The study, led by Xiaoxu Zeng, YuWei Weng, Sheng Ye, and Yifei Nie as co-first authors, with Dayan Wang of the Chinese National Influenza Center as corresponding author, was designed as a “multi-section” surveillance effort. Rather than sampling a single type of location or a single population, the team collected environmental specimens from three distinct categories of poultry-exposure settings—live poultry markets where birds are sold and slaughtered on site, commercial slaughterhouses, and poultry farms and backyard flocks—alongside respiratory specimens from patients hospitalized with pneumonia of unknown etiology, or PUE, a surveillance category used to catch severe respiratory infections whose cause is unclear at admission. The fieldwork ran from December 2021 to December 2023, spanning two full years and capturing both autumn-winter peaks and summer troughs of viral activity.

The laboratory backbone of the study was real-time reverse transcription polymerase chain reaction, or rRT-PCR, a technique that converts viral RNA into DNA and then amplifies and detects it using fluorescent probes. The researchers tested all 8,335 environmental samples for influenza A virus, the broad group that includes all avian influenza subtypes, and then ran subtype-specific assays targeting the H5, H7, H9, and H10 hemagglutinin subtypes. The hemagglutinin protein, the “H” in subtype names such as H5N1 or H9N2, is the viral surface molecule that determines which receptors the virus can bind and, largely, which species it can infect. Among human respiratory specimens, the team screened 840 samples from hospitalized PUE patients.

The headline number from the environmental sampling is striking: 32.2 percent of the 8,335 samples tested positive for influenza A virus. In other words, nearly one in three swabs taken from places where people routinely come into contact with poultry, poultry products, or poultry waste carried detectable avian influenza genetic material. The subtype breakdown was dominated by H9, which was detected in 20.1 percent of all samples—a reminder that this low-pathogenic subtype, which causes mild or asymptomatic disease in birds, is endemic in Chinese poultry production systems. H5, the subtype that includes both highly pathogenic and low-pathogenic variants, accounted for 7.1 percent of samples. H10 appeared only sporadically at 0.2 percent, and H7—the subtype behind severe human H7N9 outbreaks in China between 2013 and 2017—was not detected at all, a result consistent with the effectiveness of China’s poultry vaccination campaigns against that lineage.

Seasonality emerged as a clear pattern. Positive rates for both H5 and H9 were higher during autumn and winter months, mirroring the well-documented winter seasonality of respiratory influenza viruses and possibly reflecting cooler temperatures that prolong viral survival in the environment, along with increased poultry production and trade around winter holidays. Comparing the two study years, the median monthly positive rates for H5 and H9 were lower in 2023 than in 2022, although the researchers note that this downward trend did not reach statistical significance—a distinction that matters, because a non-significant decline could reflect sampling variability rather than a true reduction in viral circulation.

Geography within the poultry supply chain also mattered. H5 and H9 positive rates were generally higher in live poultry markets and slaughterhouses than in farms and backyard flocks. This gradient makes biological and operational sense: markets and slaughterhouses concentrate birds from many sources, creating opportunities for viral amplification and environmental contamination, while farms represent the upstream end of the chain where birds have had fewer opportunities for cross-contamination. The finding reinforces a recurring theme in influenza surveillance—live poultry markets are the critical bottleneck where avian viruses accumulate and where human exposure risk is highest.

The researchers went beyond simple positive rates by comparing specific environmental sample categories within these settings—cutting boards, drinking water, blood sewage, and other surfaces and fluids commonly sampled in poultry environments. Here the results diverged by subtype. H5 showed no statistically significant difference across sample categories (P = 0.06), but H9 did (P = 0.03), with significantly higher median positive rates found on cutting boards and in blood sewage than in drinking water. This detail has practical implications: cutting boards, where raw poultry is chopped at markets and in food preparation, and blood sewage, the contaminated liquid waste generated during slaughter, represent the environmental niches where H9 viruses persist most reliably. The finding suggests that hygiene interventions targeting these specific fomites and waste streams—disinfection of cutting surfaces, proper treatment of slaughter waste—could disproportionately reduce environmental viral load.

To understand the genetic identity and evolutionary relationships of the circulating viruses, the team sequenced the hemagglutinin genes of H5, H9, and H10 viruses detected in the environment and performed phylogenetic analysis, the computational reconstruction of evolutionary family trees from genetic sequence data. All of the viruses belonged to the Eurasian lineage, one of the two great evolutionary branches of avian influenza (the other being the North American lineage), reflecting the movement of viruses among birds across Eurasia. Critically, the H5 viruses clustered in clade 2.3.4.4b, the globally dominant H5 clade responsible for the ongoing panzootic that has devastated wild bird populations and spread to mammalian species on multiple continents. Its continued detection in Chinese poultry environments confirms that this lineage remains entrenched at the human-animal interface in the region, despite vaccination efforts.

Perhaps the most sobering finding came from the phylogenetic comparison between environmental and human viruses. Several H9 viruses recovered from environmental samples were found to be closely related to representative human H9N2 viruses—meaning the viruses circulating in market environments were genetically near neighbors of viruses known to have infected people. H9N2 is considered one of the most important zoonotic influenza lineages because it possesses receptor-binding and other genetic features compatible with human infection, and it serves as an internal gene donor in reassortment events, the process by which co-infecting influenza viruses exchange genome segments to generate novel combinations. Human infections with H9N2 are typically mild and often go undetected, which means the true spillover burden is likely underestimated—a concern amplified by the genetic proximity documented in this study.

Against this backdrop of persistent environmental contamination, the human surveillance results were, in a sense, reassuring. Of the 840 respiratory specimens from hospitalized patients with pneumonia of unknown etiology, 86 tested positive for seasonal influenza viruses—the ordinary human influenza A and B strains that cause annual epidemics. However, none of the 840 specimens tested positive for H5, H7, H9, or H10. No zoonotic spillover events were captured among severe pneumonia cases during the study period, suggesting that whatever low-level human infections with these subtypes may be occurring, they did not translate into hospitalizations with severe pneumonia in these three cities during the surveillance window.

The authors emphasize that their findings should be read as a call for continued vigilance rather than comfort. Multiple avian influenza subtypes continued to circulate in poultry-exposure environments throughout the study, with H5 clade 2.3.4.4b—an H5 lineage with demonstrated pandemic potential—still present, and H9 viruses genetically linked to known human infections persisting at high rates in the very settings where human exposure is most intense. The research team advocates for sustained, multi-site “One Health” surveillance—an integrated approach that monitors human, animal, and environmental health as a connected system—covering both poultry-exposure environments and human populations. Such surveillance, they argue, is essential to track the ongoing evolution and reassortment of avian influenza viruses and to detect zoonotic spillover events early, before they have the chance to seed larger outbreaks.

The study was approved by the Ethics Committee of the National Institute for Viral Disease Control and Prevention, with informed consent obtained from all participants, and was funded in part by China’s National Science and Technology Major Project for the Prevention and Control of Emerging and Major Infectious Diseases, along with China CDC–US CDC collaboration programs and provincial health research grants. As avian influenza viruses continue their global expansion—driven by clade 2.3.4.4b H5 viruses spreading through migratory birds and poultry trade—the study offers both a methodological template and a warning: the environment where humans and poultry meet remains heavily seeded with influenza viruses, and only continuous monitoring at that interface can reveal whether the next subtype to jump the species barrier is already quietly gathering the genetic tools to do so.

Avian influenza, commonly known as bird flu, encompasses a variety of influenza A virus subtypes that primarily infect birds but can occasionally jump to other species, including humans, with sometimes devastating consequences. The global threat posed by these viruses necessitates robust surveillance networks spanning migratory pathways and breeding grounds. However, many remote regions, especially in the global south, have historically remained data deserts due to logistical challenges and limited infrastructure. This study pioneers a paradigm shift by leveraging the rich biological information contained in bird droppings to trace viral presence and dynamics without disturbing avian hosts.

The choice of bird guano as a surveillance tool merits deeper exploration. Excreta from birds provide a concentrated matrix of viral particles shed from the gastrointestinal and respiratory tracts. Environmental stability of these viral fragments can allow for detection days after deposition, offering a non-invasive snapshot of infection prevalence. Crucially, sampling guano circumvents ethical and practical issues linked to capturing wild birds, reduces risk to field personnel, and enables repeated sampling across large and inaccessible areas. The methodological rigor demonstrated in this study includes optimized collection protocols that minimize contamination and degradation, ensuring reliable viral RNA recovery for downstream molecular analyses.

Molecular diagnostics underpin the core of this surveillance approach. Utilizing state-of-the-art quantitative reverse transcription polymerase chain reaction (qRT-PCR), researchers established sensitive and specific assays to detect various subtypes of avian influenza viruses in environmental samples. Importantly, the team validated their methods against conventional direct swab sampling from birds, confirming comparable if not superior detection rates, particularly in areas where bird trapping is impractical. Moreover, next-generation sequencing allowed the decoding of viral genomes from guano, facilitating phylogenetic analysis crucial for mapping viral evolution and potential zoonotic threats.

Fieldwork conducted in diverse remote locations—ranging from dense tropical wetlands to high-altitude migratory stopovers—demonstrates the scalability and adaptability of guano-based surveillance. The researchers engaged local communities and leveraged indigenous ecological knowledge to identify high-density roosting sites, enhancing sampling efficiency while fostering community involvement. This aspect not only enhances data quality but also aligns with ethical imperatives, promoting inclusive scientific practices and emphasizing the one-health concept by bridging wildlife, environment, and human health.

Data emerging from this extensive environmental monitoring revealed intricate patterns of viral shedding varying seasonally and geographically. For instance, higher viral loads corresponded with breeding seasons and migratory congregation events, periods known to facilitate viral spread. Interestingly, in contrast to previous assumptions, certain remote populations showed persistent low-level virus circulation even outside peak migration, suggesting endemic maintenance. These findings challenge existing models that predominantly rely on sporadic outbreak data from more accessible locales, thereby enriching our comprehension of avian influenza ecology at the global scale.

The implications for public health are profound. Early and accurate detection of emerging avian influenza strains in natural reservoirs enables proactive risk assessment and targeted interventions before spillover into domestic poultry or humans occurs. Environmental surveillance via guano sampling offers a promising sentinel system that can complement, and in some contexts replace, more laborious methodologies. The ability to monitor viral evolution in situ enhances our preparedness against potential pandemic strains that may acquire mutations facilitating human transmission.

From a virological standpoint, the study’s use of metagenomics illuminates the broader viral community interacting within avian populations, unveiling co-infections and genetic reassortment events. Reassortment, the exchange of gene segments between different influenza viruses, is a recognized driver of new viral phenotypes with pandemic potential. Detecting these events directly from environmental samples underscores the feasibility of monitoring viral genetic shifts in real time, a critical feature for dynamic global health threats.

Additionally, the research addresses environmental factors influencing virus stability and transmission. Parameters such as temperature, humidity, UV exposure, and substrate composition were systematically evaluated for their impact on viral RNA persistence in guano. This nuanced understanding enables refinement of sampling timing and locations to maximize detection probability, an essential advancement over static one-size-fits-all protocols prevalent in earlier surveillance efforts.

The study also grapples with challenges inherent to field-based molecular surveillance. Variability in viral loads, potential inhibitors in environmental matrices, and the complexity of distinguishing infectious virus particles from residual RNA fragments necessitate advanced quality controls and validation standards. Through exhaustive pilot studies and inter-laboratory comparisons, the researchers established robust operating procedures that can serve as guidelines for broader implementation.

Importantly, the longitudinal data collected foster the construction of predictive models integrating ecological, virological, and environmental variables. These models can forecast hotspots and temporal windows of increased transmission risk, guiding resource allocation. As global climate change alters migratory patterns and habitat distributions, such models are invaluable for anticipating emerging infectious disease risks under shifting ecological landscapes.

The study’s interdisciplinary approach exemplifies modern infectious disease research, combining molecular virology, ecology, environmental science, and community engagement. It underscores the necessity of expanding surveillance beyond classical paradigms to encompass environmental reservoirs, especially in areas underserved by traditional epidemiological infrastructure. By demonstrating the feasibility and value of bird guano surveillance in the global south’s remote regions, it sets a new standard for inclusive, scalable, and sensitive avian influenza monitoring.

Looking ahead, the integration of portable sequencing technologies and artificial intelligence-driven data analytics could further enhance the timeliness and precision of environmental surveillance programs. Real-time in-field diagnostics, coupled with automated data interpretation, would revolutionize outbreak detection and response, enabling rapid containment. Importantly, the lessons learned from this study extend beyond avian influenza, offering a template adaptable to other wildlife-associated viral pathogens with zoonotic potential.

In sum, this pioneering research presents a compelling case for reimagining avian influenza surveillance by focusing on the overlooked yet information-rich matrix of bird guano. It pushes the boundaries of environmental virology, emphasizing sustainability, community partnership, and technological innovation. As the world grapples with emerging infectious threats, such inventive strategies will be indispensable components of a resilient global health defense system.

Subject of Research: Surveillance of avian influenza viruses via analysis of bird guano in remote regions of the global south to understand transmission dynamics.

Article Title: Surveillance of avian influenza through bird guano in remote regions of the global south to uncover transmission dynamics.

Article References:
Wannigama, D.L., Amarasiri, M., Phattharapornjaroen, P. et al. Surveillance of avian influenza through bird guano in remote regions of the global south to uncover transmission dynamics. Nat Commun 16, 4900 (2025). https://doi.org/10.1038/s41467-025-59322-z

Image Credits: AI Generated