key: cord-0772144-gfx3qcm8 authors: Dessau, Ram B.; Lisby, Gorm; Frederiksen, Jette L. title: Coronaviruses in brain tissue from patients with multiple sclerosis date: 2001-05-25 journal: Acta Neuropathol DOI: 10.1007/s004010000331 sha: fa171dd4308d8d64a7e072fcf14e15f1d9a3ce7b doc_id: 772144 cord_uid: gfx3qcm8 Brain tissue from 25 patients with clinically definite multiple sclerosis (MS) and as controls brain tissue from 36 patients without neurological disease was tested for the presence of human coronaviral RNA. Four PCR assays with primers specific for N-protein of human coronavirus strain 229E and three PCR assays with primers specific for the nucleocapsid protein of human coronavirus strain OC43 were performed. Sporadic positive PCR assays were observed in both patients and controls in some of the PCR assays. However, these results were not reproducible and there was no difference in the proportion of positive signals from the MS patients compared to controls. Evidence for a chronic infection with the human coronaviruses strain 229E or OC43 in brain tissue from patients with MS or controls has not been found in this study. The cause of multiple sclerosis (MS) is so far unknown, but both heredity and environmental factors are considered important. Environmental factors, especially viruses, have been the subject of extensive research and discussion, but no specific agent has so far been definitely linked to MS [12] . Human coronaviruses are a worldwide cause of the common cold, but are usually not associated with more serious disease in humans [17] . The mouse hepatitis virus, a murine coronavirus, causes a MS-like demyelinating disease in the central nervous system of rodents, and this animal model has been used as a model for MS [9, 27] . Evidence for the presence of coronaviral genes in human brain tissue has been found using polymerase chain reaction (PCR) or in situ hybridization [15, 16, 25] . The aim of this study was to examine brain tissue from Danish MS patients for human coronaviral RNA sequences using reverse transcriptase (RT)-PCR. Brain tissue was obtained from 25 patients with clinically definite MS from two Danish tissue collections. Most patients had chronic progressive MS for many years. Data on the age at death was available for 20 patients (median 60 years, range 38-83 years). From the 25 patients with clinically definite MS, specimens from areas with macroscopically visible plaques were sampled from the periventricular mostly white matter. All brain specimens were stored at -70°C before and after RNA extraction. For the older tissue collection the year of death ranged from 1966 to 1981, whereas the newer brain collection was from 1987 to 1990. As controls, brain tissue was obtained from 36 consecutive routine autopsies. Autopsy of the brain was routinely performed if a patient had shown some clinical signs of possible intracerebral pathology prior to death. The pathological reports were reviewed and no patients had a history of neurological disease. The cerebral lesions were previous cerebral infarction in 2 patients and moderate cerebral arteriosclerosis 4 patients. Of the controls 27 had normal cerebral pathology. The causes of death were cancer in 13, lower respiratory tract infection in 9, acute myocardial infarction in 10, and pancreatitis in 1 of the controls. The autopsy report was not available from 3 patients. As positive control cell culture supernatant fluid containing human coronavirus strain 229E (HCV-229E, ATCC VR-740) and strain OC43 (HCV-OC43, ATCC VR-759) were used. HCV-229E was cultured on L-132 cells (ATCC CCL5) at 37°C and the cell supernatant containing a TCID 50 (50% tissue culture infective dose) of 10 6 /ml was used as positive control. HCV-OC43 was cultured on HRT-18 cells (ATCC CCL244) at 33°C and the cell supernatant containing a TCID 50 of 10 5 /ml was used as positive control. Extraction of RNA from brain tissue was performed by homogenization in guanidinium thiocyanate buffer followed by ultracentrifugation in a cesium chloride (CsCl) step gradient [22] . The extracted RNA was evaluated by absorption spectrophotometry at 260 and 280 nm, and by RT-PCR specific for myelin basic protein (MBP) mRNA. Samples negative for MBP or with an absorption ratio A 260 /A 280 lower than 1.6 were excluded from further analysis. Eight primer pairs were used in the present study (Table 1) and combined in eight different RT-PCR assays ( Table 2 ). The sensitivity of the PCR assays were tested by serial dilutions of the viral supernatants containing HCV-229E and HCV-OC43. All PCR reactions were carried out in 100-µl reaction volume and the concentration of primers, DNA-polymerase (AmpliTag, Perkin Elmer Cetus) and MgCl 2 was optimized for each assay. Extensive tables with details of primer combinations and concentrations of reagents are available from the corresponding author on request [6] . For all pre-PCR procedures filter tips and dedicated pipette sets were used. The PCR products were visualized by UV transillumination following electrophoresis on 1.5% agarose and subsequent Southern blot on Nytran filter. The blot was performed as described, with the exception that the setup was inverted [24] . After overnight transfer, the Nytran filter paper was rinsed briefly in 2× SSC, and nucleic acids were immobilized by baking at 80°C in vacuum for 2 h. Subsequently, the blots were prehybridized at 42°C for 2 h, followed by hybridization overnight with a HCV-229E or HCV-OC43 DNA probe. Stringent washing was performed in SSC buffer (saline sodium citrate) at 70°C. The probes were produced by PCR as described above from the reference strains and labeled with biotin-7-dATP by nick translation (BioNick, Life Technologies, Rockville, USA). Probe detection was performed using the PhotoGene (BioNick) detection kit according to the manufacturer's instructions. A result was registered positive when a band of the expected size was present after hybridization. Cycle sequencing was performed according to the manufacturer's instructions (Perking Elmer Applied Biosystems, California, USA) with [α-33 P]dATP as radioactive label. The reactions were performed according to the manufacturer's instruction. Electrophoresis was run on a polyacrylamide gel. The gels were dried on filter paper and X-ray films were used to visualize the radioactive bands on the dried gel. A total of 97 specimens from 61 individuals were assayed in 1063 RT-PCR assays (173 specific for MBP mRNA, 416 for HCV-229E and 474 for HCV-OC43). Twenty-one specimens had an A 260 /A 280 ratio lower than 1.6, and 4 of these were also negative for MBP. These 21 specimens were excluded, and only the results of the remaining 76 specimens are described. The 76 specimens yielded a mean of 105 µg RNA/g brain tissue, which is slightly lower than the 200-600 µg/g reported in the literature using the same method of extraction [10, 14, 21] . One MS patient was completely excluded as no RNA was detectable in any of the samples extracted. The sensitivity of the assays were in the range of below 10 for the nested PCR assays to 100 Tissue culture infective doses. Of the specimens tested, 32% were positive in at least one assay. An example of a PCR result is shown in Fig. 1 . For comparison between the MS group and controls each individual was only counted once using the first specimen extracted and assay performed (Table 3 ). In one nested PCR assay for HCV-229E, 31% of the patients were found positive. These results could not be reproduced by repeating the same PCR or on the other PCR analyses, neither on the same or different specimens. These PCR products were sequenced and found identical to the expected sequence from the human coronaviral nucleocapsid gene. There was no proportional difference in these sporadic positive signals between the MS group and controls. No patient or control was consistently positive, neither on reanalysis of RNA from the same extraction or different specimens from the same individual. The yield of RNA extraction was satisfactory, as 93 of 97 specimens contained amplifiable RNA as determined by MBP mRNA RT-PCR assay. The 4 specimens negative for MBP have been excluded as they may either have degraded RNA or contain substances inhibitory to the RT-PCR. A further 17 specimens gave a positive RT-PCR for MBP, but had a low total absorption and a low A 260 /A 280 ratio and were thus excluded due to low total RNA content. The remaining 76 specimens had optimal yield and quality. The amount of total RNA in brain tissue should not decrease significantly at least up to 48 h postmortem [10, 14, 21] ; however, some specific mRNA may have shorter half-life [18] . Premortem and postmortem factors may both increase and decrease expression of specific mRNA, but this is mainly important when attempting quantitative RT-PCR [18] . In this study MBP-RNA was amplified from most specimens including the older tissues dating back to 1966. It has been described that storage of RNA at -70°C for more than 5 years may affect the use of RNA for in vitro expression studies but sufficient partial or full-length RNA may remain for PCR studies [14] . The RT-PCR assays yielded scattered positive signals for both HCV-OC43 and HCV-229E. No simple technical explanation can be found for these inconsistent positive signals. One explanation could be a very low level presence of coronaviral target gene in only some of the specimens. If very few (in the order of 10 1 or less) copies of the target gene are present the initiation of amplification in a PCR process becomes problematic. This is due to the probability of a target gene copy being present in aliquot and the probability of the primer annealing to the specific template. A low level presence of coronaviruses in the human brain could be possible after an ordinary episode of common cold due to coronaviruses. In animal models murine coronaviruses have been found to enter the brain of mice, rats and owl monkeys after intranasal inoculation [3, 5, 19, 20] and both a hematogenous spread and direct entry via the olfactory nerve has been found. False positive results due to contamination cannot be ruled out, but extensive precautions were taken to avoid contamination [13] and UNG carry over prevention was used in some of the assays. The negative controls interspersed between the patient samples were consistently negative. When comparing the results of the present study with the literature (Table 4 ) we have found a higher frequency of positives in the control group than previously reported. This could be a bias in the previous studies due to more specimens/assays recorded in the groups of patients with MS. The two studies describing HCV genome in the human brain tissue have both counted all the patients with at least one positive signal, disregarding negative results of repeat specimens [16, 25] . Murray et al. [15] used in situ hybridization techniques and found 52% of the patients with MS positive, but only 11% of the 442 sections examined were positive. In the control group 0.8% of 376 sections were positive. In the positive sections only few cells were positive. This study is comparable to the present study in regards to the paucity of the positive signals observed. Talbot et al. [25] have examined 21 specimens from 11 patients with MS and 11 specimens from 11 controls. Not all samples from the positive patients were found positive for HCV-229E. However, the results from the positive specimens were reproducible in contrast to the present study. Weighted pooling of the data (Table 4) shows that more patients with MS were positive compared to controls. This estimate could be biased, as negative studies are less likely to be published [8] . The present results do not provide evidence for a chronic infection with the human coronaviruses strain 229E or OC43 in brain tissue from patients with MS or controls. However, few positive signals were found, which could be sporadic contamination, but low level viral expression of in brain tissue cannot be ruled out. Table 3 . Combination of the tables using weighted mean and 95% confidence intervals for the pooled differences: 26%, 12-40%, P<0.001 [2] Human coronaviruses and multiple sclerosis: detection by RT-PCR in human brains and infection of primary cultures of neural cells. 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A laboratory manual Sequence analysis of the nucleocapsid protein gene of human coronavirus 229E Detection of specific sequences among DNA fragments separated by gel electrophoresis Human coronavirus gene expression in the brains of multiple sclerosis patients The organization of the human myelin basic protein gene -comparison with the mouse gene Immunopathological aspects of coronavirus infections Acknowledgements Financial support was provided by the Scleroseforeningen (The Danish Multiple Sclerosis Society), the "Direktør Ejnar Jonasson, kaldet Johnsen og hustru's" foundation, and the "Warwara Larsens" foundation. The project was approved by Ethics Committee of Copenhagen County, Denmark. Nathalie Arbour and Pierre J. Talbot (Institut Armand-Frappier, Université de Québec, Québec, Canada) provided the positive control strains and some of the primer sequences. Jørgen Clausen, Institute of Life Science and Chemistry, Roskilde University, Denmark is thanked for providing brain tissue samples from patients with MS.