Analytical and clinical validation of a multiplex PCR assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis including simultaneous LGV serotyping on an automated high-throughput PCR system.

For effective infection control measures for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), a reliable tool for screening and diagnosis is essential. Here, we aimed to establish and validate a multiplex PCR assay on an automated system using a dual-target approach for the detection of CT/NG and differentiation between lymphogranuloma venereum (LGV) and non-LGV from genital and extra-genital specimens. Published primer/probe sets (CT: pmpH, cryptic plasmid; NG: porA, opa) were modified for the cobas 5800/6800/8800. Standards quantified by digital PCR were used to determine linearity and lower limit of detection (LLoD; eSwab, urine). For clinical validation, prospective samples (n = 319) were compared with a CE-marked in vitro diagnostics (CE-IVD) assay. LLoDs ranged from 21.8 to 244 digital copies (dcp)/mL and 10.8 to 277 dcp/mL in swab and urine, respectively. A simple linear regression analysis yielded slopes ranging from -4.338 to -2.834 and Pearson correlation coefficients from 0.956 to 0.994. Inter- and intra-run variability was <0.5 and <1 cycle threshold (ct), respectively. No cross-reactivity was observed (n = 42). Clinical validation showed a sensitivity of 94.74% (95% confidence interval (CI): 87.23%-97.93%) and 95.51% (95% CI: 89.01%-98.24%), a specificity of 99.59% (95% CI: 97.71%-99.98%) and 99.57% (95% CI: 97.58%-99.98%), positive predictive values of 89.91% (estimated prevalence: 3.7%; 95% CI: 80.91%-95.6%) and 88.61% (estimated prevalence: 3.4%; 95% CI: 80.18%-94.34%), and negative predictive values of 99.81% (95% CI: 98.14%-100%) and 99.85% (95% CI: 98.14%-100%) for the detection of CT and NG, respectively. In conclusion, we established a dual-target, internally controlled PCR on an automated system for the detectiwon of CT/NG from genital and extra-genital specimens. Depending on local regulations, the assay can be used as a screening or a confirmatory/typing assay.IMPORTANCEChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent a major global health burden, with the World Health Organization estimating that >128 million and >82 million people, respectively, were newly infected in 2020. For effective infection control measures, a reliable tool for sensitive diagnosis and screening of CT/NG is essential. We established a multiplex PCR assay for the detection of CT/NG and simultaneous discrimination between lymphogranuloma venereum (LGV) and non-LGV strains, which has been validated for genital and extra-genital specimens on a fully automated system. To increase assay sensitivity, a dual-target approach has been chosen for both pathogens. This strategy reduces false-positive results in oropharyngeal swabs due to the detection of commensal N. species that may harbor NG DNA fragments targeted in the PCR due to horizontal gene transmission following previous infection. In sum, the established assay provides a powerful tool for use as either a screening/diagnostic or a typing/confirmatory assay.

Adaptation and validation of a quantitative vanA/vanB DNA screening assay on a high-throughput PCR system.

Vancomycin resistant enterococci (VRE) are a leading cause of ICU-acquired bloodstream infections in Europe. The bacterial load in enteral colonization may be associated with a higher probability of transmission. Here, we aimed to establish a quantitative vanA/vanB DNA real-time PCR assay on a high-throughput system. Limits of detection (LOD), linear range and precision were determined using serial bacterial dilutions. LOD was 46.9 digital copies (dcp)/ml for vanA and 60.8 dcp/ml for vanB. The assay showed excellent linearity between 4.7 × 101 and 3.5 × 105 dcp/ml (vanA) and 6.7 × 102 and 6.7 × 105 dcp/ml (vanB). Sensitivity was 100% for vanA and vanB, with high positive predictive value (PPV) for vanA (100%), but lower PPV for vanB (34.6%) likely due to the presence of vanB DNA positive anerobic bacteria in rectal swabs. Using the assay on enriched VRE broth vanB PPV increased to 87.2%. Quantification revealed median 2.0 × 104 dcp/ml in PCR positive but VRE culture negative samples and median 9.1 × 104 dcp/ml in VRE culture positive patients (maximum: 107 dcp/ml). The automated vanA/B_UTC assay can be used for vanA/vanB detection and quantification in different diagnostic settings and may support future clinical studies assessing the impact of bacterial load on risk of infection and transmission.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: 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). Methods: 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. Results: 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. Conclusions: 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. Impact and implications: 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.

Rapid Adaptation of Established High-Throughput Molecular Testing Infrastructure for Monkeypox Virus Detection.

Beginning in May 2022, a rising number of monkeypox cases were reported in non-monkeypox-endemic countries in the Northern Hemisphere. We adapted 2 published quantitative PCRs for use as a dual-target monkeypox virus test on widely used automated high-throughput PCR systems. We determined analytic performance by serial dilutions of monkeypox virus reference material, which we quantified by digital PCR. We found the lower limit of detection for the combined assays was 4.795 (95% CI 3.6-8.6) copies/mL. We compared clinical performance against a commercial manual orthopoxvirus research use only PCR kit by using clinical remnant swab samples. Our assay showed 100% positive (n = 11) and 100% negative (n = 56) agreement. Timely and scalable PCR tests are crucial for limiting further spread of monkeypox. The assay we provide streamlines high-throughput molecular testing for monkeypox virus on existing broadly established platforms used for SARS-CoV-2 diagnostic testing.

Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants.

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.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.

OBJECTIVE: Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo. DESIGN: HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events. RESULTS: Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6. CONCLUSION: These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Clinical establishment of a laboratory developed quantitative HDV PCR assay on the cobas6800 high-throughput system.

BACKGROUND & AIMS: 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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. LAY SUMMARY: 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.

Strong Replication Interference Between Hepatitis Delta Viruses in Human Liver Chimeric Mice.

BACKGROUND: Hepatitis D Virus (HDV) is classified into eight genotypes with distinct clinical outcomes. Despite the maintenance of highly conserved functional motifs, it is unknown whether sequence divergence between genotypes, such as HDV-1 and HDV-3, or viral interference mechanisms may affect co-infection in the same host and cell, thus hindering the development of HDV inter-genotypic recombinants. We aimed to investigate virological differences of HDV-1 and HDV-3 and assessed their capacity to infect and replicate within the same liver and human hepatocyte in vivo. METHODS: Human liver chimeric mice were infected with hepatitis B virus (HBV) and with one of the two HDV genotypes or with HDV-1 and HDV-3 simultaneously. In a second set of experiments, HBV-infected mice were first infected with HDV-1 and after 9 weeks with HDV-3, or vice versa. Also two distinct HDV-1 strains were used to infect mice simultaneously and sequentially. Virological parameters were determined by strain-specific qRT-PCR, RNA in situ hybridization and immunofluorescence staining. RESULTS: HBV/HDV co-infection studies indicated faster spreading kinetics and higher intrahepatic levels of HDV-3 compared to HDV-1. In mice that simultaneously received both HDV strains, HDV-3 became the dominant genotype. Interestingly, antigenomic HDV-1 and HDV-3 RNA were detected within the same liver but hardly within the same cell. Surprisingly, sequential super-infection experiments revealed a clear dominance of the HDV strain that was inoculated first, indicating that HDV-infected cells may acquire resistance to super-infection. CONCLUSION: Infection with two largely divergent HDV genotypes could be established in the same liver, but rarely within the same hepatocyte. Sequential super-infection with distinct HDV genotypes and even with two HDV-1 isolates was strongly impaired, suggesting that virus interference mechanisms hamper productive replication in the same cell and hence recombination events even in a system lacking adaptive immune responses.

In Vivo Models of HDV Infection: Is Humanizing NTCP Enough?

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.

Murine hepatocytes do not support persistence of Hepatitis D virus mono-infection in vivo.

BACKGROUNDS & AIMS: As a result of the limited availability of in vivo models for hepatitis D virus (HDV), treatment options for HDV chronically infected patients are still scant. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as HDV entry receptor has enabled the development of new infection models. AIM: To comparatively assess the efficacy and persistence of HDV mono-infection in murine and human hepatocytes in vivo. METHODS: Mice with humanized NTCP (hNTCPed84-87 mice) were generated by editing amino acid residues 84-87 of murine NTCP in C57BL/6J mice. HDV infection was assessed in hNTCPed84-87 mice and in immune deficient uPA/SCID/beige (USB) mice, whose livers were reconstituted with human or murine (hNTCPed84-87 ) hepatocytes. Livers were analysed between 5 and 42 days post-HDV inoculation by qRT-PCR, immunofluorescence and RNA in situ hybridization (ISH). RESULTS: hNTCPed84-87 mice could be infected with HDV genotype 1 or 3. ISH analysis demonstrated the presence of antigenomic HDV RNA positive murine hepatocytes with both genotypes, proving initiation of HDV replication. Strikingly, murine hepatocytes cleared HDV within 21 days both in immunocompetent hNTCPed84-87 mice and in immunodeficient USB mice xenografted with murine hepatocytes. In contrast, HDV infection remained stable for at least 42 days in human hepatocytes. Intrinsic innate responses were not enhanced in any of the HDV mono-infected cells and livers. CONCLUSION: These findings suggest that in addition to NTCP, further species-specific factors limit HDV infection efficacy and persistence in murine hepatocytes. Identifying such species barriers may be crucial to develop novel potential therapeutic targets of HDV.

Hepatitis Delta Virus Acts as an Immunogenic Adjuvant in Hepatitis B Virus-Infected Hepatocytes.

Hepatitis delta virus (HDV) requires hepatitis B virus (HBV) to complete its infection cycle and causes severe hepatitis, with limited therapeutic options. To determine the prospect of T cell therapy in HBV/HDV co-infection, we study the impact of HDV on viral antigen processing and presentation. Using in vitro models of HBV/HDV co-infection, we demonstrate that HDV boosts HBV epitope presentation, both in HBV/HDV co-infected and neighboring mono-HBV-infected cells through the upregulation of the antigen processing pathway mediated by IFN-ß/λ. Liver biopsies of HBV/HDV patients confirm this upregulation. We then validate in vitro and in a HBV/HDV preclinical mouse model that HDV infection increases the anti-HBV efficacy of T cells with engineered T cell receptors. Thus, by unveiling the effect of HDV on HBV antigen presentation, we provide a framework to better understand HBV/HDV immune pathology, and advocate the utilization of engineered HBV-specific T cells as a potential treatment for HBV/HDV co-infection.

High rates of cirrhosis and severe clinical events in patients with HBV/HDV co-infection: longitudinal analysis of a German cohort.

BACKGROUND: Chronic hepatitis delta virus (HDV) infection causes severe liver disease which often leads to cirrhosis and hepatocellular carcinoma (HCC). Aim of this study was to establish the disease severity and prognostic factors for disease outcome by analysing frequencies of clinical events and their correlation with baseline virological and biochemical parameters as well as interferon and nucleos(t)ide analogue treatment choice. METHODS: We studied a single-centre cohort of 49 anti-HDAg-positive patients with HBsAg persistence for at least 6 months. Virological and biochemical parameters, interferon and nucleos(t)ide analogue treatment choice as well as clinical events during follow-up were analysed by retrospective chart review (mean follow-up time 3 years, range 0.25-7.67 years). RESULTS: Severe clinical events occurred in 11/49 hepatitis D patients, including HCC (8/49), death (8/49) or liver transplantation (2/49). HCCs only occurred secondary to liver cirrhosis and their event rates in this cohort of hepatitis D patients did not differ from a matched HBV mono-infected cohort with comparable frequency of liver cirrhosis. A stepwise multivariate logistic regression revealed low platelet count (p = 0. 0290) and older age (p = 0.0337) correlating most strongly with overall clinical events, while serum HDV RNA positivity at baseline did not correlate with any clinical outcome. Interferon-free but not nucleos(t)ide analogue-free patient care correlated with the occurrence of HCC at logistic regression, although only 3/18 interferon-treated patients demonstrated repeatedly negative HDV PCR results post therapy. CONCLUSIONS: Our data indicate that progressive liver disease at baseline plays a major role as predictive factor for overall clinical outcome of hepatitis D patients. In particular, HCC risk may not be underestimated in hepatitis D virus RNA negative hepatitis D patients with advanced liver fibrosis.

Hepatitis delta virus persists during liver regeneration and is amplified through cell division both in vitro and in vivo.

OBJECTIVE: Hepatitis delta virus (HDV) was shown to persist for weeks in the absence of HBV and for months after liver transplantation, demonstrating the ability of HDV to persevere in quiescent hepatocytes. The aim of the study was to evaluate the impact of cell proliferation on HDV persistence in vitro and in vivo. DESIGN: Genetically labelled human sodium taurocholate cotransporting polypeptide (hNTCP)-transduced human hepatoma(HepG2) cells were infected with HBV/HDV and passaged every 7 days for 100 days in the presence of the entry inhibitor Myrcludex-B. In vivo, cell proliferation was triggered by transplanting primary human hepatocytes (PHHs) isolated from HBV/HDV-infected humanised mice into naïve recipients. Virological parameters were measured by quantitative real time polymerase chain reaction (qRT-PCR). Hepatitis delta antigen (HDAg), hepatitis B core antigen (HBcAg) and cell proliferation were determined by immunofluorescence. RESULTS: Despite 15 in vitro cell passages and block of viral spreading by Myrcludex-B, clonal cell expansion permitted amplification of HDV infection. In vivo, expansion of PHHs isolated from HBV/HDV-infected humanised mice was confirmed 3 days, 2, 4 and 8 weeks after transplantation. While HBV markers rapidly dropped in proliferating PHHs, HDAg-positive hepatocytes were observed among dividing cells at all time points. Notably, HDAg-positive cells appeared in clusters, indicating that HDV was transmitted to daughter cells during liver regeneration even in the absence of de novo infection. CONCLUSION: This study demonstrates that HDV persists during liver regeneration by transmitting HDV RNA to dividing cells even in the absence of HBV coinfection. The strong persistence capacities of HDV may also explain why HDV clearance is difficult to achieve in HBV/HDV chronically infected patients.

Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection.

Chronic delta hepatitis, caused by hepatitis delta virus (HDV), is the most severe form of viral hepatitis, affecting at least 20 million hepatitis B virus (HBV)-infected patients worldwide. HDV/HBV co- or superinfections are major drivers for hepatocarcinogenesis. Antiviral treatments exist only for HBV and can only suppress but not cure infection. Development of more effective therapies has been impeded by the scarcity of suitable small-animal models. We created a transgenic (tg) mouse model for HDV expressing the functional receptor for HBV and HDV, the human sodium taurocholate cotransporting peptide NTCP. Both HBV and HDV entered hepatocytes in these mice in a glycoprotein-dependent manner, but one or more postentry blocks prevented HBV replication. In contrast, HDV persistently infected hNTCP tg mice coexpressing the HBV envelope, consistent with HDV dependency on the HBV surface antigen (HBsAg) for packaging and spread. In immunocompromised mice lacking functional B, T, and natural killer cells, viremia lasted at least 80 days but resolved within 14 days in immunocompetent animals, demonstrating that lymphocytes are critical for controlling HDV infection. Although acute HDV infection did not cause overt liver damage in this model, cell-intrinsic and cellular innate immune responses were induced. We further demonstrated that single and dual treatment with myrcludex B and lonafarnib efficiently suppressed viremia but failed to cure HDV infection at the doses tested. This small-animal model with inheritable susceptibility to HDV opens opportunities for studying viral pathogenesis and immune responses and for testing novel HDV therapeutics.

Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.

OBJECTIVE: The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo. METHODS: PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing. RESULTS: PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production. CONCLUSIONS: We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

Both interferon alpha and lambda can reduce all intrahepatic HDV infection markers in HBV/HDV infected humanized mice.

Co-infection with hepatitis B (HBV) and D virus (HDV) is associated with the most severe course of liver disease. Interferon represents the only treatment currently approved. However, knowledge about the impact of interferons on HDV in human hepatocytes is scant. Aim was to assess the effect of pegylated interferon alpha (peg-IFNα) and lambda (peg-IFNλ), compared to the HBV-polymerase inhibitor entecavir (ETV) on all HDV infection markers using human liver chimeric mice and novel HDV strand-specific qRT-PCR and RNA in situ hybridization assays, which enable intrahepatic detection of HDV RNA species. Peg-IFNα and peg-IFNλ reduced HDV viremia (1.4 log and 1.2 log, respectively) and serum HBsAg levels (0.9-log and 0.4-log, respectively). Intrahepatic quantification of genomic and antigenomic HDV RNAs revealed a median ratio of 22:1 in untreated mice, resembling levels determined in HBV/HDV infected patients. Both IFNs greatly reduced intrahepatic levels of genomic and antigenomic HDV RNA, increasing the amounts of HDAg- and antigenomic RNA-negative hepatocytes. ETV-mediated suppression of HBV replication (2.1-log) did not significantly affect HBsAg levels, HDV productivity and/or release. In humanized mice lacking adaptive immunity, IFNs but not ETV suppressed HDV. Viremia decrease reflected the intrahepatic reduction of all HDV markers, including the antigenomic template, suggesting that intracellular HDV clearance is achievable.

Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluation.

BACKGROUND & AIMS: Hepatitis E virus (HEV) is a major cause of acute hepatitis as well as chronic infection in immunocompromised individuals; however, in vivo infection models are limited. The aim of this study was to establish a small animal model to improve our understanding of HEV replication mechanisms and permit the development of effective therapeutics. METHODS: UPA/SCID/beige mice repopulated with primary human hepatocytes were used for infection experiments with HEV genotype (GT) 1 and 3. Virological parameters were determined at the serological and intrahepatic level by real time PCR, immunohistochemistry and RNA in situ hybridization. RESULTS: Establishment of HEV infection was achieved after intravenous injection of stool-derived virions and following co-housing with HEV-infected animals but not via inoculation of serum-derived HEV. GT 1 infection resulted in a rapid rise of viremia and high stable titres in serum, liver, bile and faeces of infected mice for more than 25 weeks. In contrast, viremia in GT 3 infected mice developed more slowly and displayed lower titres in all analysed tissues as compared to GT 1. HEV-infected human hepatocytes could be visualized using HEV ORF2 and ORF3 specific antibodies and HEV RNA in situ hybridization probes. Finally, six-week administration of ribavirin led to a strong reduction of viral replication in the serum and liver of GT 1 infected mice. CONCLUSION: We established an efficient model of HEV infection to test the efficacy of antiviral agents and to exploit mechanisms of HEV replication and interaction with human hepatocytes in vivo.

Hepatitis B and Delta Virus: Advances on Studies about Interactions between the Two Viruses and the Infected Hepatocyte.

The mechanisms determining persistence of hepatitis B virus (HBV) infection and long-term pathogenesis of HBV-associated liver disease appear to be multifactorial. Although viral replication can be efficiently suppressed by the antiviral treatments currently available, viral clearance is generally not achieved since HBV has developed unique replication strategies, enabling persistence of its genome within the infected hepatocytes. Moreover, no direct antiviral therapy exists for the more than 15 million people worldwide that are also coinfected with the hepatitis delta virus (HDV), a defective virus that needs the HBV envelope proteins for propagation. The limited availability of robust HBV and HDV infection systems has hindered the understanding of the complex network of virus-virus and virus-host interactions that are established in the course of infection and slowed down progress in drug development. Since chronic HBV/HDV coinfection leads to the most severe form of chronic viral hepatitis, elucidation of the molecular mechanisms regulating virus-host interplay and pathogenesis are urgently needed. This article summarizes the current knowledge regarding the interactions among HBV, HDV, and the infected target cell and discusses the dependence of HDV on HBV activity and possible future therapeutic approaches.

Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection.

BACKGROUND & AIMS: The limited availability of hepatitis Delta virus (HDV) infection models has hindered studies of interactions between HDV and infected hepatocytes. The aim was to investigate the antiviral state of HDV infected human hepatocytes in the setting of co-infection with hepatitis B virus (HBV) compared to HBV mono-infection using human liver chimeric mice. METHODS: Viral loads, human interferon stimulated genes (hISGs) and cytokines were determined in humanized uPA/SCID/beige (USB) mice by qRT-PCR, ELISA and immunofluorescence. RESULTS: Upon HBV/HDV inoculation, all mice developed viremia, which was accompanied by a significant induction of hISGs (i.e. hISG15, hSTATs, hHLA-E) compared to uninfected mice, while HBV mono-infection led to weaker hISG elevations. In the setting of chronic infection enhancement of innate defense mechanisms was significantly more prominent in HBV/HDV infected mice. Also the induction of human-specific cytokines (hIP10, hTGF-ß, hIFN-ß and hIFN-λ) was detected in HBV/HDV co-infected animals, while levels remained lower or below detection in uninfected and HBV mono-infected mice. Moreover, despite the average increase of hSTAT levels determined in HBV/HDV infected livers, we observed a weaker hSTAT accumulation in nuclei of hepatocytes displaying very high HDAg levels, suggesting that HDAg may in part limit hSTAT signaling. CONCLUSIONS: Establishment of HDV infection provoked a clear enhancement of the antiviral state of the human hepatocytes in chimeric mice. Elevated pre-treatment ISG and interferon levels may directly contribute to inflammation and liver damage, providing a rationale for the more severe course of HDV-associated liver disease. Such antiviral state induction might also contribute to the lower levels of HBV activity frequently found in co-infected hepatocytes.