Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle.

East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

Magnetic nanospheres for convenient and efficient capture and release of hepatitis B virus DNA.

Nucleic acid isolation and purification are essential steps in molecular biology. Currently-used isolation methods focus on the extraction of all the nucleic acids from crude samples, yet ignore the specific nucleic acids of interest, which may induce the loss of the specific nucleic acids and hinder their analyses. Herein, a magnetic nanospheres (MNs)-based strategy for efficient capture and release of specific nucleic acids is developed. The DNA sequence of hepatitis B virus (HBV) is taken as a model to validate this method. The MNs are modified with the complementary strand of HBV DNA for specific capture based on hybridization reaction. Then, by melting at high temperature, the captured DNAs are detached from the MNs to achieve release. The capture and release process are performed conveniently with magnetic separation. High capture efficiency (over 80%) and nearly 100% release efficiency for HBV DNA are achieved respectively via 40 min and 5 min interaction. While non-target DNAs are hardly captured, indicative of good selectivity. Moreover, after releasing DNAs, the MNs are directly regenerated and can be reused without degrading performance, which greatly reduces the operation costs. Finally, this method is applied to serum samples without any pretreatment, which exhibits similar capture and release capacity with those in the ideal samples, indicating its great application potential in practice.

Epigallocatechin gallate inhibits hepatitis B virus via farnesoid X receptor alpha.

Plants possess various natural antiviral properties. Epigallocatechin-3-gallate (EGCG), a major component of green tea, inhibits a variety of viruses. However, the clinical application of EGCG is currently hindered by a scarcity of information on its molecular mechanism of action. In the present study, we examined the anti-HBV (hepatitis B virus) effects of catechins from green tea at the transcriptional and antigen-expression levels, as well as the associated molecular mechanisms, because HBV-associated liver diseases have become a key public health issue due to their serious impact on human physical and mental health. By using fluorescence quenching and affinity binding, we demonstrated that EGCG is an important transcriptional regulator of the HBV genome, which it achieves by interacting with farnesoid X receptor alpha (FXRα). Luciferase assay showed that EGCG effectively inhibited the transcription of the HBV promoter dose-dependently when expression plasmids of FXRα and retinoid X receptor α (RXRα) were co-transfected into HEK293 cells. These results indicate that the downregulation of the HBV antigen and the decrease in the transcriptional activation of the HBV EnhII/core promoter by FXRα/RXRα are mainly due to the interaction between EGCG and FXRα. Therefore, EGCG, an antagonist of FXRα in liver cells, has the potential to be employed as an effective anti-HBV agent.

AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.

A simple QD-FRET bioprobe for sensitive and specific detection of hepatitis B virus DNA.

We report here a simple quantum dot-FRET (QD-FRET) bioprobe based on fluorescence resonance energy transfer (FRET) for the sensitive and specific detection of hepatitis B virus DNA (HBV DNA). The proposed one-pot HBV DNA detection method is very simple, rapid and convenient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide on the surface of QDs with mercaptoacetic acid (MAA). Subsequently, DNA was attached to QDs surface to form the functional QD-DNA bioconjugates by simple surface ligand exchange. After adding 6-carboxy-X-rhodamine (ROX)-modified HBV DNA (ROX-DNA) into the QD-DNA bioconjugates solution, DNA hybridization between QD-DNA bioconjugates and ROX-DNA was formed. The resulting hybridization brought the ROX fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to energy transfer from QDs to ROX. When ROX-DNA was displaced by the unlabeled HBV DNA, the efficiency of FRET was dramatically decreased. Based on the changes of both fluorescence intensities of QDs and ROX, HBV DNA could be detected with high sensitivity and specificity. Under the optimized conditions, the linear range of HBV DNA determination was 2.5 - 30 nmol L(-1), with a correlation coefficient (R) of 0.9929 and a limit of detection (3σ black) of 1.5 nmol L(-1). The relative standard deviation (R.S.D.) for 12 nmol L(-1) HBV DNA was 0.9% (n = 5). There was no interference to non-complementary DNA. Time-resolved fluorescence spectra and fluorescence images were performed to verify the validity of this method and the results were satisfying.

Preparation and characterization of zinc oxide nanoparticles and their sensor applications for electrochemical monitoring of nucleic acid hybridization.

In this study, ZnO nanoparticles (ZNP) of approximately 30 nm in size were synthesized by the hydrothermal method and characterized by X-ray diffraction (XRD), Braun-Emmet-Teller (BET) N2 adsorption analysis and transmission electron microscopy (TEM). ZnO nanoparticles enriched with poly(vinylferrocenium) (PVF+) modified single-use graphite electrodes were then developed for the electrochemical monitoring of nucleic acid hybridization related to the Hepatitis B Virus (HBV). Firstly, the surfaces of polymer modified and polymer-ZnO nanoparticle modified single-use pencil graphite electrodes (PGEs) were characterized using scanning electron microscopy (SEM). The electrochemical behavior of these electrodes was also investigated using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Subsequently, the polymer-ZnO nanoparticle modified PGEs were evaluated for the electrochemical detection of DNA based on the changes at the guanine oxidation signals. Various modifications in DNA oligonucleotides and probe concentrations were examined in order to optimize the electrochemical signals that were generated by means of nucleic acid hybridization. After the optimization studies, the sequence-selective DNA hybridization was investigated in the case of a complementary amino linked probe (target), or noncomplementary (NC) sequences, or target and mismatch (MM) mixture in the ratio of (1:1).

Anti-hepatitis B virus active lactones from the traditional Chinese herb: Swertia mileensis.

Swerilactones H-K (1-4), which are four novel lactones with an unprecedented C29 skeleton, were isolated from Swertia mileensis (Qing-Ye-Dan), an endemic Chinese herb used for treating viral hepatitis. Their structures were determined by extensive spectroscopic and X-ray crystallographic diffraction analyses. Swerilactones H-K exhibit potent anti-hepatitis B virus activity against HBV DNA replication with IC(50) values ranging from 1.53 to 5.34 µM. For the first time, a plausible biogenetic pathway for swerilactones H-K, together with the previously reported swerilactones A-D is proposed. From a biogenetic point of view, swerilactones A-D are ascribed as secoiridoid dimers, and swerilactones H-K as secoiridoid trimers.

Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence.

In this study we have used two fluorescent probes, tetrakis(diisopropylguanidino)-zinc-phthalocyanine (Zn-DIGP) and N-methylmesoporphyrin IX (NMM), to monitor the reassembly of "split" G-quadruplex probes on hybridization with an arbitrary "target" DNA. According to this approach, each split probe is designed to contain half of a G-quadruplex-forming sequence fused to a variable sequence that is complementary to the target DNA. Upon mixing the individual components, both base-pairing interactions and G-quadruplex fragment reassembly result in a duplex-quadruplex three-way junction that can bind to fluorescent dyes in a G-quadruplex-specific way. The overall fluorescence intensities of the resulting complexes were dependent on the formation of proper base-pairing interactions in the duplex regions, and on the exact identity of the fluorescent probe. Compared with samples lacking any "target" DNA, the fluorescence intensities of Zn-DIGP-containing samples were lower, and the fluorescence intensities of NMM-containing samples were higher on addition of the target DNA. The resulting biosensors based on Zn-DIGP are therefore termed "turn-off" whereas the biosensors containing NMM are defined as "turn-on". Both of these biosensors can detect target DNAs with a limit of detection in the nanomolar range, and can discriminate mismatched from perfectly matched target DNAs. In contrast with previous biosensors based on the peroxidase activity of heme-bound split G-quadruplex probes, the use of fluorescent dyes eliminates the need for unstable sensing components (H(2)O(2), hemin, and ABTS). Our approach is direct, easy to conduct, and fully compatible with the detection of specific DNA sequences in biological fluids. Having two different types of probe was highly valuable in the context of applied studies, because Zn-DIGP was found to be compatible with samples containing both serum and urine whereas NMM was compatible with urine, but not with serum-containing samples.

Treatment of hepatitis B virus-infected cells with alpha-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity.

Trimming of the N-glycans attached to the envelope proteins of hepatitis B virus (HBV) is required in different steps of the viral life cycle. Inhibition of the host enzymes alpha-glucosidases, involved in the endoplasmic reticulum (ER)-associated processing of the N-linked glycans, results in misfolding of the HBV envelope proteins, prevention of HBV secretion and accumulation of viral DNA within infected cells. However, the impact of these effects on HBV morphogenesis and infectivity of the viral particles that are still released from cells with inhibited alpha-glucosidase has not been addressed so far. Using N-butyldeoxynojirimycin (NB-DNJ), a competitive inhibitor of the ER alpha-glucosidases, we analyzed the role of these enzymes on HBV assembly and infectivity of the virions released from HepG2.2.2.15 cells. HBV secreted from drug-treated cells contained an envelope with altered composition of the disulfide-linked oligomers and no detectable middle (M) protein. These molecular changes had a significant effect on HBV infectivity, reducing it to 20% compared to controls, for the highest concentrations of NB-DNJ used. Our data show for the first time that an active alpha-glucosidase activity is crucial for production of infectious HBV and provide new insights into the controversial role of the M protein in this process.

Prevalence and risk factor analysis of GBV-C/HGV infection in prostitutes.

GB virus-C (GBV-C) and Hepatitis G virus (HGV) are variants of a recently cloned virus transmitted parenterally. It is unclear if sexual contact also transmits this virus. In this study, we detected serum GBV-C/HGV RNA in 140 prostitutes by reverse transcription polymerase chain reaction (RT-PCR) using different primers. Thirty (21%) were found with GBV-C RNA by nested PCR although only 22 (73%) had HGV RNA by single round RT-PCR. Both assays had a nearly perfect agreement (kappa value, 0.812). The prevalence of GBV-C RNA in prostitutes was significantly higher than the control group (30/140 vs. 2/40, P < 0.02). Multivariate analysis revealed that a frequency of paid sex more than 120 times per month was the only factor significantly associated with positive GBV-C RNA in prostitutes (P < 0.003). In summary, prostitutes are a high risk group and reservoir of GBV-C/HGV infection due to high frequency of paid-sex.