Mouse hepatitis virus (MHV) infection of the mouse central nervous system (CNS) is widely used to model both encephalitis and multiple sclerosis in humans. Unlike the weakly neurovirulent A59 strain of MHV, which spreads from the brain to other parts of the body, the CNS-adapted JHM isolates, which are extremely neurovirulent but replicate poorly outside the CNS, share two strain-specific and apparently independent neurovirulence traits: the ability to spread among neurons lacking the canonical MHV receptor Ceacam1a and the ability to replicate in the brain without eliciting an antigen-specific CD8+ T-cell response. Based on preliminary data, I hypothesize that both of these traits stem from the specialization of JHM for interneuronal spread, which reduces the CD8+ T-cell response by limiting viral spread to antigen-presenting cells in the CNS and/or draining lymph nodes. I propose to better define the mechanisms of this specialization in order to improve identification of potentially neurovirulent viruses, investigate the potential of JHM isolates for neuronal circui tracing, and improve our understanding of the requirements for effective presentation of CNS-derived antigen. I will first examine the neurovirulence in C57BL/6 and ceacam1a-/- mice, the abilities to spread in C57BL/6 and ceacam1a-/- neuron cultures, and the patterns of spread following intranasal (i.n.) inoculation in C57BL/6 mice of mutant JHM viruses defective for receptor-independent spread (RIS) in non- murine cells in order to determine whether Ceacam1a-independent spread in neurons requires merely RIS or a neuron-specific mechanism of spread. I will then examine the spread across synapses, release of virus from neuronal cell bodies and axons in compartmented cultures, and membrane fusion of infected neurons of JHM, A59, rA59/SJHM (a chimeric A59 virus with the JHM spike gene), and, if relevant, the RIS mutants in cultured primary C57BL/6 neurons to determine whether JHM spreads interneuronally. I will next investigate whether JHM fails to induce a CD8+ T-cell response in the brain after intracranial (i.c.) inoculation because it fails to spread to intra- or extracranial anigen-presenting cells. First, I will use a variety of RNA, protein, and functional assays to confirm tha JHM infection activates bone marrow-derived dendritic cells (DCs) for antigen presentation. Next, I will infect C57BL/6 mice adoptively transferred with lymphochoriomeningitis virus (LCMV) gp33 antigen-specific T cells simultaneously i.c. and intraperitoneally (i.p.) with LCMV gp33 epitope-expressing JHM (JHM-gp33) and evaluate the anti-gp33 CD8+ T cell response in the brain to determine whether extracranial viral replication rescues the intracranial CD8+ T cell response. Finally, I will inoculate adoptively transferred mice i.n. with wild-type JHM, inject DCs infected with JHM-gp33 i.c., and evaluate the gp33-specific T cell response in the brain to determine whether intracranial antigen presentation by infected DCs rescues the intracranial CD8+ response. These experiments will greatly enhance our understanding of neurovirulence and open new avenues of exploration of interneuronal viral spread and CNS antigen presentation.

Public Health Relevance

Mouse hepatitis virus (MHV) infection of the mouse central nervous system (CNS) is widely used to model encephalitis and multiple sclerosis in humans. Both of these disease processes are affected by the efficiency of interneuronal spread and the antiviral CD8+ T cell response in the brain. The proposed project will enhance our understanding of these model systems by defining the ways in which the adaptations of an extremely neurovirulent isolate of MHV to spread in the CNS influence both interneuronal spread and priming of the CD8+ T cell response.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Clinical Investigator Award (CIA) (K08)
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Microbiology and Infectious Diseases B Subcommittee (MID)
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Park, Eun-Chung
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University of Pennsylvania
Schools of Medicine
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Phillips, Judith M; Gallagher, Tom; Weiss, Susan R (2017) Neurovirulent Murine Coronavirus JHM.SD Uses Cellular Zinc Metalloproteases for Virus Entry and Cell-Cell Fusion. J Virol 91:
Zhang, Rong; Li, Yize; Cowley, Timothy J et al. (2015) The nsp1, nsp13, and M proteins contribute to the hepatotropism of murine coronavirus JHM.WU. J Virol 89:3598-609
Zhao, Ling; Birdwell, L Dillon; Wu, Ashley et al. (2013) Cell-type-specific activation of the oligoadenylate synthetase-RNase L pathway by a murine coronavirus. J Virol 87:8408-18
Phillips, Judith M; Kuo, I-Ting; Richardson, Chelsea et al. (2012) A novel full-length isoform of murine pregnancy-specific glycoprotein 16 (psg16) is expressed in the brain but does not mediate murine coronavirus (MHV) entry. J Neurovirol 18:138-43