Amie J. Eisfeld

Highly pathogenic avian influenza A(H5N1) (HPAI H5N1) viruses of clade 2.3.4.4b have recently been detected in U.S. dairy cattle following multiple spillover events from avian reservoirs. In December 2025, HPAI H5N1 virus was identified in a dairy herd in Wisconsin through the National Milk Testing Strategy. Here, we report the isolation of a clade 2.3.4.4b, genotype D1.1 H5N1 virus, A/dairy cow/Wisconsin/25G05743-001/2025 (WI5743-H5N1), from bulk milk associated with the affected herd, describe its phylogenetic relationships, and assess its pathogenicity in mice. Infectious virus was recovered following blind passage in embryonated chicken eggs. Phylogenetic analysis demonstrated that WI5743-H5N1 is distinct from previously reported D1.1 viruses detected in dairy cattle in Nevada and Arizona, supporting an independent introduction into cattle, and indicating a likely local avian source. Compared with closely related avian viruses, WI5743-H5N1 encoded the mammalian-adapting substitution PB2-E627K and additional amino acid differences in HA, PB1-F2, and NS1. In mice, WI5743-H5N1 replicated efficiently in respiratory tissues and was detectable in the brain but exhibited lower lethality relative to other recent clade 2.3.4.4b, genotype B3.13 viruses. Together, these findings highlight the genetic and phenotypic diversity of HPAI H5N1 viruses infecting dairy cattle and underscore the importance of continued surveillance and functional characterization of emerging strains. IMPORTANCE: Highly pathogenic avian influenza A(H5N1) viruses have recently entered U.S. dairy cattle through multiple spillover events from avian reservoirs, creating new opportunities for viral adaptation in mammals. Here, we describe the isolation and characterization of a clade 2.3.4.4b, genotype D1.1 H5N1 virus from bulk milk collected during a spillover event in Wisconsin in December 2025. Phylogenetic analyses demonstrated that this virus represents an independent introduction into dairy cattle distinct from previously reported D1.1 viruses identified in Nevada and Arizona. Although the virus encoded the mammalian-adapting PB2-E627K substitution, it exhibited comparatively low lethality in mice, highlighting the complexity of mammalian adaptation and pathogenicity in H5N1 viruses. These findings expand current understanding of the genetic and phenotypic diversity of H5N1 viruses infecting dairy cattle and emphasize the importance of continued surveillance and functional characterization of emerging strains.

The outbreak of clade 2.3.4.4b H5N1 viruses among U.S. dairy cattle has raised concerns that sustained circulation among agricultural mammals could facilitate viral adaptation toward efficient human transmission. However, the evolutionary dynamics governing such adaptation remain poorly understood. Here we investigated the evolution of two bovine-derived H5N1 B3.13 genotype viruses during infection and airborne transmission in ferrets, building on prior characterization of their robust replication and inefficient airborne transmission. Within hosts, viral genetic diversity was limited and viruses were subject to genetic drift and weak purifying selection. Transmission, when it occurred, was characterized by stringent bottlenecks that sharply reduced viral genetic diversity. We found no evidence of mammalian adaptation during infection or transmission. Together, these findings indicate that bovine-derived H5N1 viruses face evolutionary constraints during acute mammalian infection and transmission, limiting movement toward enhanced airborne spread. These constraints may help explain why efficient mammalian replication does not necessarily coincide with efficient transmission. Continued circulation of HA clade 2.3.4.4b viruses nevertheless creates repeated opportunities for rare but consequential evolutionary events, underscoring the importance of sustained surveillance and risk mitigation.

Studies on human gut microbiota have recently highlighted a significant decline in bacterial diversity associated with urbanization, driven by shifts toward processed diets, increased antibiotic usage, and improved sanitation practices. This phenomenon has been largely overlooked in the Colombian Amazon, despite rapid urbanization in the region. In this study, we investigate the composition of gut bacterial microbiota and intestinal protozoa and soil-transmitted helminths (STHs) in both urban and rural areas of Leticia, located in the southern Colombian Amazon. Despite their geographic proximity, the urban population is predominantly non-indigenous, while indigenous communities mostly inhabit the rural area, resulting in notable lifestyle differences between the two settings. Our analyses reveal a reduction in bacterial families linked to non-processed diets, such as Lachnospiraceae , Spirochaetaceae , and Succinivibrionaceae , in the urban environment compared to their rural counterparts. Prevotellaceae , typically associated with non-processed food consumption, shows a significantly higher abundance in urban Leticia. STH infections were primarily detected in rural Leticia, while intestinal protozoa were ubiquitous in both rural and urban areas. Both types of parasites were associated with higher gut bacterial richness and diversity. Additionally, microbial metabolic prediction analysis indicated differences in pathways related to unsaturated fatty acid production and aerobic respiration between rural and urban bacterial microbiomes. This suggests a tendency toward changes in the urban microbiota that may lead to increased susceptibility to non-communicable chronic diseases. These findings provide new insights into the impact of urbanization on gut microbiota dynamics in the Amazonian context and underscore the need for further research into any associated health outcomes. IMPORTANCE: Changes in the diversity and composition of gut microbiota in urban populations have been linked to the rise of non-communicable chronic diseases, such as autoimmune conditions, diabetes, and cancer. As developing countries undergo a demographic shift toward increased urbanization, accompanied by changes in diet, housing, and medication use, there is a concerning loss of microbial diversity. Therefore, it is essential to investigate microbiota changes in overlooked populations, such as indigenous communities in the Colombian Amazon basin. A better understanding of local and generalizable changes in gut microbial composition through urbanization may facilitate the development of targeted programs aimed at promoting lifestyle and diet changes to prevent diseases that healthcare systems may be ill-equipped to effectively address.

SUMMARY: Over the past 25 years, there has been an increasing number of mammalian (including human) infections caused by avian influenza A viruses that resulted in mild to severe illnesses. These viruses typically did not spread between mammals through aerosols in nature or in experimental settings. However, recently, this has changed, with several avian influenza A viruses exhibiting aerosol transmissibility among mammals, indicating that these viruses may pose a greater pandemic risk. In this review, we examine the current situation and discuss the mutations that may be necessary for avian influenza A viruses to efficiently replicate in mammals and transmit among them via aerosols.

Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

Innate immunity is protective against viruses, but also can facilitate pathological infection responses. Despite intensive research, our understanding of the mechanisms that regulate innate immunity in virus infection remains incomplete. Systems biology-based data-driven modeling approaches hold substantial promise toward discovery of crucial innate immune signaling regulators, yet model-derived predictions are almost completely unexplored. Here, we carried out systematic experimental validation of candidate regulators predicted by a transcriptional association network model of influenza virus-infected cells. We identified dozens of novel innate immune signaling regulators with potent effects on the replication of influenza and other viruses, and importantly, we established the biological relevance of a validated regulator in vivo . Collectively, these findings aid in clarifying mechanisms of influenza virus pathogenicity and might lead to innovative approaches for treating influenza virus disease. Similar data-driven modeling strategies may be applicable for the study of other pathogen systems or immunological disorders.

Significance: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are of concern, with the P.1 variants dominating in Brazil. Brazil is now seeing a record number of deaths. Here, we report that the pathogenicity in hamsters of a P.1 variant is similar to that of nonvariant SARS-CoV-2. However, it has an expanded host range as shown by its replication in mice. Prior infection with nonvariant SARS-CoV-2 strains efficiently prevented replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. Convalescent sera from patients infected with nonvariants or sera from messenger RNA vaccinees showed comparable neutralization titers among the P.1 and previously circulating strains. These results suggest that previous SARS-CoV-2 infection and vaccines based on the original SARS-CoV-2 will provide some protection against P.1 infection.