Imaging of Hepatitis B Virus Nucleic Acids: Current Advances and Challenges.

Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme viral loads, which may reach 1010 virions per mL serum, hepatitis B viruses are of low abundance and productivity in individual cells. Imaging of the infections in cells is thus a particular challenge especially for cccDNA that exists only in a few copies. The review describes the significance of microscopical approaches on genome and transcript detection for understanding hepatitis B virus infections, implications for understanding treatment outcomes, and recent microscopical approaches, which have not been applied in HBV research.

Abundance of Noncircular Intrahepatic Hepatitis B Virus DNA May Reflect Frequent Integration Into Human DNA in Chronically Infected Patients.

BACKGROUND: Hepatitis B virus (HBV) integration has implications for cancer development and surface antigen (HBsAg) production, but methods to quantify integrations are lacking. The aim of this study was to develop a droplet digital PCR (ddPCR) assay discriminating between circular and integrated HBV DNA, and to relate the distribution between the two forms to other HBV markers. METHODS: ddPCR with primers spanning the typical linearization breakpoint in the HBV genome allowed for quantification of the absolute copy numbers of total and circular HBV DNA, and calculation of linear HBV DNA. RESULTS: Analysis of 70 liver biopsies from patients with chronic HBV infection revealed that the fraction of linear HBV DNA, which includes integrations, was higher in HBeAg-negative patients than HBeAg-positive. The ratio between HBsAg and HBV DNA levels in serum correlated with the intrahepatic proportion of linear HBV DNA. Furthermore, ddPCR experiments on serum samples and experiments with nuclease indicated the contribution of encapsidated double-stranded linear DNA and replication intermediates to be limited. CONCLUSIONS: The degree of integration of intrahepatic HBV DNA in the HBeAg-negative stage may be higher than previously anticipated, and integrated DNA may explain the persistence of high HBsAg serum levels in patients with low HBV DNA levels.

Quantification of Viral RNA in Multiple Pieces of Explant Liver Tissue Shows Distinct Focal Differences in Hepatitis B Infection.

Hepatitis B virus (HBV) DNA and RNA were quantified by digital PCR assays in 20-30 tissue pieces from each of 4 liver explants with cirrhosis caused by HBV. The within-patient variability of HBV RNA levels between pieces was up to a 1000-fold. Core RNA and S RNA levels were similar and correlated strongly when replication was high, supporting that transcription was from covalently closed circular DNA (cccDNA). By contrast, enhanced expression of S RNA relative to cccDNA and core RNA in patients with medium-high or low replication supports that HBV surface antigen (HBsAg) can be expressed mainly from integrated HBV DNA in such patients.

Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.

Hepatitis B surface antigen on subviral particles reduces the neutralizing effect of anti-HBs antibodies on hepatitis B viral particles in vitro.

During hepatitis B virus (HBV) infections subviral particles (SVP) consisting mainly of hepatitis B surface antigen are present at much higher concentration than viral particles (VP) in serum. To investigate reasons for this excess of SVP production, SVP and VP were fractionated on a Nycodenz gradient and analyzed for HBV infection of HepG2-NTCP cells with and without anti-HBs antibodies. Our findings showed that SVP significantly reduced the neutralization of VP by anti-HBs, while SVP had little effect on viral entry, supporting the assumption that SVP serve as decoy facilitating cell-to-cell spread of HBV in the presence of neutralizing antibodies.

Detachment of Membrane Bound Virions by Competitive Ligand Binding Induced Receptor Depletion.

Multivalent receptor-mediated interactions between virions and a lipid membrane can be weakened using competitive nonpathogenic ligand binding. In particular, the subsequent binding of such ligands can induce detachment of bound virions, a phenomenon of crucial relevance for the development of new antiviral drugs. Focusing on the simian virus 40 (SV40) and recombinant cholera toxin B subunit (rCTB), and using (monosialotetrahexosyl)ganglioside (GM1) as their common receptor in a supported lipid bilayer (SLB), we present the first detailed investigation of this phenomenon by employing the quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy assisted 2D single particle tracking (SPT) techniques. Analysis of the QCM-D-measured release kinetics made it possible to determine the binding strength of a single SV40-GM1 pair. The release dynamics of SV40, monitored by SPT, revealed that a notable fraction of SV40 becomes mobile just before the release, allowing to estimate the distribution of SV40-bound GM1 receptors just prior to release.

A lipid zipper triggers bacterial invasion.

Glycosphingolipids are important structural constituents of cellular membranes. They are involved in the formation of nanodomains ("lipid rafts"), which serve as important signaling platforms. Invasive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium--a key process in bacterial uptake. However, it is unknown whether glycosphingolipids directly take part in the membrane invagination process. Here, we demonstrate that a "lipid zipper," which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3, triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aeruginosa. In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was sufficient to achieve complete membrane engulfment of the bacterium. In addition, theoretical modeling elucidated that the adhesion energy of the LecA-Gb3 interaction is adequate to drive the engulfment process. In cellulo experiments demonstrated that inhibition of the LecA/Gb3 lipid zipper by either lecA knockout, Gb3 depletion, or application of soluble sugars that interfere with LecA binding to Gb3 significantly lowered P. aeruginosa uptake by host cells. Of note, membrane engulfment of P. aeruginosa occurred independently of actin polymerization, thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry. Our study sheds new light on the impact of glycosphingolipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes.

The G428A nonsense mutation in FUT2 provides strong but not absolute protection against symptomatic GII.4 Norovirus infection.

In November 2004, 116 individuals in an elderly nursing home in El Grao de Castellón, Spain were symptomatically infected with genogroup II.4 (GII.4) norovirus. The global attack rate was 54.2%. Genotyping of 34 symptomatic individuals regarding the FUT2 gene revealed that one patient was, surprisingly, a non-secretor, hence indicating secretor-independent infection. Lewis genotyping revealed that Lewis-positive and negative individuals were susceptible to symptomatic norovirus infection indicating that Lewis status did not predict susceptibility. Saliva based ELISA assays were used to determine binding of the outbreak virus to saliva samples. Saliva from a secretor-negative individual bound the authentic outbreak GII.4 Valencia/2004/Es virus, but did not in contrast to secretor-positive saliva bind VLP of other strains including the GII.4 Dijon strain. Amino acid comparison of antigenic A and B sites located on the external loops of the P2 domain revealed distinct differences between the Valencia/2004/Es and Dijon strains. All three aa in each antigenic site as well as 10/11 recently identified evolutionary hot spots, were unique in the Valencia/2004/Es strain compared to the Dijon strain. To the best of our knowledge, this is the first example of symptomatic GII.4 norovirus infection of a Le(a+b-) individual homozygous for the G428A nonsense mutation in FUT2. Taken together, our study provides new insights into the host genetic susceptibility to norovirus infections and evolution of the globally dominating GII.4 viruses.

Norwalk virus-like particles bind specifically to A, H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids.

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with (125)I and then conjugated to VLPs. The (125)I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.

Human noroviruses recognize sialyl Lewis x neoglycoprotein.

The carbohydrate binding characteristics of a norovirus GII.3 (Chron1) and a GII.4 (Dijon) strain were investigated using virus-like particles (VLPs) and saliva samples from 81 individuals genotyped for FUT2 (secretor) and FUT3 (Lewis) and phenotyped for ABO and Lewis blood groups. The two VLPs showed a typical secretor-gene-dependent binding and bound significantly stronger to saliva from A, B, and AB than from O individuals (P < 0.0001 and P < 0.001) but did not bind to any samples from secretor-negative individuals. The GII.3 strain showed larger interindividual variation and bound stronger to saliva from B than from A(2) secretors (P < 0.01). When assaying for binding to neoglycoproteins, the GII.3 and GII.4 strains were compared with the Norwalk GI.1 prototype strain. Although all three strains bound to Lewis b (and H type 1 chain) glycoconjugates, only the two GII strains showed an additional binding to sialyl Lewis x. This novel binding was specific since the VLPs did not bind to structural analogs, e.g., Lewis x or sialyl Lewis a, but only to sialyl Lewis x, sialyl diLewis x and sialylated type 2 chain conjugates. In inhibition experiments, the sialyl Lewis x conjugate was the most potent inhibitor. The minimal requirement for this potential receptor structure is Neu5Ac alpha 3Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta- where Fuc is not absolutely necessary for binding. Our study shows that some human norovirus GII strains have at least two binding specificities: one secretor-gene-dependent related to alpha1,2-fucosylated carbohydrates and another related to alpha2,3-sialylated carbohydrates of the type 2 chain, e.g., sialyl Lewis x.