Hepatitis delta virus RNA decline post-inoculation in human NTCP transgenic mice is biphasic.

Chronic infection with hepatitis B and delta viruses (HDV) is the most serious form of viral hepatitis due to more severe manifestations of an accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. We characterized early HDV kinetics post-inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. We analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor-human sodium taurocholate co-transporting polypeptide (hNTCP). Kinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific-binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 35 minutes (standard error, SE: 6.3), binds to non-specific cells with a rate of 0.05 per hour (SE: 0.01), and returns as free virus with a rate of 0.11 per hour (SE: 0.02). Characterizing early HDV-host kinetics elucidates how quickly HDV is either cleared or bound depending on the immunological background and hNTCP presence. IMPORTANCE The persistence phase of HDV infection has been studied in some animal models; however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post-inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics.

Hepatitis delta virus RNA decline post inoculation in human NTCP transgenic mice is biphasic.

Background and Aims: Chronic infection with hepatitis B and hepatitis delta viruses (HDV) is considered the most serious form of viral hepatitis due to more severe manifestations of and accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no FDA-approved treatment for HDV and current interferon-alpha treatment is suboptimal. We characterized early HDV kinetics post inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. Methods: We analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor - human sodium taurocholate co-transporting peptide (hNTCP). Results: Kinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 18 minutes (standard error, SE: 2.4), binds to non-specific cells with a rate of 0.06 hour -1 (SE: 0.03), and returns as free virus with a rate of 0.23 hour -1 (SE: 0.03). Conclusions: Understanding early HDV-host kinetics will inform pre-clinical therapeutic kinetic studies on how the efficacy of anti-HDV therapeutics can be affected by early kinetics of viral decline. LAY SUMMARY: The persistence phase of HDV infection has been studied in some animal models, however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics. Understanding the kinetics of viral clearance in the blood can aid pre-clinical development and testing models for anti-HDV therapeutics.

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.

Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy.

BACKGROUND & AIMS: Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC). METHODS: Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes). RESULTS: Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion. CONCLUSIONS: The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects. LAY SUMMARY: Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.

15year fulminant hepatitis B follow-up in Belgium: Viral evolution and signature of demographic change.

Fulminant hepatitis among different clinical outcomes of hepatitis B virus infection is very rare and manifests high mortality rate, however it has not been investigated in Belgian inhabitants yet. In the frame of a retrospective study between 1995 and 2010, 80 serum samples (in some cases serial samples) archived in Biobank, were collected from 24 patients who had clinically developed fulminant infection of hepatitis B virus. In total, 33 hepatitis B virus (HBV) strains (31 full-length genome and 2 partial viral genes) of different HBV genotypes and subgenotypes including A2, B2, D1, D2, D3 and E, were amplified, sequenced and phylogenetically analyzed. HBV isolated strains from native and exotic patients were characterized by genome variations associated with viral invasiveness. Although several mutations at nucleotide and protein levels were detected, evolutionary analyses revealed a negative selective pressure over the viral genomes. This study revealed influence of immigration through a steady change in the viral epidemiological profile of the Belgian population.

HBsAg mutations related to occult hepatitis B virus infection in HIV-positive patients result in a reduced secretion and conformational changes of HBsAg.

BACKGROUND: Occult hepatitis B infection (OBI) is a frequent finding in human immunodeficiency virus (HIV)-infected patients. While several related mutations in the hepatitis B virus (HBV) genome have been reported, their distinct impact on HBsAg synthesis is largely obscure. METHODS: Thirty-one (18%) out of 172 HIV-infected patients, who were selected from HBsAg-negative patients, were positive for HBV-DNA assigned as being OBI-positive. We generated a series of expression constructs of variant HBsAg with "a" determinant amino acid substitutions including P127L, P127T, S136Y, and P127T + S136Y using site-directed mutagenesis. The expression of variant HBsAg was examined by transient transfection in hepatoma cells, followed by HBsAg immunoassay and immunofluorescence stained with specific anti-HBs antibodies. The potential impact of amino acid substitutions at different positions for conformational changes in the HBsAg was investigated using bioinformatics. RESULTS: All variants comprising either single or combined mutations resulted in significantly reduced HBsAg detection in supernatants and in cell lysates of hepatoma cells transfected with the constructs. Moreover, intracellular immunofluorescence staining of cytoblocks showed perinuclear and cytoplasmic fluorescence of HBsAg constructs with significantly diminished fluorescent intensity in comparison to the wild type. Altered protein conformations by predictive models, indicating an impaired detection by the host's immune response as well as by commercial antibody-based test assays. CONCLUSION: Mutations in the "a" determinant region of HBV as often found in OBI remarkably impair the detection of HBsAg from serum and infected cells, emphasizing the relevance of alternative methods such as HBV-DNA quantification for high-risk groups like HIV-infected individuals. J. Med. Virol. 89:246-256, 2017. © 2016 Wiley Periodicals, Inc.

Hepatocarcinogenesis associated with hepatitis B, delta and C viruses.

Globally, over half a billion people are persistently infected with hepatitis B (HBV) and/or hepatitis C viruses. Chronic HBV and HCV infection frequently lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Co-infections with hepatitis delta virus (HDV), a subviral satellite requiring HBV for its propagation, accelerates the progression of liver disease toward HCC. The mechanisms by which these viruses cause malignant transformation, culminating in HCC, remain incompletely understood, partially due to the lack of adequate experimental models for dissecting these complex disease processes in vivo.

Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C.

Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients.

Molecular interactions between hepatitis B virus and delta virus.

As a deficient virus due to the lack of envelope proteins, hepatitis D virus (HDV) causes chronic or fulminant "delta hepatitis" only in people with simultaneous hepatitis B virus (HBV) infection. HBV encodes three types of surface proteins known as small (S), medium (M) and large (L) envelope proteins. All three types of HBV surface antigens (HBsAgs) are present on HDV virions. The envelopment process of HDV occurs through interactions between the HDV ribonucleoprotein (RNP) complex and HBV HBsAgs. While HBsAg is the only protein required by HDV, the exact interaction sites between the S protein and pre-mature HDV are not well defined yet. In fact, these sites are distributed along the S protein with some hot spots for the envelopment process. Moreover, in most clinically studied samples, HDV infection is associated with a dramatically reduced HBV viral load, temporarily or permanently, while HBsAg resources are available for HDV packaging. Thus, beyond interacting with HBV envelope proteins, controlling mechanisms exist by which HDV inhibits HBV-DNA replication while allowing a selective transcription of HBV proteins. Here we discuss the molecular interaction sites between HBsAg and the HDV-RNP complex and address the proposed indirect mechanisms, which are employed by HBV and HDV to facilitate or inhibit each other's viral replication. Understanding molecular interactions between HBV and HDV may help to design novel therapeutic strategies for delta hepatitis.