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Passage-attenuated Powassan virus LI9P protects mice from lethal LI9 challenge and links envelope residue D308 to
Grace E Himmler1, Megan C Mladinich1,2, Jonas N Conde1
1Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA.
Abstract:
Powassan virus (POWV) is an emergent tick-borne flavivirus that causes lethal encephalitic disease and chronic neurologic deficits in surviving patients. POWV-LI9 is a tick-derived isolate that causes neurovirulent disease and age-dependent lethality in mice. Serial passage of VeroE6 cells infected with LI9 resulted in eight amino acid changes in a POWV strain LI9P. LI9P fails to cause neurological sequelae, or lethality in C57BL/6 mice yet elicits neutralizing POWV antibody responses and protects mice from lethal LI9 challenge. Analysis revealed that LI9, but not LI9P, is present at high levels in the CNS, suggesting that LI9P is restricted from neuroinvasion or CNS replication. LI9 and LI9P are distinguished by a D308N envelope change within a domain associated with cell attachment. We evaluated the roles of Env-Domain III residue changes in LI9 virulence and LI9P attenuation using recombinant POWVs (recPOWVs) generated by reverse genetics. Remarkably, mutating D308N in LI9 completely abolished viral lethality and neuroinvasion in 50-week-old mice, reflecting the avirulent phenotype of LI9P. Analysis of the reciprocal N308D change in LI9P only partially restored neuroinvasion and lethality to the LI9P-N308D mutant, indicating that further LI9P residue changes contribute to LI9P attenuation. Consistent with differences in neuroinvasion, we found that rapid LI9P RNA synthesis and corresponding early IFN induction may contribute to LI9P clearance. Collectively, these findings define D308 as a determinant of POWV neuroinvasion and lethality, suggest potential mechanisms for restricted LI9P CNS entry, and reveal passage-attenuated LI9P as a candidate POWV vaccine platform.
Importance:
Powassan virus (POWV) infection causes a 10% lethal encephalitis, resulting in chronic neurological symptoms in half of survivors. POWV is transmitted in as short as 15 min following tick attachment, demonstrating the need for the development of POWV vaccines and therapeutics. Mechanisms of POWV neurovirulence remain to be defined to inform vaccine and therapeutic design. Cell culture passage has successfully been used to generate live-attenuated flavivirus vaccines. Accordingly, we serially passaged POWV LI9-infected VeroE6 cells and isolated an attenuated POWV strain, LI9P, that fails to cause neurologic sequelae or murine lethality. LI9P elicits neutralizing antibody responses, protects mice from a lethal WT POWV challenge, and is a potential POWV vaccine. Analysis of attenuating mutations in LI9P revealed that changing envelope residue D308N alone in LI9 prevents POWV neurovirulence and lethality in immunocompetent mice. Altogether, this study defines viral determinants of POWV pathogenesis and attenuating mutations that inform the development of live-attenuated POWV vaccines.
Insights
Powassan virus (POWV) neurovirulence is determined by envelope residue D308. Attenuated POWV strain LI9P, lacking D308, is a promising candidate for a live-attenuated POWV vaccine.
Area of Science:
- Virology
- Immunology
- Neuroscience
Background:
- Powassan virus (POWV) is an emergent tick-borne flavivirus causing lethal encephalitis and neurological deficits.
- POWV infection has a high fatality rate and significant long-term consequences for survivors, necessitating vaccine development.
- Understanding the mechanisms of POWV neurovirulence is crucial for designing effective vaccines and therapeutics.
Purpose of the Study:
- To identify viral determinants of POWV neurovirulence and lethality.
- To investigate the potential of a cell culture-passaged POWV strain (LI9P) as a live-attenuated vaccine candidate.
- To elucidate the role of specific envelope mutations in POWV pathogenesis.
Main Methods:
- Serial passage of POWV strain LI9 in VeroE6 cells to generate an attenuated strain, LI9P.
- Generation of recombinant POWVs (recPOWVs) using reverse genetics to study specific mutations.
- Evaluation of viral neuroinvasion, replication, lethality, and immune responses in mouse models.
- Analysis of viral RNA synthesis and interferon induction.
Main Results:
- Passage-attenuated POWV strain LI9P failed to cause neurological disease or lethality in mice but elicited protective neutralizing antibodies.
- A single amino acid change (D308N) in the POWV envelope protein was identified as a key determinant of neurovirulence and lethality.
- Mutating D308N in the virulent LI9 strain abolished lethality and neuroinvasion, while the reciprocal mutation in LI9P only partially restored these phenotypes.
- LI9P exhibited restricted neuroinvasion and CNS replication compared to LI9, potentially due to rapid RNA synthesis and early interferon induction.
Conclusions:
- Envelope residue D308 is a critical determinant of POWV neuroinvasion and lethality.
- The passage-attenuated POWV strain LI9P, with specific mutations including D308N, represents a promising candidate for a live-attenuated POWV vaccine.
- Further studies are needed to fully understand the mechanisms of LI9P attenuation and its potential as a vaccine platform.
