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Coronavirus surveillance in passerines reveals novel deltacoronaviruses in Eurasian tree sparrows with implications for One Health and livestock biosecurity.

Coronaviruses (CoVs) are widespread RNA viruses infecting a broad range of avian and mammalian hosts. Although gammacoronaviruses and deltacoronaviruses (DCoVs) are common in wild birds, their presence in Eurasian passerines remains poorly understood. We screened 243 birds (35 species) at migratory stopover sites in Slovenia (2020-2021) using pan-coronavirus RT-PCR. Coronavirus RNA was detected only in four Eurasian Tree Sparrows (Passer montanus). Whole-genome sequencing yielded genomes of 26,017-26,018 bp with high internal conservation (99.95-99.98% identity). Phylogenetic analysis revealed notable evolutionary incongruence: isolates were highly related to porcine DCoVs in the ORF1ab region (95.6-96.1% amino acid identity) but clustered with divergent avian DCoVs in the spike gene (75.7-76.8% identity). RDP5 analysis provided strong evidence for a large-scale recombination event (p = 1.17 × 10-43), consistent with a mosaic genomic architecture combining an ORF1ab region closely related to porcine DCoVs with an avian-associated spike gene. This genomic pattern highlights evolutionary connectivity among DCoVs associated with different host groups and the potential role of recombination in changes in host association. The synanthropic behaviour and mobility of P. montanus facilitate contact with diverse hosts, making this species relevant for investigating DCoV ecology at wildlife-livestock interfaces. These findings represent the first genomic characterisation of DCoVs in P. montanus in Europe and support the inclusion of passerines in broader coronavirus surveillance. Genomic surveillance of underrepresented wild-bird hosts can improve our understanding of DCoV diversity, recombination, and evolution across wildlife-livestock interfaces.

Cross-species transmission↗

Avian Migration-Mediated Transmission and Recombination Driving the Diversity of Gammacoronaviruses and Deltacoronaviruses.

In the wake of pandemics like COVID-19, which have zoonotic origins, the role of wildlife as reservoirs for emerging infectious diseases has garnered heightened attention. Migratory birds, traversing continents, represent a potent but under-researched vector for the spread of infectious diseases, including novel coronaviruses. This study delves into the genetic diversity and transmission dynamics of coronaviruses in migratory birds, presenting pivotal findings. From April 2019 to April 2023, we screened 5,263 migratory bird samples collected from Shanghai, China, identifying 372 coronavirus-positive samples belonging to five avian-related coronavirus subgenera and subsequently obtaining 120 complete genome sequences. To facilitate further research with a global perspective, the study curated all available 19,000 avian-associated coronaviruses and expanded the original 12 species to 16, including three novel coronavirus species identified in our study and one re-classified species from the public domain. The study illuminates the intricate genetic evolution and transmission dynamics of birds-related coronaviruses on a global scale. A notable aspect of our research is the identification of complex recombination patterns within the spike protein across different virus species and subgenera, highlighting migratory birds as a reservoir of coronavirus. Notably, the coronaviruses found in migratory birds, predominantly from the orders Anseriformes, Charadriiformes, and Pelecaniformes, with domestic ducks from Anseriformes playing a key role in bridging the transmission of coronaviruses between migratory and non-migratory birds. These findings reveal the genetic and recombination characteristics of coronaviruses in migratory birds, emphasizing the critical role of ecologically pivotal bird species in coronavirus transmission and genetic diversity shaping.

Animals↗

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗

Rab10 coordinates SADS-CoV non-lytic egress through the ERGIC-TGN-lysosome trafficking pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-originated alphacoronavirus that causes devastating enteric disease in neonatal piglets and possesses significant potential for cross-species transmission. While the early stages of the coronavirus life cycle have been extensively characterized, the host factors indispensable for virion assembly and subsequent export remain largely enigmatic. Here, by performing a genome-wide CRISPR-Cas9 knockout screen using a recombinant icSADS-CoV-GFP reporter virus, we identified the small GTPase Rab10 as a critical host dependency factor for SADS-CoV infection. Viral life cycle analysis revealed that Rab10 is not required for viral attachment, entry, or initial genome replication, but is essential for the virion transport and non-lytic egress. Rab10 deficiency markedly reduced the extracellular release of viral RNA, viral proteins, and infectious progeny, as well as the secretion of SADS-CoV virus-like particles. Confocal imaging showed that Rab10 and viral protein-positive intracellular structures were associated with LMAN1, TGN46, and LAMP1 positive compartments. These findings support a model in which Rab10 coordinates a virus-containing vesicles trafficking pathway associated with ERGIC-TGN-lysosome compartments. Mechanistically, Rab10 facilitates the loading of the viral envelope (E) protein into transport vesicles derived from the ERGIC. Rab10 associates with the SADS-CoV E protein, and mapping analyses implicated the C-terminal PDZ-binding motif, particularly residue V75, in efficient Rab10 association and viral release. Collectively, our findings identify Rab10 as a host regulator of SADS-CoV non-lytic egress and highlight the E-Rab10 interaction and the vesicular trafficking machinery as a potential target for developing antiviral strategies.

Animals↗