Enterovirus infections of the central nervous system

Abstract

Enteroviruses (EV) frequently infect the central nervous system (CNS) and induce neurological diseases. Although the CNS is composed of many different cell types, the spectrum of tropism for each EV is considerable. These viruses have the ability to completely shut down host translational machinery and are considered highly cytolytic, thereby causing cytopathic effects. Hence, CNS dysfunction following EV infection of neuronal or glial cells might be expected. Perhaps unexpectedly given their cytolytic nature, EVs may establish a persistent infection within the CNS, and the lasting effects on the host might be significant with unanticipated consequences. This review will describe the clinical aspects of EV-mediated disease, mechanisms of disease, determinants of tropism, immune activation within the CNS, and potential treatment regimes.

Fig. 1

Neural progenitor and stem cells grown in culture are highly susceptible to coxsackievirus infection. NPSCs were isolated from the cortices of one day-old C57 BL/6 mice, cultured to form neurosphere aggregates, and infected with eGFP-CVB3 (moi = 0.1). Infected neurospheres were observed over time by fluorescence microscopy. Virus protein expression (green) was readily observed by day 1 PI. An increase in viral protein expression was seen until day 4 PI. By day 6 PI, virus protein levels were reduced, and signs of cytopathic effect (cpe) were readily observed at a higher magnification (not shown).

Fig. 2

Induction of CCL12 and the progressive extravasation of infected myeloid cells through the basement membrane as determined by confocal microscopy and Imaris 3D analysis. One day-old C57 BL/6 mice were intra-cranially infected with a recombinant coxsackievirus B3 expressing eGFP (eGFP-CVB3; 10E7 pfu). The brains were harvested 12 h later and inspected by confocal fluorescence microscopy. Myeloid cells responding to CVB3 infection were observed entering through the tight junctions of the choroid plexus epithelium. As these myeloid cells entered the lateral ventricle, they expressed high levels of viral protein (green). Also, the chemokine CCL12 (red), a potential chemoattractant molecule for infiltrating myeloid cells, was expressed within the choroid plexus and surrounding subventricular zone. The kinetics of myeloid cell migration through the basement membrane (outlined by laminin staining in red) was observed by confocal microscopy and IMARIS 3D analysis. Immunofluorescence images showed infected myeloid cell migration (green) through the tight junctions of the choroid plexus epithelium. Gray scale images of all three colors (virus-green; laminin-red, DAPI-blue) at 12 h PI revealed the intensity and organization of the myeloid cell infiltration in greater detail. In order to better visualize myeloid cell entry, IMARIS 3D with diminishing laminin label (diminishing red) was carried out, and the methodology revealed the extravasation of infected myeloid cells through the basement membrane (white arrow). Original images were obtained with a 63× Plan-Aprochromat objective at 0.3 μm interval step slices.

Fig. 3

Potential reactivation of a persistent coxsackievirus infection within quiescent neural stem cell. Coxsackieviral RNA (squiggly green lines) may persist in a latent state within quiescent type B neural stem cells in the CNS. Following cellular division reactivation of viral RNA may lead to viral protein expression and sporadic infectious virus production. The production of infectious virions may infect nearby progenitor cells. Alternatively, asymmetric division of infected type B stem cells may generate downstream progenitor cells or immature neurons that remained infected and migrate to other regions of the CNS. The outcome over the long-term of continuous virus reactivation may be virus-mediated neuropathology and virus dissemination.

Fig. 4

Increased susceptibility of young mice to coxsackievirus infection and subsequent CNS pathology. Neonatal SJL mice (days 1, 2, or 6 post-birth) were intra-cranially infected with 100 pfu CVB3. The brains of infected mice were harvested on days 11, 15 or 33 post-infection, respectively. De-paraffinized sections of the brains were stained with hematoxylin and eosin and inspected by microscopy for lesions and inflammatory cells. (A) One day-old mice suffered the greatest level of neuropathology following infection. Lesions were observed within the cortex and hippocampus (black arrows). (B) A higher magnification of (A) revealed the loss of pyramidal neurons in the hippocampus and the presence of inflammatory cells (black arrows). (C) In contrast, two day-old mice infected with an identical amount of CVB3 showed reduced signs of neuronal loss restricted to the CA3 and CA4 field of the hippocampus (black arrow). The presence of immune cell enriched perivascular cuffs were observed near the hippocampus (black arrow). (D) Six day-old mice infected with CVB3 showed little or no signs of CNS disease.

Fig. 5

Antivirals against enteroviruses: mechanisms of action. Pleconaril, ribavirin, intra-venous immunoglobulins (IVIg) and RNAi each inhibit the production of infectious virions at different steps. Pleconaril induces a conformational change in the viral VP1 capsid protein that inhibits both genome uncoating and the binding of the virus to its cognate receptor, CAR. Ribavirin, a nucleoside analog, is incorporated into the viral genome. This incorporation results in viral genomic mutations that lead to “error catastrophe”. Virus-specific IVIg may bind to virions extracellularly and inhibit the virus from entering the cell. RNAi molecules target the positive sense viral RNA strand leading to Dicer-mediated degradation of the viral genome. However, escape mutants may form that avoid the effects of RNAi. Positive-sense strand viral RNA is shown in black; negative sense strand viral RNA is shown in green. Antiviral compounds are shown in red. The replication complex is shown in blue.