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Neurocognitive deficits in patients with hepatitis C virus (HCV) infection prompted a search for HCV in brain.
HCV was present in the brains of 7 (54%) of 13 patients with viremia, as determined by 5' UTR and E1 (envelope 1) gene analysis. Brain HCV RNA consensus sequences differed from those in plasma and liver in 4 (57%) of 7 patients. The quality ofHCVRNA from postmortem brain and liver was assessed and demonstrated to be suitable for sequence analysis. Quasispecies analysis revealed that several mutations present in clones from >1 brain region were absent in clones from liver and plasma. Brain-specific mutations defined several families of related sequences. The patterns of brain-specific mutations in these families were consistent with the evolution of HCV RNA from a common ancestor. Single-nucleotide-polymorphism analysis confirmed that a prominent brain-specific mutation constituted ~10% of HCV RNA in cerebellum and medulla but that this mutation was undetectable in the liver and plasma of the same patient.
This study introduces novel methods for assessing RNA from postmortem samples. It increases the reported cases of HCV in the brain, provides the first E1 sequences from the brain, and contributes to the growing evidence that HCV replicates and evolves within the brain.
Hepatitis C virus (HCV) causes chronic infection in ~3% of the world's population. A search for HCV in brain was prompted by complaints of fatigue and cognitive dysfunction made by patients with HCV infection [1-7]. Specific cognitive deficits and nuclear magnetic resonance abnormalities have been reported in HCV-infected patients [1, 4, 8-10]. These observations, along with increasing evidence of HCV infection and replication in peripheral blood mononuclear cells (PBMCs) [11-16], suggest that the brain may also be a compartment for extrahepatic HCV replication.
To date,HCVRNAhas been amplified and sequenced from brain tissue of a limited number of subjects [17-21]. Detection of antigenomic HCV RNA has been reported in various brain regions . The population of brain HCV contains sequence variants that are absent in serum. Both the presence of antigenomic HCV RNA and the presence of brain-specific variants suggest that HCV infection and replication may occur in the brain.
Despite the importance of sequence analysis for investigations of brain HCV, no study has examined the suitability of postmortem (PM) material for sequence analysis, although, of necessity, such studies are almost universally performed on PM material. We developed a bioinformatic method to assess the quality of RNA templates from PM tissue. This method demonstrated that brain HCV RNA is a suitable template for sequence analysis. Sequence analysis was then conducted on portions of the 5' untranslated region (UTR) and the E1 (envelope 1) gene. Results of both direct sequencing and quasispecies analysis support the hypothesis that HCV replicates and evolves within the brain.
Details on sample and clinical data collection and sequence preparation can be found in appendix A, which appears only in the electronic edition of the Journal, and in tables 1 and and2.2. Sequences have been deposited in GenBank under accession numbers EU165716-EU166327.
Sequences were aligned using the ClustalW algorithm in MegAlign (DNAStar). E1 sequences were compared with 783 1a and 373 1b sequences downloaded from http://hcv.lanl.gov/components/hcv-db/combined_search/searchi.html on 19 December 2005. Substitution frequencies were tallied using custom Perl scripts. Comparisons of the average number of mutations in sequence sets were performed using 2-sample t tests. P < .05 was considered to indicate statistical significance.
Amplicons from patient 20024 were either analyzed directly or digested with Alw261 (Fermentas) in a 20-μL reaction containing 12.5 μL of gel-purified DNA. Restriction fragments were fractionated in 1.3% aga-rose 1× TBE (Tris-borate-EDTA) gels and subsequently stained with Syto 60 (Invitrogen). Gels were scanned by use of an Odyssey scanner (Licor). Bands were quantified using accompanying software.
The study population was comprised of 27 subjects (table 3). On the basis of testing of stored premortem plasma, 13 had HCV viremia, and 14 did not have HCV viremia. None received HCV-targeted therapies between determination of HCV load and death. Ten of the 13 patients with HCV viremia were positive for HIV antibodies, as were 10 of the 14 patients without HCV viremia.
In the first set of experiments, HCV was amplified from brain (frontal cortex), liver, and plasma. Amplicons were fractionated by gel electrophoresis. Specimens were scored positive if a 5' UTR and/or an E1 amplicon were present. For the 13 HCV-infected patients, 5' UTR amplicons were obtained from 12 of 13 liver, 9 of 12 plasma, and 4 of 13 brain/frontal cortex specimens (table 3); E1 amplicons were obtained from 13 of 13 liver, 11 of 12 plasma, and 7 (54%) of 13 brain/frontal cortex specimens. The average PM interval (PMI; the time between death and sample collection) was 6.6 h for brain specimens that yielded amplicons in the nonnested 5' UTR assay, whereas the PMI was 12.75 h for specimens that yielded neither 5' UTR nor E1 amplicons (P = .12). Thus, HCV RNA content may decrease during the PMI. Four of 8 brain specimens from men were positive in the 5' UTR assay, versus 0 of 5 brain specimens from women—a trend that may accord with the observation that women are more likely to clear HCV spontaneously . Brain HCV was present in 6 of 10 patients with HIV/HCV coinfection and in 1 of 3 patients with HCV monoinfection. Every amplicon except brain/frontal cortex from 40007 (denoted 40007B) was analyzed by direct sequencing. No E1 amplicon had the sequence of a laboratory strain (such as H77), ruling out plasmid contamination. No appropriately sized amplicons were obtained from 84 HCV reverse-transcription polymerase chain reactions (RT-PCRs) performed on 42 specimens from 14 control subjects without viremia; in all cases, however, a GAPDH amplicon was produced (table 3).
End-point dilution was used to compare the amount of liver and brain/frontal cortex RNA in patients 10086 and 20024. The greatest dilutions yielding E1 amplicons from 10086L and 20024L were 1:10,000 and 1:1000, respectively, whereas the greatest dilutions yielding amplicons from 10086B and 20024B were 1:10 and no dilution, respectively (figure 1). These data suggest that, at PMIs of 4 to 7.5 h, liver HCV was 1000-fold more abundant than brain/frontal cortex HCV.
Detailed analysis of brain HCV sequences would be warranted only if the extracted brain RNAs are reflective of the sequences present at the time of death. We reasoned that PM degradation might alter the template properties of the RNA (e.g., by deamination). A multicomponent bioinformatic assay was developed to assess the quality of PM RNA. This assay takes advantage of the fact that naturally occurring mutations are subject to natural selection, whereas artifactual mutations introduced after death are not. In naturally occurring sequences, natural selection reduces the ratio of nonsynonymous to synonymous (dN/dS) codon substitutions far below the ratio produced by chance. More generally, selection pressures eliminate sequences that contain lethal mutations, causing the frequency distribution of naturally occurring and artifactual mutations to differ. Sequences with artifactual mutations created by degradative processes will have an excess of mutations because they will contain both the mutations that were present at the time of death and any mutations introduced during the PMI.
For the first part of the bioinformatic assay, a 423-base-long region of the E1-encoding sequences (bases 871-1293) from our amplicons were compared with 1156 sequences from GenBank, which were primarily from premortem serum and plasma samples. The dN/dS ratio forGenBanksequences was 0.49,comparedwith 0.39 for (premortem) plasma sequences, 0.32 for PM brain/frontal cortex sequences, and 0.44 for PM liver sequences (table 4A). Thus, the proportion of nonsynonymous amino acid substitutions was actually lower in brain/frontal cortex HCV than in GenBank sequences (P = .045). By this measure, brain HCV was certainly no less constrained than GenBank HCV. This result provides evidence that brain HCV is a suitable template for sequence analysis.
Second, individual genotype 1a brain sequences and 783 genotype 1a GenBank sequences were compared with the consensus of the GenBank genotype 1a sequences, and individual genotype 1b brain/frontal cortex sequences and 373 1b GenBank sequences were compared with the consensus of the genotype 1b GenBank sequences. The average number of nucleotide mutations was 20.55 in GenBank sequences, compared with 23.0 in PM brain/frontal cortex sequences, 20.08 in PM liver sequences, and 20.51 in premortem plasma sequences (no significant difference for all comparisons) (table 4B). The average number of amino acid mutations was 6.32 in GenBank sequences, 5.83 in brain/frontal cortex sequences, 5.91 in liver sequences, and 5.90 in plasma sequences (no significant difference for all comparisons) (table 4B).
Third, mutations were scored as very rare (present in 1% or fewer GenBank sequences), rare (1%-10%), or common (10%-50%). This approach, which is an alternative to the BLOSUM score analysis , can be applied to both nucleic acid and amino acid sequences. The frequency distribution of nucleotide and amino acid mutations and a parallel analysis of BLOSUM scores are provided in table 4C and 4D. Neither PM brain/frontal cortex sequences nor PM liver HCV sequences differed significantly from GenBank sequences in any comparison. These results indicate that HCV RNA extracted from PM tissues did not contain a significantly increased proportion of unusual mutations. On the basis of results from all 3 components of the bioinformatic analysis, PM brain/frontal cortex and liver specimens are appropriate for HCV-sequencing studies.
Amplicons of the 5' UTR from 9 plasma, 12 liver, and 4 brain/frontal cortex specimens were analyzed. Five sequences contained mutations at the positions indicated in figure 2, which depicts the HCV internal ribosome entry site [24, 25]. In patients 10034, 20024, and 20028, the 5' UTR sequence from the brain did not match that from the liver or the plasma. E1 region amplicons were sequenced from 12 plasma, 13 liver, and 7 brain/frontal cortex specimens. On the basis of these sequences, most specimens contained genotype 1 HCV. Specimens 40007L and 40007P contained genotype 3a. It is likely that 40007B also contained genotype 3a HCV; however, the sequence could not be determined. No sequence was obtained from >1 patient. This result shows that specimens did not cross-contaminate each other. Sequences of the E1 protein and signal sequence appear in figure 3A-3C. Mutations in the 1a and 1b sequences are marked to indicate their abundance relative to GenBank sequences. Four putative N-linked glycosylation sites, which have the general sequence N-X-S/T , occur in this region of E1 and are indicated in the alignment. The E1 signal sequences were compared with 420 genotype 1a (figure 3A) or 629 genotype 1b sequences (figure 3B). Amplicon 20028P had an I186T substitution, whereas 20028L had an I187T substitution. Amplicon 20024P had an I187S substitution. Extensive changes in this region are known to inhibit cleavage by the signal peptide peptidase and alter localization of the core protein .
The phylogenetic relationships of the E1 nucleotide sequences are presented in figure 3D. The RNA sequences of different specimens from the same patient were more closely related to each other than to any other sequence in the set, with the exception of sequences from patient 20028. In the 2 cases indicated by asterisks—10086B and 20028B—the consensus sequence of brain HCV differed from that of liver and plasma HCV. In total, the consensus sequence from brain did not match the plasma and liver sequence in 4 of 7 patients. In the E1 region, HCV RNA from 10086 and 20028 in brain differed from that from plasma and liver. In the 5' UTR, HCV RNA from 10034, 20024, and 20028 in brain did not match that in liver and plasma.
If HCV is produced in the brain, it might be possible to find supporting evidence by cloning individual sequence variants and comparing the pattern of mutations in brain clones with that in liver and plasma clones. Quasispecies analysis was performed on a portion of the E1 gene inHCVRNAfrom 2 patients, 20024 and 10086. Plasma samples from these patients were collected 2 and 3 days before death (table 3). Three brain regions (frontal cortex, medulla, and cerebellum) were analyzed. Three separate RT-PCRs were performed on each brain region, producing 3 amplicons per region. At least 15 clones of each brain amplicon were amplified, and at least 43 clones of liver and plasma amplicons were sequenced. The mutational patterns of brain and liver sequences were compared with those of plasma sequences in a quality-control analysis similar to the one presented in table 4. Clones from PM tissues did not contain a greater number of mutations or a higher percentage of rare mutations than those from plasma (data not shown), confirming the suitability of the PM tissues for quasispecies analysis. dN/dS ratios for the quasispecies were not significantly different between compartments (table 5).
A total of 24 distinct amino acid sequence variants were cloned from patient 10086, and 34 were cloned from patient 20024 (figure 4). In both patients, a dominant sequence was shared by the brain (frontal cortex, medulla, and cerebellum combined) and plasma. In patient 10086, the topmost variant in figure 4A was dominant in all tissues; in patient 20024, the topmost variant in figure 4B was dominant in brain and plasma, whereas the 32nd variant, which is a different subgenotype, was dominant in liver.
Subsequent quasispecies analysis was focused on mutations present in clones from brain and absent in clones from plasma and liver. Mutations that were found only in clones from brain and that were present in at least 2 brain clones—but only if they did not represent the same template (i.e., the mutations were present in the products of at least 2 RT-PCRs and/or occurred in variants that differed from each other at 1 or more secondary positions)—were considered to be brain specific. These restrictions were imposed to counter the skewing of the data that can occur whenever a tiny fraction of the RNA population is cloned and template concentrations are low.
Analysis of brain-specific mutations revealed that brain HCV contained several families of related sequences (figures 5 and and6).6). Figure 5A presents a family with 14 members from the brain of patient 10086. All members of the family share at least 1 brain-specific mutation with another member of the family. All 10 of the nucleotide substitutions highlighted in figure 5A occurred only in brain HCV and were not present in liver or plasma HCV. Variant 1 can be considered the nexus of the family. It has 3 brain-specific mutations: U930C, A1062G, G1118A, and C1152U. The U930C mutation is shared by variants 2-5; the A1062G mutation is shared by variants 2, 5, 6, and 7. The C1152U mutation is shared by 6, 7, 13, and 14. The family has multiple subcomponents that form branches when phylogenetic analysis is performed (figure 5B). An explanation for the presence of families is that a sequence with a brain-specific mutation replicated locally to yield progeny that retained the original mutation and accumulated additional brain-specific mutations over time. Three additional families occurred in the clones of patient 10086, and 6 families occurred in the clones of 20024 (figure 6).
To validate the cloning and sequencing results, we used SNP analysis to measure the prevalence of one of the brain-specific mutations. The brain-specific mutation A1113G found in patient 20024 creates a novel restriction site. The amplicons of this patient were incubated with a restriction enzyme specific for this site, and the resulting restriction fragments were analyzed by gel electrophoresis (selected samples are shown in figure 7). The band resulting from cleavage at the site of the mutation (arrow C) constituted an average of 10% over the 3 cerebellum amplicons and a similar percentage of the 3 medulla amplicons. In contrast, this band was not present in digests of liver and plasma amplicons (figure 7). This SNP analysis indicates that the A113G mutation is, indeed, more prevalent in brain than in liver or plasma.
To statistically analyze the extent of sequence compartmentalization, we examined correlations between the genetic distance of the sequences and their body compartment of origin using Mantel's test. As a control for the validity of this approach, we compared sequences obtained from 2 separate RT-PCRs on the same brain region. Statistically significant compartmentalization was observed for sequences from 2 separate RT-PCRs on the cerebellum from patient 20024. Because these sequences were obtained from the same RNA population, this result demonstrates that Mantel's test cannot be used to test for sequence compartmentalization on the basis of genetic distance. For each patient, phylogenetic trees of all variants were constructed (figure 8). For patient 10086, medulla variants partially clustered. For patient 20024, most liver sequences segregated from brain and plasma. This reflects the difference in the predominant subgenotype between these compartments.
The most basic question in HCV neurobiology is whether HCV occurs in living brain outside the vasculature. An affirmative answer would provide an impetus for investigating the clinical significance of the invasion of the brain by HCV. Our investigation advances HCV brain research by presenting evidence that mutations in brain HCV do not have the aberrant features expected of mutations produced by PM degradation, supporting the use of PM brain in HCV sequencing studies. Our study also shows that the consensus sequence of HCV RNA in brain often differs from that in liver and plasma and demonstrates that families of brain sequences have shared brain-specific mutations that are consistent with HCV RNA evolution occurring within the brain. Particularly in light of the bioinformatic data demonstrating that brain HCV did not contain artifactually introduced mutations, the disparity between brain and plasma sequences effectively rules out blood contamination as the source of the brain HCV and focuses attention on the possibility that brain RNA was synthesized locally within the brain.
Our analysis also revealed a nonsignificant trend toward an inverse relationship between PMI and the detection of brain HCV. This trend suggests that the actual prevalence may exceed our observed frequency, which was 54% (7/13). A single 35-cycle protocol was used to amplify the 5' UTR. The ability to detect HCV without a “nested” PCR assay suggests that brain HCV is relatively abundant, although it is 1000-fold lower than liver HCV in PM specimens.
Direct sequencing identified mutations in the first or third putative N-linked glycosylation sites  in the brain E1 amplicon from 4 patients (10066, 20024, 20028, 10034) and in 2 clones from the medulla of patient 10086 (figure 3B and 3C). These mutations are of interest because glycans at these sites are thought to play a role in viral entry . Like the E1 amplicons, the 5' UTR amplicons of 10034B and 20028B had unusual substitutions, suggesting that in these patients both the UTR and the envelope protein may have adaptations that enhance entrance into and expression within the brain. Of the 7 patients with brain HCV, the consensus sequence did not match liver and plasma HCV in 4, contributing to the growing evidence that the brain supports replication of a separate and distinctive subpopulation of HCV.
To obtain further evidence of HCV replication and evolution within the brain, quasispecies analysis was done on HCV RNA from plasma, liver, and 3 noncontiguous brain regions of 2 patients. Specific nucleotide and amino acid mutations (i.e., brain-specific mutations) were found in clones from multiple brain regions and were not found in clones from plasma or liver of the same patient. The repeated detection of the same mutations suggests that they were not the result of polymerase or sequencing errors. Moreover, brain-specific nucleotide mutations present in clones from 2 RT-PCRs on the frontal cortex of patient 10086 were missing from the clones from cerebellum and medulla. This finding further suggests that blood contamination was not likely to be the source of brain HCV RNA, because the viral population from contaminating blood would be the same throughout the brain and would be identical to that in plasma.
Among brain sequences, several families of variants were identified that shared brain-specific mutations. Some families consisted of variants from >1 brain region and were supported by cluster analysis. To investigate the total population of molecules in amplicons (not only those that were individually cloned and sequenced), SNP analysis was performed using a restriction enzyme specific for the A1113G mutation present in the brain quasispecies of patient 20024. This analysis confirmed that the A1113G mutation was, indeed, brain specific. This finding and the presence of sequence families whose members are related to each other through shared brain-specific mutations support the hypothesis that HCV replicates and evolves within the brain.
Our results add to previously reported evidence of brain compartmentalization. The quasispecies in the gray matter of 1 (of 2) liver-transplant recipients did not match that in serum, although the consensus sequences were identical ; brain and serum 5' UTR sequences differed in 2 of 6 patients at the genotype level ; and the brain hypervariable region (HVR) 1 master sequence did not match that in serum in 1 of 2 patients . The mutations we observed in the 5' UTR of brain HCV are an interesting and nonrandom group of substitutions. The U147C mutation in 10034B has been reported in lymph node HCV , and the insertion at position 126 in 20024B and 20028B* has been reported in lymph node [18, 19] and cerebellum  HCV, suggesting that these substitutions might represent adaptations to replication in extrahepatic compartments. The insertion, however, has also been reported in serum HCV [29, 30].
Sequence analysis is currently the mainstay of molecular HCV brain research. Complementary methods, such as immunohisto-chemistry and in situ hybridization, may become more widely available in the future, but despite notable successes [20, 31-33] these techniques are extremely difficult to apply effectively and accurately to HCV [34, 35]. Minus-strand detection has often been used to identify sites of viral replication, but in the case of brain HCV this method can never be definitive because of the potential for PBMCs to portage inert HCV minus strands in and out of the brain. Proof of HCV replication in brain will probably await rigorous and unequivocal demonstration that the population of HCV in 1 or more parts of the brain is distinct from HCV in the periphery.
In conclusion, the detection of HCV in brain is not sufficient to implicate HCV in brain dysfunction; however, it is a necessary first step. The substitution patterns we detected in brain HCV provide a basis for further investigation. Occult kidney damage in patients with HCV was recently found to be prevalent and extensive , suggesting that HCV may cause more extrahepatic damage than has been appreciated. The bioinformatic approach introduced here will facilitate future studies of HCV in brain and other extrahepatic sites.
Financial support: National Institutes of Health (grants DA016156 and DK066939 to A.D.B. and grant R24MH59724 to S.M.); American Liver Foundation Scholar's Award (to F.J.E.); National Center for Research Resources (grant MO1-RR-00071).
Potential conflicts of interest: none reported