Does fever drive the evolution of antiviral genes?

Fever is an ancient, evolutionarily conserved response to pathogen sensing that is present in all extant vertebrates, suggesting that it arose when they branched from ancestral chordates, ∼600 million years ago (Kluger, 1979). Infection with diverse microbes including bacteria, fungi, parasites, and viruses results in the induction of fever, raising core body temperature with the goal of constraining pathogen replication and enhancing immune responses. In cold-blooded ectotherms, fever is achieved through temperature-seeking behavior. Behavioral fever was discovered in the lizard Dipsosaurus dorsalis, where after bacterial infection the animals sought warmer environments to raise their core body temperature (Vaughn et al., 1974). Since then, this phenomenon has been recognized in amphibians and fish with experimental evidence for a protective benefit against infections (e.g., blocking fever results in reduced protection from infection). Additionally, outside of vertebrates, physical activity in response to infection can raise temperature of not only the individual but also entire colonies, as exhibited by honeybees raising the temperature of the hive to control infection (Starks et al., 2000). Conversely, warm-blooded endotherms, such as mammals and birds, induce fever through metabolic processes that evolved independently and have been retained in these two classes of animals, suggesting its survival benefit (Ruben, 1995). Given that fever has been retained in all vertebrates further suggests a survival advantage. Many molecular pathways that lead to induction of fever response are common between endotherms and ectotherms, suggesting that they share common origins. While conserved throughout diverse animals, this deep-rooted evolutionary response to infection can be costly. For ectotherms, behavioral fever can increase the risk of predation and can be metabolically taxing for endotherms. Additionally, metabolic fever can be pathogenic, resulting in organ damage. Despite these costs, fever has been retained throughout diverse branches of life, suggesting survival benefits.

Fever can provide enhanced survival in response to infections. For ectotherms, the value of behavioral fever was elegantly demonstrated by studies in several species where fever is blocked by preventing access to warm water, resulting in reduced survival compared with those that are provided the option of moving to warm water (Covert and Reynolds, 1977; Rakus et al., 2017). In humans, this is broadly demonstrated by fever intervention therapy with antipyretics, which results in decreased antiviral responses, increased virus shedding, and mortality (Earn et al., 2014; Plaisance and Mackowiak, 2000; Schulman et al., 2005; Stanley et al., 1975; Wrotek et al., 2021). Fever’s protective benefit against infectious agents has been demonstrated through several mechanisms. The simplest mechanism is that the host’s fever can move pathogens out of their thermal optima. Induction of fever has been used to treat syphilis, and heat therapy was shown to be efficacious against poliovirus (Golden and Rogers, 2010; Vogel, 2013). A striking example of temperature impacting pathogens occurs with fungal infections where their optimal growth occurs at temperatures below most mammals’ basal body temperatures (Casadevall, 2012). This led to the intriguing hypothesis that thermal disparities between fungi and hosts contributed to the rise of mammals during the Cretaceous–Paleogene extinction event (Casadevall, 2012; Casadevall and Damman, 2020). Dust and debris in the atmosphere obscured the sun for ∼2 years. Global temperatures dropped and fungi thrived (Casadevall, 2012; Casadevall and Damman, 2020). The surviving dinosaurs were not able to achieve behavioral fever, potentially leaving them particularly susceptible to fungal infections compared with mammals who could still undergo metabolic fever, although there is some controversy, and some dinosaurs may have been endothermic (Chiarenza et al., 2024).

Fever also directly impacts the immune system, particularly in mammals (reviewed in Evans et al. [2015]). It can improve lymphocyte trafficking and neutrophil function, enhance antigen presentation and antibody production, and influence T cell differentiation and function (Evans et al., 2015; Wilander and Rathmell, 2025). However, there have only been limited studies evaluating fever’s role in intrinsic or innate immune responses to viruses. Intrinsic and innate immune responses in chordates are mediated by virus sensing through pattern recognition receptors (e.g., TLRs and RIG I like receptors) and the production of IFNs, which then signal to infected and neighboring cells, leading to the induction of hundreds of IFN-stimulated genes (ISGs). These ISGs have diverse functions, including directly blocking virus replication and altering cellular processes, to make the cell inhospitable to virus replication. There is some evidence for antiviral genes functioning differently across temperature ranges. For example, during rhinovirus infection, increased expression of IFN and ISGs and the ATPase activity of virus sensing through RIG-I and MDA5 are greater at 37°C than 33°C (Foxman et al., 2015; Foxman et al., 2016). Similarly, the human coronavirus 229E replication was impacted by elevated temperature from 32°C to 37°C, which was dependent on STAT1 signaling (Lassnig et al., 2005). Arthropod-borne viruses (arboviruses), which replicate in both vertebrates and invertebrate hosts, can replicate across a range of temperatures. In mammals, they can infect most organ systems but also replicate well in the extremities of mammals, which have lower temperatures and temperature-dependent reduction in IFN signaling (Prow et al., 2017). Although these studies did not explicitly evaluate fever temperatures, they demonstrate potentially impaired immunity at barrier surfaces or extremities, which are below core body temperatures. Other work has demonstrated that the efficacy of IFN responses was improved with fever temperatures in mouse models of arbovirus infections (Lane et al., 2018). Together, these data suggest that temperature can significantly impact the molecular response to virus infections.

There are many instances in nature where proteins have temperature-dependent function. In some mammals, temperature-sensitive mutations in tyrosinase can impact hair/coat color, where the enzyme can function at cooler temperatures in extremities, resulting in pigment production in extremities but not the torso (Aigner et al., 2000; Kwon et al., 1989). Temperature can also directly impact virus lifecycles at multiple steps. Virus fusion and entry can be temperature dependent, with fusion of paramyxovirus glycoproteins occurring more efficiently at higher temperatures (Connolly and Lamb, 2006; Wharton et al., 2000). Virus polymerases also exhibit temperature-dependent functionality, which has been exploited for live attenuated vaccine development (influenza virus permissive to replicate in lower temperature of the upper airway but restricted at core body temperature of the lower airway [Subbarao, 2021]). Enhanced protection against infection by altering immune responses, increasing gene expression, and promoting inflammation may not be the only ways in which fever can confer protection in mammals. The impact of fever on ISG function during virus infection has never been evaluated, and ISG function may change based on temperature. Here, I posit that antiviral genes may have undergone selection for their function at fever temperatures, not steady-state body temperature, where most mechanistic studies are performed (Fig. 1). Most experimental systems identifying antiviral activity or mechanisms in humans and other model mammals use cell culture systems at 37°C (or the basal physiological temperature of the host) and, therefore, may miss key interactions that occur in nature at fever temperatures. Intriguingly, many screens have shown that most ISGs do not impact replication across diverse viruses (McDougal et al., 2023; Schoggins et al., 2011; Schoggins et al., 2014). It is possible that evaluating function at temperatures lower than what these genes were selected under could be responsible. I will discuss potential impacts of fever on antiviral gene function and evolution, challenges to evaluating the role of fever, and suggestions for future experimentation.

Virus infections in chordates are sensed by pattern recognition receptors leading to the production of type I and III IFNs. IFNs then signal in an autocrine and paracrine manner inducing the expression of hundreds of ISGs. Temperature can significantly impact protein stability, often with an inverse correlation with temperature. Recently, there have been a large number of studies using machine learning to predict the impact of mutations on thermostability (Fang, 2023; Muellers et al., 2023; Rodella et al., 2024). While there is significant heterogeneity in protein attributes impacting stability, there are some common features that make mutations more likely to cause temperature-sensitive phenotypes. This includes residues that are at buried sites within a protein and the hydrophobicity of neighboring amino acids (Tan et al., 2014). Temporary reduction of function or degradation of some proteins at fever temperatures may not be detrimental to the host. However, reduced antiviral gene function or loss-of-function during the peak of virus replication could impact survival. Abundant proteins at steady state are more thermostable (Luzuriaga-Neira et al., 2023), which could buffer cell survival during fever. One hypothesis is that inducible antiviral proteins evolved to be more thermostable than other genes not under pathogen-mediated selection. Improved protein structure prediction algorithms will aid in the testing of this hypothesis. Protein stability at fever temperatures could also be impacted by heat shock proteins. This class of genes is conserved across all domains of life and is induced during cellular stress, including temperature shifts, infection, and chemical and physical insults. Heat shock proteins aid in protein folding to prevent damage from accumulation of misfolded proteins and target misfolded proteins for degradation. Heat shock proteins may play a critical role in ensuring antiviral protein function at fever temperatures. There is some evidence that they can impact the function of immune genes. For example, HSP90 binding to α4 integrins causes a conformation change at fever temperatures to the active form, enhancing lymphocyte trafficking (Lin et al., 2019). Could heat shock proteins play a similar role in ISG folding? For example, HSP21 supports antiviral sensing by stabilizing IRF3 and blocking dephosphorylation (Xu et al., 2022), and there may be additional mechanisms for other heat shock proteins as yet to be uncovered.

Temperature increases due to fever may also impact the function of ISG protein products. ISGs are highly diverse and span many protein classes. The simplest impact of temperature on ISG function could be enzyme rate of action according to the Arrhenius equation. Several enzymatic ISGs have demonstrated temperature-dependent functions. For example, RNaseL functions more efficiently at 37°C than 33°C (Foxman et al., 2016), but this has not been evaluated at fever temperatures. Additionally, the RNA helicase activity of RIG-I was impaired at 42°C while that of MDA5 activity was unaffected (Foxman et al., 2015). Given that enzyme functions can be altered by temperatures, it is possible that enzymes that confer posttranslational modifications (PTMs) may be impacted by temperature. Importantly, many ISGs’ functions have been demonstrated to be dependent on PTMs, including palmitoylation of IFITM3, SUMOylation of MXA, phosphorylation of PKR, and glycosylation of BST2 (Carroll et al., 1993; Chamontin et al., 2021; Maarifi et al., 2016; Perez-Caballero et al., 2009; Yount et al., 2010). Altered PTM due to fever could impact antiviral protein function. It would be intriguing if some ISGs were only modified and functioned at fever temperature. This could help to mitigate potential toxic or aberrant impacts of ISG expression in the absence of a viral threat. Interestingly, there are also temperature-dependent impacts on immune genes in insects that lack IFN but, in some cases, can exhibit behavioral fever (e.g., honeybees raising the temperature of the hive through increased activity). There is evidence that RNA interference, the adaptive antiviral immune system in plants and in some worms and insects, require higher temperatures for optimal functionality (Adelman et al., 2013).

In both endotherms and ectotherms, fever is induced after sensing of pathogens by pattern recognition receptors, driving metabolic fever and temperature-seeking behavior, respectively. In mammals, pathogen sensing leads to the production of prostaglandin E2, which then signals in neurons in the hypothalamus releasing noradrenaline and acetylcholine. Noradrenaline the drives the physiological changes responsible for core body temperature increase, including vasoconstriction, brown adipose tissue thermogenesis, and acetylcholine drives increased metabolism and energy release from muscles (Evans et al., 2015). Pyrogenic cytokines downstream of pathogen sensing like IL-6 also stimulate the production of prostaglandin E2 and help initiate and sustain fever responses (Evans et al., 2015). This process takes time, and after acute viral infection in mammals, fever is induced within ∼48 h with peak fever often occurring at peak virus load. Given these kinetics, it is intriguing to speculate fever would have a greater impact on “late” acting ISGs, critical to control the outcome of infections. There are also temporal differences in IFNs with IFN-I acting earlier than IFN-III. While largely overlapping, there are also some transcriptional differences in the ISGs induced by IFN-I versus -III (Forero et al., 2019; Lazear et al., 2019). Would ISGs induced specifically by IFN-III be more likely to have evolved fever-dependent function? IFN-I and -III also have different structure and binding properties to their receptors (Mendoza et al., 2017; Thomas et al., 2011). Is it possible that IFN-III evolved to be more thermal stable or have more thermal stable interactions with its receptor since it predominantly signals during fever? Viruses encode potent IFN antagonists and can evade IFN induction and signaling at early stages. However, at later stages of infection when fever has set in, virus stealth fails, and antiviral genes and immune cells are able to control the infection. Viruses have also evolved to block fever, delaying this response. For example, cyprinid herpesvirus 3 of carp has been recently shown to express a decoy TNF receptor which delays behavioral fever, enhancing viral replication (Rakus et al., 2017). Interestingly, influenza virus replication at fever temperatures results in the production of more RIG-I ligands, increasing IFN production (Bisht et al., 2025, Preprint), yet another complication to studying temperature-dependent effects on antiviral genes. There has likely been an ongoing evolutionary arms race between fever temperature and in the viral regulation of induction and regulation of fever responses.

While fever can be beneficial acutely, it can also drive significant pathology. This dichotomy has led to the interesting hypothesis that fever may be a way to accelerate death of infected individuals (Mackowiak, 1994). In this model, fever either helps clear the infection or it accelerates the demise of the individual to protect the population (Mackowiak, 1994). Given the potential pathogenesis and high metabolic costs, fever must be tightly regulated. Additionally, failure to regulate IFN responses can lead to interferonopathies. Therefore, it is possible that fever temperatures may serve to regulate modulators of the IFN response to prevent interferonopathies. For example, would suppressors of cytokine signaling 1 and 3 that directly inhibit JAK enzymatic activity downstream of IFN receptors function more efficiently at ambient temperature and with reduced function at fever, thereby promoting IFN signaling during infection? Autoimmune diseases are often accompanied by fever; could increased antiviral gene protein function, activated in the absence of infection, exacerbate the disease? One might also expect that because fever and the IFN response happen concomitantly, IFN and ISGs may regulate fever. IFN treatment increases prostaglandin E2 synthesis and Cox2 expression and prostaglandin E2 signaling can dampen, demonstrating cross talk between the systems (Blanco et al., 2000; Boraschi et al., 1984; Coulombe et al., 2014; Fuse et al., 1982). However, these studies did not evaluate fever itself. It is somewhat surprising that there is not more evidence for ISGs regulating the induction or suppression of fever responses.

Animals with IFN responses have a wide range of basal body temperatures. Comparative analysis of protein sequence and structure could be used to identify temperature-dependent signatures of selection. Mammals and even some birds and fish possess a shared core set of genes induced by IFNs (Levraud et al., 2019; Shaw et al., 2017). However, these genes have been under heavy selection, and their sequences are highly divergent. Did species like rabbits, which have higher basal body temperatures (39°C) than naked mole rats (31°C), need to adapt mutations in ISGs to improve their stability or function at higher fever temperatures? Lifestyle can also impact basal body temperatures. Mammals have gone back to the water at least seven independent times (Uhen, 2007). This resulted in a significant environmental change where heat is conducted more efficiently than air, requiring adaptations to thermoregulation. This environment also lively presented exposure to new pathogens. There is evidence that this environmental shift has driven adaptive changes in TLRs in cetaceans (whales, dolphins, and porpoises) (Ishengoma and Agaba, 2017; Shen et al., 2012). This likely because of the aquatic environment and exposure to new pathogens, but could colder temperatures have had an impact as well? This is an area that deserves more study. Another lifestyle that can impact body temperature is flight. Flight is energy intensive, and bats and birds have higher basal temperatures than flightless mammals. Interestingly, bats’ basal temperatures during flight are near the fever temperatures of many mammals, which spawned the “flight as fever” hypothesis (O'Shea et al., 2014). It is possible that ISGs selected for enhanced function at fever temperatures before the order Chiroptera (bats) emerged would then be operating at the optimal selective temperature during flight, even in the absence of fever. This could be a mechanism for enhanced control of virus infection that has been proposed to exist in bats. However, other mammalian species also exhibit increased body temperature basally and during activity so flight as fever may not be a strong driver of increased tolerance to infections observed in bats (Levesque et al., 2021). In addition to the flight as fever hypotheses, another proposed mechanism is reduced inflammation and tolerance permitting high replication with more limited pathology (Demian et al., 2024). Some bats may also have constitutive expression of IFN-I and have expansion and diversification of ISGs (Bondet et al., 2021; Zhang and Irving, 2023). Bats are incredibly diverse and represent ∼20% of all mammals. A better understanding of how bats interact with viruses and if fever, altered metabolism, and body temperature are involved will be crucial to understanding how viruses evolve and emerge from this reservoir.

Given the deep evolutionary conservation of fever, it may seem surprising that there are limited studies evaluating the impact of fever temperature on antiviral protein function. However, there are many challenges to studying the impact of temperature on antiviral gene function. Temperature can have impacts on the pathogen itself, global cellular transcription, protein stability, and cell physiology. These caveats make it difficult to isolate the specific impact of fever temperatures on virus replication. One approach to address this would be using temperature-sensitive virus mutants that can grow across a range of temperatures to isolate fever effects to virus or hosts. Additionally, cells could be treated with IFN, or IFN blockade, at physiological temperatures and then shifted to fever temperatures to control for temperature-dependent expression differences. Additionally, rationally designed mutations that only disrupt protein function during fever, but not basal physiological temperatures, could define impacts of fever on specific antiviral genes. Studies have demonstrated that up to 6% of amino acid substitutions can drive temperature-sensitive phenotypes (Shiraishi et al., 2004). Further, advances in protein thermal stability structure-function prediction algorithms will be needed to aid in these analyses. Screening for temperature-dependent functional residues by deep mutational scanning could elucidate antiviral mechanisms during fever. In vivo experiments can capture impacts of fever on antiviral gene function (Lane et al., 2018). However, it is difficult to disentangle fever’s pleiotropic effects in vivo. Combinations of in vivo and reductionist systems will be needed to identify fever-dependent mechanisms of antiviral genes. As evidence of the antiviral effects of fever, viruses have evolved fever antagonists (Rakus et al., 2017). In systems where fever antagonists are active, mutations would need to be made to prevent antagonism to identify fever-dependent antiviral mechanisms. Virus suppression of fever would only be functional in whole organisms, and in vitro systems could be used to determine virus–host interactions at fever temperatures.

Fever is an evolutionarily conserved response to pathogens, which provides survival benefits to hosts from insects to mammals. Fever does not come without costs. A 1°C rise in body temperature requires a 10–12.5% increase in metabolic rate, which can be energetically taxing for the host (Kluger, 1979; Muchlinski, 1985). Behavioral fever also comes at the risk for increased predation (Otti et al., 2012). Despite these drawbacks, fever has been maintained across many independent branches of life and may have shaped the evolution of intrinsic antiviral defenses. ISGs may have evolved to function better at increased temperatures. While this is speculative and a challenge to study, new computational and high-throughput molecular methodologies will aid in testing these hypotheses. Temperature changes between basal and fever levels are small, especially when considering the extreme range of temperatures of life (extremophiles can be active from −15°C to 120°C [Cowen, 2004; Raymond-Bouchard et al., 2018]). Even within a species, there can be temperature changes across sex, life span, anatomical location, and time of day or year. However, evolutionary conservation and that selection for antiviral gene function may occur at fever temperatures suggest that small improvements in protein stability or function could have been selected over 600 million years of fever’s existence. Host fever responses may also have an impact on the evolution of the pathogen. Interestingly, pathogens that evolve to replicate at fever temperatures may be less fit when transmitting to individuals with baseline body temperatures (Clint and Fessler, 2016).

The average human body temperature has been decreasing over time, likely due to improved hygiene and infectious disease interventions (Protsiv et al., 2020). Does this put us at greater risk for infection as our ISGs evolved to function at higher temperatures? Mice raised in specific pathogen–free environments also have lower body temperatures compared with those with normalized infection histories (Hild et al., 2021). Could this impact interpretation of antiviral gene function in the most prevalent animal model of infection? There is a strong link between human genetics and the outcome of infectious diseases (Casanova and Abel, 2024). Most variants in the human population are likely neutral, and methods are used to down select on those that may be the most deleterious. These currently do not take temperature into account but could. Variants may exist that impact responses at fever temperature but have no impact at basal temps. These would not be prioritized for further study under current screening cutoffs. Validation studies of mutant antiviral gene function performed at 37°C would also miss genes with altered function at fever temperature. If fever has driven the selection of antiviral genes, then we currently have a significant blind spot in our understanding of which antiviral genes control infections and potentially their mechanisms of action.

I would like to thank Drs. Lauren Aguado, Christopher Brooke, Jean-Laurent Casanova, Emily Hemann, and Frances Shepherd for their thoughtful comments on this piece.

This work was supported by the University of Minnesota Institute on Infectious Diseases and the National Institutes of Health grant R01AI173043.

Author contributions: Ryan A. Langlois: conceptualization, funding acquisition, writing—original draft, review, and editing.