Currently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability. A primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60). The LLOD of the HDV utility-channel (HDV_UTC) assay was determined as 3.86 IU/ml (95% CI 2.95–5.05 IU/ml) with a linear range from 10–10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R² from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV_UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R² 0.8733; 95% CI 0.8914–0.9609; p value <0.0001). The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring. The hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results.

The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a hepatitis B (HBV) and delta virus (HDV) entry receptor has encouraged the development of new animal models of infection. This review provides an overview of the different in vivo models that are currently available to study HDV either in the absence or presence of HBV. By presenting new advances and remaining drawbacks, we will discuss human host factors which, in addition to NTCP, need to be investigated or identified to enable a persistent HDV infection in murine hepatocytes. Detailed knowledge on species-specific factors involved in HDV persistence also shall contribute to the development of therapeutic strategies.

Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Background: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. Methods: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. Results: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9–132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7–334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3–158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7–129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). Conclusion: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures.

The role of hepatocytes in producing chemokines and triggering liver inflammation and damage in chronic hepatitis D (CHD) is not fully understood. Herein, we investigated the contribution of primary human hepatocytes (PHHs) infected with HDV in triggering inflammation by producing the chemokines CXCL9–11. We performed quantitative PCR, RNA in situ hybridisation, activation-induced marker (AIM) assays, and FACS analysis to investigate the CXCR3/CXCL9–11 receptor/ligand axis of T cells in peripheral blood and livers from patients with chronic hepatitis B (n = 27 and 18, respectively) and CHD (n = 20 and 18, respectively). Chemokine expression was investigated in cultured HDV-infected PHHs and in livers of HBV- or HBV/HDV-infected humanised mice in the presence or absence of adoptively transferred human immune cells (n = 35 in total). In patient and chimeric mouse livers, higher expression levels of CXCL9–11 were found in an HBV/HDV-coinfected vs. HBV-mono-infected setting. Similarly, high levels of CXCL9–11 were observed in HDV-infected PHHs in vitro. Analysis by RNA in situ hybridisation on patient livers revealed that HDV-infected hepatocytes were a significant contributor to the chemokine expression. The corresponding chemokine receptor CXCR3 was found upregulated specifically on peripheral bulk CD4 T cells of patients with CHD. CXCR3 upregulation was unspecific and was not detected on HDAg- or HBsAg-specific CD4 T cells by activation-induced marker assay. Lastly, adoptive transfer of human T cells in humanised mice led to recruitment of non-HBV/HDV-specific CD4+ T cells only in the setting of HBV/HDV coinfection, but not in HBV-mono-infected mice. HDV infection upregulated the intrahepatic expression of the CXCL9–11/CXCR3 receptor/ligand axis. Higher amounts of HBV/HDV-unspecific CD4 T cells expressing CXCR3 may contribute to the aggravated liver inflammation frequently observed in patients with CHD. Chronic hepatitis D (CHD) causes the most severe form of viral hepatitis, and treatment options are still limited; therefore, a more precise understanding of CHD immunopathology is needed. In this study, we demonstrated that HDV infection triggers CXCL9–11 expression in hepatocytes and liver infiltration of CXCR3-expressing CD4 T cells in preclinical models as well as patient biopsies. Because recruitment of Th1-polarised CD4 T cells to the liver has been also described for other severe liver diseases, such as autoimmune hepatitis, it may represent an important mechanism of aggravating liver diseases. The data of this study set hereby the basis for future studies analysing phenotype and function of intrahepatic T cells in CHD.

Recently, cases of human infection with rat hepatitis E virus (ratHEV, Rocahepevirus ratti ) have been reported worldwide. Due to the significant genetic differences between ratHEV and human HEV genotypes 1–4 ( Paslahepevirus balayani ), current HEV diagnostic assays are unable to detect ratHEV. The aim was to establish and validate a laboratory‐developed ratHEV RT‐qPCR assay for use with human plasma and stool samples on a fully automated, high‐throughput platform. Published primers and probes were optimized for use on cobas 5800/6800/8800 systems using European Union In Vitro Diagnostics Regulation (IVDR)‐grade reagents, including an RNA full‐process inhibition control. Analytical sensitivity (21 repeats), linear range (five repeats) and precision (three repeats over 3 days) were evaluated using viral particles from cell culture (MN450851.1). The inclusivity was verified using DNA oligonucleotides and known positive samples (rat liver and human serum). The limits of detection were 98.9 copies/ml in plasma and 60.3 copies/ml in stool, and the assay showed excellent linearity over at least 5 log (r ² : 0.991 in plasma and 0.9989 in stool) and high precision (< 0.62 ct). The assay reliably detected different ratHEV C1 subgenotypes, returning positive results for all 11 rat liver samples and one known ratHEV RNA‐positive human plasma sample, while no false positives were detected in the broad cross‐reactivity set ( n = 41). In the pilot ratHEV surveillance cohort, 1.1% of plasma samples ( n = 1999) were positive for HEV RNA, but none were positive for ratHEV RNA. Our new, fully automated, lab‐developed ratHEV assay can be used in compliance with the IVDR for routine human diagnostics. Further studies are needed to determine the clinical relevance in different human cohorts.