Primary Peripheral Epstein-Barr Virus Infection Can Lead to CNS Infection and Neuroinflammation in a Rabbit Model: Implications for Multiple Sclerosis Pathogenesis

Abstract

Epstein-Barr virus (EBV) is a common herpesvirus associated with malignant and non-malignant conditions. An accumulating body of evidence supports a role for EBV in the pathogenesis of multiple sclerosis (MS), a demyelinating disease of the CNS. However, little is known about the details of the link between EBV and MS. One obstacle which has hindered research in this area has been the lack of a suitable animal model recapitulating natural infection in humans. We have recently shown that healthy rabbits are susceptible to EBV infection, and viral persistence in these animals mimics latent infection in humans. We used the rabbit model to investigate if peripheral EBV infection can lead to infection of the CNS and its potential consequences. We injected EBV intravenously in one group of animals, and phosphate-buffered saline (PBS) in another, with and without immunosuppression. Histopathological changes and viral dynamics were examined in peripheral blood, spleen, brain, and spinal cord, using a range of molecular and histopathology techniques. Our investigations uncovered important findings that could not be previously addressed. We showed that primary peripheral EBV infection can lead to the virus traversing the CNS. Cell associated, but not free virus in the plasma, correlated with CNS infection. The infected cells within the brain were found to be B-lymphocytes. Most notably, animals injected with EBV, but not PBS, developed inflammatory cellular aggregates in the CNS. The incidence of these aggregates increased in the immunosuppressed animals. The cellular aggregates contained compact clusters of macrophages surrounded by reactive astrocytes and dispersed B and T lymphocytes, but not myelinated nerve fibers. Moreover, studying EBV infection over a span of 28 days, revealed that the peak point for viral load in the periphery and CNS coincides with increased occurrence of cellular aggregates in the brain. Finally, peripheral EBV infection triggered temporal changes in the expression of latent viral transcripts and cytokines in the brain. The present study provides the first direct in vivo evidence for the role of peripheral EBV infection in CNS pathology, and highlights a unique model to dissect viral mechanisms contributing to the development of MS.

What do animal models tell us about the role of EBV in the pathogenesis of multiple sclerosis?

“…These animals displayed focal CNS cellular aggregates composed of densely clustered blood-derived macrophages surrounded by activated microglia and astrocytes, neutrophils, CD8 + T lymphocytes, dispersed EBI2 + cells and PCNA + , IgM + , and IgG + B lymphocytes ( Figure 1 ). Remarkably, the cell aggregates showed myelin damage in the center ( Figure 1 ) ( 56 ). This may model the early stages of lesions that progress into MS smoldering active plaques ( 78 ).…”

“…A recent study examined if EBV, following primary infection, could enter the rabbit CNS and by what mechanism ( 56 ). The study revealed that, at day 14 of infection, the levels of viral DNA were highest in both peripheral and CNS compartments.…”

“… Inflammatory cellular aggregates in brain of EBV-infected rabbits. Brain sections were stained for: (A) H&E; (B) EBV latent nuclear protein, EBNA-1; (C) Blood-derived macrophage marker, RAM11; (D) Microglia marker, Iba1; (E) Neutrophil marker, RPN3/57; (F) B-cell marker, CD79a; (G) pan-T-cell marker, CD3; (H) Cytotoxic T-cell marker, CD8; (I) Astrocyte marker, GFAP; (J) Myelin marker, MBP; (K) Counter stain, DAPI; (L) MBP/DAPI merge showing areas of demyelination [adapted from ( 56 )]. The figure shows non-serial sections from the same block.…”

“…Importantly, EBV latent transcripts, most notably EBV-encoded RNAs (EBERs) correlate positively with the proinflammatory cytokines IL1β and IL6 in the brain and spleen ( 56 ). EBER2 expression in vitro is associated with high levels of IL-6 produced by infected B cells ( 82 ).…”