Autologous concentrated growth factor mediated accelerated bone healing in root-end microsurgery: A multicenter randomized clinical trial.

Introduction: Concentrated growth factor (CGF) is a new-generation autologous platelet concentrate that promotes tissue regeneration and has anti-inflammatory properties. This randomized multicenter trial aimed to evaluate the effects of CGF on bone healing in combination with root-end microsurgery. Methods: Healthy adult patients indicated for root-end microsurgery were randomly assigned to either the CGF or control (no CGF implantation) groups. CGF was implanted into the bone cavity after root-end filling with mineral trioxide aggregate. Clinical and periapical radiographic evaluations were conducted at 1, 3, 6, and 12 months postoperatively, with follow-up cone-beam computed tomography (CBCT) at 6 months. The lesion volume reduction rate was calculated based on data from the preoperative and follow-up CBCT images. Results: A total of 24 patients were enrolled. The treatment success rate was 91.7% and 83.3% on 12-month periapical radiography and 6-month CBCT, respectively, without a significant difference between the two groups. The lesion volume reduction rate in the CGF group (75.6%) was significantly higher than that in the control (61.0%) group. Conclusions: Autologous CGF in conjunction with root-end microsurgery accelerated lesion reduction as observed on CBCT. Administering autologous blood products to stimulate healing in addition to removing the source of infection appears to be a promising treatment option for root-end microsurgery.

Irrigation with reduced sodium hypochlorite solution concentration using laser-induced cavitation is effective and safe in rat intraradicular biofilm model.

This study aimed to investigate the optimal sodium hypochlorite solution (NaOCl) concentration to effectively remove the root canal biofilm without stimulating periradicular inflammation using coronal laser-activated irrigation (CLAI). To compare the efficacy of different NaOCl concentrations combined with CLAI in removing the biofilm, an in vivo intraradicular biofilm rat model was used. Root canals were irrigated using an Er:YAG laser with either 5% or 0.5% NaOCl. Biofilm removal efficacy of CLAI was compared to that of conventional needle irrigation using scanning electron microscopy (SEM) and quantitative polymerase chain reaction (qPCR). Histological observation of CLAI-associated periradicular inflammation was also conducted. In both the 5% and 0.5% CLAI groups, SEM observation showed the opening of the dentin tubules and biofilm removal. qPCR analysis indicated that the residual bacteria counts after cleaning were significantly lower in the 5% and 0.5% CLAI groups than in the conventional needle irrigation and positive control groups (Tukey test, p < 0.05), and no significant difference was observed between the 5% and 0.5% CLAI groups (p > 0.05). Periapical inflammation in the 5% CLAI group revealed the most severe, including significant neutrophilic and lymphocytic infiltration with abscess formation, while only mild vasodilation was observed in the 0.5% CLAI group. CLAI can remove the biofilm independently of chemical action, which avoids the risks associated with high NaOCl concentrations. Therefore, this root canal irrigation technique ensures safety and effectiveness, promising to contribute to new treatment strategies intended to remove intraradicular biofilm.

Governance paradox: implications from Japan's national parks for managing complex protected areas.

Herein, we discuss the governance implications for emerging protected areas with complexity in the 2020s by analyzing public-private partnership frameworks in Japan's national parks. First, we summarize previous literature to elucidate the characteristics of Japan's national park management as "weak government" represented by a lack of administrative resources and weak regulatory power. Second, we identify the weak implementation of two legal public-private partnership frameworks from questionnaires and interviews: the Park Management Organization and the Scenic Area Protection Agreement. We discuss the high transaction costs and lack of sufficient benefits to the private sector as the main reasons behind weak implementation. We identify this mismatch as a "governance paradox" and argue that sufficient administrative support and institutional design are indispensable for active partnership implementation.

Development and Characterization of Efficient Cell Culture Systems for Genotype 1 Hepatitis E Virus and Its Infectious cDNA Clone.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition.

Spillover-mediated harvesting competition: Effects of fishing ground configuration on fisheries targeting transboundary species.

Overfishing is the main threat to sustainable fisheries and the loss of marine biodiversity. The race-to-fish phenomenon is a central driver of overfishing, and it prevails from small-scale to large-scale management regardless of whether spatial-right-based fisheries management has been implemented to ensure the ownership of fishery resources. In practice, the fishing grounds of resource users create complex configurations. Systematic understanding of harvesting competition across these configurations is necessary to promote sustainable fisheries management. Here, we developed a spatially-explicit model to analyze various scenarios of harvesting competition between two user groups using a game-theoretic approach. We found that realized harvesting competition was largely determined by the configuration of fishing grounds and an ecological mechanism where the ecological rescue effect could escalate harvesting competition, leading to a low population size. Our results also suggested that the implementation of voluntary no-take marine protected areas could largely mitigate harvesting competition. This suggests that the coordination of user groups is essential to resolve the race-to-fish.

Er:YAG laser-induced cavitation can activate irrigation for the removal of intraradicular biofilm.

We investigated the biofilm removal effects of laser activated irrigation (LAI) using a pig model, focusing on the impact of the fiber tip position, and used a high-speed camera to observe the occurrence and positioning of the cavitation associated with laser irradiation. A total of 16 roots of deciduous mandibular second premolars from 4 pigs were used. After a pulpectomy, the canals were left open for 2 weeks and sealed for 4 weeks to induce intraradicular biofilm. Root canal irrigation was then performed with Er:YAG laser activation. The fiber tip was inserted at two different positions, i.e., into the root canal in the intracanal LAI group and into the pulp chamber in the coronal LAI group. Intracanal needle irrigation with saline or 5% NaOCl was utilized in the positive control and conventional needle irrigation (CNI) groups. SEM and qPCR were carried out to evaluate treatment efficacy. Statistical analysis was performed using ANOVA and a Tukey-Kramer post-hoc test for qPCR and with a Steel-Dwass test to compare the SEM scores, with α = 0.05. A high-speed camera was used to observe the generation of cavitation bubbles and the movement of the induced bubbles after laser irradiation. The intracanal and coronal LAI groups showed significantly lower amounts of bacteria than either the positive control or CNI groups. There was no significant difference found between the intracanal and coronal LAI groups. SEM images revealed opened dentinal tubules with the destruction of biofilm in both LAI groups. High-speed camera images demonstrated cavitation bubble production inside the root canal after a single pulse irradiation pulse. The generated bubbles moved throughout the entire internal multi-rooted tooth space. Coronal LAI can generate cavitation in the root canal with a simply placed fiber inside the pulp chamber, leading to effective biofilm removal. This method could thus contribute to the future development of endodontic treatments for refractory apical periodontitis caused by intraradicular biofilm.

An experimental intraradicular biofilm model in the pig for evaluating irrigation techniques.

BACKGROUND: We established an in vivo intraradicular biofilm model of apical periodontitis in pigs in which we compared the efficacy of different irrigant activation techniques for biofilm removal. METHODS: Twenty roots from the deciduous mandibular second premolar of 5 male pigs were used. After pulpectomy, canals were left open for 2 weeks and then sealed for 4 weeks to enable the development of an intracanal biofilm. The intraradicular biofilms was evaluated using SEM and bacterial 16S rRNA gene-sequencing. To investigate the efficacy of biofilm removal, root canal irrigations were performed using conventional needle, passive ultrasonic, subsonic, or laser-activated irrigation. Real-time PCR was conducted to quantitate the remaining biofilm components. Statistical analysis was performed using ANOVA followed by a Tukey kramer post-hoc test with α = 0.05. RESULTS: The pulp exposure model was effective in inducing apical periodontitis and SEM analysis revealed a multi-layer biofilm formation inside the root canal. 16S rRNA sequence analysis identified Firmicutes, Bacteroidetes, and Fusobacteria as the predominant bacterial phyla components, which is similar to the microbiome profile seen in humans. None of the tested irrigation techniques completely eradicated the biofilm components from the root canal, but the subsonic and laser-activated irrigation methods produced the lowest bacterial counts (p < 0.05). CONCLUSIONS: An experimental intraradicular biofilm model has been successfully established in pigs. Within the limitations of the study, subsonic or laser-activated irrigation demonstrated the best biofilm removal results in the pig system.

Analysis of the Governance Structures in Japan's Biosphere Reserves: Perspectives from Bottom-Up and Multilevel Characteristics.

This paper analyzes the governance structures of Biosphere Reserves (BRs) in Japan by focusing on six criteria that elucidate the main characteristics therein: general information (nomination process, year of designation, size, and population), legal frameworks, stakeholder identification, and decision-making processes (number of municipalities and role of consociation), administrative institutions (human resources, budgetary situation, and expense distribution), executed BR implementation activities, and participatory/collaborative frameworks. This research consists of a literature review, a questionnaire administered to the secretariats of seven existing BRs and follow-up interviews. Three main characteristics of BRs were identified. First, a responsible local government(s) is nominated to manage the BR rather than the central government. Consequently, BR implementation in Japan is led by those municipalities that have strong motivations for regional development using the BR concept. Second, two types of BR governance structures exist in Japan: the single municipality type and the multi-municipality type. All BRs have so called Kyougikai, a consociation for decision-making, consultation and/or collaboration among stakeholders. In the single municipality structure, the consociation includes diverse actors from private and community sectors, while in the multi-municipality structure, consociations are based in more diplomatic settings and only include members of the public sector. Third, gaps between pre/post-Seville BR implementation sites were identified. The motivations for the formation of pre-Seville BRs, which were designated in 1980 in a top-down fashion prior to an awareness of BRs, varied greatly from those BRs nominated by municipalities after 2010. The authors identified fewer administrative resources and activities associated with the pre-Seville sites.

Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone.

To characterize the genomic mutations of hepatitis E virus (HEV) during consecutive passages associated with adaptation to growth in cell culture, a cloned genotype 3 HEV [pJE03-1760F/wt, starting virus (SV)] was passaged 10 times in A549 cells, and the entire genomic sequence of the passage 10 (P10) progeny was determined. Compared to SV, P10 virus possessed two non-synonymous (T2808C and A5054G) and four synonymous mutations (C1213T, T2557C, C3118T and C4435T) in the ORF1. Full-length infectious cDNA clones with a single, double (T2808C and A5054G), or all six mutations, identical to P10, were constructed, and their replication capacity was compared. Four (C1213T, T2557C, T2808C and A5054G) of the six viruses with a single mutation grew more efficiently than SV. The P10 virus propagated more rapidly and grew more efficiently than SV and T2808C+A5054G and reached a higher viral load (95.1- and 8.5-fold, respectively) at 20days post-inoculation. An immunofluorescence analysis revealed that a high percentage (>80%) of cells inoculated with the P10 virus expressed ORF2 proteins, while relatively low percentages (nearly 30% or 5%) inoculated with T2808C+A5054G or SV, respectively, expressed ORF2 proteins. We found that not only non-synonymous but also synonymous HEV mutations are independently associated with increased virus production.

Sulfurized limonite as material for fast decomposition of organic compounds by heterogeneous Fenton reaction.

Rapid decomposition of wastewater contaminants using sulfurized limonite (S-limonite) was investigated. Limonite is used for desulfurization of biogases, and S-limonite is obtained from desulfurization plants as solid waste. In this work, the profitable use of S-limonite in water treatment was examined. The divalent Fe in S-limonite was expected to produce OH radicals, as Fe(2+) ions and limonite thermally treated with H2 do. Methylene blue was used for batch-wise monitoring of the decomposition performance. The decomposition rate was fast and the methylene blue solution color disappeared in only 10s when a small amount of H2O2 was added (1mM in the sample solution) in the presence of S-limonite. The OH radicals were formed by a heterogeneous reaction on the S-limonite surface and Fenton reaction with dissolved Fe(2+). The decomposition of pentachlorophenol was also examined; it was successfully decomposed in batch-wise tests. The surfaces of limonite before sulfurization, S-limonite, and S-limonite after use for water treatment were performed using scanning electron microscopy and X-ray photoelectron spectroscopy. The results show that S-limonite reverted to limonite after being used for water treatment.

Flow-based ammonia gas analyzer with an open channel scrubber for indoor environments.

A robust and fully automated indoor ammonia gas monitoring system with an open channel scrubber (OCS) was developed. The sample gas channel dimensions, hydrophilic surface treatment to produce a thin absorbing solution layer, and solution flow rate of the OCS were optimized to connect the OCS as in-line gas collector and avoid sample humidity effects. The OCS effluent containing absorbed ammonia in sample gas was injected into a derivatization solution flow. Derivatization was achieved with o-phthalaldehyde and sulfite in pH 11 buffer solution. The product, 1-sulfonateisoindole, is detected with a home-made fluorescence detector. The limit of detection of the analyzer based on three times the standard deviation of baseline noise was 0.9 ppbv. Sample gas could be analyzed 40 times per hour. Furthermore, relative humidity of up to 90% did not interfere considerably with the analyzer. Interference from amines was not observed. The developed gas analysis system was calibrated using a solution-based method. The system was used to analyze ammonia in an indoor environment along with an off-site method, traditional impinger gas collection followed by ion chromatographic analysis, for comparison. The results obtained using both methods agreed well. Therefore, the developed system can perform on-site monitoring of ammonia in indoor environments with improved time resolution compared with that of other methods.

A549 and PLC/PRF/5 cells can support the efficient propagation of swine and wild boar hepatitis E virus (HEV) strains: demonstration of HEV infectivity of porcine liver sold as food.

Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs and wild boars to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. However, no efficient cell culture system for swine and boar HEV strains has been established. We inoculated A549 cells with 12 swine and boar HEV strains of liver, feces, or serum origin at an HEV load of ≥2.0 × 10(4) copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of ~10(8) copies/ml. However, the HEV load in the culture medium at 30 days post-inoculation differed markedly by inoculum, ranging from 1.0 × 10(2) to 1.1 × 10(7) copies/ml upon inoculation at a lower load of approximately 10(5) copies per well. All progeny were passaged successfully onto A549 and PLC/PRF/5 cells. In sharp contrast, no progeny viruses were detectable in the culture supernatant upon inoculation with 13 swine and boar HEV strains at an HEV load of <1.8 × 10(4) copies per well. The present study also demonstrates that swine liver sold as food can be infectious, supporting the risk of zoonotic food-borne HEV infection.

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.

Cells isolated from inflamed periapical tissue express mesenchymal stem cell markers and are highly osteogenic.

INTRODUCTION: We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues. METHODS: Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model. RESULTS: Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice. CONCLUSIONS: Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.

A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells.

We have previously demonstrated that the release of hepatitis E virus (HEV) from infected cells depended on ORF3 protein, which harbours one or two PSAP motifs. To elucidate the PSAP motif(s) in the ORF3 protein during virion egress, five PSAP mutants derived from an infectious genotype 3 cDNA clone of pJE03-1760F/wt that can grow efficiently in PLC/PRF/5 cells were analysed. Four mutants, including mutLSAP, mutPSAL, mutLSAL (the substituted amino acids in the authentic PSAP motif are underlined) and mutPLAP/PSAP (the changed amino acid in the additional PSAP motif is underlined) generated progenies as efficiently as the wild-type virus. Conversely, the HEV RNA level in the culture supernatant of mutPLAP/LSAL RNA-transfected cells was significantly lower than in cells transfected with the wild-type RNA, similar to an ORF3-null mutant. Consistent with the ORF3-deficient mutant, the mutPLAP/LSAL mutant with no intact PSAP motifs banded at 1.26-1.27 g ml(-1) in sucrose, and was captured by anti-ORF2, but not by anti-ORF3, with or without prior treatment with detergent (0.1 % sodium deoxycholate). The absence of the ORF3 protein on the mutant particles in the culture supernatant was confirmed by Western blotting, despite the expression of ORF3 protein in the RNA-transfected cells, as detected by immunofluorescence and Western blotting. Therefore, at least one of the two intact PSAP motifs in the ORF3 protein is required for the formation of membrane-associated HEV particles possessing ORF3 proteins on their surface, thus suggesting that the PSAP motif plays a role as a functional domain for HEV budding.

Miniature open channel scrubbers for gas collection.

An open channel scrubber is proposed as a miniature fieldable gas collector. The device is 100mm in length, 26 mm in width and 22 mm in thickness. The channel bottom is rendered hydrophilic and liquid flows as a thin layer on the bottom. Air sample flows atop the appropriately chosen flowing liquid film and analyte molecules are absorbed into the liquid. There is no membrane at the air-liquid interface: they contact directly each other. Analyte species collected over a 10 min interval are determined by fluorometric flow analysis or ion chromatography. A calculation algorithm was developed to estimate the collection efficiency a priori; experimental and simulated results agreed well. The characteristics of the open channel scrubber are discussed in this paper from both theoretical and experimental points of view. In addition to superior collection efficiencies at relatively high sample air flow rates, this geometry is particularly attractive that there is no change in collection performance due to membrane fouling. We demonstrate field use for analysis of ambient SO(2) near an active volcano. This is basic investigation of membraneless miniature scrubber and is expected to lead development of an excellent micro-gas analysis system integrated with a detector for continuous measurements.

Hepatitis E Virus (HEV) strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation.

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 and A549 cells, using fecal specimens from HEV-infected patients. Since transfusion-associated hepatitis E has been reported, we examined PLC/PRF/5 and A549 cells for the ability to support replication of HEV in various serum samples obtained from 23 patients with genotype 1, 3, or 4 HEV. HEV progenies emerged in culture media of PLC/PRF/5 cells, regardless of the coexistence of HEV antibodies in serum but dependent on the load of HEV inoculated (31% at 2.0 x 10(4) copies per well and 100% at >or=3.5 x 10(4) copies per well), and were successfully passaged in A549 cells. HEV particles in serum, with or without HEV antibodies, banded at a sucrose density of 1.15 to 1.16 g/ml, which was markedly lower than that for HEV particles in feces, at 1.27 to 1.28 g/ml, and were nonneutralizable by immune sera in this cell culture system. An immuno-capture PCR assay of HEV virions treated with or without detergent indicated that HEV particles in serum are associated with lipids and HEV ORF3 protein, similar to those in culture supernatant. By immunoprecipitation, it was found that >90% of HEV particles in the circulation exist as free virions not complexed with immunoglobulins, despite the coexistence of HEV antibodies. These results suggest that our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.

Determination of the 5'-terminal sequence of subgenomic RNA of hepatitis E virus strains in cultured cells.

Using RNA preparations extracted from PLC/PRF/5 cells transfected with infectious genotype 3 hepatitis E virus (HEV) cDNA clones or inoculated with a fecal suspension containing a genotype 4 HEV, the 5'-terminal sequence of a 2.2-kb subgenomic RNA of genotype 3 and 4 HEVs was determined. Despite an insertion of T after nucleotide 5116 or an ORF3-null mutation in genotype 4 HEV and/or one of the genotype 3 variants, it was found that the subgenomic RNA of genotype 3 and 4 HEVs initiates exclusively at nucleotide 5122 with the common sequence of 5'-GC, which is identical to that of the prototype genotype 1 HEV.

ORF3 protein of hepatitis E virus is essential for virion release from infected cells.

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (DeltaORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The DeltaORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of DeltaORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of DeltaORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the DeltaORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15-1.16 g ml(-1), in contrast with the DeltaORF3 mutant in culture supernatant, which banded at 1.27-1.28 g ml(-1), similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.

Development and characterization of a genotype 4 hepatitis E virus cell culture system using a HE-JF5/15F strain recovered from a fulminant hepatitis patient.

We developed an efficient cell culture system for genotype 4 hepatitis E virus using the HE-JF5/15F strain recovered from a fulminant hepatitis patient. The sixth-passage virus in the culture supernatant reached 1.5 x 10(8) copies/ml at 10 days postinoculation and possessed 10 nucleotide mutations with four amino acid changes.