Deep Sequencing of Hepatitis C Virus Reveals Genetic Compartmentalization in Cerebrospinal Fluid From Cognitively Impaired Patients

Damien C. Tully; Simon Hjerrild; Peter D. Leutscher; Signe G. Renvillard; Colin B. Ogilvie; David J. Bean; Poul Videbech; Todd M. Allen; Jane A. McKeating; Nicola F. Fletcher


Liver International. 2016;36(10):1418-1424. 

In This Article

Abstract and Introduction


Background & Aims: Hepatitis C virus (HCV) causes neuropsychiatric impairment and fatigue with recent studies suggesting HCV invasion of the central nervous system (CNS). Our previous finding that endothelial cells from the blood–brain barrier support HCV infection warrants further investigation to elucidate whether the CNS can serve as a reservoir for independent HCV evolution.

Methods: Cerebrospinal fluid (CSF) and plasma from six HCV-infected patients without liver disease or co-morbidities together with plasma from six healthy subjects were profiled for markers of immune activation and viral quasispecies measured by deep sequencing. Unsupervised data analyses were used to identify any associations between cytokine activation markers and clinical outcomes.

Results: Four of six HCV-infected patients showed significant evidence of cognitive dysfunction and fatigue. Deep sequencing revealed independent viral evolution within the CNS of two cognitively impaired patients. Principal component analysis of peripheral cytokines demonstrated that individuals without cognitive impairment clustered together while a distinct cytokine pattern emerged with patients exhibiting cognitive dysfunction and fatigue.

Conclusions: Deep sequencing demonstrated unique viral variants in the CSF of two cognitively impaired patients consistent with CNS replication or sequestration. Meanwhile, compartmentalization was absent in infected patients with no neurocognitive impairment. Examination of cytokine profiles in HCV-infected patients with cognitive dysfunction revealed elevated peripheral cytokine levels resulting in a distinct cytokine profile that may be related to cognitive impairment or viral penetration into the CNS. Further studies to determine the significance of unique HCV variants within the CNS are warranted.


Hepatitis C virus (HCV) infects approximately 180 million people worldwide. While the primary cellular reservoir for HCV infection is liver hepatocytes, infection is associated with extrahepatic symptoms including central nervous system (CNS) abnormalities, cognitive dysfunction, fatigue and depression.[1] It is unclear whether HCV-associated CNS symptoms are a function of systemic disease, impaired hepatic function or infection of the CNS.[2] Fatigue is the most commonly reported neuropsychiatric symptom associated with HCV infection, with 65–80% of infected patients complaining of fatigue that is independent of liver dysfunction.[1] Recent studies reporting ex vivo infection of cells within the CNS and the detection of HCV RNA in brain biopsies[2] support a model where HCV infects the brain. This raises questions as to whether HCV can evolve within the CNS and if this is associated with neurological symptoms in the absence of confounding factors such as liver disease, HIV co-infection or prior interferon treatment.

Genetic compartmentalization of a virus within the brain has been observed for other infections, including HIV,[3] and this has implications for treatment since antiviral therapies for HIV and HCV are substrates of p-glycoprotein, a multidrug transporter expressed at the blood–brain barrier (BBB),[4] that limits the penetration of many therapies into the CNS. Studies have reported evidence of independent HCV evolution in the CNS compared to the liver.[5–7] However, these studies used tissue from patients diagnosed with cirrhosis, fibrosis or HIV co-infection: co-morbidities that are independently associated with cognitive dysfunction. Furthermore, genetic analyses of HCV variants were restricted to small fragments of the viral genome because of the difficulties in amplifying viral RNA from brain biopsies following RNA degradation with post-mortem tissue combined with the reduced viral burden in the brain compared to liver.[2,5] Consequently, it has been difficult to establish a link between neuropsychiatric symptoms and HCV infection of the CNS. Furthermore, studies to date have been limited by Sanger sequencing with typically only a few virus clones selected. As a result the true genetic structure of the virus population could be obscured and minor variants not detected. To overcome this problem we have implemented a deep sequencing approach that allows for a sensitive and comprehensive understanding of the intrapatient viral quasispecies. This sequencing technology coupled with a unique cohort of HCV-infected subjects without severe liver disease, HIV co-infection or prior interferon therapy allows us to investigate the impact of CNS invasion by HCV.