Wild birds play a significant role in maintaining the natural circulation of numerous pathogens, including some with zoonotic potential. In fact, it is estimated that approximately 20% of zoonotic pathogens of relevance for human health can be transmitted by wild birds (Woolhouse et al., 2012). One of the most prominent examples is influenza A viruses, where wild birds, primarily waterfowl and shorebirds (i.e., the orders Anseriformes and Charadriiformes), serve as natural reservoirs and a source of large virus subtype and strain diversity (Swayne, 2008). In these hosts, avian influenza viruses (AIV) representing 17 of the 19 known subtypes (H1–H16 and H19) have been detected (Swayne, 2008; Olsen et al., 2006; Bellido-Martín et al., 2026). In addition, influenza A viruses have a high mutation rate and a segmented genome that promotes reassortment, which makes them prone to cross host species barriers (Swayne, 2008). One worrying example is the current near-global epizootic of highly pathogenic avian influenza (HPAI) in wild birds, caused by a H5N1 virus that infects a range of waterfowl and seabirds and with spillover infections in poultry, domestic and wild carnivores, marine mammals, and ruminants (Peacock et al., 2025), underscoring the risk of emergence of a pandemic strain (Bellido-Martín et al., 2026; Lowen et al., 2025) and highlighting the substantial economic impact AIVs have on health and agricultural sectors (Alexakis et al., 2024; Seeger et al., 2021).

Birds other than Anseriformes and Charadriiformes have received significantly less attention regarding their role in influenza virus circulation, including the Passeriformes, despite it being the most diverse avian order (comprising over 6,000 species in approximately 140 families), present in literally every ecosystem, and accounting for around 60% of the global bird population (Williams et al., 2023). Some passerine species are abundant, and several are synanthropic (Shriner and Root, 2020), living in close association with human-modified environments (particularly species from families such as Corvidae, Hirundinidae, Sturnidae, Turdidae, Passeridae, and Fringillidae), which may enhance the risk of pathogen exchange between humans, domestic birds, animals, and birds (Gibb et al., 2020). Moreover, in seasonal environments, a large proportion of Passeriformes species are migratory, potentially capable of transporting pathogens along the migration and contributing to the spread of disease into new geographic areas.

The role of Passeriformes in the ecology of influenza viruses remains a controversial topic (Causey and Edwards, 2008; Peterson et al., 2008; Cumming et al., 2011). Some researchers assert that passerines do not participate in, nor play any role in, the ecology of AIVs (Morishita et al., 1999; Munster et al., 2007), while others argue that they may indeed play a role (Shriner and Root, 2020; Peterson et al., 2008; Chang et al., 2014; Fuller et al., 2010; Kou et al., 2005; Lipkind et al., 1982; Račnik et al., 2008). So far only limited experimental evidence exists, but infection experiments have demonstrated that various low- and highly pathogenic avian influenza virus strains can infect some passerine species, confirming the virus’s ability to replicate in their bodies. Experimental infection studies using two low pathogenic AI strains showed that European starlings Sturnus vulgaris could be infected, shed virus, and seroconvert (Qin et al., 2011). Experiments with Red-billed queleas Quelea quelea and blackcaps Sylvia atricapilla shows virus reproduction, virus shedding after oculo-oronasally inoculation of HPAI H5N1 (Breithaupt et al., 2011) as well as American robins Turdus migratorius after experimental infection of HPAI H5N2 and H5N8 (Root et al., 2018). Given their abundance in farm environments, passerines have been hypothesized as potential “bridge species” allowing virus transmission among different bird groups (Caron et al., 2014), including domestic poultry, wild waterfowl, chickens, and turkeys (Ayala et al., 2020). Literature data indicates that approximately 0.1% of passerines had detectable antibodies to influenza viruses (Slusher et al., 2014), although other reports cite much higher seropositivity levels ranging from 6 to 76% (Hadipour et al., 2011). Virus isolation studies have shown an average prevalence of 0.5% among passerines (Slusher et al., 2014), while PCR-based detection rates ranged from 0.1–1% (Fuller et al., 2010; Slusher et al., 2014; Hesterberg et al., 2009; Cappelle et al., 2012; Kou et al., 2009; Williams et al., 2012) to 2.3–17.5% in other studies (Peterson et al., 2008; Cumming et al., 2011; Qin et al., 2011; Borovská et al., 2011; Gronesova et al., 2008; Gronesova et al., 2008; Thinh et al., 2012), and in some cases as high as 50% (Fuller et al., 2010). Most positive results of isolation (77%) were associated with sampling of birds in an unnatural setting (recognized poultry outbreaks of LPAI or HPAI, live-bird markets, or when samples only included peridomestic species). A minority of the AIV isolations (23%) were from terrestrial birds in natural settings. For PCR surveillance most of positive samples were obtained from a natural setting but some of them were associated with outbreaks (Slusher et al., 2014). New wave of broad-scale surveillance of wild birds, including Passeriformes, has intensified during the global panzootic of H5 highly pathogenic avian influenza (HPAI) since 2020. During this period, between 1.1 and 6.5% of Passeriformes tested positive for influenza virus by PCR, and in most cases these detections involved H5 HPAI viruses (European Food Safety Authority, European Centre for Disease Prevention and Control, 2022; Günther et al., 2023).

Research on the ecology of influenza viruses in wild birds in Ukraine began in the early 2000s and has primarily focused on wild waterfowl and shorebirds (Muzyka et al., 2012; Muzyka et al., 2016; Muzyka et al., 2016; Muzyka et al., 2019). Circulation of this pathogen among other ecological bird groups has received little attention. Currently, the order Passeriformes in Ukraine includes 165 species that are widely distributed and inhabit a variety of ecosystems, including urbanized and synanthropic environments. Given the current epizootic situation, the widespread circulation of HPAI viruses among wild birds, poultry, and mammals, and the potential threat to human health, interest has grown in identifying new sources and reservoirs that may contribute to the natural circulation of pathogens (Williams et al., 2023). In this context, we have renewed our focus on wild passerine birds. This article summarizes the results of 20 years of research on Passeriformes in Ukraine.

The results of our 20-year-long serological, virological, and PCR surveillance of Passeriformes in Ukraine confirm a low-level circulation of influenza A virus among these birds. In total, 65 passerine species (of the 165 registered species in Ukraine), representing a variety of migratory statuses and ecological characteristics, were examined. The study encompassed diverse regions of Ukraine and included samples collected during different stages of the birds’ life cycle (migration, breeding, and wintering). Overall, only a small proportion of the tested birds showed the positive results for influenza A virus: 1.2% of blood serum samples, 8.9% of egg yolk samples, 0.15% from virus isolation, and 3.6% by PCR. Of the 20 families studied, only eight tested positive for influenza A virus: Corvidae (serologically), Emberizidae (serologically), Muscicapidae (serologically, virologically, PCR), Sylviidae (serologically, PCR), Paridae (serologically, yolks, PCR), Passeridae (serologically, yolks, PCR), Fringillidae (PCR), Panuridae (PCR). In our studies, conducted using different methods, we also obtained divergent results. Virological examinations showed a low positivity rate in Passeriformes (0.15%) compared to PCR results, where the proportion of positive samples reached 3.6%. In our view, there are several explanations for this. First, PCR is a more sensitive and specific method than virological techniques (Qian et al., 2025). In addition, virological studies detect only the circulation of viable influenza viruses capable of replication in laboratory biological systems. Similar discrepancies between the results of virological testing and PCR have also been reported by other researchers in wild birds (Qin et al., 2011; Kim et al., 2019). Given the fact that influenza viruses of subtypes H1, H3, and H7 are of epidemiological importance to human health, their monitoring is essential. Subtypes H1 and H3 cause seasonal influenza epidemics in humans; moreover, the first fatal human infection with an avian influenza H3N8 virus has recently been reported (Yang et al., 2022). Influenza viruses of the H7 subtype are also capable of causing human disease, ranging from conjunctivitis to severe pneumonia with fatal outcomes (Fouchier et al., 2004). Therefore, surveillance of these influenza virus subtypes, especially in new and atypical natural hosts, is of great importance. In our study, we did not identify any relationship between the H1, H3, and H7 influenza viruses circulating among Passeriformes in Ukraine and human influenza viruses of the same subtypes. Nevertheless, our findings highlight the need for continued research and more in-depth investigation of the genetic and antigenic characteristics of viruses circulating in atypical hosts.

Our findings are consistent with other studies indicating that certain Passeriformes families are more frequently associated with the potential transmission of influenza viruses (Williams et al., 2023). Williams R., summarizing data on multiple avian pathogens (including influenza), identified the following families as among the most frequently infected of them: Turdidae (which includes widespread and abundant species such as Common Blackbirds and European Robins), Estrildidae, Fringillidae (with many common species), Passeridae, Corvidae, Sturnidae, and Paridae, many of which are widely distributed and include synanthropic species (Callaghan et al., 2021). In the United States, during 2005–2008, a study of 22 passerine species (comprising over 4,000 samples) found 0.8% PCR-positive for AIVs (Fuller et al., 2010). Five species tested positive, with prevalence ranging from 9.0 to 50%, which is in line with our prevalence estimates for certain species. In China, a study conducted during 2005–2007 found that 2.3% of Passeriformes from the families Emberizidae, Motacillidae, Muscicapidae, Paridae, Passeridae, Sylviidae, Timaliidae, and Zosteropidae were AIV PCR-positive (Peterson et al., 2008). Among these, 4.8% samples from migratory species and 1.8% from non-migratory were positive. The difference in prevalence between open-habitat and forest birds was minimal: 2.9% vs. 2.4%, respectively. None of the tested samples were positive for the H5 subtype. In our study, seroprevalence was ranging from 1.96 to 12.5% per species However, according to some published sources, seroprevalence among synanthropic Passeriformes may reach 6–79% (Slusher et al., 2014; Hadipour et al., 2011). At the same time, other studies have reported no positive samples: for example, research conducted in Northern Europe between 1998 and 2006 involving over 4,500 Passeriformes from 16 families yielded no positive detections for influenza A virus (Munster et al., 2007).

Analyzing the results of our studies in Ukraine, we detected influenza virus antibodies in birds of the family Corvidae, which are widely distributed both globally and within Ukraine. These birds are generally omnivorous and are frequently observed near poultry and livestock farms (Winkler et al., 2020). Although previous studies (Shriner and Root, 2020) did not detect influenza antibodies in Corvidae, our findings revealed the presence of antibodies in a small number of individuals inhabiting areas in close proximity to poultry farms. At the same time, we did not detect the virus itself or any PCR-positive samples from Corvidae in our investigations. However, influenza virus infections (mainly HPAI H5) have been reported in Corvidae in India (Nagarajan et al., 2010), Bangladesh (Khan et al., 2014), and Germany (Globig et al., 2017). These findings support the certain involvement of Corvidae in the outbreaks of HPAI, although data on their role in the ecology of LPAI viruses remain limited (Ferrazzi et al., 2007).

In analyzing the obtained results, we will take a closer look at several species and families of Passeriformes and their ecological characteristics that may have epidemiological significance for the circulation of influenza A. We found that 1.42% of House Sparrows Passer domesticus were positive for AIV in PCR, and 10.0% had AIV antibodies in egg yolk samples. Positive individuals were detected in urban areas and near poultry farms. This species, along with the Eurasian Tree Sparrow Passer montanus, belongs to the Passeridae family and is a well-known synanthropic bird. Both species are commonly found in urbanized landscapes and frequently inhabit areas in close proximity to farms and human settlements. According to published data comprising 13 studies (Shriner and Root, 2020), the seroprevalence of these sparrows reaches 11.4%, while the PCR-based prevalence is significantly lower, at 0.6%. Additionally, a substantial number of experimental studies have demonstrated successful infections of sparrows with various subtypes of influenza viruses, including both low and highly pathogenic strains, confirming the ability of the virus to at least replicate in these birds (Shriner and Root, 2020). In this study, we detected influenza A virus in Passeriformes that are not typically considered synanthropic. Birds of the family Turdidae generally do not exhibit synanthropic behavior, yet they are commonly observed in urbanized landscapes and in proximity to poultry and livestock farms. In the United States, some Turdidae species (e.g., American Robins and Swainson’s Thrushes Catharus ustulatus) have been reported as seropositive for influenza A virus, with viral RNA also detected via PCR. (Shriner and Root, 2020) Furthermore, experimental infection studies have shown that members of this family are susceptible to HPAI viruses (Shriner and Root, 2020). In our studies conducted in Ukraine, we not only isolated two LPAI viruses (H7N1 and H1N1) from Fieldfare in 2021, but also detected PCR-positive samples from four additional Turdidae species: Common Blackbird, Collared Flycatcher, European Robin, and Song Thrush, confirming the presence of influenza viruses within this family. The family Fringillidae represents a large group of primarily non-synanthropic birds, though some individuals, such as the Eurasian Chaffinch, may display limited synanthropic behavior. There are relatively few reports of influenza detection in this group, some PCR-positive individuals have been reported in California (USA), whereas no positives were found in Germany (Shriner and Root, 2020). In contrast, in our research we identified three Fringillidae species as PCR-positive for influenza A virus, with prevalence rates ranging from 4.8 to 6.6%: European Chaffinch, European Goldfinch, and European Greenfinch.

In summary, while Passeriformes are not considered a new reservoir for influenza A viruses, they remain a poorly studied group, and current evidence suggests the need for further in-depth investigations. Our data clearly confirm the presence of influenza virus in wild passerine birds in Ukraine and underscore the importance of continuing and expanding surveillance to include non-waterfowl and non-aquatic ecological groups of wild birds. Moreover, our findings highlight the need to improve sampling methodologies, ensuring sufficient quantity and volume of materials (e.g., swabs, feces, blood samples) for testing, or to explore alternative sampling and diagnostic approaches, particularly for small-bodied bird species that are often excluded from monitoring due to sampling difficulties. In addition, the observed low seroprevalence in contrast to higher PCR detection rates indicates the necessity for further studies and/or improvements in serological diagnostic tools. While the detection of antibodies, viral isolates, and/or influenza viral RNA provides strong evidence for virus circulation, it does not yet elucidate the ecological dynamics of natural influenza virus circulation in Passeriformes. This gap in knowledge represents a promising direction for future research.

Study limitations. This article presents summarized results of study conducted using various methods over a 20-year period, which may impose certain limitations. First, the aim of the study was not to compare different methods, but rather to present the results obtained through these approaches. The choice of diagnostic methods was determined by the availability of resources, test-kits, and reagents at the time of researcher. Second, during field studies, only a small number of samples were obtained for certain species at some locations. In several cases these few samples tested positive (50–100% prevalence), making it impossible to perform robust statistical analyses on these data. Third, given that most of the birds are small-bodied species, the amount of biological material available for testing (feces/cloacal swabs) was limited. As a result, the quantity of viral RNA in PCR-positive samples was often low, which prevented performing full subtype identification by PCR and conducting whole-genome sequencing. At the same time, these limitations do not affect the overall results or the main conclusions of the study. Rather, they underscore the need for continued research on monitoring the natural circulation of influenza A viruses among atypical hosts, as well as for improving methods for detecting influenza antibodies and viral genetic material.