Abstract
Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1–2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a “hit”. Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4–10 million sequences (“reads”) each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.
Highlights
We report the results of deep sequencing of diseased brain tissue taken from deceased patients with multiple sclerosis
The 17 sequenced Multiple sclerosis (MS) specimens came from deceased patients that carried the diagnoses of primary progressive MS (3), chronic progressive multiple sclerosis (5), secondary progressive MS (5), relapsing-remitting MS (2), or multiple sclerosis, subtype not specified (2)
Human and ribosomal reads were subtracted from the dataset, providing 0.9–7.4 million screened reads for analysis
Summary
Multiple sclerosis (MS) is a chronic demyelinating disease of unknown cause, which affects the brain and spinal cord of about 400,000 individuals in the U.S A number of viral infections of the CNS can lead to demyelination, including distemper (dogs), measles (SSPE, humans), and influenza (humans). [1] Viruses have long been suspected as causative agents in MS based on the epidemiology of the disease including geographic patterns, isolated outbreaks, and migration studies. [2,3,4,5] Novel viruses that cause human disease continue to be discovered using molecular techniques including hepatitis C (1989), corona virus NL63 (2004), bocavirus (2005), and rhinovirus C group(2007). [6,7,8,9] New human polyoma and arenaviruses, identified by high-throughput or ‘‘deep’’ sequencing of pathologic specimens, were recently discovered as causes of serious human diseases(2008). [10,11]To date most studies into the possible viral etiology of MS have focused on human DNA viruses. [2,3,4,5] Novel viruses that cause human disease continue to be discovered using molecular techniques including hepatitis C (1989), corona virus NL63 (2004), bocavirus (2005), and rhinovirus C group(2007). The completion of the Human Genome Project allows subtraction of human sequences from the dataset resulting in a large number of plausibly-nonhuman sequences that can be compared to existing databases such as GenBank. This technique was recently employed to discover novel picornaviruses linked to respiratory illness and diarrhea in children. This technique was recently employed to discover novel picornaviruses linked to respiratory illness and diarrhea in children. [12,13]
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