Three Distinct Reporter Systems of Hepatitis E Virus and Their Utility as Drug Screening Platforms.

The hepatitis E virus (HEV) is increasingly acknowledged as the primary cause of acute hepatitis. While most HEV infections are self-limiting, cases of chronic infection and fulminant hepatitis necessitate the administration of anti-HEV medications. However, there is a lack of specific antiviral drugs designed for HEV, and the currently available drug (ribavirin) has been associated with significant adverse effects. The development of innovative antiviral drugs involves targeting distinct steps within the viral life cycle: the early step (attachment and internalization), middle step (translation and RNA replication), and late step (virus particle formation and virion release). We recently established three HEV reporter systems, each covering one or two of these steps. Using these reporter systems, we identified various potential drug candidates that target different steps of the HEV life cycle. Through rigorous in vitro testing using our robust cell culture system with the genotype 3 HEV strain (JE03-1760F/P10), we confirmed the efficacy of these drugs, when used alone or in combination with existing anti-HEV drugs. This underscores their significance in the quest for an effective anti-HEV treatment. In the present review, we discuss the development of the three reporter systems, their applications in drug screening, and their potential to advance our understanding of the incompletely elucidated HEV life cycle.

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.

Development of Recombinant Infectious Hepatitis E Virus Harboring the nanoKAZ Gene and Its Application in Drug Screening.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

Superinfection of hepatitis A virus in hepatocytes infected with hepatitis B virus.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis including acute liver failure. Hepatitis B infection (HBV) occurs worldwide, with the highest rates in Asian and African countries, and there are several reports that HAV infection may have a more severe clinical course in patients with chronic HBV infection. We previously demonstrated that Japanese miso extracts have inhibitory effects on HAV replication. In the present study, we examined the replication of HAV and HBV in a hepatocyte superinfection model and the inhibitory effects of Japanese miso extracts on both viruses. According to the results, HAV infection inhibited HBV replication in superinfected hepatocytes, and Japanese rice-koji miso extracts had inhibitory effects on HAV replication. Our findings provide useful information for clinicians in managing HAV infection in patients with chronic HBV infection.

Screening of novel drugs for inhibiting hepatitis E virus replication.

Hepatitis E, which is caused by hepatitis E virus (HEV), is generally a self-limiting, acute, and rarely fatal disease. It is sometimes fulminant and lethal, especially during pregnancy. Indeed, it occasionally takes a chronic course in immunocompromised individuals. To cure hepatitis E patients, the broad-spectrum antivirals (ribavirin and pegylated interferon α) are used. However, this treatment is insufficient and unsafe in some patients due to embryoteratogenic effects, leukopenia, and thrombocytopenia. In this study, we constructed an HEV replication reporter system with Gaussia luciferase for comprehensively screening anti-HEV drug candidates, and developed a cell-culture system using cells robustly producing HEV to validate the efficacy of anti-HEV drug candidates. We screened anti-HEV drug candidates from United States Food and Drug Administration-approved drugs using the established HEV replication reporter system, and investigated the selected candidates and type III interferons (interferon λ1-3) using the cell-culture system. In conclusion, we constructed an HEV replicon system for anti-HEV drug screening and a novel cell-culture system to strictly evaluate the replication-inhibitory activities of the obtained anti-HEV candidates. Our findings suggested that interferon λ1-3 might be effective for treating hepatitis E.

Inhibitory effect of Japanese rice-koji miso extracts on hepatitis A virus replication in association with the elevation of glucose-regulated protein 78 expression.

Hepatitis A virus (HAV) infection is one of the major causes of acute hepatitis and acute liver failure in developing and developed countries. Although effective vaccines for HAV infection are available, outbreaks of HAV infection still cause deaths, even in developed countries. One approach to control HAV infection is prevention through diet, which can inhibit HAV propagation and replication. Glucose-regulated protein 78 (GRP78) is a member of the heat shock protein 70 family of molecular chaperone required for endoplasmic reticulum stress and stress-induced autophagy. We previously showed that the elevation of GRP78 expression inhibits HAV replication. It has been reported that Japanese miso extracts, which was made from rice-koji, enhance GRP78 expression. In the present study, we used human hepatoma Huh7 cells and human hepatocyte PXB cells to examine the efficacy of Japanese miso extracts as antiviral agents against HAV. Japanese miso extracts enhanced GRP78 expression and inhibited HAV replication in human hepatocytes. Together, these results demonstrate that Japanese miso extracts may partly modulate GRP78 expression and additively or synergistically work as antivirals against HAV infection. Japanese miso extracts can be used as effective dietary supplements for severe hepatitis A.

A prospective study of shortened vitamin supplementation prior to cisplatin-pemetrexed therapy for non-small cell lung cancer.

BACKGROUND: Prior supplementation with folic acid and vitamin B12 is required to reduce pemetrexed therapy toxicity; the recommended lead-in time is at least 7 days. On the basis of previous pharmacokinetic and clinical studies, we hypothesized that the lead-in time could be shortened to 24 hours, enabling earlier commencement of standard chemotherapy; thus, we planned the first prospective trial of this regimen. METHODS: Patients with advanced nonsquamous non-small cell lung cancer who had not previously received cytotoxic chemotherapy were enrolled. After measurement of homocysteine concentrations, the patients received 1,000 µg of vitamin B12 by intramuscular injection and began taking 350-500 µg of oral folic acid daily. Starting 24-48 hours after the vitamin B12 injection, the patients received intravenous 500 mg/m(2) pemetrexed and 75 mg/m(2) cisplatin for 4 cycles at 3 weekly intervals. The primary endpoint was the proportion of patients who developed neutropenia grade ≥3. RESULTS: Thirty patients received chemotherapy starting within 48 hours of the vitamin B12 injection. No treatment-related deaths or grade 4 toxicity occurred. Neutropenia grade ≥3, other laboratory toxicities grade ≥3, and nonlaboratory toxicities grade ≥3 occurred in 6.7%, 13%, and 13% of patients, respectively. The baseline homocysteine concentrations were not higher in patients with grade ≥3 toxicities than in the remainder of the cohort (mean values, 8.6 and 10.7 µmol/L, respectively). The response rate to chemotherapy was 43%. CONCLUSION: The shortened vitamin supplementation was well tolerated and retained antitumor efficacy. Analysis of baseline homocysteine concentrations confirmed the efficacy of short-term vitamin supplementation.