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


To our knowledge, this is the first study to use deep sequencing technologies to investigate HCV genetic compartmentalization between plasma and CSF from patients where the levels of fatigue and cognitive impairment are known. Importantly, virus was detected in the CSF from subjects without severe liver disease, HIV co-infection or prior interferon therapy, all of which are known to cause fatigue or neurological impairment. HCV RNA was previously reported to be present in the brain tissue from HCV/HIV co-infected patients[2,5] and analysis of the HVR sequences demonstrated unique variants in 4 of 7 subjects.[5] This study relied on bulk Sanger sequencing which is insufficient to detect low-frequency variants. Our study confirms and extends these observations and sampled the CSF from HIV-negative, cognitively impaired subjects with modest liver disease. Moreover, as a result of the intact nature of RNA obtained from the CSF, it was possible to amplify the structural half of the HCV genome and to deep sequence viral RNA species and to compare to peripheral circulating variants in the plasma. Without deep sequencing, it is likely that many of these variants would have gone undetected. Viral compartmentalization in CSF associated with a higher plasma viral load, suggesting that increased viral burden in the periphery may facilitate invasion of the CNS. These studies show clear evidence for genetic compartmentalization between plasma and CSF, supporting a model where HCV establishes a productive infection within the CNS. Although the number of patients studied was small, compartmentalization occurred in two of three subjects with cognitive impairment (patients 4 and 6) while patient 5 exhibiting normal cognitive dysfunction showed no evidence of viral evolution within the CSF. Schnell and colleagues reported transient HIV compartmentalization in CNS and blood[16] that may reflect asynchronous rates of virus seeding. Future studies involving larger sample sizes are required to replicate these findings and should include longitudinal sampling of CSF and plasma compartments to define the dynamics of HCV compartmentalization.

Quantification of 27 inflammatory cytokines revealed there was a trend towards higher cytokine levels in plasma from cognitively impaired compared with normal cognitive patients, which warrants further investigation with larger cohorts. However, larger studies on chronically infected HCV patients examining the association between serum cytokine levels and the severity of neuropsychiatric symptoms have found conflicting results.[17,18] Furthermore, CSF cytokines do not appear to be elevated and parallel the perturbations in the periphery at least from the few cytokines where responses were detected. Inflammatory cytokines, including interferons, are known to enter the brain in a variety of disease states and induce fatigue and other neurological symptoms.[19] Importantly, the patients enrolled in our study had not received interferon, which can complicate studies of cognitive impairment. Recent studies show that inflammatory cytokines, including IL-1β and TNF-α, promote HCV particle entry into target cells,[20] which may result in increased viral load in patients with high levels of circulating pro-inflammatory cytokines. These cytokines disrupt BBB integrity and may facilitate viral invasion of the brain.[20]

In summary, this study demonstrates compartmentalization of HCV genetic variants between CSF and plasma in chronically infected patients with no significant liver disease or HIV co-infection. This study highlights that direct-acting antivirals (DAAs) for treating hepatitis C may need to penetrate the BBB and that drug levels in the brain may be subtherapeutic leading to development of viral 'reservoirs' that have the potential to seed resistance mutations into the periphery as previously reported in HIV infection.[3]