Neuroinvasion by human respiratory coronaviruses

Abstract

Human coronaviruses (HCoV) cause common colds but can also infect neural cell cultures. To provide definitive experimental evidence for the neurotropism and neuroinvasion of HCoV and its possible association with multiple sclerosis (MS), we have performed an extensive search and characterization of HCoV RNA in a large panel of human brain autopsy samples. Very stringent reverse transcription-PCR with two primer pairs for both viral strains (229E and OC43), combined with Southern hybridization, was performed on samples from 90 coded donors with various neurological diseases (39 with MS and 26 with other neurological diseases) or normal controls (25 patients). We report that 44% (40 of 90) of donors were positive for 229E and that 23% (21 of 90) were positive for OC43. A statistically significant higher prevalence of OC43 in MS patients (35.9%; 14 of 39) than in controls (13.7%; 7 of 51) was observed. Sequencing of nucleocapsid protein (N) gene amplicons revealed point mutations in OC43, some consistently found in three MS patient brains and one normal control but never observed in laboratory viruses. In situ hybridization confirmed the presence of viral RNA in brain parenchyma, outside blood vessels. The presence of HCoV in human brains is consistent with neuroinvasion by these respiratory pathogens. Further studies are needed to distinguish between opportunistic and disease-associated viral presence in human brains.

FIG. 1

Northern hybridization for HCoV-OC43 on RNA extracted from human brains (lanes 1 to 3) or from positive-control HCoV-OC43-infected HRT-18 cells (lane V). HCoV-OC43 subgenomic RNAs are as follows, from top to bottom (estimated sizes and encoded proteins are indicated): RNA 2 (11.6 kb; ns2 protein); RNA 2-1 (8.8 kb; HE protein); RNA 3 (7.1 kb; S protein); RNA 4 (4.0 kb; ns4 protein); RNA 5 (3.3 kb; ns5 protein); RNA 5-1 (3.0 kb; E protein); RNA 6 (2.5 kb; M protein); RNA 7 (1.8 kb; N).

FIG. 2

Photomicrographs showing in situ hybridization for HCoV-OC43 RNA in human brain sections, using a radiolabeled riboprobe for the viral N gene. Results shown in panels A and B and in panels C and D were taken from two different blocks from the same MS patient. (E and F) Negative control; slides adjacent to those used for panels A and B; samples were treated by RNase A prior to in situ hybridization. Photographs shown in panels A, C, and E were taken under bright-field exposure, whereas those shown in panels B, D, and F were taken at the same site under dark-field exposure. Magnification = ×100.