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In this study, the anti-severe acute respiratory syndrome coronavirus-2 (anti-SARS-CoV-2) activity of mycophenolic acid (MPA) and IMD-0354 was analyzed. These compounds were chosen based on their antiviral activities against other coronaviruses. Because they also inhibit dengue virus (DENV) infection, other anti-DENV compounds/drugs were also assessed. On SARS-CoV-2-infected VeroE6/TMPRSS2 monolayers, both MPA and IMD-0354, but not other anti-DENV compounds/drugs, showed significant anti-SARS-CoV-2 activity. Although MPA reduced the viral RNA level by only approximately 100-fold, its half maximal effective concentration was as low as 0.87 µ m, which is easily achievable at therapeutic doses of mycophenolate mofetil. MPA targets the coronaviral papain-like protease and an in-depth study on its mechanism of action would be useful in the development of novel anti-SARS-CoV-2 drugs.

Dengue virus (DENV) infections are a major cause of morbidity and mortality in tropical and subtropical areas. Several compounds that act against DENV have been studied in clinical trials to date; however, there have been no compounds identified that are effective in reducing the severity of the clinical manifestations. To explore anti-DENV drugs, we examined small molecules that interact with DENV NS1 and inhibit DENV replication. Cyclofenil, which is a selective estrogen receptor modulator (SERM) and has been used clinically as an ovulation-inducing drug, showed an inhibitory effect on DENV replication in mammalian cells but not in mosquito cells. Other SERMs also inhibited DENV replication in mammalian cells, but cyclofenil showed the weakest cytotoxicity among these SERMs. Cyclofenil also inhibited the replication of Zika virus. A time-of-addition assay suggested that cyclofenil may interfere with two stages of the DENV life cycle: the translation-RNA synthesis and assembly-maturation stages. However, the level of intracellular infectious particles decreased more drastically after treatment with cyclofenil than the viral RNA level did, indicating that the assembly-maturation stage might be the main target of cyclofenil. In electron microscopy analysis, many aggregated particles were detected in DENV-infected cells in the presence of cyclofenil, supporting the possibility that cyclofenil impedes the process of assembly and maturation of DENV.

The chemically synthesized endoperoxide compound N-89 and its derivative N-251 were shown to have potent antimalarial activity. We previously demonstrated that N-89 and N-251 potently inhibited the RNA replication of hepatitis C virus (HCV), which belongs to the Flaviviridae family. Since antimalarial and anti-HCV mechanisms have not been clarified, we were interested whether N-89 and N-251 possessed the activity against viruses other than HCV. In this study, we examined the effects of N-89 and N-251 on other flaviviruses (dengue virus and Japanese encephalitis virus) and hepatitis viruses (hepatitis B virus and hepatitis E virus). Our findings revealed that N-89 and N-251 moderately inhibited the RNA replication of Japanese encephalitis virus and hepatitis E virus, although we could not detect those anti-dengue virus activities. We also observed that N-89 and N-251 moderately inhibited the replication of hepatitis B virus at the step after viral translation. These results suggest the possibility that N-89 and N-251 act on some common host factor(s) that are necessary for viral replications, rather than the possibility that N-89 and N-251 directly act on the viral proteins except for HCV. We describe a new type of antiviral reagents, N-89 and N-251, which are applicable to multiple different viruses., Highlights • We evaluated the effects of N-89 and N-251 on 4 viruses other than HCV. • N-89 and N-251 moderately inhibited the replication of Japanese encephalitis virus. • N-89 and N-251 moderately inhibited the replication of hepatitis B and E viruses. • We found the antiviral effects of N-89 and N-251 to 4 viruses including HCV.

In July 2018, a Japanese traveler returning from Saudi Arabia was diagnosed with dengue. The dengue virus type 2 gene was detected from a whole blood sample. Phylogenetic analysis revealed that the strain was clustered with isolates from Singapore and India. Travelers to Saudi Arabia should be cautious about mosquito bites.

Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromocriptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z' factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-β-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4'-thio and 4'-seleno derivatives of EICAR and EICNR 3-6 were prepared. The resulting 4'-thioEICAR and 4'-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.

Dengue virus (DENV) is the most prevalent human arthropod-borne virus and causes severe problems worldwide, mainly in tropical and sub-tropical regions. However, there is no specific antiviral drug against DENV infection. We and others recently reported that stearoyl-CoA desaturase-1 (SCD1) inhibitor showed potent suppression of hepatitis C virus replication. In this study, we examined the impact of SCD1 on DENV replication. We found that SCD1 inhibitors (MK8245 and #1716) dramatically suppressed DENV replication in a dose-dependent manner without cytotoxicity. This anti-DENV efficacy was observed against all four DENV serotypes and other flaviviruses, including Zika virus and Japanese encephalitis virus. A subgenomic replicon system of DENV was used to confirm that SCD1 inhibitor suppressed viral RNA replication. Interestingly, exogenous supplementation of unsaturated fatty acids resulted in recovery of the DENV titer even in the presence of SCD1 inhibitor, suggesting that fatty acid biosynthesis contributes to DENV genome replication. These findings indicate that SCD1 is a novel host factor required for DENV replication, and SCD1 inhibitor is a potential candidate for treating dengue fever.

In June 2017, dengue virus type 2 infection was diagnosed in 2 travelers returned to Japan from Sri Lanka, where the country’s largest dengue fever outbreak is ongoing. Travelers, especially those previously affected by dengue fever, should take measures to avoid mosquito bites.

Since April 2017, a dengue fever outbreak has been ongoing in Côte d’Ivoire. We diagnosed dengue fever (type 2 virus) in a traveler returning to Japan from Côte d’Ivoire. Phylogenetic analysis revealed strain homology with the Burkina Faso 2016 strain. This case may serve as an alert to possible disease spread outside Africa.

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1-4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.

In the growth kinetics analysis of flaviviruses in Aedes albopictus C6/36 cell lines obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank and the European Collection of Authenticated Cell Culture (ECACC), these two cells line showed different viral susceptibility for Zika virus (ZIKV), Dengue virus (DENV), and Japanese encephalitis virus (JEV). Next-generation sequencing (NGS) analysis revealed that the C6/36 JCRB strain was persistently infected with two viruses without showing any cytopathic effects. The complete sequence analysis demonstrated that the one virus was Menghai rhabdovirus (MERV), which has been found from Aedes albopictus mosquito. The other virus was a novel virus, designated as Shinobi tetravirus (SHTV). Interestingly, the viral susceptibility of these two strains was almost even for Sindbis virus and Getah virus. We cloned SHTV and MERV from JCRB C6/36 cell line and then re-infected them into another C6/36 cell line, resulting in the reproduction of persistent infection with each virus. ZIKV growth was suppressed in SHTV and/or MERV re-infected C6/36 cells also. To our knowledge, this is the first demonstration that persistent infection with rhabdovirus and/or permutotetravirus suppressed flavivirus replication in mosquito cells.

Dengue virus (DENV) causes a wide range of illnesses in humans, including dengue fever and dengue haemorrhagic fever. Current animal models of DENV infection are limited for understanding infectious diseases in humans. Bonnet monkeys (Macaca radiata), a type of Old World monkey, have been used to study experimental and natural infections by flaviviruses, but Old World monkeys have not yet been used as DENV infection models. In this study, the replication levels of several DENV strains were evaluated using peripheral blood mononuclear cells. Our findings indicated that DENV-4 09-48 strain, isolated from a traveller returning from India in 2009, was a highly replicative virus. Three bonnet monkeys were infected with 09-48 strain and antibody responses were assessed. DENV nonstructural protein 1 antigen was detected and high viraemia was observed. These results indicated that bonnet monkeys and 09-48 strain could be used as a reliable primate model for the study of DENV.

Dengue virus (DENV) is transmitted to humans by Aedes mosquitoes and is a public health issue worldwide. No antiviral drugs specific for treating dengue infection are currently available. To identify novel DENV inhibitors, we analyzed a library of 95 compounds and 120 extracts derived from crude drugs (herbal medicines). In the primary screening, A549 cells infected with DENV-1 were cultured in the presence of each compound and extract at a final concentration of 10 μM (compound) and 100 μg/mL (extract), and reduction of viral focus formation was assessed. Next, we eliminated compounds and extracts which were cytotoxic using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hirsutine, an indole alkaloid of Uncaria rhynchophylla, was identified as a potent anti-DENV compound exhibiting high efficacy and low cytotoxicity. Hirsutine showed antiviral activity against all DENV serotypes. Time-of-drug-addition and time-of-drug-elimination assays indicated that hirsutine inhibits the viral particle assembly, budding, or release step but not the viral translation and replication steps in the DENV lifecycle. A subgenomic replicon system was used to confirm that hirsutine does not restrict viral genome RNA replication. Hirsutine is a novel DENV inhibitor and potential candidate for treating dengue fever.

Replicon systems have been used for high-throughput screening of anti-dengue virus (anti-DENV) inhibitors and for understanding mechanisms of viral replication. In the present study, we constructed novel DENV-1 replicons encoding Gaussia luciferase that was secreted into the culture medium. Two types of constructs were generated: RNA-based and DNA-based. Each type was translated in an internal ribosome entry site (IRES)-dependent or IRES-independent manner. Among these constructs, the DNA-based replicon employing IRES-dependent translation (DGL2) produced the highest titer. Luciferase levels in the culture medium revealed that the DGL2 replicon was inhibited by ribavirin (a well-known DENV inhibitor) at levels similar to those measured for drug inhibition of multi-round DENV-1 infection. These results indicate that the DNA-based IRES-driven DENV-1 replicon may facilitate studies on viral replication and antiviral compound discovery.

Dengue virus (DENV) infectious disease is a major public health problem worldwide; however, licensed vaccines or specific antiviral drugs against this infection are not available. To identify novel anti-DENV compounds, we screened 1280 pharmacologically active compounds using focus reduction assay. Bromocriptine (BRC) was found to have potent anti-DENV activity and low cytotoxicity (half maximal effective concentration [EC50], 0.8–1.6 μM; and half maximal cytotoxicity concentration [CC50], 53.6 μM). Time-of-drug-addition and time-of-drug-elimination assays suggested that BRC inhibits translation and/or replication steps in the DENV life cycle. A subgenomic replicon system was used to verify that BRC restricts RNA replication step. Furthermore, a single amino acid substitution (N374H) was detected in the NS3 protein that conferred resistance to BRC. In summary, BRC was found to be a novel DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.

We would like to correct the information on the antibody used in this study. In Fig. 5 of the article, cellular β-actin was detected as an internal control using anti-β-actin antibody (Fujifilm Wako Pure Chemicals, #017-24573).

To investigate the potential role of non-human primates (NHPs) in a dengue virus (DENV) epidemic, we conducted serological and genomic studies using plasma samples collected from 100 cynomolgus monkeys (Macaca fascicularis) in an animal breeding facility in the Philippines. An ELISA revealed 21 monkeys with a positive IgM reaction and 19 positive for IgG. Five of the monkeys were positive for both IgM and IgG. Of the 21 IgM-positive samples, a neutralization assay identified seven containing DENV-specific antibodies. We amplified the viral non-structural 1 (NS1) gene in two and the envelope (E) gene in one of these seven samples by RT-PCR. Phylogenetic analyses revealed that these DENV genes belonged to the epidemic DENV-2 family, not the sylvatic DENV family. These results suggest that NHPs may serve as a reservoir of epidemic DENV; therefore, the ecology of the urban DENV infection cycle should be investigated in these animals in detail.