Respiratory pathogens are expected to spread through social contacts, but outbreaks often occur quickly and unpredictably, making it challenging to simultaneously record social contact and disease incidence data, especially in wildlife. Thus, the role of social contacts in the spread of infectious disease is often treated as an assumption in disease simulation studies, and few studies have empirically demonstrated how pathogens spread through social networks. In July–August 2015, an outbreak of respiratory disease was observed in a wild chimpanzee community in Kibale National Park, Uganda, during an ongoing behavioral study of male chimpanzees, offering a rare opportunity to evaluate how social behavior affects individual exposure to socially transmissible diseases. From May to August 2015, we identified adult and adolescent male chimpanzees displaying coughs and rhinorrhea and recorded 5-m proximity data on males (N = 40). Using the network k-test, we found significant relationships between male network connectivity and the distribution of cases within the network, supporting the importance of short-distance contacts for the spread of the respiratory outbreak. Additionally, chimpanzees central to the network were more likely to display clinical signs than those with fewer connections. Although our analyses were limited to male chimpanzees, these findings underscore the value of social connectivity data in predicting disease outcomes and elucidate a potential evolutionary cost of being social.

Patterns of epistasis and shapes of fitness landscapes are of wide interest because of their bearings on a number of evolutionary theories. The common phenomena of slowing fitness increases during adaptations and diminishing returns from beneficial mutations are believed to reflect a concave fitness landscape and a preponderance of negative epistasis. Paradoxically, fitness decreases tend to decelerate and harm from deleterious mutations shrinks during the accumulation of random mutations—patterns thought to indicate a convex fitness landscape and a predominance of positive epistasis. Current theories cannot resolve this apparent contradiction. Here, we show that the phenotypic effect of a mutation varies substantially depending on the specific genetic background and that this idiosyncrasy in epistasis creates all of the above trends without requiring a biased distribution of epistasis. The idiosyncratic epistasis theory explains the universalities in mutational effects and evolutionary trajectories as emerging from randomness due to biological complexity. Theory, simulations and empirical data are used to show that the phenotypic effect of a mutation varies substantially depending on the specific genetic background, thereby resolving an apparent contradiction between predictions of convex and concave fitness landscapes.

Influenza remains a persistent public health challenge, because the rapid evolution of influenza viruses has led to marginal vaccine efficacy, antiviral resistance, and the annual emergence of novel strains. This evolvability is driven, in part, by the virus's capacity to generate diversity through mutation and reassortment. Because many new traits require multiple mutations and mutations are frequently combined by reassortment, epistatic interactions between mutations play an important role in influenza virus evolution. While mutation and epistasis are fundamental to the adaptability of influenza viruses, they also constrain the evolutionary process in important ways. Here, we review recent work on mutational effects and epistasis in influenza viruses.

The substitution rates of transitions are higher than expected by chance relative to those of transversions. Many have argued that selection disfavors transversions, as nonsynonymous transversions are less likely to conserve biochemical properties of the original amino acid. Only recently has it become feasible to directly test this selective hypothesis by comparing the fitness effects of a large number of transition and transversion mutations. For example, a recent study of six viruses and one beta-lactamase gene did not find evidence supporting the selective hypothesis. Here, we analyze the relative fitness effects of transition and transversion mutations from our recently published genome-wide study of mutational fitness effects in influenza virus. In contrast to prior work, we find that transversions are significantly more detrimental than transitions. Using what we believe to be an improved statistical framework, we also identify a similar trend in two HIV data sets. We further demonstrate a fitness difference in transition and transversion mutations using four deep mutational scanning data sets of influenza virus and HIV, which provided adequate statistical power. We find that three of the most commonly cited radical/conservative amino acid categories are predictive of fitness, supporting their utility in studies of positive selection and codon usage bias. We conclude that selection is a major contributor to the transition:transversion substitution bias in viruses and that this effect is only partially explained by the greater likelihood of transversion mutations to cause radical as opposed to conservative amino acid changes.

Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus's high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.

Numerous virulence factors expressed by C. neoformans (C. neo) modulate host defenses by promoting non-protective Th2-biased adaptive immune responses. Prior studies demonstrate that the HSP70 homologue, Ssa1, significantly contributes to serotype-D C. neo virulence through the induction of laccase, a Th2-skewing and CNS-tropic factor. In the current study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A (serA) strain of C. neo (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 days. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase-deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate, but not the adaptive phase of the immune response. We conclude that Ssa1’s virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neo, but ultimately has no effect on cryptococcal control by adaptive immunity.

Deletions of cryptococcal PIK1, RUB1, and ENA1 genes independently rendered defects in yeast survival in human CSF and within macrophages. We evaluated virulence potential of these genes by comparing wild-type Cryptococcus neoformans strain H99 with deletant and complement strains in a BALB/c mouse model of pulmonary infection. Survival of infected mice; pulmonary cryptococcal growth and pathology; immunological parameters; dissemination kinetics; and CNS pathology were examined. Deletion of each PIK1, RUB1, and ENA1 differentially reduced pulmonary growth and dissemination rates of C. neoformans and extended mice survival. Furthermore, pik1Δ induced similar pathologies to H99, however, with significantly delayed onset; rub1Δ was more efficiently contained within pulmonary macrophages and was further delayed in causing CNS dissemination/pathology; whereas ena1Δ was progressively eliminated from the lungs and did not induce pathological lesions or disseminate into the CNS. The diminished virulence of mutant strains was associated with differential modulation of pulmonary immune responses, including changes in leukocyte subsets, cytokine responses, and macrophage activation status. Compared to H99 infection, mutants induced more hallmarks of a protective Th1 immune response, rather than Th2, and more classical, rather than alternative, macrophage activation. The magnitude of immunological effects precisely corresponded to the level of virulence displayed by each strain. Thus, cryptococcal PIK1, RUB1, and ENA1 differentially contribute to cryptococcal virulence, in correlation with their differential capacity to modulate immune responses.

C. neoformans is a leading cause of fatal mycosis linked to CNS dissemination. Laccase, encoded by the LAC1 gene, is an important virulence factor implicated in brain dissemination yet little is known about the mechanism(s) accounting for this observation. Here, we investigated whether the presence or absence of laccase altered the local immune response in the lungs by comparing infections with the highly virulent strain, H99 (which expresses laccase) and mutant strain of H99 deficient in laccase (lac1Δ) in a mouse model of pulmonary infection. We found that LAC1 gene deletion decreased the pulmonary fungal burden and abolished CNS dissemination at weeks 2 and 3. Furthermore, LAC1 deletion lead to: 1) diminished pulmonary eosinophilia; 2) increased accumulation of CD4+ and CD8+ T cells; 3) increased Th1 and Th17 cytokines yet decreased Th2 cytokines; and 4) lung macrophage shifting of the lung macrophage phenotype from M2- towards M1-type activation. Next, we used adoptively transferred CD4+ T cells isolated from pulmonary lymph nodes of mice infected with either lac1Δ or H99 to evaluate the role of laccase-induced immunomodulation on CNS dissemination. We found that in comparison to PBS treated mice, adoptively transferred CD4+ T cells isolated from lac1Δ-infected mice decreased CNS dissemination, while those isolated from H99-infected mice increased CNS dissemination. Collectively, our findings reveal that immune modulation away from Th1/Th17 responses and towards Th2 responses represents a novel mechanism through which laccase can contribute to cryptococcal virulence. Furthermore, our data support the hypothesis that laccase-induced changes in polarization of CD4+ T cells contribute to CNS dissemination.