Identification of Coronaviruses in Migratory Birds
In order to minimize the risk of missing coronavirus strains in the samples, this study utilized two sets of screening primers specifically designed for CoVs, conducting an in-depth investigation of 5,263 samples from migratory birds. A total of 317 samples were found to be CoV positive, representing 6.0% of the total samples. (Table S2). These positive samples were primarily distributed across three orders: Anseriformes (n = 265), Charadriiformes (n = 97), and Pelecaniformes (n = 10), encompassing 4 families, 17 genera, and 32 species of birds. Notably, the Anseriformes such as common teals (n = 81), mallards (n = 101), spot-billed duck (n = 37), and Eurasian wigeons (n = 11); the Charadriiformes like great knots (n = 43) and whimbrels (Numenius phaeopus, n = 20); and the Pelecaniformes such as little egrets (Egretta garzetta, n = 4) had a higher number of positive samples (Fig. 2 and Table S3). It's noteworthy that the detection of positive samples exhibited distinct seasonal characteristics, especially from September to December each year during the autumn and winter seasons, where the detection rate of CoVs was significantly higher than in other months, despite larger sampling volumes in some months like April 2019, February to April 2020, April to May 2021, and August 2022, but with relatively fewer positive samples (Supplementary Fig. 3). Moreover, the positive rates of CoVs across different species revealed that the majority of migratory birds had positive rates ranging between 1.6%-24.3%, for instance, common teals at 10.1% (95% CI: 8.2%-12.3%), mallards at 24.3% (95% CI: 20.5%-28.7%), and great knots at 11.8% (95% CI: 8.9%-15.5%). However, it should be noted that some species such as spot-billed ducks and Eastern great egrets exhibited a positive rate of 100% (95% CI: 64.6%-100% and 43.9%-100% respectively), but due to the small sample sizes, these high positivity rates may not fully reflect the actual situation. (Supplementary Fig. 3 and Table S4).
In this study, a preliminary classification was conducted by performing sequence alignment using the partial RNA-dependent RNA polymerase (RdRp) (440bp) from the 317 positive samples (Table S3). The sequence alignment indicated that the identified CoV samples belonged entirely to the DeltaCoV and GammaCoV genera. Specifically, the newly identified virus strains were assigned into the subgenera Andecovirus (n = 22), Buldecovirus (n = 32), and Herdecovirus (n = 11) of DeltaCoV, as well as the subgenera Brangacovirus (n = 2) and Igacovirus (n = 250) of GammaCoV, with no viruses detected from the subgenus Cegacovirus (Table S5). Notably, the study identified at least two unknown evolutionary lineages, suggesting the need for whole-genome sequence analysis of these viruses for a deeper understanding of their genetic characteristics and evolutionary relationships.
To efficiently obtain the whole-genome sequences of the newly identified CoVs, high-throughput sequencing was conducted. The sequencing generated approximately 4,331 GB of clean data, a total of 12,591,633,958 reads. Among these reads, 21,331,910,202 were identified as virus-related, with 3,439,101,672 being CoV-related. Additional reads associated with other virus families, such as Paramyxoviridae, Orthomyxoviridae, and Picornaviridae, were also detected. Notably, Picobirnaviridae presented the highest detection rate, while the most novel viruses were identified within the family Picornaviridae (Fig. 3a and 3b). Combining the results from the RdRp-based classification and the CoV-related reads obtained from high-throughput sequencing, 131 representative strains were selected for whole-genome amplification. Ultimately, 122 complete CoV genome sequences were obtained (Table S5).
Classification of Migratory Bird-Associated Deltacoronavirus and Gammacoronavirus on a global scale
To maximize our understanding for the diversity and ecological characteristics of all known avian-associated CoVs, this study collected publicly available virus sequences within GammaCoVs and DeltaCoVs. In addition to viral sequences, relevant meta-information concerning the samples, such as host species, sampling time/location and related literature, underwent manual curation as lineated in the ‘Methods’ section. All the information was organized according to our predesigned generic data schema, previously employed in the ZOVER databases14, and stored in a local MySQL system to form the background datasets (http://www.mgc.ac.cn/cgi-bin/ZOVER/aCoV/main.cgi) (Supplementary Fig. 4). As of May 24, 2024, the database includes 16,491 sequences identified from more than 300 animal species in approximately 90 counties globally, covering 1,042 complete sequences and 15,449 partial sequences.
Based on the latest CoV classification standards set by the International Committee on Taxonomy of Viruses (ICTV), all known 1,183 complete/partial sequences that contains 5 conserved domains in the ORF1ab region were first identified (Fig. 3c). Compared with public dataset, the phylogenies revealed that the 120 CoV whole-genome sequences obtained here encompass 7 coronavirus species, including 3 newly identified species named Deltacoronavirus_anatis (DuCoV_NL1), Deltacoronavirus_Sandpipers (SandCoV_NL2), and Gammacoronavirus_Anatiscopes (DuCoV_NL3). Additionally, from sequences sourced from chickens in public databases (MK778364 and MK778365), a misclassified virus species was emerged from the water and named Gammacoronavirus_Novel-IBV (GaCoV_NL4). It is particularly noteworthy that DuCoV_NL3, initially thought to belong to Igacovirus under DuCoV_2714 and first found in domestic ducks, was later extensively detected in migratory birds from the orders Anseriformes and Charadriiformes. To date, only one whole-genome sequence (NC_048214) of this virus species is available in public databases, yet this study achieved a breakthrough by obtaining 87 complete genome sequences related to DuCoV_2714. However, strict classification criteria indicated that DuCoV_2714 identified in domestic ducks (NC_048214) and the strains identified in migratory birds should be classified into two distinct species. Despite the close phylogenetic relationship in the RdRp region between DuCoV_NL3 and DuCoV_2714, the genomic differences at the whole-genome level are significant. The circulating strains of DuCoV_2714 in migratory birds are likely all related to DuCoV_NL3, whereas DuCoV_2714 may result from cross-species transmission. Subsequent genomic structure and evolutionary analysis further revealed the differences between DuCoV_NL3 and DuCoV_2714.
Evolutionary History of Migratory Bird-Associated Deltacoronavirus and Gammacoronavirus
To delve into the evolutionary history of CoVs carried by migratory birds, this study initially extracted all available full-length and partial RdRp, and S protein sequences from our database. After 95% de-duplicating IBV and PDCoV sequences, the IQ-TREE 2 software was used, employing the maximum likelihood method, to construct phylogenetic trees. These analyses aimed to unveil the genetic diversity of CoVs within migratory birds and their evolutionary relationships within the GammaCoV and DeltaCoV (Fig. 4a, 4b, 4c). Phylogenetic analysis based on the RdRp indicated that among the 16 classified CoV species within the DeltaCoV and GammaCoV genera, 9 CoV species primarily host migratory birds. These include the DuCoV_NL3 and DuCoV_2714 of the subgenus Igacovirus, BcanCoV_CB17 of the Brangacovirus, DuCoV_NL1 and WiCoV_HKU20 of the Andecovirus, SandCoV_NL2, WECoV_HKU16, and CMCoV_HKU21 of the Buldecovirus, and NHCoV_HKU19 of the Herdecovirus. Analysis based on the S protein phylogenetic tree revealed complex genetic evolutionary relationships between CoVs carried by migratory birds and those from non-migratory birds, including the IBV and GaCoV_NL4 of the Igacovirus, and PoCoV_HKU15 and BulCV_HKU11 of the Buldecovirus in the S protein tree. Furthermore, the phylogenetic tree based on partial RdRp showed two clusters of unclassified viruses within the Igacovirus, one cluster from black-headed gulls (Chroicocephalus ridibundus) of Charadriiformes, named Igacovirus sp1, and another primarily from rock pigeons (Columba livia) of Columbiformes, named Igacovirus sp2. Additionally, a cluster of unclassified viruses from the black-faced spoonbills (Platalea minor) of Pelecaniformes was identified within the subgenus Herdecovirus and named Herdecovirus sp (Fig. 4c and Table S5).
In GammaCoV, this study obtained 87 complete genome sequences of CoVs belonging to DuCoV_NL3 of Igacovirus, with Whole-genome sequence homology ranging from 69.26–100% among them (Table S5 and S7). The closest known CoV related to DuCoV_NL3 is DuCoV_2714 (NC_048214), with the highest whole-genome sequence homology of 75.24% (Table S7). This newly classified CoV species, DuCoV_NL3, along with DuCoV_2714, exhibits a complex genetic evolutionary relationship, primarily hosting migratory birds from the orders Anseriformes and Charadriiformes. Phylogenetic analysis based on the RdRp revealed that DuCoV_NL3 related CoVs and DuCoV_2714 form a closely related evolutionary cluster in RdRp, sharing 94.46%-97.49% sequence homology (Fig. 4a, 4b and Table S8). However, analysis of the S protein tree revealed significant genetic differentiation in DuCoV_NL3 related CoVs based on host diversity, resulting in two distinct clustering groups, DuCoV_NL3 clade1 and clade2. CoVs in DuCoV_NL3 clade1 primarily originate from migratory birds within the order Anseriformes, such as northern pintails (Anas acuta), common teal, spot-billed ducks, and mallards. Meanwhile, DuCoV_NL3 clade2 is divided into Anseriformes and Charadriiformes clusters based on different hosts, with representative hosts including spot-billed ducks and great knots. Notably, IBV related CoVs also formed two evolutionary clusters with host differences in the S protein tree, IBV clade1 and clade2. IBV clade1 is more closely related to both DuCoV_NL3 clade1 and clade2, while IBV clade2 clusters with DuCoV_2714, sharing 58.31%-69.01% sequence homology (Table S9). Although strains related to BcanCoV_CB17 were detected in bean geese (Anser fabalis) and tundra swans (Cygnus columbianus), whole-genome sequences were not obtained. BcanCoV_CB17 strains, primarily found in Canada geese (Branta canadensis), bean geese, and mute swans (Cygnus olor), are divided into two evolutionary clusters in the S protein, BcanCoV_CB17 clade1 and clade2. The former clusters with DuCoV_NL3 clade1, sharing 50.57–61.68% sequence homology, while the latter clusters with IBV clade2, sharing 53.02%-56.73% sequence homology (Table S9). Moreover, the newly classified GaCoV_NL4 related CoVs from domestic fowls (Gallus gallus), while forming an independent branch closer to DuCoV_NL3 in RdRp, cluster with IBV clade1 in the S protein, sharing 60%-95.38% sequence homology (Fig. 4a, 4b and Table S9).
In DeltaCoV, this study obtained three whole-genome sequences of CoVs from the Andecovirus, two of which belong to a newly classified CoV species, DuCoV_NL1, with a whole-genome sequence homology of 78.36% (Fig. 4a, 4b and Table S5, S7). One sequence belongs to WiCoV_HKU20, with a sequence homology of 76.69% to known viruses of this species (Table S5 and S7). CoVs in the Andecovirus, primarily originating from Anseriformes migratory birds such as spot-billed ducks and common teals for DuCoV_NL1 and Eurasian wigeons for WiCoV_HKU20, showed no significant changes in evolutionary clustering in the RdRp and S protein. In Buldecovirus, 24 whole-genome sequences of CoVs were obtained, 21 of which belong to the newly classified SandCoV_NL2, and 3 to WECoV_HKU16 (Table S5). CoVs of SandCoV_NL2, mainly from great knots and ruddy turnstones (Arenaria interpres) of Charadriiformes, shared a whole-genome sequence homology of 75.43%-100% and displayed stable classification trends in the RdRp and S protein trees (Fig. 4a, 4b and Table S7). WECoV_HKU16 related CoVs differentiated into three evolutionary clusters with host differences in the S protein tree, WECoV_HKU16 clade1, clade2, and clade3. WECoV_HKU16 clade1 strains from sharp-tailed sandpipers (Calidris acuminata) clustered with SandCoV_NL2, sharing 71.76%-98.18% sequence homology (Table S8). WECoV_HKU16 clade2 exhibited host diversity, including migratory birds from Charadriiformes and non-migratory birds from Falconiformes and Otidiformes. WECoV_HKU16 clade3's hosts are mainly from Swinhoe's white-eyes (Zosterops simplex) of Passeriformes, clustering with a strain from Eurasian magpies (Pica pica) (MW349841) in PoCoV_HKU15, sharing 71.94% sequence homology (Table S9). No strains related to CMCoV_HKU21, detected in common moorhens and previously found only by a research team in Hong Kong, were found. Its sequence is closest to SandCoV_NL2 in the RdRp tree, with a sequence homology of 85.47%-86.01%, but it shows a closer relationship with non-migratory bird strains from Passeriformes in the S protein tree, including BulCV_HKU11 and some strains in PoCoV_HKU15. In Herdecovirus, six whole-genome sequences were obtained, all belonging to NHCoV_HKU19, with hosts primarily from Pelecaniformes migratory birds such as little egrets, black-crowned night-herons (Nycticorax nycticorax), and Western cattle egrets (Bubulcus ibis). The genetic clustering of CoVs belonging to NHCoV_HKU19 appeared stable in both RdRp and S protein trees, with no divergence in clustering trends (Fig. 4a and 4b).
To trace the ancestral hosts of CoVs within the GammaCoV and DeltaCoV genera and to explain the diversification of these viruses over time, we conducted an analysis using complete RdRp sequences, employing a Bayesian ancestral reconstruction approach (Fig. 4d and Table S5). The analysis with the Maximum Clade Credibilty (MCC) tree indicated that CoVs of the GammaCoV primarily originated from birds of the Gallus genus within the Galliformes order. Over time, these viruses gradually diversified into three subgenera, with virus hosts also diversifying. Notably, the Cegacovirus subgenus comprises only one virus species, BdCoV_HKU22, which shifted hosts from Gallus genus birds to marine mammals of the Delphinapterus subgenus within the Artiodactyla order. In the Igacovirus subgenus, CoVs related to IBV and GaCoV_NL4 primarily host Gallus genus birds. However, the primary hosts of DuCoV_NL3 related CoVs shifted to migratory birds of the Anas genus within the Anseriformes order, and viruses within this species further diversified into migratory birds of the Calidris genus within the Charadriiformes order. It is noteworthy that DuCoV_2714 shares a diversification trend with DuCoV_NL3, but considering its close phylogenetic relationship with IBV in other genomic regions, it is inferred that DuCoV_2714 might have originated from multiple recombination events between DuCoV_NL3 and IBV-related CoVs during evolution. The Brangacovirus has only one virus species, BcanCoV_CB17, whose primary hosts shifted from Gallus genus birds to migratory birds of the Anser genus within the Anseriformes order. Moreover, CoVs of DeltaCoV mainly originated from migratory birds of the Mareca subgenus within the Anseriformes order. Over time, hosts diversified at two temporal points into migratory birds of the Anas genus within the Anseriformes order. After the first diversification into Anas genus migratory birds, further diversification occurred into migratory birds of the Calidris genus within the Charadriiformes order and the Egretta genus within the Pelecaniformes order. CoVs carried by migratory birds of the Calidris genus formed two virus species, SandCoV_NL2 and WECoV_HKU16, with SandCoV_NL2 related CoVs differentiating into migratory birds of the Gallinula genus within the Gruiformes order to form CMCoV_HKU21. In contrast, WECoV_HKU16 diversified into several species of the Passeriformes order, forming three virus genera primarily hosted by non-migratory birds, BulCV_HKU11, MunCV_HKU13, and PoCoV_HKU15. Notably, PoCoV_HKU15-related CoVs have recently diversified into Sus scrofa within the Artiodactyla order. Viruses carried by migratory birds of the Mareca subgenus, during the second diversification into Anas genus migratory birds, formed WiCoV_HKU20, which still primarily hosts Mareca genus migratory birds, while viruses that diversified into Anas genus migratory birds formed DuCoV_NL1.
Recombination Analysis for Migratory Bird-Associated Deltacoronavirus and Gammacoronavirus
Building on the insights from the phylogenetic analyses of the RdRp and S protein trees discussed in the previous section, where potential recombination signals were identified, this section focuses on delineating the recombination characteristics of CoVs in migratory birds. To this end, this study utilized our database to construct phylogenetic trees based on different genomic regions and extracted features such as clustering and genetic distances from these trees. Subsequently, t-SNE was applied to display changes in clustering trends across different genomic regions of these coronaviruses (Fig. 5a and Supplementary Fig. 5). Across multiple regions of the CoV genome, we identified anomalous clustering phenomena within different groups of the same species, between species, and even across subgenera, particularly in the S protein region. This indicates complex genetic recombination relationships among CoVs carried by migratory birds, as well as between migratory and non-migratory birds. For instance, the DuCoV_2714 strain (NC_048214) clusters with the DuCoV_NL3 Group1 strains in the RdRp region, with the DuCoV_NL3 Group4 strains in ORF1a, with the DuCoV_NL3 Group3 strains in the ORF1b, with Infectious Bronchitis Virus (IBV) and BcanCoV_CB17 strains in the S protein, with IBV strains in E protein, and again with the DuCoV_NL3 strains in the M protein and N protein. Further analysis using Simplot revealed changes in sequence consistency across different genomic regions between DuCoV_2714 and strains of DuCoV_NL3, IBV, and BcanCoV_CB17 (Fig. 5b). The identification of DuCoV_2714 in domestic ducks, featuring genomic elements of DuCoV_NL3 from migratory birds and related strains like the domestic chicken's IBV, not only reveals the complex genetic exchange and recombination mechanisms among CoVs across hosts but also underscores the key role of migratory birds in the diversity and transmission dynamics of CoVs.
Transmission Characteristics of Migratory Bird-Associated Deltacoronavirus and Gammacoronavirus
To comprehensively reveal the transmission characteristics of CoVs carried by migratory birds both within and between host species, this study built upon the foundation of 317 positive CoV results, collecting and organizing 2,826 sequences of CoVs belonging to the DeltaCoV and GammaCoV genera from our database. After correcting the host information from the public data, we obtained 3,212 sequences with clear host designation and 2 virus sequences with hosts identified at the genus level (Table S5). Analysis of the data revealed that most CoV species have their preferred host species, such as common teals for DuCoV_NL1 and Eurasian wigeons and Northern shovelers (Anas clypeata) for WiCoV_HKU20 (Supplementary Fig. 6a). However, some CoV species exhibit significant host diversity, such as DuCoV_NL3 and IBV, suggesting these viruses have likely undergone multiple cross-species transmission events. Further analysis indicated that these cross-species transmission events occurred at the levels of animal species, genera, families, and even orders. An examination of the host characteristics of 16 classified virus species within the DeltaCoV and GammaCoV genera revealed that 13 classified CoV species were found in hosts spanning more than two species and genera, 10 virus species were discovered in hosts across more than two families, and 6 virus species were identified in hosts from more than two orders (Fig. 6a and 6b).
After analyzing whether the cross-species transmission of CoVs is associated with migratory birds, it was found that among the 12 classified CoV species that have undergone cross-species transmission, seven of which have migratory birds as their primary hosts, and 3 CoV species primarily hosted by non-migratory birds and then transmitted to migratory birds (Fig. 6a and 6b). Notably, the newly identified DuCoV_NL3-related CoV in this study has been found in 58 host species, marking it as the CoV species with the broadest known range of host species, which includes both migratory and non-migratory birds. Following DuCoV_NL3, WECoV_HKU16 related CoV has the most extensive host range at the order level, circulating among both migratory and non-migratory birds (Fig. 6b and Table S5). Additionally, NHCoV_HKU19 also exhibits circulation among both migratory and non-migratory birds.
To delve deeper into the transmission characteristics of viruses within the DeltaCoV and GammaCoV among different hosts, this study constructed host-virus networks at both the host family and host species levels (Fig. 6c, Supplementary Fig. 6b and 6c). Through network analysis, we discovered that host animals within the families Anatidae, Phasianidae, and Columbidae generate the most connections among different virus species, indicating these hosts play a pivotal role in the cross-species transmission of viruses. Analyzing the unique ecological niches occupied by birds in these three families revealed that birds within Anatidae straddle the ecological niches of migratory birds and poultry, birds in Phasianidae are primarily in the poultry niche, and pigeons in Columbidae occupy an ecological niche between non-poultry domesticated birds and resident birds. Together, they form the interface of contact among migratory birds, resident birds, and poultry. Moreover, the host-virus network at the host species level revealed that viruses related to DuCoV_NL3 exhibit the most significant host diversity, highlighting their potential for transmission among different hosts. In this study, using DuCoV_2714 as an example, a pathogen transmission model of CoVs among migratory birds, resident birds, and poultry was constructed around key hosts.
DuCoV_2714, identified in domestic ducks, exhibits recombination characteristics closely related to the DuCoV_NL3 strain found in wild ducks and the IBV strain in chickens. This formation process reflects, to a certain extent, the transmission dynamics of CoVs between migratory birds and poultry. Here, the formation process of DuCoV_2714 was speculated (Fig. 7). Initially, domestic ducks carrying DuCoV_2714 might have acquired it through contact with migratory birds of the Anatidae family, such as spot-billed ducks or mallards, or possibly domestic pigeons from the family Columbidae could have acquired it from spot-billed ducks or mallards and subsequently transmitted it to domestic ducks upon returning to the farm. Later, in mixed-species rearing environments with chickens, ducks infected with DuCoV_NL3 might have co-infected with IBV carried by chickens, leading to the recombination and formation of the DuCoV_2714 strain. Given that multiple recombination events were detected in the genome sequence of DuCoV_2714, and most recombinant regions show varying genetic distances to known strains of IBV or DuCoV_NL3, such cross-species transmission may have occurred repeatedly over an extended period. Since no strains closely related to DuCoV_2714 at the genomic level have been detected in wild ducks, it remains uncertain whether this strain has further spread among migratory birds. This necessitates enhanced monitoring of viruses carried by migratory birds to more comprehensively analyze the cross-species transmission dynamics and patterns of coronaviruses between migratory birds and poultry.
Factors Influencing Cross-Species Transmission of Migratory Bird-Associated Deltacoronavirus and Gammacoronavirus
To delve deeper into understanding the key factors influencing cross-species transmission among virus species, this study employed Generalized Additive Models (GAMs) for modeling analysis. We used the data on cross-species transmission of viruses at the species, genus, family, and order levels of host as the dependent variable, while the number of each virus species, host diversity (encompassing species, genus, family, and order levels), and the number of migratory birds among the hosts of virus species were used as independent variables. Model evaluation was based on the Akaike Information Criterion (AIC) to determine the best-fitting model (Fig. 8a, 8b and Supplementary Fig. 7a, 7b), where the best-fitting models could respectively explain 98.2%, 97.2%, 95.7%, and 94.5% of the variance in virus cross-species transmission across species, genus, family, and order of host (Fig. 8a, 8b and Supplementary Fig. 7a, 7b, Table S10). Quantile-Quantile Plots (QQ plots) further validated the good performance of the models (Fig. 8c).
The findings of this study indicate a nonlinear relationship between the number of each virus species and the risk of cross-species transmissions. As the number of each virus species increases, the incidence of cross-species transmission initially shows an upward trend, reaching a peak, and then begins to decrease. This phenomenon is consistently observed across four levels of cross-species transmission models (cross-species transmission of host species, genus, family, and order level) (Fig. 8a, 8b and Supplementary Fig. 7a, 7b). Despite the observation that higher diversity of host species associated with a virus (regardless of whether the diversity is at the host species, genus, family, or order level) correlates with an increase in the number of cross-species transmission events (Fig. 8d, Supplementary Fig. 7c, 7d), it is only in the model focusing on cross-species transmission at the host species level that the diversity of species associated with a virus significantly influences cross-species transmission (P < 0.01). Moreover, the models indicate that an increase in the number of migratory bird species serving as hosts for a virus is associated with an increase in cross-species transmission events, although this increase tends to plateau as the number of bird species grows (Fig. 8a, 8b and Supplementary Fig. 7a, 7b). Intriguingly, significance analysis reveals that the increase in the number of host species that are migratory birds significantly impacts cross-species transmission numbers only at cross-species transmission of the host species level (P < 0.001) and at the genus level (P < 0.05) (Table S10).
In the comparative analysis of cross-species, cross-genus, cross-family, and cross-order transmission among various virus species, significant disparities were identified (Fig. 8e, 8f, 8g, 8h). Particularly in cross-species transmission, viruses associated with IBV (0.90 ± 0.69) exhibited a significantly higher number of transmissions compared to BcanCoV_CB17 (0.54 ± 0.46), PoCoV_HKU15 (0.59 ± 0.50), and WECoV_HKU16 (0.42 ± 0.22) (Mean ± std) (p < 0.05, p < 0.001). Additionally, the number of cross-species transmissions by DuCoV_NL3 (0.74 ± 0.55) was significantly higher than that by WECoV_HKU16 (0.42 ± 0.22) (p < 0.001) (Table S11). Similarly, SandCoV_NL2 (0.67 ± 0.41) demonstrated a significantly greater capability for cross-species transmission than WECoV_HKU16 (0.42 ± 0.22) (p < 0.05) (Table S11). Further analysis revealed that viruses in IBV have a significantly greater capacity for transmission across genera, families, and orders of host than WECoV_HKU16 viruses (p < 0.05) (Table S11). These results highlight significant differences in the cross-species transmission capabilities among different virus species, providing crucial scientific evidence for understanding the dynamics of virus cross-species transmission.