Molecular Characterization of Complete Genome Sequence of an Avian Coronavirus Identified in a Backyard Chicken from Tanzania.

A complete genome sequence of an avian coronavirus (AvCoV; 27,663 bp excluding 3' poly(A) tail) was determined using nontargeted next-generation sequencing (NGS) of an oropharyngeal swab from a backyard chicken in a live bird market in Arusha, Tanzania. The open reading frames (ORFs) of the Tanzanian strain TZ/CA127/19 are organized as typical of gammaCoVs (Coronaviridae family): 5'UTR-[ORFs 1a/1b encoding replicase complex (Rep1ab) non-structural peptides nsp2-16]-[spike (S) protein]-[ORFs 3a/3b]-[small envelop (E) protein]-[membrane (M) protein]-[ORFs 4a/4c]-[ORFs 5a/5b]-[nucleocapsid (N) protein]-[ORF6b]-3'UTR. The structural (S, E, M and N) and Rep1ab proteins of TZ/CA127/19 contain features typically conserved in AvCoVs, including the cleavage sites and functional motifs in Rep1ab and S. Its genome backbone (non-spike region) is closest to Asian GI-7 and GI-19 infectious bronchitis viruses (IBVs) with 87.2-89.7% nucleotide (nt) identities, but it has a S gene closest (98.9% nt identity) to the recombinant strain ck/CN/ahysx-1/16. Its 3a, 3b E and 4c sequences are closest to the duck CoV strain DK/GD/27/14 at 99.43%, 100%, 99.65% and 99.38% nt identities, respectively. Whereas its S gene phylogenetically cluster with North American TCoVs and French guineafowl COVs, all other viral genes group monophyletically with Eurasian GI-7/GI-19 IBVs and Chinese recombinant AvCoVs. Detection of a 4445 nt-long recombinant fragment with breakpoints at positions 19,961 and 24,405 (C- and N-terminus of nsp16 and E, respectively) strongly suggested that TZ/CA127/19 acquired its genome backbone from an LX4-type (GI-19) field strain via recombination with an unknown AvCoV. This is the first report of AvCoV in Tanzania and leaves unanswered the questions of its emergence and the biological significance.

Gulls as a host for both gamma and deltacoronaviruses.

The coronaviruses (CoV) are ubiquitous pathogens found in wide variety of hosts that constantly pose a threat to human and animal health as a result of their enormous capacity to generate genetic changes. Constant monitoring of virus reservoirs can constitute an early-warning tool and control the spread and evolution of the virus. Coronaviruses are common in wild birds, globally, and birds of the Charadriiformes in particular have been demonstrated to be carriers of delta- (dCoV) and gammacoronaviruses (gCoV). In this paper, we present the genetic characterisation of five CoV strains from black-headed (Chroicocephalus ridibundus) and common (Larus canus) gulls. Whole genome sequence analysis showed high similarity of detected dCoV in gulls to previously identified strains from falcon, houbara, pigeon and gulls from Asia (UAE, China). However, phylogenetic analysis revealed bifurcation within a common branch. Furthermore, the accumulation of numerous amino acid changes within the S-protein was demonstrated, indicating further evolution of dCoV within a single gull host. In turn, phylogenetic analysis for the most of the structural and non-structural genes of identified gCoV confirmed that the strain belongs to the duck coronavirus 2714 (DuCoV2714) species within Igacovirus subgenera, while for the spike protein it forms a separate branch not closely related to any gCoV species known to date. The current study provides new and significant insights into the evolution and diversification of circulating coronaviruses in members of Laridae family.

Genetic diversity, recombination and cross-species transmission of a waterbird gammacoronavirus in the wild.

Viruses emerging from wildlife can cause outbreaks in humans and domesticated animals. Predicting the emergence of future pathogens and mitigating their impacts requires an understanding of what shapes virus diversity and dynamics in wildlife reservoirs. In order to better understand coronavirus ecology in wild species, we sampled birds within a coastal freshwater lagoon habitat across 5 years, focussing on a large population of mute swans (Cygnus olor) and the diverse species that they interact with. We discovered and characterised the full genome of a divergent gammacoronavirus belonging to the Goose coronavirus CB17 species. We investigated the genetic diversity and dynamics of this gammacoronavirus using untargeted metagenomic sequencing of 223 faecal samples from swans of known age and sex, and RT-PCR screening of 1632 additional bird samples. The virus circulated persistently within the bird community; virus prevalence in mute swans exhibited seasonal variations, but did not change with swan age-class or epidemiological year. One whole genome was fully characterised, and revealed that the virus originated from a recombination event involving an undescribed gammacoronavirus species. Multiple lineages of this gammacoronavirus co-circulated within our study population. Viruses from this species have recently been detected in aquatic birds from both the Anatidae and Rallidae families, implying that host species habitat sharing may be important in shaping virus host range. As the host range of the Goose coronavirus CB17 species is not limited to geese, we propose that this species name should be updated to 'Waterbird gammacoronavirus 1'. Non-invasive sampling of bird coronaviruses may provide a tractable model system for understanding the evolutionary and cross-species dynamics of coronaviruses.

Does Avian Coronavirus Co-Circulate with Avian Paramyxovirus and Avian Influenza Virus in Wild Ducks in Siberia?

Avian coronaviruses (ACoV) have been shown to be highly prevalent in wild bird populations. More work on avian coronavirus detection and diversity estimation is needed for the breeding territories of migrating birds, where the high diversity and high prevalence of Orthomyxoviridae and Paramyxoviridae have already been shown in wild birds. In order to detect ACoV RNA, we conducted PCR diagnostics of cloacal swab samples from birds, which we monitored during avian influenza A virus surveillance activities. Samples from two distant Asian regions of Russia (Sakhalin region and Novosibirsk region) were tested. Amplified fragments of the RNA-dependent RNA-polymerase (RdRp) of positive samples were partially sequenced to determine the species of Coronaviridae represented. The study revealed a high presence of ACoV among wild birds in Russia. Moreover, there was a high presence of birds co-infected with avian coronavirus, avian influenza virus, and avian paramyxovirus. We found one case of triple co-infection in a Northern Pintail (Anas acuta). Phylogenetic analysis revealed the circulation of a Gammacoronavirus species. A Deltacoronavirus species was not detected, which supports the data regarding the low prevalence of deltacoronaviruses among surveyed bird species.

Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison.

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and ß showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and ß-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10-5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, ß-, and γ-.

Diversity of Coronaviruses in Wild Representatives of the Aves Class in Poland.

The revealed prevalence of coronaviruses in wild bird populations in Poland was 4.15% and the main reservoirs were birds from orders Anseriformes and Charadriiformes, with a prevalence of 3.51% and 5.59%, respectively. Gammacoronaviruses were detected more often than deltacoronaviruses, with detection rates of 3.5% and 0.7%, respectively. Gammacoronaviruses were detected in birds belonging to six orders, including Anseriformes, Charadriiformes, Columbiformes, Galliformes, Gruiformes, and Passeriformes, indicating a relatively wide host range. Interestingly, this was the only coronavirus detected in Anseriformes (3.51%), while in Charadriiformes, the prevalence was 3.1%. The identified gammacoronaviruses belonged to the Igacovirus and Brangacovirus subgeneras. Most of these were igacoviruses and formed a common phylogenetic group with a Duck Coronavirus 2714 and two with an Avian Coronavirus/Avian Coronavirus9203, while the viruses from the pigeons formed a distinct "pigeon-like" group, not yet officially represented. The presence of deltacoronaviruses was detected in birds belonging to three orders, Charadriiformes, Galliformes, and Suliformes indicating a narrower host range. Most identified deltacoronaviruses belonged to the Buldecovirus subgenus, while only one belonged to Herdecovirus. Interestingly, the majority of buldecoviruses were identified in gulls, and they formed a distinct phylogenetic lineage not represented by any officially ratified virus species. Another separate group of buldecoviruses, also not represented by the official species, was formed by a virus identified in a common snipe. Only one identified buldecovirus (from common pheasant) formed a group with the ratified species Coronavirus HKU15. The results obtained indicate the high diversity of detected coronaviruses, and thus also the need to update their taxonomy (establishing new representative virus species). The serological studies performed revealed antibodies against an infectious bronchitis virus in the sera of white storks and mallards.

Recombinant turkey coronavirus: are some S gene structures of gammacoronaviruses especially prone to exchange?

The objective of the present study was to characterize the atypical turkey coronavirus strain detected in a commercial meat turkey farm in Poland. Using the viral metagenomics approach, we obtained a complete genome sequence of coronavirus, isolated from duodenum samples of animals suffering from acute enteritis. The nearly full-length genome consisted of 27,614 nucleotides and presented a typical genetic organization similar to that of Polish infectious bronchitis virus (IBV) or French turkey coronavirus/guinea fowl coronavirus strains. Phylogenetic analysis based on both the full-length genome and the whole S gene suggested that gCoV/Tk/Poland/G160/2016 is related to turkey and guinea fowl coronavirus and not IBV strains. Sequence analysis of the genome revealed unique genetic characteristics of the present strain, demonstrating that the virus emerged as a result of the exchange of the S gene of IBV GI-19 lineage with the S gene related to the North American turkey coronaviruses and French guinea fowl coronaviruses. Analysis of earlier, similar recombinations suggests that both the S gene structures may be particularly mobile, willingly switching between different gammacoronavirus genomic backbones. The identified recombinant caused a severe course of the disease, which may imply that it is in the first phase of breaking the barriers between different bird species.

Emergence of Avian coronavirus genotype GI-11 in Colombia.

Avian coronavirus (AvCoV/IBV) is a virus with high morbidity, which can cause respiratory, digestive, renal, and reproductive diseases in chickens. Molecular detection and sequencing are the main tool for identification and classification of AvCoV. Thirty-six samples were collected in three broiler farms from different regions in Colombia, due to mortality increase; ten samples were positive using RT-qPCR targeted to the 5' UTR of AvCoV, and one sample was positive and had its partial S gene sequenced. Phylogenetic analysis revealed that this strain belongs to the GI-11 lineage, similar to the Brazilian cluster. Several lineages have already been described in Colombia but, to the best of our knowledge, this is the first time that GI-11 has been detected in this country, which suggests that this subtype may be more widespread in South America than previously thought.

Detection and Characterization of New Coronavirus in Bottlenose Dolphin, United States, 2019.

We characterized novel coronaviruses detected in US bottlenose dolphins (BdCoVs) with diarrhea. These viruses are closely related to the other 2 known cetacean coronaviruses, Hong Kong BdCoV and beluga whale CoV. A deletion in the spike gene and insertions in the membrane gene and untranslated regions were found in US BdCoVs (unrelated to severe acute respiratory syndrome coronavirus 2).

miRNA repertoire and host immune factor regulation upon avian coronavirus infection in eggs.

Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.

Genetic, antigenic and pathogenic characterization of avian coronaviruses isolated from pheasants (Phasianus colchicus) in China.

Two viruses were isolated in 2017 from commercial pheasants with severe clinical signs and mortality in Shandong and Anhui provinces, China, respectively. We examined the pathogenic effects of the viruses in chicken embryos and the size and morphology of the virus particles, performed phylogenetic analysis based on the S1 gene and complete genomic sequences, and examined the antibody responses against infectious bronchitis virus (IBV). The results suggested that the viruses I0623/17 and I0710/17 were avian coronaviruses and were identified as pheasant coronaviruses (PhCoV), with greatest similarity to IBV. Further investigations of the antigenicity, complete genome organization, substitutions in multiple genes, and viral pathogenicity, replication, and shedding in chickens and pheasants showed obvious differences between PhCoV and IBV in terms of antigenicity, and viral pathogenicity, replication, and shedding in chickens and pheasants. The close genetic relationship, but obvious differences between PhCoVs and IBVs suggested the IBVs could be the ancestors of PhCoVs, and that PhCoVs isolated from different outbreaks may have evolved independently from IBVs circulating in the specific region by adaption in pheasants. This hypothesis was supported by analysis of the S1 gene fragments of the two PhCoVs isolated in the current study, as well as PhCoVs isolated in the UK and selected IBV strains. Such analyses indicated different evolution patterns and different tissue tropisms between PhCoVs isolated in different outbreaks. Further studies are needed to confirm this hypothesis by studying the complete genomic sequences of PhCoVs from different outbreaks and the pathogenicity of IBVs in pheasants to compare and clarify the relationships between PhCoVs and IBVs.

Epidemiology of Deltacoronaviruses (δ-CoV) and Gammacoronaviruses (γ-CoV) in Wild Birds in the United States.

Porcine deltacoronavirus (δ-CoV) is the object of extensive research in several countries including the United States. In contrast, the epidemiology of δ-CoVs in wild birds in the US is largely unknown. Our aim was to comparatively assess the prevalence of δ- and γ-CoVs in wild migratory terrestrial and aquatic birds in Arkansas, Illinois, Indiana, Maryland, Mississippi, Missouri, Ohio, Tennessee and Wisconsin. A total of 1236 cloacal/fecal swabs collected during the period 2015-2018 were tested for γ- and δ-CoVs using genus-specific reverse transcription-PCR assays. A total of 61 (4.99%) samples were γ-CoV positive, with up to 29 positive samples per state. In contrast, only 14 samples were positive for δ-CoV (1.14%) with only 1-4 originating from the same state. Thus, unlike previous reports from Asia, γ-CoVs are more prevalent than δ-CoVs in the US, suggesting that δ-CoVs may spread in birds with lower efficiency. This may indicate δ-CoV emerging status and incomplete adaptation to new host species limiting its spread. Phylogenetic analysis of the partial N gene revealed that the newly identified δ-CoV strains were most closely related to the HKU20 (wigeon) strain. Further studies are necessary to investigate the role of aquatic bird δ-CoVs in the epidemiology of δ-CoVs in swine and terrestrial birds.

Discovery and Characterization of Novel RNA Viruses in Aquatic North American Wild Birds.

Wild birds are recognized viral reservoirs but our understanding about avian viral diversity is limited. We describe here three novel RNA viruses that we identified in oropharyngeal/cloacal swabs collected from wild birds. The complete genome of a novel gull metapneumovirus (GuMPV B29) was determined. Phylogenetic analyses indicated that this virus could represent a novel avian metapneumovirus (AMPV) sub-group, intermediate between AMPV-C and the subgroup of the other AMPVs. This virus was detected in an American herring (1/24, 4.2%) and great black-backed (4/26, 15.4%) gulls. A novel gull coronavirus (GuCoV B29) was detected in great black-backed (3/26, 11.5%) and American herring (2/24, 8.3%) gulls. Phylogenetic analyses of GuCoV B29 suggested that this virus could represent a novel species within the genus Gammacoronavirus, close to other recently identified potential novel avian coronaviral species. One GuMPV-GuCoV co-infection was detected. A novel duck calicivirus (DuCV-2 B6) was identified in mallards (2/5, 40%) and American black ducks (7/26, 26.9%). This virus, of which we identified two different types, was fully sequenced and was genetically closest to other caliciviruses identified in Anatidae, but more distant to other caliciviruses from birds in the genus Anas. These discoveries increase our knowledge about avian virus diversity and host distributions.

Guinea Fowl Coronavirus Diversity Has Phenotypic Consequences for Glycan and Tissue Binding.

Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-N-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.

Divergent coronaviruses detected in wild birds in Brazil, including a central park in São Paulo.

Coronaviruses are single-stranded positive-sense RNA viruses associated with important avian diseases. Their relatively high rates of mutation and recombination frequencies allow them to adapt to new hosts and ecological niches. Although Brazil has 18% of global avian species diversity, studies regarding the presence of avian viral diseases in wild birds in South America are scarce. In this study, we performed a retrospective analysis of the presence of CoVs in 746 wild birds. Oropharyngeal and cloacal swabs were obtained and placed together in vials containing VTM transport medium collected in different regions of Brazil between 2006 and 2013. Screening for viral nucleic acid was performed using conventional RT-PCR and pancoronavirus nested PCR. Positive samples were characterized by partial sequencing of the RNA-dependent RNA polymerase (RdRp) gene, and ensuing phylogenetic analysis was performed to investigate the association between virus epidemiology and bird migration routes. Coronavirus RNA were detected and sequenced from six samples, in which three were related to gammacoronaviruses group and the other three to deltacoronavirus group. Our study documents the presence of CoVs related to avian gamma- and deltacoronaviruses circulating in both urban- and poultry-farm regions of Brazil, implicating wild birds as potential carriers of CoVs which may represent a risk to poultry farms and public health in Brazil.

Genetic Diversity and Phylodynamics of Avian Coronaviruses in Egyptian Wild Birds.

Avian coronaviruses (ACoVs) are continuously evolving and causing serious economic consequences in the poultry industry and around the globe. Owing to their extensive genetic diversity and high mutation rates, controlling ACoVs has become a challenge. In this context, the potential contribution of wild birds in the disease dynamics, especially in domesticated birds, remains largely unknown. In the present study, five hundred fifty-seven (n = 557) cloacal/fecal swabs were collected from four different wild bird species from eight Egyptian governorates during 2016 and a total of fourteen positive isolates were used for phylodynamics and evolutionary analysis. Genetic relatedness based on spike (S1) gene demonstrated the clustering of majority of these isolates where nine isolates grouped within Egy/variant 2 (IS/885 genotype) and five isolates clustered within Egy/variant 1 (IS/1494/06 genotype). Interestingly, these isolates showed noticeable genetic diversity and were clustered distal to the previously characterized Egy/variant 1 and Egy/variant 2 in Egyptian commercial poultry. The S1 gene based comparison of nucleotide identity percentages revealed that all fourteen isolates reported in this study were genetically related to the variant GI-23 lineage with 92⁻100% identity. Taken together, our results demonstrate that ACoVs are circulating in Egyptian wild birds and highlight their possible contributions in the disease dynamics. The study also proposes that regular monitoring of the ACoVs in wild birds is required to effectively assess the role of wild birds in disease spread, and the emergence of ACoVs strains in the country.

Avian coronavirus isolated from a pigeon sample induced clinical disease, tracheal ciliostasis, and a high humoral response in day-old chicks.

The detection of avian coronaviruses (AvCoV) in wild birds and the emergence of new AvCoV have increased in the past few years. In the present study, the pathogenicity of three AvCoV isolates was investigated in day-old chicks. One AvCoV isolated from a pigeon, which clustered with the Massachusetts vaccine serotype, and two AvCoV isolated from chickens, which grouped with a Brazilian genotype lineage, were used. Clinical signs, gross lesions, histopathological changes, ciliary activity, viral RNA detection, and serology were evaluated during 42 days post infection. All AvCoV isolates induced clinical signs, gross lesions in the trachea, moderate histopathological changes in the respiratory tract, and mild changes in other tissues. AvCoV isolated from the pigeon sample caused complete tracheal ciliostasis over a longer time span. Specific viral RNA was detected in all tissues, but the highest RNA loads were detected in the digestive tract (cloacal swabs and ileum). The highest antibody levels were also detected in the group infected with an isolate from the pigeon. These results confirm the pathogenicity of Brazilian variants, which can cause disease and induce gross lesions and histopathological changes in chickens. Our results suggest that non-Galliformes birds can also play a role in the ecology of AvCoV.

Reverse spillover of avian viral vaccine strains from domesticated poultry to wild birds.

Transmission of viruses from the commercial poultry to wild birds is an emerging paradigm of livestock-wildlife interface. Here, we report the identification and isolation of vaccine strains of avian paramyxovirus serotype 1 (APMV1) and avian coronaviruses (ACoV) from different wild bird species across eight Egyptian governorates between January 2014 and December 2015. Surveillance of avian respiratory viruses in free-ranging wild birds (n=297) identified three species that harboured or excreted APMV1 and ACoVs. Genetic characterization and phylogenetic analysis of recovered viruses revealed a close association with the most widely utilized vaccine strains in the country. These results highlight the potential spillover of vaccine-viruses probably due to extensive use of live-attenuated vaccines in the commercial poultry, and close interaction between domesticated and wild bird populations. Further exploring the full spectrum of vaccine-derived viral vaccine strains in wild birds might help to assess the emergence of future wild-birds origin viruses.

Urbanization and the dynamics of RNA viruses in Mallards (Anas platyrhynchos).

Urbanization is intensifying worldwide, and affects the epidemiology of infectious diseases. However, the effect of urbanization on natural host-pathogen systems remains poorly understood. Urban ducks occupy an interesting niche in that they directly interact with both humans and wild migratory birds, and either directly or indirectly with food production birds. Here we have collected samples from Mallards (Anas platyrhynchos) residing in a pond in central Uppsala, Sweden, from January 2013 to January 2014. This artificial pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. Nine hundred and seventy seven (977) fecal samples were screened for RNA viruses including: influenza A virus (IAV), avian paramyxovirus 1, avian coronavirus (CoV), and avian astrovirus (AstroV). This intra-annual dataset illustrates that these RNA viruses exhibit similar annual patterns to IAV, suggesting similar ecological factors are at play. Furthermore, in comparison to wild ducks, autumnal prevalence of IAV and CoV are lower in this urban population. We also demonstrate that AstroV might be a larger burden to urban ducks than IAV, and should be better assessed to demonstrate the degree to which wild birds contribute to the epidemiology of these viruses. The presence of economically relevant viruses in urban Mallards highlights the importance of elucidating the ecology of wildlife pathogens in urban environments, which will become increasingly important for managing disease risks to wildlife, food production animals, and humans.