Human Pegivirus in Patients with Encephalitis of Unclear Etiology, Poland.

Human pegivirus (HPgV), previously called hepatitis G virus or GB virus C, is a lymphotropic virus with undefined pathology. Because many viruses from the family Flaviviridae, to which HPgV belongs, are neurotropic, we studied whether HPgV could infect the central nervous system. We tested serum and cerebrospinal fluid samples from 96 patients with a diagnosis of encephalitis for a variety of pathogens by molecular methods and serology; we also tested for autoantibodies against neuronal antigens. We found HPgV in serum and cerebrospinal fluid from 3 patients who had encephalitis of unclear origin; that is, all the markers that had been tested were negative. Single-strand confirmation polymorphism and next-generation sequencing analysis revealed differences between the serum and cerebrospinal fluid-derived viral sequences, which is compatible with the presence of a separate HPgV compartment in the central nervous system. It is unclear whether HPgV was directly responsible for encephalitis in these patients.

A Cluster of Fatal Tick-borne Encephalitis Virus Infection in Organ Transplant Setting.

Background: Tick-borne encephalitis virus (TBEV) infection has become a major health problem in Europe and is currently a common cause of viral brain infection in many countries. Encephalitis in transplant recipients, althrough rare, is becoming a recognized complication. Our study provides the first description of transmission of TBEV through transplantation of solid organs. Methods: Three patients who received solid organ transplants from a single donor (2 received kidney, and 1 received liver) developed encephalitis 17-49 days after transplantation and subsequently died. Blood and autopsy tissue samples were tested by next-generation sequencing (NGS) and reverse transcription polymerase chain reaction (RT-PCR). Results: All 3 recipients were first analyzed in autopsy brain tissue samples and/or cerebrospinal fluid by NGS, which yielded 24-52 million sequences per sample and 9-988 matched TBEV sequences in each patient. The presence of TBEV was confirmed by RT-PCR in all recipients and in the donor, and direct sequencing of amplification products corroborated the presence of the same viral strain. Conclusions: We demonstrated transmission of TBEV by transplantation of solid organs. In such a setting, TBEV infection may be fatal, probably due to pharmacological immunosuppression. Organ donors should be screened for TBEV when coming from or visiting endemic areas.

No evidence of West Nile virus infection among Polish patients with encephalitis.

West Nile virus (WNV) infection usually causes mild febrile illness, but in a small proportion of patients it can lead to encephalitis. Epidemiological studies of WNV indicate fast spread of infection worldwide and in Europe, but there have been no comprehensive studies of WNV infection among encephalitis patients in Poland. Here we present the results of WNV RNA and anti-WNV testing in serum and cerebrospinal fluid (CSF) samples in 80 patients with the clinical diagnosis of viral encephalitis. WNV RNA was not detected in any of the analyzed samples. Anti-WNV IgG and IgM were not present in CSF in any of the investigated patients, but anti-WNV IgM were unexpectedly detected in serum of 14 subjects. The latter represented false positive results are probably related to cross reactivity of antibodies. Although there was no evidence of WNV infection in any of our patients, epidemiological situation in the neighbouring countries warrants vigilance and appropriate measures, including introduction of specific diagnostic tools into clinical practice, seem necessary.

The correlation between pretreatment cytokine expression patterns in peripheral blood mononuclear cells with chronic hepatitis C outcome.

BACKGROUD: Cytokine response against hepatitis C virus (HCV) is likely to determine the natural course of infection as well as the outcome of antiviral treatment. However, the role of particular cytokines remains unclear. The current study analyzed activation of cytokine response in chronic hepatitis C patients undergoing standard antiviral treatment. METHODS: Twenty-two patients were treated with pegylated interferon and ribavirin. Twenty-six different cytokine transcripts were measured quantitatively in peripheral blood mononuclear cells (PBMC) before and after therapy and correlated with therapy outcome as well as with clinical and liver histological data. RESULTS: We found that patients who achieved sustained virological response (SVR) showed higher pretreatment cytokine response when compared to subjects in whom therapy was unsuccessful. The differentially expressed factors included IL-8, IL-16, TNF-α, GM-CSF, MCP-2, TGF-ß, and IP-10. Serum ALT activity and/or histological grading also positively correlated with the expression of IL-1α, IL-4, IL-6, IL-10, IL-12, IL-15, GM-CSF, M-CSF, MCP-2 and TGF-ß. CONCLUSION: Pretreatment activation of the immune system, as reflected by cytokines transcripts upregulation, positively correlates with treatment outcome and closely reflects liver inflammatory activity.

Evidence for immune activation in patients with residual hepatitis C virus RNA long after successful treatment with IFN and ribavirin.

Low-level hepatitis C virus (HCV) RNA may persist in PBMCs after successful treatment of chronic hepatitis C, but the consequences of this phenomenon are unclear. Forty-nine patients who achieved a sustained virological response (SVR) after pegylated IFN and ribavirin therapy were analysed 52-66 months after the SVR. HCV RNA was detected in PBMCs from 18 patients (47.4 %), and PBMCs in two patients stained positive for non-structural protein 3 (NS3). Quantification of various cytokine and chemokine transcripts in PBMCs revealed that levels of IL-6, IL-8, IL-12, TNF-α and macrophage inflammatory protein 1ß were significantly higher in HCV-positive patients than in HCV-negative individuals. In conclusion, persistence of HCV RNA in PBMCs of patients with a SVR appears to be associated with immune activation.

Deep sequencing of hepatitis C virus hypervariable region 1 reveals no correlation between genetic heterogeneity and antiviral treatment outcome.

BACKGROUND: Hypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable part of HCV genome and its translation product is a major target for the host immune response. Variability within HVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study was to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of PEG-IFN-α (pegylated interferon α) and ribavirin treatment. METHODS: HVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype 1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche). Reads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using three different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mann-Whitney and Fisher's exact tests. RESULTS: Complexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic substitutions were not significantly different between responders and non-responders, when analyzing viral populations at any of the three frequencies (≥1%, ≥2% and ≥5%). When clonal sample was used to determine pyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a frequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous variant present at frequency of 0.5%. CONCLUSIONS: While deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients, there was no correlation between treatment outcome and any of the analyzed quasispecies parameters.

[Comparison of 5'UTR and HVR1 of hepatitis c virus (HCV) in serum and peripheral blood mononuclear cells in the early phase of interferon and ribavirin treatment]. / Analiza porównawcza regionu 5'UTR oraz HVR1 wirusa zapalenia watroby typu C (HCV) w surowicy i komorkach jednojadrzastych krwi obwodowej (PBMC) we wczesnej fazie leczenia interferonem i rybawiryna.

Genetic heterogeneity is a characteristic feature of hepatitis C virus (HCV) and it reflects selection mechanisms affecting the virus. It is considered a major factor contributing the viral persistence and drug resistance. The following work presents the preliminary results of 5'UTR and E2/HVR1 genetic variation analysis and comparison in serum and peripheral blood mononuclear cells (PBMC) of 7 patients before and during the early phase of pegylated interferon alfa (PEG-IFN-alpha) and ribavirin therapy. Single strand conformational polymorphism (SSCP) analysis revealed genetic stability of 5'UTR in all but one patient who did not respond to treatment (SVR-), where new genetic variants appeared. E2/HVR1 genetic changes were characteristic in patients displaying treatment failure (SVR-) and usually reflected fluctuations in complexity and appearance of new genetic HCV variants. Genetic changes (reduction in complexity) were found in one of three sustained virological responders (SVR+ patients). Comparatory analysis of the HCV quasispecies present in serum and PBMC showed differences in at least one analysed region in all non-responders. Presented results suggest the independent forces driving genetic changes in analysed regions. They also point out the presence of genetic compartmentalization possibly having an impact on antiviral treatment result.

Seronegative hepatitis C virus infection.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. The routine diagnostics identifying HCV infection include testing for specific anti-HCV antibodies by enzyme-linked immnunosorbent assay and viral genetic material in serum or plasma. However, a small proportion of patients persistently infected with HCV, in whom anti-HCV are undetectable, constitute a serious diagnostic and possibly epidemiologic problem, as they could facilitate pathogen spread in the population. This type of infection is termed seronegative or serosilent. Seronegative HCV infection is currently of great interest to both scientists and physicians. The review presents epidemiological data concerning the prevalence of seronegative HCV infection in HIV/HCV co-infected individuals, hemodialysis patients, and blood and organ donors. The possible mechanisms behind this atypical course of infection are discussed. Furthermore, the differences between seronegative and occult infections and prolonged seroconversion are explained.