Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.

4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

Effects of testosterone on urogenital tract morphology and androgen receptor expression in immature Eastern Fence lizards (Sceloporus undulatus).

In non-avian reptiles, the onset of sexual dimorphism of the major structures of the urogenital tract varies temporally relative to gonadal differentiation, more so than in other amniote lineages. In the current study, we used tonic-release implants to investigate the effects of exogenous testosterone (T) on postnatal development of the urogenital tract in juvenile Eastern Fence Lizards (Sceloporus undulatus) to better understand the mechanisms underlying the ontogeny of sexual differentiation in reptiles. We examined gonads, mesonephric kidneys and ducts (male reproductive tract primordia), paramesonephric ducts (oviduct primordia), sexual segments of the kidneys (SSKs), and hemiphalluses to determine which structures were sexually dimorphic independent of T treatment and which structures exhibited sexually dimorphic responses to T. To better understand tissue-level responsiveness to T treatment, we also characterized androgen receptor (AR) expression by immunohistochemistry. At approximately 4 months after hatching in control animals, gonads were well differentiated but quiescent; paramesonephric ducts had fully degenerated in males; mesonephric kidneys, mesonephric ducts, and SSKs remained sexually undifferentiated; and hemiphalluses could not be everted in either sex. Exogenous T caused enlargement, regionalization, and secretory activity of the mesonephric ducts and SSKs in both sexes; enlargement and regionalization of the oviducts in females; and enlargement of male hemipenes. The most responsive tissues exhibited moderate but diffuse staining for AR in control lizards and intense nuclear staining in T-treated lizards, suggestive of autoregulation of AR. The similarity between sexes in the responsiveness of the mesonephric ducts and SSK to T indicates an absence of sexually dimorphic organizational effects in these structures prior to treatment, which was initiated approximately 2 months after hatching. In contrast, the sex-specific responses in oviducts and hemipenes indicate that significant organization and/or differentiation had taken place prior to treatment.

Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity.

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA.

Species differences in hormonally mediated gene expression underlie the evolutionary loss of sexually dimorphic coloration in Sceloporus lizards.

Phenotypic sexual dimorphism often involves the hormonal regulation of sex-biased expression for underlying genes. However, it is generally unknown whether the evolution of hormonally mediated sexual dimorphism occurs through upstream changes in tissue sensitivity to hormone signals, downstream changes in responsiveness of target genes, or both. Here, we use comparative transcriptomics to explore these possibilities in 2 species of Sceloporus lizards exhibiting different patterns of sexual dichromatism. Sexually dimorphic S. undulatus develops blue and black ventral coloration in response to testosterone, while sexually monomorphic S. virgatus does not, despite exhibiting similar sex differences in circulating testosterone levels. We administered testosterone implants to juveniles of each species and used RNAseq to quantify gene expression in ventral skin. Transcriptome-wide responses to testosterone were stronger in S. undulatus than in S. virgatus, suggesting species differences in tissue sensitivity to this hormone signal. Species differences in the expression of genes for androgen metabolism and sex hormone-binding globulin were consistent with this idea, but expression of the androgen receptor gene was higher in S. virgatus, complicating this interpretation. Downstream of androgen signaling, we found clear species differences in hormonal responsiveness of genes related to melanin synthesis, which were upregulated by testosterone in S. undulatus, but not in S. virgatus. Collectively, our results indicate that hormonal regulation of melanin synthesis pathways contributes to the development of sexual dimorphism in S. undulatus, and that changes in the hormonal responsiveness of these genes in S. virgatus contribute to the evolutionary loss of ventral coloration.

Reproductive trade-offs and phenotypic selection change with body condition, but not with predation regime, across island lizard populations.

Trade-offs between reproduction and survival are central to life-history theory and are expected to shape patterns of phenotypic selection, but the ecological factors structuring these trade-offs and resultant patterns of selection are generally unknown. We manipulated reproductive investment and predation regime in island populations of brown anole lizards (Anolis sagrei) to test (1) whether previously documented increases in the survival of experimentally non-reproductive females (OVX = ovariectomy) reflect the greater susceptibility of reproductive females (SHAM = control) to predation and (2) whether phenotypic selection differs as a function of reproductive investment and predation regime. OVX females exceeded SHAM controls in growth, mass gain and body condition, indicating pronounced energetic costs of reproduction. Although mortality was greatest in the presence of bird and snake predators, differences in survival between OVX and SHAM were unrelated to predation regime, as were patterns of natural selection on body size. Instead, we found that body condition at the conclusion of the experiment differed significantly across populations, suggesting that local environments varied in their ability to support mass gain and positive energy balance. As mean body condition improved across populations, the magnitude of the survival cost of reproduction increased, linear selection on body size shifted from positive to negative, and quadratic selection shifted from stabilizing to weakly disruptive. Our results suggest that reproductive trade-offs and patterns of phenotypic selection in female brown anoles are more sensitive to inferred variation in environmental quality than to experimentally induced variation in predation.

Evolution of hormone-phenotype couplings and hormone-genome interactions.

When selection favors a new relationship between a cue and a hormonally mediated response, adaptation can proceed by altering the hormonal signal that is produced or by altering the phenotypic response to the hormonal signal. The field of evolutionary endocrinology has made considerable progress toward understanding the evolution of hormonal signals, but we know much less about the evolution of hormone-phenotype couplings, particularly at the hormone-genome interface. We briefly review and classify the mechanisms through which these hormone-phenotype couplings likely evolve, using androgens and their receptors and genomic response elements to illustrate our view. We then present two empirical studies of hormone-phenotype couplings, one rooted in evolutionary quantitative genetics and another in comparative transcriptomics, each focused on the regulation of sexually dimorphic phenotypes by testosterone (T) in the brown anole lizard (Anolis sagrei). First, we illustrate the potential for hormone-phenotype couplings to evolve by showing that coloration of the dewlap (an ornament used in behavioral displays) exhibits significant heritability in its responsiveness to T, implying that anoles harbor genetic variance in the architecture of hormonal pleiotropy. Second, we combine T manipulations with analyses of the liver transcriptome to ask whether and how statistical methods for characterizing modules of co-expressed genes and in silico techniques for identifying androgen response elements (AREs) can improve our understanding of hormone-genome interactions. We conclude by emphasizing important avenues for future work at the hormone-genome interface, particularly those conducted in a comparative evolutionary framework.

Propagule size and sex ratio influence colonisation dynamics after introduction of a non-native lizard.

The composition of founding populations plays an important role in colonisation dynamics and can influence population growth during early stages of biological invasion. Specifically, founding populations with small propagules (i.e. low number of founders) are vulnerable to the Allee effect and have reduced likelihood of establishment compared to those with large propagules. The founding sex ratio can also impact establishment via its influence on mating success and offspring production. Our goal was to test the effects of propagule size and sex ratio on offspring production and annual population growth following introductions of a non-native lizard species (Anolis sagrei). We manipulated propagule composition on nine small islands, then examined offspring production, population growth and survival rate of founders and their descendants encompassing three generations. By the third reproductive season, per capita offspring production was higher on islands seeded with a relatively large propagule size, but population growth was not associated with propagule size. Propagule sex ratio did not affect offspring production, but populations with a female-biased propagule had positive growth, whereas those with a male-biased propagule had negative growth in the first year. Populations were not affected by propagule sex ratio in subsequent years, possibly due to rapid shifts towards balanced (or slightly female biased) population sex ratios. Overall, we show that different components of population fitness have different responses to propagule size and sex ratio in ways that could affect early stages of biological invasion. Despite these effects, the short life span and high fecundity of A. sagrei likely helped small populations to overcome Allee effects and enabled all populations to successfully establish. Our rare experimental manipulation of propagule size and sex ratio can inform predictions of colonisation dynamics in response to different compositions of founding populations, which is critical in the context of population ecology and invasion dynamics.

The evolution of monogamy is associated with reversals from male to female bias in the survival cost of parasitism.

The extent to which parasites reduce host survival should depend upon how hosts balance trade-offs between reproduction and survival. For example, parasites are predicted to impose greater survival costs under polygynous or promiscuous mating systems in which competition for mates favours increased reproductive investment, particularly in males. We provide, to our knowledge, the first comparative test of the hypothesis that the mating system of the host is an important determinant of (i) the extent to which parasites reduce survival, and (ii) the extent to which males and females differ in the survival cost of parasitism. Using meta-analysis of 85 published estimates of the survival cost of parasitism from 72 studies of 64 species representing diverse animal lineages, we show that parasites impose a mean 3.5-fold increase in the odds of mortality on their hosts. Although this survival cost does not differ significantly across monogamous, polygynous and promiscuous mating systems, females incur a greater survival cost than males in monogamous species, whereas males incur a greater survival cost than females in polygynous and promiscuous species. Our results support the idea that mating systems shape the relative extent to which males and females invest in reproduction at the expense of defence against parasites.

Identification and Characterization of a Small-Molecule Rabies Virus Entry Inhibitor.

Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry.IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

Development of an allosteric inhibitor class blocking RNA elongation by the respiratory syncytial virus polymerase complex.

Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design.IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family.

Rapid evolution of testis size relative to sperm morphology suggests that post-copulatory selection targets sperm number in Anolis lizards.

Post-copulatory sexual selection is thought to be responsible for much of the extraordinary diversity in sperm morphology across metazoans. However, the extent to which post-copulatory selection targets sperm morphology versus sperm production is generally unknown. To address this issue, we simultaneously characterized the evolution of sperm morphology (length of the sperm head, midpiece and flagellum) and testis size (a proxy for sperm production) across 26 species of Anolis lizards, a group in which sperm competition is likely. We found that the length of the sperm midpiece has evolved 2-3 times faster than that of the sperm head or flagellum, suggesting that midpiece size may be the most important aspect of sperm morphology with respect to post-copulatory sexual selection. However, testis size has evolved faster than any aspect of sperm morphology or body size, supporting the hypothesis that post-copulatory sexual selection acts more strongly upon sperm production than upon sperm morphology. Likewise, evolutionary increases in testis size, which typically indicate increased sperm competition, are not associated with predictable changes in sperm morphology, suggesting that any effects of post-copulatory selection on sperm morphology are either weak or variable in direction across anoles. Collectively, our results suggest that sperm production is the primary target of post-copulatory sexual selection in this lineage.

Sperm morphology and count vary with fine-scale changes in local density in a wild lizard population.

Given that sperm production can be costly, theory predicts that males should optimally adjust the quantity and/or quality of their sperm in response to their social environment to maximize their paternity success. Although experiments demonstrate that males can alter their ejaculates in response to manipulations of the social environment and studies show that ejaculate traits covary with social environment across populations, it is unknown whether individual variation in sperm traits corresponds to natural variation found within wild populations. Using an island population of brown anole lizards (Anolis sagrei), we tested the prediction that sperm traits (sperm count, sperm morphology, sperm velocity) respond to natural variation in the risk of sperm competition, as inferred from the local density and operational sex ratio (OSR) of conspecifics. We found that males living in high-density areas of the island produced relatively larger sperm midpieces, smaller sperm heads, and lower sperm counts. Sperm traits were unrelated to OSR after accounting for the covariance between OSR and density. Our findings broaden the implications of sperm competition theory to intrapopulation social environment variation by showing that sperm count and sperm morphology vary with fine-scale differences in density within a single wild population.

Sex-Specific Selection and the Evolution of Between-Sex Genetic Covariance.

Because the sexes share a genome, traits expressed in males are usually genetically correlated with the same traits expressed in females. On short timescales, between-sex genetic correlations (rmf) for shared traits may constrain the evolution of sexual dimorphism by preventing males and females from responding independently to sex-specific selection. However, over longer timescales, rmf may evolve, thereby facilitating the evolution of dimorphism. Although it has been suggested that sexually antagonistic selection may reduce rmf, we lack a general theory for the evolution of rmf and its multivariate analog, the between-sex genetic covariance matrix (B). Here, we derive a simple analytical model for the within-generation change in B due to sex-specific directional selection. We present a single-trait example demonstrating that sex-specific directional selection may either increase or decrease between-sex genetic covariance, depending on the relative strength of selection in each sex and on the current value of rmf. Although sexually antagonistic selection can reduce between-sex covariance, it will only do so when selection is much stronger in one sex than in the other. Counterintuitively, sexually antagonistic selection that is equal in strength in the 2 sexes will maintain positive between-sex covariance. Selection acting in the same direction on both sexes is predicted to reduce between-sex covariance in many cases. We illustrate our model numerically using empirical measures of sex-specific selection and between-sex genetic covariance from 2 populations of sexually dimorphic brown anole lizards (Anolis sagrei) and discuss its importance for understanding the resolution of intralocus sexual conflict.

Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses.

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

Thermal physiology and thermoregulatory behaviour exhibit low heritability despite genetic divergence between lizard populations.

Ectothermic species are particularly sensitive to changes in temperature and may adapt to changes in thermal environments through evolutionary shifts in thermal physiology or thermoregulatory behaviour. Nevertheless, the heritability of thermal traits, which sets a limit on evolutionary potential, remains largely unexplored. In this study, we captured brown anole lizards (Anolis sagrei) from two populations that occur in contrasting thermal environments. We raised offspring from these populations in a laboratory common garden and compared the shape of their thermal performance curves to test for genetic divergence in thermal physiology. Thermal performance curves differed between populations in a common garden in ways partially consistent with divergent patterns of natural selection experienced by the source populations, implying that they had evolved in response to selection. Next, we estimated the heritability of thermal performance curves and of several traits related to thermoregulatory behaviour. We did not detect significant heritability in most components of the thermal performance curve or in several aspects of thermoregulatory behaviour, suggesting that contemporary selection is unlikely to result in rapid evolution. Our results indicate that the response to selection may be slow in the brown anole and that evolutionary change is unlikely to keep pace with current rates of environmental change.

Hormonally Mediated Increases in Sex-Biased Gene Expression Accompany the Breakdown of Between-Sex Genetic Correlations in a Sexually Dimorphic Lizard.

The evolution of sexual dimorphism is predicted to occur through reductions in between-sex genetic correlations (rmf) for shared traits, but the physiological and genetic mechanisms that facilitate these reductions remain largely speculative. Here, we use a paternal half-sibling breeding design in captive brown anole lizards (Anolis sagrei) to show that the development of sexual size dimorphism is mirrored by the ontogenetic breakdown of rmf for body size and growth rate. Using transcriptome data from the liver (which integrates growth and metabolism), we show that sex-biased gene expression also increases dramatically between ontogenetic stages bracketing this breakdown of rmf. Ontogenetic increases in sex-biased expression are particularly evident for genes involved in growth, metabolism, and cell proliferation, suggesting that they contribute to both the development of sexual dimorphism and the breakdown of rmf. Mechanistically, we show that treatment of females with testosterone stimulates the expression of male-biased genes while inhibiting the expression of female-biased genes, thereby inducing male-like phenotypes at both organismal and transcriptomic levels. Collectively, our results suggest that sex-specific modifiers such as testosterone can orchestrate sex-biased gene expression to facilitate the phenotypic development of sexual dimorphism while simultaneously reducing genetic correlations that would otherwise constrain the independent evolution of the sexes.

Natural selection on thermal performance in a novel thermal environment.

Tropical ectotherms are thought to be especially vulnerable to climate change because they are adapted to relatively stable temperature regimes, such that even small increases in environmental temperature may lead to large decreases in physiological performance. One way in which tropical organisms may mitigate the detrimental effects of warming is through evolutionary change in thermal physiology. The speed and magnitude of this response depend, in part, on the strength of climate-driven selection. However, many ectotherms use behavioral adjustments to maintain preferred body temperatures in the face of environmental variation. These behaviors may shelter individuals from natural selection, preventing evolutionary adaptation to changing conditions. Here, we mimic the effects of climate change by experimentally transplanting a population of Anolis sagrei lizards to a novel thermal environment. Transplanted lizards experienced warmer and more thermally variable conditions, which resulted in strong directional selection on thermal performance traits. These same traits were not under selection in a reference population studied in a less thermally stressful environment. Our results indicate that climate change can exert strong natural selection on tropical ectotherms, despite their ability to thermoregulate behaviorally. To the extent that thermal performance traits are heritable, populations may be capable of rapid adaptation to anthropogenic warming.

Does adaptive radiation of a host lineage promote ecological diversity of its bacterial communities? A test using gut microbiota of Anolis lizards.

Adaptive radiations provide unique opportunities to test whether and how recent ecological and evolutionary diversification of host species structures the composition of entire bacterial communities. We used 16S rRNA gene sequencing of faecal samples to test for differences in the gut microbiota of six species of Puerto Rican Anolis lizards characterized by the evolution of distinct 'ecomorphs' related to differences in habitat use. We found substantial variation in the composition of the microbiota within each species and ecomorph (trunk-crown, trunk-ground, grass-bush), but no differences in bacterial alpha diversity among species or ecomorphs. Beta diversity analyses revealed subtle but significant differences in bacterial composition related to host phylogeny and species, but these differences were not consistently associated with Anolis ecomorph. Comparison of a trunk-ground species from this clade (A. cristatellus) with a distantly related member of the same ecomorph class (A. sagrei) where the two species have been introduced and are now sympatric in Florida revealed pronounced differences in the alpha diversity and beta diversity of their microbiota despite their ecological similarity. Comparisons of these populations with allopatric conspecifics also revealed geographic differences in bacterial alpha diversity and beta diversity within each species. Finally, we observed high intraindividual variation over time and strong effects of a simplified laboratory diet on the microbiota of A. sagrei. Collectively, our results indicate that bacterial communities are only weakly shaped by the diversification of their lizard hosts due to the strikingly high levels of bacterial diversity and variation observed within Anolis species.