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Livestock animals housed in close proximity to humans can act as sources or intermediate hosts facilitating animal-to-human transmission of zoonotic diseases. Understanding virus diversity in livestock is important for identifying potential zoonotic threats and for ensuring animal health and safe livestock production. Here, we report viral metagenomic characterization of chicken feces (N=51) and paired farm dust samples (N=13) using metagenomic deep sequencing. Samples were collected at 4-5 time points in three broiler farms in the Netherlands. Viruses in the Parvoviridae and Picornaviridae families were the most prevalent, detected in all feces and dust samples and in all feces and 85% of dust samples, respectively. Virus composition found in chicken feces and corresponding dust samples were similar. Great genomic diversity was identified in Picornaviridae and 46 sequences from five picornavirus genera (Sicinivirus, Megrivirus, Anatvirus, Gallivirus and Avisvirus) were detected. For calicivirus, Basovirus and an unclassified novel chicken calicivirus were identified in 13 fecal and 1 dust samples. Two distinct types of chicken astroviruses were identified. Phylogenetic analyses of identified virus sequences from Picornaviridae, Astroviridae and Caliciviridae suggested that viral sequences obtained from different farms are often more closely related to each other than global reference sequences, and sequences from feces and paired dust samples also clustered together. Importantly, our sequencing methodology enabled the recover viral genome sequences from farm dusts, allowing the tracking of virus chatter between livestock animals and their farm environment. This study, albeit relative sample size, does expand current knowledge of virus communities in chickens and surrounding dusts. IMPORTANCEChickens may harbor various zoonotic pathogens, some of which can cause severe clinical outcomes in animals and humans. Farm dust can act as vector to facilitate zoonoses transmission. Here, we report the metagenomic characterization of virus communities of chicken feces and paired farm dust samples collected at multiple time points during the production cycle in broiler farms in the Netherlands. Parvoviridae and Picornaviridae were most frequently detected. We also identified novel astrovirus and calicivirus sequences that would inform future virus taxonomy classification. This is the first study to characterize virus communities in farmed chickens and paired farm dust samples. We also describe a dust sequencing strategy that can be adapted for future dust metagenomic characterization. Our study could help setting up a surveillance baseline for tracking virus flow between chickens and their farm environment which could guide zoonotic outbreak preparedness and health risk assessment of farm exposure.

In this study all currently known chicken picornaviruses including a novel one (chicken phacovirus 1, KT880670) were identified by viral metagenomic and RT-PCR methods from a single specimen of a diarrheic chicken suffering from a total of eight picornavirus co-infections, in Hungary. The complete genomes of six picornaviruses were determined and their genomic and phylogenetic characteristics and UTR RNA structural models analyzed in details. Picornaviruses belonged to genera Sicinivirus (the first complete genome), Gallivirus, Tremovirus, Avisivirus and "Orivirus" (two potential genotypes). In addition, the unassigned phacoviruses were also detected in multiple samples of chickens in the USA. Multiple co-infections promote and facilitate the recombination and evolution of picornaviruses and eventually could contribute to the severity of the diarrhea in chicken, in one of the most important food sources of humans.

While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in poultry. We discovered a previously unknown diversity of picornaviruses in chickens in Hong Kong. Picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by reverse transcription-PCR, with their partial 3Dpol gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornaviruses, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (Avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses, especially in the P1 and P2 regions, including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and Duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses that may cross avian species barriers through mutation or recombination.