A mouse-adapted Yezo virus model for antiviral testing in immunocompetent mice

1. Problem Statement

• Emerging Threat: Yezo virus (YEZV), a tick-borne orthornairovirus, is an emerging human pathogen causing fever, thrombocytopenia, leukopenia, and severe liver injury. However, its pathogenesis and therapeutic options remain poorly understood.
• Model Limitations: Existing animal models have significant drawbacks. The AG129 mouse model (deficient in type I/II interferon receptors) recapitulates liver damage but fails to mimic the clinical course of human infection due to the lack of a functional immune system. Furthermore, wild-type YEZV strains (e.g., T-HLJ01) cause only transient, subclinical infections in immunocompetent mice, rendering them unsuitable for studying pathogenesis or screening antivirals.
• Need: There is an urgent need for a robust, immunocompetent mouse model that faithfully recapitulates human YEZV disease to facilitate mechanistic studies and antiviral drug development.

2. Methodology

• Virus Adaptation: The researchers serially passaged the tick-derived YEZV strain (T-HLJ01) in C57BL/6J immunocompetent mice via intraperitoneal (i.p.) inoculation. Passages were conducted using liver homogenates collected at 3-day intervals (later shortened to 2.5 days due to mortality).
• Characterization:
  • In Vivo: Mice were challenged with varying doses (1, 10², 10⁴ TCID₅₀) of the adapted strain (MA-YEZV) to assess lethality, weight loss, and tissue tropism across different mouse strains (C57BL/6J, BALB/c, Kunming) and infection routes (i.p., s.c., i.n.).
  • Genomic Analysis: Whole-genome sequencing of the parental and adapted strains was performed to identify non-synonymous mutations associated with adaptation.
  • Pathology & Immunology: Histopathology (H&E, IHC), immunofluorescence (co-staining for NP and cell markers), serum biochemistry, hematology, and cytokine profiling (serum and liver) were conducted to characterize disease progression.
  • Antiviral Testing: The efficacy of Ribavirin and Remdesivir (and its metabolite GS-441524) was evaluated in vitro (Vero cells) and in vivo. Mice were treated with these drugs starting 2 hours post-infection.
  • Transmission Studies: Experiments were conducted to assess horizontal (contact) and vertical (mother-to-offspring) transmission capabilities.

3. Key Contributions

• Development of MA-YEZV: Successfully generated a mouse-adapted Yezo virus strain (MA-YEZV) that causes lethal infection in immunocompetent C57BL/6J mice, a first for this pathogen.
• Clinical Fidelity: The model replicates key human clinical features, including thrombocytopenia, leukopenia, and severe hepatocellular necrosis, which were absent in previous immunodeficient models.
• Therapeutic Validation: Demonstrated that Ribavirin is highly effective against MA-YEZV, whereas Remdesivir is ineffective, providing a critical tool for preclinical antiviral screening.
• Genomic Insights: Identified 31 non-synonymous mutations acquired during adaptation, offering clues to viral virulence and host adaptation mechanisms.

4. Key Results

• Adaptation and Virulence:
  • MA-YEZV (isolated at passage 40) caused 100% mortality in C57BL/6J mice at doses as low as 10² TCID₅₀, with a median survival time of 7–9 days.
  • The virus retained hepatotropism but exhibited enhanced systemic infectivity compared to the parental strain.
  • The model was effective across multiple mouse strains (BALB/c and Kunming), though susceptibility varied.
• Pathogenesis:
  • Liver Injury: MA-YEZV caused severe hepatitis with widespread hepatocellular necrosis, inflammatory infiltration, and elevated liver enzymes (ALT, AST, LDH).
  • Cellular Tropism: The virus infected hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells.
  • Immune Response: Infection triggered a robust inflammatory response characterized by a "cytokine storm" (elevated IFN-γ, TNF-α, IL-6) and inflammasome activation (NLRP3, pyroptosis markers), which correlated with tissue damage.
• Genomic Findings:
  • Serial passaging resulted in 31 non-synonymous mutations.
  • Key mutations were found in the Large (L) segment (RdRp and OTU protease domains) and Medium (M) segment (Gc glycoprotein), suggesting adaptations in replication, immune evasion, and cell entry.
• Antiviral Efficacy:
  • Ribavirin: Treatment (50 or 100 mg/kg) completely prevented mortality, normalized liver enzymes, reduced viral loads to undetectable levels, and mitigated histopathological damage.
  • Remdesivir: Showed no efficacy in vivo; treated mice exhibited mortality and viral loads comparable to vehicle controls.
• Transmission: Despite high viral shedding in oropharyngeal and rectal swabs, no horizontal (contact) or vertical (maternal-fetal) transmission was observed in the mouse model.

5. Significance

• Research Platform: The MA-YEZV model fills a critical gap in orthonairovirus research by providing an immunocompetent system to study host-virus interactions, immune-mediated pathology, and viral evolution.
• Therapeutic Development: The study establishes Ribavirin as a promising candidate for treating YEZV infections, while ruling out Remdesivir. This provides a clear direction for clinical management and further drug optimization.
• Public Health Preparedness: As YEZV is an emerging tick-borne threat with a broad geographic range, this model enables rapid evaluation of countermeasures against current and future orthonairovirus outbreaks.
• Mechanistic Insight: The identification of specific adaptive mutations offers a roadmap for understanding how arboviruses evolve to become more virulent in mammalian hosts.