ArticleViruses2026
Fluorescent Powassan Reporter Viruses Infect Neuron, Astrocyte and Microglial Cell Lines Independent of Attenuating D308N Envelope Protein Modification.
Article in Viruses, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The Powassan virus (POWV) is a neurovirulent tick-borne virus that causes age-associated lethality and long-term neurologic sequelae in 50% of survivors. In aged mice the POWV strain LI9 mirrors human lethality and neuropathology; however, an avirulent POWV mutant, LI9-D308N, fails to enter the CNS or cause lethal disease. The D308N mutation is present in an envelope protein domain associated with cell attachment, yet the role of D308N mutations in cell tropism and neuroinvasion remains to be resolved. Here, we engineered fluorescent mScarlet3 and mNeonGreen reporter genes into WT LI9, and avirulent LI9-D308N viruses and assessed their ability to infect CNS cells in vitro. In addition, we generated replication-defective reporter POWVs that only replicate in NS1-expressing cells by replacing NS1 with fluorescent genes. Similar to WT LI9, fluorescent reporter POWVs spread focally and nonlytically, are stable following passage and reach high titers 2-5 dpi. In NS1-expressing VeroE6 cells, LI9-ΔNS1-FL reporters exhibited robust fluorescence 24 h post-infection (hpi), while fluorescence from LI9-reporter infections was first observed ~32 hpi. Comparing LI9-mScarlet3 and avirulent LI9-D308N-mScarlet3 viruses revealed no difference in their ability to infect human brain microvascular endothelial cells, pericytes, astrocytes, microglia or neuronal cells in vitro. Notably, LI9-mScarlet3 viruses productively and persistently infected differentiated, neuron-like, SH-SY5Y cells without apparent cytotoxicity. These findings indicate that LI9-D308N is capable of infecting blood-brain-barrier and CNS cells, and suggest that neuroinvasion is restricted prior to LI9-D308N engaging CNS cells. These results are consistent with clearance of LI9-D308N from the blood, or the D308N mutation interfering with potential routes of POWV neuroinvasion. Collectively, fluorescent POWV reporter viruses provide insight into the mechanism of POWV neuroinvasion, permit analysis of replication-defective POWVs as vaccines and provide a means of analyzing POWV cell tropism, antivirals and cell-to-cell spread in BSL2 and BSL3 settings.
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