The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC₅₀ values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

The current antiviral agents for the treatment of chronic infection with hepatitis B virus (HBV) do not completely remove covalently closed circular DNA (cccDNA) and integrated viral DNA fragments from patients. Berberine is an isoquinoline alkaloid extracted from various plants and has been reported to inhibit the replication of various types of DNA. In this study, we tested the effects of berberine and its derivatives on HBV infection. Berberine inhibited viral core promoter activity at the highest level among the compounds tested and suppressed HBV production and cccDNA synthesis in primary human hepatocytes and HBV-infected HepG2-NTCP cells at an EC50 value of 3.6 mu M and a CC50 value of over 240.0 mu M. Compared with other viral promoter activities, berberine treatment potently downregulated core promoter activity and reduced protein levels, but not RNA levels, of hepatic nuclear factor 4 alpha (HNF4 alpha), which primarily enhances enhancer II/core promoter activity. Furthermore, berberine treatment enhanced K48-linked, but not K63-linked, polyubiquitination and subsequent proteasomedependent degradation of HNF4 alpha. These results suggest that berberine enhances the polyubiquitination- and proteasome-dependent degradation of HNF4 alpha and then inhibits HBV replication via the suppression of core promoter activity. The development of antiviral agents based on berberine may contribute to the amelioration of HBV-related disorders, regardless of the presence of residual cccDNA or integrated viral DNA fragments.

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.

Approximately 58 million people worldwide are believed to be infected with hepatitis C virus (HCV), a major causative agent of chronic liver diseases. Hepacivirus ratti strain rn-1, which was discovered from Rattus norvegicus (Norway rat) and designated Norway rat hepacivirus 1 (NRHV1), shares similar properties with HCV in terms of genetic homology, target cell tropism, pathogenicity, and the immune response. In vivo infection systems for NRHV1 will help overcome the challenges in HCV research for vaccine development. However, the virological characteristics of NRHV1, such as the mechanisms of cell entry, remain largely unexplored, in part owing to a paucity of cell culture systems for NRHV1. Here, we identified the host factors that facilitate NRHV1 entry by profiling the gene expression of two cell lines with different susceptibilities to NRHV1 infection. NRHV1 employs rodent orthologues of HCV entry factors, including scavenger receptor class BI, CD81, and occludin, and utilizes claudin-3 (CLDN3) but not claudin-1. The expression of rat and mouse, but not human, CLDN3 facilitates the entry of NRHV1 into murine cell lines that are nonsusceptible to NRHV1 infection. The host-specific cell entry of CLDN3 is determined by two amino acid residues, Ile ⁴⁴ and Trp ⁴⁶ , in extracellular loop 1. These findings suggest that CLDN3 serves as an entry factor for rat hepacivirus.

In manufacturing industry, CAM (Computer aided manufacturing) software is utilized to generate to tool paths for the CNC (Computer numerical controlled) machine tools. However, there are still many parameters requiring human decision. Especially in free form surface machining field, the diversity and complexity of the surface make it challenging to achieve both the surface quality and machining efficiency. In the proposed method the freeform surfaces are subdivided into different groups of patches according to the surface normal vector value. The neighboring areas on the freeform surface that approximately have the same normal vectors are clustered into one group of patch and tool path is generated on each patch. This method is implemented on a freeform surface model. The result of the proposed method is compared with the traditional tool path generation method and evaluated by the machining time.