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Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection., Author summary Microsporidia infect a wide range of hosts: from economically important invertebrates such as silkworms and honey bees, to vertebrates including humans, where infection in immunocompromised patients can be fatal. In order to infect the host, microsporidia utilize a unique harpoon-like invasion organelle called the polar tube (PT), which serves as a pathway for transport of infectious cargo to the host. Knowledge of how the long PT is packaged in the spore, the kinetics of its firing and how the cargo is transported through the PT are still poorly understood. We use electron microscopy techniques to generate 3D reconstructions of intact spores, which reveal the configuration of the PT and other organelles within. Using high-speed live-cell imaging, we capture and quantify the kinetics of PT firing, and how cargo is deformed during transport through the narrow PT. Our study provides new insights into architecture and dynamics of the PT, which serve as foundations for our understanding of microsporidia infection.

Most HIV-1 infected individuals do not know their infection dates. Precise infection timing is crucial information for studies that document transmission networks or drug levels at infection. To improve infection timing, we used the prospective RV217 cohort where the window when plasma viremia becomes detectable is narrow: the last negative visit occurred a median of four days before the first detectable HIV-1 viremia with an RNA test, referred below as diagnosis. We sequenced 1,280 HIV-1 genomes from 39 participants at a median of 4, 32 and 170 days post-diagnosis. HIV-1 infections were dated by using sequence-based methods and a viral load regression method. Bayesian coalescent and viral load regression estimated that infections occurred a median of 6 days prior to diagnosis (IQR: 9–3 and 11–4 days prior, respectively). Poisson-Fitter, which analyzes the distribution of hamming distances among sequences, estimated a median of 7 days prior to diagnosis (IQR: 15–4 days) based on sequences sampled 4 days post-diagnosis, but it did not yield plausible results using sequences sampled at 32 days. Fourteen participants reported a high-risk exposure event at a median of 8 days prior to diagnosis (IQR: 12 to 6 days prior). These different methods concurred that HIV-1 infection occurred about a week before detectable viremia, corresponding to 20 days (IQR: 34–15 days) before peak viral load. Together, our methods comparison helps define a framework for future dating studies in early HIV-1 infection., Author summary HIV-1 infected individuals rarely know when they became infected but knowing when an infection occurred provides critical information regarding HIV-1 pathogenesis and epidemiology. Using a unique cohort in which infection was known to have occurred in a narrow interval, we investigated methods to estimate the timing of infections. Several methods suggested that HIV-1 infection typically occurs a median of one week before the infection can be detected by HIV-1 RNA testing. Going forward, we provide a strategy that can be used to elucidate the origin of an acute/early infection.

Brucellosis remains the most common zoonotic disease globally. Currently no vaccines for humans exist, and conventional brucellosis vaccines for livestock fail to confer complete protection; hence, an improved vaccine is needed. Although Brucella infections primarily occur following a mucosal exposure, vaccines are administered parenterally. Few studies have considered mucosal vaccinations, or even targeting of tissue-resident memory T (TRM) cells. TRM cells protect against viral infections, but less is known of their role in bacterial infections, and even less for brucellosis. Oral prime, nasal boost with a newly developed Brucella abortus double mutant (znBAZ) confers nearly complete protection against pulmonary challenge with wild-type (wt) B. abortus 2308, and its protective efficacy is >2800-fold better than the RB51 vaccine. Vaccination with znBAZ potently stimulated CD8+ T cells, whereas mucosal vaccination with RB51 induced mostly CD4+ T cells. Subsequent analysis revealed these pulmonary CD44+ CD69+ CD8+ T cells to be either CD103+ or CD103- TRM cells, and these sequestered to the lung parenchyma as CXCR3lo and to the airways as CXCR3hi. Both CD8+ TRM subsets contained single-positive IFN-γ and TNF-α, as well as, polyfunctional cells. IL-17-producing CD8+ TRM cells were also induced by znBAZ vaccination, but in vivo IL-17 neutralization had no impact upon protection. In vivo depletion of CD4+ T cells had no impact upon protection in znBAZ-vaccinated mice. In contrast, CD4+ T cell depletion reduced RB51’s protective efficacy in spleens and lungs by two- and three-logs, respectively. Although anti-CD8 mAb-treated znBAZ-vaccinated mice showed a significantly reduced pulmonary efficacy, this treatment failed to completely deplete the lung CD8+ T cells, leaving the CD103+ and CD103- CD8+ TRM cell ratios intact. Only znBAZ-vaccinated CD8-/- mice were fully sensitive to pulmonary challenge with virulent wt B. abortus 2308 since CD8+ TRM cells could not be induced. Collectively, these data demonstrate the key role of mucosal vaccination for the generation of CD8+ TRM cells in protecting against pulmonary challenge with virulent B. abortus., Author summary Brucellosis is the most common zoonotic disease worldwide and is transmitted via the consumption of unpasteurized dairy products or exposure to Brucella-laden aerosols. In fact, mucosal exposure is the most common route of infection for humans and animals, yet parenteral vaccination of livestock remains the preferred route of immunization. To determine whether development of a mucosal vaccination regimen could effectively generate immunity against pulmonary challenge with virulent B. abortus, a double-mutant B. abortus vaccine was administered mucosally, and found to induce CD8+ TRM cells. These conferred complete protection against pulmonary infection and prevented systemic brucellae spread even in the absence of immune recirculating CD8+ T cells. These data show that mucosal vaccination can stimulate the induction of TRM cells, which should be considered as a more effective means to protect against brucellosis. Furthermore, brucellosis needs to be considered a mucosal pathogen to warrant development of approaches different from conventional methods to protect humans and livestock against this disease.

The HIV latent reservoir exhibits slow decay on antiretroviral therapy (ART), impacted by homeostatic proliferation and activation. How these processes contribute to the total dynamic while also producing the observed profile of sampled latent clone sizes is unclear. An agent-based model was developed that tracks individual latent clones, incorporating homeostatic proliferation of cells and activation of clones. The model was calibrated to produce observed latent reservoir dynamics as well as observed clonal size profiles. Simulations were compared to previously published latent HIV integration data from 5 adults and 3 children. The model simulations reproduced reservoir dynamics as well as generating residual plasma viremia levels (pVL) consistent with observations on ART. Over 382 Latin Hypercube Sample simulations, the median latent reservoir grew by only 0.3 log10 over the 10 years prior to ART initiation, after which time it decreased with a half-life of 15 years, despite number of clones decreasing at a faster rate. Activation produced a maximum size of genetically intact clones of around one million cells. The individual simulation that best reproduced the sampled clone profile, produced a reservoir that decayed with a 13.9 year half-life and where pVL, produced mainly from proliferation, decayed with a half-life of 10.8 years. These slow decay rates were achieved with mean cell life-spans of only 14.2 months, due to expansion of the reservoir through proliferation and activation. Although the reservoir decayed on ART, a number of clones increased in size more than 4,000-fold. While small sampled clones may have expanded through proliferation, the large sizes exclusively arose from activation. Simulations where homeostatic proliferation contributed more to pVL than activation, produced pVL that was less variable over time and exhibited fewer viral blips. While homeostatic proliferation adds to the latent reservoir, activation can both add and remove latent cells. Latent activation can produce large clones, where these may have been seeded much earlier than when first sampled. Elimination of the reservoir is complicated by expanding clones whose dynamic differ considerably to that of the entire reservoir., Author summary The HIV latent reservoir decreases slowly on antiretroviral therapy (ART). However there are cellular processes operating within this reservoir that can expand or contract subpopulations. This means that what is happening at the macro level may not be reflected at the micro level. To investigate this, we analysed published data on HIV latent clone sizes. By constructing an agent model incorporating the processes of cellular activation and proliferation, we were able to show that activation can expand clone sizes significantly even while on ART. Homeostatic proliferation also plays a role in maintaining the reservoir but these clones, though more frequent, are much smaller in size. Our calculations also show that activation and proliferation of the intact latent reservoir can lead to some of these cells becoming virally productive to a level consistent with observed residual viremia during ART. This analysis explains how normal cellular processes restructure the make-up of the latent reservoir and contribute to residual viremia.

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to “bumpy” surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become “bumpy”. These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus., Author summary DENV2 particles have been shown to change their morphologies (compact smooth to loose bumpy surfaced) when temperature is switched from 28°C to 37°C. We used two DENV2 viruses both belonging to the same strain designation but with a different passage history—one of which exhibited the smooth surfaced morphology while the other was bumpy surfaced, observed by cryoEM. We mutated residues in the E protein of the DENV2 infectious clone that has the smooth surfaced morphology to determine if any could result in a bumpy morphology. Results showed several different mutations could lead to this change. Using molecular dynamics simulations, we showed how these mutations likely destabilize the E protein dimeric interactions. We investigated whether the bumpy morphology also occurs in DENV2 clinical isolates, and showed that these viruses can exhibit both morphologies, indicating that vaccine and therapeutics development should target both virus forms.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition., Author summary Control of primary pathogens that infect phagocytes often requires adaptive immunity, but the mechanisms that convert host cells from permissive to antimicrobial states are only partially understood. The intracellular fungal pathogen Histoplasma capsulatum resides and proliferates within the macrophage phagosome. During innate immunity, macrophages which normally control fungi prove ineffective against H. capsulatum yeasts. At this stage, the phagosome of unactivated macrophages has ample copper that facilitates intracellular growth of Histoplasma but does not cause copper toxicity. However, the onset of adaptive immunity and the subsequent activation of macrophages decreases phagosomal copper and macrophages become less permissive to Histoplasma proliferation. IFN-γ acts as a key cytokine for switching the macrophage strategy by changing phagosomes from a copper-sufficient to a copper-depleted state in order to control intracellular pathogens. In such activated macrophages, H. capsulatum yeasts upregulate expression of the Ctr3 copper transporter to enable continued acquisition of essential copper.

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes., Author summary HIV-1 Env glycoprotein is produced by a precursor gp160 that has a signal peptide at its N-terminus. The SP is highly diverse among the HIV-1 isolates. This study presents site-specific analyses of N-linked glycosylation on HIV-1 envelope glycoproteins from infectious viruses produced with different envelope signal peptides. We show that signal peptide swapping alters the envelope glycan shield, including the conserved N156 and N160 glycans located in the V1V2 region on the trimer apex, to impact Env recognition and virus neutralization by antibodies. The data offer crucial insights into the role of signal peptide in the interplay between HIV-1 and antibodies and its potential utility to control Env glycosylation in the development of Env-based HIV-1 vaccine.

Helminth infection and dietary intake can affect the intestinal microbiota, as well as the immune system. Here we analyzed the relationship between fecal microbiota and blood profiles of indigenous Malaysians, referred to locally as Orang Asli, in comparison to urban participants from the capital city of Malaysia, Kuala Lumpur. We found that helminth infections had a larger effect on gut microbial composition than did dietary intake or blood profiles. Trichuris trichiura infection intensity also had the strongest association with blood transcriptional profiles. By characterizing paired longitudinal samples collected before and after deworming treatment, we determined that changes in serum zinc and iron levels among the Orang Asli were driven by changes in helminth infection status, independent of dietary metal intake. Serum zinc and iron levels were associated with changes in the abundance of several microbial taxa. Hence, there is considerable interplay between helminths, micronutrients and the microbiota on the regulation of immune responses in humans., Author summary Parasitic intestinal worms and gut bacteria occupy the same space, but we do not understand the nature and scope of their interaction. This is further complicated by dietary effects on the gut bacteria, as well as the immune responses of the host. To better understand these complex interactions, we compared individuals living in indigenous communities in Malaysia, where worm infections are common, with people living in the capital of Malaysia, who were not infected with worms. Data collected included burden of infection, a dietary survey, clinical tests, RNA profiles on blood samples and gut bacteria composition. By collecting data before and after treating the indigenous Malaysians with deworming medication, we could determine what was associated with changes in worm burden following deworming. We found that worm infection had a larger effect on gut bacteria composition than did dietary intake or blood profiles. Worm burden also had the strongest association with blood RNA profiles. We found that zinc and iron levels in the blood were associated with changes in helminth infection status, independent of dietary metal intake. Our results suggest that there is considerable interplay between intestinal worms and gut bacteria with zinc and iron levels in infected people.

To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This operon encodes a predicted transcriptional regulator (ClnR) and an ABC transporter system (ClnAB), all of which are required for function. Analyses of a clnR mutant indicate that ClnR is a pleiotropic regulator that directly binds to LL-37 and controls expression of numerous genes, including many involved in metabolism, cellular transport, signaling, gene regulation, and pathogenesis. The data suggest that ClnRAB is a novel regulatory mechanism that senses LL-37 as a host signal and regulates gene expression to adapt to the host intestinal environment during infection., Author summary C. difficile is a major nosocomial pathogen that causes severe diarrheal disease. Though C. difficile is known to inhabit the human gastrointestinal tract, the mechanisms that allow this pathogen to adapt to the intestine and survive host defenses are not known. In this work, we investigated the response of C. difficile to the host defense peptide, LL-37, to determine the mechanisms underlying host adaptation and survival. Expression analyses revealed a previously unknown locus, which we named clnRAB, that is highly induced by LL-37 and acts as a global regulator of gene expression in C. difficile. Mutant analyses indicate that ClnRAB is a novel regulatory system that senses LL-37 as a host signal to regulate adaptation to the intestinal environment.

Regulation of capsid disassembly is crucial for efficient HIV-1 cDNA synthesis after entry, yet host factors involved in this process remain largely unknown. Here, we employ genetic screening of human T-cells to identify maternal embryonic leucine zipper kinase (MELK) as a host factor required for optimal uncoating of the HIV-1 core to promote viral cDNA synthesis. Depletion of MELK inhibited HIV-1 cDNA synthesis with a concomitant delay of capsid disassembly. MELK phosphorylated Ser-149 of the capsid in the multimerized HIV-1 core, and a mutant virus carrying a phosphorylation-mimetic amino-acid substitution of Ser-149 underwent premature capsid disassembly and earlier HIV-1 cDNA synthesis, and eventually failed to enter the nucleus. Moreover, a small-molecule MELK inhibitor reduced the efficiency of HIV-1 replication in peripheral blood mononuclear cells in a dose-dependent manner. These results reveal a previously unrecognized mechanism of HIV-1 capsid disassembly and implicate MELK as a potential target for anti-HIV therapy., Author summary Phosphorylation of the HIV-1 capsid has long been known to regulate viral uncoating and cDNA synthesis processes, but the cellular kinases responsible for this have remained unidentified. Here, we report that a host cell kinase MELK dictates optimal capsid disassembly through phosphorylation of Ser-149 in the multimerized HIV-1 core, which leads to efficient viral cDNA synthesis in target cells. The phosphorylation-mimetic capsid mutation of Ser-149 caused aberrant capsid disassembly and too-early completion of reverse transcription, and impeded nuclear entry of HIV-1 cDNA, suggesting the importance of well-ordered capsid disassembly in the early stages of viral replication. This discovery will facilitate understanding of the functional link among virus uncoating, reverse transcription and nuclear entry, and is expected to contribute to developing a novel strategy for AIDS therapy.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3ʹ-Cre). CHIKV-3ʹ-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3ʹ-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3ʹ-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease., Author summary Chikungunya virus (CHIKV) is spread by mosquitoes and causes severe joint and muscle pain. Approximately 30 to 60 percent of people infected with CHIKV continue to experience joint pain for months to years after the initial infection. However, the cause of this persistent joint pain is unclear, as replicating virus cannot be detected during the chronic phase. We have developed a reporter virus system to permanently mark cells infected by CHIKV. Using this system, we show in mice that marked cells surviving CHIKV infection are present for at least 112 days after initial virus inoculation. The marked cells are a mixture of muscle and skin cells. We also show that treatment of mice with an antibody that blocks receptor engagement and CHIKV infection reduces the number of marked cells in the muscle and skin. Finally, we have demonstrated that surviving tdTomato+ cells contain most of the persistent CHIKV RNA. This reporter virus system represents a useful tool for identifying and isolating cells that harbor chronic viral RNA in order to study chronic disease pathogenesis.

Salmonella exploit host-derived nitrate for growth in the lumen of the inflamed intestine. The generation of host-derived nitrate is dependent on Nos2, which encodes inducible nitric oxide synthase (iNOS), an enzyme that catalyzes nitric oxide (NO) production. However, the cellular sources of iNOS and, therefore, NO-derived nitrate used by Salmonella for growth in the lumen of the inflamed intestine remain unidentified. Here, we show that iNOS-producing inflammatory monocytes infiltrate ceca of mice infected with Salmonella. In addition, we show that inactivation of type-three secretion system (T3SS)-1 and T3SS-2 renders Salmonella unable to induce CC- chemokine receptor-2- and CC-chemokine ligand-2-dependent inflammatory monocyte recruitment. Furthermore, we show that the severity of the pathology of Salmonella- induced colitis as well as the nitrate-dependent growth of Salmonella in the lumen of the inflamed intestine are reduced in mice that lack Ccr2 and, therefore, inflammatory monocytes in the tissues. Thus, inflammatory monocytes provide a niche for Salmonella expansion in the lumen of the inflamed intestine., Author summary Salmonella exploit gut inflammation to edge out competing microbes in the intestinal lumen and establish infection. Notably, Salmonella use inflammation-derived nitrate for growth in the intestinal lumen. The generation of inflammation-derived nitrate is dependent on Nos2, which encodes inducible nitric oxide synthase (iNOS), an enzyme that catalyzes nitric oxide production. However, the cellular sources of iNOS and, therefore, the nitric oxide-derived nitrate used by Salmonella for growth in the inflamed intestine remain unidentified, presenting an important, long-standing open question. Here, we show that iNOS-producing inflammatory monocytes, which are phagocyte precursors that play a critical role in immunity and host defense, promote nitrate-dependent Salmonella expansion in the inflamed intestine, providing new insights into how Salmonella exploit gut inflammation to establish infection.

The sexually transmitted bacterium Neisseria gonorrhoeae has developed resistance to all antibiotic classes that have been used for treatment and strains resistant to multiple antibiotic classes have evolved. In many countries, there is only one antibiotic remaining for empirical N. gonorrhoeae treatment, and antibiotic management to counteract resistance spread is urgently needed. Understanding dynamics and drivers of resistance spread can provide an improved rationale for antibiotic management. In our study, we first used antibiotic resistance surveillance data to estimate the rates at which antibiotic-resistant N. gonorrhoeae spread in two host populations, heterosexual men (HetM) and men who have sex with men (MSM). We found higher rates of spread for MSM (0.86 to 2.38 y−1, mean doubling time: 6 months) compared to HetM (0.24 to 0.86 y−1, mean doubling time: 16 months). We then developed a dynamic transmission model to reproduce the observed dynamics of N. gonorrhoeae transmission in populations of heterosexual men and women (HMW) and MSM. We parameterized the model using sexual behavior data and calibrated it to N. gonorrhoeae prevalence and incidence data. In the model, antibiotic-resistant N. gonorrhoeae spread with a median rate of 0.88 y−1 in HMW and 3.12 y−1 in MSM. These rates correspond to median doubling times of 9 (HMW) and 3 (MSM) months. Assuming no fitness costs, the model shows the difference in the host population’s treatment rate rather than the difference in the number of sexual partners explains the differential spread of resistance. As higher treatment rates result in faster spread of antibiotic resistance, treatment recommendations for N. gonorrhoeae should carefully balance prevention of infection and avoidance of resistance spread., Author Summary More and more infectious disease treatments fail because the causative pathogens are resistant to the drugs used for treatment. For the treatment of Neisseria gonorrhoeae, a sexually transmitted bacterium, drug resistance is a particularly big problem: there is only a single antibiotic left that is recommended for treatment. We aimed to understand how antibiotic-resistant N. gonorrhoeae spread in a sexually active host population and how the spread of resistance can be slowed. From antibiotic resistance surveillance data, we first estimated the rate at which antibiotic-resistant N. gonorrhoeae spread. Second, we reproduced the observed dynamics in a mathematical model describing the transmission between hosts. We found that antibiotic-resistant N. gonorrhoeae spread faster in host populations of men who have sex with men than in host populations of heterosexuals. We could attribute the faster spread of resistant pathogens to higher treatment rates. This finding implies that promoting screening to control antibiotic-resistant N. gonorrhoeae could in fact accelerate their spread.

Most individuals exposed to hepatitis C virus (HCV) become persistently infected while a minority spontaneously eliminate the virus. Although early immune events influence infection outcome, the cellular composition, molecular effectors, and timeframe of the host response active shortly after viral exposure remain incompletely understood. Employing specimens collected from people who inject drugs (PWID) with high risk of HCV exposure, we utilized RNA-Seq and blood transcriptome module (BTM) analysis to characterize immune function in peripheral blood mononuclear cells (PBMC) before, during, and after acute HCV infection resulting in spontaneous resolution. Our results provide a detailed description of innate immune programs active in peripheral blood during acute HCV infection, which include prominent type I interferon and inflammatory signatures. Innate immune gene expression rapidly returns to pre-infection levels upon viral clearance. Comparative analyses using peripheral blood gene expression profiles from other viral and vaccine studies demonstrate similarities in the immune responses to acute HCV and flaviviruses. Of note, both acute dengue virus (DENV) infection and acute HCV infection elicit similar innate antiviral signatures. However, while transient in DENV infection, this signature was sustained for many weeks in the response to HCV. These results represent the first longitudinal transcriptomic characterization of human immune function in PBMC during acute HCV infection and identify several dynamically regulated features of the complex response to natural HCV exposure., Author summary Hepatitis C virus (HCV) is a leading cause of liver disease, with an estimated 71 million people infected worldwide. Following exposure, a subset of individuals spontaneously clears the virus while a majority progress to chronic infection. The immune functions active during the initial, acute infection by HCV are not well understood, due in part to difficulties associated with studying the early stages of this disease in humans. Most individuals acutely infected with HCV are asymptomatic and do not initially seek medical care, which presents challenges in conducting prospective longitudinal studies. Here, by employing specimens from a rare research cohort of individuals sampled before infection, during infection, and after spontaneous viral clearance, we use RNA-Seq to characterize the early immune processes active in acute HCV. Our analysis identified a robust innate antiviral gene signature that corresponds with HCV viremia. In addition, comparisons to other immune transcriptomics datasets demonstrated that the immune response to acute HCV shares many features with responses to flaviviruses. These results offer a detailed longitudinal description of immune function active during the spontaneous resolution of acute HCV infection and provide insight into the early events that may contribute to viral clearance.

Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities., Author summary Chikungunya virus (CHIKV) and Ross River virus are mosquito-borne alphaviruses that cause epidemics of human arthritic disease that usually last from weeks to months. Arthropathy predominantly manifests in the joints of limbs and the joints at the extremities (e.g. hand and feet). Herein we show a surprisingly large reduction in viral loads and foot arthropathy in mice when animals were housed at 30°C rather than the conventional 22°C, with the feet of the former mice being ≈3–4°C warmer. Using RNA-Seq analyses of mice feet, we illustrate that this small increase in temperature results in a significant increase in both the unfolded protein response and anti-viral type I interferon responses. Taken together these results suggest that the predominantly peripheral alphaviral arthropathy is due to the usually slightly lower temperature of limbs and extremities, which results in less effective type I interferon responses and a subsequent increase in viral loads and ensuing arthritic disease.

Our ability to respond appropriately to infectious diseases is enhanced by identifying differences in the potential for transmitting infection between individuals. Here, we identify epidemiological traits of self-limited infections (i.e. infections with an effective reproduction number satisfying ) that correlate with transmissibility. Our analysis is based on a branching process model that permits statistical comparison of both the strength and heterogeneity of transmission for two distinct types of cases. Our approach provides insight into a variety of scenarios, including the transmission of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the Arabian peninsula, measles in North America, pre-eradication smallpox in Europe, and human monkeypox in the Democratic Republic of the Congo. When applied to chain size data for MERS-CoV transmission before 2014, our method indicates that despite an apparent trend towards improved control, there is not enough statistical evidence to indicate that has declined with time. Meanwhile, chain size data for measles in the United States and Canada reveal statistically significant geographic variation in , suggesting that the timing and coverage of national vaccination programs, as well as contact tracing procedures, may shape the size distribution of observed infection clusters. Infection source data for smallpox suggests that primary cases transmitted more than secondary cases, and provides a quantitative assessment of the effectiveness of control interventions. Human monkeypox, on the other hand, does not show evidence of differential transmission between animals in contact with humans, primary cases, or secondary cases, which assuages the concern that social mixing can amplify transmission by secondary cases. Lastly, we evaluate surveillance requirements for detecting a change in the human-to-human transmission of monkeypox since the cessation of cross-protective smallpox vaccination. Our studies lay the foundation for future investigations regarding how infection source, vaccination status or other putative transmissibility traits may affect self-limited transmission., Author Summary The goal of this paper is to identify epidemiological factors that correlate with either an increased or decreased risk of transmitting a particular disease. We are particularly interested in identifying such factors for diseases that are self-limited (meaning that infections tend to occur in isolated clusters), because targeted control of these diseases can facilitate public health goals for minimizing the risk of disease emergence or promoting disease elimination. For example, we show that there is a significant difference in the transmission of measles between the United States and Canada. In contrast, we find that an observed decrease in the transmission of Middle East respiratory syndrome coronavirus during the latter half of 2013 cannot be ascertained with sufficient confidence. We then quantify the degree to which control was effective in eradicating smallpox in Europe. We also consider how the transmission of monkeypox in humans depends on whether the infection source is an animal or a human. Finally, we demonstrate how our approach can be used by surveillance programs to detect changes in transmission that may occur over time.

Hill et al. provide a critique[1] of our recent paper, in which we developed two novel methods for estimating the rate of reactivation from latency directly from existing clinical data [2]. Our goal was to use minimal assumptions and be guided by the data. We found that across four cohorts, the best-fit frequency of HIV reactivation from latency was once every 5–8 days. Hill et al.’s modeling demonstrates an alternative approach, in which they start with a fixed rate of reactivation (four events per day, derived from modeling of time to drug resistance under therapy [3,4]) and then adjust the model and other parameters to fit their fixed reactivation rate. Importantly, amongst the various fitting described by Hill et al., they never actually fit the reactivation rate, instead always fixing it at their preferred value and adjusting the model and fitting other parameters around it. Hill et al. state that the data can be fit with “modest variation” of the reactivation rate. However, the parameters used require that ≈8% of patients reactivate less than once every 6 days (the mean rate we estimated in [2]) and, similarly, ≈8% reactivate more than 100 times per day (see S1 Methods). Below, we show that once the reactivation rate is fitted to all datasets, the results strongly support that the median frequency of HIV reactivation from latency is once every 5–8 days. The first criticism by Hill et al. is that we did not incorporate multiple reactivation events or a distribution of reactivation rates into our model. They use simulation to incorporate such a distribution because of “lack [of] an analytical expression for the model” of multiple reactivation events. In response, we now derive an analytical approximation incorporating multiple reactivation events and apply it to all four available datasets (see S1 Methods). In each case, we fitted reactivation rate and initial viral load (V0), plus the distribution in reactivation rate. Because viral growth is a measureable parameter, we fixed this in the model, firstly using Hill et al.’s chosen viral growth rate (0.4/day) and then using a more realistic value obtained directly from the data (0.8/day) (see S1 Methods). Fig 1 shows fitting to all four datasets of (i) our original two-parameter model (panels A–D), (ii) the model with multiple reactivation events using Hill et al.’s preferred low growth rate (E–H), and (iii) a model with a more realistic growth rate (I–L). We note that only in 1 out of 12 fits (cohort 3 with a low growth rate, panel G, Fig 2) did we obtain a reactivation rate more frequent than once every 2 days. In all cases with realistic growth rates, the estimated rate of HIV reactivation from latency was very similar to our original model (once every 5–8 days) even once a distribution was included (Fig 1I–1L; Fig 2). Thus, the results presented by Hill et al. are highly dependent on the choice of a low viral growth rate and require an extremely wide distribution in reactivation rates. Fig 1 Estimating HIV reactivation rate using different models. Fig 2 Frequency of HIV reactivation from latency estimated using different models: The mean frequency of reactivation estimated using the original model (red circles, corresponding to panels A–D of Fig 1), Hill’s model with slow growth rate ... Hill et al. raise additional arguments about distributions in viral growth rate. In our previous publication, we found no significant correlation between growth rate and time-to-detection. We agree with Hill et al. that there is always a risk of a type II error in such analyses. However, the simulation performed by Hill et al. significantly misrepresents the probability of this error. Stating they observed no significant correlation (p > 0.05) in 50% of simulations grossly overstates the probability of observing the correlation seen in the data. The correlation between growth rate and time-to-detection in cohort 3 was in fact positive (r = 0.029, p = 0.9) despite an expected negative correlation. Thus, it is more appropriate to ask either, “In what proportion of simulations did we see an r value of 0.029 or greater?” or, “In what proportion of simulations was the p value greater than 0.9?” Using the parameters suggested in Hill et al.’s Fig 1D [1], we find that less than 2% of simulations were as poorly correlated as the observed data. Thus, we maintain our view that although growth rate must affect time-to-detection, based on the data this contribution is likely small. Hill et al.’s primary argument is that there is strong prior evidence of high reactivation rates, and they cite four papers in support (references 1, 3–5 in their comment). None of the papers they cite in support of their reactivation rate of four-per-day actually analyses data on “reactivation from latency after treatment interruption” (the title of our earlier publication [2]). Two of these papers [3,5] base their estimates on time-to-drug-resistance under therapy, using a wide variety of assumptions on HIV replication under therapy, mutation rates, and drug selection. The third paper [6] measures T cell activation, not latent cell reactivation, so it does not provide an estimate. The fourth estimate comes from Hill et al.’s own previous work[4] but is derived directly from the estimate of reference [3] (see S1 Methods). Thus, the evidence for a reactivation rate of 4 per day hinges upon reference [3]’s use of a 13-parameter model of time to drug resistance, in which many of the input parameters are described as “roughly based on the literature”[3]. Hill et al. also argue that reactivation must be high because of a lack of correlation between reservoir size and rebound time in the literature. This appears to ignore recent publications showing such a correlation [7–9] as well as the apparent wide variation between different measures of reservoir size from peripheral blood [10]. Hill et al. have gone to some lengths to try to show that their previous estimate of HIV reactivation of 4 times a day could be made compatible with the data from the cohorts presented in Pinkevych et al. However, the failure to actually fit the key parameter significantly undermines these arguments. Once rigorous quantitative fitting is applied, the parameters suggested by Hill et al. do not provide the best fit to the data. Indeed, the additional analyses undertaken herein provide strong support for our previous estimate that HIV reactivates from latency approximately once every 5–8 days. The derivation of the analytical approximation and a detailed explanation of the modeling are provided in S1 Methods because of word limits on this reply.

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries., Author summary Shigella sonnei is predominantly responsible for dysentery in developed countries, and is replacing Shigella flexneri in areas undergoing economic development and improvements in water quality. Using Shigella infection of zebrafish (in vivo) and human neutrophils (in vitro), we discover that S. sonnei is more virulent than S. flexneri because of neutrophil tolerance mediated by its O-antigen oligosaccharide acquired from the environmental bacteria Plesiomonas shigelloides. To inspire new approaches for S. sonnei control, we show that increased phagolysosomal acidification or innate immune training can promote S. sonnei clearance by neutrophils in vivo. These findings have major implications for our evolutionary understanding of Shigella, and may explain why exposure to P. shigelloides in low and middle-income countries (LMICs) can protect against dysentery incidence.