Human coronavirus OC43 infection induces chronic encephalitis leading to disabilities in BALB/C mice

Abstract

The notion that an infectious respiratory pathogen can damage the central nervous system (CNS) and lead to neurological disease was tested using a human respiratory coronavirus, the OC43 strain of human coronavirus (HCoV-OC43). First, primary cell cultures were used to determine the susceptibility of each type of neural cells to virus infection. Neurons were the target cells, undergoing degeneration during infection, in part due to apoptosis. Second, neuropathogenicity was investigated in susceptible mice. Intracerebral inoculation of HCoV-OC43 into BALB/c mice led to an acute encephalitis with neuronal cell death by necrosis and apoptosis. Infectious virus was apparently cleared from surviving animals, whereas viral RNA persisted for several months. Some of the animals surviving to acute encephalitis presented an abnormal limb clasping reflex and a decrease in motor activity starting several months post-infection. These results suggest that viral persistence could be associated with an increased neuronal degeneration leading to neuropathology and motor deficits in susceptible individuals.

Fig. 1

Susceptibility of each type of neural cells to HCoV-OC43 infection. Immunofluorescent labeling demonstrated that HCoV-OC43 proteins were found in almost pure cultures of astrocytes (A), oligodendrocytes (B), microglial cells (C), and neurons (D).

Fig. 2

Viral infection and cytopathic effects of DRG-N cultures. At 2 days post-infection (dpi), numerous DRG-neurons were present (A), and only a few were infected (C). At 8 dpi, neurons were still present in the infected culture (B) and were densely packed. Numerous neurons were infected at 8 dpi (D). (E) A succession of expansions and constrictions can be seen in infected axons of these unmyelinated fibers (arrowheads point towards axonal beading). (F) Noninfected culture showing less densely packed neurons, with numerous axonal processes as compared to the infected culture (B). Original magnification: ×100, except panel E: original magnification: ×400.

Fig. 3

HCoV-OC43 tropism and induction of apoptosis in mouse cortical or hippocampal cultures. (Panels A and B) Neuronal cell cultures were double stained for HCoV-OC43 (in green) and for astrocytes (in red). At 1 day post-infection (A), only neuronal cells were positive for virus, whereas at 3 dpi (B), several astrocytes were also double stained. (C) An activated microglial cell (in green) was in close proximity with infected axonal prolongations (in red). (D) Glial cortical cultures contained numerous astrocytes (in red) with some of them double stained with MAbs against HCoV-OC43 (in green). (E–K) Apoptotic death of primary CNS neurons. (E–H) Cortical cell cultures containing numerous infected cells (E) and some activated caspase-3-positive cells (F). The merged picture in panel G shows that most activated caspase-3-positive cells were infected (yellow). Adding DAPI staining to the merged picture shown in panel G (H) showed that numerous noninfected cells were also present in this field. (I–K) Primary hippocampal cell cultures infected by HCoV-OC43. Activated caspase-3 staining and fragmented nuclei revealed by DAPI-staining were perfectly colocalized (I). In the same field, merging activated caspase-3 (red) and antiviral staining (green) revealed that most of the activated caspase-3-positive cells were infected (yellow), although some were not (appearing red, arrowheads). In panel K, merging of neuronal staining (green) and antiviral staining (red) confirmed that infected cells (arrowheads) were neurons. Original magnification: ×100 for panels A, B, E–H, ×200 for panels D, I–J and ×400 for panels C and K.

Fig. 4

Viral replication and release of TNF-α by CNS cells. (A) Infectious viral titers in infected neuronal or glial cortical cell cultures after HCoV-OC43 ATCC and HCoV-OC43 Paris infections. Replication levels in neuronal cells increased during the first days of infection, reaching a maximum at around 3 days post-infection. At that time, cytopathic effects in cultures increased and numerous neurons were dead. Replication levels in glial cell cultures were less pronounced than in neuronal cell cultures for both HCoV-OC43 strains. (B) Infectious viral titers in supernatant versus intracellular bodies of infected neuronal (N) or glial (G) cortical cell cultures after HCoV-OC43 ATCC and HCoV-OC43 Paris infection. (C) TNF-α release after HCoV-OC43 infection of neuronal cells. HCoV-OC43 ATCC infected neuronal cell cultures released a high amount of TNF-α in the supernatant.

Fig. 5

Apoptosis during acute encephalitis. TUNEL staining (A) at 11 days post-infection (dpi) was evident in the CA1 hippocampal layers, and the same region was positive for viral antigen (B), and the merged picture (C) illustrates that some neurons (arrowheads) underwent apoptosis. At a higher magnification, some infected neurons were positive for TUNEL staining (arrowheads, panel D), whereas some noninfected cells with hallmarks of apoptotic death (arrowheads, panel E) were located close to infected cells (in red, panel E). In CA3 hippocampal layers, some infected cells (F) also exhibited apoptotic cell death as shown on the merged figure (arrowheads, panel G). Original magnification: ×100 for panels A and B, ×400 for panels C to D.

Fig. 6

HCoV-OC43-mediated pathogenesis in BALB/c mice. When suspended by the tail, asymptomatic HCoV-OC43-infected BALB/c mice (A) extended their legs, whereas symptomatic HCoV-OC43-infected mice presented abnormal flexion of the four limbs (C). (B and D) Cresyl violet staining of a hippocampus from a control (B) and an infected (D) mouse. Animals surviving the acute HCoV-OC43 infection were histologically examined 1 year later and showed a hippocampus smaller than noninfected or asymptomatic animals (Py: pyramidal cell layers; DG: dentate gyrus; fi: fimbria of hippocampus; cc: corpus callosum; LV lateral ventricle; 3V: third ventricle). (E) Motor activity (means ± SEM) of control, asymptomatic and symptomatic groups of mice in the open field test. The observer registered the number of times the animals entered completely into each square (counts/5 min). (F) Detection of HCoV-OC43 RNA in the brain of symptomatic BALB/c mice inoculated intracerebrally with 10^4.5 TCID₅₀ of HCoV-OC43. Although RT-PCR analysis with primer pairs O1–O3 did not detect HCoV-OC43 RNA, nested primer pairs O1.1–O3.1 did show the presence of HCoV-OC43 RNA. Lanes 1, 2, 3, and 6, 7, 8: surviving mice 1 year post-infection; lanes 4 and 9: noninfected mice; lanes 5 and 10: positive-control HCoV-OC43 infected HRT-18 cells.

Fig. 7

Chronic encephalitis and neuronal loss in hippocampus, 6 months post-infection. Scattered damage of CA1 neuronal cell layers was observed (arrows). Disruption of the neuronal CA1 layer is more evident at higher magnification (right panel). Original magnification, ×40 (left panel) and ×200 (right panel).

Fig. 8

Chronic encephalitis and neuronal loss in hippocampus, 1 year post-infection. (A) Cresyl violet staining revealed neuronal loss in CA1 and CA3 hippocampal cell layers (arrows). Original magnification, ×40. Magnification of damaged regions (a–c) illustrates neuronal loss on CA1 and CA3 hippocampal layers and the presence of infiltrating cells (magnification, ×200). Panel C and magnification on panel D: cluster of activated microglia. Original magnification, ×100 (C) and ×200 (D).