An Emerging DTMUV Variant Remains Restricted in Mammals but Acquires Olfactory Route-Mediated Neuroinvasion Following Limited Adaptive Mutations

Abstract

Duck Tembusu virus (DTMUV), a mosquito-borne flavivirus, has caused widespread outbreaks in poultry and has exhibited an expanding host range among avian species, accompanied by ongoing genetic variation and adaptation. As many mosquito-borne flaviviruses can infect mammals and cross species barriers, it remains unclear whether recently emerged DTMUV variants have acquired similar potential. To address this, we assessed whether the circulating variant AQ-19 can establish infection in mammals via peripheral routes. Despite its enhanced virulence in avian species, this variant failed to establish infection in mice following peripheral inoculation, indicating that a substantial barrier still restricts cross-species transmission to mammals. We next investigated whether additional adaptive changes are required for DTMUV to overcome this barrier. Through serial intracranial passage in Institute of Cancer Research (ICR) mice, we generated a mouse-adapted strain (F13) that exhibited markedly enhanced virulence and, notably, acquired the ability to invade the central nervous system (CNS) via intranasal (i.n.) inoculation, resulting in lethal infection. This neuroinvasive phenotype was strictly route-dependent, as infection remained ineffective via intraperitoneal (i.p.) and subcutaneous (s.c.) routes. We found that the adapted virus invades the CNS via the olfactory route and disseminates broadly, exhibiting strong neurotropism and inducing severe neural damage. Functional disruption of the olfactory epithelium using ZnSO₄ significantly delayed disease onset and reduced viral burden in the brain, supporting its role as a major route for nasal-to-brain transmission. Importantly, genomic analysis identified only four nonsynonymous amino acid substitutions in the adapted strain, suggesting that a limited number of mutations may be sufficient to confer enhanced neurovirulence and enable neuroinvasion in a mammalian host. Collectively, these findings demonstrate that the emerging DTMUV variant AQ-19 remains restricted in mammals, whereas limited adaptive changes acquired during experimental mouse adaptation are associated with the acquisition of neuroinvasive capacity, providing new insights into the evolutionary potential of DTMUV for mammalian adaptation.