Characterization of Viral Diversity in Synanhropic Birds of Southwestern Siberia

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Synanthropic birds, which live in close proximity to humans and domestic animals, can act as crucial bridge hosts for viral transmission between wild and domestic populations. This study aimed to assess the prevalence and diversity of four key viral families—Orthomyxoviridae, Paramyxoviridae, Coronaviridae, and Astroviridae—in synanthropic birds from the south of Western Siberia, an important convergence zone of major migratory flyways for many bird species. Using molecular detection methods, we identified avian coronaviruses (ACoV) and avastroviruses (AAstV), but found no evidence of avian influenza virus (AIV) or avian paramyxoviruses (APMV). Overall, 3.8% of birds tested positive for at least one of the studied viruses, with ACoV detected in 1.4% and AAstV in 2.4% of samples. Phylogenetic analysis revealed that the detected coronaviruses belong to the genus Deltacoronavirus and form a distinct clade with a previously identified virus from North Siberia, suggesting a stable, regional corvid-associated lineage. The detected astroviruses were highly diverse, falling within a broad group of unclassified passerine-associated avastroviruses with phylogenetic links to viruses from Kazakhstan and China, reflecting the region's role as a migratory crossroads. The absence of AIV and APMV may reflect low prevalence at the time of sampling or host-specific factors like low susceptibility or immunocompetence that suppress viral replication. These findings highlight that synanthropic birds in this key ecological region harbor novel and diverse viruses and represent important, though often overlooked, subjects for expanding our understanding of viral diversity in surveillance programs. Figures Figure 1 Figure 2 Figure 3 1. Introduction Avian infectious diseases in both wild and domestic bird populations are a global One Health concern due to their impact on poultry farming, food security, and biodiversity of wild birds. Many avian viruses can spread across species boundaries, including occasional transmission to mammals, making their control a complex challenge [ 1 ]. Synanthropic birds – wild birds that thrive in human-modified environments – frequently come into contact with both wildlife and poultry. This positions them as potential “bridge hosts” that can facilitate bidirectional virus exchange between wild and domestic birds [ 2 – 5 ]. Indeed, species such as Eurasian tree sparrows, European starlings, and house crows have been shown to transmit avian influenza viruses from wild waterfowl to domestic poultry and vice versa [ 6 – 8 ]. These dynamics underscore the need to better understand the viral diversity carried by synanthropic birds in order to assess risks to agriculture and public health. Avian influenza A viruses (AIV) are perhaps the most infamous avian RNA viruses given their ability to cause devastating outbreaks in poultry and occasional zoonotic infections in humans. Wild waterfowl and shorebirds serve as the natural reservoir for a great diversity of influenza A subtypes, and these viruses periodically spill over into domestic bird populations [ 9 – 12 ]. Highly pathogenic avian influenza (HPAI virus) strains (e.g., H5N1, H5N8) continue to emerge, causing mass mortality in poultry and wild birds [ 13 – 15 ]. Synanthropic species can play a role in AIV ecology: for example, passerines and columbids (pigeons and doves) are generally less susceptible to infection, but have occasionally been found carrying HPAI strains during outbreaks [ 16 , 17 ]. House sparrows, tree sparrows, and common starlings have demonstrated susceptibility to various AIV subtypes in experimental studies [ 18 – 20 ], and die-offs of corvids (crows) have been linked to H5N1 outbreaks in poultry [ 21 – 23 ]. These findings illustrate that even though waterfowl are the primary influenza reservoir, synanthropic land birds can become infected and potentially spread influenza viruses at the wild–domestic interface [ 1 , 12 ]. Newcastle disease virus (NDV), or Orthoavulavirus javaense , causes a highly contagious and often lethal disease in poultry. It remains a major economic concern for the poultry industry [ 24 ]. NDV and other avian paramyxoviruses also circulate in wild birds: low-virulence NDV strains have been isolated from waterfowl, shorebirds, and cormorants [ 25 – 28 ]. A distinct variant, pigeon paramyxovirus type 1 (PPMV-1), adapts to Columbiformes and has caused NDV outbreaks in feral and domestic pigeons as well as spillover to wild doves [ 29 – 31 ]. Notably, even some synanthropic passerines (e.g. house sparrows) and corvids have been found seropositive or experimentally susceptible to NDV, suggesting they could contribute to interspecies transmission [ 3 , 32 ]. Urban-adapted birds often exhibit strong immune competence against such viruses, yet they still may carry and shed pathogens without severe disease [ 3 , 33 ]. The ubiquity of paramyxoviruses across bird taxa and the adaptation of certain NDV strains to synanthropic species highlight the need for surveillance in these bridge hosts. Coronaviruses infect a broad range of hosts and include important poultry pathogens. Avian coronavirus infections in domestic birds are exemplified by infectious bronchitis virus (IBV) of chickens, which causes respiratory and renal disease leading to reduced meat and egg production [ 34 , 35 ]. IBV and related gamma-coronaviruses also infect turkeys, pheasants, quail, and ducks [ 36 – 41 ]. Until recently, it was understood that gammacoronaviruses and deltacoronaviruses primarily infect birds, while alphacoronaviruses and betacoronaviruses are mainly found in mammals. However, several exceptions have been identified—for example, gammacoronaviruses have been detected in mammals such as beluga whales and bottlenose dolphins, and deltacoronaviruses in pigs [ 42 ]. Furthermore, recent studies have found coronaviruses in synanthropic birds, including feral pigeons, suggesting a more complex pattern of host range than previously recognized [ 43 , 44 ]. In addition, the Deltacoronavirus genus, initially identified in wild birds, was found to include a virus in pigs (porcine deltacoronavirus), indicating cross-order transmission [ 45 ]. A striking example is the recently discovered pigeon deltacoronavirus (PiDCoV), which is closely related to porcine deltacoronavirus and to a sparrow deltacoronavirus, suggesting that avian coronaviruses may jump between distant hosts under the right conditions [ 4 , 46 , 47 ]. The ability of some avian coronaviruses to evolve and infect mammals underscores their zoonotic potential and highlights the importance of monitoring these viruses in bird populations associated with human habitats. Astroviruses are small, non-enveloped RNA viruses known to cause gastroenteric disease in many mammals and birds. Avian astroviruses (genus Avastrovirus ) have been associated with enteritis, growth depression, and kidney and liver diseases in poultry including chickens, turkeys, ducks, and geese [ 48 – 50 ]. For example, severe outbreaks in ducklings and goslings have been attributed to novel astrovirus strains [ 51 , 52 ]. Despite their presence in a wide range of domestic and wild birds [ 53 – 55 ], astroviruses in synanthropic species are poorly studied. There are only a few reports to date: detections in urban rock pigeons and in wild crows have demonstrated that synanthropic birds can carry avastroviruses [ 56 , 57 ]. Some genetic analyses even suggest past interspecies transmission of astroviruses among different bird orders [ 58 ]. The diversity of avian astroviruses and their propensity for cross-species infection raise concerns that synanthropic birds could serve as reservoirs or mixing vessels for emergent strains [ 56 – 58 ]. In summary, avian RNA viruses from four major families ( Orthomyxoviridae , Paramyxoviridae , Coronaviridae , and Astroviridae ) each exhibit distinct ecological reservoirs and transmission routes. Viruses maintained by waterfowl, such as avian influenza virus, primarily spread across broad geographic areas through migratory flyways and waterborne routes. In contrast, viruses like Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and astroviruses tend to transmit more locally in farms and urban environments through direct contact, aerosols, or fecal contamination. Synanthropic birds, by frequenting both farm environments and natural wetlands or urban refuse sites, may be exposed to a variety of pathogens and subsequently carry them into new contexts. Their role as a bridge between wild and domestic bird populations has been highlighted in ecological studies [ 1 , 12 , 16 ]. Understanding the viral diversity in synanthropic birds is therefore critical. It can provide insight into how viruses might spread to poultry or even jump to mammalian hosts, and it informs surveillance strategies needed to mitigate emerging disease threats in a One Health framework. The south of Western Siberia is a globally important region where the Black Sea-Mediterranean, West Asia-East African, Central Asian and the East Asian-Australasian migratory flyways intersect. This area serves as a crucial stopover and breeding ground for millions of migratory birds, creating a potential "melting pot" for the exchange of pathogens from different continents. While the viral diversity in the waterfowl of this region has been extensively studied [ 59 – 63 ], the virome of the resident synanthropic bird populations, which interact with both migratory birds and local poultry, remains largely uncharacterized. This represents a critical gap in our understanding of regional virus ecology. Therefore, the purpose of this study was to conduct targeted molecular surveillance to assess the prevalence and characterize the genetic diversity of avian influenza viruses, coronaviruses, paramyxoviruses, and astroviruses in key synanthropic wild bird species in the south of Western Siberia. 2. Materials and methods 2.1. Ethic issue The study was approved by the Biomedical Ethics Committee of the FRC FTM, Novosibirsk (protocols No. 2021-10). Samples from Eurasian magpie ( Pica pica ), Eurasian tree sparrow ( Passer montanus ), Eurasian skylark ( Alauda arvensis ), Common starling ( Sturnus vulgarius ) and Rock dove ( Columba livia ) were collected from caught individuals using misnets. Samples from Hooded crow ( Corvus cornix ), Western jackdaw ( Coloeus monedula ) and Rook ( Corvus frugilegus ) were collected during the hunting season under license from the regional ministries of ecology and natural resources as part of the annual collection of biological material (the program for the study of infectious diseases of wild animals, FRC FTM, Novosibirsk). 2.2. Sample collection Cloacal swabs from wild birds in the Karasuk district (Novosibirsk region, 53°37′N, 77°35′E) were collected between April 18 and May 3, 2023, in individual 2 ml tubes containing 1 ml of viral transport medium. For transportation to the laboratory and subsequent analysis, the tubes were stored in liquid nitrogen .[ 64 ]. 2.3. RNA Extraction, Reverse Transcription, and PCR RNA was isolated from cloacal swabs using a column-based RNA extraction kit (Biolabmix, Russia) following manufacture protocol. The resulting RNA was used to detect avian influenza viruses, astroviruses, coronaviruses and avian paramyxoviruses. Avian influenza virus The presence of conservative M gene regions of the influenza A virus was determined by real-time PCR using the Influenza A virus Real-Time RT-PCR kit (Medical-Biological Union LLC, Russia), adapted to detect both human and avian influenza virus. Avian Paramyxoviruses RNA was used in the reverse transcription reaction using the REVERTA-L kit (AmpliSens, Russia). To detect avian paramyxoviruses family-wide oligonucleotides (Table 1 ) were used. Oligonucleotides were diluted to a concentration of 50 pmol/µl. A reaction mixture was prepared using 25 µl of HS-Taq PCR-Color (2×) Master Mix (Biolabmix, Russia), 1 µl of forward and reverse oligonucleotides, and 5 µl of cDNA. Water was then added to achieve a final volume of 50 µl. The reaction mixture was incubated at 95°C for 5 min, then for 40 cycles at 95°C for 15 s, at 41°C for 30 s, at 72°C for 15 s, and then a final extension at 72°C for 7 min. The reaction products were visualized by electrophoresis in 1.5% agarose gel in the gel documentation system "E-Box CX5" (VILBER, Germany). To estimate the amplicon size, a 100-bp DNA ladder DNA marker Step100 (Biolabmix, Russia) was used. Table 1 Amplification primers for virus detection Virus type Name of primer Primer sequence Fragment size (nt) Product Reference Avian astroviruses AAstV-F 5’-GAYTGGACNMGNT WYGAYGGNACNATNCC-3’ 430 Part of RdRp 1 gene [ 65 ] AAstV-R 5’-YTTNACCCACATNCCRAA-3’ Avian coronaviruses AC-CoV-F 5′-GGTTGGGATTATCCWAARTGT G-3′ 602 Part of RdRp 1 gene [ 66 ] AC-CoV-R 5′-TGYTGTGARCAAAAYTCRTG-3′ Avian paramyxoviruses PMX-1 5′-GAR-GGI-YII-TGY-CAR-AAR-NTN-TGG-AC-3′ 121 Part of RdRp 1 gene [ 67 ] PMX-2 5′-TIA-YIG-CWA-TIR-IYT-GRT-TRT-CNC-C-3′ 1 - RdRP, RNA-dependent RNA-polymerase. Astroviruses Reverse transcription was performed using the RNAScribe kit (Biolabmix, Russia), namely, 4 µl of RT buffer, 3 µl of dH2O, 1 µl of reverse transcriptase, 2 µl of reverse oligonucleotide (50 pmol) and 10 µl of RNA. The reaction took place under the following conditions: 50°С for 40 min, 85°С for 5 min. The obtained cDNA was used in real-time PCR to identify avian astroviruses. The oligonucleotides used are presented in Table 1 . PCR was performed in a 50 µL reaction system consisting of 5 µL sterile water, 25 µL BioMaster HS-qPCR SYBR Blue 2× (Biolabmix, Russia), 5 µL forward and reverse primers (diluted to 10 pmol/µL) and 10 µL cDNA. Amplification involved a 4 min denaturation step at 95°C, followed by 44 cycles of denaturation at 95°C for 30 s, primer annealing for 30 s, and extension at 72°C for 45 s, after a final extension step of 3 min at 72°C. In this work, touchdown PCR was used, so the annealing temperature was reduced each 3 cycles by 2°C (from 58 to 46°C). The stage 95°C for 30 s.; 46°C for 30 s.; 72°C for 45 s”. was repeated for 26 cycles. The reaction products were visualized by agarose gel electrophoresis as described above. Samples in which amplicons of the target size were detected were prepared for Sanger sequencing. Avian coronaviruses Reverse transcription using 100 pmol of reverse oligonucleotide (Table 1 ), 4 µL of RT buffer, 1 µL of reverse transcriptase, and 10 µL of RNA was implemented using a RNAScribe kit (Biolabmix, Russia) in the following conditions: 50°C for 40 min, 85°C for 5 min. PCR with a SYBR Blue HS-qPCR kit (Biolabmix, Russia) was carried out mixing 1 µL of H2O, 5 µL of BiomasterMix, 1 pM forward, and 1 pM reverse oligonucleotides. PCR in the following conditions was implemented: at 95°C for 30 s, annealing for 30 s and at 72°C for 45 s following final elongation at 72°C for 3 min. Annealing temperature decreased every 3 cycles by 2°C from 60°C to 48°C. The main phase at the 48°C annealing temperature had 30 cycles. Melting curves were constructed according to the following conditions: at 95°C for 15 s, at 60°C for 1 min, and at 60°C to 95°C, with 0.05°C/s increments. The reaction products were visualized by agarose gel electrophoresis as described above. Samples in which amplicons of the target size were detected were prepared for Sanger sequencing. 2.4 Fragment sequencing of avian astroviruses and coronaviruses Recognized amplicons were excised from the gel and extracted using the GeneJet Gel Extraction kit (Thermo Fisher Scientific, USA) according to the protocol. The isolated amplified DNA was used for Sanger sequencing reaction using the BigDye V3.1 kit (Thermo Fisher Scientific, USA). Fragments were sequenced using an ABI 3130XL Genetic Analyser (Applied Biosystems, USA) in accordance with the manufacturer's instructions at the Genomics Core Facility of the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS, Novosibirsk, Russia). 2.5. Phylogenetic analysis Phylogenetic analysis was performed to determine the species affiliation of viruses. For the analysis, the most relevant and reference sequences were added in the NCBI GenBank database. Sequence alignment was performed using the ClustalW algorithm in the MEGA X program. Maximum likelihood phylogenetic trees were constructed using the T92 + G + I (for astroviruses) and GTR + G (for coronaviruses) substitution models with a bootstrap test for 1000 iterations (the search for the substitution model was performed using MEGA X). 3. Results 3.1. Virus Detection A total of 209 cloacal swabs were collected from eight species of wild birds representing five families ( Corvidae , Passeridae , Alaudidae , Sturnidae , Columbidae ) in the Karasuk district of the Novosibirsk region (Fig. 1 ). The majority of samples were from species in the Corvidae family (n = 161), with rooks ( Corvus frugilegus ) being the most sampled species (n = 145). Using PCR-based methods, we detected avian astroviruses (AAstV) and avian coronaviruses (ACoV). No samples were positive for avian influenza virus (AIV) or avian paramyxoviruses (APMV). A total of five samples were positive for AAstV, and three were positive for ACoV. Overall, 3.8% of birds tested positive for at least one of the studied viruses (8/209). The virus-specific prevalence was 2.4% (5/209) for AAstV and 1.4% (3/209) for ACoV. All positive samples originated from birds of the order Passeriformes, specifically from the families Corvidae (hooded crow, rook) and Passeridae (Eurasian tree sparrow). The distribution of positive samples among the species is detailed in Table 2 . Table 2 Sample size and results of virus detection of birds in the Novosibirsk region Host Family Host Species Samples N (% of total) AAstV positive ACoV positive APMV positive AIV positive Corvidae Hooded crow ( Corvus cornix ) 14 (6.7%) 2 1 0 0 Western jackdaw ( Coloeus monedula ) 1 (0.5%) 0 0 0 0 Rook ( Corvus frugilegus ) 145 (69.4%) 2 2 0 0 Eurasian magpie ( Pica pica ) 1 (0.5%) 0 0 0 0 Passeridae Eurasian tree sparrow ( Passer montanus ) 4 (1.9%) 1 0 0 0 Alaudidae Eurasian skylark ( Alauda arvensis ) 2 (0.9%) 0 0 0 0 Sturnidae Common starling ( Sturnus vulgaris ) 18 (8.6%) 0 0 0 0 Columbidae Rock dove ( Columba livia ) 24 (11.5%) 0 0 0 0 Total (8 spp.) 209 (100%) 5 3 0 0 3.2. Phylogenetic Analysis of Avastroviruses Partial RdRp gene sequences, ranging from 293 to 379 nucleotides due to quality variations, were successfully obtained from all five AAstV-positive samples. BLAST analysis confirmed that all detected viruses belong to the genus Avastrovirus (GenBank accession numbers: PQ789233–PQ789237). Phylogenetic analysis demonstrated that these viruses do not group with any of the three officially designated Avastrovirus species ( Avastrovirus galli , Avastrovirus intestini , Avastrovirus meleagridis ). Instead, they form a distinct, large clade composed exclusively of unclassified astroviruses previously detected in birds of the order Passeriformes (Fig. 2 ). Within this passerine-associated clade, the viruses from this study exhibited further substructuring. Two isolates from hooded crows and one from a rook grouped together with a reference sequence from a house crow, forming a Corvidae-specific subgroup. One of the remaining rook isolates clustered with a group of viruses from Kazakhstan, while the Eurasian tree sparrow isolate was most closely related to astroviruses identified in passerine birds from China. 3.3. Phylogenetic Analysis of Avian Coronaviruses Partial RdRp gene sequences (602 nt) were obtained for the three ACoV-positive samples, which were isolated from two rooks and one hooded crow (GenBank accession numbers: PQ661954 – PQ661956). Phylogenetic analysis revealed that all three sequences belong to the genus Deltacoronavirus (Fig. 3 ). The isolates from this study formed a monophyletic group with a high bootstrap support value, clustering tightly with a deltacoronavirus sequence previously obtained from a hooded crow in North Siberia in 2020(GenBank ON6058555). This grouping forms a distinct clade that can be described as "Siberian Corvidae Unclassified Deltacoronavirus". Discussion This study represents the first targeted viral surveillance of synanthropic bird populations in the south of Western Siberia, a region of global significance for avian migration. The investigation revealed the circulation of two important viral groups: deltacoronaviruses and avastroviruses. Specifically, we identified what appears to be a distinct regional lineage of deltacoronaviruses in corvids ( Corvus cornix and C. frugilegus ) and a diverse array of unclassified avastroviruses in both corvids and a Eurasian tree sparrow ( Passer montanus ). An important negative finding was the absence of detectable avian influenza virus and avian paramyxovirus in the 209 samples analyzed, despite sampling species known to be susceptible. The detection of deltacoronaviruses in rooks and hooded crows provides new insight into the viral ecology of this genus. Phylogenetic analysis revealed that our three isolates form a tight, monophyletic cluster with a previously reported deltacoronavirus from a hooded crow in North Siberia. The strong statistical support for this clade, which is not closely related to other known deltacoronaviruses, can suggest that these are not merely incidental infections. Rather, it indicates the presence of a stable, widespread, and potentially corvid-adapted deltacoronavirus lineage circulating across the vast expanse of Siberia.This finding transforms a local detection into evidence for an established host-virus system that has likely persisted over time and space. However, the number of available sequences is limited yet to support clearly this hypothesis. This observation is significant within the broader context of deltacoronavirus biology. The genus is known for its broad host range in birds and mammals. The potential for interspecies transmission has been underscored by recent findings of pigeon deltacoronaviruses that are genetically close to both porcine and sparrow deltacoronavirus strains. The identification of a stable deltacoronavirus lineage circulating in synanthropic corvids—birds that frequently interact with both agricultural landscapes and human settlements—highlights a potential One Health risk pathway that warrants closer scrutiny, particularly in regions with mixed farming systems where poultry and swine may be in proximity. Our detection of avastroviruses in corvids and a Eurasian tree sparrow adds to the growing body of evidence that passerine birds are significant hosts for this viral family. All five astroviruses identified in this study fall within a large, phylogenetically distinct group of "unclassified" astroviruses associated exclusively with the order Passeriformes. While classified astroviruses are known to cause disease in poultry, the pathogenic potential and ecological significance of these unclassified passerine-associated viruses remain largely unknown. The genetic diversity observed even within our small sample of five isolates is remarkable and highlights to the complex epidemiology of these viruses in the study region. The phylogenetic tree revealed at least three distinct transmission dynamics at play. The clustering of AAstV isolates from hooded crows and a rook with a reference house crow sequence suggests a corvid-specific transmission cycle. In contrast, another rook isolate grouped with viruses from Kazakhstan, while the sparrow isolate was most closely related to viruses from China. This linkage between molecular data and large-scale bird ecology suggests that synanthropic birds at this migratory crossroads are not merely passive carriers but actively sample from and contribute to a vast, geographically distinct pool of viral diversity. They appear to be involved in complex, multi-directional transmission networks connecting local ecosystems with distant geographic regions, though they may not be the sole representatives within these networks. The failure to detect AIV or APMV in any of the 209 samples is a scientifically significant result that requires careful interpretation. These viruses are of major concern for poultry health, and synanthropic species like pigeons, crows, and sparrows are known to be susceptible to infection. However, the absence of detection does not necessarily equate to an absence of risk. Experimental studies have shown that some synanthropic species, such as pigeons, exhibit low susceptibility to certain AIV subtypes and demonstrate limited viral shedding, which would make detection in the field challenging [ 19 , 20 , 68 , 69 ]. For paramyxoviruses like NDV, recent research has indicated that urban populations of crows and sparrows can possess high immunocompetence [ 3 ]. This robust immune response could lead to rapid viral clearance or suppression of viral replication to levels below the detection limit of standard PCR assays. Therefore, our negative result likely indicates that active circulation or outbreaks of AIV and APMV were not occurring in these specific populations at the time of sampling. It does not, however, rule out the possibilities of very low prevalence, transient infections with short shedding periods, or the presence of population-level immunity that masks ongoing low-level circulation. Influenza viruses and paramyxoviruses are known to circulate less actively in synanthropic birds [ 2 , 70 ]. The low prevalence reported in the literature may explain their absence in our study, which involved a relatively small sample size. It is important to consider the characteristics of the study area, which is sparsely populated and not highly urbanized, with no large poultry farms. However, the region contains numerous reservoirs supporting a high density of migrating waterfowl, alongside private farms raising chickens and ducks. These factors are critical for understanding viral ecology in this context. This study provides a valuable first snapshot of the viral landscape in synanthropic birds of this region, but several limitations should be acknowledged. The cross-sectional design captures only a single time point and may miss temporal or seasonal fluctuations in viral prevalence [ 55 , 70 ]. The sample sizes for some species were small, which limits the statistical power to detect rare viruses. Furthermore, the reliance on cloacal swabs for detection may miss pathogens that preferentially replicate in the respiratory tract or other tissues. Future research should aim to address these limitations. Longitudinal surveillance is needed to understand how viral prevalence changes seasonally and in response to the influx of migratory birds[ 16 , 70 , 71 ]. Full-genome sequencing of the detected deltacoronaviruses and astroviruses would provide much deeper evolutionary insights into their origin, adaptation [ 47 , 51 ]. Crucially, serological surveys should be conducted to assess the history of exposure and determine the level of population immunity to viruses like AIV and APMV [ 72 ]. Such data would be invaluable for interpreting negative PCR results and for building more accurate risk assessment models. Conclusion This study provides the first molecular evidence for the circulation of novel deltacoronavirus and diverse avastrovirus lineages within the synanthropic bird community of Southwestern Siberia. Our findings identify a potentially stable, regional clade of corvid-associated deltacoronaviruses and demonstrate that local passerines are integrated into large-scale astrovirus transmission networks linked to avian ecology. This work underscores the critical role of these "bridge host" species as reservoirs of viral diversity at a key ecological nexus. Continuous and expanded surveillance in these often-overlooked populations is essential for a proactive, evidence-based One Health approach to predicting, preventing, and managing future viral emergence events at the wildlife-livestock-human interface. Declarations Competing interests: The authors declare no competing interests. Acknowledgments The authors gratefully appreciate the who contributed to genome sequences information provided to GenBank. The authors also thank the sequencing facility at Genomics Core Facility of the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS, Novosibirsk, Russia) for technical assistance. This work was supported by the following sources: RSF project 23-44-00026, state-funded budget project 225020408196-1. References Caron A, Cappelle J, Cumming GS, et al (2015) Bridge hosts, a missing link for disease ecology in multi-host systems. Vet Res 46:1–11. https://doi.org/10.1186/S13567-015-0217-9/TABLES/2 Shriner SA, Root JJ (2020) A Review of Avian Influenza A Virus Associations in Synanthropic Birds. Viruses 2020, Vol 12, Page 1209 12:1209. https://doi.org/10.3390/V12111209 Habib M, Ul-Rahman A, Zia-ur-Rehman, et al (2023) Comparative immunocompetence and interspecies transmission of avian orthoavulavirus-1 in feral birds originating from rural and urban settings. 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Arch Virol 147:1287–1302. https://doi.org/10.1007/S00705-002-0818-2/METRICS Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Archives of Virology → Version 1 posted Reviewers invited by journal 11 Sep, 2025 Editor assigned by journal 10 Sep, 2025 First submitted to journal 10 Sep, 2025 Editorial decision: Minor Revision 07 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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10:20:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7550152/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7550152/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-026-06534-3","type":"published","date":"2026-02-19T15:58:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91698903,"identity":"971034af-237f-42c4-a991-e95f96f375bd","added_by":"auto","created_at":"2025-09-19 10:06:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":895503,"visible":true,"origin":"","legend":"\u003cp\u003eMap of sampling site with viruses detected in this study. The Novosibirsk region is indicated in green, the Karasuk district is indicated in red (the upper right sector of the figure).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7550152/v1/365ba33102d77c32049996be.png"},{"id":91698904,"identity":"30d1682a-aae6-490f-bdbf-94a70c9e41e7","added_by":"auto","created_at":"2025-09-19 10:06:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":238840,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7550152/v1/c49d099b8151fcddc5d8024a.png"},{"id":91698906,"identity":"b1558498-59cd-4f19-abc3-9f42ce994b8b","added_by":"auto","created_at":"2025-09-19 10:06:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":367091,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7550152/v1/2f0534e7bbb37b48238e69cc.png"},{"id":103251388,"identity":"28da192a-ed3b-4eae-8a25-938e5ad81e81","added_by":"auto","created_at":"2026-02-23 16:08:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1916435,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7550152/v1/c5c63a38-0b55-4d5e-9a33-8a84b1246bf5.pdf"}],"financialInterests":"","formattedTitle":"Characterization of Viral Diversity in Synanhropic Birds of Southwestern Siberia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAvian infectious diseases in both wild and domestic bird populations are a global One Health concern due to their impact on poultry farming, food security, and biodiversity of wild birds. Many avian viruses can spread across species boundaries, including occasional transmission to mammals, making their control a complex challenge [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Synanthropic birds – wild birds that thrive in human-modified environments – frequently come into contact with both wildlife and poultry. This positions them as potential “bridge hosts” that can facilitate bidirectional virus exchange between wild and domestic birds [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e–\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Indeed, species such as Eurasian tree sparrows, European starlings, and house crows have been shown to transmit avian influenza viruses from wild waterfowl to domestic poultry and vice versa [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These dynamics underscore the need to better understand the viral diversity carried by synanthropic birds in order to assess risks to agriculture and public health.\u003c/p\u003e\u003cp\u003eAvian influenza A viruses (AIV) are perhaps the most infamous avian RNA viruses given their ability to cause devastating outbreaks in poultry and occasional zoonotic infections in humans. Wild waterfowl and shorebirds serve as the natural reservoir for a great diversity of influenza A subtypes, and these viruses periodically spill over into domestic bird populations [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Highly pathogenic avian influenza (HPAI virus) strains (e.g., H5N1, H5N8) continue to emerge, causing mass mortality in poultry and wild birds [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Synanthropic species can play a role in AIV ecology: for example, passerines and columbids (pigeons and doves) are generally less susceptible to infection, but have occasionally been found carrying HPAI strains during outbreaks [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. House sparrows, tree sparrows, and common starlings have demonstrated susceptibility to various AIV subtypes in experimental studies [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and die-offs of corvids (crows) have been linked to H5N1 outbreaks in poultry [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings illustrate that even though waterfowl are the primary influenza reservoir, synanthropic land birds can become infected and potentially spread influenza viruses at the wild–domestic interface [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNewcastle disease virus (NDV), or \u003cem\u003eOrthoavulavirus javaense\u003c/em\u003e, causes a highly contagious and often lethal disease in poultry. It remains a major economic concern for the poultry industry [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. NDV and other avian paramyxoviruses also circulate in wild birds: low-virulence NDV strains have been isolated from waterfowl, shorebirds, and cormorants [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e–\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A distinct variant, pigeon paramyxovirus type 1 (PPMV-1), adapts to Columbiformes and has caused NDV outbreaks in feral and domestic pigeons as well as spillover to wild doves [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Notably, even some synanthropic passerines (e.g. house sparrows) and corvids have been found seropositive or experimentally susceptible to NDV, suggesting they could contribute to interspecies transmission [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Urban-adapted birds often exhibit strong immune competence against such viruses, yet they still may carry and shed pathogens without severe disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The ubiquity of paramyxoviruses across bird taxa and the adaptation of certain NDV strains to synanthropic species highlight the need for surveillance in these bridge hosts.\u003c/p\u003e\u003cp\u003eCoronaviruses infect a broad range of hosts and include important poultry pathogens. Avian coronavirus infections in domestic birds are exemplified by infectious bronchitis virus (IBV) of chickens, which causes respiratory and renal disease leading to reduced meat and egg production [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. IBV and related gamma-coronaviruses also infect turkeys, pheasants, quail, and ducks [\u003cspan additionalcitationids=\"CR37 CR38 CR39 CR40\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e–\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Until recently, it was understood that gammacoronaviruses and deltacoronaviruses primarily infect birds, while alphacoronaviruses and betacoronaviruses are mainly found in mammals. However, several exceptions have been identified—for example, gammacoronaviruses have been detected in mammals such as beluga whales and bottlenose dolphins, and deltacoronaviruses in pigs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Furthermore, recent studies have found coronaviruses in synanthropic birds, including feral pigeons, suggesting a more complex pattern of host range than previously recognized [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, the Deltacoronavirus genus, initially identified in wild birds, was found to include a virus in pigs (porcine deltacoronavirus), indicating cross-order transmission [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A striking example is the recently discovered pigeon deltacoronavirus (PiDCoV), which is closely related to porcine deltacoronavirus and to a sparrow deltacoronavirus, suggesting that avian coronaviruses may jump between distant hosts under the right conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The ability of some avian coronaviruses to evolve and infect mammals underscores their zoonotic potential and highlights the importance of monitoring these viruses in bird populations associated with human habitats.\u003c/p\u003e\u003cp\u003eAstroviruses are small, non-enveloped RNA viruses known to cause gastroenteric disease in many mammals and birds. Avian astroviruses (genus \u003cem\u003eAvastrovirus\u003c/em\u003e) have been associated with enteritis, growth depression, and kidney and liver diseases in poultry including chickens, turkeys, ducks, and geese [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e–\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. For example, severe outbreaks in ducklings and goslings have been attributed to novel astrovirus strains [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Despite their presence in a wide range of domestic and wild birds [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e–\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], astroviruses in synanthropic species are poorly studied. There are only a few reports to date: detections in urban rock pigeons and in wild crows have demonstrated that synanthropic birds can carry avastroviruses [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Some genetic analyses even suggest past interspecies transmission of astroviruses among different bird orders [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The diversity of avian astroviruses and their propensity for cross-species infection raise concerns that synanthropic birds could serve as reservoirs or mixing vessels for emergent strains [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e–\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn summary, avian RNA viruses from four major families (\u003cem\u003eOrthomyxoviridae\u003c/em\u003e, \u003cem\u003eParamyxoviridae\u003c/em\u003e, \u003cem\u003eCoronaviridae\u003c/em\u003e, and \u003cem\u003eAstroviridae\u003c/em\u003e) each exhibit distinct ecological reservoirs and transmission routes. Viruses maintained by waterfowl, such as avian influenza virus, primarily spread across broad geographic areas through migratory flyways and waterborne routes. In contrast, viruses like Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and astroviruses tend to transmit more locally in farms and urban environments through direct contact, aerosols, or fecal contamination. Synanthropic birds, by frequenting both farm environments and natural wetlands or urban refuse sites, may be exposed to a variety of pathogens and subsequently carry them into new contexts. Their role as a bridge between wild and domestic bird populations has been highlighted in ecological studies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Understanding the viral diversity in synanthropic birds is therefore critical. It can provide insight into how viruses might spread to poultry or even jump to mammalian hosts, and it informs surveillance strategies needed to mitigate emerging disease threats in a One Health framework. The south of Western Siberia is a globally important region where the Black Sea-Mediterranean, West Asia-East African, Central Asian and the East Asian-Australasian migratory flyways intersect. This area serves as a crucial stopover and breeding ground for millions of migratory birds, creating a potential \"melting pot\" for the exchange of pathogens from different continents. While the viral diversity in the waterfowl of this region has been extensively studied [\u003cspan additionalcitationids=\"CR60 CR61 CR62\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e–\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], the virome of the resident synanthropic bird populations, which interact with both migratory birds and local poultry, remains largely uncharacterized. This represents a critical gap in our understanding of regional virus ecology. Therefore, the purpose of this study was to conduct targeted molecular surveillance to assess the prevalence and characterize the genetic diversity of avian influenza viruses, coronaviruses, paramyxoviruses, and astroviruses in key synanthropic wild bird species in the south of Western Siberia.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Ethic issue\u003c/h2\u003e\u003cp\u003e The study was approved by the Biomedical Ethics Committee of the FRC FTM, Novosibirsk (protocols No. 2021-10). Samples from Eurasian magpie (\u003cem\u003ePica pica\u003c/em\u003e), Eurasian tree sparrow (\u003cem\u003ePasser montanus\u003c/em\u003e), Eurasian skylark (\u003cem\u003eAlauda arvensis\u003c/em\u003e), Common starling (\u003cem\u003eSturnus vulgarius\u003c/em\u003e) and Rock dove (\u003cem\u003eColumba livia\u003c/em\u003e) were collected from caught individuals using misnets. Samples from Hooded crow (\u003cem\u003eCorvus cornix\u003c/em\u003e), Western jackdaw (\u003cem\u003eColoeus monedula\u003c/em\u003e) and Rook (\u003cem\u003eCorvus frugilegus\u003c/em\u003e) were collected during the hunting season under license from the regional ministries of ecology and natural resources as part of the annual collection of biological material (the program for the study of infectious diseases of wild animals, FRC FTM, Novosibirsk).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Sample collection\u003c/h2\u003e\u003cp\u003eCloacal swabs from wild birds in the Karasuk district (Novosibirsk region, 53\u0026deg;37\u0026prime;N, 77\u0026deg;35\u0026prime;E) were collected between April 18 and May 3, 2023, in individual 2 ml tubes containing 1 ml of viral transport medium. For transportation to the laboratory and subsequent analysis, the tubes were stored in liquid nitrogen .[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.3. RNA Extraction, Reverse Transcription, and PCR\u003c/h2\u003e\u003cp\u003eRNA was isolated from cloacal swabs using a column-based RNA extraction kit (Biolabmix, Russia) following manufacture protocol. The resulting RNA was used to detect avian influenza viruses, astroviruses, coronaviruses and avian paramyxoviruses.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAvian influenza virus\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe presence of conservative M gene regions of the influenza A virus was determined by real-time PCR using the Influenza A virus Real-Time RT-PCR kit (Medical-Biological Union LLC, Russia), adapted to detect both human and avian influenza virus.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAvian Paramyxoviruses\u003c/em\u003e\u003c/p\u003e\u003cp\u003eRNA was used in the reverse transcription reaction using the REVERTA-L kit (AmpliSens, Russia). To detect avian paramyxoviruses family-wide oligonucleotides (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were used. Oligonucleotides were diluted to a concentration of 50 pmol/\u0026micro;l. A reaction mixture was prepared using 25 \u0026micro;l of HS-Taq PCR-Color (2\u0026times;) Master Mix (Biolabmix, Russia), 1 \u0026micro;l of forward and reverse oligonucleotides, and 5 \u0026micro;l of cDNA. Water was then added to achieve a final volume of 50 \u0026micro;l. The reaction mixture was incubated at 95\u0026deg;C for 5 min, then for 40 cycles at 95\u0026deg;C for 15 s, at 41\u0026deg;C for 30 s, at 72\u0026deg;C for 15 s, and then a final extension at 72\u0026deg;C for 7 min.\u003c/p\u003e\u003cp\u003eThe reaction products were visualized by electrophoresis in 1.5% agarose gel in the gel documentation system \"E-Box CX5\" (VILBER, Germany). To estimate the amplicon size, a 100-bp DNA ladder DNA marker Step100 (Biolabmix, Russia) was used.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAmplification primers for virus detection\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVirus type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eName of primer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrimer sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFragment size (nt)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eProduct\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAvian astroviruses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAstV-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-GAYTGGACNMGNT\u003c/p\u003e\u003cp\u003eWYGAYGGNACNATNCC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e430\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePart of RdRp\u003csup\u003e1\u003c/sup\u003e gene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAAstV-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026rsquo;-YTTNACCCACATNCCRAA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAvian coronaviruses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAC-CoV-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GGTTGGGATTATCCWAARTGT G-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePart of RdRp\u003csup\u003e1\u003c/sup\u003e gene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAC-CoV-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TGYTGTGARCAAAAYTCRTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAvian paramyxoviruses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePMX-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GAR-GGI-YII-TGY-CAR-AAR-NTN-TGG-AC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePart of RdRp\u003csup\u003e1\u003c/sup\u003e gene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePMX-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TIA-YIG-CWA-TIR-IYT-GRT-TRT-CNC-C-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e- RdRP, RNA-dependent RNA-polymerase.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAstroviruses\u003c/em\u003e\u003c/p\u003e\u003cp\u003eReverse transcription was performed using the RNAScribe kit (Biolabmix, Russia), namely, 4 \u0026micro;l of RT buffer, 3 \u0026micro;l of dH2O, 1 \u0026micro;l of reverse transcriptase, 2 \u0026micro;l of reverse oligonucleotide (50 pmol) and 10 \u0026micro;l of RNA. The reaction took place under the following conditions: 50\u0026deg;С for 40 min, 85\u0026deg;С for 5 min.\u003c/p\u003e\u003cp\u003eThe obtained cDNA was used in real-time PCR to identify avian astroviruses. The oligonucleotides used are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. PCR was performed in a 50 \u0026micro;L reaction system consisting of 5 \u0026micro;L sterile water, 25 \u0026micro;L BioMaster HS-qPCR SYBR Blue 2\u0026times; (Biolabmix, Russia), 5 \u0026micro;L forward and reverse primers (diluted to 10 pmol/\u0026micro;L) and 10 \u0026micro;L cDNA. Amplification involved a 4 min denaturation step at 95\u0026deg;C, followed by 44 cycles of denaturation at 95\u0026deg;C for 30 s, primer annealing for 30 s, and extension at 72\u0026deg;C for 45 s, after a final extension step of 3 min at 72\u0026deg;C. In this work, touchdown PCR was used, so the annealing temperature was reduced each 3 cycles by 2\u0026deg;C (from 58 to 46\u0026deg;C). The stage 95\u0026deg;C for 30 s.; 46\u0026deg;C for 30 s.; 72\u0026deg;C for 45 s\u0026rdquo;. was repeated for 26 cycles.\u003c/p\u003e\u003cp\u003eThe reaction products were visualized by agarose gel electrophoresis as described above. Samples in which amplicons of the target size were detected were prepared for Sanger sequencing.\u003c/p\u003e\u003cp\u003e\u003cem\u003eAvian coronaviruses\u003c/em\u003e\u003c/p\u003e\u003cp\u003eReverse transcription using 100 pmol of reverse oligonucleotide (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 4 \u0026micro;L of RT buffer, 1 \u0026micro;L of reverse transcriptase, and 10 \u0026micro;L of RNA was implemented using a RNAScribe kit (Biolabmix, Russia) in the following conditions: 50\u0026deg;C for 40 min, 85\u0026deg;C for 5 min. PCR with a SYBR Blue HS-qPCR kit (Biolabmix, Russia) was carried out mixing 1 \u0026micro;L of H2O, 5 \u0026micro;L of BiomasterMix, 1 pM forward, and 1 pM reverse oligonucleotides. PCR in the following conditions was implemented: at 95\u0026deg;C for 30 s, annealing for 30 s and at 72\u0026deg;C for 45 s following final elongation at 72\u0026deg;C for 3 min. Annealing temperature decreased every 3 cycles by 2\u0026deg;C from 60\u0026deg;C to 48\u0026deg;C. The main phase at the 48\u0026deg;C annealing temperature had 30 cycles. Melting curves were constructed according to the following conditions: at 95\u0026deg;C for 15 s, at 60\u0026deg;C for 1 min, and at 60\u0026deg;C to 95\u0026deg;C, with 0.05\u0026deg;C/s increments.\u003c/p\u003e\u003cp\u003eThe reaction products were visualized by agarose gel electrophoresis as described above. Samples in which amplicons of the target size were detected were prepared for Sanger sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Fragment sequencing of avian astroviruses and coronaviruses\u003c/h2\u003e\u003cp\u003eRecognized amplicons were excised from the gel and extracted using the GeneJet Gel Extraction kit (Thermo Fisher Scientific, USA) according to the protocol. The isolated amplified DNA was used for Sanger sequencing reaction using the BigDye V3.1 kit (Thermo Fisher Scientific, USA). Fragments were sequenced using an ABI 3130XL Genetic Analyser (Applied Biosystems, USA) in accordance with the manufacturer's instructions at the Genomics Core Facility of the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS, Novosibirsk, Russia).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Phylogenetic analysis\u003c/h2\u003e\u003cp\u003ePhylogenetic analysis was performed to determine the species affiliation of viruses. For the analysis, the most relevant and reference sequences were added in the NCBI GenBank database. Sequence alignment was performed using the ClustalW algorithm in the MEGA X program. Maximum likelihood phylogenetic trees were constructed using the T92\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;I (for astroviruses) and GTR\u0026thinsp;+\u0026thinsp;G (for coronaviruses) substitution models with a bootstrap test for 1000 iterations (the search for the substitution model was performed using MEGA X).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Virus Detection\u003c/h2\u003e\u003cp\u003eA total of 209 cloacal swabs were collected from eight species of wild birds representing five families (\u003cem\u003eCorvidae\u003c/em\u003e, \u003cem\u003ePasseridae\u003c/em\u003e, \u003cem\u003eAlaudidae\u003c/em\u003e, \u003cem\u003eSturnidae\u003c/em\u003e, \u003cem\u003eColumbidae\u003c/em\u003e) in the Karasuk district of the Novosibirsk region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe majority of samples were from species in the \u003cem\u003eCorvidae\u003c/em\u003e family (n\u0026thinsp;=\u0026thinsp;161), with rooks (\u003cem\u003eCorvus frugilegus\u003c/em\u003e) being the most sampled species (n\u0026thinsp;=\u0026thinsp;145). Using PCR-based methods, we detected avian astroviruses (AAstV) and avian coronaviruses (ACoV). No samples were positive for avian influenza virus (AIV) or avian paramyxoviruses (APMV).\u003c/p\u003e\u003cp\u003eA total of five samples were positive for AAstV, and three were positive for ACoV. Overall, 3.8% of birds tested positive for at least one of the studied viruses (8/209). The virus-specific prevalence was 2.4% (5/209) for AAstV and 1.4% (3/209) for ACoV. All positive samples originated from birds of the order Passeriformes, specifically from the families \u003cem\u003eCorvidae\u003c/em\u003e (hooded crow, rook) and \u003cem\u003ePasseridae\u003c/em\u003e (Eurasian tree sparrow). The distribution of positive samples among the species is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSample size and results of virus detection of birds in the Novosibirsk region\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHost Family\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHost Species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSamples N (% of total)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAAstV positive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eACoV positive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAPMV positive\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAIV positive\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCorvidae\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHooded crow (\u003cem\u003eCorvus cornix\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14 (6.7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWestern jackdaw (\u003cem\u003eColoeus monedula\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (0.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRook (\u003cem\u003eCorvus frugilegus\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e145 (69.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEurasian magpie (\u003cem\u003ePica pica\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (0.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePasseridae\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEurasian tree sparrow (\u003cem\u003ePasser montanus\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (1.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAlaudidae\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEurasian skylark (\u003cem\u003eAlauda arvensis\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (0.9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSturnidae\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCommon starling (\u003cem\u003eSturnus vulgaris\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18 (8.6%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eColumbidae\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRock dove (\u003cem\u003eColumba livia\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24 (11.5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal (8 spp.)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e209 (100%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Phylogenetic Analysis of Avastroviruses\u003c/h2\u003e\u003cp\u003ePartial RdRp gene sequences, ranging from 293 to 379 nucleotides due to quality variations, were successfully obtained from all five AAstV-positive samples. BLAST analysis confirmed that all detected viruses belong to the genus \u003cem\u003eAvastrovirus\u003c/em\u003e (GenBank accession numbers: PQ789233\u0026ndash;PQ789237). Phylogenetic analysis demonstrated that these viruses do not group with any of the three officially designated \u003cem\u003eAvastrovirus\u003c/em\u003e species (\u003cem\u003eAvastrovirus galli\u003c/em\u003e, \u003cem\u003eAvastrovirus intestini\u003c/em\u003e, \u003cem\u003eAvastrovirus meleagridis\u003c/em\u003e). Instead, they form a distinct, large clade composed exclusively of unclassified astroviruses previously detected in birds of the order Passeriformes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within this passerine-associated clade, the viruses from this study exhibited further substructuring. Two isolates from hooded crows and one from a rook grouped together with a reference sequence from a house crow, forming a Corvidae-specific subgroup. One of the remaining rook isolates clustered with a group of viruses from Kazakhstan, while the Eurasian tree sparrow isolate was most closely related to astroviruses identified in passerine birds from China.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Phylogenetic Analysis of Avian Coronaviruses\u003c/h2\u003e\u003cp\u003ePartial RdRp gene sequences (602 nt) were obtained for the three ACoV-positive samples, which were isolated from two rooks and one hooded crow (GenBank accession numbers: PQ661954 \u0026ndash; PQ661956). Phylogenetic analysis revealed that all three sequences belong to the genus \u003cem\u003eDeltacoronavirus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The isolates from this study formed a monophyletic group with a high bootstrap support value, clustering tightly with a deltacoronavirus sequence previously obtained from a hooded crow in North Siberia in 2020(GenBank ON6058555). This grouping forms a distinct clade that can be described as \"Siberian Corvidae Unclassified Deltacoronavirus\".\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study represents the first targeted viral surveillance of synanthropic bird populations in the south of Western Siberia, a region of global significance for avian migration. The investigation revealed the circulation of two important viral groups: deltacoronaviruses and avastroviruses. Specifically, we identified what appears to be a distinct regional lineage of deltacoronaviruses in corvids (\u003cem\u003eCorvus cornix\u003c/em\u003e and \u003cem\u003eC. frugilegus\u003c/em\u003e) and a diverse array of unclassified avastroviruses in both corvids and a Eurasian tree sparrow (\u003cem\u003ePasser montanus\u003c/em\u003e). An important negative finding was the absence of detectable avian influenza virus and avian paramyxovirus in the 209 samples analyzed, despite sampling species known to be susceptible.\u003c/p\u003e\u003cp\u003eThe detection of deltacoronaviruses in rooks and hooded crows provides new insight into the viral ecology of this genus. Phylogenetic analysis revealed that our three isolates form a tight, monophyletic cluster with a previously reported deltacoronavirus from a hooded crow in North Siberia. The strong statistical support for this clade, which is not closely related to other known deltacoronaviruses, can suggest that these are not merely incidental infections. Rather, it indicates the presence of a stable, widespread, and potentially corvid-adapted deltacoronavirus lineage circulating across the vast expanse of Siberia.This finding transforms a local detection into evidence for an established host-virus system that has likely persisted over time and space. However, the number of available sequences is limited yet to support clearly this hypothesis.\u003c/p\u003e\u003cp\u003eThis observation is significant within the broader context of deltacoronavirus biology. The genus is known for its broad host range in birds and mammals. The potential for interspecies transmission has been underscored by recent findings of pigeon deltacoronaviruses that are genetically close to both porcine and sparrow deltacoronavirus strains. The identification of a stable deltacoronavirus lineage circulating in synanthropic corvids\u0026mdash;birds that frequently interact with both agricultural landscapes and human settlements\u0026mdash;highlights a potential One Health risk pathway that warrants closer scrutiny, particularly in regions with mixed farming systems where poultry and swine may be in proximity.\u003c/p\u003e\u003cp\u003eOur detection of avastroviruses in corvids and a Eurasian tree sparrow adds to the growing body of evidence that passerine birds are significant hosts for this viral family. All five astroviruses identified in this study fall within a large, phylogenetically distinct group of \"unclassified\" astroviruses associated exclusively with the order Passeriformes. While classified astroviruses are known to cause disease in poultry, the pathogenic potential and ecological significance of these unclassified passerine-associated viruses remain largely unknown.\u003c/p\u003e\u003cp\u003eThe genetic diversity observed even within our small sample of five isolates is remarkable and highlights to the complex epidemiology of these viruses in the study region. The phylogenetic tree revealed at least three distinct transmission dynamics at play. The clustering of AAstV isolates from hooded crows and a rook with a reference house crow sequence suggests a corvid-specific transmission cycle. In contrast, another rook isolate grouped with viruses from Kazakhstan, while the sparrow isolate was most closely related to viruses from China. This linkage between molecular data and large-scale bird ecology suggests that synanthropic birds at this migratory crossroads are not merely passive carriers but actively sample from and contribute to a vast, geographically distinct pool of viral diversity. They appear to be involved in complex, multi-directional transmission networks connecting local ecosystems with distant geographic regions, though they may not be the sole representatives within these networks.\u003c/p\u003e\u003cp\u003eThe failure to detect AIV or APMV in any of the 209 samples is a scientifically significant result that requires careful interpretation. These viruses are of major concern for poultry health, and synanthropic species like pigeons, crows, and sparrows are known to be susceptible to infection. However, the absence of detection does not necessarily equate to an absence of risk.\u003c/p\u003e\u003cp\u003eExperimental studies have shown that some synanthropic species, such as pigeons, exhibit low susceptibility to certain AIV subtypes and demonstrate limited viral shedding, which would make detection in the field challenging [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. For paramyxoviruses like NDV, recent research has indicated that urban populations of crows and sparrows can possess high immunocompetence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This robust immune response could lead to rapid viral clearance or suppression of viral replication to levels below the detection limit of standard PCR assays. Therefore, our negative result likely indicates that active circulation or outbreaks of AIV and APMV were not occurring in these specific populations at the time of sampling. It does not, however, rule out the possibilities of very low prevalence, transient infections with short shedding periods, or the presence of population-level immunity that masks ongoing low-level circulation. Influenza viruses and paramyxoviruses are known to circulate less actively in synanthropic birds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The low prevalence reported in the literature may explain their absence in our study, which involved a relatively small sample size.\u003c/p\u003e\u003cp\u003eIt is important to consider the characteristics of the study area, which is sparsely populated and not highly urbanized, with no large poultry farms. However, the region contains numerous reservoirs supporting a high density of migrating waterfowl, alongside private farms raising chickens and ducks. These factors are critical for understanding viral ecology in this context.\u003c/p\u003e\u003cp\u003eThis study provides a valuable first snapshot of the viral landscape in synanthropic birds of this region, but several limitations should be acknowledged. The cross-sectional design captures only a single time point and may miss temporal or seasonal fluctuations in viral prevalence [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The sample sizes for some species were small, which limits the statistical power to detect rare viruses. Furthermore, the reliance on cloacal swabs for detection may miss pathogens that preferentially replicate in the respiratory tract or other tissues. Future research should aim to address these limitations. Longitudinal surveillance is needed to understand how viral prevalence changes seasonally and in response to the influx of migratory birds[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Full-genome sequencing of the detected deltacoronaviruses and astroviruses would provide much deeper evolutionary insights into their origin, adaptation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Crucially, serological surveys should be conducted to assess the history of exposure and determine the level of population immunity to viruses like AIV and APMV [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Such data would be invaluable for interpreting negative PCR results and for building more accurate risk assessment models.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first molecular evidence for the circulation of novel deltacoronavirus and diverse avastrovirus lineages within the synanthropic bird community of Southwestern Siberia. Our findings identify a potentially stable, regional clade of corvid-associated deltacoronaviruses and demonstrate that local passerines are integrated into large-scale astrovirus transmission networks linked to avian ecology. This work underscores the critical role of these \"bridge host\" species as reservoirs of viral diversity at a key ecological nexus. Continuous and expanded surveillance in these often-overlooked populations is essential for a proactive, evidence-based One Health approach to predicting, preventing, and managing future viral emergence events at the wildlife-livestock-human interface.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests:\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors gratefully appreciate the who contributed to genome sequences information provided to GenBank. The authors also thank the sequencing facility at Genomics Core Facility of the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS, Novosibirsk, Russia) for technical assistance. This work was supported by the following sources: RSF project 23-44-00026, state-funded budget project 225020408196-1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCaron A, Cappelle J, Cumming GS, et al (2015) Bridge hosts, a missing link for disease ecology in multi-host systems. Vet Res 46:1\u0026ndash;11. https://doi.org/10.1186/S13567-015-0217-9/TABLES/2\u003c/li\u003e\n\u003cli\u003eShriner SA, Root JJ (2020) A Review of Avian Influenza A Virus Associations in Synanthropic Birds. 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Journal of Veterinary Medical Science 84:378\u0026ndash;389. https://doi.org/10.1292/JVMS.21-0608\u003c/li\u003e\n\u003cli\u003eTrogu T, Bellini S, Canziani S, et al (2024) Surveillance for Avian Influenza in Wild Birds in the Lombardy Region (Italy) in the Period 2022\u0026ndash;2024. Viruses 16:1668. https://doi.org/10.3390/V16111668/S1\u003c/li\u003e\n\u003cli\u003eStanislawek WL, Wilks CR, Meers J, et al (2002) Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand. Arch Virol 147:1287\u0026ndash;1302. https://doi.org/10.1007/S00705-002-0818-2/METRICS\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7550152/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7550152/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInfectious diseases in birds represent a significant threat to poultry economies, wild bird biodiversity, and public health, underscoring the importance of surveillance within a One Health framework. Synanthropic birds, which live in close proximity to humans and domestic animals, can act as crucial bridge hosts for viral transmission between wild and domestic populations. This study aimed to assess the prevalence and diversity of four key viral families\u0026mdash;Orthomyxoviridae, Paramyxoviridae, Coronaviridae, and Astroviridae\u0026mdash;in synanthropic birds from the south of Western Siberia, an important convergence zone of major migratory flyways for many bird species. Using molecular detection methods, we identified avian coronaviruses (ACoV) and avastroviruses (AAstV), but found no evidence of avian influenza virus (AIV) or avian paramyxoviruses (APMV). Overall, 3.8% of birds tested positive for at least one of the studied viruses, with ACoV detected in 1.4% and AAstV in 2.4% of samples. Phylogenetic analysis revealed that the detected coronaviruses belong to the genus \u003cem\u003eDeltacoronavirus\u003c/em\u003e and form a distinct clade with a previously identified virus from North Siberia, suggesting a stable, regional corvid-associated lineage. The detected astroviruses were highly diverse, falling within a broad group of unclassified passerine-associated avastroviruses with phylogenetic links to viruses from Kazakhstan and China, reflecting the region's role as a migratory crossroads. The absence of AIV and APMV may reflect low prevalence at the time of sampling or host-specific factors like low susceptibility or immunocompetence that suppress viral replication. These findings highlight that synanthropic birds in this key ecological region harbor novel and diverse viruses and represent important, though often overlooked, subjects for expanding our understanding of viral diversity in surveillance programs.\u003c/p\u003e","manuscriptTitle":"Characterization of Viral Diversity in Synanhropic Birds of Southwestern Siberia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 10:06:38","doi":"10.21203/rs.3.rs-7550152/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2025-09-11T18:31:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T03:15:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-09-10T04:57:01+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor Revision","date":"2025-09-08T03:25:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c561940c-6413-472b-8ff6-8a1d35ebbf27","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:05:35+00:00","versionOfRecord":{"articleIdentity":"rs-7550152","link":"https://doi.org/10.1007/s00705-026-06534-3","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2026-02-19 15:58:50","publishedOnDateReadable":"February 19th, 2026"},"versionCreatedAt":"2025-09-19 10:06:38","video":"","vorDoi":"10.1007/s00705-026-06534-3","vorDoiUrl":"https://doi.org/10.1007/s00705-026-06534-3","workflowStages":[]},"version":"v1","identity":"rs-7550152","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7550152","identity":"rs-7550152","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}