Masitinib Inhibits Hepatitis A Virus Replication.

The hepatitis A virus (HAV) infection causes acute hepatitis. HAV also induces acute liver failure or acute-on-chronic liver failure; however, no potent anti-HAV drugs are currently available in clinical situations. For anti-HAV drug screening, more convenient and useful models that mimic HAV replication are needed. In the present study, we established HuhT7-HAV/Luc cells, which are HuhT7 cells stably expressing the HAV HM175-18f genotype IB subgenomic replicon RNA harboring the firefly luciferase gene. This system was made by using a PiggyBac-based gene transfer system that introduces nonviral transposon DNA into mammalian cells. Then, we investigated whether 1134 US Food and Drug Administration (FDA)-approved drugs exhibited in vitro anti-HAV activity. We further demonstrated that treatment with tyrosine kinase inhibitor masitinib significantly reduced both HAV HM175-18f genotype IB replication and HAV HA11-1299 genotype IIIA replication. Masitinib also significantly inhibited HAV HM175 internal ribosomal entry-site (IRES) activity. In conclusion, HuhT7-HAV/Luc cells are adequate for anti-HAV drug screening, and masitinib may be useful for the treatment of severe HAV infection.

Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication.

Hepatitis A virus (HAV) infection often causes acute hepatitis, which results in a case fatality rate of 0.2% and fulminant hepatitis in 0.5% of cases. However, no specific potent anti-HAV drug is available on the market to date. In the present study, we focused on inhibition of HAV internal ribosomal entry site (IRES)-mediated translation and investigated novel therapeutic drugs through drug repurposing by screening for inhibitors of HAV IRES-mediated translation and cell viability using a reporter assay and cell viability assay, respectively. The initial screening of 1,158 drugs resulted in 77 candidate drugs. Among them, nicotinamide significantly inhibited HAV HA11-1299 genotype IIIA replication in Huh7 cells. This promising drug also inhibited HAV HM175 genotype IB subgenomic replicon and HAV HA11-1299 genotype IIIA replication in a dose-dependent manner. In the present study, we found that nicotinamide inhibited the activation of activator protein 1 (AP-1) and that knockdown of c-Jun, which is one of the components of AP-1, inhibited HAV HM175 genotype IB IRES-mediated translation and HAV HA11-1299 genotype IIIA and HAV HM175 genotype IB replication. Taken together, the results showed that nicotinamide inhibited c-Jun, resulting in the suppression of HAV IRES-mediated translation and HAV replication, and therefore, it could be useful for the treatment of HAV infection. IMPORTANCE Drug screening methods targeting HAV IRES-mediated translation with reporter assays are attractive and useful for drug repurposing. Nicotinamide (vitamin B3, niacin) has been shown to effectively inhibit HAV replication. Transcription complex activator protein 1 (AP-1) plays an important role in the transcriptional regulation of cellular immunity or viral replication. The results of this study provide evidence that AP-1 is involved in HAV replication and plays a role in the HAV life cycle. In addition, nicotinamide was shown to suppress HAV replication partly by inhibiting AP-1 activity and HAV IRES-mediated translation. Nicotinamide may be useful for the control of acute HAV infection by inhibiting cellular AP-1 activity during HAV infection processes.

Amantadine and Rimantadine Inhibit Hepatitis A Virus Replication through the Induction of Autophagy.

Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. Furthermore, HAV causes acute liver failure or acute-on-chronic liver failure. However, no potent anti-HAV drugs are currently available in the clinical situations. There have been some reports that amantadine, a broad-spectrum antiviral, suppresses HAV replication in vitro. Therefore, we examined the effects of amantadine and rimantadine, derivates of adamantane, on HAV replication, and investigated the mechanisms of these drugs. In the present study, we evaluated the effects of amantadine and rimantadine on HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in cell culture infection systems. Amantadine and rimantadine significantly inhibited HAV replication at the post-entry stage in Huh7 cells. HAV infection inhibited autophagy by suppressing the autophagy marker light chain 3 and reducing number of lysosomes. Proteomic analysis on HAV-infected Huh7 cells treated by amantadine and rimantadine revealed the changes of the expression levels in 42 of 373 immune response-related proteins. Amantadine and rimantadine inhibited HAV replication, partially through the enhancement of autophagy. Taken together, our results suggest a novel mechanism by which HAV replicates along with the inhibition of autophagy and that amantadine and rimantadine inhibit HAV replication by enhancing autophagy. IMPORTANCE Amantadine, a nonspecific antiviral medication, also effectively inhibits HAV replication. Autophagy is an important cellular mechanism in several virus-host cell interactions. The results of this study provide evidence indicating that autophagy is involved in HAV replication and plays a role in the HAV life cycle. In addition, amantadine and its derivative rimantadine suppress HAV replication partly by enhancing autophagy at the post-entry phase of HAV infection in human hepatocytes. Amantadine may be useful for the control of acute HAV infection by inhibiting cellular autophagy pathways during HAV infection processes.

Favipiravir Inhibits Hepatitis A Virus Infection in Human Hepatocytes.

Hepatitis A virus (HAV) is a causative agent of acute hepatitis and can occasionally induce acute liver failure. However, specific potent anti-HAV drug is not available on the market currently. Thus, we investigated several novel therapeutic drugs through a drug repositioning approach, targeting ribonucleic acid (RNA)-dependent RNA polymerase and RNA-dependent deoxyribonucleic acid polymerase. In the present study, we examined the anti-HAV activity of 18 drugs by measuring the HAV subgenomic replicon and HAV HA11-1299 genotype IIIA replication in human hepatoma cell lines, using a reporter assay and real-time reverse transcription polymerase chain reaction, respectively. Mutagenesis of the HAV 5' untranslated region was also examined by next-generation sequencing. These specific parameters were explored because lethal mutagenesis has emerged as a novel potential therapeutic approach to treat RNA virus infections. Favipiravir inhibited HAV replication in both Huh7 and PLC/PRF/5 cells, although ribavirin inhibited HAV replication in only Huh7 cells. Next-generation sequencing demonstrated that favipiravir could introduce nucleotide mutations into the HAV genome more than ribavirin. In conclusion, favipiravir could introduce nucleotide mutations into the HAV genome and work as an antiviral against HAV infection. Provided that further in vivo experiments confirm its efficacy, favipiravir would be useful for the treatment of severe HAV infection.

Involvement of the Interferon Signaling Pathways in Pancreatic Cancer Cells.

BACKGROUND/AIM: To examine interferon (IFN) signaling pathways in human pancreatic cancer cells and their therapeutic application for pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We examined the effects of IFNα on cytotoxicity, migration, as well as on the levels of toll-like receptor (TLR) signaling pathway-associated genes expression in pancreatic cancer cells. We also examined the additive effects of IFNα and poly(I-C) on tyrosine kinase inhibitor (TKI)-induced cytotoxicity. We performed transcriptome analysis (RNA-Seq) of clinical samples and compared the profile between pancreatic intraepithelial neoplasias (PanINs) and PDACs. RESULTS: IFNα suppressed cell viability and cell migration, and affected TLR signaling pathways, in pancreatic cancer cells. TLR3 is one of the potential genes involved in IFN-treated pancreatic cancer cells. Furthermore, similar to IFN, extracellular addition of poly(I-C) enhanced TKI-induced cytotoxicity in pancreatic cancer cells. RNA-Seq analysis demonstrated that IFN signaling is one of the potential pathways involved in the progression of PanIN to PDAC. CONCLUSION: IFN signaling may be involved in the development of PDAC. Treatments that target the IFN and TLR3 signaling pathways may be therapeutic options against PDAC.

Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 Cells by HBV Sequence Capture-Based Next-Generation Sequencing.

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC) worldwide. The integration of HBV genomic DNA into the host genome occurs randomly, early after infection, and is associated with hepatocarcinogenesis in HBV-infected patients. Therefore, it is important to analyze HBV genome integration. We analyzed HBV genome integration in human hepatoma PLC/PRF/5 cells by HBV sequence capture-based next-generation sequencing (NGS) methods. We confirmed the results by using Sanger sequencing methods. We observed that HBV genotype A is integrated into the genome of PLC/PRF/5 cells. HBV sequence capture-based NGS is useful for the analysis of HBV genome integrants and their locations in the human genome. Among the HBV genome integrants, we performed functional analysis and demonstrated the automatic expression of some HBV proteins encoded by HBV integrants from chromosomes 3 and 11 in Huh7 cells transfected with these DNA sequences. HBV sequence capture-based NGS may be a useful tool for the assessment of HBV genome integration into the human genome in clinical samples and suggests new strategies for hepatocarcinogenesis in HBV infection.

Follow-up Results of HCV GT2 Patients After Sofosbuvir/Ribavirin Therapy: Careful Attention to Occurrence of HCC.

BACKGROUND: We examined treatment the efficacy and data on long-term outcomes in real-world Japanese patients infected with hepatitis C virus (HCV) genotype 2 treated with 12-week sofosbuvir/ribavirin combination therapy. PATIENTS AND METHODS: In a total of 86 patients who were treated with sofosbuvir/ribavirin, sustained virological response (SVR) rates and long-term-outcomes were retrospectively analyzed. RESULTS: The adherence to this combination therapy was 98.8%. The rates of SVR at week 24 (SVR24) achieved with this treatment according to the 'intention-to-treat' and 'per-protocol' analyses were 89.5% and 96.2%, respectively. Two patients who experienced relapse did not have any previously reported resistance-associated substitutions in the HCV non-structural protein 5B (NS5B) polymerase region. We did not observe any patients who experienced late relapse but did observe that 50% and 1.3% of patients with and without a previous history of hepatocellular carcinoma (HCC), respectively, developed HCC after achieving SVR24 (with a mean follow-up period of 2.7±0.8 years). CONCLUSION: Patients with SVR should be carefully followed-up to screen for the occurrence of HCC, although it is infrequent.

Overexpression of a virus-like particle influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion.

Ecological investigations of silkworms have revealed that Eri silkworms (Samia cynthia ricini) possess useful morphological and ecological characteristics for virus-like particle (VLP) production, namely non-seasonal breeding, longer lengths, and heavier weights than Bombyx mori silkworms. Furthermore, when vector DNA from Bombyx mori nuclear polyhedrosis virus (BmNPV), which is unable to replicate in Sf9 cells from Eri silkworms, was replaced with the Autographa californica nuclear polyhedrosis virus (AcNPV) vector, three improved AcNPV influenza virus recombinants capable of replication in Sf9 cells were obtained. Although VLP antigens produced previously in silkworms were not evaluated individually, the present recombinant Fukushima (FkH5) and Anhui (AnH7) VLP antigens were detected in tissue fluids and fat bodies of Eri silkworms. Here, we aimed to determine the function of the AcNPV vector and P143 gene by expressing recombinants in Sf9 cells and eri silkworm pupae. The FkH5 recombinant produced high yields of haemagglutinin (HA)-positive VLPs, showing a mean HA titre of 1.2 million. Similarly, high production of H7 HA VLPs was observed in the fat bodies of eri silkworm pupae. Antigenic analysis and electron microscopy examination of Eri-silkworm-produced H5 HA VLPs showed characteristic antigenicity and morphology similar to those of the influenza virus. Although FkH5 recombinants possessing the AcNPV vector did not replicate in Bm-N cells, the introduction of the helicase p143 gene from BmNPV resulted in their production in Bm-N and Sf9 cells.

Apoptosis and non-alcoholic fatty liver diseases.

The number of patients with nonalcoholic fatty liver diseases (NAFLD) including nonalcoholic steatohepatitis (NASH), has been increasing. NASH causes cirrhosis and hepatocellular carcinoma (HCC) and is one of the most serious health problems in the world. The mechanism through which NASH progresses is still largely unknown. Activation of caspases, Bcl-2 family proteins, and c-Jun N-terminal kinase-induced hepatocyte apoptosis plays a role in the activation of NAFLD/NASH. Apoptotic hepatocytes stimulate immune cells and hepatic stellate cells toward the progression of fibrosis in the liver through the production of inflammasomes and cytokines. Abnormalities in glucose and lipid metabolism as well as microbiota accelerate these processes. The production of reactive oxygen species, oxidative stress, and endoplasmic reticulum stress is also involved. Cell death, including apoptosis, seems very important in the progression of NAFLD and NASH. Recently, inhibitors of apoptosis have been developed as drugs for the treatment of NASH and may prevent cirrhosis and HCC. Increased hepatocyte apoptosis may distinguish NASH from NAFLD, and the improvement of apoptosis could play a role in controlling the development of NASH. In this review, the association between apoptosis and NAFLD/NASH are discussed. This review could provide their knowledge, which plays a role in seeing the patients with NAFLD/NASH in daily clinical practice.

Functional growth inhibition of influenza A and B viruses by liquid and powder components of leaves from the subtropical plant Melia azedarach L.

We evaluated the anti-influenza-virus effects of Melia components and discuss the utility of these components. The effects of leaf components of Melia azedarach L. on viruses were examined, and plaque inhibition tests were performed. The in vivo efficacy of M. azedarach L. was tested in a mouse model. Leaf components of Melia azedarach L. markedly inhibited the growth of various influenza viruses. In an initial screening, multiplication and haemagglutination (HA) activities of H1N1, H3N2, H5, and B influenza viruses were inactivated by the liquid extract of leaves of M. azedarach L. (MLE). Furthermore, plaque inhibition titres of H1N1, H3N2, and B influenza viruses treated with MLE ranged from 103.7 to 104.2. MLE possessed high plaque-inhibitory activity against pandemic avian H5N1, H7N9, and H9N2 vaccine candidate strains, with a plaque inhibition titre of more than 104.2. Notably, the buoyant density decreased from 1.175 to 1.137 g/cm3, and spikeless particles appeared. We identified four anti-influenza virus substances: pheophorbide b, pheophorbide a, pyropheophorbide a, and pheophytin a. Photomorphogenesis inside the envelope may lead to removal of HA and neuraminidase spikes from viruses. Thus, MLE could efficiently remove floating influenza virus in the air space without toxicity. Consistent with this finding, intranasal administration of MLE in mice significantly decreased the occurrence of pneumonia. Additionally, leaf powder of Melia (MLP) inactivated influenza viruses and viruses in the intestines of chickens. MLE and MLP may have applications as novel, safe biological disinfectants for use in humans and poultry.

Addition of an EGFP-tag to the N-terminal of influenza virus M1 protein impairs its ability to accumulate in ND10.

A previous report demonstrated that influenza virus infection induces accumulation of EGFP-tagged M1 protein (EGFP-M1) in the sub-nuclear domain ND10. Here, we show that the transfection of four viral protein (NP, PB2, PB1, PA) expression vectors and eight RNA segment expression vectors induced the formation of nuclear dots of EGFP-M1 as seen in virus infections. Omission of the segment 7 RNA expression vector, however, abolished the nuclear dots of EGFP-M1. This result suggests an essential role for authentic M1 protein and/or M2 protein, both of which are encoded in segment 7, in the formation of nuclear dots of EGFP-M1. Co-expression of M1 protein but not M2 protein with EGFP-M1 induced the formation of nuclear dots of EGFP-M1. The dots co-localized with PML protein, which is an indicator of ND10. When only M1 protein was expressed, immunostaining of M1 protein clearly revealed the nuclear dots and their colocalization with PML protein. These results demonstrate that the accumulation in ND10 is an intrinsic characteristic of M1 protein and EGFP addition abolishes this characteristic. The addition of EGFP to M1 protein induced a defect in M1 protein.

The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae.

We successfully established a mass production system for an influenza virus-like particle (VLP) vaccine using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation, the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million hemagglutination units (HAU), approximately 2,000 µg HA protein per pupa, more than 50-fold higher than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameter and covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic influenza HA spike. Detailed electron micrographs indicated that the VLP spike density was similar to that of authentic influenza virus particles. The results clearly show that the expression of a single HA gene can efficiently produce VLPs in silkworm pupae. When chickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in their sera reached values of 2,048-8,192 after approximately 1 month. This is the first report demonstrating that a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae. Our system might be useful for future vaccine development against other viral diseases.

H1N1/09 influenza A virus infection of immortalized first trimester human trophoblast cell lines.

PROBLEM: Although high maternal mortality was reported in the most recent pandemic of swine-origin influenza A H1N1/09 (H1N1/09), its direct effects on the feto-placental unit are unknown. In the present study, we examined the susceptibility of immortalized human trophoblasts to clinical isolates of pandemic 2009 H1N1 influenza A (H1N1/09) virus. METHOD OF STUDY: The H1N1/09 virus was isolated from a patient with influenza, sequenced and identified as the A/Narita/2009 (H1N1) strain. The trophoblast cell lines Swan71 and HTR8 were challenged with the virus and examined for the expression of H1N1/09 viral RNA and proteins. RESULTS: Viral RNA and proteins were observed 24 hr after inoculation. However, viral release was not detected. CONCLUSION: First trimester human trophoblast cell lines were susceptible to the H1N1/09 influenza A virus. However, viral release and cytopathic effects were minimal. Our data suggest that placental damage by the H1N1/09 virus may be limited.

Pleomorphic adenoma originating from the trachea showing the appearance of a follicular tumor of the thyroid on ultrasonography.

Pleomorphic adenoma (PA) is the most common tumor of the salivary gland. This report presents a case of a PA originating from the trachea that looked like a thyroid neoplasm on ultrasonography, showing a well-circumscribed, hypovascular, solid, and hypoechoic tumor within the thyroid. The tumor was resected with the right lobe of the thyroid and the first tracheal ring, which revealed a PA impacted within the thyroid. PAs originating outside of the salivary glands are rare, and there have been no reports of PAs arising from the lateral side of the trachea. This report describes the first reported, and unique, case of this type of tumor.

H3N2 influenza A virus replicates in immortalized human first trimester trophoblast cell lines and induces their rapid apoptosis.

PROBLEM: Epidemiological data suggested that pandemic influenza increased the risks of spontaneous abortion and premature labor, while seasonal influenza also increased the risk of schizophrenia in adolescence. However, their pathogenesis is so far unknown. METHOD OF STUDY: The first trimester trophoblast cell lines, namely, Swan71 and HTR8 cells were challenged with A/Udorn/72 influenza virus (H3N2). At indicated time points, cells were examined for expression of influenza proteins. Viral replication in culture media, apoptosis and the expression of human leukocyte antigen (HLA)-G were also examined. RESULTS: Intracellular localization of viral proteins was observed. Twenty-four hours after inoculation, virus was detected in culture media while most cells fell into apoptosis. During apoptosis, expression of HLA-G was unchanged. CONCLUSION: We revealed replication of low pathogenic influenza virus in the first trimester trophoblast cell lines. Placental damages are likely to be induced by direct cytopathic effects of influenza virus and subsequent apoptosis rather than down regulation of HLA-G expression and subsequent rejection by maternal immune system.

Immunofluorescence imaging of the influenza virus M1 protein is dependent on the fixation method.

The distribution of the matrix (M1) protein of influenza virus in infected cells was examined using immunostaining. The fixation method influenced strongly the immunofluorescence pattern of the M1 protein. The M1 protein was distributed uniformly in both the cytoplasm and in nuclei when cells that had been infected with virus were fixed with paraformaldehyde. In cells that had been fixed with methanol, however, nuclear dots of the M1 protein were clearly visible. The dots were evident at 8h post-inoculation. Up to 6h post-inoculation, only a diffuse distribution of the M1 protein was observed. The dots were co-localized with promyelocytic leukemia (PML) protein, a major component of nuclear domain 10 (ND10), also called PML oncogenic domains (PODs) or PML-nuclear bodies (NBs). These results indicate that the nuclear dots of the M1 protein in cells that had been fixed with methanol are not artifacts of the fixation method. Furthermore, methanol fixation is preferred for localization of the influenza M1 protein in nuclei using immunostaining.

Anti-influenza virus activity of Chaenomeles sinensis.

AIM OF THE STUDY: This investigation evaluated anti-influenza virus activity of 50% ethanol extract of the fruit of Chaenomeles sinensis K(OEHNE), which is widely used as a traditional Chinese medicine to treat throat diseases. MATERIAL AND METHODS: Type A and B influenza viruses were treated with the extract at various concentrations for 1h at room temperature; then the plaque titers of the treated viruses were determined. The neutralizing component in the extract was partially purified using HP20 column chromatography. RESULTS: Treatment with the extract at concentrations greater than 5mg/ml reduced the plaque titers of the both viruses to less than 10% of those of untreated viruses. The treatment inhibited viral hemagglutination activity, too. When the 50mg/ml extract was added to the culture medium after inoculation of the virus, viral NS2 protein synthesis was selectively inhibited and progeny virus was not detected in the infected cell medium. Partial purification showed that the neutralizing component consisted of high molecular weight polyphenols. CONCLUSION: High molecular weight polyphenols in the fruits of C. sinensis neutralizes influenza virus by inhibiting hemagglutination activity and by suppressing NS2 protein synthesis.

A case of malignant eccrine spiradenoma metastatic to intramammary lymph node.

Eccrine spiradenoma (ES) is a fairly common, benign, cutaneous tumor originating from the sweat glands. In contrast, the malignant counterpart of ES, malignant eccrine spiradenoma (MES), is extremely rare. A long-standing lesion rarely begins to enlarge rapidly. A growth that results in ulceration or discoloration may be associated with malignant transformation. We present the first reported case of this tumor metastasizing to an intramammary lymph node (IMLN). The uncommon metastasizing focus of the periumbilical MES and its histopathological similarity with a primary breast carcinoma made the diagnosis difficult.

Prognostic significance of gastric cancer metastasis in second-tier lymph nodes detected on reverse transcriptase-polymerase chain reaction and immunohistochemistry.

To determine the prognostic significance of the methods used to determine the presence of metastasis in second-tier lymph nodes of patients with gastric cancer, the authors studied lymph nodes surgically removed from 100 patients with gastric cancer (55 with early cancer, 45 with progressive). The results of HE staining were compared with those of immunohistochemistry using the anticytokeratin (CK) antibody and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. Lymph node 7 or 8a was obtained intraoperatively, then mRNA was extracted using an immunobeads method, and RT-PCR with CK19 mRNA was performed. The P for Cox regression analysis for metastasis detected by HE staining, CK staining, and RT-PCR of all 100 cases was 0.312, 0.426, and 0.021, respectively, while for second-tier lymph nodes it was 0.154, 0.013, and 0.006, respectively. In conclusion, RT-PCR and CK staining for detection of metastasis in second-tier lymph nodes were more reliable prognostic indicators than conventional HE staining.