B virus is an alphaherpesvirus, endemic to macaque monkeys, capable of deadly human zoonosis with an 80% mortality rate in untreated cases. The macaque monkey is widely used in biomedical research and the threat of B virus poses an occupational hazard to researchers, veterinarians, and animal handlers. B virus establishes a life-long latent infection in sensory neurons of the peripheral nervous system (PNS) in the natural host. In human infections, B virus readily transits to the central nervous system (CNS) and destroys brain tissues. Identifying immune correlates of B virus infection in the PNS of the natural host is critical in understanding viral lethality in the human host. The lack of an accurate animal model and restrictions on handling potentially infected nervous tissue previously limited studies of B virus infection in macaque ganglia. To address this barrier, a long-lived mixed neuron/glia cell culture model was established from macaque DRG explants using a novel methodology that relied on cellular migration from whole tissues. Utilizing this model, the hypothesis tested was that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation. Chemokines were upregulated in B virus-infected cultures and infected cell media induced leukocyte chemotaxis. Leukocytes were less effectively activated by media from infected cells when compared to media from mock-infected cells. To identify factors responsible for this, focused microarrays were performed and cytokine profiles were quantified from B virus and mock-infected culture supernatants. IL-6 protein levels were significantly reduced in B virus infected cultures. This observation led to the hypothesis that IL-6 downregulation impairs leukocyte activation and, indeed, when IL-6 was added to B virus-infected culture supernatants to control levels, these cultures were far more effective at eliciting leukocyte activation when compared with mock-infected cultures. Collectively, these data support the hypothesis that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation and identifies a potential viral mechanism to impair leukocyte functionality. Additionally, this work presents a novel methodology for establishing long-lived mixed neuron/glia cultures from postnatal/adult macaque DRGs.

Plasmacytoid dendritic cells (pDC) are a specialized group of circulating dendritic cells that respond to viral nucleic acids with Type I IFN production as well as other cytokine and chemokines. These pDC responses lead to the production of antiviral molecules and recruitment of defense cells. During zoonotic B virus infection, a simplex virus of the subfamily Alphaherpesviridae, our lab has observed that infected individuals who succumb to infection have little-to-no-antibody or cell-mediated defenses. To identify whether this was partly due to failure of pDCs to produce antiviral interferon responses or produce chemokine and cytokines, we tested the hypothesis that B virus modulates the IFN response during zoonotic infection by blocking pDC activation and subsequent IFN signaling pathways to circumvent host defenses, while these pathways remain intact in the macaque hosts. We showed that human pDCs respond to B virus through the production of IFN-a, IL-1a, IL-6, TNF-a, MIP-1a/b and IP-10. Human pDCs co-cultured with B virus infected fibroblasts produced fewer cytokines and at lower levels. The macaque response to B virus was measured using PBMCs, as there are no specific reagents available to enrich macaque pDCs. Human and macaque PBMCs produced IFN-a when exposed directly to B virus infected lysates. Co-cultures of PBMCs with B virus infected fibroblasts from both hosts failed to produce any significant amounts of IFN-a. To quantify the antiviral effects of PBMC induced IFN-a, we measured B virus titers after exposure to supernatants from B virus exposed PBMCs, PBMC co-cultures with infected fibroblasts and exogenous recombinant Type I IFN. Our data further suggest that B virus resistance was not due to virus specific blockade of the Type I IFN signaling pathway because STAT-1 was activated in infected fibroblasts when treated with Type I IFNs. These data demonstrate for the first time that B virus replication is unimpeded in the presence of any source of IFN-a in either host cell type. In conclusion, this dissertation shows that the IFN-a production by both hosts in response to B virus is similar and that IFN-a treatment of B virus infected fibroblasts did not reduce B virus replication.