The flavivirus family has many members (for example West Nile or Japanese B) with well-documented neurovirulence, capable of eliciting both significant neurologic and neuropathologic sequellae (McMinn, 1997
; Sejvar et al, 2003
). Typical of viral encephalitidies, the pathogenesis of these disorders is often inflammatory, with acute to subacute neurologic progression. In contrast, the major clinical manifestation of HCV is chronic liver disease, with hepatitis evolving to cirrhosis in a variable proportion of individuals infected (Hoofnagle, 2002
). However, although HCV’s main clinical sequellae are hepatitis and cirrhosis, it is clearly not anatomically restricted to liver. The virus has been detected in multiple organs, including lymph node, bone marrow, pancreas, thyroid, adrenal, and spleen (reviewed in Gowans, 2000
). Positive-sense HCV RNA has been detected in the CSF and brains of limited numbers of chronically infected patients with variable clinical and neuropathological abnormalities (Laskus et al, 2002
; Morsica et al, 1997
; Maggi et al, 1999
; Bagaglio et al, 2005
). Evidence of replication in brain and CSF compartments, with negative-sense HCV RNA detection and sequence divergence between CNS and systemic quasispecies, has also been published in limited numbers of subjects (Forton et al, 2004a
; Radkowski et al, 2002
; Vargas et al, 2002
). The present study adds 7 patients to the previous literature documenting HCV in brain tissues of 10 patients with chronic infection, and attempts to determine whether there are any clinical characteristics in the setting of HIV that are associated with or contribute to this localization. Unexamined is whether nervous system HCV infection is an isolated extrahepatic phenomenon, or part of a disseminated HCV disease process.
This study demonstrates that brain HCV sequences are commonly found in patients with HIV/HCV coinfection. They were detected in 60% of HIV-infected patients with untreated plasma/liver HCV. Prior studies of coinfected patients from which CNS HCV prevalence can be estimated have been restricted to paired CSF and peripheral blood, and thus represent analysis of a different CNS compartment with regard to cellular trafficking and viral dynamics. Nevertheless, our rate of brain HCV sequence detection is the same as that found by Laskus and colleagues in CSF. In their study, HCV was found in 60% (6/10) of CSF samples from coinfected individuals with advanced HIV (Laskus et al, 2002
). In other prior studies of HIV/HCV-coinfected patients, rates of CSF HCV detection have ranged from 24% (5/21 patients) to 100% (5/5 patients) (Morsica et al, 1997
; Maggi et al, 1999
; Bagaglio et al, 2005
). The reasons for this variability are unclear, and underscore the need for larger numbers of patients to be studied, and an enhanced understanding of the factors that allow HCV penetration into CNS fluids and tissues. Such factors may include perturbations of blood-brain and blood-CSF barriers, potential neurotropic HCV variants, viral burden and duration, HCV RNA detection methods, immunologic and HIV virologic status of the coinfected patients, or other comorbid risks or demographies.
With regard to factors that may be important for CNS pathogenesis, the present pilot is the first to suggest an association between poorly controlled HIV infection and brain localization of HCV. Although the magnitude of HCV plasma viremia did not correlate with its presence in brain, there was a trend for patients with brain HCV to be more frequently off antiretroviral therapy and have higher plasma HIV loads. A significant association was seen between brain HCV and detection of HIV in a premortem sample of CSF, consistent with the hypothesis that HIV may contribute to abnormalities favoring HCV brain penetration. Abnormalities of blood-brain barrier have been documented with HIV infection, and literature is accumulating to demonstrate that HIV enhances the migration of leukocytes into the brain (Eugenin et al, 2006
). HCV can be detected in peripheral blood mononuclear cells (PBMCs) (Laskus et al, 1998
; for a review, see Dammacco et al, 2000
), and HIV facilitates HCV infection of naive human macrophages in vitro
(Laskus et al, 2004
). Thus, uncontrolled HIV disease may be hypothesized to contribute to a state of enhanced brain mononuclear cell migration, facilitating cell-associated HCV brain entry. On the other hand, an alternative explanation for the association of brain HCV and uncontrolled HIV may also be postulated: it is possible that brain localization of HCV enhances HIV expression in the CNS. The concept of “synergistic copathogenicity”—where replication of one virus results in elevation of HIV—has been demonstrated for other organ systems (Corey, 2007
). It would be possible to postulate that HCV may potentiate HIV in the CNS compartment. Further studies will be necessary to confirm our observation and determine its relevance towards HCV and HIV neuropathogenesis.
Prior studies have suggested that PBMCs comprise the pertinent blood fraction with regard to CNS penetration, with reports of two brain- and four CSF-derived HCV virotypes more comparable to PBMC or lymph node than plasma or serum (Forton et al, 2004a
; Laskus et al, 2002
). It may be that HCV brain penetration follows a mechanism similar to the “Trojan horse” monocyte-directed access of HIV. However, this has not been definitively established, and a comprehensive model of HCV neurobiology based on observation of in vivo
phenomena is not extant. Furthermore, given the “Trojan horse” hypothesis, it is important to distinguish brain migration of leukocytes from brain accumulation. Neuropathologies were examined in the present study, and there was no indication at the level of hematoxylin and eosin (H&E) histology that inflammatory or necrotizing lesions were associated with the presence of brain HCV sequences. Indeed, CNS inflammation or necrosis was seen in three out of six patients with HCV in liver only (patients 10001, 10027, 20015), and in three out of seven with HCV in brain (patients 10016, 10086, 10034). This is consistent with prior literature, in which HCV has not generally been associated with encephalitis or myelitis. Only rare case reports have described individuals with acute inflammatory syndromes involving brain or spinal cord, in which HCV RNA has been demonstrated in brain or CSF (Bolay et al, 1996
; Fujita et al, 1999
; Gazzola et al, 2001
; Sacconi et al, 2001
). A caveat with our analysis is that it was performed only through light microscopic examination of routine H&E stains, and thus only pronounced accumulations of inflammatory cells would have been appreciated. It is possible that enhanced inflammatory cell infiltration or activation, detectable by expression of inflammatory cell markers, but not by increased nuclear density, could have been present. This has been well demonstrated for patients with HIV, who can demonstrate elevations of brain inflammatory mediators in the absence of overtly encephalitic pathologies (Glass et al, 1993
). Importantly, in some magnetic resonance spectroscopy (MRS) studies of HCV mono-and HIV/HCV-coinfected patients, there has been evidence of white matter inflammation as indicated by increased choline neurometabolites (McAndrews et al, 2005
; reviewed in Forton et al, 2006
The neuropathologic finding of Alzheimer type 2 gliosis tended to be present more often in patients with HCV than in those without, although this was not specific to those with brain localization. This pathology is typically seen in patients with decompensated liver disease, and is thought to reflect the profound metabolic CNS abnormalities induced by biochemical abnormalities in liver failure. Interestingly, one patient with brain HCV but without liver fibrosis or cirrhosis (10066) had Alzheimer type 2 gliosis. Further studies will be warranted to determine whether HCV contributes directly in some manner to this histopathology.
Although chronic HCV may lack a characteristic neuropathology, there is accumulating evidence that it is associated with cognitive dysfunction. In monoinfected as well as coinfected cohorts, HCV has been associated with deficits in abstraction and executive functioning, speed of information processing, visuospatial construction, and concentration (Cherner et al, 2005
; Forton et al, 2002
; Hilsabeck et al, 2002
; Perry et al, 2005
; Richardson et al, 2005
; Ryan et al, 2004
). Individuals with chronic HCV may have significantly prolonged P300 latencies on electrophysiologic testing (Kramer et al, 2002
). MRS abnormalities distinct from those encountered in liver failure have been noted in patients with HCV (Forton et al, 2001
). In all these studies, nervous system function has been correlated with the presence or absence of HCV indicators or load in peripheral blood. However, in contrast to these studies, there are investigators who have been unable to demonstrate HCV-specific deficits that go beyond the cognitive perturbations accompanying hepatic decompensation (Edwin et al, 1999
; Hilsabeck et al, 2002
; Soogoor et al, 2006
). This has left unresolved an important question: are the cognitive abnormalities detected in these patients specific to HCV in the nervous system, or a function of systemic disease and/or impairments in hepatic function?
The current pilot study does not resolve this question, as it is underpowered with regard to cognitive analysis. It does suggest that the presence of brain HCV is a factor in producing cognitive deficits in abstraction and executive functioning, with a descending hierarchy of performance from better to worse in those with HIV infection alone, HIV/HCV coinfection restricted to systemic locations, and HIV/HCV coinfection with brain HCV. Caveats must be considered: the brains analyzed derive from our larger clinical cohort, in which prior study of 116 HIV-infected individuals showed deficits in abstraction and executive function correlated with positive HCV serology (Ryan et al, 2004
). Furthermore, given numerical limitations, statistical analysis suggested trends but lacked significance. Analysis of larger numbers of brains from HIV-infected subjects with and without HCV will be necessary to confirm this suggestion of our pilot study.
Finally, the sequence analyses in the current study provide more evidence for an HCV subpopulation in brain. In four of six patients, predominant brain species did not match those in plasma or liver. The presence of a predominant sequence in brain that is different from the predominant sequence in plasma and liver suggests that the brain HCV is not produced in the liver; however, it is possible that the brain isolate is a minority population that is produced in the liver that has a propensity to accumulate in the brain. Further compartmental studies with quasispecies analysis are warranted.
In summary, the current pilot study adds seven patients to the literature documenting HCV sequences in brain, and begins an analysis of clinicopathologic correlates of this localization, demonstrating uncontrolled HIV infection in those with brain HCV. Future studies on larger numbers of subjects will be necessary to determine whether a clinically relevant phenotype can be related to this phenomenon, and to elucidate a neurobiology of HCV that is relevant not only to function, but also to general disease progression and treatment.