Experimental studies in guinea pigs demonstrated that influenza virus transmission is strongly modulated by temperature and humidity. A number of epidemiological studies have followed up on these findings and revealed robust associations between influenza incidence in temperate regions and local conditions of humidity and temperature, offering a long-awaited explanation for the wintertime seasonality of influenza in these locales. Despite recent progress, important questions remain as to the mechanism(s) by which humidity and/or temperature affects transmission.

Influenza A viruses (IAV) have caused more documented global pandemics in human history than any other pathogen^1,2. High pathogenicity avian influenza (HPAI) viruses belonging to the H5N1 subtype are a leading pandemic risk. Two decades after H5N1 “bird flu” became established in poultry in Southeast Asia, its descendants have resurged³, setting off an H5N1 panzootic in wild birds that is fueled by (a) rapid intercontinental spread, reaching South America and Antarctica for the first time^4,5; (b) fast evolution via genomic reassortment⁶; and (c) frequent spillover into terrestrial^7,8 and marine mammals⁹. The virus has sustained mammal-to-mammal transmission in multiple settings, including European fur farms^10,11, South American marine mammals^12–15, and US dairy cattle^16–19, raising questions about whether humans are next. Historically, swine are considered optimal intermediary hosts that help avian influenza viruses (AIV) adapt to mammals before jumping to humans²⁰. However, the altered ecology of H5N1 has opened the door to new evolutionary pathways. Could dairy cattle, farmed mink, or South American sea lions serve as new mammalian gateways to humans? Here we explore the molecular and ecological factors driving H5N1’s sudden expansion in host range and assess the likelihood of different zoonotic pathways leading to an H5N1 pandemic.

The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

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 are urgently needed that are efficacious against major drivers of influenza-like disease including influenza viruses and respiratory syncytial virus (RSV). 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 origin. 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 inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36-56%, 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/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAV in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burden 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 a promising candidate for future clinical development as a treatment option for influenza-like diseases.

Reassortment is fundamental to the evolution of influenza viruses and plays a key role in the generation of epidemiologically significant strains. Previous studies indicate that reassortment is restricted by segment mismatch, arising from functional incompatibilities among components of two viruses. Additional factors that dictate the efficiency of reassortment remain poorly characterized. Thus, it is unclear what conditions are favorable for reassortment and therefore under what circumstances novel influenza A viruses might arise in nature. Herein, we describe a system for studying reassortment in the absence of segment mismatch and exploit this system to determine the baseline efficiency of reassortment and the effects of infection dose and timing. Silent mutations were introduced into A/Panama/2007/99 virus such that high-resolution melt analysis could be used to differentiate all eight segments of the wild-type and the silently mutated variant virus. The use of phenotypically identical parent viruses ensured that all progeny were equally fit, allowing reassortment to be measured without selection bias. Using this system, we found that reassortment occurred efficiently (88.4%) following high multiplicity infection, suggesting the process is not appreciably limited by intracellular compartmentalization. That co-infection is the major determinant of reassortment efficiency in the absence of segment mismatch was confirmed with the observation that the proportion of viruses with reassortant genotypes increased exponentially with the proportion of cells co-infected. The number of reassortants shed from co-infected guinea pigs was likewise dependent on dose. With 10⁶ PFU inocula, 46%–86% of viruses isolated from guinea pigs were reassortants. The introduction of a delay between infections also had a strong impact on reassortment and allowed definition of time windows during which super-infection led to reassortment in culture and in vivo. Overall, our results indicate that reassortment between two like influenza viruses is efficient but also strongly dependent on dose and timing of the infections. Reassortment is the process by which influenza viruses, which carry RNA genomes comprising eight segments, exchange genetic material. Reassortment of the genome segments of two differing influenza strains has the potential to vastly increase the diversity of circulating influenza viruses. Despite its importance to influenza virus evolution, the frequency with which reassortment occurs in a cell or an animal infected with two or more variant viruses is unclear. Toward determining how readily reassortment can occur, we assessed the incidence of reassortment during experimental infection in cultured cells and in guinea pigs. We found that reassortment can occur with high efficiency in both systems, but that that efficiency is dependent on i) the dose of each virus added to the cells or taken up by the host and ii) the relative timing with which each virus infects. These results suggest that influenza A virus reassortment may be more prevalent in nature than one might expect based on the results of surveillance studies.

Influenza A viruses are constantly changing. This change accounts for seasonal epidemics, infrequent pandemics, and zoonotic outbreaks. A major mechanism underlying the genetic diversification of influenza A virus is reassortment of intact gene segments between coinfecting viruses. This exchange is possible because of the segmented nature of the viral genome. Here, I first consider the constraints and drivers acting on influenza A virus reassortment, including the likelihood of coinfection at the host and cellular levels, mixing and assembly of heterologous gene segments within coinfected cells, and the fitness associated with reassortant genotypes. I then discuss the implications of reassortment for influenza A virus evolution, including its classically recognized role in the emergence of genetically “shifted” pandemic strains as well as its potential role as a catalyst of genetic drift.