Development of an HR2-Derived Fusion Inhibitor Against Canine Coronavirus; in vitro and in silico approaches

Introduction: Zoonoses are infectious diseases transmitted from animals to humans and pose a significant global public health threat due to our close contact with animals. Approximately two-thirds of emerging infectious diseases in humans originate from animals, including Ebola, avian influenza, COVID-19, and hantavirus. Among coronaviruses, Canine coronavirus (CCoV), an alphacoronavirus, causes mild enteritis in dogs and other carnivores; however, its high recombination rate has led to hypervirulent strains with increased pathogenicity and potential for cross-species transmission. Recently, a novel CCoV strain, CCoV-HuPn-2018, was identified in patients with pneumonia in Malaysia, Haiti, and Vietnam, highlighting the zoonotic potential of CCoV-like viruses, although their epidemiological implications remain under investigation.

Aim: Innovative solutions are needed from a One Health perspective to address growing challenges. In this study, to prevent or block CCoV replication, we designed a fusion-inhibiting peptide derived from the heptad repeat domain (HRC) of the CCoV S glycoprotein, a crucial region for membrane fusion and viral infection.

Material and methods: In vitro TCID₅₀ antiviral assays, cell-cell fusion assays, and Real-Time PCR, together with in silico analyses and molecular dynamics simulations, were conducted to evaluate the peptide's interaction with the S protein fusion core.

Results: At non-toxic concentrations (3–50 µM), the peptide effectively blocked S protein-mediated fusion and markedly reduced live-virus infection in vitro at 35 and 25 µM, respectively. Molecular detection showed a dose-dependent reduction in gene expression of the S glycoprotein. These results were supported by molecular docking and dynamics simulations, which revealed that the peptide, mimicking the HR2 region, binds strongly to the HR1 region, thereby hindering viral protein binding and fusion.

Conclusion: Overall, these results provide a strong foundation for developing peptide-based treatments to inhibit CCoV fusion and replication.
Additional research is needed to examine the chemical properties of the peptide and its safety in animal models.