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The testis is a reservoir for viruses that can cause persistent infection and adversely affect male reproductive health, an observation commonly attributed to deficiencies in inducible antiviral defence mechanisms. In this study, we demonstrate that interferon-epsilon (IFNε), a type I interferon initially discovered in female reproductive epithelia, is constitutively expressed by meiotic and post-meiotic spermatogenic cells, Leydig cells and macrophages in mouse testes. A similar distribution pattern was observed in human testes. Mice lacking IFNɛ were more susceptible to Zika virus-induced inflammation and damage of the testis and epididymis compared to wild-type mice. Exogenous IFNε treatment reduced the viral infection burden in cultured human testicular cells by inducing interferon-stimulated gene expression, and reducing inflammatory gene expression and cell damage. Treatment was more effective when administered prior to infection. These data indicate a critical role for constitutively-expressed IFNɛ in limiting viral infection and inflammatory damage in the male reproductive tract. Author summary: Many clinically significant viruses can infect the testis, affecting male fertility, and the health of the partners and children of infected men. Crucially, many viruses are transmitted via semen, because infections can elude the immune system by hiding in the testis, presumably because of its unique "immunologically privileged" environment. However, our understanding of anti-viral defences in the testis is lacking. We found that under normal physiological conditions, Interferon epsilon (IFNε), a novel antiviral molecule initially identified in the female reproductive tract, is expressed in testicular immune cells, but more significantly in cells that develop into sperm in both mice and humans. In this paper, we show that IFNε plays a crucial role in limiting Zika virus induced damage to the male reproductive system. [ABSTRACT FROM AUTHOR]

Drug-resistant tuberculosis (DR-TB) threatens progress in the control of TB. Mathematical models are increasingly being used to guide public health decisions on managing both antimicrobial resistance (AMR) and TB. It is important to consider bacterial heterogeneity in models as it can have consequences for predictions of resistance prevalence, which may affect decision-making. We conducted a systematic review of published mathematical models to determine the modelling landscape and to explore methods for including bacterial heterogeneity. Our first objective was to identify and analyse the general characteristics of mathematical models of DR-mycobacteria, including M. tuberculosis. The second objective was to analyse methods of including bacterial heterogeneity in these models. We had different definitions of heterogeneity depending on the model level. For between-host models of mycobacterium, heterogeneity was defined as any model where bacteria of the same resistance level were further differentiated. For bacterial population models, heterogeneity was defined as having multiple distinct resistant populations. The search was conducted following PRISMA guidelines in five databases, with studies included if they were mechanistic or simulation models of DR-mycobacteria. We identified 195 studies modelling DR-mycobacteria, with most being dynamic transmission models of non-treatment intervention impact in M. tuberculosis (n = 58). Studies were set in a limited number of specific countries, and 44% of models (n = 85) included only a single level of "multidrug-resistance (MDR)". Only 23 models (8 between-host) included any bacterial heterogeneity. Most of these also captured multiple antibiotic-resistant classes (n = 17), but six models included heterogeneity in bacterial populations resistant to a single antibiotic. Heterogeneity was usually represented by different fitness values for bacteria resistant to the same antibiotic (61%, n = 14). A large and growing body of mathematical models of DR-mycobacterium is being used to explore intervention impact to support policy as well as theoretical explorations of resistance dynamics. However, the majority lack bacterial heterogeneity, suggesting that important evolutionary effects may be missed. Author summary: The emergence of drug-resistant tuberculosis (DR-TB), where the causative bacterium Mycobacterium tuberculosis is resistant to key antibiotics such as rifampicin and isoniazid, poses a significant threat to TB control efforts. To gain a broader understanding of the challenges surrounding DR-TB, mathematical models are increasingly being employed to estimate the impact of interventions, effectiveness of treatment, and to predict the evolution of drug-resistance. However, pragmaticism surrounding model construction often means that important aspects, such as bacterial heterogeneity, are overlooked. We undertook a systematic review of the existing DR-mycobacterium modelling literature, with the specific aim of capturing methods for including bacterial heterogeneity. Our analysis revealed that most models of drug-resistance in mycobacteria primarily focus on intervention strategies and cost-effectiveness analyses, with minimal attention to bacterial heterogeneity. Where heterogeneity is included it mostly consisted of different fitness costs for resistance. [ABSTRACT FROM AUTHOR]

The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world's population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EVJC+). The restriction was not absolute and small amounts of virus or EVJC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EVJC+ penetration leading us to hypothesize that virus and EVJC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment. Author summary: In patients with compromised immune systems due to underlying immunosuppressive diseases or due to treatment with immunomodulatory drugs, JC virus (JCPyV) invades the brain and causes a rapidly progressing and often fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The brain is protected from invading pathogens by two major barriers, the blood-brain barrier (BBB), and the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is composed of choroid plexus epithelial cells that are directly exposed to the blood. The epithelial cells in the choroid plexus are held together by tight junctions that limit and regulate the movement of molecules and cells from the blood to the cerebral spinal fluid. In this paper we used an established adult choroid plexus epithelial cell line to model the blood-cerebrospinal fluid barrier and found that it was highly restrictive of virus penetration. The restriction was not absolute and small amounts of virus penetrated and established infection in underlying human glial cells. We also show a second mechanism of virus invasion involving direct infection of choroid plexus epithelial cells by JCPyV and directional release of infectious virions across the barrier. Both mechanisms likely contribute to the neuroinvasiveness of JCPyV. [ABSTRACT FROM AUTHOR]

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis. Author summary: Blood-stage trypanosome parasites have a specialised invagination on the cell surface named the flagellar pocket (FP), where invariant essential nutrient receptors are located. The pocket houses diverse proteins, including a transferrin receptor (TfR), which facilitates uptake of host transferrin-bound iron for survival. Several FP proteins, including TfR, are linked to complex sugar molecules (carbohydrates), the functions of which are not well understood. Complex carbohydrates are made by enzymes called glycosyltransferases (GTs) and previously we partially inhibited complex carbohydrate synthesis by deletion of either TbGT8 or TbGT10. However, mutant parasites lacking either one of these enzymes survived, suggesting functional redundancy. Here, we created a parasite mutant that lacks both TbGT8 and TbG10 to understand the combined effect of losing both enzymes. The mutant parasites showed a decreased ability to uptake tomato lectin, a protein that specifically binds to these sugar conjugates in the FP, indicating a reduction in carbohydrate complexity. Despite reduced complexity in the sugar structures attached to TfR, its critical function in transferrin/iron uptake remained effective. Furthermore, the mutants remained viable in culture and in animal models, challenging previous assumptions about the necessity and function of these carbohydrate conjugates. Our findings imply a greater flexibility and redundancy in the carbohydrate complex roles than previously appreciated. [ABSTRACT FROM AUTHOR]

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process. Author summary: Hepatitis C virus (HCV) is a hepatotropic RNA virus. An estimated 58 million people are chronically infected with HCV, while HCV-associated hepatocellular carcinoma and cirrhosis accounted for 290, 000 deaths in 2019. HCV entry into hepatocytes is highly complex with distinct stages and multiple host cofactors. Perturbing the interaction of HCV with entry host factors is potential strategy for therapeutic and vaccine-based anti-HCV approaches. However, the precise functions of most of the host factors in HCV entry are not fully appreciated. Moreover, studies of entry using polarized cell culture that physiologically resembles hepatocytes in vivo are limited. In this paper, we evaluate the roles of three entry factors in polarized hepatoma spheroids: epidermal growth factor receptor (EGFR) and tight junction proteins claudin-1 (CLDN1) and occludin (OCLN). We observe that EGFR performs multiple functions to regulate HCV internalization and trafficking to endosomes. The process involves EGFR phosphorylation-dependent and -independent roles and downstream RAF-MEK-ERK signaling pathway. We also show that CLDN1 and OCLN, despite both localizing at the tight junction, function at two distinct stages of HCV entry. CLDN1 is required for the accumulation of HCV virions at the tight junction from the basolateral membrane, while OCLN regulates HCV internalization. These findings provide insights into the mechanism of HCV entry and highlight potential interventional strategies. [ABSTRACT FROM AUTHOR]

Innate immune priming increases an organism's survival of a second infection after an initial, non-lethal infection. We used Drosophila melanogaster and an insect-derived strain of Enterococcus faecalis to study transcriptional control of priming. In contrast to other pathogens, the enhanced survival in primed animals does not correlate with decreased E. faecalis load. Further analysis shows that primed organisms tolerate, rather than resist infection. Using RNA-seq of immune tissues, we found many genes were upregulated in only primed flies, suggesting a distinct transcriptional program in response to initial and secondary infections. In contrast, few genes continuously express throughout the experiment or more efficiently re-activate upon reinfection. Priming experiments in immune deficient mutants revealed Imd is largely dispensable for responding to a single infection but needed to fully prime. Together, this indicates the fly's innate immune response is plastic—differing in immune strategy, transcriptional program, and pathway use depending on infection history. Author summary: Most animals, like the fruit fly Drosophila melanogaster, lack an adaptive immune system and react to infection using only an innate immune response. In this paper, we study how previous infection with the bacterium Enterococcus faecalis changes the immune response to a second infection with the same bacterium, through a phenomenon called immune priming. We find that primed flies tend to survive more, tolerate a higher bacterial load, and undergo priming-specific gene expression reprogramming compared to non-primed flies. We also find that eliminating a key component of the Imd pathway, which is not canonically related to response to E. faecalis lowered priming ability in flies. These experiments highlight the true complexity of fly immune response and provide a basis for further exploring the interrelatedness of multiple known innate immune pathways in regulating a complex phenomenon like immune priming. [ABSTRACT FROM AUTHOR]

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical YATPmax of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle. Author summary: Cells use ATP as the main energy currency for the synthesis, organization and maintenance of their macromolecules and cellular structures, in order to stay alive and proliferate. For this purpose, ATP is produced from external nutrients, and is spent by cells in the many processes that are necessary for maintenance and building up new cells. Despite its relevance and the impressive quantity of biological data available, very little is known about how much ATP is required for maintaining and duplicating a cell. In this paper, we present a calculation on how much of the ATP produced by catabolism of the nutrient glucose is used to energize the different processes known to occur during the cell cycle of the infective form of the trypanosomatid parasite that causes human sleeping sickness, the bloodstream form of Trypanosoma brucei. [ABSTRACT FROM AUTHOR]

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and 'locked' C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing. Author summary: In this paper, we focus on how complement proteins, an essential part of the immune system, kill Gram-negative bacteria via so-called membrane attack complex (MAC) pores. The MAC is a large pore that consists of five different proteins. The final component, C9, assembles a ring of up to 18 C9 molecules that damages the bacterial cell envelope. Here, we aimed to better understand if this polymeric-C9 ring is necessary to kill bacteria and if bacteria can interfere in its assembly. We uncover that polymerization of C9 increased the damage to the entire bacterial cell envelope, which resulted in more rapid killing of several Gram-negative species. We also show that some clinical Escherichia coli strains can block polymerization of C9 and survive MAC-dependent killing by modifying sugars in the bacterial cell envelope, namely the O-antigen of lipopolysaccharide. These insights help us to better understand how the immune system kills bacteria and how pathogenic bacteria can survive killing. [ABSTRACT FROM AUTHOR]

Candia auris is an emerging human pathogenic yeast; yet, despite phenotypic attributes and genomic evidence suggesting that it probably emerged from a natural reservoir, we know nothing about the environmental phase of its life cycle and the transmission pathways associated with it. The thermotolerant characteristics of C. auris have been hypothesised to be an environmental adaptation to increasing temperatures due to global warming (which may have facilitated its ability to tolerate the mammalian thermal barrier that is considered a protective strategy for humans against colonisation by environmental fungi with pathogenic potential). Thus, C. auris may be the first human pathogenic fungus to have emerged as a result of climate change. In addition, the release of antifungal chemicals, such as azoles, into the environment (from both pharmaceutical and agricultural sources) is likely to be responsible for the environmental enrichment of resistant strains of C. auris; however, the survival and dissemination of C. auris in the natural environment is poorly understood. In this paper, we critically review the possible pathways through which C. auris can be introduced into the environment and evaluate the environmental characteristics that can influence its persistence and transmission in natural environments. Identifying potential environmental niches and reservoirs of C. auris and understanding its emergence against a backdrop of climate change and environmental pollution will be crucial for the development of effective epidemiological and environmental management responses. Author summary: The natural environment can play a significant role in the epidemiology of infectious diseases as pathogens persist and evolve in environmental niches from where they can be transferred to new hosts. Therefore, understanding the interaction of pathogens with potential environmental reservoirs is crucial to the control of pathogenic diseases. Pathogenic Candida species have been widely isolated from nonclinical environmental sources such as soil, freshwater, seawater, and animals such as birds. The persistence and dissemination of C. auris, a recently emerged human pathogenic yeast, in the environment is still poorly understood. Here, we identify the potential pathways through which C. auris can be introduced into the natural environment and the characteristics that can influence its survival in various environmental matrices. In conclusion, we review the current knowledge gaps and identify the future research needed to understand the epidemiological implications of C. auris in the natural environment. [ABSTRACT FROM AUTHOR]

Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11's interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs. Author summary: (+)RNA viruses have to co-opt numerous host proteins to support their replication in infected cells. These viruses induce the biogenesis of viral replication organelles (VROs), the sites of replication, in cells. However, what the mechanism of subversion for most of the cytosolic host proteins is not yet dissected. In this paper, the authors used a plant (+)RNA virus, tomato bushy stunt virus (TBSV), to study the role of a cellular proteasomal protein, called Rpn11 (POH1), as a protein interaction hub. They show that knockdown of Rpn11 or retargeting Rpn11 into the nucleus and destruction of actin filaments diminishes TBSV replication in yeast and plant cells. This effect is due to diminished recruitment of pro-viral metabolic enzymes into VROs. Overall, data presented support a novel viral recruitment strategy for cytosolic host factors. Via the small viral replication protein, TBSV targets the cytosolic proteasomal Rpn11 protein interaction hub protein and the co-opted and stabilized actin filaments. The combined and coordinated subversion of Rpn11 and the actin network allows tombusviruses to gain access to abundant cytosolic proteins, such as the glycolytic and fermentation enzymes, which are then efficiently delivered to perform pro-viral functions within the VROs. [ABSTRACT FROM AUTHOR]

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen micro evolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes (Lm) is a food-borne pathogen that causes severe bacterial gastroenteritis, with high rates of hospitalization and mortality. Lm is ubiquitous in soil, water and livestock, and can survive and proliferate at low temperatures. Following oral ingestion of contaminated food, Lm crosses the epithelium through intestinal goblet cells in a mechanism mediated by Lm InlA binding host E-cadherin. Importantly, human infections typically occur with Lm growing at or below room temperature, which is flagellated and motile. Even though many important human bacterial pathogens are flagellated, little is known regarding the effect of Lm motility on invasion and immune evasion. Here, we used complementary imaging and computer modeling approaches to test the hypothesis that bacterial motility helps Lm locate and engage target cells permissive for invasion. Imaging explanted mouse and human intestine, we showed that Lm grown at room temperature uses motility to scan the epithelial surface and preferentially attach to target cells. Furthermore, we integrated quantitative parameters from our imaging experiments to construct a versatile "layered" cellular Potts model (L-CPM) that simulates host-pathogen dynamics. Simulated data are consistent with the hypothesis that bacterial motility enhances invasion by allowing bacteria to search the epithelial surface for their preferred invasion targets. Indeed, our model consistently predicts that motile bacteria invade twice as efficiently over the first hour of infection. We also examined how bacterial motility affected interactions with host cellular immunity. In a mouse model of persistent infection, we found that neutrophils migrated to the apical surface of the epithelium 5 hours post infection and interacted with Lm. Yet in contrast to the view that neutrophils "hunt" for bacteria, we found that these interactions were driven by motility of Lm—which moved at least ~50x faster than neutrophils. Furthermore, our L-CPM predicts that motile bacteria maintain their invasion advantage even in the presence of host phagocytes, with the balance between invasion and phagocytosis governed almost entirely by bacterial motility. In conclusion, our simulations provide insight into host pathogen interaction dynamics at the intestinal epithelial barrier early during infection. Author summary: Many important human bacterial pathogens are motile, yet for many it remains unclear how this motility affects their ability to cause disease. Here, we sought to answer this question for Listeria monocytogenes (Lm), a food-borne bacterium that can cause severe gut infections leading to hospitalization or even death. After being ingested, Lm must engage specific "target cells" in the gut before it can cross the gut lining and cause infection. By imaging Lm interacting with mouse and human intestines, we found that Lm motility facilitates this process: motile Lm could more easily reach and move along the gut lining, allowing them to locate target cells faster than non-motile Lm. To further understand these dynamics, we built a computer model of Lm gut infection and explored how phagocytes, specifically neutrophils, might interfere with this process. Again, motile bacteria more efficiently located and invaded target cells narrowing the window for neutrophils to capture them. But our simulations also challenge the commonly held view that phagocytes "hunt" bacteria, which move orders of magnitude faster. Instead, phagocytes in our simulations act like "fly paper" to capture bacteria. These findings provide new insights into the early dynamics of bacterial gut infections. [ABSTRACT FROM AUTHOR]

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts. Author summary: Defining the host factors responsible for controlling viral population dynamics during infection is critical for establishing a thorough understanding of viral transmission, dissemination, pathogenesis, and immune evasion. Here, we employed a barcoded virus strategy to interrogate how host factors modulate viral diversity of CR6, a persistent strain of murine norovirus. By evaluating barcode levels in tissues and stool of wild-type mice, mice lacking critical innate immune response genes, and mice treated with cytokine to enhance susceptible tuft cell levels, we found that both the availability of tuft cells and viral replication limitations imposed by interferon signaling serve as critical bottlenecks for CR6 diversity. Our studies also indicated that stool virus is likely predominantly derived from the colon, and that extraintestinal dissemination of CR6 in immunodeficient mouse strains likely occurs independently of intestinal infection. Our study thus revealed key constraints regulating norovirus population dynamics and provided additional insights into the mechanisms of viral shedding and dissemination. [ABSTRACT FROM AUTHOR]

The severity of Entamoeba histolytica infection is determined by host immunology, pathogen virulence, and the intestinal environment. Conventional research for assessing pathogen virulence has been mainly performed using laboratory strains, such as a virulent HM-1: IMSS (HM-1) and an avirulent Rahman, under various artificial environmental conditions because of the difficulties of axenic isolation of the clinical strains. However, it is still unclear whether scientific knowledge based on laboratory strains are universally applicable to the true pathogenesis. Hereby, we performed transcriptomic analysis of clinical strains from patients with different degrees of disease severity, as well as HM-1 under different conditions. Even after several months of axenization, Clinical strains show the distinct profile in gene expression during in vitro passage, moreover, difference between any 2 of these strains was much greater than the changes on the liver challenge. Interestingly, 26 DEGs, which were closely related to the biological functions, were oppositely up- or down regulated between virulent Ax 19 (liver abscess) and avirulent Ax 11 (asymptomatic carrier). Additionally, RNAseq using laboratory strain (HM1) showed more than half of genes were differently expressed between continuously in vitro passaged HM1 (in vitro HM1) and periodically liver passaged HM1 (virulent HM1), which was much greater than the changes on the liver passage of virulent HM1. Also, transcriptomic analysis of a laboratory strain revealed that continuous environmental stress enhances its virulence via a shift in its gene expression profile. Changes in gene expression patterns on liver abscess formation were not consistent between clinical and laboratory strains. Author summary: Various genotypes of Entamoeba histolytica are prevalent in the field. Some papers suggest the association between genotypes and disease severity. However, most studies for assessing pathogen virulence were performed using laboratory strains, such as virulent HM1: IMSS (HM1) and avirulent Rahman, because axenic isolation from clinical specimen is technically complex and time consuming. This transcriptomic analysis using clinical strains from the patients with different clinical severity, as well as the laboratory strain HM1 under different conditions showed unique gene expression patterns. Following things were confirmed; 1. Virulent clinical strain maintains its virulence with unique gene expression pattern after axenic isolation, 2. Continuous environmental stress enhances its virulence via the accumulation of altered gene expressions, and 3. Changes in gene expression on the liver abscess formation are not always the same amongst strains. For an accurate understanding the pathogenesis, comprehensive analyses of various clinical strains under different environmental conditions should be promoted. [ABSTRACT FROM AUTHOR]

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites. Author summary: Cryptosporidium parvum and C. hominis are waterborne parasites that are the second to third leading cause of diarrheal disease, and a major contributor to childhood deaths worldwide. Traditionally these intestinal parasites have proven difficult to culture for more than 2–3 days, which hampers long term in vitro studies. We reasoned that cultures of intestinal epithelial cells as monolayers in static plates results in the production of unpolarized epithelial cells. Utilizing hollow fiber technology, we have developed a method for producing intestinal epithelial cell growth that simulates the body resulting in polarized intestinal epithelial cells that have basal and apical surfaces, tight junctions, and develop functional villi. Using this system, we have maintained in vitro cultures of C. parvum that produce all life cycle stages for 20 months. Long-term in vitro culture of parasites often results in the development of a phenotype that is no longer pathogenic to the host; In this publication we show that using a calf model C. parvum BGF-T20HF after 20 months of in vitro culture was unchanged with respect to diarrhea output, parasite load, and clinical scores from the isolate used to initiate the culture (BGF-T0). In addition, we can show that the genome of the cultured parasites (BGF-T20HF) has undergone a similar genomic drift as the parent isolate (BGF-T0) used to start the inoculum that had been maintained by passage through fetal calves. Collectively the data supports the use of the in vitro cultured isolate, BGF-T20HF, for human trials, and provides a long-term model for the development of novel chemotherapeutic drugs to treat this disease. [ABSTRACT FROM AUTHOR]

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH. Author summary: People living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes. [ABSTRACT FROM AUTHOR]

In this essay, we show that 3 distinct approaches to immunological exhaustion coexist and that they only partially overlap, generating potential misunderstandings. Exploring cases ranging from viral infections to cancer, we propose that it is crucial, for experimental and therapeutic purposes, to clarify these approaches and their interconnections so as to make the concept of exhaustion genuinely operational. Author summary: In this essay, we have written a critical review on immunological exhaustion. We believe that this widely used concept often remains in fact imprecise because there exist 3 different approaches to exhaustion, namely, in terms of dysfunction, cause, and marker, and those are not sufficiently well distinguished and articulated in most scientific papers. We also propose to talk about "exhaustion" for and only for the phenomena in which all 3 approaches are aligned. This can be called the "convergence strategy": T cells would be described as "exhausted" if and only if they are simultaneously dysfunctional, express given markers such as programmed cell death protein 1 (PD-1), and induced by a specific cause such as chronicity. This strategy could be perfectly reasonable, and we see much value in adopting it, because it would force everyone to be more specific and rigorous when talking about exhaustion. Clarifying the characteristics of cells defined as "exhausted," and using a convergent strategy to define T-cell exhaustion could have major experimental and clinical consequences, including for viral infections such as the Coronavirus Disease 2019 (COVID-19). [ABSTRACT FROM AUTHOR]

SARS-CoV-2 spike protein (SARS-2-S) induced cell–cell fusion in uninfected cells may occur in long COVID-19 syndrome, as circulating SARS-2-S or extracellular vesicles containing SARS-2-S (S-EVs) were found to be prevalent in post-acute sequelae of COVID-19 (PASC) for up to 12 months after diagnosis. Although isolated recombinant SARS-2-S protein has been shown to increase the SASP in senescent ACE2-expressing cells, the direct linkage of SARS-2-S syncytia with senescence in the absence of virus infection and the degree to which SARS-2-S syncytia affect pathology in the setting of cardiac dysfunction are unknown. Here, we found that the senescent outcome of SARS-2-S induced syncytia exacerbated heart failure progression. We first demonstrated that syncytium formation in cells expressing SARS-2-S delivered by DNA plasmid or LNP-mRNA exhibits a senescence-like phenotype. Extracellular vesicles containing SARS-2-S (S-EVs) also confer a potent ability to form senescent syncytia without de novo synthesis of SARS-2-S. However, it is important to note that currently approved COVID-19 mRNA vaccines do not induce syncytium formation or cellular senescence. Mechanistically, SARS-2-S syncytia provoke the formation of functional MAVS aggregates, which regulate the senescence fate of SARS-2-S syncytia by TNFα. We further demonstrate that senescent SARS-2-S syncytia exhibit shrinked morphology, leading to the activation of WNK1 and impaired cardiac metabolism. In pre-existing heart failure mice, the WNK1 inhibitor WNK463, anti-syncytial drug niclosamide, and senolytic dasatinib protect the heart from exacerbated heart failure triggered by SARS-2-S. Our findings thus suggest a potential mechanism for COVID-19-mediated cardiac pathology and recommend the application of WNK1 inhibitor for therapy especially in individuals with post-acute sequelae of COVID-19. Author summary: In this paper, we directly linked SARS-2-S-triggered syncytium formation in the absence of infection with the ensuing induction of cellular senescence and its pathophysiological contribution to heart failure. We propose that both SARS-2-S expression and SARS-2-S protein internalization were sufficient to induce senescence in nonsenescent ACE2-expressing cells. This is important because of the persistent existence of SARS-2-S or extracellular vesicles containing SARS-2-S during the post-acute stages of SARS-CoV-2 infection in human subjects. In searching for the underlying molecular mechanisms determining syncytial fate, the formation of functional MAVS aggregates dependent on RIG-I was observed at an early stage during fusion and regulated the anti-death to senescence fate of SARS-2-S syncytia through the TNFα-TNFR2 axis. We also found impaired cardiac metabolism in SARS-2-S syncytia induced by condensed WNK1. However, syncytium formation or cellular senescence observed with the wild-type fusogenic S protein does not occur with the spike proteins produced by currently approved COVID-19 mRNA vaccines. Importantly, SARS-2-S-exacerbated heart failure could be largely rescued by WNK1 inhibitor, anti-syncytial drug or senolytic agent. Together, we suggest that rescuing metabolism dysfunction in senescent SARS-2-S syncytia should be taken into consideration in individuals with post-acute sequelae of COVID-19 (PASC). [ABSTRACT FROM AUTHOR]

Avian influenza A viruses (IAVs) pose risks to public, agricultural, and wildlife health. Bridge hosts are spillover hosts that share habitat with both maintenance hosts (e.g., mallards) and target hosts (e.g., poultry). We conducted a comprehensive assessment of European starlings (Sturnus vulgaris), a common visitor to both urban and agricultural environments, to assess whether this species might act as a potential maintenance or bridge host for IAVs. First, we experimentally inoculated starlings with a wild bird IAV to investigate susceptibility and replication kinetics. Next, we evaluated whether IAV might spill over to starlings from sharing resources with a widespread IAV reservoir host. We accomplished this using a specially designed transmission cage to simulate natural environmental transmission by exposing starlings to water shared with IAV-infected mallards (Anas platyrhynchos). We then conducted a contact study to assess intraspecies transmission between starlings. In the initial experimental infection study, all inoculated starlings shed viral RNA and seroconverted. All starlings in the transmission study became infected and shed RNA at similar levels. All but one of these birds seroconverted, but detectable antibodies were relatively transient, falling to negative levels in a majority of birds by 59 days post contact. None of the contact starlings in the intraspecies transmission experiment became infected. In summary, we demonstrated that starlings may have the potential to act as IAV bridge hosts if they share water with IAV-infected waterfowl. However, starlings are unlikely to act as maintenance hosts due to limited, if any, intraspecies transmission. In addition, starlings have a relatively brief antibody response which should be considered when interpreting serology from field samples. Further study is needed to evaluate the potential for transmission from starlings to poultry, a possibility enhanced by starling's behavioral trait of forming very large flocks which can descend on poultry facilities when natural resources are scarce. Author summary: Besides causing seasonal influenza, influenza A viruses (IAVs) are important because they can become pathogenic and threaten human, livestock, or wildlife health. Wild birds are the primary reservoir of IAVs which are generally low pathogenic, but when wild bird viruses spill over into poultry, they can evolve to be highly pathogenic to poultry and sometimes to wild birds or humans. Thus, understanding how viruses move from wild birds into poultry is important. Aquatic birds such as ducks and geese are commonly infected with IAVs, but in many regions, these birds are uncommon on farms. Therefore, species that use both aquatic and agricultural areas may pose a risk by moving IAVs from aquatic birds to poultry. In this paper we evaluated whether European starlings, a species commonly found in both aquatic and agricultural habitats, can be infected by sharing water with IAV-infected ducks. We found that starlings can become infected when exposed to contaminated water, but IAV does not readily transmit between starlings. Consequently, starlings may pose a risk for spillover of IAVs to farms but are unlikely to maintain infections without exposure to other species. [ABSTRACT FROM AUTHOR]

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics. Author summary: For a SARS-CoV-2 virion to enter a cell, the spike protein displayed at its surface must be recognized by the host ACE2 receptor. Serum neutralizing antibodies, shown to develop upon natural SARS-CoV-2 infection or vaccination, bind spike protein preventing the recognition by ACE2 and, consequently, infection. However, SARS-CoV-2 virus is constantly evolving, and can acquire mutations in spike that render this protein resistant to neutralizing antibodies and make vaccines ineffective. In this paper, we tested how single and a combination of mutations naturally occurring in spike, including in variants of concern, would synergize to affect antibody neutralizing capacity. We then integrated these findings with in silico analyses of amino acids binding to ACE2 and antibodies, and distributed them in a grid as amino acids important for binding to ACE2 or antibodies, or both. We found that changes in amino such as E484 and S494, which frequently interact with antibodies but not with ACE2, promptly evolve immune escape mutants, elicited by infection or vaccination, if the mutation severely alters the binding specificity of the antibody. Our work also suggests that the combination of these mutations with others promoting ACE2 binding, such as N501Y, increases their ability to escape neutralizing-antibody responses. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development. Author summary: SARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research. [ABSTRACT FROM AUTHOR]

The APOBEC3 (A3) genes encode cytidine deaminase proteins with potent antiviral and anti-retroelement activity. This locus is characterized by duplication, recombination, and deletion events that gave rise to the seven A3s found in primates. These include three single deaminase domain A3s (A3A, A3C, and A3H) and four double deaminase domain A3s (A3B, A3D, A3F, and A3G). The most potent of the A3 proteins against HIV-1 is A3G. However, it is not clear if double deaminase domain A3s have a generalized functional advantage to restrict HIV-1. In order to test whether superior restriction factors could be created by genetically linking single A3 domains into synthetic double domains, we linked A3C and A3H single domains in novel combinations. We found that A3C/A3H double domains acquired enhanced antiviral activity that is at least as potent, if not better than, A3G. Although these synthetic double domain A3s package into budding virions more efficiently than their respective single domains, this does not fully explain their gain of antiviral potency. The antiviral activity is conferred both by cytidine-deaminase dependent and independent mechanisms, with the latter correlating to an increase in RNA binding affinity. T cell lines expressing this A3C-A3H super restriction factor are able to control replicating HIV-1ΔVif infection to similar levels as A3G. Together, these data show that novel combinations of A3 domains are capable of gaining potent antiviral activity to levels similar to the most potent genome-encoded A3s, via a primarily non-catalytic mechanism. Author summary: Antiviral genes are encoded by all organisms to help protect them from viral infections, including proteins encoded by primates to protect them from viruses similar to HIV-1. These antiviral proteins are also called "restriction factors". Some restriction factors are broadly acting, while others are very specific. During the course of evolution, some of these genes have expanded into multiple copies and rearranged in different versions to give them new activities. In this paper, we validated the hypothesis that one particular antiviral gene family, called the APOBEC3 family, has the capability of making novel combinations of antiviral human genes with as great, or greater, potency against HIV-1 as the most potent natural member of this family. By combining parts of the APOBEC3 proteins into novel combinations, we created potent antiviral versions that act through a mechanism distinct from existing APOBEC3 proteins. [ABSTRACT FROM AUTHOR]

Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6 to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6 cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins. Author summary: Legionella pneumophila causes a sever pneumonia called Legionnaires' disease and a threat of this disease has increased on a world-wide scale. As a feature of L. pneumophila, it secrets over 300 bacterial effectors to adapt and survive inside the host and many of effectors modify the host proteins in a unique manner. L. pneumophila is known to travel inside the host and final destination of this pathogens is the host ER. In the initial step of this travel, L. pneumophila subverts host early vesicular trafficking to remodel the membrane composition of Legionella-containing vacuole (LCV). Although this remodeling process has been well characterized, the molecular machinery of association of remodeled vacuoles with the ER is still obscure. This paper shows that the host GTPases Rab6 and Rab33B, both of which control Golgi-to-ER traffic, are recruited to the LCV in a cascade manner and are required for the association of LCVs with the ER through the interaction between Rab6 and ER-resident t-SNARE proteins. Of note, we demonstrate that a bacteria-specific Rab33B modification called phosphoribosyl-ubiquitination by Legionella effectors proteins of the SidE family is essential for the recruitment of Rab33B to the LCV. [ABSTRACT FROM AUTHOR]

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system. Author summary: In this paper we identified how different players of the human immune system cooperate to degrade the complex cell envelope of Gram-negative bacteria. The outer membrane of Gram-negative bacteria forms an impermeable barrier for various antimicrobial proteins of the immune system. Here we show that complement-dependent Membrane Attack Complex (MAC) formation permeabilizes this barrier, allowing otherwise impermeable antimicrobial proteins to reach their targets underneath the outer membrane. Specifically, we show that outer membrane damage by the MAC allows lysozyme to degrade the peptidoglycan layer, and secreted phospholipase A2-IIA to hydrolyze the bacterial inner membrane. MAC formation also sensitizes Gram-negative bacteria to more efficient degradation and killing inside human neutrophils. Altogether, this knowledge may guide the development of new antimicrobial strategies to treat infections caused by Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Environments such as light condition influence the spread of infectious diseases by affecting insect vector behavior. However, whether and how light affects the host defense which further affect insect preference and performance, remains unclear, nor has been demonstrated how pathogens co-adapt light condition to facilitate vector transmission. We previously showed that begomoviral βC1 inhibits MYC2-mediated jasmonate signaling to establish plant-dependent mutualism with its insect vector. Here we show red-light as an environmental catalyzer to promote mutualism of whitefly-begomovirus by stabilizing βC1, which interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) transcription factors. PIFs positively control plant defenses against whitefly by directly binding to the promoter of terpene synthase genes and promoting their transcription. Moreover, PIFs interact with MYC2 to integrate light and jasmonate signaling and regulate the transcription of terpene synthase genes. However, begomovirus encoded βC1 inhibits PIFs' and MYC2' transcriptional activity via disturbing their dimerization, thereby impairing plant defenses against whitefly-transmitted begomoviruses. Our results thus describe how a viral pathogen hijacks host external and internal signaling to enhance the mutualistic relationship with its insect vector. Author summary: Climate change is driving disease rapidly spread, esp. for global distribution of insect-borne diseases. This paper reports red-light as an environmental factor to promote insect vector olfactory orientation behavior and increase viral disease transmission. Plant virus adapts the supplemental red lighting practice in modern agricultural greenhouse production under protection, therefore enhancing disease spreading globally. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses. Author summary: Positive-stranded RNA viruses are major pathogens of plants, humans and animals. These viruses hijack and deform intracellular membranes to build viral replication compartments, which support virus replication. In this paper, the authors have identified the critical roles of cellular membrane-shaping proteins and a unique lipid in the formation of the replicase complex for tomato bushy stunt virus (TBSV). TBSV co-opts and retargets the endosomal sorting nexin-BAR proteins into the large TBSV-induced replication compartment. TBSV also promotes the enrichment of PI(3)P phosphoinositide within the subverted membranes. The interplay between the membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable membranous structures around the viral replicase, which provide the essential protection of the viral RNAs against the host antiviral responses. [ABSTRACT FROM AUTHOR]

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses. Author summary: Epstein-Barr virus (EBV), one of the human gamma-herpesviruses, is a well-defined viral agent that strongly associates with malignancies of lymphoid and epithelial origin. EBV-associated gastric carcinoma (EBVaGC) is the most common malignancy caused by EBV infection. In this paper, we identified that beyond tumor suppressor genes, immune related genes (IRGs) are another group of host genes that are extensively DNA hypermethylated and epigenetically silenced in EBVaGC comparing to non-EBV GC. For certain IRGs, such as metallothionein-1 (MT1) and homeobox A (HOXA) genes, the whole gene clusters undergo DNA hypermethylation in EBVaGC, while it also occurs for isolated individual IRGs, such as IRAK2 and MAL. Considering the critical role of innate immunity in antiviral control, we expected that silencing of IRGs would benefit EBV viral replication and propagation. Indeed, our further investigation of several IRGs (MT1G, HOXA10, IRAK2, and MAL) confirmed their antiviral activities against EBV lytic replication. Some of them also suppress lytic replication of another human gamma-herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), indicating their broad antiviral spectrum. Collectively, our results demonstrated that epigenetic silencing of antiviral IRGs is an efficient viral strategy utilized by oncoviruses, such as EBV, to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis. [ABSTRACT FROM AUTHOR]

An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing. Author summary: In this paper we focus on how the complement system, an essential part of the immune system, kills bacteria via so-called membrane attack complex (MAC) pores. The MAC is a large, ring-shaped pore that consists of five different proteins, which is assembled when the complement system is activated on the bacterial surface. Here, we aimed to better understand how MAC pores are assembled on Escherichia coli and how clinical E. coli strains resist killing by MAC pores. We uncover that rapid recruitment of one of the MAC proteins, namely C7, is crucial to efficiently anchor the MAC precursor to the bacterial surface and ensure killing of a variety of E. coli strains via MAC pores. Furthermore, we reveal that some clinical E. coli strains prevent this efficient anchoring of MAC precursors and thereby resist bacterial killing. These insights help us to better understand how the immune system kills bacteria and how pathogenic bacteria evade this. [ABSTRACT FROM AUTHOR]

We previously demonstrated that the flagellin of intracellular Vibrio splendidus AJ01 could be specifically identified by tropomodulin (Tmod) and further mediate p53-dependent coelomocyte apoptosis in the sea cucumber Apostichopus japonicus. In higher animals, Tmod serves as a regulator in stabilizing the actin cytoskeleton. However, the mechanism on how AJ01 breaks the AjTmod-stabilized cytoskeleton for internalization remains unclear. Here, we identified a novel AJ01 Type III secretion system (T3SS) effector of leucine-rich repeat-containing serine/threonine-protein kinase (STPKLRR) with five LRR domains and a serine/threonine kinase (STYKc) domain, which could specifically interact with tropomodulin domain of AjTmod. Furthermore, we found that STPKLRR directly phosphorylated AjTmod at serine 52 (S52) to reduce the binding stability between AjTmod and actin. After AjTmod dissociated from actin, the F-actin/G-actin ratio decreased to induce cytoskeletal rearrangement, which in turn promoted the internalization of AJ01. The STPKLRR knocked out strain could not phosphorylated AjTmod and displayed lower internalization capacity and pathogenic effect compared to AJ01. Overall, we demonstrated for the first time that the T3SS effector STPKLRR with kinase activity was a novel virulence factor in Vibrio and mediated self-internalization by targeting host AjTmod phosphorylation dependent cytoskeleton rearrangement, which provided a candidate target to control AJ01 infection in practice. Author summary: Vibrio splendidus AJ01 is the major pathogen for skin ulcer syndrome (SUS) in Apostichopus japonicus, nevertheless, its pathogenic mechanism remains unknown. Eukaryotic-like factors play a crucial role in bacterial virulence by targeting hosts. Despite their significance, the eukaryotic factors in Vibrio splendidus have not been studied, and the mechanism by which they interact with the host remains unclear. In this study, we found an eukaryotic-like factor STPKLRR of V. splendidus for the first time. Furthermore, STPKLRR, selected by T3SS, can phosphorylate Tmod at S52, dissociate the Tmod/actin complex, cause cytoskeleton rearrangement and promote the internalization of V. splendidus. Our findings provide insight into the mechanisms underlying the internalization of V. splendidus and advance our knowledge of the general biology of pathogen-host interactions. [ABSTRACT FROM AUTHOR]

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling. Author summary: Human Immunodeficiency Virus type 1 (HIV-1) remains a global health threat. Drug therapies can help manage HIV-1 infection and prevent its spread, but without treatment, the immune system fails to control the virus. In part, this is because HIV-1 is thought to avoid detection by immune cells during entry. Once inside a target cell, the virus copies its RNA genome into double-stranded DNA within a viral protein shell called the capsid. In this study, we developed a novel cell-free method to study how the HIV-1 capsid shields viral DNA from detection. We found that the capsid normally functions as a molecular cloak that hides the viral DNA from innate immune sensors. We also identified experimental conditions and drug treatments that can "break" the capsid and allow the viral DNA to be detected, both in vitro and in cells. We anticipate that future work using cell-free systems will reveal how host factors regulate innate detection of HIV-1, possibly guiding new therapeutic strategies that block infection and engage antiviral immune responses simultaneously. [ABSTRACT FROM AUTHOR]

We have demonstrated that the cellular protein M-Sec promotes the transmission of human T-cell leukemia virus type 1 (HTLV-1) in vitro and in vivo. Here, we show how HTLV-1 utilizes M-Sec for its efficient transmission. HTLV-1-infected CD4+ T cells expressed M-Sec at a higher level than uninfected CD4+ T cells. The ex vivo culture of the infected cells upregulated the expression of M-Sec, the level of which was sustained for a long time. The viral structural protein Gag is distributed in a punctate pattern in cells. M-Sec promoted the accumulation of large intracellular Gag puncta. This accumulation was dependent on phosphatidylinositol 4,5-bisphosphate (PIP2), since it was lost upon the removal of PIP2 binding motifs in M-Sec or the depletion of cellular PIP2. The viral envelope protein Env co-localized with the large Gag puncta induced by M-Sec. Furthermore, viral particles produced by M-Sec-expressing cells contained a higher amount of Env. Given that M-Sec alters the cellular distribution of PIP2, these results suggest that M-Sec promotes the formation of infectious viral particles through PIP2. Since the expression of M-Sec is mediated by HTLV-1 Tax protein, M-Sec appears to function in a positive feedback loop that ensures efficient HTLV-1 transmission. Author summary: Human T-cell leukemia virus type 1 (HTLV-1) causes an aggressive blood cancer known as ATL and a neurodegenerative condition known as HAM/TSP. HTLV-1 infects CD4+ T lymphocytes, and induces or upregulates the expression of a wide array of cellular proteins, including M-Sec. Our previous in vitro and in vivo studies have demonstrated that M-Sec mediates an efficient cell-to-cell transmission of HTLV-1. In this study, we discovered that M-Sec can also facilitate virus spread by promoting the formation of infectious viral particles. Mechanistically, our data suggest that through redistribution of phosphatidylinositol 4,5-bisphosphate, M-Sec promotes accumulation of a viral structural protein Gag and an envelope protein Env to large intracellular compartments, and facilitates the incorporation of Env into progeny viral particles. Thus, the current study reveals that M-Sec and its downstream effectors are key host components that govern both the virion infectivity and cell-to-cell transmission and thereby potentially serve as targets of anti-HTLV-1 strategies. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome is a fundamental component of the innate immune system, yet its excessive activation is intricately associated with viral pathogenesis. Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), belonging to the family Arteriviridae, triggers dysregulated cytokine release and interstitial pneumonia, which can quickly escalate to acute respiratory distress and death. However, a mechanistic understanding of PRRSV-2 progression remains unclear. Here, we screen that PRRSV-2 nsp2 activates the NLRP3 inflammasome, thereby instigating a state of hyperinflammation. Mechanistically, PRRSV-2 nsp2 interacts with the nucleotide-binding and oligomerization (NACHT) domain of NLRP3, augmenting IKKβ recruitment to driving NLRP3 translocation to the dispersed trans-Golgi network (dTGN) for oligomerization. This process facilitates ASC polymerization, culminating in the activation of the NLRP3 inflammasome. In addition, the IKKβ-dependent NLRP3 translocation to the dTGN is pivotal for pseudorabies virus (PRV) and encephalomyocarditis virus (EMCV)-induced inflammatory responses. Collectively, these results elucidate a novel mechanism of NLRP3 inflammasome activation during PRRSV-2 infection, providing valuable insights into PRRSV-2 pathogenesis. Author summary: Hyperactivation of the NLRP3 inflammasome triggers excessive proinflammatory cytokine release, skewing the host response toward hyperinflammation and severe tissue damage. PRRSV-2, a positive-stranded RNA virus that causes significant economic losses in the swine industry, triggers severe inflammation and cytopathology during infection. Gaining a deep understanding of its pathogenesis is crucial for developing effective strategies to combat the disease. Here, we show that PRRSV-2 nsp2 plays a crucial role in activating the NLRP3 inflammasome by interacting with NLRP3 and facilitating the recruitment of IKKβ, which drives the translocation of NLRP3 to the dTGN for oligomerization, ultimately leading to the activation of the NLRP3 inflammasome. This process is also crucial for the inflammatory responses in both PRV and EMCV infections. Our findings uncover a novel function of PRRSV-2 nsp2 in the activation of inflammatory responses, advancing our understanding of PRRSV-2 pathogenesis, and highlight the importance of IKKβ-dependent translocation of NLRP3 to the dTGN in virus-induced NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

With the rapid increase in the number of implant operations, the incidence of bone infections has increased. Methicillin-resistant Staphylococcus aureus (S. aureus) and other emerging fully drug-resistant strains make the management of bone infections even more challenging. Bone infections are mainly caused by S. aureus and require extensive surgical intervention and long-term antibiotic therapy. The host autophagy response is critical to the elimination of S. aureus infections. In this study, we demonstrate that a circular RNA (circRNA), circSyk, is a potential biological target for the treatment of S. aureus-induced bone infection. Most importantly, S. aureus regulates circSyk to block autophagy and promote bone destruction via the circSyk/miR-5106/Sik3 axis in a nonclassical pathway, which is involved in the S. aureus infection process through a competitive endogenous RNA network. In summary, this study proposes a novel perspective on the immune escape of S. aureus in bone infections, based on circRNA. Author summary: Bone infection is mainly caused by Staphylococcus aureus, and results in progressive inflammation and bone destruction, requiring extensive surgical interventions and long-term antibiotic treatment. However, evidence has shown that S. aureus can survive inside a variety of cells, including osteoclasts, which is considered to underlie the persistent bone infections caused by S. aureus. Circular RNAs are noncoding RNAs that play important roles in bone infection. Following from our previous transcriptomic analysis of circRNA expression in response to the infection of osteoclasts by S. aureus, we show that S. aureus upregulates circSyk to block osteoclast autophagy and promote bone destruction via the circSyk/miR-5106/Sik3 axis. [ABSTRACT FROM AUTHOR]

Lenacapavir (LEN) is a highly potent, long-acting antiretroviral medication for treating people infected with muti-drug-resistant HIV-1 phenotypes. The inhibitor targets multifaceted functions of the viral capsid protein (CA) during HIV-1 replication. Previous studies have mainly focused on elucidating LEN's mode of action during viral ingress. Additionally, the inhibitor has been shown to interfere with mature capsid assembly during viral egress. However, the mechanism for how LEN affects HIV-1 maturation is unknown. Here, we show that pharmacologically relevant LEN concentrations do not impair proteolytic processing of Gag in virions. Instead, we have elucidated the primary mechanism for highly potent inhibition of HIV-1 maturation by sub-stoichiometric LEN:CA ratios. The inhibitor exerts opposing effects on formation of CA pentamers versus hexamers, the key capsomere intermediates in mature capsid assembly. LEN impairs formation of pentamers, whereas it induces assembly of hexameric lattices by imposing an opened CA conformation and stabilizing a dimeric form of CA. Consequently, LEN treatment results in morphologically atypical virus particles containing malformed, hyper-stable CA assemblies, which fail to infect target cells. Moreover, we have uncovered an inverse correlation between inhibitor potency and CA levels in cell culture assays, which accounts for LEN's ability to potently (with picomolar EC50 values) inhibit HIV-1 maturation at clinically relevant drug concentrations. Author summary: Lenacapavir (LEN) is used to treat adults with multi-drug-resistant HIV-1 infection. LEN is the first in class, highly potent and long-acting antiretroviral, which works by a unique mechanism of targeting the viral capsid protein. LEN is administered through subcutaneous injections twice-yearly in combination with other HIV-1 medications. The long-acting antiviral activity relies on the ability of sub-nanomolar LEN concentrations to inhibit HIV-1 replication. Accordingly, cell culture-based mechanistic studies are expected to focus on elucidating highly potent antiviral modes of action of LEN. We and others have previously reported the primary mechanism of inhibition of HIV-1 ingrees by LEN. Our studies here have deciphered previously undescribed mechanistic and structural bases for a highly potent antiviral activity of LEN during viral egress. Specifically, we show that clinically relevant LEN concentrations induce aberrant capsid protein assembly during virus maturation and consequently yield non-infectious HIV-1 paricles. These findings will inform clinical applications of LEN as a potent HIV-1 maturation inhibitor and aid the development of second-generation inhibitors targeting assembly of the mature viral capsid. [ABSTRACT FROM AUTHOR]

The coronavirus HCoV-OC43 circulates continuously in the human population and is a frequent cause of the common cold. Here, we generated a high-resolution atlas of the transcriptional and translational landscape of OC43 during a time course following infection of human lung fibroblasts. Using ribosome profiling, we quantified the relative expression of the canonical open reading frames (ORFs) and identified previously unannotated ORFs. These included several potential short upstream ORFs and a putative ORF nested inside the M gene. In parallel, we analyzed the cellular response to infection. Endoplasmic reticulum (ER) stress response genes were transcriptionally and translationally induced beginning 12 and 18 hours post infection, respectively. By contrast, conventional antiviral genes mostly remained quiescent. At the same time points, we observed accumulation and increased translation of noncoding transcripts normally targeted by nonsense mediated decay (NMD), suggesting NMD is suppressed during the course of infection. This work provides resources for deeper understanding of OC43 gene expression and the cellular responses during infection. Author summary: RNA viruses are under constant evolutionary pressure to maintain compact and highly-streamlined genomes. Consequently, these viruses frequently employ unusual gene expression strategies to maximize the coding potential of their relatively small genomes. In this work, we investigated the gene expression of the human coronavirus HCoV-OC43. OC43 generally causes mild common cold symptoms, but the species is closely related to more pathogenic coronaviruses such as SARS-CoV-2. Here, we used ribosome profiling and RNA sequencing to determine the expression kinetics and translation efficiencies of both viral and cellular genes. We uncovered several unexpected features in the OC43 genome, including short open reading frames upstream of major genes, and a putative alternative translation start site within the M gene. In parallel, we surveyed the cellular response to OC43 infection. Genes associated with the endoplasmic reticulum (ER) stress response were strongly induced late in infection. By contrast, conventional antiviral genes remained largely unaltered throughout the course of infection, suggesting OC43 effectively evades or disables the host antiviral response. [ABSTRACT FROM AUTHOR]

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cell's transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 recruits the host protein CTCF to viral episomes to control the differentiation-dependent viral transcriptional programme. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18 may also manipulate CTCF to contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression. Author summary: Infection with oncogenic HPV is the cause of numerous cancer types, which generally arise after persistent HPV infection. Upon infection, HPV alters the gene expression profile of infected cells to facilitate virus replication and persistence. Multiple mechanisms of HPV-induced host cell reprogramming have been previously suggested. Here, we show that HPV infection induces rearrangement of specific genomic loci by altering the chromatin binding of the host cell protein CTCF, an important regulator of chromatin architecture. Loss of CTCF binding to a cluster of binding sites at the CADM1 locus on chromosome 11 is coincident with epigenetic reprogramming and disruption of long-range chromatin interactions, resulting in transcriptional repression of CADM1. Our data show that repression of CADM1 is an early event in HPV-driven disease, preceding hypermethylation of the CADM1 transcriptional promoter that is frequently observed in HPV-driven cancers, demonstrating a novel mechanism of HPV-induced host cell transcriptional reprogramming. [ABSTRACT FROM AUTHOR]

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage, and visualised the effects of structural changes in the polar plugs, leading to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing 'hatch-ready' L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes. Author summary: Human whipworms are parasites that cause the gastrointestinal disease trichuriasis in millions of people around the world. Infections occur when whipworm eggs, ingested in contaminated food and water, hatch in the intestine in response to gut bacteria (microbiota). The egg encloses a larva within an egg-shell and has a plug at each end. Hatching liberates the larva that burrows inside the cells that line the gut. Interactions between the microbiota of the gut and whipworm eggs are needed for hatching, but are poorly understood. In this study, using the natural mouse whipworm as an infection model, we show that bacteria bind the whipworm egg plugs during the initial stages of hatching, resulting in their degradation and leading to larval exit. We further show that disintegration of the egg plugs is caused by protein-degrading enzymes produced by the bacteria and the larvae. The production of those enzymes by the parasite is dependent on the full development of the larva inside the whipworm egg. These new mechanistic insights pave the way for future studies to understand human whipworm infection and develop new tools to tackle these globally important parasites. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 continues to transmit and evolve in humans and animals. White-tailed deer (Odocoileus virginianus) have been previously identified as a zoonotic reservoir for SARS-CoV-2 with high rates of infection and probable spillback into humans. Here we report sampling 1,127 white-tailed deer (WTD) in Pennsylvania, and a genomic analysis of viral dynamics spanning 1,017 days between April 2021 and January 2024. To assess viral load and genotypes, RNA was isolated from retropharyngeal lymph nodes and analyzed using RT-qPCR and viral whole genome sequencing. Samples showed a 14.64% positivity rate by RT-qPCR. Analysis showed no association of SARS-CoV-2 prevalence with age, sex, or diagnosis with Chronic Wasting Disease. From the 165 SARS-CoV-2 positive WTD, we recovered 25 whole genome sequences and an additional 17 spike-targeted amplicon sequences. The viral variants identified included 17 Alpha, 11 Delta, and 14 Omicron. Alpha largely stopped circulating in humans around September 2021, but persisted in WTD as recently as March of 2023. Phylodynamic analysis of pooled genomic data from Pennsylvania documents at least 12 SARS-CoV-2 spillovers from humans into WTD, including a recent series of Omicron spillovers. Prevalence was higher in WTD in regions with crop coverage rather than forest, suggesting an association with proximity to humans. Analysis of seasonality showed increased prevalence in winter and spring. Multiple examples of recurrent mutations were identified associated with transmissions, suggesting WTD-specific evolutionary pressures. These data document ongoing infections in white-tailed deer, probable onward transmission in deer, and a remarkable rate of new spillovers from humans. Author summary: SARS-CoV-2 continues to evolve and circulate in humans and animals. Sometimes accumulating genetic changes give rise to new viral variants, the origins of which are incompletely understood and may involve transmissions between humans and animals. White-tailed deer (Odocoileus virginianus) is one of several host species known to harbor SARS-CoV-2 at high prevalence. We thus studied circulation and evolutionary dynamics of SARS-CoV-2 in 1,127 white-tailed deer in Pennsylvania from 2021 to 2024. Prevalence was higher in white-tailed deer in regions with crop coverage rather than forest. Analysis of seasonality showed increased prevalence in winter and spring. Our viral genome sequence analysis identified white-tailed deer as a SARS-CoV-2 reservoir of ancestral and highly divergent lineages consistent with genetic distances similar to new variants that have emerged in the human population. We found multiple examples of recurrent mutations in independent suspected spillover events, consistent with selective pressures in white-tailed deer. Together, these findings provide a unique longitudinal perspective on SARS-CoV-2 circulation, persistence, and evolution in Pennsylvania wild white-tailed deer. [ABSTRACT FROM AUTHOR]

Pathogenic protists are responsible for many diseases that significantly impact human and animal health across the globe. Almost all protists possess mitochondria or mitochondrion-related organelles, and many contain plastids. These endosymbiotic organelles are crucial to survival and provide well-validated and widely utilised drug targets in parasitic protists such as Plasmodium and Toxoplasma. However, mutations within the organellar genomes of mitochondria and plastids can lead to drug resistance. Such mutations ultimately challenge our ability to control and eradicate the diseases caused by these pathogenic protists. Therefore, it is important to understand how organellar genomes, and the resistance mutations encoded within them, are inherited during protist sexual reproduction and how this may impact the spread of drug resistance and future therapeutic approaches to target these organelles. In this review, we detail what is known about mitochondrial and plastid inheritance during sexual reproduction across different pathogenic protists, often turning to their better studied, nonpathogenic relatives for insight. [ABSTRACT FROM AUTHOR]

Prion diseases, particularly sporadic cases, pose a challenge due to their complex nature and heterogeneity. The underlying mechanism of the spontaneous conversion from PrPC to PrPSc, the hallmark of prion diseases, remains elusive. To shed light on this process and the involvement of cofactors, we have developed an in vitro system that faithfully mimics spontaneous prion misfolding using minimal components. By employing this PMSA methodology and introducing an isoleucine residue at position 108 in mouse PrP, we successfully generated recombinant murine prion strains with distinct biochemical and biological properties. Our study aimed to explore the influence of a polyanionic cofactor in modulating strain selection and infectivity in de novo-generated synthetic prions. These results not only validate PMSA as a robust method for generating diverse bona fide recombinant prions but also emphasize the significance of cofactors in shaping specific prion conformers capable of crossing species barriers. Interestingly, once these conformers are established, our findings suggest that cofactors are not necessary for their infectivity. This research provides valuable insights into the propagation and maintenance of the pathobiological features of cross-species transmissible recombinant murine prion and highlights the intricate interplay between cofactors and prion strain characteristics. Author summary: Prion diseases are rare and complex neurodegenerative disorders that can occur spontaneously, through a poorly understood conformational or structural change of normal, physiological prion protein. This abnormally structured form, known as prion, acquires the capacity to induce the same transformation to surrounding prion proteins, leading to disease. In our study, we take advantage of a recently developed methodology that closely mimics this process in a test tube using extremely simple components. Applying this system and modifying a specific part of the mouse prion protein, we were able to generate different prion strains with unique characteristics spontaneously. This process is greatly enhanced using an additional molecule called cofactor, that has been proposed to affect the variability and infectivity (capacity to cause disease in an animal model) of these prions. Our findings show that while cofactors facilitate the spontaneous formation of prions and may influence their final characteristics, they are not necessary for their infectivity nor for their spontaneous formation. This research gives new insights into the role cofactors play in the spontaneous generation of prions and their behavior in animal models. [ABSTRACT FROM AUTHOR]

The viral protein mutations can modify virus-host interactions during virus evolution, and thus alter the extent of infection or pathogenicity. Studies indicate that nucleocapsid (N) protein of SARS-CoV-2 participates in viral genome assembly, intracellular signal regulation and immune interference. However, its biological function in viral evolution is not well understood. SARS-CoV-2 N protein mutations were analyzed in Delta, Omicron, and original strains. Two mutations with a methionine (M) residue at site 203 and a tyrosine (Y) residue at site 377 of the N protein were found in Delta strain but not in Omicron and original strains, and promoted SARS-CoV-2 infection therein. Those mutations, R203M and D377Y, enhanced the inhibitory impact of N protein on the impairment of RIG-I-mediated antiviral signaling, such as IRF3 phosphorylation and IFN-β activation. The viral RNA-binding activity of N protein was promoted by these mutations, effectively attenuating the recognition and interaction of RIG-I with viral RNA compared to the original or other variants. The R203M/D377Y mutations thus enhanced the suppressive activity of the N protein on RIG-I-mediated interferon induction both in vitro and in vivo, which in turn promoted viral replication. This study helps to understand the variability of SARS-CoV-2 in regulating host immunity. Author summary: Recently, the World Health Organization declared that the COVID-19 pandemic is no longer a Public Health Emergency of International Concern (PHEIC). However, the SARS-CoV-2 virus continues to infect and transmit, with ongoing mutations leading to the emergence of multiple variants that still pose a threat to human health. Among these, compared to the original strain, the Delta variant demonstrates faster transmission speed, higher viral load and a higher likelihood of causing severe disease. In contrast, the Omicron variant and its sublineages (including XBB, BQ.1.1, and CH.1.1) exhibit weaker replication ability but possess stronger immune escape and transmission capabilities. Therefore, closely monitoring different mutant strains of SARS-CoV-2 and investigating the impact of mutations on the virus is of significant scientific importance. The N protein of SARS-CoV-2 is closely related to viral replication. We have found that the R203M and D377Y mutations in the N protein of the Delta variant can competitively bind to viral RNA with RIG-I, thereby inhibiting interferon production regulated by the RIG-I-MAVS signaling pathway and promoting viral replication. Our study elucidates the biological function of the N protein during the evolution of SARS-CoV-2 and provides new insights into the regulatory pathways of viral replication in the virus's evolutionary process. [ABSTRACT FROM AUTHOR]

Gram-negative bacterial pathogens inject effector proteins inside plant cells using a type III secretion system. These effectors manipulate plant cellular functions and suppress the plant immune system in order to promote bacterial proliferation. Despite the fact that bacterial effectors are exogenous threatening proteins potentially exposed to the protein degradation systems inside plant cells, effectors are relative stable and able to perform their virulence functions. In this work, we found that RipE1, an effector protein secreted by the bacterial wilt pathogen, Ralstonia solanacearum, undergoes phosphorylation of specific residues inside plant cells, and this promotes its stability. Moreover, RipE1 associates with plant ubiquitin proteases, which contribute to RipE1 deubiquitination and stabilization. The absence of those specific phosphorylation sites or specific host ubiquitin proteases leads to a substantial decrease in RipE1 protein accumulation, indicating that RipE1 hijacks plant post-translational modification regulators in order to promote its own stability. These results suggest that effector stability or degradation in plant cells constitute another molecular event subject to co-evolution between plants and pathogens. Author summary: Most bacterial plant pathogens inject effector proteins inside host cells to suppress immune responses and manipulate other plant functions in order to cause disease. Even though these effector proteins could be targeted by the protein degradation systems in plant cells, they are able to perform their virulence functions, which suggests that they are relatively stable, even though the mechanisms leading to this stability remain poorly understood. In this work, we found that RipE1, an effector protein secreted by the bacterial wilt pathogen, Ralstonia solanacearum, has the potential to be ubiquitinated and degraded in plant cells. However, RipE1 hijacks plant kinases and undergoes phosphorylation of specific residues inside plant cells, and this counteracts its ubiquitination and promotes its stability. Moreover, RipE1 associates with plant ubiquitin proteases, which contribute to RipE1 deubiquitination and stabilization. Our study suggests that effector stability or degradation in plant cells constitute another molecular event subject to co-evolution between plants and pathogens, and that pathogen effectors hijack plant post-translational modification regulators in order to promote their own stability. [ABSTRACT FROM AUTHOR]

Orthohantaviruses are emerging zoonotic viruses that can infect humans via the respiratory tract. There is an unmet need for an in vivo model to study infection of different orthohantaviruses in physiologically relevant tissue and to assess the efficacy of novel pan-orthohantavirus countermeasures. Here, we describe the use of a human lung xenograft mouse model to study the permissiveness for different orthohantavirus species and to assess its utility for preclinical testing of therapeutics. Following infection of xenografted human lung tissues, distinct orthohantavirus species differentially replicated in the human lung and subsequently spread systemically. The different orthohantaviruses primarily targeted the endothelium, respiratory epithelium and macrophages in the human lung. A proof-of-concept preclinical study showed treatment of these mice with a virus neutralizing antibody could block Andes orthohantavirus infection and dissemination. This pan-orthohantavirus model will facilitate progress in the fundamental understanding of pathogenesis and virus-host interactions for orthohantaviruses. Furthermore, it is an invaluable tool for preclinical evaluation of novel candidate pan-orthohantavirus intervention strategies. Author summary: Orthohantaviruses are rodent-borne pathogens that can be transmitted from rodents to humans by inhalation of aerosolized virus-contaminated rodent excreta. While orthohantaviruses generally do not cause overt clinical signs in rodents, orthohantavirus infection can lead to severe disease with case fatality rates up to 40%. The pathogenesis of associated hemorrhagic fever with renal syndrome (HFRS) mainly in Europe and Asia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas remains poorly understood. Particularly the understanding of the early steps of pathogenesis occurring in the human lungs is limited. In addition, assessment of the efficacy of cross-reactive anti-orthohantavirus antibodies is severely hampered by the limited availability of in vivo models that allow for replication of both HFRS- and HCPS-associated orthohantaviruses within the same animal model. Therefore, we present a human lung xenograft mouse model to study early events of orthohantavirus pathogenesis in the human lung. We demonstrated that multiple orthohantaviruses replicate inside the human lung xenografts, and characterized the tropism and host responses within these human lung xenografts. Also, as a proof-of-principle, we showed that the model can be utilized to assess the protective efficacy of an existing monoclonal antibody against Andes virus infection. Overall, this pan-orthohantavirus model allows for the study of orthohantavirus pathogenesis and will aid as a novel platform to test anti-orthohantavirus countermeasures. [ABSTRACT FROM AUTHOR]

Viral infections of the central nervous system (CNS) are a major cause of morbidity largely due to lack of prevention and inadequate treatments. While mortality from viral CNS infections is significant, nearly two thirds of the patients survive. Thus, it is important to understand how the human CNS can successfully control virus infection and recover. Since it is not possible to study the human CNS throughout the course of viral infection at the cellular level, here we analyzed a non-lethal viral infection in the CNS of nonhuman primates (NHPs). We inoculated NHPs intracerebrally with a high dose of La Crosse virus (LACV), a bunyavirus that can infect neurons and cause encephalitis primarily in children, but with a very low (≤ 1%) mortality rate. To profile the CNS response to LACV infection, we used an integrative approach that was based on comprehensive analyses of (i) spatiotemporal dynamics of virus replication, (ii) identification of types of infected neurons, (iii) spatiotemporal transcriptomics, and (iv) morphological and functional changes in CNS intrinsic and extrinsic cells. We identified the location, timing, and functional repertoire of optimal transcriptional and translational regulation of the primate CNS in response to virus infection of neurons. These CNS responses involved a well-coordinated spatiotemporal interplay between astrocytes, lymphocytes, microglia, and CNS-border macrophages. Our findings suggest a multifaceted program governing an optimal CNS response to virus infection with specific events coordinated in space and time. This allowed the CNS to successfully control the infection by rapidly clearing the virus from infected neurons, mitigate damage to neurophysiology, activate and terminate immune responses in a timely manner, resolve inflammation, restore homeostasis, and initiate tissue repair. An increased understanding of these processes may provide new therapeutic opportunities to improve outcomes of viral CNS diseases in humans. Author summary: Viral infections of the brain and spinal cord can be devastating and even fatal in some patients, while others survive and recover. Understanding how the human brain and spinal cord can successfully control virus infection may lead to improved treatments and help to determine the optimal timing for their use. Here, we studied how the brain and spinal cord of nonhuman primates respond to virus infection after inoculation of La Crosse virus directly into the brain. We identified the location, timing, and functional repertoire of optimal host responses in the primate brain and spinal cord that led to rapid virus clearance from infected neurons. These host responses were carefully regulated at the levels of gene expression and cellular functions to mitigate neurological damage, resolve inflammation, restore homeostasis, and initiate tissue repair. A better understanding of the processes that govern optimal responses to viral infection in the brain and spinal cord may improve outcomes of viral neurological diseases and allow full recovery in a higher proportion of patients. [ABSTRACT FROM AUTHOR]

Background: Orthohantaviruses are negative-sense RNA viruses that can cause hantavirus cardiopulmonary syndrome (HCPS) in humans. In the United States, Sin Nombre orthohantavirus (SNV) is the primary cause of HCPS, with a fatality rate of 36% and most cases occuring in the southwestern states. The western deer mouse, Peromyscus sonoriensis, is the primary reservoir for SNV; however, it remains unclear if alternative reservoirs exist. Results: We conducted an extensive survey of SNV genetic prevalence in wild-caught small mammal communities throughout New Mexico and observed that 27% of all animals were positive for SNV. Through longitudinal trapping at a site of patient exposure, we found that SNV circulates at a high rate in multiple species over time. Furthermore, we isolated live SNV from tissues and feces from multiple small mammal species, demonstrating infectious virus in alternative and novel reservoirs. Significance: Altogether, this work shows that SNV is widely prevalent and persistent throughout New Mexico in multiple small mammal reservoirs that can harbor and shed infectious virus. This encourages future work for additional surviellance efforts and revaluates host-species dynamics for New World hantaviruses. Author summary: The first reported hantavirus cases in the United States occurred in the Four Corners region (Arizona, New Mexico, Utah and Colorado) and was found to be transmitted by the droppings of western deer mice. This led to discovery of Sin Nombre virus (SNV). Unlike hantaviruses found through Europe and Asia, SNV causes hantavirus cardiopulmonary syndrome (HCPS), which has a high mortality rate. It is generally thought that only one small mammal host is capable of carrying and transmitting hantavirus to humans. However, our group and others have found hantavirus genetic material in multiple small mammal species, but these studies have been fairly limited and did not determine if the virus could replicate in these species and be transmitted through them. Here, we conducted a large study to look for SNV in wild-caught rodents throughout New Mexico in counties with and without reported human HCPS cases. We found that SNV is widespread in all areas of NM and stably circulates in rodents at a site of human infection. Importantly, we show that multiple small mammal species can carry and shed infectious SNV. Altogether, our study provides a novel shift in the understanding of host reservoir dynamics for SNV. [ABSTRACT FROM AUTHOR]

Hybridisation is a source of genetic diversity, can drive adaptation to new niches and has been found to be a frequent event in lineages harbouring pathogenic fungi. However, little is known about the genomic implications of hybridisation nor its impact on pathogenicity-related traits. A common limitation for addressing these questions is the narrow representativity of sequenced genomes, mostly corresponding to strains isolated from infected patients. The opportunistic human pathogen Candida metapsilosis is a hybrid that descends from the crossing between unknown parental lineages. Here, we sequenced the genomes of five new C. metapsilosis isolates, one representing the first African isolate for this species, and four environmental isolates from marine niches. Our comparative genomic analyses, including a total of 29 sequenced strains, shed light on the phylogenetic relationships between C. metapsilosis hybrid isolates and show that environmental strains are closely related to clinical ones and belong to different clades, suggesting multiple independent colonisations. Furthermore, we identify a new diverging clade likely emerging from the same hybridisation event that originated two other previously described hybrid clades. Lastly, we evaluate phenotypes relevant during infection such as drug susceptibility, thermotolerance or virulence. We identify low drug susceptibility phenotypes which we suggest might be driven by loss of heterozygosity events in key genes. We discover that thermotolerance is mainly clade-dependent and find a correlation with the faecal origin of some strains which highlights the adaptive potential of the fungus as commensal. Author summary: Yeast pathogens of the Candida genus are estimated to cause almost a million deaths each year globally. Although most infections are caused by the five most common Candida species, the frequency of more rarely isolated species is increasing in the last few years. This is the case of Candida metapsilosis, a hybrid fungus of uncertain origin. Here we sequenced the genomes of several new strains, including the first African isolate and several environmental ones, to unravel the complex evolutionary history of this species. Our analysis revealed that environmental and clinical strains are closely related, suggesting that this fungus can easily transition between these habitats. We identified several divergent clades of hybrids, and to understand the implications of this genetic diversity, we investigated several traits relevant to infection, such as drug susceptibility and thermotolerance. We found that certain genetic variations can lead to reduced drug susceptibility, while thermotolerance seems to be largely influenced by the strain's evolutionary history. Our findings shed light on the complex biology of C. metapsilosis and its potential for causing infections. This knowledge can contribute to the development of more effective strategies for prevention and treatment. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) are small, icosahedral viruses that cause serious clinical symptoms in livestock. The FMDV VP1 protein is a key structural component, facilitating virus entry. Here, we find that the E3 ligase RNF5 interacts with VP1 and targets it for degradation through ubiquitination at the lys200 of VP1, ultimately inhibiting virus replication. Mutations at this lysine site have been found to increase the replication of FMDV in mice. Importantly, the RNF5 pharmacological activator Analog-1 alleviates disease development in a mouse infection model. Furthermore, RNF5 recognizes the VP1 protein from several picornaviruses, suggesting that targeting RNF5 may be a broad-spectrum antiviral strategy. These findings shed light on the role of the ubiquitin-proteasome system in controlling virus replication, offering potential new strategies for treating viral infections. Author summary: Elucidation of the host factors that modulate the FMDV lifecycle is vital for developing antiviral drugs and vaccines. In this study, we show that RNF5 acts as a cell-intrinsic antiviral effector by inhibiting multiple picornaviruses through targeting the viral VP1 protein for ubiquitination and subsequent degradation by the proteasome. Further investigation revealed that the Lys200 of VP1 is a specific site for RNF5-mediated ubiquitination. Disruption of this ubiquitination site led to increased replication of FMDV both in vitro and in vivo. The use of Analog-1, a pharmacological activator of RNF5, was shown to mitigate disease progression in a mouse infection model. These findings unveil a strategy for targeting picornavirus replication and offer insights that could aid in the development of new therapeutics for picornavirus infections. [ABSTRACT FROM AUTHOR]

Interaction with host cell receptors initiates internalization of Kaposi's sarcoma-associated herpesvirus (KSHV) particles. Fusion of viral and host cell membranes, which is followed by release of the viral capsid into the cytoplasm, is executed by the core fusion machinery composed of glycoproteins H (gH), L (gL), and B (gB), that is common to all herpesviruses. KSHV infection has been shown to be sensitive to inhibitors of vacuolar acidification, suggestive of low pH as a fusion trigger. To analyze KSHV entry at the single particle level we developed dual-fluorescent recombinant KSHV strains that incorporate fluorescent protein-tagged glycoproteins and capsid proteins. In addition, we generated a hybrid rhesus monkey rhadinovirus (RRV) that expresses KSHV gB in place of RRV gB to analyze gB-dependent differences in infection pathways. We demonstrated lytic reactivation and infectivity of dual-fluorescent KSHV. Confocal microscopy was used to quantify co-localization of fluorescently-tagged glycoproteins and capsid proteins. Using the ratio of dual-positive KSHV particles to single-positive capsids as an indicator of fusion events we established KSHV fusion kinetics upon infection of different target cells with marked differences in the "time-to-fusion" between cell types. Inhibition of vesicle acidification prevented KSHV particle-cell fusion, implicating low vesicle pH as a requirement. These findings were corroborated by comparison of RRV-YFP wildtype reporter virus and RRV-YFP encoding KSHV gB in place of RRV gB. While RRV wt infection of receptor-overexpressing cells was unaffected by inhibition of vesicle acidification, RRV-YFP expressing KSHV gB was sensitive to Bafilomycin A1, an inhibitor of vacuolar acidification. Single- and dual-fluorescent KSHV strains eliminate the need for virus-specific antibodies and enable the tracking of single viral particles during entry and fusion. Together with a hybrid RRV expressing KSHV gB and classical fusion assays, these novel tools identify low vesicle pH as an endocytotic trigger for KSHV membrane fusion. Author summary: All herpesviruses encode a fusion protein, gB, which fuses the virus membrane with host membranes. Membrane fusion is essential for infection by enveloped viruses. Under conditions where gB proteins from other herpesviruses fuse cells into syncytia, KSHV gB does not readily fuse cells, and KSHV infection was previously shown to be inhibited by substances that raise the pH of endocytotic vesicles. We therefore sought to test whether fusion of KSHV viral particles with cellular membranes is a pH-dependent step. We developed two tools to test this hypothesis. The first tool is a dual-fluorescent KSHV with differently colored protein tags at the viral envelope and the capsid, which lose colocalization upon fusion and release of the capsid from the membrane, but not in the presence of an inhibitor of vesicle acidification. The second tool is a hybrid RRV strain that expresses KSHV gB instead of RRV gB. RRV infected receptor-overexpressing cells in a manner that was not inhibited by an inhibitor of vesicle acidification. The hybrid RRV expressing KSHV gB on the other hand was sensitive to inhibition of vesicle acidification. Together, these findings show that KSHV gB dictates a low-pH, endocytotic route of infection. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that has re-emerged to cause large outbreaks of human infections worldwide. There are currently no approved antivirals for treatment of CHIKV infection. Recently, we reported that the ribonucleoside analog 4′-fluorouridine (4′-FlU) is a highly potent inhibitor of CHIKV replication, and targets the viral nsP4 RNA dependent RNA polymerase. In mouse models, oral therapy with 4′-FlU diminished viral tissue burdens and virus-induced disease signs. To provide critical evidence for the potential of 4′-FlU as a CHIKV antiviral, here we selected for CHIKV variants with decreased 4′-FlU sensitivity, identifying two pairs of mutations in nsP2 and nsP4. The nsP4 mutations Q192L and C483Y were predominantly responsible for reduced sensitivity. These variants were still inhibited by higher concentrations of 4′-FlU, and the mutations did not change nsP4 fidelity or provide a virus fitness advantage in vitro or in vivo. Pathogenesis studies in mice showed that the nsP4-C483Y variant caused similar disease and viral tissue burden as WT CHIKV, while the nsP4-Q192L variant was strongly attenuated. Together these results support the potential of 4′-FlU to be an important antiviral against CHIKV. Author summary: The alphavirus chikungunya virus (CHIKV) is spread by mosquito vectors and currently causes millions of cases of human infections around the world. Initial symptoms of CHIKV infection include fever, rash, and severe joint and muscle pain. In a high proportion of cases infection progresses to chronic painful arthritis, and can even cause fatal encephalitis. There are no approved antiviral treatments for CHIKV infection. We recently demonstrated that the orally available compound 4′-fluorouridine (4′-FlU) strongly inhibits CHIKV replication in cell culture and decreases viral disease symptoms in a mouse model of CHIKV infection. In order to advance 4′-FlU as a potential antiviral treatment, here we asked if 4′-FlU could select for CHIKV resistance or increased viral pathogenesis. We identified 2 mutations that decreased the sensitivity of CHIKV to inhibition by 4′-FlU. However, CHIKV variants containing those mutations were still inhibited by higher concentrations of 4′-FlU. In addition, the variants did not show increased virus growth and were not more pathogenic in mice. Together our data support the potential of 4′-FIU as an antiviral against CHIKV and related alphaviruses. [ABSTRACT FROM AUTHOR]

Plasmodium vivax is the predominant malaria parasite in Latin America. Its colonization history in the region is rich and complex, and is still highly debated, especially about its origin(s). Our study employed cutting-edge population genomic techniques to analyze whole genome variation from 620 P. vivax isolates, including 107 newly sequenced samples from West Africa, Middle East, and Latin America. This sampling represents nearly all potential source populations worldwide currently available. Analyses of the genetic structure, diversity, ancestry, coalescent-based inferences, including demographic scenario testing using Approximate Bayesian Computation, have revealed a more complex evolutionary history than previously envisioned. Indeed, our analyses suggest that the current American P. vivax populations predominantly stemmed from a now-extinct European lineage, with the potential contribution also from unsampled populations, most likely of West African origin. We also found evidence that P. vivax arrived in Latin America in multiple waves, initially during early European contact and later through post-colonial human migration waves in the late 19th-century. This study provides a fresh perspective on P. vivax's intricate evolutionary journey and brings insights into the possible contribution of West African P. vivax populations to the colonization history of Latin America. Author summary: Our study investigates the origins and spread of Plasmodium vivax in Latin America, the primary malaria parasite in this region. By analyzing genetic data from 620 P. vivax samples worldwide, including new samples from West Africa, the Middle East, and Latin America, we were able to reconstruct the evolutionary history of the parasite in this region. Our findings indicate that the current P. vivax strains in Latin America mainly descended from a now-extinct European lineage. Additionally, we found evidence suggesting that several migration waves following human migrations waves may have contributed to the parasite diversity, with possible contributions from West African populations during the transatlantic slave trade (16th to 19th century) and further input from Europe during post-colonial human migrations in the late 19th century. This study offers a fresh perspective on how historical human migrations have shaped the genetic landscape of P. vivax in Latin America. These insights are valuable for understanding the parasite evolution and may help inform strategies for malaria control and prevention in the future. [ABSTRACT FROM AUTHOR]