Deep sequencing for the detection of virus-like sequences in the brains of patients with multiple sclerosis: detection of GBV-C in human brain

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.

Figure 1. Representational Analysis.

Virus-like sequences overrepresented in MS brain specimens compared with controls are displayed. Bonferroni corrected p-values beginning with 0.05 are displayed. Each row (labeled, N = 64) represents an overrepresented viral taxon. Each column (N = 17) represents a different MS brain specimen. The shaded yellow boxes represent significant hits. The GBV-C sequences confirmed by PCR in one of the specimens is circled in red.

Figure 2. Amplification of GBV-C RNA from the brain of MS-afflicted subject MS-6.

Following the failure of published primers to amplify sequences in this specimen, specific primers were constructed based on the sequences of specific reads obtained from the Illumina GAII sequencing of this specimen. A. Four regions of the GBV-C genome were selected for amplification including 2 sites in the 5′ non-translated region, the E2 (envelope protein) gene, and NS3 (non-structural). B. Amplicons were derived from each of these primer sets, including + (genome) and − (replication intermediate) strands. Sequencing of 3 of these amplicons spanning 906 bp revealed identity with >100 published GBV-C isolates, indicating that this subject had a novel strain of GBV-C in her brain tissue which appeared to be replicating at the time of her death.

Figure 3. Assembly of GBV-C sequences from the deep sequencing reads.

Sanger sequencing results from brain specimen MS-6 are shown in green. Deep sequencing reads from subject MS-6 that aligned perfectly (36/36 bp matches) to the GBV-C genome are displayed in orange.