Evidence of Mpox clade IIb infection in primary human alveolar epithelium

Monkeypox virus (Mpox) has been recognized for causing distinct skin lesions and is primarily transmitted through skinand sexual contact. To date, the transmissibility and pathogenesis of the Mpox virus in distal human lung has never beencompletely explored. Here the transmission pathways and Mpox tropism on patient-derived air-liquid epithelium (ALE)model fabricated using isolated primary human alveolar epithelial cells (hAECs) were investigated. hAECs were culturedand exposed to the Mpox virus clade IIb isolated from the patient. DNA, proteins, and the tropism were elucidated usingpolymerase chain reaction (PCR), Western blot, and high-content fluorescent imaging. Transmission electron microscopy(TEM) was employed to systematically observe the cellular distribution of viral particles. Viral titres were determined byTCID50 assay. Innate immune response and inflammatory mediators were measured using Milliplex® multiplex and ELISAanalysis. Pathology at alveolar barrier integrity was determined using transepithelial electrical resistance (TEER) analysis.The study included mock-infected cells as control. Mpox virus significantly infected 42.82% of total hAEC populations.The prominent observed pathology included a significant reduction in TEER values, loss of tight junction protein,presence of tunnelling nanotubes (TNTs), and syncytium morphology. Four stages of Mpox biogenesis were clearlyobserved without significant activation of IL-6, MIP1alpha, TNF-α, and Galectin-9, although IL-1β were subtlypromoted. The developed patient-derived ALE is a versatile model for Mpox virus clade IIb infection reflectingrespiratory transmission competence of the Mpox. Postinfection lung pathogenesis demonstrated alveolar barrierdamage without significant inflammation, raising concerns about possible immune evasion by the virus.