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.

Facilitating the deployment of Japanese human resources for responding global outbreaks of emerging and Re-emerging infectious diseases: A cross-sectional study.

INTRODUCTION: In response to global outbreaks of infectious diseases, the need for support from organizations such as the World Health Organization Global Outbreak Alert and Response Network (GOARN) is increasing. Identifying the obstacles and support needs for applicants could increase GOARN deployments from Japan. METHODS: This cross-sectional study involved a web-based, self-administered questionnaire survey targeting Japanese participants in the GOARN Tier 1.5 training workshop, held in Tokyo in December 2019. RESULTS: All 47 Japanese participants in the workshop responded to the survey. Most responders were male and in their 30s and 40s. Participants specialized in case management (42.6%), infection prevention and control (25.6%), epidemiology and surveillance (19.1%). Only two participants (4.6%) had experienced a GOARN deployment. Their motivations for joining the GOARN training workshop were "Desire to be part of an international emerging infectious disease response team" (44.6%), "Interest in making an international contribution" (19.1%), and "Interest in working for the Japanese government in the field of international infectious diseases" (14.9%). Obstacles to GOARN deployments were "Making time for deployments" (45.7%) and "Lack of required professional skills and knowledge" (40.4%). The support needs for GOARN deployments constituted "Periodic simulation training" (51.1%), "Financial support during deployments" (44.7%), and "Technical support for deployments" (40.4%). CONCLUSIONS: Our study revealed the obstacles and support needs of Japanese candidates for GOARN deployment. Making time and upskilling for GOARN deployment were the main obstacles. More practical training (like GOARN Tier 2.0) with other supports are needed. The national framework is desirable to realize these supports.

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.).

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.

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.

Identification of the human herpesvirus 6A gQ1 domain essential for its functional conformation.

Human herpesvirus 6 is a T lymphotropic herpesvirus, long classified into variants A and B (HHV-6A and HHV-6B) based on differences in sequence and pathogenicity. Recently, however, HHV-6A and HHV-6B were reclassified as different species. Here, we isolated a neutralizing monoclonal antibody (Mab) named AgQ 1-1 that was specific for HHV-6A glycoprotein Q1 (AgQ1), and we showed that amino acid residues 494 to 497 of AgQ1 were critical for its recognition by this Mab. This region was also essential for AgQ1's complex formation with gH, gL, and gQ2, which might be important for viral binding to the cellular receptor, CD46. In addition, amino acid residues 494 to 497 are essential for viral replication. Interestingly, this sequence corresponds to the domain on HHV-6B gQ1 that is critical for recognition by an HHV-6B-specific neutralizing Mab. Within this domain, only Q at position 496 of HHV-6A is distinct from the HHV-6B sequence; however, the mutant AgQ1(Q496E) was still clearly recognized by the Mab AgQ 1-1. Surprisingly, replacement of the adjacent amino acid, in mutant AgQ1(C495A), resulted in poor recognition by Mab AgQ 1-1, and AgQ1(C495A) could not form the gH/gL/gQ1/gQ2 complex. Furthermore, the binding ability of mutant AgQ1(L494A) with CD46 decreased, although it could form the gH/gL/gQ1/gQ2 complex and it showed clear reactivity to Mab AgQ 1-1. These data indicated that amino acid residues 494 to 497 of AgQ1 were critical for the recognition by Mab AgQ 1-1 and essential for AgQ1's functional conformation.

Human herpesvirus-6A gQ1 and gQ2 are critical for human CD46 usage.

Based on genetic and antigenic differences and on their cell tropism, human herpes virus-6 (HHV-6) has been classified into two variants, HHV-6A and HHV-6B. Recently, these variants were re-classified as two different species. The HHV-6A glycoprotein complex, gH/gL/gQ1/gQ2 binds to its cellular receptor, CD46; however, the corresponding complex in HHV-6B rarely binds to CD46. To determine which viral molecules in the glycoprotein complex determine HHV-6A-CD46 binding, each molecule of the HHV-6A complex (i.e., gH, gL, gQ1, or gQ2) was replaced with the corresponding HHV-6B molecule, and the ability of the replaced protein to be incorporated into the complex and the ability of the complex to bind CD46 were examined. It was found that when all four glycoproteins were expressed, they were able to form a tetrameric complex. However, a complex formed by HHV-6A gH/gL/gQ1/gQ2 complexes replaced with HHV-6B gQ1 or gQ2 scarcely bind CD46, whereas HHV-6A complexes in which gH or gL was replaced with the HHV-6B molecules did bind it. These results indicate that HHV-6A gQ1 and gQ2 play an important role in CD46 binding.

Pathological and virological findings of type I interferon receptor knockout mice upon experimental infection with Heartland virus.

Heartland virus (HRTV) causes generalized symptoms, severe shock, and multiple organ failure. We previously reported that interferon-α/ß receptor knockout (IFNAR-/-) mice infected intraperitoneally with 1 × 107 tissue culture-infective dose (TCID50) of HRTV died, while those subcutaneously infected with the same dose of HRTV did not. The pathophysiology of IFNAR-/- mice infected with HRTV and the mechanism underlying the difference in disease severity, which depends on HRTV infection route, were analyzed in this study. The liver, spleen, mesenteric and axillary lymph nodes, and gastrointestinal tract of intraperitoneally (I.P.) infected mice had pathological changes; however, subcutaneously (S.C.) infected mice only had pathological changes in the axillary lymph node and gastrointestinal tract. HRTV RNA levels in the mesenteric lymph node, lung, liver, spleen, kidney, stomach, intestine, and blood were significantly higher in I.P. infected mice than those in S.C. infected mice. Chemokine ligand-1 (CXCL-1), tumor necrosis factor (TNF)-α, interleukin (IL)-12, interferon (IFN)-γ, and IL-10 levels in plasma of I.P. infected mice were higher than those of S.C. infected mice. These results indicated that high levels of viral RNA and the induction of inflammatory responses in HRTV-infected IFNAR-/- mice may be associated with disease severity.

Growth, Pathogenesis, and Serological Characteristics of the Japanese Encephalitis Virus Genotype IV Recent Strain 19CxBa-83-Cv.

Genotype IV Japanese encephalitis (JE) virus (GIV JEV) is the least common and most neglected genotype in JEV. We evaluated the growth and pathogenic potential of the GIV strain 19CxBa-83-Cv, which was isolated from a mosquito pool in Bali, Indonesia, in 2019, and serological analyses were also conducted. The growth ability of 19CxBa-83-Cv in Vero cells was intermediate between that of the genotype I (GI) strain Mie/41/2002 and the genotype V (GV) strain Muar, whereas 19CxBa-83-Cv and Mie/41/2002 grew faster than Muar in mouse neuroblastoma cells. The neuroinvasiveness of 19CxBa-83-Cv in mice was higher than that of Mie/41/2002 but lower than that of Muar; however, there were no significant differences in neurovirulence in mice among the three strains. The neutralizing titers of sera from 19CxBa-83-Cv- and Mie/41/2002-inoculated mice against 19CxBa-83-Cv and Mie/41/2002 were similar, whereas the titers against Muar were lower than those of the other two viruses. The neutralizing titers of JE vaccine-inoculated mouse pool serum against 19CxBa-83-Cv and Muar were significantly lower than those against Mie/41/2002. The neutralizing titers against the three viruses were similar in three out of the five serum samples from GI-infected JE patients, although the titers against Mie/41/2002 were higher than those against 19CxBa-83-Cv and Muar in the remaining two sera samples. In summary, we identified the basic characteristics of 19CxBa-83-Cv, but further studies are needed to better understand GIV JEV.

Human herpesvirus 6 glycoprotein complex formation is required for folding and trafficking of the gH/gL/gQ1/gQ2 complex and its cellular receptor binding.

Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. A glycoprotein (g) complex that is unique to HHV-6, gH/gL/gQ1/gQ2, is a viral ligand for its cellular receptor, human CD46. However, whether complex formation or one component of the complex is required for CD46 binding and how the complex is transported in cells are open questions. Furthermore, in HHV-6-infected cells the gQ1 protein modified with N-linked glycans is expressed in two forms with different molecular masses: an 80-kDa form (gQ1-80K) and a 74-kDa form (gQ1-74K). Only gQ1-80K, but not gQ1-74K, forms the complex with gQ2, gH, and gL, and this four-component complex is incorporated into mature virions. Here, we characterized the molecular context leading to the maturation of gQ1 by expressing combinations of the individual gH/gL/gQ1/gQ2 components in 293T cells. Surprisingly, only when all four molecules were expressed was a substantial amount of gQ1-80K detected, indicating that all three of the other molecules (gQ2, gH, and gL) were necessary and sufficient for gQ1 maturation. We also found that only the tetrameric complex, and not its subsets, binds to CD46. Finally, a gQ2-null virus constructed in the BAC (bacterial artificial chromosome) system could not be reconstituted, indicating that gQ2 is essential for virus growth. These results show that gH, gL, gQ1, and gQ2 are all essential for the trafficking and proper folding of the gH/gL/gQ1/gQ2 complex and, thus, for HHV-6 infection.

Analysis of a neutralizing antibody for human herpesvirus 6B reveals a role for glycoprotein Q1 in viral entry.

Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.

Susceptibility of Type I Interferon Receptor Knock-Out Mice to Heartland Bandavirus (HRTV) Infection and Efficacy of Favipiravir and Ribavirin in the Treatment of the Mice Infected with HRTV.

Heartland bandavirus (HRTV) is an emerging tick-borne virus that is distributed in the United States and that causes febrile illness with thrombocytopenia and leukocytopenia. It is genetically close to Dabie bandavirus, which is well known as severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV). The mortality rate of human HRTV infection is approximately 10%; however, neither approved anti-HRTV agents nor vaccines exist. An appropriate animal model should be developed to evaluate the efficacy of antiviral agents and vaccines against HRTV. The susceptibility of IFNAR-/- mice with HRTV infection was evaluated using subcutaneous, intraperitoneal, and retro-orbital inoculation routes. IFNAR-/- mice intraperitoneally infected with HRTV showed the most severe clinical signs, and the 50% lethal dose was 3.2 × 106 TCID50. Furthermore, to evaluate the utility of a novel lethal IFNAR-/- mice model, IFNAR-/- mice were orally administered favipiravir, ribavirin, or a solvent for 5 days immediately after a lethal dose of HRTV inoculation. The survival rates of the favipiravir-, ribavirin-, and solvent-administered mice were 100, 33, and 0%, respectively. The changes in bodyweights and HRTV RNA loads in the blood of favipiravir-treated IFNAR-/- mice were the lowest among the three groups, which suggests that favipiravir is a promising drug candidate for the treatment of patients with HRTV infection.

Immunogenicity and Protective Ability of Genotype I-Based Recombinant Japanese Encephalitis Virus (JEV) with Attenuation Mutations in E Protein against Genotype V JEV.

Genotype V (GV) Japanese encephalitis virus (JEV) has emerged in Korea and China since 2009. Recent findings suggest that current Japanese encephalitis (JE) vaccines may reduce the ability to induce neutralizing antibodies against GV JEV compared to other genotypes. This study sought to produce a novel live attenuated JE vaccine with a high efficacy against GV JEV. Genotype I (GI)-GV intertypic recombinant strain rJEV-EXZ0934-M41 (EXZ0934), in which the E region of the GI Mie/41/2002 strain was replaced with that of GV strain XZ0934, was introduced with the same 10 attenuation substitutions in the E region found in the live attenuated JE vaccine strain SA 14-14-2 to produce a novel mutant virus rJEV-EXZ/SA14142m-M41 (EXZ/SA14142m). In addition, another mutant rJEV-EM41/SA14142m-M41 (EM41/SA14142m), which has the same substitutions in the Mie/41/2002, was also produced. The neuroinvasiveness and neurovirulence of the two mutant viruses were significantly reduced in mice. The mutant viruses induced neutralizing antibodies against GV JEV in mice. The growth of EXZ/SA14142m was lower than that of EM41/SA14142m. In mouse challenge tests, a single inoculation with a high dose of the mutants blocked lethal GV JEV infections; however, the protective efficacy of EXZ/SA14142m was weaker than that of EM41/SA14142m in low-dose inoculations. The lower protection potency of EXZ/SA14142m may be ascribed to the reduced growth ability caused by the attenuation mutations.

Leu-to-Phe substitution at prM146 decreases the growth ability of Zika virus and partially reduces its pathogenicity in mice.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes.

Dengue Virus Serotype 1 Exported to Japan from Côte d'Ivoire, 2019.

Dengue fever outbreaks have been repeatedly reported in Côte d'Ivoire. During the 2019 outbreak, DENV-1 was the predominant strain and phylogenetic analysis of the DENV-1 genome obtained from the present patient who returned to Japan in January 2019 revealed a high homology with the 2013-2014 Southeast Asian strains. In a previous outbreak in 2017, DENV-1 accounted for 5% of the DENV serotypes. The endemic DENV-1 strain in Abidjan in 2019 could be a strain that was imported from Southeast Asia. Dengue virus can spread globally, and imported dengue fever cases could serve as an alert for outbreaks in the exporting country.