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The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier., Author Summary Previous studies of HIV transmission have yielded conflicting results regarding the genetic heterogeneity of the virus establishing infection in the newly infected individual. In this study of populations from Zambia and Rwanda that are infected by two distinct viral genetic subtypes, we compared viral sequences that encode the entry-mediating envelope glycoproteins from newly infected individuals (recipients) and their spouses (donors) very early after infection, as well as newly infected individuals infected by someone other than their spouse. In spite of the genetically diverse virus population in the donor, approximately 90% of newly infected individuals were infected by a single viral variant, while the rest were infected by multiple viral variants. The homogeneity of the virus population in the newly infected recipient, as well as the presence, in some cases, of identical virus variants in the donor, allowed us to precisely identify the transmitted variant. We were able to examine the clinical history of each newly infected individual and observed that all individuals infected by multiple variants also showed evidence of inflammatory genital infections. Our results suggest that the genital mucosa provides a natural barrier to infection by multiple genetic variants of HIV-1, but that this barrier can be lowered by inflammatory genital infections.

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller's ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study., Author Summary The ability of arboviruses to perpetuate in nature given that they must infect two disparate hosts (the mosquito vector and the vertebrate host) remains a mystery. We studied how viral genetic diversity is impacted by the dual host transmission cycle. Our studies of an enzootic cycle using Venezuelan equine encephalitis virus (VEEV) and its natural mosquito, Culex taeniopus, revealed the stages of infection that result in a viral population bottleneck. Using a set of marked VEEV clones and repeated sampling at various time points following C. taeniopus infection, we determined the number of clones in various mosquito tissues culminating in transmission. Bottlenecks were identified but the stage of occurrence was dependent on the dose that initiated infection. Understanding the points at which mosquito-borne viruses are constrained will shed light on the ways in which virus diversity varies, leading to selection of mutants that may result in host range changes or alterations in virulence.

The 3′ untranslated genome region (UTR) of arthropod-borne viruses is characterized by enriched direct repeats (DRs) and stem-loop structures. Despite many years of theoretical and experimental study, on-going positive selection on the 3′UTR had never been observed in ‘real-time,’ and the role of the arbovirus 3′UTR remains poorly understood. We observed a lineage-specific 3′UTR sequence pattern in all available Asian lineage of the mosquito-borne alphavirus, chikungunya virus (CHIKV) (1958–2009), including complicated mutation and duplication patterns of the long DRs. Given that a longer genome is usually associated with less efficient replication, we hypothesized that the fixation of these genetic changes in the Asian lineage 3′UTR was due to their beneficial effects on adaptation to vectors or hosts. Using reverse genetic methods, we examined the functional importance of each direct repeat. Our results suggest that adaptation to mosquitoes, rather than to mammalian hosts, is a major evolutionary force on the CHIKV 3′UTR. Surprisingly, the Asian 3′UTR appeared to be inferior to its predicted ancestral sequence for replication in both mammals and mosquitoes, suggesting that its fixation in Asia was not a result of directional selection. Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia. We propose that this introduction of a 3′UTR with deletions led to genetic drift and compensatory mutations associated with the loss of structural/functional constraints, followed by two independent beneficial duplications and fixation due to positive selection. Our results provide further evidence that the limited epidemic potential of the Asian CHIKV strains resulted from founder effects that reduced its fitness for efficient transmission by mosquitoes there., Author Summary The 3′ untranslated genome region (UTR) of arbovirus (arthropod-borne virus) RNA genomes is characterized by enriched direct repeats (DRs) and stem-loop structures, which are thought to play a specific role in maintaining efficient transmission in multiple hosts. However, this hypothesis is vague and has little experimental support. Based on our observation of a distinct, lineage-specific sequence structure in the chikungunya virus (CHIKV) Asian lineage 3′UTR, we tried to understand the underlying driving forces on the arboviral 3′UTR evolution. Specifically, we sought to determine whether the dramatic genetic changes in the Asian lineage 3′UTR were the result of adaptation into the new host or vector populations. Using reverse genetic methods, we obtained results that suggested that the DRs have a significant effect on the viral fitness level in mosquitoes but not in mammals. Interestingly, instead of a directional selection from its ancestral state, our results suggest that the evolution of the CHIKV Asian lineage 3′UTR involved a population bottleneck with a deleterious deletion of DRs, followed by accumulation of mutations due to the loss of structural/functional constraints. Later, the duplication of two regions that are beneficial in mosquitoes led to the fixation of this 3′UTR sequence in Asian lineage, possibly facilitating the 1958 Thailand outbreak.

The pathogenesis of bacteraemia after challenge with one million pneumococci of three isogenic variants was investigated. Sequential analyses of blood samples indicated that most episodes of bacteraemia were monoclonal events providing compelling evidence for a single bacterial cell bottleneck at the origin of invasive disease. With respect to host determinants, results identified novel properties of splenic macrophages and a role for neutrophils in early clearance of pneumococci. Concerning microbial factors, whole genome sequencing provided genetic evidence for the clonal origin of the bacteraemia and identified SNPs in distinct sub-units of F0/F1 ATPase in the majority of the ex vivo isolates. When compared to parental organisms of the inoculum, ex-vivo pneumococci with mutant alleles of the F0/F1 ATPase had acquired the capacity to grow at low pH at the cost of the capacity to grow at high pH. Although founded by a single cell, the genotypes of pneumococci in septicaemic mice indicate strong selective pressure for fitness, emphasising the within-host complexity of the pathogenesis of invasive disease., Author Summary Decades of research on bacterial sepsis have been devoted to analysing the steps that lead from a local event, either carriage or a localised infection, to systemic disease. Our work analyses in depth the events determining systemic infection by one of the main human pathogens, Streptococcus pneumoniae. Consistent with similar findings on the pathogenesis of bacteraemia due to other commensal pathogens, our results show that after an intravenous inoculum of a million pneumococci, the resulting septicaemia is often founded by a single bacterial cell. Investigation into the nature of this monoclonal infection identified strong within-host selective pressure for metabolic fitness during outgrowth of the bacterial population.

Antibiotic treatment has emerged as a promising strategy to sterilize and kill filarial nematodes due to their dependence on their endosymbiotic bacteria, Wolbachia. Several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity. However, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. Concerns about resistance arising from mass drug administration necessitate a careful exploration of potential Wolbachia recrudescence. In the present study, we investigated the long-term effects of the FDA-approved antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Genome sequence analyses of Wolbachia revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of densely packed Wolbachia within the worm’s ovarian tissues were observed by confocal microscopy and remained in worms treated with rifampicin, suggesting that they may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. To our knowledge, these clusters have not been previously described and may be the source of the Wolbachia rebound., Author summary Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are two neglected tropical diseases caused by filarial nematodes, which harbor the endosymbiotic bacteria, Wolbachia. Major efforts to discover new drugs to treat these diseases have led to the discovery of novel compounds including those that target Wolbachia. We investigated the long-term effects of rifampicin on the filarial nematode, Brugia pahangi, and its endosymbiont, Wolbachia, in an in vivo rodent model of infection. Initially, Wolbachia titers were significantly reduced by 95% and female fecundity was impaired shortly after treatment. 8 months later however, Wolbachia rebounded and embryogenesis returned to normal. Sequence analysis of the Wolbachia genome revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of Wolbachia were observed within the ovaries of female worms throughout the entire 8-month study. These clusters may sequester Wolbachia and allow the bacteria to persist during antibiotic treatment, thereby enabling them to repopulate ovarian tissues and ensuring their vertical transmission to future generations of microfilariae.

Plasmodium sporozoites are the infective stage of the malaria parasite. Though this is a bottleneck for the parasite, the quantitative dynamics of transmission, from mosquito inoculation of sporozoites to patent blood-stage infection in the mammalian host, are poorly understood. Here we utilize a rodent model to determine the probability of malaria infection after infectious mosquito bite, and consider the impact of mosquito parasite load, blood-meal acquisition, probe-time, and probe location, on infection probability. We found that infection likelihood correlates with mosquito sporozoite load and, to a lesser degree, the duration of probing, and is not dependent upon the mosquito’s ability to find blood. The relationship between sporozoite load and infection probability is non-linear and can be described by a set of models that include a threshold, with mosquitoes harboring over 10,000 salivary gland sporozoites being significantly more likely to initiate a malaria infection. Overall, our data suggest that the small subset of highly infected mosquitoes may contribute disproportionally to malaria transmission in the field and that quantifying mosquito sporozoite loads could aid in predicting the force of infection in different transmission settings., Author summary Malaria is a leading cause of death in many parts of the world. Infection is initiated when infected Anopheles mosquitoes inject sporozoites as they look for blood. Though transmission is a bottleneck for the parasite and thus a good point for intervention, many aspects of transmission remain poorly understood. In this study, using a rodent model of malaria, we found that the majority of infective bites do not result in malaria infection. Furthermore, we found that the bites of mosquitoes with heavy parasite burdens are significantly more likely to result in blood stage infection. These data have important implications for designing interventions targeting transmission stages of the malaria parasite as they suggest that reducing parasite loads, even without completely eliminating them, could be effective against disease spread. We also found that mosquitoes that probe but do not succeed in finding blood are equally likely to initiate infection, an important finding for human vaccine trials. Overall this work contributes to our understanding of the epidemiology of malaria and should aid in the development of malaria elimination strategies.

HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants’ samples was sequenced close to transmission (median 21 days post infection, IQR 18–41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF., Author summary The development of a safe and effective HIV-1 vaccine alongside cure strategies is a major public health concern and requires in-depth knowledge of the HIV-1 populations that establish infection. Here we sequenced the HIV genetic populations present during the acute stage of HIV-1 infection in 38 Men who have sex with men (MSM) and identified the transmitted/founder variant’s sequence. We then cloned the gag gene from each patient’s transmitted/founder gag sequences in both single and multiple variant infection into a common, lab-adapted virus and measured the replicative capacity it conferred on this virus. Finally, cellular immune responses were compared between single variant and multiple variant infection at 0–3, 6–9 and 24–30 months after infection. We observed a higher frequency of multivariant infection in MSM than has been previously described in heterosexually infected participants, and this was associated with faster decline of CD4+ T cells and perturbances in the CD4+ T cell and the B cell compartment. Moreover, the replicative capacity conferred by gag was lower in multivariant infection, suggesting a less stringent transmission bottleneck that allowed for less fit variants to establish infection. These results suggest that strategies to tighten the stringency of the transmission bottleneck may be of benefit to patients by reducing the likelihood of multivariant transmission and potentially slowing down disease progression.

In the majority of cases, human immunodeficiency virus type 1 (HIV-1) infection is transmitted through sexual intercourse. A single founder virus in the blood of the newly infected donor emerges from a genetic bottleneck, while in rarer instances multiple viruses are responsible for systemic infection. We sought to characterize the sequence diversity at early infection, between two distinct anatomical sites; the female reproductive tract vs. systemic compartment. We recruited 72 women from Uganda and Zimbabwe within seven months of HIV-1 infection. Using next generation deep sequencing, we analyzed the total genetic diversity within the C2-V3-C3 envelope region of HIV-1 isolated from the female genital tract at early infection and compared this to the diversity of HIV-1 in plasma. We then compared intra-patient viral diversity in matched cervical and blood samples with three or seven months post infection. Genetic analysis of the C2-V3-C3 region of HIV-1 env revealed that early HIV-1 isolates within blood displayed a more homogeneous genotype (mean 1.67 clones, range 1–5 clones) than clones in the female genital tract (mean 5.7 clones, range 3–10 clones) (p, Author summary During chronic HIV-1 infection, high viral diversity can be found in the blood and semen of donors. However, a single HIV-1 clone establishes productive infection in the recipient following heterosexual transmission. To investigate the genetic bottleneck occurring at the earliest stages of heterosexual HIV-1 transmission, we characterized the HIV-1 envelope sequence diversity at very early and early stages of infection in the female reproductive tract and matched plasma samples from a cohort of Ugandan and Zimbabwean women. A more diverse viral population was observed in the endocervical swab samples compared to plasma. Endocervical samples harbored a larger number of viral clones, while in the majority of plasma samples only a single clone was present early in infection. Interestingly, these observations were independent of HIV-1 subtype, hormonal contraceptive use or the number of sex acts and partners. Furthermore, in the cases of higher HIV-1 diversity in the blood during early infection, faster CD4 T cell decline were observed during chronic disease suggesting faster disease progression. Our findings provide novel in vivo evidence for the existence of an intra-patient genetic bottleneck restricting the HIV-1 from the vaginal tract to the blood during early heterosexual HIV-1 transmission.

Herein, we studied a virulent isolate of the leading bacterial pathogen Streptococcus pneumoniae in an infant mouse model of colonization, disease and transmission, both with and without influenza A (IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion., Author Summary Many discrete steps are involved in the progression of infectious diseases. Bottlenecks represent key points where the population size/genetic diversity is at a minimum and the pathogen is most vulnerable to intervention strategies. Our study used an infant mouse model for a comprehensive analysis of bottlenecks in infection by the major pathogen Streptococcus pneumoniae. In our model, we also considered influenza A virus, a clinically important and common co-infection. The main findings reveal i) a single cell bottleneck during host-to-host transmission and ii) the bottleneck in transmission occurs during events between bacterial exit from one host and establishment in another host. We manipulated innate immune responses involved in viral control and inflammation to show that viral co-infection allows the bottleneck in transmission to be overcome by increasing bacterial exit. Finally, we demonstrate that a specific host response stimulated by influenza A is sufficient to recapitulate effects of viral co-infection. Thus, our study identifies key vulnerable stages during S. pneumoniae infection and provides mechanistic understanding for how viral infection promotes bacterial contagion.

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection., Author Summary The earliest stage of any infection takes place when a pathogen enters the body (inoculation) at an initial site of contact. From this point, the pathogen can spread into deeper tissues where the pathogen itself and the immune responses against it cause disease. Very little is known about the events that follow inoculation and how pathogens move from the initial site of contact into deeper tissues. A better understanding of this process can potentially result in strategies to control or prevent disease. We studied the highly infectious bacterium that causes bubonic plague (Yersinia pestis) and how it spreads inside the body, from the skin into lymph nodes. We found that movement from the skin is highly restricted as only a small fraction of the bacteria that are deposited into this tissue are found in lymph nodes. While it is currently thought that Y. pestis spreads from the skin inside trafficking cells of the innate immune response, our work suggests these cells are not required for the bacteria to move into lymph nodes. Our findings can influence vaccine development efforts as these strategies are based on the study of early pathogen interactions with cells of the immune response.

Identification of cellular receptors and characterization of viral tropism in animal models have vastly improved our understanding of morbillivirus pathogenesis. However, specific aspects of viral entry, dissemination and transmission remain difficult to recapitulate in animal models. Here, we used three virologically identical but phenotypically distinct recombinant (r) canine distemper viruses (CDV) expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets (Mustela putorius furo). Six donor ferrets simultaneously received three rCDVs expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc), intra-nasal (IN) or intra-tracheal (IT) inoculation. Two days post-inoculation sentinel ferrets were placed in physically separated adjacent cages to assess airborne transmission. All donor ferrets developed lymphopenia, fever and lethargy, showed progressively increasing systemic viral loads and were euthanized 14 to 16 days post-inoculation. Systemic replication of virus inoculated via the Oc, IN and IT routes was detected in 2/6, 5/6 and 6/6 ferrets, respectively. In five donor ferrets the IT delivered virus dominated, although replication of two or three different viruses was detected in 5/6 animals. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections. Transmission occurred efficiently and all recipient ferrets showed evidence of infection between 18 and 22 days post-inoculation of the donor ferrets. In all cases, airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in the donor ferret. This study demonstrates that morbilliviruses can use multiple entry routes in parallel, and co-infection of cells during viral dissemination in the host is common. Airborne transmission was efficient, although transmission of viruses expressing a single color suggested a bottleneck event. The identity of the transmitted virus was not determined by the site of inoculation but by the viral dominance during dissemination., Author summary Canine distemper virus (CDV) infection of ferrets is a tractable animal model for measles. Ferrets are highly susceptible to CDV, and inoculation with a low dose leads to lethal disease. We performed in vivo competition experiments to study virus entry, dissemination and transmission. Ferrets were simultaneously inoculated with CDV via the conjunctival, intra-nasal and intra-tracheal routes. The viruses were identical except for the fluorescent reporter protein encoded by the viral genome. By detecting cells expressing the different fluorescent reporter proteins at various sites in the host, we determined that CDV can enter the host in parallel at multiple sites. Virus spread in the ferret occurred via infected lymphocytes, which often turned out to be double- or triple-infected. Sentinel ferrets, placed in physically separated adjacent cages, became infected by airborne transmission. Transmission of the dominant single color despite replication of multicolor viruses in the upper respiratory tract suggested a bottleneck event.

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs., Author Summary Staphylococcus aureus is a major cause of human disease, made even more notable due to the spread of antibiotic resistance. We used a combination of animal models to study the spread of bacteria between organs during an infection and the resulting effect of antibiotic intervention. We found that S. aureus infection is highly clonal, following a “bottleneck” in which very few bacterial cells found each abscess. Despite previous in vitro research, the effect of antibiotics on S. aureus infection was poorly understood. We utilized our systemic infection models to study intervention with sub-curative antibiotic doses, such as one might encounter upon failing to complete an antibiotic course. We have shown that such doses are able to support the preferential expansion of antibiotic-resistant organisms during a mixed infection. This selection is due to the clonal pattern of infection, occurring despite a lack of effect on growth rate or on the spontaneous generation of resistance. Furthermore, it is generic to multiple pathogen species, including Pseudomonas aeruginosa, and antibiotic classes, such as with methicillin-resistant S. aureus (MRSA) in the presence of oxacillin. Given the current debate in the field, our results have important implications for the design of properly-controlled treatment regimes.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently., Author Summary Most new HIV-1 infections worldwide are caused by the sexual transmission of subtype C viruses, which are prevalent in Asia and southern Africa. While chronically infected individuals harbor a genetically diverse set of viruses, most new infections are established by single variants, termed transmitted/founder (T/F) viruses. This raises the question whether certain viral variants have particular properties allowing them to more efficiently overcome the transmission bottleneck. Preferential binding of the viral envelope (Env) to the integrin α4β7 has been hypothesized as one important feature of transmitted viruses. Here, we compared Envs from subtype C viruses that were transmitted to those that were prevalent in chronic infections for efficiency in utilizing α4β7, CD4 and CCR5 for cell entry and replication. We found that transmitted and chronic Envs engaged CD4 and CCR5 with equal efficiency, and that blocking the interaction between Env and α4β7 failed to inhibit replication of T/F as well as control viruses. While the search for determinants of transmission fitness remains an important goal, preferential CD4, CCR5 or α4β7 interactions do not appear to represent distinguishing features of T/F viruses.

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection., Author Summary Primary hepatitis C (HCV) infection is typically asymptomatic and commonly results in persistent infection. The characteristics of early infection remain undefined. Four subjects were studied longitudinally from within a few weeks of transmission until resolution of outcome, via a full genome analysis of viral evolution. In the acute phase (

Listeria monocytogenes is a facultative intracellular bacterial pathogen that can infect the placenta, a chimeric organ made of maternal and fetal cells. Extravillous trophoblasts (EVT) are specialized fetal cells that invade the uterine implantation site, where they come into direct contact with maternal cells. We have shown previously that EVT are the preferred site of initial placental infection. In this report, we infected primary human EVT with L. monocytogenes. EVT eliminated ∼80% of intracellular bacteria over 24-hours. Bacteria were unable to escape into the cytoplasm and remained confined to vacuolar compartments that became acidified and co-localized with LAMP1, consistent with bacterial degradation in lysosomes. In human placental organ cultures bacterial vacuolar escape rates differed between specific trophoblast subpopulations. The most invasive EVT—those that would be in direct contact with maternal cells in vivo—had lower escape rates than trophoblasts that were surrounded by fetal cells and tissues. Our results suggest that EVT present a bottleneck in the spread of L. monocytogenes from mother to fetus by inhibiting vacuolar escape, and thus intracellular bacterial growth. However, if L. monocytogenes is able to spread beyond EVT it can find a more hospitable environment. Our results elucidate a novel aspect of the maternal-fetal barrier., Author Summary Infection of the placenta and fetus is an important cause of pregnancy complications and fetal and neonatal morbidity and mortality. Listeria monocytogenes is an intracellular bacterial pathogen that causes pregnancy-related infections in humans. The pathogenesis of listeriosis during pregnancy is poorly understood. We have previously shown that transmission of L. monocytogenes from maternal cells and tissues to fetal cells occurs in the uterine implantation site, and that a small subpopulation of specialized fetal cells called extravillous trophoblasts are the preferred initial site of infection. Here we use primary human placental organ and cell culture systems to characterize the intracellular fate of L. monocytogenes in extravillous trophoblasts. We found that these cells entrap bacteria in vacuolar compartments where they are degraded and therefore reduce bacterial dissemination into deeper structures of the placenta. Our study provides new insights into the nature of the maternal-fetal barrier. Extravillous trophoblasts that are accessible to infection with intracellular pathogens from infected maternal cells have host defense mechanisms that constitute a bottleneck in maternal-fetal transmission.

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA’s c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that ‘ectopic’ production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete., Author summary Herein, we examined the roles of c-di-GMP and PlzA throughout the Borrelia burgdorferi lifecycle. Using a plzA point mutant that is unable to bind c-di-GMP, we confirmed that the protective function(s) of PlzA in feeding ticks and mammals are c-di-GMP-dependent and -independent, respectively. Contrary to previous studies, loss of PlzA did not affect expression of rpoS or RpoS-dependent genes, which are important for host-adaptation and persistence in the mammal. Host-adaptation prior to needle-inoculation of C3H mice or infection of scid mice substantially restored infectivity in the absence of PlzA. Using a Borrelia strain that synthesizes c-di-GMP constitutively, this secondary messenger was shown to be tick-phase specific and detrimental to host-adaptation via PlzA-mediated antagonism of RpoS function. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA provide insight into the dramatic conformational changes upon c-di-GMP binding that likely underlie its differential function: liganded-PlzA supports survival within feeding ticks, while unliganded-PlzA promotes an RpoS-independent facet of host adaptation, enabling spirochetes to evade early immune clearance by the host.

Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4+ T cells. Low-level viral replication occurs initially in mucosal CD4+ T cells, but within days high-level replication occurs in Peyer's patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α4β7 +/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin- α4β7. High-affinity for integrin α4β7, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α4β7 affinity is mediated by sequences encoded in gp120 V1/V2. α4β7-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α4β7 +/CD4+ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine., Author Summary In the first days following sexual transmission, HIV replication occurs initially at relatively low levels in mucosal tissues because of a paucity of CD4+ T cell targets for the virus to infect. After a period of days, virus accesses specific gut tissues that are enriched in activated CD4+ T cells, where near-exponential replication ensues. The period of time before HIV accesses gut tissues represents a window of opportunity where a microbicide, pre-exposure and/or post-exposure antiretroviral prophylaxis or a vaccine-induced immune response could block infection. We previously reported that the HIV envelope protein gp120 binds to integrin α4β7 on the surface of CD4+ T cells. α4β7 mediates the homing of CD4+ T cells into the gut tissues where HIV can replicate exponentially. Here we report that the genotypic features that distinguish viruses isolated within the first month after infection, termed early-transmitting isolates, promote increased steady-state reactivity with α4β7. This property likely provides these viruses with enhanced transmission-fitness. These results suggest that the infection of α4β7 +/CD4+ T cells can play an important role early in HIV transmission. These findings have potentially important implications in the design of interventions to block the mucosal transmission of HIV.

Virions are a common antigen source for many viral vaccines. One limitation to using virions is that the antigen abundance is determined by the content of each protein in the virus. This caveat especially applies to viral-based influenza vaccines where the low abundance of the neuraminidase (NA) surface antigen remains a bottleneck for improving the NA antibody response. Our systematic analysis using recent H1N1 vaccine antigens demonstrates that the NA to hemagglutinin (HA) ratio in virions can be improved by exchanging the viral backbone internal genes, especially the segment encoding the polymerase PB1 subunit. The purified inactivated virions with higher NA content show a more spherical morphology, a shift in the balance between the HA receptor binding and NA receptor release functions, and induce a better NA inhibitory antibody response in mice. These results indicate that influenza viruses support a range of ratios for a given NA and HA pair which can be used to produce viral-based influenza vaccines with higher NA content that can elicit more balanced neutralizing antibody responses to NA and HA., Author summary Influenza vaccines are produced on a large scale to meet the annual U.S. and global demand. To efficiently produce the required number of influenza vaccine doses, virions are commonly used as the antigen source due to their high viral protein content. A draw-back to using virions is that the final antigen composition of the vaccine is determined by the inherent properties of the vaccine virus. While this approach for influenza vaccines is beneficial for the more abundant HA antigen, it likely limits the protective response generated by the less abundant NA antigen. Our results demonstrate that the NA and HA content in vaccine viruses can be optimized by changing the internal genes of the vaccine virus, thereby preserving the surface antigens. The increase in the virion NA content that was achieved elicited higher NA antibody titres and generated more balanced neutralizing antibody responses to HA and NA. Since HA and NA neutralizing antibodies are both protective, this approach could help to improve the suboptimal efficacy of current influenza vaccines and to generate vaccines that provide broader coverage against circulating strains.

RNA viruses such as poliovirus have high mutation rates, and a diverse viral population is likely required for full virulence. We previously identified limitations on poliovirus spread after peripheral injection of mice expressing the human poliovirus receptor (PVR), and we hypothesized that the host interferon response may contribute to the viral bottlenecks. Here, we examined poliovirus population bottlenecks in PVR mice and in PVR mice that lack the interferon α/β receptor (PVR-IFNAR−/−), an important component of innate immunity. To monitor population dynamics, we developed a pool of ten marked polioviruses discriminated by a novel hybridization-based assay. Following intramuscular or intraperitoneal injection of the ten-virus pool, a major bottleneck was observed during transit to the brain in PVR mice, but was absent in PVR-IFNAR−/− mice, suggesting that the interferon response was a determinant of the peripheral site-to-brain bottleneck. Since poliovirus infects humans by the fecal–oral route, we tested whether bottlenecks exist after oral inoculation of PVR-IFNAR−/− mice. Despite the lack of a bottleneck following peripheral injection of PVR-IFNAR−/− mice, we identified major bottlenecks in orally inoculated animals, suggesting physical barriers may contribute to the oral bottlenecks. Interestingly, two of the three major bottlenecks we identified were partially overcome by pre-treating mice with dextran sulfate sodium, which damages the colonic epithelium. Overall, we found that viral trafficking from the gut to other body sites, including the CNS, is a very dynamic, stochastic process. We propose that multiple host barriers and the resulting limited poliovirus population diversity may help explain the rare occurrence of viral CNS invasion and paralytic poliomyelitis. These natural host barriers are likely to play a role in limiting the spread of many microbes., Author Summary RNA viruses are highly error prone, and can use their replication infidelity to adapt to complex environments within an infected host. However, viral populations may experience bottlenecks, which limit their diversity and potentially reduce their virulence. We hypothesized that natural barriers may limit the spread of RNA viruses within an infected host. To test this hypothesis, we engineered a pool of ten marked polioviruses identifiable by a novel assay, infected susceptible mice by injection or oral inoculation, and determined the percentage of the ten viruses that successfully spread to various body sites, including the brain. We found that, on average, only 10%–20% of the input viruses were found in most tissues, suggesting that barriers prevented the spread of the whole population. The importance of one such physical barrier, the colonic epithelium, was demonstrated in experiments where the colon was damaged prior to oral inoculation. Under these conditions, 30%–50% of the input viruses successfully spread to various body sites. We propose that host barriers limit viral spread, and this could possibly explain the rare incidence of paralytic poliomyelitis due to central nervous system invasion.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche., Author summary Streptococcus pneumoniae, the main causative agent of pneumonia, triggers inflammation and tissue damage by producing a pore-forming toxin, pneumolysin (Ply). Ply-induced inflammation drives pneumococcal transmission from nasopharynx (its primary reservoir), but also contributes to host mortality, limiting its occupiable habitats. Here, we uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways. This commensal-like lifestyle, resembling that of members of the mitis group of Streptococci, might have evolved within ST306 by loss of function ply mutations, compensating for limited nasopharyngeal carriage capacity by facilitating adaptation to an alternate niche.

Infection by large dsDNA viruses can lead to a profound alteration of host transcriptome and metabolome in order to provide essential building blocks to support the high metabolic demand for viral assembly and egress. Host response to viral infection can typically lead to diverse phenotypic outcome that include shift in host life cycle and activation of anti-viral defense response. Nevertheless, there is a major bottleneck to discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidic setup that captures individual algal cells and multiplex quantitate PCR. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and susceptible cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies., Author summary Almost all of our current understanding of the molecular mechanisms that govern host-pathogen interactions in the ocean is derived from experiments carried out at the population level, neglecting any heterogeneity. Here we used a single cell approach to unmask the phenotypic heterogeneity produced within infected populations of the cosmopolitan bloom-forming alga Emiliania huxleyi by its specific lytic virus. We found high variability in expression of viral genes among individual cells. This heterogeneity was used to map cells into their infection state and allowed to uncover a yet unrecognized host response. We also provide evidence that variability in host metabolic states provided a sensitive tool to decipher between susceptible and resistant cells.

Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium’s infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito’s innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito’s miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs’ influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes’ antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes., Author summary Malaria is caused by the Plasmodium parasite that is transmitted by Anopheles mosquitoes. The mosquito’s innate immune system plays an important role in controlling parasite infection. We have identified mosquito microRNAs (miRNAs) that are involved in regulating mosquito immunity to parasite infection. Transgenic mosquitoes that deplete the immunity-related miRNAs aga-miR-14 or aga-miR-305 through miRNA sponges, show increased resistance to both human and rodent parasite infection, and enhanced antibacterial defenses. Depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes, and the overall fitness cost of transgenic mosquitoes upon depletion of aga-miR-14 or aga-miR-305 was negligible. We show that targeting mosquito miRNA by transgenic expression of miRNA sponges may have potential for the development of malaria control through genetically engineered mosquitoes.

Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have addressed the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation (n = 3), a group pretreated with a high-neutralizing potency HIG preparation (n = 3), and an untreated control group (n = 5). Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different between control, standard-potency, and high-potency pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it had no apparent impact on intrahost viral genetic diversity at the investigated loci. Interestingly, some minor haplotypes present in fetal and maternal-fetal interface tissues were also identified as minor haplotypes in corresponding maternal tissues, providing evidence for a loose RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies., Author summary Human cytomegalovirus (CMV) is the most common infectious cause of birth defects worldwide. Knowledge gaps remain regarding how maternal immunity impacts the genetic composition of CMV populations and the incidence of congenital virus transmission. Addressing these gaps is important to inform vaccine development efforts. Using viral DNA isolated from a monkey model of congenital CMV infection, we investigated the impact of passively-administered maternal antibodies on the genetic composition of the maternal virus population and that transmitted to the fetus. Our analysis focused on regions of two CMV genes that encode glycoproteins known to facilitate viral cellular entry and to be targeted by CMV-specific antibodies. We investigated viral haplotypes across sampled maternal tissues and amniotic fluid, finding no impact of preexisting CMV-specific antibodies on CMV genetic diversity despite the observation that antibodies reduce viral load and can confer protection against congenital transmission. We further found that some minor haplotypes identified in fetal and maternal-fetal interface tissues were also present in paired maternal tissues. This finding indicates that a large number of viral particles likely passed from dam to fetus during congenital transmission.

The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.

Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control., Author Summary Cytomegaloviruses (CMVs) infect most people and are a common cause of fetal damage. We lack an effective vaccine. Our knowledge of human CMV is largely limited to chronic infection, which is hard to treat. Vaccination must target early infection. Related animal viruses therefore provide a vital source of information. Lymph nodes are a bottleneck in murine CMV spread from local to systemic infection. We show that viral passage through lymph nodes is restricted by interferons and NK cells. These defences alone cannot contain infection, but boosting their recruitment by vaccination has the potential to keep infection locally contained.

Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis., Author Summary Mycobacterium tuberculosis is amongst the most successful and enigmatic pathogens that has burdened humanity for thousands of years. The success of this pathogen depends on unique strategies employed to adapt during infection. Understanding these strategies is key to decipher the complexity of M. tuberculosis and crucial for epidemiological predictions and drug treatments. However, little is known on the impact of transmission population bottlenecks and host immune pressures on sequence diversity of mycobacteria. By combining deep sequencing and parallel evolution, we characterized the evolution of M. tuberculosis genetic diversity during the course of pulmonary infection in mice, and in disseminated M. bovis BCG infection in humans. We found that under in vitro rich culture conditions, multiple adaptive mutations arise, but none of these generate lasting allele diversity in vivo. We found that this phenomenon was not uniquely dictated by transmission bottleneck and that within host evolution contribute to select for a dominant genotype during M. tuberculosis infection. Finally, we used a immuno-deficient mouse model to show that T cells are contributor rather than limitator of genetic diversity during infection. Together, our results emphasize the complexity and uniqueness of M. tuberculosis and contribute understanding M. tuberculosis genetic adaptation.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential., Author Summary Despite the available HIV-1 diversity present in a chronically infected individual, single viral variants are transmitted in 80–90% of heterosexual transmission events. These breakthrough viruses may have unique properties that confer a higher capacity to transmit. Determining these properties could help inform the rational design of vaccines and enhance our understanding of viral transmission. We isolated the transmitted variant and a set of related non-transmitted variants from the transmitting partner near the estimated date of transmission from six epidemiologically linked transmission pairs to investigate viral correlates of transmission. The simplest explanation that transmitted variants are inherently more infectious or faster replicators in vitro did not hold true. In addition, transmitted variants did not replicate more efficiently than their non-transmitted counterparts in dendritic cells or in the presence of interferon-alpha in vitro, suggesting that they are not uniquely adapted to these components of the innate immune system. More ancestral genomes that were relatively sensitive to antibody neutralization tended to transmit, supporting previous reports that mutational escape away from the adaptive immune response likely reduces the ability to transmit. Our investigation into the traits of transmitted HIV-1 variants adds to the understanding of viral determinants of transmission.

Topological, chemical and immunological barriers are thought to limit infection by enteropathogenic bacteria. However, in many cases these barriers and their consequences for the infection process remain incompletely understood. Here, we employed a mouse model for Salmonella colitis and a mixed inoculum approach to identify barriers limiting the gut luminal pathogen population. Mice were infected via the oral route with wild type S. Typhimurium (S. Tm) and/or mixtures of phenotypically identical but differentially tagged S. Tm strains (“WITS”, wild-type isogenic tagged strains), which can be individually tracked by quantitative real-time PCR. WITS dilution experiments identified a substantial loss in tag/genetic diversity within the gut luminal S. Tm population by days 2–4 post infection. The diversity-loss was not attributable to overgrowth by S. Tm mutants, but required inflammation, Gr-1+ cells (mainly neutrophilic granulocytes) and most likely NADPH-oxidase-mediated defense, but not iNOS. Mathematical modelling indicated that inflammation inflicts a bottleneck transiently restricting the gut luminal S. Tm population to approximately 6000 cells and plating experiments verified a transient, inflammation- and Gr-1+ cell-dependent dip in the gut luminal S. Tm population at day 2 post infection. We conclude that granulocytes, an important clinical hallmark of S. Tm-induced inflammation, impose a drastic bottleneck upon the pathogen population. This extends the current view of inflammation-fuelled gut-luminal Salmonella growth by establishing the host response in the intestinal lumen as a double-edged sword, fostering and diminishing colonization in a dynamic equilibrium. Our work identifies a potent immune defense against gut infection and reveals a potential Achilles' heel of the infection process which might be targeted for therapy.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68–1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68–1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68–1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68–1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68–1 and 68–1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics., Author Summary Nonhuman primates are excellent models of HCMV infection and immunity, and their CMVs closely resemble HCMV in genomic organization and coding potential. Here, we expand the existing models by isolating new full-length, non-human primate CMV strains that more closely resemble primary HCMV isolates. In addition, we identify determinants needed for recombinant clones of RhCMV to infect MCM both in vitro and in vivo. Given their simplified immunogenetics, these results enable the use of MCM to deepen our understanding of CMV immunology.

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures., Author Summary Hepatitis C virus infects as many as 170 million people worldwide. Globally, there are seven major genotypes of HCV that differ by approximately 30% in nucleotide sequence. Importantly, the natural history of HCV infection is variable, ranging from spontaneous resolution to persistent viremia and chronic disease. Factors responsible for this variability in clinical outcome are unknown but likely involve a combination of viral and host determinants. To this end, a precise molecular identification of transmitted HCV genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of plasma viral RNA to identify transmitted viral genomes and their progeny in 17 subjects with acute infection. Numbers of transmitted viruses leading to productive clinical infection ranged from 1 to 37 or more (median = 4). Surprisingly, we found evidence of high multiplicity acute-to-acute HCV transmission in 3 of 17 subjects, which suggests that clinical transmission of HCV, like that of HIV-1, may be enhanced in early infection when virus titers are highest and neutralizing antibodies are absent. These results provide novel insight into HCV transmission and early virus diversification key to our understanding of virus natural history and response to drug selection and immune pressure.

The midgut epithelium of the mosquito malaria vector Anopheles is a hostile environment for Plasmodium, with most parasites succumbing to host defenses. This study addresses morphological and ultrastructural features associated with Plasmodium berghei ookinete invasion in Anopheles gambiae midguts to define the sites and possible mechanisms of parasite killing. We show by transmission electron microscopy and immunofluorescence that the majority of ookinetes are killed in the extracellular space. Dead or dying ookinetes are surrounded by a polymerized actin zone formed within the basal cytoplasm of adjacent host epithelial cells. In refractory strain mosquitoes, we found that formation of this zone is strongly linked to prophenoloxidase activation leading to melanization. Furthermore, we identify two factors controlling both phenomena: the transmembrane receptor frizzled-2 and the guanosine triphosphate–binding protein cell division cycle 42. However, the disruption of actin polymerization and melanization by double-stranded RNA inhibition did not affect ookinete survival. Our results separate the mechanisms of parasite killing from subsequent reactions manifested by actin polymerization and prophenoloxidase activation in the A. gambiae–P. berghei model. These latter processes are reminiscent of wound healing in other organisms, and we propose that they represent a form of wound-healing response directed towards a moribund ookinete, which is perceived as damaged tissue., Synopsis A dangerous journey awaits malaria Plasmodium parasites ingested by a mosquito. Most parasites are destroyed by host responses in the midgut, and in parasite-resistant refractory strains of mosquito the mortality can reach 100%. This midgut “bottleneck” represents an appealing target for reducing malaria transmission by the genetic control of wild mosquitoes. However, the killing mechanisms are still unclear. In this study, electron microscopical analyses followed the entire midgut invasion process in mosquitoes to identify the major site(s) and ultrastructural features of Plasmodium killing. The authors found that invasion can be divided into two steps: a swift passage through a midgut cell, followed by establishment of the parasite in the basal extracellular space, where it becomes an important target for destruction by soluble immunity factors. In refractory mosquitoes, dead parasites are associated with the formation of organelle-free zones of actin in adjacent midgut cells, and melanin deposition on the parasite surface. The authors identify two genes, called frizzled-2 and cell division cycle 42, that control these phenomena. Actin zone formation and melanization are generally thought to be killing mechanisms; however, the authors show by gene silencing that neither is lethal to Plasmodium. Instead, these mechanisms may represent a form of mosquito wound-healing response that is triggered by the presence of a moribund parasite.

Like other arthropod-borne viruses (arboviruses), mosquito-borne dengue virus (DENV) is maintained in an alternating cycle of replication in arthropod and vertebrate hosts. The trade-off hypothesis suggests that this alternation constrains DENV evolution because a fitness increase in one host usually diminishes fitness in the other. Moreover, the hypothesis predicts that releasing DENV from host alternation should facilitate adaptation. To test this prediction, DENV was serially passaged in either a single human cell line (Huh-7), a single mosquito cell line (C6/36), or in alternating passages between Huh-7 and C6/36 cells. After 10 passages, consensus mutations were identified and fitness was assayed by evaluating replication kinetics in both cell types as well as in a novel cell type (Vero) that was not utilized in any of the passage series. Viruses allowed to specialize in single host cell types exhibited fitness gains in the cell type in which they were passaged, but fitness losses in the bypassed cell type, and most alternating passages, exhibited fitness gains in both cell types. Interestingly, fitness gains were observed in the alternately passaged, cloned viruses, an observation that may be attributed to the acquisition of both host cell–specific and amphi-cell-specific adaptations or to recovery from the fitness losses due to the genetic bottleneck of biological cloning. Amino acid changes common to both passage series suggested convergent evolution to replication in cell culture via positive selection. However, intriguingly, mutations accumulated more rapidly in viruses passed in Huh-7 cells than in those passed in C6/36 cells or in alternation. These results support the hypothesis that releasing DENV from host alternation facilitates adaptation, but there is limited support for the hypothesis that such alternation necessitates a fitness trade-off. Moreover, these findings suggest that patterns of genetic evolution may differ between viruses replicating in mammalian and mosquito cells., Author Summary Dengue virus (DENV) is an arthropod-borne virus of immense public health concern. Recent studies suggest that the rate of DENV evolution has accelerated, as well as the disease severity (virulence), most likely driven by the increased transmission and selection by the vector, Aedes aegypti, over the last 50 years. Despite the nearly global impact of DENV, little is known about the forces that influence its host range evolution, a deficiency confounded by the absence of a practical vertebrate model. We used vertebrate (Huh-7) and invertebrate (C6/36) cells lines as surrogate hosts for studying DENV host range evolution. Using genetically homogeneous or heterogeneous DENV for serial passages (specialization) in either cell line, or alternating passage, we demonstrated that (i) viruses allowed to specialize in single host cells exhibited fitness gains but fitness losses in the bypassed cell line; (ii) most viruses passaged in alternating cycles exhibited detectable fitness gains in both cell lines; (iii) amino acid changes common in both passage series suggested convergent evolution via positive selection; and (iv) there was limited support that DENV evolution is constrained by alternate host transmission and replication. These results provide insights into the likelihood and extent of shifts towards higher DENV virulence.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance., Author Summary Amphibian species are facing a current global extinction crisis of unprecedented magnitude. The major factors causing their decline are the emerging disease chytridiomycosis and habitat destruction. Chytridiomycosis is caused by the aquatic fungus Batrachochytrium dendrobatidis and has been linked to species extinctions and population declines in montane regions including Australia, Panama, North America, and Spain. Currently, it is debated whether the recent emergence of the pathogen is largely the result of environmental factors triggering an outbreak of an endemic pathogen or if the epidemic has been caused by widespread introduction of the pathogen into naïve host populations (“pathogen pollution”). We studied the population genetics of chytridiomycosis using DNA sequences from a global panel of strains. These data showed evidence of a strong genetic bottleneck in the history of the pathogen, and the epidemic appears traceable to the widespread dispersal of a single genotype. Populations were not structured by host-origin, and the same lineage was detected in populations of both resistant and highly sensitive species. The data suggest that the chytridiomycosis epidemic is caused by the emergence of a novel pathogen but that disease outcome is contingent on host resistance and environmental factors.

Despite the potential for infectious agents harbored by other species to become emerging human pathogens, little is known about why some agents establish successful cross-species transmission, while others do not. The simian immunodeficiency viruses (SIVs), certain variants of which gave rise to the human HIV-1 and HIV-2 epidemics, have demonstrated tremendous success in infecting new host species, both simian and human. SIVsm from sooty mangabeys appears to have infected humans on several occasions, and was readily transmitted to nonnatural Asian macaque species, providing animal models of AIDS. Here we describe the first in-depth analysis of the tremendous SIVsm quasispecies sequence variation harbored by individual sooty mangabeys, and how this diverse quasispecies adapts to two different host species—new nonnatural rhesus macaque hosts and natural sooty mangabey hosts. Viral adaptation to rhesus macaques was associated with the immediate amplification of a phylogenetically related subset of envelope (env) variants. These variants contained a shorter variable region 1 loop and lacked two specific glycosylation sites, which may be selected for during acute infection. In contrast, transfer of SIVsm to its natural host did not subject the quasispecies to any significant selective pressures or bottleneck. After 100 d postinfection, variants more closely representative of the source inoculum reemerged in the macaques. This study describes an approach for elucidating how pathogens adapt to new host species, and highlights the particular importance of SIVsm env diversity in enabling cross-species transmission. The replicative advantage of a subset of SIVsm variants in macaques may be related to features of target cells or receptors that are specific to the new host environment, and may involve CD4-independent engagement of a viral coreceptor conserved among primates., Synopsis Why do some infectious agents establish successful cross-species transmission while others do not? Despite the clear potential for diseases harbored by animals to become emerging human pathogens, this question remains unanswered. Certain simian immunodeficiency viruses (SIVs) responsible for the human HIV-1 and HIV-2 epidemics have succeeded in infecting new host species, including humans. This study provides clues to how an SIV adapts to a new host in an experimental cross-species transmission. Indeed, many emerging diseases are caused by highly mutation-prone RNA viruses like SIV, which exist not as a single species, but rather as a population of genetic variants within a single infection. The presence of numerous viral variants in an infected animal increases the chance that variants with the ability to enter into or multiply in a new host species are present. This study describes how an SIV population from a natural reservoir host, the sooty mangabey, adapts to a new monkey species, the rhesus macaque. A limited subset of SIV variants containing unique viral surface proteins appears well suited to multiply in the new host. This study documents how viral variation facilitates cross-species transmission, and highlights the particular importance of immunodeficiency virus envelope variants in infecting new hosts.

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success., Synopsis Mycobacterium tuberculosis, the agent of tuberculosis, is a highly successful human pathogen and kills nearly 3 million persons each year. This pathogen and its close relatives sum up in a single and compact clonal group dating back only a few tens of thousands of years. Using genetic data, the researchers have discovered that human tubercle bacilli from East Africa represent extant bacteria of a much broader progenitor species from which the M. tuberculosis clonal group evolved. They estimate that this progenitor species is as old as 3 million years. This suggests that our remote hominid ancestors may well have already suffered from tuberculosis. In addition, the researchers show that tubercle bacilli are able to exchange parts of their genome with other strains, a process that is known to play a crucial role in adaptation of pathogens to their hosts. Thus, the M. tuberculosis genome appears to be a composite assembly, resulting from ancient horizontal DNA exchanges before its clonal expansion. These findings open novel perspectives for unraveling the origin and the molecular bases of M. tuberculosis evolutionary success, and lead to reconsideration of the impact of tuberculosis on human natural selection.

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration., Author Summary The arrival of bacilli in the lymph node is a bottleneck for initiating T cell responses to mycobacteria but remains poorly studied. To address this we used a mouse model to track the entry of cells and bacteria into the lymph node during skin infection with Mycobacterium bovis BCG, the live tuberculosis vaccine. We identified a population of migratory Dendritic cells that transport bacilli from the skin into the lymph node and which engage CD4+ T cells therein. The mobilization of Dendritic cells from the skin, and with these cells the transport of mycobacteria into the lymph node, was regulated by cytokines, in particular Interleukin–1. This was also dependent on MyD88, an adaptor molecule downstream of the Interleukin–1 receptor. We also found a requirement for MyD88 in driving Dendritic cells to the lymph node that was both inherent and extrinsic to the migrating cell. These findings bare consequences for our understanding of how T-cell responses are initiated during microbial challenge in the skin and hold promises for improving vaccines of low-to-modest efficacy such as BCG, which rely on such responses.

By combining high-throughput sequencing (HTS) with experimental evolution, we can observe the within-host dynamics of pathogen variants of biomedical or ecological interest. We studied the evolutionary dynamics of five variants of Potato virus Y (PVY) in 15 doubled-haploid lines of pepper. All plants were inoculated with the same mixture of virus variants and, variant frequencies were determined by HTS in eight plants of each pepper line, at each of six sampling dates. We developed a method for estimating the intensities of selection and genetic drift in a multi-allelic Wright-Fisher model, applicable whether these forces are strong or weak, and in the absence of neutral markers. This method requires variant frequency determination at several time points, in independent hosts. The parameters are the selection coefficients for each PVY variant and four effective population sizes N e at different time-points of the experiment. Numerical simulations of asexual haploid Wright-Fisher populations subjected to contrasting genetic drift (N e 2 [10, 2000]) and selection (|s| 2 [0, 0.15]) regimes were used to validate the method proposed. The experiment in closely related pepper host genotypes revealed that viruses experienced a considerable diversity of selection and genetic drift regimes. The resulting variant dynamics were accurately described by Wright-Fisher models. The fitness ranks of the variants were almost identical between host genotypes. By contrast, the dynamics of N e were highly variable, although a bottleneck was often identified during the systemic movement of the virus. We demonstrated that, for a fixed initial PVY population, virus effective population size is a heritable trait in plants. These findings pave the way for the breeding of plant varieties exposing viruses to stronger genetic drift, thereby slowing virus adaptation. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.