In vitro preparation of uniform and nucleic acid free hepatitis B core particles through an optimized disassembly-purification-reassembly process.

Structure heterogeneity and host nucleic acids contamination are two major problems for virus-like particles (VLPs) produced by various host cells. In this study, an in vitro optimized disassembly-purification-reassembly process was developed to obtain uniform and nucleic acid free hepatitis B core (HBc) based VLPs from E. coli fermentation. The process started with ammonium sulfate precipitation of all heterogeneous HBc structures after cell disintegration. Then, dissolution and disassembly of pellets into basic subunits were carried out under the optimized disassembly condition. All contaminants, including host nucleic acids and proteins, were efficiently removed with affinity chromatography. The purified subunits reassembled into VLPs by final removal of the chaotropic agent. Two uniform and nucleic acid free HBc-based VLPs, truncated HBc149 and chimeric HBc183-MAGE3 I, were successfully prepared. It was found that disassembly degree of HBc-based VLPs had a great influence on the protein yield, nucleic acid removal and reassembly efficiency. 4 M urea was optimal because lower concentration would not disassemble the particles completely while higher concentration would further denature the subunits into disordered aggregate and could not be purified and reassembled efficiently. For removal of strong binding nucleic acids such as in the case of HBc183-MAGE3 I, benzonase nuclease was added to the disassembly buffer before affinity purification. Through the optimized downstream process, uniform and nucleic acid free HBc149 VLPs and HBc183-MAGE3 I VLPs were obtained with purities above 90% and yields of 55.2 and 43.0 mg/L, respectively. This study would be a reference for efficient preparation of other VLPs.

On-treatment monitoring of liver fibrosis with serum hepatitis B core-related antigen in chronic hepatitis B.

BACKGROUND: Non-invasive evaluation for liver fibrosis is clinically important, especially in patients with undetectable hepatitis B virus (HBV) DNA treated with nucleoside analogs. AIM: To clarify the monitoring power of hepatitis B core-related antigen (HBcrAg) for hepatic histologic changes in patients with chronic hepatitis B (CHB) treated with entecavir. METHODS: This prospective multicenter study used multiple ordinal and multivariate logistics regression analysis to assess variables associated with Ishak fibrosis score and regression for fibrosis regression, respectively, in 403 CHB patients, including 374 with entecavir for 72 weeks (291 underwent paired liver biopsy) and 29 as controls. RESULTS: Level of HBcrAg correlated negatively with liver fibrosis staging (γ = -0.357, P < 0.001) in hepatitis B e antigen (HBeAg)-positive patients, and positively with liver fibrosis staging in HBeAg-negative patients. Higher HBcrAg concentration was associated with younger age, HBeAg positive status, high HBV DNA loads, high level of hepatitis B surface antigen (HBsAg) and higher necroinflammation, but not with HBV genotype. Serum concentration of HBcrAg, basal core promoter/precore (BCP/PC) mutant, quantitation of HBsAg (qHBsAg) and platelet counts were independently associated with Ishak fibrosis score on multiple ordinal regression. HBV DNA was undetectable in 88.37% of patients treated with entecavir at week 72, while their level of HBcrAg was still detectable. A greater reduction in post-treatment HBcrAg concentration was associated with the regression of hepatic fibrosis and histological improvement. HBcrAg concentration > 6.33 log IU/mL at baseline and logarithmic reduction > 1.03 log IU/mL at week 72 were associated with a higher chance of regression of liver fibrosis and histological improvement, respectively. CONCLUSION: HBcrAg level is associated with liver fibrosis progression. HBcrAg is an excellent monitor of hepatic histological changes, especially in CHB patients treated with nucleoside analogs.

Enhanced sentinel surveillance for hepatitis B infection in 200 counties in China, 2013-2016.

Hepatitis B infection is a major public health challenge in China. Clinicians report hepatitis B cases to the National Notifiable Disease Reporting System. A 2007 study found that only 35% of hepatitis B cases that had been reported as acute infections met a rigorous case definition of acute hepatitis B, implying overreporting of new-onset infections. To increase the accuracy of reported acute hepatitis B infections, in 2013, we initiated enhanced hepatitis B surveillance in 200 sentinel counties. We compared incidences and proportions of different stages of hepatitis B infection before and after implementation of enhanced surveillance. We checked the accuracy of reported data and re-diagnosed hepatitis B cases reported as acute infection according to the enhanced diagnostic criteria and calculated positive predictive value(PPV) of acute hepatitis B reports. Compared to previous surveillance, with enhanced surveillance, the incidence of reported acute hepatitis B infection decreased by 53.7% and the proportion of unclassified hepatitis B infection was reduced by 79.4%. From 2013 to 2016, the PPV of acute hepatitis B increased (55.8% to 71.0%); PPV rates in western and rural areas were lower than in other areas. We recommend enhancing hepatitis B surveillance nationwide using these new standards, and raising western and rural areas clinicians' diagnostic and reporting capacity, and ensuring sufficient resources for IgM anti-HBc testing.

Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

BACKGROUND: Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. RESULTS: The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. CONCLUSIONS: These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.

Measurement of the hepatitis B core-related antigen is valuable for predicting the pathological status of liver tissues in chronic hepatitis B patients.

The objective of this study was to evaluate the validities of serum hepatitis B core-related antigen (HBcrAg) for predicting the pathological status of liver tissues of chronic hepatitis B (CHB). A total of 205 Chinese patients with CHB, including 121 HBeAg-positive and 84 HBeAg-negative patients, were enrolled in this study. In HBeAg-positive patients, AUCs of serum HBcrAg for predicting severe necro-inflammation and advanced fibrosis were greater than 0.70; using serum HBcrAg<=4.81×10(4)kUmL(-1) and <=2.45×10(4)kUmL(-1) as cutoffs, the sensitivities, specificities, accuracies for predicting severe necro-inflammation and advanced fbrosis were 0.697, 0.716, 0.711 and 0.818, 0.778, 0.785, respectively. In HBeAg-negative patients, the AUCs of serum HBcrAg for predicting significant necro-inflammation and significant fibrosis were greater than 0.70; using serum HBcrAg>=1.70×10(2)kUmL(-1) and >=4.02kUmL(-1) as cutoffs, the sensitivities, specificities, accuracies for predicting significant necro-inflammation and significant fibrosis were 0.929, 0.964, 0.952 and 1.000, 1.000, 1.000, respectively. These results indicated favorable performances of serum HBcrAg for predicting severe necro-inflammation and advanced fibrosis in HBeAg-positive patients and significant necro-inflammation and significant fibrosis in negative patients.

Negative chromatography purification of hepatitis B virus-like particles using poly(oligo(ethylene glycol) methacrylate) grafted cationic adsorbent.

Poly(oligo(ethylene glycol) methacrylate) (POEGMA), an inert polymer was grafted onto an anion exchange adsorbent for the exclusion of relatively larger hepatitis B virus-like particles (HB-VLPs) from the anion exchange ligand (Q) and at the same time this process allowed the selective adsorption of smaller size Escherichia coli host cell proteins (HCPs). The chain lengths of the POEGMA grafted were modulated by varying the amount of monomers used in the polymer grafting. The purification factor and yield of the HB-VLPs obtained from the flow-through of negative chromatography were 2.3 and 66.0±3.1%, respectively, when shorter chain length of POEGMA (SQ) was grafted. Adsorbent grafted with longer chain of POEGMA (LQ) excluded some HCPs that are larger in size together with the HB-VLPs, reducing the purity of the recovered HB-VLPs. Further heat-treatment of the flow-through pool from SQ followed by centrifugation increased the purity of heat stable HB-VLPs to 87.5±1.1%. Heat-treatment of the flow through sample resulted in thermal denaturation and aggregation of HCPs, while the heat stable HB-VLPs still remained intact as observed under a transmission electron microscope. The performance of the negative chromatography together with heat treatment in the purification of HB-VLPs is far better than the reported bind-and-elute techniques.

Hepatitis B virus peptide inhibitors: solution structures and interactions with the viral capsid.

Hepatitis B virus (HBV) infection remains a health problem globally despite the availability of effective vaccines. In the assembly of the infectious virion, both the preS and S regions of the HBV large surface antigen (L-HBsAg) interact synergistically with the viral core antigen (HBcAg). Peptides preS and S based on the L-HBsAg were demonstrated as potential inhibitors to block the viral assembly. Therefore, the objectives of this study were to determine the solution structures of these peptides and study their interactions with HBcAg. The solution structures of these peptides were solved using (1)H, (13)C, and (15)N NMR spectroscopy. Peptide preS has several structured regions of ß-turns at Ser7-Pro8-Pro9, Arg11-Thr12-Thr13 and Ser22-Thr23-Thr24 sequences whereas peptide S has only one structured region observed at Ser3-Asn4-His5. Both peptides contain bend-like structures surrounding the turn structures. Docking studies revealed that both peptides interacted with the immunodominant region of HBcAg located at the tip of the viral capsid spikes. Saturation Transfer Difference (STD) NMR experiments identified several aromatic residues in peptides preS and S that interact with HBcAg. This study provides insights into the contact regions of L-HBsAg and HBcAg at atomic resolution which can be used to design antiviral agents that inhibit HBV morphogenesis.

Occult hepatitis B virus infection in chacma baboons, South Africa.

During previous studies of susceptibility to hepatitis B virus (HBV) infection, HBV DNA was detected in 2/6 wild-caught baboons. In the present study, HBV DNA was amplified from 15/69 wild-caught baboons. All animals were negative for HBV surface antigen and antibody against HBV core antigen. Liver tissue from 1 baboon was immunohistochemically negative for HBV surface antigen but positive for HBV core antigen. The complete HBV genome of an isolate from this liver clustered with subgenotype A2. Reverse transcription PCR of liver RNA amplified virus precore and surface protein genes, indicating replication of virus in baboon liver tissue. Four experimentally naive baboons were injected with serum from HBV DNA-positive baboons. These 4 baboons showed transient seroconversion, and HBV DNA was amplified from serum at various times after infection. The presence of HBV DNA at relatively low levels and in the absence of serologic markers in the baboon, a nonhuman primate, indicates an occult infection.

Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles.

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM) than previously reported. The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particles (VLPs) using bioorthogonal azide-alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold.

Hepatitis B virus infection among first-time blood donors in Italy: prevalence and correlates between serological patterns and occult infection.

BACKGROUND: A prospective, 1-year study was performed among Italian first-time, volunteer blood donors, who account for 12% of all donations, in order to assess the frequency and serological patterns of hepatitis B virus infection and the presence of occult infection. MATERIALS AND METHODS: Consecutive donors (n=31,190) from 21 blood transfusion centres, from age classes not subjected to universal HBV vaccination, were tested for HBsAg and anti-HBc by commercial immunoassays. Other HBV serological markers were searched for and qualitative and quantitative assessments of HBV-DNA were made in HBsAg and/or anti-HBc-positive individuals. RESULTS: Of the 31,190 donors studied, 100 (0.32%) were positive for both HBsAg and anti-HBc, 2 for HBsAg (0.01%) alone, and 2,593 (8.3%) for anti-HBc. Of these last, 86.7% were also positive for anti-HBs (with or without anti-HBe), 2.9% were positive for anti-HBe without anti-HBs and 10.4% had no other HBV markers (anti-HBc alone). A general north-south increasing gradient of HBV prevalence was observed. Circulating HBV-DNA was found in 96.8% of HBsAg-positive subjects as compared to 0.55% (12/2,186) of anti-HBc-positive/HBsAg-negative subjects, with higher frequencies among anti-HBs-negative than among anti-HBs-positive ones (1.68% vs. 0.37%; p <0.01) and among the 57 cases positive for both anti-HBc and anti-HBe (7%). HBV-DNA levels were significantly higher in HBsAg-positive subjects than in HBsAg-negative ones (median: 456 IU/mL vs. 38 IU/mL). CONCLUSIONS: The prevalence of HBV infection among Italian first-time blood donors is much lower than in the past. The presence of occult infections in this group was confirmed (frequency: 1 in 2,599), supporting the hypothesis of long-term persistence of HBV infection after clearance of HBsAg. HBsAg and nucleic acid amplification testing for blood screening and vaccination against HBV are crucial in order to further reduce the risk of transfusion-transmitted HBV towards zero.

Native agarose gel electrophoresis and electroelution: A fast and cost-effective method to separate the small and large hepatitis B capsids.

Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.

Influencia de la positividad del anti-HBc en el donante de órganos en el trasplante cardiaco / Influence of HBcAb positivity in the organ donor in heart transplantation

Objetivo: Existe alto riesgo de transmitir una infección por virus B con el hígado de donantes AgHBs (-), anti-HBc, sin embargo, este riesgo está poco estudiado en el trasplante cardiaco. Los objetivos son conocer la influencia del anti-HBc (+) en la aceptación del corazón para trasplante y hacer una puesta al día de los trabajos publicados. Diseño: Encuesta y revisión bibliográfica en bases médicas desde 1994 hasta octubre del 2011. Ámbito: Equipos españoles de trasplante cardiaco. Pacientes: No aplicable. Intervenciones: Ninguna. Variables de interés: Aceptación para trasplante del corazón de donantes anti-Hbc (+). Resultados: Doce de 15 equipos encuestados vacunan contra el VHB y 2 cuantifican los títulos anti-HBs. Siete solicitan el anti-HBc del donante. En caso de positividad, 2 no aceptan la oferta, 2 aceptan si el donante es también anti-HBs (+), uno selecciona el receptor en situación de urgencia y 3 emplean profilaxis farmacológica de forma aislada o complementaria a las anteriores. Solo se ha publicado un caso de hepatitis B en un receptor anti-HBc (-), anti-HBs (-) que no recibió medidas profilácticas. Hay descritas seroconversiones de los marcadores anti-HBc y anti-HBs de dudosa etiología. Conclusiones: El anti-HBc (+) del donante influye para aceptar el corazón aunque hay disparidad de criterios. Existe escasa información publicada sobre la evolución de los receptores. Hasta el momento se ha descrito un caso de infección postrasplante. Aunque poco frecuente, un donante anti-HBc (+) puede albergar infección oculta por VHB. El riesgo puede prevenirse con títulos anti-HBs adecuados o con medidas farmacológicas (AU)

Objective: There is a significant risk of hepatitis B transmission from HBsAg (-), HBcAb (+) donors in liver transplantation, but there is little information about hepatitis B transmission from HBcAb heart donors. The present study examines the influence of HBcAb presence in relation to heart donor acceptance and offers an update of the published studies. Design: Survey and medical database update from 1994 to October 2011. Setting: Spanish heart transplantation teams. Patients: Not applicable. Study variables: Acceptance of heart transplant from an HBcAb (+) organ donor. Results: Twelve out of 15 surveyed teams were seen to vaccinate against HBV, and two quantify HBsAb titers. Seven teams specifically request donor HBcAb status. If the latter proves positive, two do not accept transplantation, two accept if the donor is also HBsAb (+), one selects the receptor under emergency conditions, and three use drug prophylaxis isolatedly or complementary to the above. Only one case of hepatitis B has been reported in a HBcAb (-) and HBsAb (-) receptor that did not receive prophylactic measures. There have been reports of seroconversion of the HBcAb and HBsAb markers, though with an uncertain etiology. Conclusions: HBcAb seropositivity influences acceptance of a heart donor, but agreement is lacking. There is limited information on receptor evolution. To date there has been one reported case of hepatitis B after heart transplant. Although rare, an HBcAb (+) donor can harbor occult HBV infection. The risk of infection can be prevented with appropriate HBsAb titers following vaccination or by pharmacological measures (AU)

DNase-mediated single-cycle selection of aptamers for proteins blotted on a membrane.

We describe a single-cycle DNA aptamer selection strategy that is able to obtain high affinity aptamers (K(d) of sub-nM) directly from a protein blotted on membrane. The key to the success of this strategy is the unique use of DNase I digestion to remove unwanted ssDNA from the membrane, leaving only the strongest bound aptamers. A crude Hepatitis B virus core protein (HBcAg) was separated using polyacrylamide gel electrophoresis (PAGE) and electro-blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane strip containing HBcAg and a second membrane strip containing human serum proteins were coincubated with a ssDNA library consisting of ∼10 copies each of 10(15) random sequences. Unbound and weakly bound sequences were efficiently removed from the membrane containing HBcAg using DNase I digestion and gradient wash with urea buffers. The remaining ssDNA bound to the target consisted of approximately 500 molecules, from which two aptamers with high affinity (K(d) ∼100 and 200 pM) were identified. This technique can be potentially used for selection of aptamers directly from multiple proteins that are separated by gel electrophoresis from a biological mixture.

Purification of recombinant hepatitis B core antigen from unclarified Escherichia coli feedstock using phage-immobilized expanded bed adsorption chromatography.

Fusion M13 phage with disulfide constrained heptapeptide, C-WSFFSNI-C, inserted into the minor coat protein (gpIII), has been selected in the current study as ligand in direct purification of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli (E. coli) feedstock. The selected fusion phage showed strong association with the surface of the core particle. In the present study, this fusion M13 phage was immobilized onto Streamline base matrix via epoxy activation and used as adsorbent to capture HBcAg from crude E. coli homogenate. The maximum binding capacity for the adsorbent was 3.76 mg/mL with equilibrium coefficient of 1.83 mg/mL. Due to the slow uptake rate of HBcAg by M13 phage-immobilized adsorbents, a modified EBAC operation with recirculation of feedstock into the expanded bed has been investigated in this study. The introduction of feedstock recirculation has led to an 18% increase in yield; however, the purity of the eluted product was reduced by 15% compared with typical EBAC operation. The level of antigenicity exhibited by the core particles purified by both EBAC operations employed in the present study was comparable to that purified using sucrose ultracentrifugation.

Chimeric Hepatitis B core antigen virus-like particles displaying the envelope domain III of dengue virus type 2.

BACKGROUND: Dengue is a global public health problem for which no drug or vaccine is available. Currently, there is increasing interest in developing non-replicating dengue vaccines based on a discrete antigenic domain of the major structural protein of dengue viruses (DENVs), known as envelope domain III (EDIII). The use of bio-nanoparticles consisting of recombinant viral structural polypeptides, better known as virus-like particles (VLPs), has emerged as a potential platform technology for vaccine development. This work explores the feasibility of developing nanoparticles based on E. coli-expressed recombinant Hepatitis B virus core antigen (HBcAg) designed to display EDIII moiety of DENV on the surface. FINDINGS: We designed a synthetic gene construct encoding HBcAg containing an EDIII insert in its c/e1 loop. The fusion antigen HBcAg-EDIII-2 was expressed in E. coli, purified to near homogeneity using Ni+2 affinity chromatography and demonstrated to assemble into discrete 35-40 nm VLPs by electron microscopy. Competitive ELISA analyses showed that the EDIII-2 moieties of the VLPs are accessible to anti-EDIII-2-specific monoclonal and polyclonal antibodies, suggesting that they are surface-displayed. The VLPs were highly immunogenic eliciting high titer anti-EDIII-2 antibodies that were able to recognize, bind and neutralize infectious DENV based on ELISA, immunofluorescence and virus-neutralization assays. CONCLUSION: This work demonstrates that HBcAg-derived nanoparticles can serve as a useful platform for the display of DENV EDIII. The EDIII-displaying nanoparticles may have potential applications in diagnostics/vaccines for dengue.

Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris.

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0mg per 1g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.

[Occult HBV infection in patients with anti-HBc positive alone].

OBJECTIVE: This study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection. METHODS: Sera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive. RESULTS: DNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples. CONCLUSIONS: Occult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.

Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography.

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).

Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis.

A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.

N-terminally His-tagged hepatitis B core antigens: construction, expression, purification and antigenicity.

The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.