N-glycosylation of viral glycoprotein is a novel determinant for the tropism and virulence of highly pathogenic tick-borne bunyaviruses.

Severe fever with thrombocytopenia syndrome (SFTS) virus, a tick-borne bunyavirus, causes a severe/fatal disease termed SFTS; however, the viral virulence is not fully understood. The viral non-structural protein, NSs, is the sole known virulence factor. NSs disturbs host innate immune responses and an NSs-mutant SFTS virus causes no disease in an SFTS animal model. The present study reports a novel determinant of viral tropism as well as virulence in animal models, within the glycoprotein (GP) of SFTS virus and an SFTS-related tick-borne bunyavirus. Infection with mutant SFTS viruses lacking the N-linked glycosylation of GP resulted in negligible usage of calcium-dependent lectins in cells, less efficient infection, high susceptibility to a neutralizing antibody, low cytokine production in macrophage-like cells, and reduced virulence in Ifnar-/- mice, when compared with wildtype virus. Three SFTS virus-related bunyaviruses had N-glycosylation motifs at similar positions within their GP and a glycan-deficient mutant of Heartland virus showed in vitro and in vivo phenotypes like those of the SFTS virus. Thus, N-linked glycosylation of viral GP is a novel determinant for the tropism and virulence of SFTS virus and of a related virus. These findings will help us understand the process of severe/fatal diseases caused by tick-borne bunyaviruses.

Reverse Genetics System for Heartland Bandavirus: NSs Protein Contributes to Heartland Bandavirus Virulence.

Heartland bandavirus (HRTV), which is an emerging tick-borne virus first identified in Missouri in 2009, causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. HRTV is genetically close to Dabie bandavirus, which is the causative agent of severe fever with thrombocytopenia syndrome (SFTS) in humans and is known as SFTS virus (SFTSV). The generation of infectious HRTV entirely from cloned cDNAs has not yet been reported. The absence of a reverse genetics system for HRTV has delayed efforts to understand its pathogenesis and to generate vaccines and antiviral drugs. Here, we developed a reverse genetics system for HRTV, which employs an RNA polymerase I-mediated expression system. A recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO) was generated. We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. The rHRTV-NSsKO was highly attenuated, indicated by the apparent absence of symptoms in a mouse model of HRTV infection. Moreover, mice immunized with rHRTV-NSsKO survived a lethal dose of HRTV. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV. IMPORTANCE Heartland bandavirus (HRTV) is a tick-borne virus identified in the United States in 2009. HRTV causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. FDA-approved vaccines and antiviral drugs are unavailable. The lack of a reverse genetics system hampers efforts to develop such antiviral therapeutics. Here, we developed a reverse genetics system for HRTV that led to the generation of a recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO). We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. Furthermore, rHRTV-NSsKO was highly attenuated and immunogenic in a mouse model. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/ß receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766's virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

Analysis of cross-reactivity among flaviviruses using sera of patients with dengue showed the importance of neutralization tests with paired serum samples for the correct interpretations of serological test results for dengue.

Dengue is a febrile illness caused by the dengue virus (DENV) that belongs to the genus Flavivirus in the family Flaviviridae. Cross-reactivity between flaviviruses poses a challenge while interpreting serological test results. In the present study, the cross-reactivity of sera of the patients with dengue, who traveled from Japan to DENV-endemic countries, was analyzed by using an enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). Sixteen serum samples were collected from patients with dengue and were tested for: i) IgM antibodies against Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) using IgM ELISA, ii) IgG antibody against TBEV using IgG ELISA, and iii) neutralizing antibody against ZIKV, WNV, TBEV, and JEV. Among the 16 samples tested using ELISA, seven samples were IgM-positive for at least one of the other flaviviruses, and nine samples were IgG-positive for TBEV. Neutralizing antibody titers (NATs) against ZIKV, WNV, and TBEV were one-fourth or lower than those against the causative DENV in all samples. The NATs against JEV were one-fourth or lower than those against the causative DENV in six convalescent-phase serum sample among the seven convalescent-phase serum samples. The NAT against DENV of the residual one convalescent-phase serum was similar to that against JEV and that against JEV of its relevant acute-phase serum sample. These results showed that NTs with paired serum samples are important to correctly interpret the serological test results for DENV.

Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon.

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection.

Virological and genomic analysis of SARS-CoV-2 from a favipiravir clinical trial cohort.

INTRODUCTION: Several clinical studies have reported the efficacy of favipiravir in reducing viral load and shortening the duration of symptoms. However, the viability of SARS-CoV-2 in the context of favipiravir therapy and the potential for resistance development is unclear. METHODS: We sequenced SARS-CoV-2 in nasopharyngeal specimens collected from patients who participated in a randomized clinical trial of favipiravir at hospitals across Japan between March and May 2020. Paired genomes were sequenced from those who remained RT-PCR-positive 5-8 days into favipiravir therapy. Daily nasopharyngeal specimens from 69 patients who were RT-PCR-positive at randomization were examined for a cytopathic effect (CPE). RESULTS: Some strains early in the trial belonged to clade 19 B, whereas the majority belonged to clade 20 B. The median time from the disease onset to negative CPE was 9 days. CPE was strongly correlated with the time from disease onset, viral load, age, and male sex. Among 23 patients for whom paired genomes were available, all except one had identical genomes. Two mutations were observed in one patient who received favipiravir, neither in the RdRp gene. CONCLUSIONS: The SARS-CoV-2 genome distribution in this clinical trial conducted in Japan reflected the early influx of strains from China followed by replacement by strains from Europe. CPE was significantly associated with age, male sex, and viral loads but not with favipiravir therapy. There was no evidence of resistance development during favipiravir therapy.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

A Prospective, Randomized, Open-Label Trial of Early versus Late Favipiravir Therapy in Hospitalized Patients with COVID-19.

Favipiravir is an oral broad-spectrum inhibitor of viral RNA-dependent RNA polymerase that is approved for treatment of influenza in Japan. We conducted a prospective, randomized, open-label, multicenter trial of favipiravir for the treatment of COVID-19 at 25 hospitals across Japan. Eligible patients were adolescents and adults admitted with COVID-19 who were asymptomatic or mildly ill and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients were randomly assigned at a 1:1 ratio to early or late favipiravir therapy (in the latter case, the same regimen starting on day 6 instead of day 1). The primary endpoint was viral clearance by day 6. The secondary endpoint was change in viral load by day 6. Exploratory endpoints included time to defervescence and resolution of symptoms. Eighty-nine patients were enrolled, of whom 69 were virologically evaluable. Viral clearance occurred within 6 days in 66.7% and 56.1% of the early and late treatment groups (adjusted hazard ratio [aHR], 1.42; 95% confidence interval [95% CI], 0.76 to 2.62). Of 30 patients who had a fever (≥37.5°C) on day 1, times to defervescence were 2.1 days and 3.2 days in the early and late treatment groups (aHR, 1.88; 95% CI, 0.81 to 4.35). During therapy, 84.1% developed transient hyperuricemia. Favipiravir did not significantly improve viral clearance as measured by reverse transcription-PCR (RT-PCR) by day 6 but was associated with numerical reduction in time to defervescence. Neither disease progression nor death occurred in any of the patients in either treatment group during the 28-day participation. (This study has been registered with the Japan Registry of Clinical Trials under number jRCTs041190120.).

Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication.

Two viral nonstructural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication, presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with the HSP90 inhibitors 17-allylamino-17-desmethoxygeldanamycin (17-AAG) and ganetespib suppressed RUBV genome replication. HSP90α physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrate that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication.IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genomes. However, the host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrate that the HSP90 molecular chaperone is needed for the efficient replication of the RUBV genome. Further, we reveal that HSP90 interacts with RUBV nonstructural protein p150 and its precursor polyprotein, p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of nonstructural proteins for RUBV genome replication. These findings provide novel insight into this host-virus interaction.

An estrogen antagonist, cyclofenil, has anti-dengue-virus activity.

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.

Correction to: An estrogen antagonist, cyclofenil, has anti-dengue-virus activity.

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

Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis.

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.

A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

BACKGROUND: JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required. METHODS: A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML. RESULTS: The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 100 to 3.2 × 106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay. CONCLUSIONS: The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.

Correction to: A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

In the original publication of article [1], '20 × 101 copies', which is in the sentence 'As seen in Fig. 4, the sensitivity of the specimens containing equal to or more than 20 × 101 copies in 2 µL of extracted DNA (equivalent to ≥3.0 × 103 copies/mL CSF) was 100% (29/29)' changes to '2.0 × 101 copies' in results section. The publisher apologizes to the readers and authors for the inconvenience.

Genotype-specific and cross-reactive neutralizing antibodies induced by dengue virus infection: detection of antibodies with different levels of neutralizing activities against homologous and heterologous genotypes of dengue virus type 2 in common marmosets (Callithrix jacchus).

BACKGROUND: A vaccine against all four dengue virus (DENV) serotypes includes the formulation of one genotype of each serotype. Although genetic similarities among genotypes within a serotype are higher as compared to those among serotypes, differences in the immunogenicity of the included genotypes would be a critical issue in maximizing successful dengue vaccine development. Thus, we determined the neutralizing antibody responses against three genotypes of dengue virus serotype 2 (DENV-2), namely Cosmopolitan, Asian I, and Asian/American, after primary and secondary inoculation with DENV-2 in a dengue animal model, the common marmoset (Callithrix jacchus). METHODS: A total of fifty-four plasma samples were obtained from thirty-four marmosets that were inoculated with clinically-isolated DENV strains or DENV candidate vaccines, were used in this study. Plasma samples were obtained from marmosets after primary inoculation with DENV-2 infection, secondary inoculation with homologous or heterologous genotypes, and tertiary inoculation with heterologous DENV. Neutralizing antibody titers against DENV-2 (Cosmopolitan, Asian I, and Asian/American genotypes) and DENV-1 were determined using a conventional plaque reduction neutralization assay. RESULTS: In marmosets that were inoculated with the Cosmopolitan genotype in primary infection, neutralizing antibody neutralized 3 genotypes, and the titers to Asian I genotype were significantly higher than those to homologous Cosmopolitan genotype. After secondary DENV-2 infection with heterologous genotype (Asian I in primary and Asian/American in secondary), neutralizing antibody titers to Asian/American genotype was significantly higher than those against Cosmopolitan and Asian I genotypes. Following tertiary infection with DENV-1 following DENV-2 Asian I and Cosmopolitan genotypes, neutralizing antibody titers to Asian/American were also significantly higher than those against Cosmopolitan and Asian I genotypes. CONCLUSION: The present study demonstrated that different levels of neutralizing antibodies were induced against variable DENV-2 genotypes after primary, secondary and tertiary infections, and that neutralizing antibody titers to some heterologous genotypes were higher than those to homologous genotypes within a serotype. The results indicate that heterogeneity and homogeneity of infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced in following infections. The results also suggest that certain genotypes may possess advantage in terms of breakthrough infections against vaccination.

Association of the Emergence of Acyclovir-Resistant Herpes Simplex Virus Type 1 With Prognosis in Hematopoietic Stem Cell Transplantation Patients.

Background: Antiviral-resistant herpes simplex virus type 1 (HSV-1) has been recognized as an emerging clinical problem among patients undergoing hematopoietic stem cell transplantation (HSCT). Methods: A prospective observational study was conducted at a hematological center over a 2-year period. Oropharyngeal swab samples were serially collected each week from 1 week before and up to 100 days after HSCT and were tested for virus isolation. The HSV-1 isolates were tested for sensitivity to acyclovir (ACV). The prognosis of patients with ACV-resistant (ACVr) HSV-1 and the genetic background of the ACVr HSV-1 isolates were assessed. Results: Herpes simplex virus type 1 was isolated in 39 of 268 (15%) HSCT patients within 100 days after transplantation. Acyclovir-resistant HSV-1 emerged in 11 of these 39 patients (28%). The 100-day death rates of HSCT patients without HSV-1 shedding, those with only ACV-sensitive HSV-1 shedding, and those with ACVr HSV-1 shedding were 31%, 39%, and 64%, respectively. Patients with HSV-1, including ACVr HSV-1, shedding showed a significantly higher mortality rate. Relapsed malignancies were a significant risk factor for the emergence of ACVr HSV-1. Acyclovir resistance was attributable to viral thymidine kinase and DNA polymerase mutations in 6 and 5 patients, respectively. Conclusions: Herpes simplex virus type 1, including ACVr HSV-1, shedding was associated with poorer outcome in HSCT patients, even if HSV disease did not always occur. Patients with relapsed malignancies were at especially high risk for the emergence of ACVr HSV-1.

[The Recent Epidemic Spread of Zika Virus Disease].

Zika virus (ZIKV) is one of the members of the Spondweni serocomplex within the genus Flavivirus of the family Flaviviridae. The virus was first isolated from a serum specimen from a sentinel non-human primate in the Zika forest of Uganda in 1947. ZIKV is transmitted by Aedes aegypti and A. albopictus in an urban cycle and maintained in a sylvatic cycle between Aedes mosquitoes and monkeys in Africa and Asia. Initially, the virus was thought to cause only mild and nonspecific clinical symptoms in humans. However, ZIKV became a serious public health concern in recent years due to an association with congenital malformation known as microcephaly in newborns as well as Guillain-Barré syndrome and other neurologic disorders in adults. The severe nature of complications of ZIKV infection have led to an urgent need for a safe and effective vaccine worldwide including Japan. The first large outbreak of disease caused by ZIKV infection was reported from the island of Yap, Micronesia in 2007. It was followed by outbreaks in French Polynesia, Cook Islands, Ester Island, and New Caledonia in 2013 and 2014. In 2015, ZIKV outbreak was reported in Brazil and has spread across the Latin America, and the Caribbean. The exact prevalence of ZIKV infection has not been reported because of the absence of a standardized protocol for differential diagnosis and its clinical resemblance to dengue virus and other flavivirus infections. In Japan, the first human case of ZIK fever, who developed illness soon after returning from French Polynesia, was reported in 2013, and until 2017, 20 imported cases were documented. Currently, research on ZIKV has progressed remarkably thus this article aims to review recent progress in virology, epidemiology, and pathology of ZIKV infection.

Dengue Virus Type 2 in Travelers Returning to Japan from Sri Lanka, 2017.

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.

Importation of Zika Virus from Vietnam to Japan, November 2016.

We report a case of Zika virus infection that was imported to Japan by a traveler returning from Vietnam. We detected Zika virus RNA in the patient's saliva, urine, and whole blood. In the Zika virus strain isolated from the urine, we found clearly smaller plaques than in previous strains.

Dengue Virus Exported from Côte d'Ivoire to Japan, June 2017.

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.