Christian G. Schüttler

microRNA-122 stimulates translation of hepatitis C virus RNA

Hepatitis C virus (HCV) is a positive strand RNA virus that propagates primarily in the liver. We show here that the liver‐specific microRNA‐122 (miR‐122), a member of a class of small cellular RNAs that mediate post‐transcriptional gene regulation usually by repressing the translation of mRNAs through interaction with their 3′‐untranslated regions (UTRs), stimulates the translation of HCV. Sequestration of miR‐122 in liver cell lines strongly reduces HCV translation, whereas addition of miR‐122 stimulates HCV translation in liver cell lines as well as in the non‐liver HeLa cells and in rabbit reticulocyte lysate. The stimulation is conferred by direct interaction of miR‐122 with two target sites in the 5′‐UTR of the HCV genome. With a replication‐defective NS5B polymerase mutant genome, we show that the translation stimulation is independent of viral RNA synthesis. miR‐122 stimulates HCV translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage. In conclusion, the liver‐specific miR‐122 may contribute to HCV liver tropism at the level of translation.

Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein

BACKGROUND/AIMS Epidemiological studies have shown that coinfection or superinfection with hepatitis B virus (HBV) and C virus (HCV) frequently leads to the suppression of hepatitis B virus replication. The mechanism of this phenomenon is still unclear. Shih et al. [J Virol 1993;67:5823] reported a direct suppression of HBV replication by the core protein of HCV. The target structure of HCV core protein in this system remained unclear. METHODS As HCV core protein has been shown to influence expression from transcriptional elements, we studied whether HCV core protein altered the activity of the two HBV enhancers 1 and 2. Luciferase vectors for HBV enhancers 1 or 2 were cotransfected with expression constructs for HCV core protein in murine and human hepatocyte lines. RESULTS Full-length HCV core protein suppressed the HBV enhancer 1 up to 11-fold, the enhancer 2 3-4-fold. Suppression of HBV enhancer 1 by HCV core from genotype 1b was stronger than by HCV core of genotypes 3a or 1a. Carboxyterminally truncated core proteins had lower or no suppression activity. CONCLUSIONS These data suggest that HCV core protein may directly repress transcription of the HBV RNAs. This trans-repression may contribute to suppression of HBV replication in patients coinfected with both viruses.

Hepatitis C virus transmission by a blood donation negative in nucleic acid amplification tests for viral RNA

Hepatitis C virus (HCV) was transmitted by transfusion of a platelet concentrate made from an anti-HCV and HCV-PCR-negative blood donation. Even a negative nucleic acid amplification test cannot completely prevent transmission of HCV.

Occult hepatitis B virus infection: detection and significance.

The Taormina Consensus Conference defined ‘occult hepatitis B virus (HBV) infection’ (OBI) as the ‘presence of HBV DNA in the liver of individuals testing HBsAg-negative with currently available assays’. Most occult is the so-called ‘window period’ after exposure before HBV DNA appears in the blood. We identified two blood donors whose donations tested HBsAg- and HBV DNA-negative, but transmitted HBV. Both subsequently developed HBsAg and acute hepatitis. However, such cases are not considered as true OBI. A true transient OBI remains HBsAg-negative during the entire course. One case of acute OBI showed a peak viremia of 15,000 IU/ml HBV DNA and sub-borderline HBsAg, suggesting a ratio of virions to subviral particles of 1:10, whereas ‘normal’ cases show at peak viremia a ratio of 1:3,000. Blood donors with OBI may transmit HBV. We studied 5 blood donors with OBI and 55 of their recipients. In 22 recipients, transmission was probable, but they remained healthy. However, in 3 recipients, who were immunosuppressed at the time of transfusion, fatal fulminant hepatitis B developed. The majority of anti-HBc-positive healthy individuals have HBV DNA in the liver which may start replication under severe immunosuppression. Nine such cases are described here. OBI or reactivated HBV infections often lead to selection of HBsAg escape mutations as we could show in 11 of 14 cases. Infection of vaccinated individuals favors development of OBI as we observed in 6 blood donors. HB vaccination may solve the problem of overt HBV infection but may favor OBI.

Deficiencies in the standardization and sensitivity of diagnostic tests for hepatitis B virus.

Summary.  The patterns of hepatitis B virus (HBV) markers described in textbooks apply to acute and chronic infection with wild‐type HBV. Deviations from these patterns occur in the very early phase, in low‐level (or occult) infection and under immunosuppression. Variability may originate from the virus, the host or the test kits. In order to obtain a reliable diagnosis under these conditions, tests for all three markers of HBV infection have to be applied: HBsAg, HBV DNA and anti‐HBc. All tests should be as sensitive as feasible, but even then occult infection may be missed. Reliable detection of occult or mutated HBV is particularly important in blood and organ donors and in patients before or with immunosuppression.

A multiplex-PCR to identify hepatitis B virus—genotypes A–F

Eight genotypes (A-H) of hepatitis B virus (HBV) are known with variations in nucleotide sequences greater than 8%. Several recent publications found that the clinical course and outcome of antiviral therapy depended on the genotype of the infecting HBV strain. Large epidemiological studies will require the availability of a system which is rapid, reliable and can be performed on a large number of samples. We have developed a multiplex-PCR assay which uses genotype-specific primer pairs for HBV genotypes A-F. These primer pairs specifically amplified HBV DNA of the respective genotype, either in single or in multiplex-PCR. Sensitivity of the assay was in the range of 10(4) genome equivalents.

Variable Ratio of Hepatitis C Virus RNA to Viral Core Antigen in Patient Sera

ABSTRACT Quantification of hepatitis C virus (HCV) core antigen and RNA in serum samples leads to a highly variable ratio of both. It is not clear whether this is due to the inaccuracy of RNA quantification or whether both are independent parameters in a certain range. We established a real-time reverse transcription (RT)-PCR for HCV RNA that combines very high sensitivity with a large dynamic range and minimal standard deviations. The assay was calibrated with the first international standard, 96/790, and the international genotype panel for HCV from the National Institute of Biological Standardisation and Control. A linear readout was obtained between 200 and 5 × 107 IU/ml. The detection limit was 80 IU/ml, the reproducibility was <0.05 log, and the standard error within one run was <0.01. Comparison of the method with the Roche Monitor competitive RT-PCR revealed its high accuracy. The core protein concentration was determined within a range from 1.5 to 400 pg/ml by using the preliminary trak-C assay from Ortho Clinical Diagnostics. Correlating the HCV RNA levels with core antigen concentrations in 197 serum samples from 23 interferon-treated patients, a average ratio of 7,900 IU of HCV RNA per pg of core antigen was estimated, but the variability of this ratio exceeded largely the variability of the two assays, ranging from 50 to 20,000 IU/pg. Theoretically, HCV should contain ca. 43,000 IU of RNA/pg core. In conclusion, the core antigen assay seems to detect, in addition to complete virions, RNA-free core protein structures, which enhances its sensitivity (98% in this group). The variable ratio of RNA and core protein is not mainly due to standard deviations of quantification but could be an additional parameter for treatment follow-up and state of viral replication.

Acute Epididymitis Revisited: Impact of Molecular Diagnostics on Etiology and Contemporary Guideline Recommendations

BACKGROUND Acute epididymitis is a common infectious disease of unknown etiology in about 30% of cases with guidelines based on studies published >15 yr ago. OBJECTIVE To investigate the etiology of acute epididymitis using state-of-the-art methods and to provide rational data for antimicrobial therapy and clinical management. DESIGN, SETTING, AND PARTICIPANTS Between 2007 and 2013, 237 patients (150 antimicrobially naive and 87 antibiotically pretreated) with acute epididymitis underwent comprehensive investigation comprising microbiologic cultures, polymerase chain reaction (PCR) for sexually transmitted infections (STIs), 16S ribosomal DNA (rDNA) analysis, and PCR detection of 23 viruses. Clinical management followed international guidelines. OUTCOME MEASURES AND STATISTICAL ANALYSIS Etiology, clinical management, and outcome after 3 mo were assessed. RESULTS AND LIMITATIONS A causative pathogen, predominantly Escherichia coli (56%), was identified in 132 antibiotic-naive patients (88%) and 44 pretreated patients (51%); 16S rDNA analysis increased the detection rate by 10%. STIs were present in 34 cases (14%) (25 patients with Chlamydia trachomatis) and were not restricted to a specific age group. Enteroviruses were found in only two patients (1%). In naive patients, cultured bacteria were susceptible to fluoroquinolones and group 3 cephalosporins in >85% of cases (preateted patients: 42% and 67%, respectively). Primary empirical therapy was continued in 88% of naive patients for 11 d and in 77% of pretreated patients for 13 d with indwelling urinary catheters, rendering patients as high risk for switching. Only six patients (2.5%) underwent semicastration. Prostate-specific antigen levels halved within 3 mo, except in patients who were antibiotic naive and without detected pathogens. Study limitations included a lack of susceptibility testing in cases of STIs. CONCLUSIONS Even in antimicrobially pretreated patients, acute epididymitis is mainly of bacterial origin. STIs are not limited to patients aged <35 yr. Viral epididymitis seems a rare condition. Current guideline recommendations on empirical antimicrobial therapy are adequate. PATIENT SUMMARY Patients with acute epididymitis should receive appropriate diagnostics and antimicrobial therapy for safe conservative management.

Semen quality in HIV patients under stable antiretroviral therapy is impaired compared to WHO 2010 reference values and on sperm proteome level.

Objectives:To investigate semen quality in HIV patients under stable antiretroviral therapy (ART) compared with WHO 2010 reference values and on the sperm proteome level. Design:Between 2011 and 2013, we prospectively enrolled 116 HIV-positive men without hepatitis B or C co-infections from our outpatient department for infectious diseases. Methods:Patients received a comprehensive andrological work-up. Complete semen analysis was performed according to WHO 2010 recommendations, with each semen variable of the study population being compared with the WHO reference group (n ∼ 2000). Correlation analysis was done to investigate the influence of HIV surrogate parameters on semen quality. Two-dimensional gel electrophoresis and subsequent protein identification was performed to determine any differences in the sperm protein composition of the 15 HIV-positive patients and that of 15 age-matched healthy men. Results:Median values of all assessed semen parameters were within a normal range. However, for each semen variable, about 25% of patients had values below the fifth percentile of the WHO 2010 reference group. Disease-related parameters (CD4+ cell count, viral load, CDC stage, duration of disease, duration of ART, number and type of antiretroviral drugs) were not significantly correlated with any sperm parameter. Sperm proteome analysis identified 14 downregulated proteins associated with sperm motility and fertility. Conclusion:This is the first study that compares all standard semen parameters in HIV-positive patients under ART to WHO 2010 reference values. It provides evidence of impaired conventional semen parameters and altered sperm protein composition. Finally, HIV surrogate parameters are not suitable for predicting semen quality.

The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.