phenotypes of Lyme disease in humans. [ABSTRACT FROM AUTHOR]

The genetic causes of phenotypic variation often differ depending on the population examined, particularly if the populations were founded by relatively small numbers of genotypes. Similarly, the genetic causes of phenotypic variation among similar traits (resistance to different xenobiotic compounds or pathogens) may also be completely different or only partially overlapping. Differences in genetic causes for variation in the same trait among populations suggests context dependence for how selection acts on those traits. Similarities in the genetic causes of variation for different traits, on the other hand, suggests pleiotropy which would also influence how natural selection shapes variation in a trait. We characterized immune defense against a natural Drosophila pathogen, the Gram-positive bacterium Lysinibacillus fusiformis, in three different populations and found almost no overlap in the genetic architecture of variation in survival post infection. However, when comparing our results to a similar experiment with the fungal pathogen, B. bassiana, we found a convincing shared QTL peak for both pathogens. This peak contains the Bomanin cluster of Drosophila immune effectors. Loss of function mutants and RNAi knockdown experiments confirms a role of some of these genes in immune defense against both pathogens. This suggests that natural selection may act on the entire cluster of Bomanin genes (and the linked region under the QTL) or specific peptides for specific pathogens. Author summary: Like most traits, the way individuals respond to infection vary among individuals within a population. Some of this variation is caused by genetic differences in the host. Over the past decade, two prominent resources were developed to assess genetic variation for complex traits of the fruit fly, Drosophila melanogaster and map the genetic variants responsible. We recently described a strain of Lysinibacillus fusiformis bacteria that was isolated from fruit flies and is moderately virulent when flies are infected via septic injury. We mapped genetic variation in resistance L. fusiformis using these mapping resources. We find that among the resources, different genetic changes were associated with immune defense. However, we also found that within a resource, the same region of the genome was associated with resistance to both L. fusiformis and a fungal pathogen. These results suggest that different populations adapt differently to the same pathogens, but two distinct pathogens share similar causes of genetic variation within a single population. [ABSTRACT FROM AUTHOR]

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2–12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions. Author summary: There is heterogeneity in the rates of clinical disease progression in antiretroval therapy-naïve people living with HIV (PLWH). In heterosexual HIV-1 transmission, only a single or very few viral strains, called transmitted/founder (T/F) viruses establish infection in a newly infected host. The long terminal repeat (LTR) is the viral promoter that drives viral gene transcription and is important for the HIV-1 life cycle. In this study we investigated the impact of HIV-1 subtype C T/F virus LTR genetic variation on transcriptional activity, clinical disease outcomes and response to cell activation. Our data show inter-patient T/F LTR genetic variation and limited intrahost evolution by 12 months post infection. T/F LTR variants exhibit differential basal LTR transcriptional activity, which is significanly increased in the presence of the Transactivator of transcription (Tat) protein. Furthermore, we show that T/F LTR transcription activity significanly correlates positively with viral load and viral load set point but negatively with CD4 T cell count. Lastly, we show that T/F LTR variants exhibit differential responses to cell activators PMA, TNF-α, Prostratin and SAHA. Taken together our data suggest that T/F viruses LTR genetic variation and functional heterogeneity are important determinants of clinical outcomes and virus reactivation potential. [ABSTRACT FROM AUTHOR]

Interactions between coinfecting pathogens have the potential to alter the course of infection and can act as a source of phenotypic variation in susceptibility between hosts. This phenotypic variation may influence the evolution of host-pathogen interactions within host species and interfere with patterns in the outcomes of infection across host species. Here, we examine experimental coinfections of two Cripaviruses–Cricket Paralysis Virus (CrPV), and Drosophila C Virus (DCV)–across a panel of 25 Drosophila melanogaster inbred lines and 47 Drosophilidae host species. We find that interactions between these viruses alter viral loads across D. melanogaster genotypes, with a ~3 fold increase in the viral load of DCV and a ~2.5 fold decrease in CrPV in coinfection compared to single infection, but we find little evidence of a host genetic basis for these effects. Across host species, we find no evidence of systematic changes in susceptibility during coinfection, with no interaction between DCV and CrPV detected in the majority of host species. These results suggest that phenotypic variation in coinfection interactions within host species can occur independently of natural host genetic variation in susceptibility, and that patterns of susceptibility across host species to single infections can be robust to the added complexity of coinfection. Author summary: The susceptibility of a host to infection can be influenced by the presence of co-occurring (i.e., coinfecting) pathogens, which can interact by suppressing and activating host immunity, and competing for host resources. The influence of coinfections on host susceptibility could have ramifications for the evolution of hosts and pathogens, and this may be seen within a single host species as a change in the contribution of host genetics to susceptibility during coinfections, and across host species as a change in the ability of evolutionary relationships to explain similarities in susceptibility. Here, we test these possibilities by measuring susceptibility to two different Cripaviruses during single infections and coinfections across different insect host genotypes and host species. We find that these two viruses interact during coinfections within the fruit fly Drosophila melanogaster, altering host susceptibility, but that variation in this interaction didn't show any evidence of being influenced by host genetics. Similarly, we find little evidence of the explanatory power of evolutionary relationships across host species changing between single infections and coinfections. These results suggest that variation in interactions between pathogens can occur without the influence of host genetics, and that it is possible for patterns in susceptibility across host species measured during single infection to be maintained during coinfection. [ABSTRACT FROM AUTHOR]

Paratyphoid fever caused by S. Paratyphi A is endemic in parts of South Asia and Southeast Asia. The proportion of enteric fever cases caused by S. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory of S. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study "Surveillance of Enteric Fever in India (SEFI)." In the study period (2017–2020), S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis highlights that the genome degradation events and gene acquisitions or losses are key molecular events in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 hypothetically disrupted coding sequences (HDCS) or pseudogenes-forming mutations possibly associated with the emergence of lineages were identified. The pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We have identified six characteristic missense mutations associated with lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A, however, these mutations confer only a low structural impact and possibly have minimal impact on vaccine effectiveness. Since S. Paratyphi A is human-restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and monitoring by means of genomic surveillance would be constantly needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today. Author summary: Salmonella enterica serovar Paratyphi A remains one of the most common causes of enteric fever in the endemic regions of Asia. Within-host evolution of this human-restricted pathogen has led to the emergence of multiple lineages and sub-lineages through significant genomic rearrangement and pseudogene formation. Our study provides a snapshot overview of the gene flux and genome degradation events that shaped the evolution of S. Paratyphi A over the last 300 years. We leveraged the ongoing enteric fever surveillance program in India to collect 441 S. Paratyphi A clinical isolates, of which 152 were whole-genome sequenced. We carefully screened every internal node and cataloged mutations and gene flux at every stage of divergence/lineages throughout the phylogeny, revealing the role of key genetic changes in the evolutionary adaptation of S. Paratyphi A. Though several studies have previously reported the evolutionary trajectory of S. Paratyphi A, our study provides additional signatures with the most plausible evidence to date, backed by a molecular basis of adaptive mutations. The data from this study would merit further attention and interest in validating vaccines and developing novel diagnostics against S. Paratyphi A infections. [ABSTRACT FROM AUTHOR]

Treatment of parasitic nematode infections in humans and livestock relies on a limited arsenal of anthelmintic drugs that have historically reduced parasite burdens. However, anthelmintic resistance (AR) is increasing, and little is known about the molecular and genetic causes of resistance for most drugs. The free-living roundworm Caenorhabditis elegans has proven to be a tractable model to understand AR, where studies have led to the identification of molecular targets of all major anthelmintic drug classes. Here, we used genetically diverse C. elegans strains to perform dose-response analyses across 26 anthelmintic drugs that represent the three major anthelmintic drug classes (benzimidazoles, macrocyclic lactones, and nicotinic acetylcholine receptor agonists) in addition to seven other anthelmintic classes. First, we found that C. elegans strains displayed similar anthelmintic responses within drug classes and significant variation across drug classes. Next, we compared the effective concentration estimates to induce a 10% maximal response (EC10) and slope estimates of each dose-response curve of each strain to the laboratory reference strain, which enabled the identification of anthelmintics with population-wide differences to understand how genetics contribute to AR. Because genetically diverse strains displayed differential susceptibilities within and across anthelmintics, we show that C. elegans is a useful model for screening potential nematicides before applications to helminths. Third, we quantified the levels of anthelmintic response variation caused by genetic differences among individuals (heritability) to each drug and observed a significant correlation between exposure closest to the EC10 and the exposure that exhibited the most heritable responses. These results suggest drugs to prioritize in genome-wide association studies, which will enable the identification of AR genes. Author summary: Parasitic nematodes infect most animal species and significantly impact human and animal health. Control of parasitic nematodes in host species relies on a limited collection of anthelmintic drugs. However, anthelmintic resistance is widespread, which threatens our ability to control parasitic nematode populations. Here, we used the non-parasitic roundworm Caenorhabditis elegans as a model to study anthelmintic resistance across 26 anthelmintics that span ten drug classes. We leveraged the genetic diversity of C. elegans to quantify anthelmintic responses across a range of doses, estimate dose-response curves, fit strain-specific model parameters, and calculate the contributions of genetics to these parameters. We found that genetic variation within a species plays a considerable role in anthelmintic responses within and across drug classes. Our results emphasize how the incorporation of genetically diverse C. elegans strains is necessary to understand anthelmintic response variation found in natural populations. These results highlight drugs to prioritize in future mapping studies to identify genes involved in anthelmintic resistance. [ABSTRACT FROM AUTHOR]

Transmissions of simian viruses to humans has originated the different groups of HIV-1. We recently identified a functional motif (CLA), in the C-terminal domain of the integrase, essential for integration in HIV-1 group M. Here, we found that the motif is instead dispensable in group O isolates, because of the presence, in the N-terminal domain of HIV-1 O of a specific sequence, Q7G27P41H44, that we define as the NOG motif. Alterations of reverse transcription and of 3' processing observed by mutating the CLA motif of IN M are fully rescued to wt levels by inserting the sequence of the NOG motif in the N-ter of the protein. These results indicate that the two motifs (CLA and NOG) functionally complement each other and a working model accounting for these observations is proposed. The establishment of these two alternative motifs seems to be due to the different phylogenetic origin and history of these two groups. Indeed, the NOG motif is already present in the ancestor of group O (SIVgor) while it is absent from SIVcpzPtt, the ancestor of group M. The CLA motif, instead, seems to have emerged after SIVcpzPtt has been transferred to humans, since no conservation is found at the same positions in these simian viruses. These results show the existence of two-group specific motifs in HIV-1 M and O integrases. In each group, only one of the motifs is functional, potentially leading the other motif to diverge from its original function and, in an evolutionary perspective, assist other functions of the protein, further increasing HIV genetic diversity. Author summary: HIV-1 establishes a permanent infection by integrating its genome in the one of the host cell. This key step is achieved by the viral integrase (IN), which is composed of three domains: N-terminal (NTD), catalytic core (CCD), and C-terminal (CTD). We recently reported, in the CTD, the existence of a functional motif (CLA) that is essential for integration in HIV-1 group M. Here we show that in HIV-1 group O, the function exerted by the integrase M CLA motif is instead ensured by another motif (NOG), located in the NTD of the protein. We show that these two motifs can functionally complement each other by acting on the same steps of the viral cycle, highlighting for the first time such an important functional duality between HIV-1 integrases. We also show that, while the NOG motif in HIV-1 O was likely inherited from its simian ancestor (SIVgor), the CLA motif seems to have emerged in the human host, probably in the process of adaptation to the new host of the simian virus on its way to become HIV-1 M. Such a requirement did not seem to have existed for HIV-1 O, possibly because of the presence of the NOG motif. [ABSTRACT FROM AUTHOR]

RNA viruses can exchange genetic material during coinfection, an interaction that creates novel strains with implications for viral evolution and public health. Influenza A viral genetic exchange can occur when genome segments from distinct strains reassort in coinfected cells. Predicting potential genomic reassortment between influenza strains has been a long-standing goal. Experimental coinfection studies have shed light on factors that limit or promote reassortment. However, determining the reassortment potential between diverse Influenza A strains has remained elusive. To address this challenge, we developed a high throughput genotyping approach to quantify reassortment among a diverse panel of human influenza virus strains encompassing two pandemics (swine and avian origin), three specific epidemics, and both circulating human subtypes A/H1N1 and A/H3N2. We found that reassortment frequency (the proportion of reassortants generated) is an emergent property of specific pairs of strains where strain identity is a predictor of reassortment frequency. We detect little evidence that antigenic subtype drives reassortment as intersubtype (H1N1xH3N2) and intrasubtype reassortment frequencies were, on average, similar. Instead, our data suggest that certain strains bias the reassortment frequency up or down, independently of the coinfecting partner. We observe that viral productivity is also an emergent property of coinfections, but uncorrelated to reassortment frequency; thus viral productivity is a separate factor affecting the total number of reassortants produced. Assortment of individual segments among progeny and pairwise segment combinations within progeny generally favored homologous combinations. These outcomes were not related to strain similarity or shared subtype but reassortment frequency was closely correlated to the proportion of both unique genotypes and of progeny with heterologous pairwise segment combinations. We provide experimental evidence that viral genetic exchange is potentially an individual social trait subject to natural selection, which implies the propensity for reassortment is not evenly shared among strains. This study highlights the need for research incorporating diverse strains to discover the traits that shift the reassortment potential to realize the goal of predicting influenza virus evolution resulting from segment exchange. Author summary: Influenza A viruses are global pathogens that cause significant human morbidity and mortality through seasonal epidemics and pandemics that arise every few decades. These viruses have remained a public health threat in large part due to their high genetic variability, notably through exchange of their eight genome segments when two viruses coinfect a cell. Thus, determining the potential for genetic exchange has been an urgent goal, but has been difficult to achieve. Here we developed a novel method to quantify genetic exchange in a diverse collection of human influenza A viruses. We show that patterns of genetic exchange show strong strain-dependence, and do not necessarily track similarity between coinfecting strains or their subtype (H1N1 or H3N2). Our data support that specific strains can consistently bias genetic exchange, increasing or decreasing exchange independent of the other coinfecting strain. Our study suggests the propensity for reassortment is not evenly shared among strains and that identifying high-reassortment strains could improve pandemic preparedness or reveal new treatments. [ABSTRACT FROM AUTHOR]

Biofilms of the fungal pathogen Candida albicans include abundant long filaments called hyphae. These cells express hypha-associated genes, which specify diverse virulence functions including surface adhesins that ensure biofilm integrity. Biofilm formation, virulence, and hypha-associated gene expression all depend upon the transcription factor Efg1. This transcription factor has been characterized extensively in the C. albicans type strain SC5314 and derivatives, but only recently has its function been explored in other clinical isolates. Here we define a principal set of Efg1-responsive genes whose expression is significantly altered by an efg1Δ/Δ mutation across 17 clinical isolates. This principal gene set includes 68 direct Efg1 targets, whose 5' regions are bound by Efg1 in five clinical isolates, and 42 indirect Efg1 targets, whose 5' regions are not detectably bound by Efg1. Three direct Efg1 target genes encode transcription factors—BRG1, UME6, and WOR3 –whose increased expression in an efg1Δ/Δ mutant restores expression of multiple indirect and direct principal targets, as well as biofilm formation ability. Although BRG1 and UME6 are well known positive regulators of hypha-associated genes and biofilm formation, WOR3 is best known as an antagonist of Efg1 in the sexual mating pathway. We confirm the positive role of WOR3 in biofilm formation with the finding that a wor3Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo biofilm model. Positive control of Efg1 direct target genes by other Efg1 direct target genes–BRG1, UME6, and WOR3 –may buffer principal Efg1-responsive gene expression against the impact of genetic variation in the C. albicans species. Author summary: Biofilm growth of the fungal pathogen Candida albicans is a prevalent source of infection. Biofilm gene regulation is well understood in derivatives of the type strain SC5314, but few studies have examined regulatory features in other clinical isolates. Here we characterize the phenotypic and gene regulatory impact of deletion of master regulatory gene EFG1 in seventeen clinical isolates that represent a range of clades and infection sites. The mutation abolishes biofilm formation in all isolates, but has widely variable gene expression impact, even among members of a clade. Fewer than 20% of the gene expression targets in any one isolate are principal targets, which are shared among all isolates. One principal target encodes the transcription factor Wor3, an antagonist of Efg1 in the sexual mating pathway. Our analysis shows that Wor3 has an unexpected role in which it promotes biofilm formation and the expression of principal Efg1-activated genes. [ABSTRACT FROM AUTHOR]

The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring less genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to changes in drug regime. Using newly sequenced whole genomes, we measure relatedness between 166 parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore past selection events in the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in small populations, as will occur in any region nearing malaria elimination. Author summary: Malaria caused by Plasmodium falciparum is a leading cause of mortality in young children, primarily in sub-Saharan Africa. Safe and effective antimalarials have spurred decades of declining prevalence, but drug resistance threatens global elimination efforts. In the Americas, P. falciparum transmission and genetic diversity are low. The region accounts for only a small proportion of global infections and mortality, yet it has historically contributed to the emergence and dissemination of P. falciparum antimalarial drug resistance. Genomic surveillance in the region can detect emerging drug resistance, dissect its evolutionary dynamics, and impede its spread to highly endemic regions. In this work, we generated and analyzed whole genome sequence data from samples collected in the Pacific Coast region of Colombia and Ecuador, a hotspot of P. falciparum malaria. This region exhibits a large proportion of single-clone infections as well as long-term clonal persistence. Our study analyzes this inbred population structure using relatedness estimates calculated as identity-by-descent. We describe the spatial and temporal dynamics of clonal transmission and outcrossing, demonstrating that selection is still effective in this small, inbred population. These findings will support future drug resistance surveillance in regions with intense interventions and declining prevalence. [ABSTRACT FROM AUTHOR]

Kaposi sarcoma (KS), a common HIV-associated malignancy, presents a range of clinicopathological features. Kaposi sarcoma-associated herpesvirus (KSHV) is its etiologic agent, but the contribution of viral genomic variation to KS development is poorly understood. To identify potentially influential viral polymorphisms, we characterized KSHV genetic variation in 67 tumors from 1–4 distinct sites from 29 adults with advanced KS in Kampala, Uganda. Whole KSHV genomes were sequenced from 20 tumors with the highest viral load, whereas only polymorphic genes were screened by PCR and sequenced from 47 other tumors. Nine individuals harbored ≥1 tumors with a median 6-fold over-coverage of a region centering on K5 and K6 genes. K8.1 gene was inactivated in 8 individuals, while 5 had mutations in the miR-K10 microRNA coding sequence. Recurring inter-host polymorphisms were detected in K4.2 and K11.2. The K5-K6 region rearrangement breakpoints and K8.1 mutations were all unique, indicating that they arise frequently de novo. Rearrangement breakpoints were associated with potential G-quadruplex and Z-DNA forming sequences. Exploratory evaluations of viral mutations with clinical and tumor traits were conducted by logistic regression without multiple test corrections. K5-K6 over-coverage and K8.1 inactivation were tentatively correlated (p<0.001 and p = 0.005, respectively) with nodular rather than macular tumors, and with individuals that had lesions in ≤4 anatomic areas (both p≤0.01). Additionally, a trend was noted for miR-K10 point mutations and lower survival rates (HR = 4.11, p = 0.053). Two instances were found of distinct tumors within an individual sharing the same viral mutation, suggesting metastases or transmission of the aberrant viruses within the host. To summarize, KSHV genomes in tumors frequently have over-representation of the K5-K6 region, as well as K8.1 and miR-K10 mutations, and each might be associated with clinical phenotypes. Studying their possible effects may be useful for understanding KS tumorigenesis and disease progression. Author summary: How chronic KSHV infection leads to the development of KS is poorly understood, however, evaluation of KSHV mutations found in vivo may assist this understanding. Here, targeted and whole genome sequencing of KSHV in multiple tumors from individuals in Uganda revealed an increase in copy number of the region of the genome encompassing the K5 and K6 genes or inactivation of the K8.1 gene in at least a third of the individuals. In an exploratory analysis these mutations were found more in tumors of nodular than macular morphotype and in individuals with fewer lesions. These mutations were unique across individuals, indicating that they arise frequently de novo. Point mutations in the miR-K10 micro-RNA sequence were weakly associated with lower survival. Occasionally, distinct tumors within an individual shared the same viral mutation, suggesting metastases or transmission of the aberrant viruses within the host. Clustering of genome rearrangement breakpoints were associated with potential G-quadruplex and Z-DNA forming regions in the viral DNA. The viral genetic differences uncovered and their associated clinical phenotypes call for targeted surveys of these genes to potentially establish a prognostic or treatment-selective biomarker and more studies into their roles in tumorigenesis. [ABSTRACT FROM AUTHOR]

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be. Author summary: HIV-1 sexual transmission is surprisingly inefficient. In the relatively unusual event of successful transmission, there is typically a severe genetic bottleneck, such that infection is established by just a single (or small number) of genetic variants, described as transmitted founder (TF) viruses. Such limited transmission is considered to arise from both physical and immunological barriers that restrict viruses, such as the interferon system. TF viruses have been described as more interferon resistant than viruses present in chronically infected individuals. Here we found that an IFN-resistant transmitted HIV-1 virus is uniformly resistant to multiple interferon-stimulated genes, which led us to investigate the underlying mechanism. Our characterisation of a single TF and chronic virus pair (closely matched in replicative fitness) revealed that small differences in replicative growth rates could possibly explain the broad interferon resistance frequently associated with transmitted HIV-1, although further studies are required to confirm this. Our study provides insight into how relatively small differences in growth rate could still be phenotypically significant, and that a nonspecific mechanism could possibly explain the interferon resistance of transmitted HIV-1. [ABSTRACT FROM AUTHOR]

The lung is an understudied site of HIV persistence. We isolated 898 subgenomic proviral sequences (nef) by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 8% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harboured at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. The extent of blood proviral diversity on ART was also a strong indicator of diversity in lung (Spearman's ρ = 0.98, p<0.0001). For three participants, insufficient lung sequences were recovered to reliably investigate genetic compartmentalization. Of the remainder, only two participants showed statistically significant support for compartmentalization when analysis was restricted to distinct proviruses per site, and the extent of compartmentalization was modest in both cases. When all within-host sequences (including duplicates) were considered, the number of compartmentalized datasets increased to four. Thus, while a subset of individuals harbour somewhat distinctive proviral populations in blood and lung, this can simply be due to unequal distributions of clonally-expanded sequences. For two participants, on-ART proviruses were also phylogenetically analyzed in context of plasma HIV RNA populations sampled up to 18 years prior, including pre-ART and during previous treatment interruptions. In both participants, on-ART proviruses represented the most ancestral sequences sampled within-host, confirming that HIV sequences can persist in the body for decades. This analysis also revealed evidence of re-seeding of the reservoir during treatment interruptions. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individual's proviral composition. Personalized HIV remission and cure strategies may be needed to overcome these challenges. Author summary: HIV persists in the body despite long-term antiretroviral therapy (ART). Much of our knowledge about the HIV reservoir comes from studying blood proviruses on ART, but a fundamental question is whether these are distinct from those in tissue. The lung could theoretically engender genetically distinctive HIV populations, but this remains understudied. Our analysis of nearly 900 subgenomic proviral sequences from blood and lung of individuals receiving long-term HIV therapy revealed substantial within-host heterogeneity, yet some common patterns. Identical sequences (consistent with clonal expansion of infected cells) were observed in everyone, though at varying (8–86%) frequencies. The same sequence was often recovered from both blood and lung (77% of participants). Only a subset of individuals exhibited blood-lung genetic compartmentalization, and only modestly so, where this compartmentalization was sometimes due to differential frequencies of identical sequences, not the presence of truly genetically distinctive populations, across sites. Blood proviral diversity mirrored that in lung, indicating that strategies to limit the former (e.g. early ART) should also limit the latter. Results also revealed evidence that blood and lung proviruses can persist for >20 years within-host, and that viruses emerging in blood during treatment interruptions can re-seed both lung and blood reservoirs. [ABSTRACT FROM AUTHOR]

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source. [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is an arboviral disease of humans and livestock responsible for severe economic and human health impacts. In humans, RVF spans a variety of clinical manifestations, ranging from an acute flu-like illness to severe forms of disease, including late-onset encephalitis. The large variations in human RVF disease are inadequately represented by current murine models, which overwhelmingly die of early-onset hepatitis. Existing mouse models of RVF encephalitis are either immunosuppressed, display an inconsistent phenotype, or develop encephalitis only when challenged via intranasal or aerosol exposure. In this study, the genetically defined recombinant inbred mouse resource known as the Collaborative Cross (CC) was used to identify mice with additional RVF disease phenotypes when challenged via a peripheral foot-pad route to mimic mosquito-bite exposure. Wild-type Rift Valley fever virus (RVFV) challenge of 20 CC strains revealed three distinct disease phenotypes: early-onset hepatitis, mixed phenotype, and late-onset encephalitis. Strain CC057/Unc, with the most divergent phenotype, which died of late-onset encephalitis at a median of 11 days post-infection, is the first mouse strain to develop consistent encephalitis following peripheral challenge. CC057/Unc mice were directly compared to C57BL/6 mice, which uniformly succumb to hepatitis within 2–4 days of infection. Encephalitic disease in CC057/Unc mice was characterized by high viral RNA loads in brain tissue, accompanied by clearance of viral RNA from the periphery, low ALT levels, lymphopenia, and neutrophilia. In contrast, C57BL/6 mice succumbed from hepatitis at 3 days post-infection with high viral RNA loads in the liver, viremia, high ALT levels, lymphopenia, and thrombocytopenia. The identification of a strain of CC mice as an RVFV encephalitis model will allow for future investigation into the pathogenesis and treatment of RVF encephalitic disease and indicates that genetic background makes a major contribution to RVF disease variation. Author summary: Rift Valley fever virus (RVFV) is a negative sense RNA arbovirus of clinical relevance found throughout Africa and into the Middle East. In humans, RVF disease exhibits various clinical manifestations including acute febrile illness, hepatitis, hemorrhagic fever, and encephalitis. Unfortunately, existing murine models have been limited to studies of acute lethal hepatitis. Here, we identify a mouse model in which all animals survive acute hepatitis and go on to succumb to late onset encephalitis. A key feature of this new experimental system is administration of the virus in the footpad (to mimic mosquito bite), to model a natural route of infection. Importantly in this work, we used the Collaborative Cross recombinant inbred mouse resource to identify this RVFV CNS model. Therefore, our study demonstrates that host genetics can modulate RVF disease manifestations. The identification of this model also enables the field for high throughput, in vivo studies of central nervous system pathogenesis as well as in vivo evaluation of vaccines and therapeutics targeted against RVF encephalitis. [ABSTRACT FROM AUTHOR]

To manage and treat chronic fungal diseases effectively, we require an improved understanding of their complexity. There is an increasing appreciation that chronic infection populations are often heterogeneous due to diversification and drift, even within a single microbial species. Genetically diverse populations can contribute to persistence and resistance to treatment by maintaining cells with different phenotypes capable of thriving in these dynamic environments. In chronic infections, fungal pathogens undergo prolonged challenges that can drive trait selection to convergent adapted states through restricted access to critical nutrients, assault by immune effectors, competition with other species, and antifungal drugs. This review first highlights the various genetic and epigenetic mechanisms that promote diversity in pathogenic fungal populations and provide an additional barrier to assessing the actual heterogeneity of fungal infections. We then review existing studies of evolution and genetic heterogeneity in fungal populations from lung infections associated with the genetic disease cystic fibrosis. We conclude with a discussion of open research questions that, once answered, may aid in diagnosing and treating chronic fungal infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Murante, Hogan. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The pathogenesis of HIV-1 infection is governed by a highly dynamic, time-dependent interaction between the host and the viral genome. In this study, we developed a novel systematic approach to assess the host-virus interaction, using average pairwise viral diversity as a proxy for time since infection, and applied this method to nearly whole viral genome sequences (n = 4,464), human leukocyte antigen (HLA) genotyping data (n = 1,044), and viral RNA load (VL) measurements during the untreated chronic phase (n = 829) of Swiss HIV Cohort Study participants. Our systematic genome-wide screen revealed for 98 HLA/viral-variant pairs a signature of immune-driven selection in the form of an HLA-dependent effect of infection time on the presence of HIV amino acid variants. Of these pairs, 12 were found to have an effect on VL. Furthermore, 28/58 pairs were validated by time-to-event analyses and 48/92 by computational HLA-epitope predictions. Our diversity-based approach allows a powerful and systematic investigation of the interaction between the virus and cellular immunity, revealing a notable subset of such interaction effects. From an evolutionary perspective, these observations underscore the complexity of HLA-mediated selection pressures on the virus that shape viral evolution and pathogenesis., Competing Interests: K.J.M. has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, GlaxoSmithKline, Merck Sharp & Dohme, Bristol-Myers Squibb, ViiV, and Abbott; and the University of Zurich has received research grants from Gilead Science, Novartis, Roche, and Merck Sharp & Dohme for studies in which K.J.M. serves as principal investigator, and advisory board honoraria from Gilead Sciences and ViiV. A.R. reports support to his institution for advisory board and/or travel grants from MSD, Gilead Sciences, Pfizer, and Moderna, and an investigator-initiated trial (IIT) grant from Gilead Sciences. All honoraria went to his home institution, not to A.R. personally, and all honoraria were provided outside of the submitted work. M.S. reports advisory board consultations from Gilead, ViiV, MSD, paid to his institution, and travel grants for conferences from Gilead, paid to his institution. E.B.’s institution has received research grants from Gilead and Merck unrelated to this work; E.B.’s institution has also received consultancy fees and travel grants from Gilead, ViiV, Merck, Pfizer, Astra Zeneca, Moderna, Abbvie, and Ely Lilly. J.N. has received travel grants from Gilead. H.F.G. has received grants from the Yvonne-Jacob Foundation, the Clinical Research Priority Program of the University of Zurich, and Gilead Sciences, and, outside of this study, grants for unrestricted research from the Swiss HIV Cohort Study, the Swiss National Science Foundation, the National Institutes of Health, the Bill and Melinda Gates Foundation, Gilead, and ViiV; and personal fees as a consultant for Merck, ViiV Healthcare, and Gilead Sciences and as a member of the Data and Safety Monitoring Board for Merck. H.F.G.’s institution has received educational grants unrelated to this work from Gilead, ViiV, MSD, Abbvie, Pfizer, and Sandoz. C.P. has received fellowships from the Collegium Helveticum. R.D.K. has received grants from Gilead Sciences, the National Institutes of Health, the Swiss National Science Foundation, and the Swiss HIV Cohort Study. N.N.J. has received support from the Swiss National Science Foundation and the Swiss HIV Cohort Study. All other authors report no potential conflicts of interest., (Copyright: © 2024 Neuner-Jehle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Competing Interests: The authors have declared that no competing interests exist.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. Author summary: Chronic HCV infections remain an important global health issue, despite the availability of highly efficient therapies. So far no protective vaccine is available, which is in part due to the high divergence of HCV variants and the limited possibility to mirror this genetic diversity in cell culture. It has been proven particularly difficult to grow infectious virus in cell culture, requiring extensive adaptation with multiple mutations, which in turn affect infectivity of the adapted variants in vivo. Here we have isolated a genotype 1b variant from a very high titer serum of a patient after liver transplantation (German Liver Transplant 1, GLT1), showing an outstanding genome replication efficiency in cultured hepatoma cells. We were able to adapt this isolate to production of infectious virus, therefore generating a novel full-replication cycle cell culture model for the highly prevalent HCV genotype 1b. Despite multiple mutations required, adapted GLT1 was still infectious in vivo. GLT1 therefore is an important development facilitating future efforts in vaccine development. [ABSTRACT FROM AUTHOR]

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203–205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection. Author summary: Since its emergence, SARS-CoV-2 has continued to adapt for human infection resulting in the emergence of variants with unique genetic profiles. Most studies of genetic variation have focused on spike, the target of currently available vaccines, leaving the importance of variation elsewhere understudied. Here, we characterize a highly variable motif at residues 203–205 in nucleocapsid. Recreating the prominent nucleocapsid R203K+G204R mutation in an early pandemic background, we show that this mutation is alone sufficient to enhance SARS-CoV-2 replication and pathogenesis. We also link augmentation of SARS-CoV-2 infection by the R203K+G204R mutation to its modulation of nucleocapsid phosphorylation. Finally, we characterize an analogous alanine double substitution at positions 203–204. This mutant was found to mimic R203K+G204R, suggesting augmentation of infection occurs by disrupting the ancestral sequence. Together, our findings illustrate that mutations outside of spike are key components of SARS-CoV-2's adaptation to human infection. [ABSTRACT FROM AUTHOR]

Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, on the available data we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north. Author summary: A result of the ever-growing poultry industry is a dramatic global increase in the incidence of high pathogenicity avian influenza virus outbreaks. In contrast, wild birds are believed to be the main reservoir for low pathogenic avian influenza A virus. Due to intensive research and surveillance of AIV in waterfowl in the Northern Hemisphere, we have a better understanding of AIV ecology and evolution in that region compared to the Southern Hemisphere, which are characterised by different patterns of avian migration and ecological conditions. We analysed 333 unique AIV genomes collected from wild birds in Australia to understand how Australia fits into global AIV dynamics and how viruses are maintained and dispersed within the continent of Australia. We show that the Southern Hemisphere experiences differing evolutionary dynamics to those seen in Northern Hemisphere with Australia representing a global sink for AIV. [ABSTRACT FROM AUTHOR]

Serratia marcescens is a versatile opportunistic pathogen that can cause a variety of infections, including bacteremia. Our previous work established that the capsule polysaccharide (CPS) biosynthesis and translocation locus contributes to the survival of S. marcescens in a murine model of bacteremia and in human serum. In this study, we determined the degree of capsule genetic diversity among S. marcescens isolates. Capsule loci (KL) were extracted from >300 S. marcescens genome sequences and compared. A phylogenetic comparison of KL sequences demonstrated a substantial level of KL diversity within S. marcescens as a species and a strong delineation between KL sequences originating from infection isolates versus environmental isolates. Strains from five of the identified KL types were selected for further study and electrophoretic analysis of purified CPS indicated the production of distinct glycans. Polysaccharide composition analysis confirmed this observation and identified the constituent monosaccharides for each strain. Two predominant infection-associated clades, designated KL1 and KL2, emerged from the capsule phylogeny. Bacteremia strains from KL1 and KL2 were determined to produce ketodeoxynonulonic acid and N-acetylneuraminic acid, two sialic acids that were not found in strains from other clades. Further investigation of KL1 and KL2 sequences identified two genes, designated neuA and neuB, that were hypothesized to encode sialic acid biosynthesis functions. Disruption of neuB in a KL1 isolate resulted in the loss of sialic acid and CPS production. The absence of sialic acid and CPS production also led to increased susceptibility to internalization by a human monocytic cell line, demonstrating that S. marcescens phagocytosis resistance requires CPS. Together, these results establish the capsule genetic repertoire of S. marcescens and identify infection-associated clades with sialic acid CPS components. Author summary: Serratia marcescens is a bacterial species with a broad natural distribution that is capable of causing infections, particularly in individuals with underlying health conditions. One consideration for organisms, such as S. marcescens, that can thrive in diverse environments is whether specific genetic lineages are better suited to causing infection than others. The capsular polysaccharide of S. marcescens is a variable surface structure that is important for bacterial survival in an experimental model of bloodstream infection and we sought to determine the spectrum of genetic diversity within the capsule-encoding genetic region among both infection and environmental isolates. Here, we demonstrate that S. marcescens strains originating from infection and environmental sources segregate into distinct clades based on capsule gene phylogeny. The majority of infection isolates analyzed were further differentiated into two types. Strains from both of the primary infection-associated capsule types produce sialic acids, a previously unrecognized trait that is likely to have implications in capsule polysaccharide function and host immune system recognition. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited β-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited β-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel β-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable β-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes. Author summary: Human cytomegalovirus (HCMV) is a prevalent herpesvirus infecting an estimated 60% of the human population worldwide. It is commonly transmitted during early childhood and leads to life-long latency, where viral reactivation can cause severe complications in case of host immune suppression. Furthermore, HCMV is the leading cause of congenital infections. Circulating HCMV strains exhibit great genetic diversity unusual for DNA viruses. One of its most diverse genes is UL146, which encodes a chemokine that facilitates viral dissemination by exploiting the human immune system through mimicry of key immunity components. In this study, we investigate how the diversity of UL146 affects its signaling and structural properties to understand why its genetic diversity is maintained across human populations. We find that certain genotypes that lack key structural domains present in the human homologs nonetheless exert similar functions in the virus-host relationship. Furthermore, many of the UL146 genotypes contain novel structural elements critical for correct protein folding and with the potential to provide HCMV with additional immune modulatory and evasive features. Together, our data highlight a considerable degree of host-adaptation by HCMV and propose novel structural interactions with implications for the virus-host interplay. [ABSTRACT FROM AUTHOR]

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C—40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes. Author summary: Arthropod-borne viruses (arboviruses) have emerged in recent decades due to complex factors that include increases in international travel and trade, the breakdown of public health infrastructure, land use changes, and many others. Climate change also has the potential to shift the geographical ranges of arthropod vectors, consequently increasing the global risk of arbovirus infection. Changing temperatures may alter the virus-host interaction, ultimately resulting in the emergence of new viruses and virus genotypes in new areas. Therefore, we sought to characterize how temperature (both constant and fluctuating) alters the ability of Aedes aegypti and Aedes albopictus to transmit Zika virus, and how it influences virus populations within mosquitoes. We found that intermediate temperatures maximize virus transmission compared to more extreme and fluctuating temperatures. Constant temperatures increased positive selection on virus genomes, while fluctuating temperatures strengthened purifying selection. Our studies provide evidence that in addition to altering vector competence, temperature significantly influences natural selection within mosquitoes. [ABSTRACT FROM AUTHOR]

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5' region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease. Author summary: The human bacterial pathogen Neisseria meningitidis (the meningococcus) causes sepsis and meningitis worldwide. Paradoxically, it is carried harmlessly in the nose and throat far more commonly than it causes invasive disease, raising the question of whether the bacteria causing disease are genetically the same as those that do not. We looked for systematic differences in the DNA of the bacteria from invasive disease and those that were carried harmlessly. Whilst controlling for population structure as a confounder, we identified multiple genes apparently contributing to invasion and identified a signal around the gene encoding an important component of some meningococcal vaccines, called factor H binding protein (fHbp). This protein helps the meningococcus evade killing by the human immune response by binding to a human defence protein called complement factor H. We validated this result in an independent population of meningococci and identified that DNA variants that control the amount of fHbp produced by the bacteria were important, with high levels of fHbp expression associated with invasion. Given that human DNA variation in complement factor H increases the risk of meningococcal invasion, this highlights the important connection between human and bacterial genetic variation and helps explain why some people infected with meningococci suffer meningococcal disease whilst others do not. [ABSTRACT FROM AUTHOR]

Competing Interests: The authors have declared that no competing interests exist.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Salmonella enterica (S. enterica) has infected humans for a long time, but its evolutionary history and geographic spread across Eurasia is still poorly understood. Here, we screened for pathogen DNA in 14 ancient individuals from the Bronze Age Quanergou cemetery (XBQ), Xinjiang, China. In 6 individuals we detected S. enterica. We reconstructed S. enterica genomes from those individuals, which form a previously undetected phylogenetic branch basal to Paratyphi C, Typhisuis and Choleraesuis–the so-called Para C lineage. Based on pseudogene frequency, our analysis suggests that the ancient S. enterica strains were not host adapted. One genome, however, harbors the Salmonella pathogenicity island 7 (SPI-7), which is thought to be involved in (para)typhoid disease in humans. This offers first evidence that SPI-7 was acquired prior to the emergence of human-adapted Paratyphi C around 1,000 years ago. Altogether, our results show that Salmonella enterica infected humans in Eastern Eurasia at least 3,000 years ago, and provide the first ancient DNA evidence for the spread of a pathogen along the Proto-Silk Road. Author summary: Recent studies that use DNA extracted from ancient human remains have shown that the bacterial pathogen Salmonella enterica has infected humans since prehistoric times. Expanding the knowledge about its geographic spread and genetic diversity we reconstructed six ancient Salmonella genomes from individuals excavated at the Bronze Age Quanergou cemetery in Xinjiang, China. Our analysis shows that this form of Salmonella was closely related to a type that today specifically infects humans and/or pigs. However, based on our genetic assessment we determined that our ancient strains were not host adapted yet. Furthermore, we could show that in the course of its evolution the bacterium acquired some important virulence factors earlier than previously thought. Interpreting our results within an archaeological context we suggest that in Bronze Age Eastern Eurasia the spread of this pathogen was likely promoted through trade networks referred to as the Proto-Silk Road. [ABSTRACT FROM AUTHOR]

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus. Author summary: Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving disease pathology. In this study, we aimed to determine if genetic variation of ECE1 existed across a collection of C. albicans clinical isolates. Using a targeted sequencing approach, we identified a conserved variant candidalysin isoform. We then constructed isogenic chimeric strains harboring swapped Ece1p peptides or HiBiT tags between representative isoforms and showed impaired secretion with the variant that was associated with reduced virulence in vitro and in vivo. We also elucidated a conserved pattern between key sequences near lysine-arginine cleavage sites involved in Ece1p polypeptide processing by the Kex2p enzyme that correlate with predicted and experimental candidalysin secretion. Therefore, we present a novel mechanism for attenuation of C. albicans virulence at the ECE1 locus that may partly underlie differential clinical outcomes and contribute to the balance between commensal and pathogen at the vaginal mucosa. [ABSTRACT FROM AUTHOR]

Influenza A virus [IAV] genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins [vRNPs]. Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virusproduction. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes [TNTs]as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion. Author summary: Influenza A viruses infect epithelial cells of the upper and lower respiratory tract in humans. Infection is propagated by the generation of viral particles from infected cells, which disseminate within the tissue. Disseminating particles can encounter obstacles in the extracellular environment, including mucus, ciliary movement, antibody neutralization and uptake by phagocytic immune cells. An alternative mode of spread, which avoids these hazards, involves directtransport of viral components between cells. This cell-cell spread of infection is not a well understood process. In this study we demonstrate thatthe host factor Rab11a mediates the transport of viral genomes in the cell-cell spread of infection. Rab11a is already known to play a pro-viral role in the transport of viral genomes to the plasma membrane for assembly into virus particles. Here, we see that this same transport mechanism is co-opted for direct cell-cell spread through cellular connections called tunneling nanotubes. We show that complexes of Rab11a and viral components can be trafficked across tunneling nanotubes, transmitting infection without the formation of virus particles. Importantly, this route of spread often seeds viral genomes from multiple donor cells into recipient cells, which in turn increases viral genetic diversity. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue. Author summary: Herpesviruses are highly human pathogens that establish latency in neurons of the peripheral nervous system. Colonization of nerve endings is required for herpes simplex virus (HSV) persistence and pathogenesis. HSV-1 global prevalence is much higher than HSV-2, in addition to their preferential tendency to infect the oronasal and genital areas, respectively. How these closely related viruses have been adapting and evolving to replicate and colonize these two different anatomical areas remains unclear. Herpesviruses were presumed to mutate much less than viruses with RNA genomes, due to the higher fidelity of the DNA polymerase and proofreading mechanisms when replicating. However, the worldwide accessibility and development of high-throughput sequencing technologies have revealed the heterogenicity and high diversity present in viral populations clinically isolated. Here we show that HSV-2 mutates much faster than HSV-1, when compared under similar and controlled cell culture conditions. This high mutation rate is translated into an increase in coding diversity, since the great majority of these new mutations lead to nonconservative changes in viral proteins. Understanding how herpesviruses differentially mutate under similar selective pressures is critical to prevent resistance to anti-viral drugs. [ABSTRACT FROM AUTHOR]

The emergence of divergent SARS-CoV-2 lineages has raised concern that novel variants eliciting immune escape or the ability to displace circulating lineages could emerge within individual hosts. Though growing evidence suggests that novel variants arise during prolonged infections, most infections are acute. Understanding how efficiently variants emerge and transmit among acutely-infected hosts is therefore critical for predicting the pace of long-term SARS-CoV-2 evolution. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely-infected individuals. We find that within-host diversity is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household, and are rarely detected along phylogenetically linked infections in the broader community. These findings suggest that most variation generated within-host is lost during transmission. Author summary: RNA viruses generate diversity within individual, infected hosts. This genetic diversity can be used to trace how viruses evolve during the course of infection within individuals, and transmission between them. To investigate how SARS-CoV-2 diversity is generated and propagated, we deep sequenced 133 SARS-CoV-2 genomes isolated from acutely infected individuals in Wisconsin. We capitalize on a large dataset of consensus genomes from Wisconsin to investigate how variants are transmitted within the surrounding community, and use a unique household dataset to estimate the number of viruses that are transmitted between epidemiologically linked individuals. We find that most SARS-CoV-2 infections are characterized by limited within-host diversity, and that the vast majority of intra-host single nucleotide variants (iSNVs) are lost during transmission. We do not find evidence that variation is frequently propagated along phylogenetically linked infections, and estimate that most infections are founded by very few unique virions. The combination of limited within-host diversity and tight transmission bottlenecks may slow the pace of SARS-CoV-2 evolution in the future, and suggests that extensive within-host evolution is likely rare. [ABSTRACT FROM AUTHOR]

Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards. Little is known about the virus diversity in wild citrus species. Here, we used a metatranscriptomics approach to characterize the virus diversity in a wild citrus habitat within the proposed center of the origin of citrus plants. We discovered a total of 44 virus isolates that could be classified into species Citrus tristeza virus and putative species citrus associated ampelovirus 1, citrus associated ampelovirus 2, and citrus virus B within the family Closteroviridae, providing important information to explore the factors facilitating outbreaks of citrus viruses and the evolutionary history of the family Closteroviridae. We found that frequent horizontal gene transfer, gene duplication, and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae. Recombination frequently occurred among distinct Closteroviridae members, thereby facilitating the evolution of Closteroviridae. Given the potential emergence of similar wild-citrus-originated novel viruses as pathogens, the need for surveillance of their pathogenic and epidemiological characteristics is of utmost priority for global citrus production. Author summary: Closterovirids are principal plant pathogens for citrus trees and other plants, as they sometimes cause new or re-emerging diseases. However, the closterovirid diversity in natural plant hosts, especially citrus plants, is unclear. Here, we describe three novel species and Citrus tristeza virus within the family Closteroviridae that were sampled from wild citrus trees growing in their natural habitat in southwestern China. The presence of three different taxon classes of the family Closteroviridae indicates the geographical uniqueness of the sampling region for citrus closterovirid evolution. Our analysis shows that frequent horizontal gene transfer, gene duplication, alteration of expression strategy, and recombination have been important evolutionary processes in the diversification of the family Closteroviridae. Our study also shows the significance of natural reserves as potential sources of disease agents endangering cultivated crop plants. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management. Author summary: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), bacteria that are able to persist inside the patient for many months or years, thus requiring long antibiotic treatments. Here we focused on TB patients with delayed response to treatment and we performed genetic characterization of Mtb isolates to search for sub-populations that may tolerate anti-TB drugs. We found that Mtb cultured from 9/15 patients contained different sub-populations, and in vitro drug exposure revealed Mtb sub-populations in 6/15 isolates, none related to known drug-resistance mechanisms. By contrast, drug exposure revealed Mtb sup-populations in 2/20 isolates in the control cohort of patients with fast culture conversion. Furthermore, we characterized a Mtb variant isolated from a sub-population growing in the presence of rifampicin (RIF), a major anti-TB drug. We found that this variant featured a modified lipidic envelope, and that it was able to develop in the presence of RIF and inside human macrophage cells. We performed pharmacological modelling and found that this kind of variant may be related to a poor response to treatment. In conclusion, searching for particular Mtb sub-populations may help to detect patients at risk of treatment failure and provide additional guidance for TB management. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection. Author summary: Understanding how human pathogens, such as Pseudomonas aeruginosa (PA), adapt to the host environment during prolonged infections can provide important information for the treatment of disease. In this study, we compared the genome sequences of PA isolates from cystic fibrosis (CF) and non-CF patients and identified genetic variation that was significantly associated with CF disease. CF-associated genetic variations included: (i) the deletion of three amino acids in a transcriptional regulator (PA5438), which leads to decreased antibiotic susceptibility, enhanced virulence expression and sustained biofilm production in the presence of transferrin, and (ii) the insertion of three amino acids in a cystine transporter (PA0313), which results in decreased pyochelin production. Furthermore, single nucleotide replacement in a promoter was identified and shown to upregulate the cellular process for iron acquisition. Our bioinformatics analyses, linked with experimental validation via genetic manipulation and phenotypic investigation of mutants, provides novel insights into the genomic plasticity of PA during chronic CF infection. [ABSTRACT FROM AUTHOR]

I albicans i cells to mainline echinocandin (ECN) drugs to aneuploidies of chromosomes 5 (Ch5) and Ch2. As an example, Ch5 contains two key genes of interest, I TAC1 i , a positive regulator of I CDR1 i and I CDR2 i genes encoding fluconazole efflux pumps on Ch3, and I ERG11 i , a target gene for fluconazole (Fig 2A) [[27]]. I albicans i genome contains two genes, I FKS2 i and I FKS3 i , which have considerable sequence identity with the key ECN resistance gene I FKS1 i but act as negative regulators of I FKS1 i [[39]]. Note that genes in red encode negative regulators of ECN susceptibility [[17]], while genes in blue encode positive regulators of fluconazole susceptibility [[33]]. [Extracted from the article]

Meningococcal disease is a life-threatening illness caused by the human-restricted bacterium Neisseria meningitidis. Outbreaks in the USA involve at least two cases in an organization or community caused by the same serogroup within three months. Genome comparisons, including phylogenetic analysis and quantification of genome distances can provide confirmatory evidence of pathogen transmission during an outbreak. Interpreting genome distances depends on understanding their distribution both among isolates from outbreaks and among those not from outbreaks. Here, we identify outbreak strains based on phylogenetic relationships among 141 N. meningitidis isolates collected from 28 outbreaks in the USA during 2010–2017 and 1516 non-outbreak isolates collected through contemporaneous meningococcal surveillance. We show that genome distance thresholds based on the maximum SNPs and allele distances among isolates in the phylogenetically defined outbreak strains are sufficient to separate most pairs of non-outbreak isolates into separate strains. Non-outbreak isolate pairs that could not be distinguished from each other based on genetic distances were concentrated in the clonal complexes CC11, CC103, and CC32. Within each of these clonal complexes, phylodynamic analysis identified a group of isolates with extremely low diversity, collected over several years and multiple states. Clusters of isolates with low genetic diversity could indicate increased pathogen transmission, potentially resulting in local outbreaks or nationwide clonal expansions. Author summary: Meningococcal disease is a life-threatening illness caused by the bacterium Neisseria meningitidis. Meningococcal disease outbreaks occur when the same serogroup of N. meningitidis causes multiple cases of disease over a short time period in a population such as a community, college, or prison. As with many other pathogens, genome sequencing can reveal genetic relationships among N. meningitidis based on genomic changes that accumulated as the bacteria were transmitted from person to person. Here, we review 28 outbreaks that occurred over eight years in the United States and identify outbreak strains based on how the N. meningitidis isolated from these outbreaks relate to each other and to N. meningitidis isolated from hundreds of other cases from across the country. We show that pairs of isolates from the same outbreak strain have much higher genome similarity than is typical for pairs of isolates that are not from outbreaks; therefore, genome similarity can help delimit outbreak strains during future outbreak investigations. We also identify groups of N. meningitidis that had similar genomes despite being collected over several years and in multiple states; illustrating how changes in meningococcal disease epidemiology could be affected by the spread of these bacteria. [ABSTRACT FROM AUTHOR]

Despite recent advances in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains poorly understood. Aiming to understand if S. Typhi becomes genetically adapted for long-term colonisation in the gallbladder, we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneously sampled sequences from organisms isolated from the blood of acute patients within the same population. We found that S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes, but was largely reflective of S. Typhi circulating in the general population. However, gallbladder isolates showed a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p = 2.8x10-9), respectively. Within gallbladder isolates of the predominant H58 genotype, variation was associated with a higher prevalence of nonsense mutations. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Specifically, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to the 134Kb deletion of SPI-7. S. Typhi is under strong selective pressure in the human gallbladder, which may be reflected phylogenetically by long terminal branches that may distinguish organisms from chronic and acute infections. Our work shows that selective pressures asserted by the hostile environment of the human gallbladder generate new antigenic variants and raises questions regarding the role of carriage in the epidemiology of typhoid fever. Author summary: Salmonella Typhi is the bacterium that causes typhoid. Salmonella Typhi is infamous for being able to be carried in the gallbladder, with Typhoid Mary being the best-known example of a typhoid carrier. Despite having new tools for typhoid control, we have made little progress in understanding this disease process. Aiming to understand if Salmonella Typhi is adapted for long-term survival in the gallbladder, we sequenced the genomes of 24 Salmonella Typhi isolated from the gallbladders of typhoid carriers. We compared these genomes to Salmonella Typhi from acute typhoid patients within the same population. The carriage of Salmonella Typhi was not restricted to any specific genotype or resistance to antibiotics, but reflective of the organisms causing acute disease. However, gallbladder isolates had higher genetic variability than acute isolates, with a higher frequency of mutations changing the amino acid sequences of hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. We identified several gallbladder-specific mutations involved in polysaccharide synthesis on the bacterial surface. Our work shows that selective pressures asserted by the hostile environment of the human gallbladder generates genetic variation, which is not observed in acute isolates, raising questions regarding the role of carriage in the epidemiology of typhoid. [ABSTRACT FROM AUTHOR]