Encephalitis is a severe neurological syndrome usually caused by viruses. Despite significant progress in diagnostic techniques, the causative agent remains unidentified in the majority of cases. The aim of the present study was to test an alternative approach for the detection of putative pathogens in encephalitis using next-generation sequencing (NGS). RNA was extracted from cerebrospinal fluid (CSF) from a 60-year-old male patient with encephalitis and subjected to isothermal linear nucleic acid amplification (Ribo-SPIA, NuGen) followed by next-generation sequencing using MiSeq (Illumina) system and metagenomics data analysis. The sequencing run yielded 1,578,856 reads overall and 2579 reads matched human herpesvirus I (HHV-1) genome; the presence of this pathogen in CSF was confirmed by specific PCR. In subsequent experiments we found that the DNAse I treatment, while lowering the background of host-derived sequences, lowered the number of detectable HHV-1 sequences by a factor of 4. Furthermore, we found that the routine extraction of total RNA by the Chomczynski method could be used for identification of both DNA and RNA pathogens in typical clinical settings, as it results in retention of a significant amount of DNA. In summary, it seems that NGS preceded by nucleic acid amplification could supplement currently used diagnostic methods in encephalitis.

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.

The long-term consequences of T cell responses’ impairment in chronic HCV infection are not entirely characterized, although they may be essential in the context of the clinical course of infection, re-infection, treatment-mediated viral clearance and vaccine design. Furthermore, it is unclear whether a complete reinvigoration of HCV-specific T cell response may be feasible. In most studies, attempting to reverse the effects of compromised immune response quality by specific blockades of negative immune regulators, a restoration of functional competence of HCV-specific T cells was shown. This implies that HCV-induced immune dysfunction may be reversible. The advent of highly successful, direct-acting antiviral treatment (DAA) for chronic HCV infection instigated investigation whether the treatment-driven elimination of viral antigens restores T cell function. Most of studies demonstrated that DAA treatment may result in at least partial restoration of T cell immune function. They also suggest that a complete restoration comparable to that seen after spontaneous viral clearance may not be attained, pointing out that long-term antigenic stimulation imprints an irreversible change on the T cell compartment. Understanding the mechanisms of HCV-induced immune dysfunction and barriers to immune restoration following viral clearance is of utmost importance to diminish the possible long-term consequences of chronic HCV infection.

During chronic hepatitis C virus (HCV) infection, both CD4⁺and CD8⁺T-cells become functionally exhausted, which is reflected by increased expression of programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and elevated anti-inflammatory interleukin 10 (IL-10) plasma levels. We studied 76 DAA-treated HCV-positive patients and 18 non-infected controls. Flow cytometry measured pretreatment frequencies of CD4⁺PD-1⁺, CD4⁺PD-1⁺Tim-3⁺and CD8⁺PD-1⁺Tim-3⁺T-cells and IL-10 levels measured by ELISA were significantly higher and CD4⁺PD-1⁻Tim-3⁻and CD8⁺PD-1⁻Tim-3⁻T-cells were significantly lower in patients than in controls. Treatment resulted in significant decrease of CD4⁺Tim-3⁺, CD8⁺Tim-3⁺, CD4⁺PD-1⁺Tim-3⁺and CD8⁺PD-1⁺Tim-3⁺T-cell frequencies as well as IL-10 levels and increase in CD4⁺PD-1⁻Tim-3⁻and CD8⁺PD-1⁻Tim-3⁻T-cells. There were no significant changes in the frequencies of CD4⁺PD-1⁺T-cells, while CD8⁺PD-1⁺T-cells increased. Patients with advanced liver fibrosis had higher PD-1 and lower Tim-3 expression on CD4⁺T-cells and treatment had little or no effect on the exhaustion markers. HCV-specific CD8⁺T-cells frequency has declined significantly after treatment, but their PD-1 and Tim-3 expression did not change. Successful treatment of chronic hepatitis C with DAA is associated with reversal of immune exhaustion phenotype, but this effect is absent in patients with advanced liver fibrosis.

Background and Aims: During chronic hepatitis C virus (HCV) infection, CD8⁺ T-cells become functionally exhausted, undergoing progressive phenotypic changes, i.e., overexpression of “inhibitory” molecules such as PD-1 (programmed cell death protein 1) and/or Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3). The extreme intrahost genetic diversity of HCV is a major mechanism of immune system evasion, facilitating epitope escape. The aim of the present study was to determine whether T-cell exhaustion phenotype in chronic HCV infection is related to the sequence repertoire of NS3 viral immunodominant epitopes.Methods: The study population was ninety prospective patients with chronic HCV genotype 1b infection. Populations of peripheral blood CD8⁺ T-cells expressing PD-1/Tim-3 were assessed by multiparametric flow cytometry, including HCV-specific T-cells after magnetic-based enrichment using MHC-pentamer. Autologous epitope sequences were inferred from next-generation sequencing. The correction of sequencing errors and genetic variants reconstruction was performed using Quasirecomb.Results: There was an interplay between the analyzed epitopes sequences and exhaustion phenotype of CD8⁺ T-cells. A predominance of NS3₁₄₀₆ epitope sequence, representing neither prototype KLSGLGLNAV nor cross-reactive variants (KLSSLGLNAV, KLSGLGINAV or KLSALGLNAV), was associated with higher percentage of HCV-specific CD8⁺PD-1⁺Tim-3⁺ T-cells, P=0.0102. Variability (at least two variants) of NS3₁₄₀₆ epitope sequence was associated with increased frequencies of global CD8⁺PD-1⁺Tim-3⁺ T-cells (P=0.0197) and lower frequencies of CD8⁺PD-1⁻Tim-3⁻ T-cells (P=0.0079). In contrast, infection with NS3₁₀₇₃ dominant variant epitope (other than prototype CVNGVCWTV) was associated with lower frequency of global CD8⁺PD-1⁺Tim-3⁺ T-cells (P=0.0054).Conclusions: Our results indicate that PD-1/Tim-3 receptor expression is largely determined by viral epitope sequence and is evident for both HCV-specific and global CD8⁺ T-cells, pointing to the importance of evaluating autologous viral epitope sequences in the investigation of CD8⁺ T-cell exhaustion in HCV infection.

Genetic variability of hepatitis C virus (HCV) determines pathogenesis of infection, including viral persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR1) of a chronically infected patient by ultradeep 454 sequencing strategy. Three independent sequencing error correction methods were applied. First correction method (Method I) implemented cut-off for genetic variants present in less than 1%. In the second method (Method II), a condition to call a variant was bidirectional coverage of sequencing reads. Third method (Method III) usedShort Read Assembly into Haplotypes(ShoRAH) program. After the application of these three different algorithms, HVR1 population consisted of 8, 40, and 186 genetic haplotypes. The most sensitive method was ShoRAH, allowing to reconstruct haplotypes constituting as little as 0.013% of the population. The most abundant genetic variant constituted only 10.5%. Seventeen haplotypes were present in a frequency above 1%, and there was wide dispersion of the population into very sparse haplotypes. Our results indicate that HCV HVR1 heterogeneity andquasispeciespopulation structure may be reconstructed by ultradeep sequencing. However, credible analysis requires proper reconstruction methods, which would distinguish sequencing error from real variabilityin vivo.