An efficient and versatile reverse genetics system for bovine coronavirus facilitates antiviral evaluation
Abstract
Bovine coronavirus (BCoV), a primary etiological agent of calf diarrhea, exhibits dual tropism for the respiratory and enteric tracts. However, research on BCoV gene function and pathogenesis has been limited by the lack of convenient and flexible reverse genetics tools. Herein, we report a robust reverse genetics system for BCoV based on an optimized circular polymerase extension reaction (CPER) methodology. We successfully rescued recombinant BCoV (rBCoV) in HRT-18G cells and engineered recombinant BCoV expressing ZsGreen fluorescent protein (rBCoV/Zs-T2A-ns2). Phenotypic analysis demonstrated that both unmodified rBCoV and rBCoV/Zs-T2A-ns2 maintained growth kinetics comparable to the wild-type BCoV. Importantly, the reporter virus demonstrated remarkable genetic stability for over ten passages, identifying the ns2 locus as a suitable insertion site for stable foreign gene expression without compromising viral fitness or sequence integrity. As a proof-of-concept application, the rBCoV/Zs-T2A-ns2 was employed for antiviral compound evaluation, successfully identifying four compounds—camostat, nafamostat, niclosamide, and nelfinavir—capable of effectively inhibiting BCoV replication. Further time-of-addition assays revealed that nelfinavir and niclosamide targeted the BCoV replication stage, while camostat and nafamostat interfered with BCoV internalization. This versatile CPER-based reverse genetics system provides a critical platform for advanced BCoV pathogenesis research and accelerated development of novel vaccines and effective antiviral therapeutics, ultimately contributing to enhanced bovine health and sustainable livestock production.
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BibTeX
@article{zeng2026efficient,
title = {{An efficient and versatile reverse genetics system for bovine coronavirus facilitates antiviral evaluation}},
author = {Yiran Zeng and Hanqiong Gao and Yuanlu Lu and Menglu Fan and Zhiyuan Liu and Yue Li and Linjuan Wu and Jun Xia and Jihui Ping},
journal = {Veterinary Research},
year = {2026},
month = oct,
doi = {10.1186/s13567-026-01851-6},
url = {https://hal.science/hal-05793445v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - An efficient and versatile reverse genetics system for bovine coronavirus facilitates antiviral evaluation
AU - Yiran Zeng
AU - Hanqiong Gao
AU - Yuanlu Lu
AU - Menglu Fan
AU - Zhiyuan Liu
AU - Yue Li
AU - Linjuan Wu
AU - Jun Xia
AU - Jihui Ping
PY - 2026
DA - 2026/10/11
JO - Veterinary Research
DO - 10.1186/s13567-026-01851-6
UR - https://hal.science/hal-05793445v1
AB - Bovine coronavirus (BCoV), a primary etiological agent of calf diarrhea, exhibits dual tropism for the respiratory and enteric tracts. However, research on BCoV gene function and pathogenesis has been limited by the lack of convenient and flexible reverse genetics tools. Herein, we report a robust reverse genetics system for BCoV based on an optimized circular polymerase extension reaction (CPER) methodology. We successfully rescued recombinant BCoV (rBCoV) in HRT-18G cells and engineered recombinant BCoV expressing ZsGreen fluorescent protein (rBCoV/Zs-T2A-ns2). Phenotypic analysis demonstrated that both unmodified rBCoV and rBCoV/Zs-T2A-ns2 maintained growth kinetics comparable to the wild-type BCoV. Importantly, the reporter virus demonstrated remarkable genetic stability for over ten passages, identifying the ns2 locus as a suitable insertion site for stable foreign gene expression without compromising viral fitness or sequence integrity. As a proof-of-concept application, the rBCoV/Zs-T2A-ns2 was employed for antiviral compound evaluation, successfully identifying four compounds—camostat, nafamostat, niclosamide, and nelfinavir—capable of effectively inhibiting BCoV replication. Further time-of-addition assays revealed that nelfinavir and niclosamide targeted the BCoV replication stage, while camostat and nafamostat interfered with BCoV internalization. This versatile CPER-based reverse genetics system provides a critical platform for advanced BCoV pathogenesis research and accelerated development of novel vaccines and effective antiviral therapeutics, ultimately contributing to enhanced bovine health and sustainable livestock production.
SN - 0928-4249
SN - 1297-9716
ER -