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Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro-and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types. [ABSTRACT FROM AUTHOR]

The bacterial microbiome has a major impact on health and can shape metabolism, host tolerance, immune responses, and the outcome of future infections. The bacterial microbiome is highly variable between individuals. Specific pathogen-free animals have reduced microbiome diversity, making it difficult to evaluate the impact of infection-induced microbiome disruption that would be observed in free-living animals, including people. Mice are commonly used as a preclinical model but unfortunately often fail to predict translation success or failure, particularly for immune and infectious disease-targeting therapies. Here, we utilize pet store mouse cohoused "dirty" mice with diverse microbial experience to explore how host variability and infection may be interacting to drive unique microbiome changes. We found that cohoused animals had significantly increased bacterial diversity in the small intestine and cecum but not in the large intestine. There were differentially abundant taxa between clean and dirty animals in all three tissues. After infection with influenza A virus, samples clustered by both housing condition and infection status in the cecum and large intestine, while small intestine samples clustered predominantly by infection. Altogether, these results highlight the differential impact of housing, infection, and interaction between the two in dictating community composition across the gastrointestinal microbiome.IMPORTANCETraditionally housed pathogen-free mouse models do not fully capture the natural variability observed among human microbiomes, which may underlie their poor translationally predictive value. Understanding the difference between pathogen-induced shifts in the bacterial microbiome and natural microbiome variance is a major hurdle to determining bacterial biomarkers of disease. It is also critical to understand how diverse baseline microbiomes may be differentially impacted by infection and contribute to disease. Pet store cohoused "dirty" mice have diverse microbial experiences and microbiomes, allowing us to evaluate how baseline variation, infection, and interaction between the two impact the microbiome.

Zoonotic viruses are an omnipresent threat to global health. Influenza A virus (IAV) transmits between birds, livestock, and humans. Proviral host factors involved in the cross-species interface are well known. Less is known about antiviral mechanisms that suppress IAV zoonoses. We observed CpG dinucleotide depletion in human IAV relative to avian IAV. Notably, human ZAP selectively depletes CpG-enriched viral RNAs with its cofactor KHNYN. ZAP is conserved in tetrapods but we uncovered that avian species lack KHNYN. We found that chicken ZAP does not affect IAV (PR8) or CpG enriched IAV. Human ZAP or KHNYN independently restricted CpG enriched IAV by overexpression in chicken cells or knockout in human cells. Additionally, mammalian ZAP-L and KHNYN also independently restricted an avian retrovirus (ROSV). Curiously, platypus KHNYN, the most divergent from eutherian mammals, was also capable of direct restriction of multiple diverse viruses. We suggest that mammalian KHNYN may be a bona fide restriction factor with cell-autonomous activity. Furthermore, we speculate that through repeated contact between avian viruses and mammalian hosts, protein changes may accompany CpG-biased mutations or reassortment to evade mammalian ZAP and KHNYN., Competing Interests: DECLARATION OF INTERESTS The authors have no competing interests to declare.

When a virus crosses from one host species to another, the consequences can be devastating. However, animal models to empirically evaluate cross-species transmission can fail to recapitulate natural transmission routes, physiologically relevant doses of pathogens, and population structures of naturally circulating viruses. Here, we present a new model of cross-species transmission where deer mice ( Peromyscus maniculatus ) are exposed to the natural virome of pet store mice ( Mus musculus ). Using RNA sequencing, we tracked viral transmission via fecal-oral routes and found the evidence of transmission of murine astroviruses, coronaviruses, and picornaviruses. Deep sequencing of murine kobuvirus revealed tight bottlenecks during transmission and purifying selection that leaves limited diversity present after transmission from Mus to Peromyscus . This work provides a structure for studying viral bottlenecks across species while keeping natural variation of viral populations intact and a high resolution look at within-host dynamics that occur during the initial stages of cross-species viral transmission.IMPORTANCEViral spillover events can have devastating public health consequences. Tracking cross-species transmission in real-time and evaluating viral evolution during the initial spillover event are useful for understanding how viruses adapt to new hosts. Using our new animal model and next generation sequencing, we develop a framework for understanding intrahost viral evolution and bottleneck events, which are very difficult to study in natural transmission settings., Competing Interests: The authors declare no conflict of interest.