Neuroinvasion by Human Respiratory Coronaviruses

Frozen coded brain samples from MS patients and control donors were received from the following brain banks: The Multiple Sclerosis Society Tissue Bank, Institute of Neurology, University of London, London, United Kingdom (Jia Newcombe); Laboratoire de Neuropathologie Raymond Escourolle, Groupe Hospitalier Pitié-Salpétrière, Paris, France (Jean-Jacques Hauw and Danielle Seilhean); Rocky Mountain Multiple Sclerosis Center Tissue Bank, Englewood, Colo. (Catalin Butunoi and Ronald S. Murray); Multiple Sclerosis Human Neurospecimen Bank, West Los Angeles VA Medical Center, Los Angeles, Calif.) (Virginia Sanders and Wallace W. Tourtellotte); Douglas Hospital Research Center Brain Bank, McGill University, Verdun, Québec, Canada (Danielle Cécyre, Yvan Dumont, and Rémi Quirion). Samples were stored at −70°C.

Total cellular RNA from human brain tissues was extracted as already described (4). Briefly, tissues were lysed with guanidinium isothiocyanate buffer and then layered onto a cesium chloride cushion for a 12- to 20-h centrifugation at 150,000 × g. The RNA pellet was resuspended in H₂O. The pairs of primers used for amplification of the RNA of HCoV-OC43, HCoV-229E, and MBP (internal gene control) are described in Table1. Forty picomoles of the inversely complementary primer was incubated with total cellular RNA at 65°C for 5 min to denature RNA; this was followed by a slow cool down to 37°C for annealing. Reverse transcription with Moloney murine leukemia virus reverse transcriptase (38 U; Pharmacia, Baie d'Urfé, Québec, Canada) was performed at 42°C for 90 min in the presence of a mixture of 48 to 75 U of RNA Guard (Pharmacia), 0.4 mM (each) deoxynucleoside triphosphate (dNTP) (Pharmacia), and 1× reverse transcriptase buffer (50 mM Tris-HCl [pH 8.0], 62.5 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol [DTT]). Optimal PCR conditions were determined with cDNA transcribed from positive-control RNAs. For PCR, one-fifth of the synthesized cDNA was incubated in the presence of either 20 (O1 and O3, O7 and O9, and M1 and M3 primer pairs) or 50 pmol (E7 and E9 and E11 and E13 primer pairs) of the sense and antisense primers, with either 2.0 (E7 and E9 and O7 and O9 primer pairs), 2.5 (O1 and O3 and M1 and M3 primer pairs), or 4.0 mM (E11 and E13 primer pair) MgCl₂ (Roche Diagnostics, Laval, Québec, Canada), 1× PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl; Roche Diagnostics), dNTP (0.2 mM dATP, dCTP, and dGTP and 0.6 mM dUTP; Roche Diagnostics), and 2 U of uracil DNA glycosylase (UNG; Roche Diagnostics) at 20°C for 10 min (UNG elimination of any potential contamination from a previous PCR) and then at 94°C for 10 min (inactivation of UNG and denaturation of DNA), and finally at 60°C for another 5 min (annealing). After the addition of Taq DNA polymerase (2.5 U; Roche Diagnostics) 30 cycles of 2 min at 72°C, 1 min at 94°C, and 2 min at 60°C were performed, with a final elongation step of 10 min at 72°C. DNA amplicons were separated by electrophoresis in a 1.5% (wt/vol) agarose gel. Southern hybridization was performed on amplicons according to standard techniques using a³²P-labeled internal oligonucleotide (O2, O8, E8, E12, or M2; Table 1). The suitability of RNA for RT-PCR amplification was verified by an RT-PCR specific for an internal control, MBP RNA. We worked in very stringent conditions in order to avoid any contamination from laboratory viruses or previous PCR amplicons. Positive-displacement pipettes and the UNG carryover prevention system were used. Moreover, a strict physical separation of pre- and post-PCR areas was enforced. RNA extracted from infected cells (HRT-18 cells infected by HCoV-OC43 and L-132 cells infected by HCoV-229E) were used as positive controls in our RT-PCR. A negative control consisting of master mixture solution without added RNA or cDNA was included in each reaction of RT and PCR, respectively, and put also on each agarose gel that was eventually transferred onto a nylon membrane and hybridized with a radiolabeled oligonucleotide. The sensitivity of the combination of the RT-PCR and Southern testing was estimated by preparing in vitro-transcribed RNA from the N gene cloned in pGEM-T vector as described below (“Preparation of riboprobes”). The RNA was purified from an agarose gel, quantified by spectrometry measurements, and then diluted and used to perform the RT-PCR and Southern hybridization under the same conditions used for brain RNA. The sensitivity of our RT-PCR/Southern hybridization test was estimated at 10²copies of the viral N gene (data not shown). We do not consider it possible that the viral sequences detected by RT-PCR could have been DNA since the MBP-specific RT-PCR encompassed different exons. A DNA contamination would have been detected by a larger PCR product than expected without introns, which we have never experienced with the numerous samples we have handled.

Since PCR products were most of the time not detectable on an agarose gel, a nested PCR was performed on amplicons that were positive for the first round as detected by Southern hybridization, in order to obtain enough material for cloning and sequencing. PCR products yielding a positive signal in Southern blotting were used for nested PCR to allow sequencing of specific amplicons. Ten microliters of a 1/100 dilution of the first PCR product was incubated with 50 pmol (O1.1 and O3.1 and E7.1 and E9.1 primer pairs) of sense and antisense primers–0.4 mM (each) dNTP–1× PCR buffer–2.5 mM MgCl₂ for 5 min at 94°C and then for 5 min at 60°C. Taq DNA polymerase was then added, and tubes were incubated for 30 cycles as described above. PCR products were cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and XL-1 blue bacteria and sequenced with an automated sequencer (ALF; Pharmacia) in both directions; at least three clones were used for each cloning reaction. Nucleotide sequences and predicted amino sequences were analyzed with Geneworks software, version 2.5.1 for Macintosh (Oxford Molecular Ltd., Oxford, United Kingdom).

RNA from infected cells (HRT-18 cells infected with HCoV-OC43 and L-132 cells infected with HCoV-229E; both laboratory HCoV strains were originally obtained from the American Type Culture Collection) was used to amplify a fragment of the N-coding RNA of each viral strain with primers described in Table 1. RT was performed as described above. PCR was done as for nested PCR described above; 1/10 of the cDNA was used with 20 pmol of sense and antisense primers. PCR products were cloned into the pGEM-T vector (Promega; distributed by Fisher Scientific Ltd., Nepean, Ontario, Canada) and then partially sequenced with an automated sequencer (ALF; Pharmacia) in both directions to verify the integrity of each sequence as well as its orientation. Plasmids were then linearized, and 1 to 10 μg of DNA was transcribed into RNA for 2 h at 37°C in the presence of a mixture consisting of SP6 or T7 RNA polymerase (20 U; Roche Diagnostics), 40 mM Tris-HCl (pH 7.4), 6 mM MgCl₂, 20 mM DTT, 4 mM spermidine, RNase inhibitor (40 U; Roche Diagnostics), and 0.5 mM ATP, CTP, and GTP and 170 μCi of [³²P]UTP (800 Ci/mmol, 20 mCi/ml; Amersham) for the riboprobes to be used for Northern hybridization or 0.5 mM ATP and GTP and 250 μCi of³⁵S-UTP and ³⁵S-CTP (10 mCi/ml, >1,000 Ci/mmol; Amersham) for the riboprobes to be used for in situ hybridization. Plasmid DNA was digested with RNase-free DNase (10 U; Roche Diagnostics) in the presence of a mixture of 25 μg of yeast tRNA, 40 mM Tris-HCl (pH 7.4), 6 mM MgCl₂, 10 mM DTT, 4 mM spermidine, and RNase inhibitor (40 U; Roche Diagnostics) for 15 min at 37°C. A phenol-chloroform extraction was performed, and the upper phase was transferred onto a G-50 Sephadex column to eliminate nonincorporated radiolabeled nucleotides. For in situ hybridization, probes were hydrolyzed in the presence of 40 mM NaHCO₃–60 mM Na₂CO₃ for 15 min at 60°C, followed by a neutralization and precipitation step with 91 mM sodium acetate–0.5% acetic acid–15 μg of yeast tRNA–608 mM ammonium acetate to which was added 2.5 volumes of cold ethanol. The RNA pellet was resuspended in H₂O. Probes labeled with ³⁵S were maintained under reducing conditions by adding a final concentration of 10 mM DTT.

For Northern hybridization, ³²P-labeled riboprobes were diluted in the hybridization buffer (5% [wt/vol] sodium dodecyl sulfate [SDS], 400 mM NaPO₄, 1 mM EDTA, 1 mg/ml bovine serum albumin, 50% (vol/vol) formamide). For in situ hybridization,³⁵S-labeled riboprobes were diluted in the in situ hybridization buffer (75% [vol/vol] formamide, 3× SSC [1× SSC is 0.15 M NaCl plus 0.017 M sodium citrate], 0.1× Denhardt's solution, 200 μg of yeast tRNA/ml, 50 mM NaPO₄ [pH 7.4], 0.1 g of dextran sulfate/ml) at a concentration of 3.3 × 10⁴ cpm/μl.

Five micrograms of denatured total RNA was separated by electrophoresis on a 1.5% (wt/vol) agarose–20 mM HEPES–1 mM EDTA–6% (vol/vol) formaldehyde gel. Bands were transferred overnight to a positively charged nylon membrane (Nytran; Xymotech, Mont-Royal, Québec, Canada) by capillary action in 10× SSC buffer (1.5 M NaCl, 0.17 M sodium citrate, pH 7.0). The membrane was dried at 65°C for 1 h, and then RNA was fixed with UV light. The membrane was stained with methylene blue to verify adequate transfer. Prehybridization (prehybridization buffer: 5% [wt/vol] SDS, 400 mM NaPO₄, 1 mM EDTA, 1 mg of bovine serum albumin/ml, 50% [vol/vol] formamide) for at least 2 h at 60°C preceded overnight hybridization at 60°C with ³²P-labeled riboprobes. Blots were washed for 20 min and then twice for 1 h in 0.1× SSC–0.1% (wt/vol) SDS–1 mM EDTA at 70°C and exposed to Kodak X-OMAT autoradiography film (17).

In situ hybridization was performed with a slight modification of a published protocol (46). Tissue sections 5 to 10 μm thick were fixed in 4% (vol/vol) phosphate-buffered paraformaldehyde solution for 1 h at room temperature and then washed twice in phosphate-buffered saline, pH 7.4. A proteinase K treatment of 10 min at 37°C was performed (0.1 μg/ml) followed by the following washes: 5 min in H₂O, 5 min in 0.1 M triethanolamine, 10 min in 0.1 M triethanolamine containing 0.25% (vol/vol) acetic anhydride, and then 5 min in 2× SSC. The washes were followed by dehydration in graded ethanol baths (50 to 95% [vol/vol]). A ³⁵S-labeled (10⁶-cpm) riboprobe was added onto each tissue section, and an overnight incubation at 55°C in a humid chamber with 75% (vol/vol) formamide was performed. Sections were washed with 2× SSC to remove excess hybridization buffer and then treated for 45 min at 37°C with RNase A (40 μg/ml), and finally the following washes were performed: three 10-min washes at room temperature in 2×, 1×, and 0.5× SSC and then one wash for 45 min at 60°C in 0.1× SSC. Sections were dehydrated in graded ethanol (50 to 100% [vol/vol]) prior to coating with K-5 nuclear emulsion (Illford), exposition for 1 to 2 months at 4°C, and then development with D19 (Kodak) for 2 min and fixation for 4 min with sodium thiosulfate (30% [wt/vol]). Tissues were dehydrated in graded ethanol baths and counterstained with cresyl violet. The counterstain performed after the hybridization procedure does not result in a typical Nissls staining. Neuron nuclei were weakly stained and are large and pale, while glial cell nuclei were stained strongly purple-blue and are smaller in size (17). Slides were observed with an Eclipse 800 Nikon microscope and a Darklite illuminator (Micro Video Instruments Inc., Avon, Mass.). A positive signal was characterized by a significant number of silver grains developed over cells compared with background.

HCoV or murine-like coronavirus RNA has been previously detected in human brain samples, with an apparent preferential association with MS (37, 51). However, the establishment of the statistical significance of these observations justified the requirement for a larger number of patients. Therefore, we verified the presence of HCoV RNA within a large panel of human brain autopsy samples. We looked for viral RNA and not viral protein or infectious virions since it has been established that during persistent MHV infections of murine CNS, viral proteins and virions could not be detected for periods as long as could viral RNA (22). Coded brain samples were treated blindly, and only when experiments were completed were the codes broken to ensure complete objectivity in the interpretation of results. We amplified by RT-PCR segments of the nucleocapsid protein (N) gene of HCoV since this RNA is expressed in larger amounts during a viral infection and also because its sequence is present on all subgenomic and genomic viral RNAs (32). For each viral strain, two distinct regions of the N gene were amplified. In order to increase both the sensitivity and specificity of our assay, a Southern hybridization was performed on PCR amplicons. An experiment was considered successful and was included in our final results when both negative controls (RT and PCR) did not show any signal and also when the positive control showed a very strong hybridization signal. A brain donor was considered positive for one viral strain only when both RT-PCR primer pairs for that specific strain showed a positive signal on the Southern blot or RNA from two distinct extractions amplified by the same primer pair yielded positive Southern hybridization signals.

A description of patients is presented in Table2. The time postdeath prior to brain removal varied between samples but did not influence the suitability of RNA for amplification by RT-PCR since we were able to amplify MBP RNA for at least one block from every patient from which a sample was obtained. A description of the histological localization of positive signals for viral detection by donors is presented in Table3, when this information was available from brain banks. A positive signal for HCoV detection was often observed in more than one brain tissue block obtained from the same patient. Signals were observed in plaque samples but also in grey and white matter from both controls and MS patients. There was no apparent correlation between parts of the brain used for HCoV detection and results obtained.

Overall, 48% of all donors were positive for either one or both HCoV (Table 4). Considering that we have used on average a 200-mg tissue sample per block and that a human brain weighs between 750 g and 1 kg, we used less than 1/1,000 of the total brain. Given these considerations, we conclude that the proportion of human brain samples which were positive for one or both HCoV (48% of all) was relatively large. Statistical analysis of our results showed that the 229E strain was present in all groups and did not show a preferential association for women versus men or for patients with MS versus those with OND or normal controls. However, the OC43 strain was preferentially present in MS patients compared to those with OND (P = 0.0169) or compared to all controls (P = 0.0137) as calculated by chi-square and Fisher's exact tests, respectively. Normal controls did not show a statistically different proportion of HCoV-OC43-positive brains compared to MS patients.

To ascertain the coronaviral origin and also to verify the possible molecular adaptation of these viruses in human brain samples compared to laboratory viruses, we sequenced HCoV amplicons from human brains. It has been shown for the murine counterpart of HCoV, MHV, that point mutations and deletions arise during a persistent in vivo infection of the murine CNS (1, 44). Moreover, certain point mutations were mainly observed in animals experiencing chronic demyelinating disease and rarely in asymptomatic animals (42). The viral protein N was previously shown to have mutated in the CNS of the viral host (1). We compared sequences obtained to already-published sequences for each viral strain. We did not observe point mutations or deletions among the human brain amplicons for the 229E strain compared to viral amplicons obtained from our positive control (RNA from infected cells). Two point mutations for the OC43 strain, one leading to an amino acid change (position 398; AGA→GGA; R→G) and the other silent (position 442; CTG→CTA) were observed in four different brains: one normal control and three brains of MS patients. Moreover, an additional point mutation (position 261; GTA→GCA; V→A) was observed in one of the MS patient brains. These mutations were never observed in any positive controls (viral RNA from acutely infected patient cell lines). We conclude that these point mutations were probably representative of those in viral RNA present in human brains. Indeed, these changes suggest that HCoV could have adapted to the CNS, unlike the laboratory viruses, which we obtained from the American Type Culture Collection and which were initially isolated from the upper respiratory tract. Moreover, relative consistency of the viral sequence for the N gene strongly suggests that the amplified RNA from human brains was really from HCoV and not from an unknown source.

In order to evaluate the amount of viral RNA and whether a sufficient amount was detectable prior to attempting in situ hybridization experiments, we performed Northern hybridization on RNA taken from some human brains (four samples were tested, three of which were positive by RT-PCR and two of which yielded signals in Northern hybridization). Representative results obtained are shown in Fig.1. Expected viral subgenomic RNAs, all of which retain the sequence of the N gene, were detected in the positive control (RNA from virus-infected cells). As expected (32), the subgenomic viral RNA encoding the N gene was expressed in the largest amounts in infected cells. Lanes 2, 3, and 4 represent hybridization to three different brain RNAs. We were able to detect RNA corresponding to the N gene of the OC43 strain on two of three RNA preparations. For the 229E strain, even though the positive control showed a strong signal for all subgenomic viral RNAs, we did not detect any signal in the human brain RNA tested (data not shown). Thus, viral RNA could be detected in human brains even without amplification by RT-PCR. Since this technique is less sensitive than RT-PCR coupled to Southern hybridization but has a sensitivity comparable to that of Northern blotting, this suggested that viral RNA may be present in sufficient amounts to be detected by in situ hybridization, which has even been reported to be more sensitive than Northern blotting when RNA is localized in a small portion of the tissue (3).

In order to verify the localization of viral RNA in the CNS, an essential confirmation of the suspected neuroinvasion, we performed in situ hybridization on human brain sections, using the same probe as for the Northern blot showing specific hybridization to viral RNA. In situ hybridization performed on immortalized human neuronal cells revealed radiolabeled riboprobe signals only on infected cells and not on noninfected cells (M. Viau, N. Arbour, and P. J. Talbot, unpublished observations). These experiments were also performed blindly, in order to provide absolute objectivity in the interpretation of results. At least two slides of the same tissue block had to be positive in order to consider the block positive. Samples from seven donors were selected on the basis of RT-PCR results. We did not observe any signal for the 229E strain, most likely because the amount of viral RNA was not sufficient for detection by this technique (data not shown). Indeed, we did not detect HCoV-229E RNA in human brains by Northern blotting, corroborating these in situ hybridization results (data not shown). On the other hand, we detected positive signals for the OC43 strain on two blocks from one MS patient and on one block from a healthy control. Samples from both of these donors were positive for HCoV-OC43 by RT-PCR. We did not detect any signal when slides adjacent to the positive ones were pretreated with RNase A prior to hybridization (data not shown). We also did not observe any positive signal with the sense probe, suggesting that the negative-strand viral RNA was not present in detectable amounts (data not shown). Hybridization of slides just adjacent to the positive ones with the 229E strain riboprobe did not yield any signal (data not shown). Examples of hybridization signals observed are shown in Fig.2. Dark-field pictures clearly show that positive signals were really distinct from normal background. Three or four positive regions were randomly distributed on each positive slide. We often observed that more than one cell was positive in the same region. Signals obtained were never seen in blood vessels or grey matter but only in the white matter parenchyma.