Crisis management for the future: Building a platform to provide information on emerging and re-emerging infectious diseases from normal times in Japan.

At the beginning of the mpox (disease caused by monkey pox) epidemic, there was no platform in Japan to provide appropriate information on emerging and re-emerging infectious diseases (EIDs), and the number of accesses to bioterrorism-related information sites increased rapidly. Even though the interest in mpox was much smaller than in coronavirus infectious disease, emerged in late 2019 (COVID-19), the increase in the number of views were much greater than during the COVID-19 epidemic. This may not be because mpox is bioterrorism-related as an analog of smallpox, but rather because there were no other websites providing information on mpox. For future crisis management, there should be a platform to provide information on possible epidemics of EIDs from normal times in Japan.

The Association between COVID-19-Related Discrimination and Probable Post-Traumatic Stress Disorder among Patients with COVID-19 in Sapporo, Japan.

BackgroundDisasters such as earthquakes, terrorism, and pandemics have triggered post-traumatic stress disorder (PTSD), and discrimination against the affected individuals has been linked to the development of PTSD. However, there is limited evidence regarding the association between discrimination against coronavirus disease 2019 (COVID-19) patients and probable PTSD in Japan.MethodsWe conducted a cross-sectional study utilizing a web-based questionnaire targeting individuals who had contracted the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Sapporo City. A total of 4247 individuals with laboratory-confirmed SARS-CoV-2 infection spanning from February 2020 to February 2022 completed the questionnaire (response rate: 15.9%). Probable PTSD was measured using the three-item Posttraumatic Diagnostic Scale. The stratified exact logistic regression was applied to calculate the odds ratios (OR) of probable PTSD for COVID-19-related discrimination with adjusted factors.ResultsThis study included 3626 patients who had a history of SARS-CoV-2 infection. Among them, 321 patients (8.9%) experienced COVID-19-related discrimination. The prevalence of probable PTSD was 19.6% (63/321) among the patients who experienced COVID-19-related discrimination, and 4.6% (152/3305) among those who had not encountered such discrimination. The adjusted OR of COVID-19-related discrimination for probable PTSD was 4.68 (95% confidence interval [95% CI], 3.36-6.53). The population attributable fraction of probable PTSD attributable to COVID-19-related discrimination among COVID-19 patients was estimated to be 23.4% (95% CI, 21.5-25.3).ConclusionThe comprehensive epidemiological survey of COVID-19 patients in Japan showed that COVID-19-related discrimination was associated with a higher prevalence of probable PTSD. Mitigating discrimination could be helpful to attenuate PTSD in future pandemics.

Mice, myeloid cells, and dengue: a new model for unraveling vascular leakage mysteries.

Introduction: Severe dengue is thought to be caused by an excessive host immune response. Methods: To study the pathogenesis of severe dengue, we developed a novel model using LysM Cre+Ifnarflox/flox mice carrying depleted Ifnar expression only in subsets of murine myeloid cells. Results: Although dengue virus (DENV) clinical isolates were not virulent in LysM Cre+Ifnarflox/flox mice, mouse-adapted DV1-5P7Sp and DV3P12/08P4Bm, which were obtained by passaging the spleen or bone marrow of mice, demonstrated 100% lethality with severe vascular leakage in the liver and small intestine. DV1-5P7Sp and DV3P12/08P4Bm harbored five and seven amino acid substitutions, respectively. Infection also induced neutrophil infiltration in the small intestine, and increased expression of IL-6 and MMP-8 and blockade of TNF-α signaling protected the mice, as demonstrated in a previous severe dengue mouse model using C57/BL6 mice lacking both IFN-α/ß and IFN-γ receptors. Notably, the new models with DV1-5P7Sp and DV3P12/08P4Bm showed an increased proliferative capacity of the adapted viruses in the thymus and bone marrow. Discussion: These observations suggest that myeloid cell infection is sufficient to trigger cytokine storm-induced vascular leakage. This model can refine the factors involved in the pathology of severe dengue leading to vascular leakage.

Traditional and Social Media Usage Associated With COVID-19 Vaccine Uptake in Sapporo, Japan.

This study aimed to determine the relationship between specific information source usage and uptake of the coronavirus disease 2019 (COVID-19) vaccine. We analyzed 3348 participants aged 20 to 65 years who were not diagnosed with COVID-19 in a case-control study in Sapporo, Japan. The most prevalent information source on COVID-19 was television (TV; 87.8%), followed by online news sites (74.3%), newspapers (38.7%), websites of public institutions (30.9%), and families (29.7%). Multivariate logistic regression showed that the adjusted odds ratios of incompletion of second vaccinations for users of TV and newspaper to gather COVID-19 information were 0.31 (95% confidence interval [CI] [0.21, 0.44]) and 0.32 (95% CI [0.20, 0.50]), respectively, whereas those for users of books, commercial video sites, Facebook, and "personal blog or bulletin board system" were 3.34 (95% CI [1.58, 7.06]), 2.22 (95% CI [1.44, 3.43]), 2.36 (95% CI [1.24, 4.48]), and 4.81 (95% CI [2.72, 8.48]), respectively. Social media use among older or male participants was associated with lower vaccine uptake.

Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.

Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus Mammarenavirus, family Arenaviridae and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.

Patient Characteristics and Public Health Office Factors Associated With Long Reporting Delay of COVID-19 Cases in Sapporo City, Japan.

BACKGROUND: For therapeutic efficacy, molnupiravir and nirmatrelvir-ritonavir must be started to treat patients within 5 days of disease onset to treat patients with novel coronavirus disease 2019 (COVID-19). However, some patients spend more than 5 days from disease onset before reporting to the Public Health Office. This study aimed to clarify the characteristics of patients with reporting delay. METHODS: This study included data from 12,399 patients with COVID-19 who reported to the Public Health Office from March 3rd, 2021 to June 30th, 2021. Patients were stratified into "linked" (n = 7,814) and "unlinked" (n = 4,585) cases depending on whether they were linked to other patients. A long reporting delay was defined as the difference between the onset and reporting dates of 5 days or more. Univariate and multivariate analyses were performed using log-binomial regression to identify factors related to long reporting delay, and prevalence ratios with corresponding 95% confidence intervals were calculated. RESULTS: The proportion of long reporting delay was 24.4% (1,904/7,814) and 29.3% (1,344/4,585) in linked and unlinked cases, respectively. Risks of long reporting delay among linked cases were living alone and onset on the day with a higher 7-day daily average confirmed cases or onset on weekends; whereas, risks for unlinked cases were age over 65 years, without occupation, and living alone. CONCLUSION: Our results suggest the necessity to establish a Public Health Office system that is less susceptible to the rapid increase in the number of patients, promotes educational activities for people with fewer social connections, and improves access to health care.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

IMPORTANCE: Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Macacine alphaherpesvirus 1 (B Virus) Infection in Humans, Japan, 2019.

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients.

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.

Construction of a recombinant vaccine expressing Nipah virus glycoprotein using the replicative and highly attenuated vaccinia virus strain LC16m8.

Nipah virus (NiV) is a highly pathogenic zoonotic virus that causes severe encephalitis and respiratory diseases and has a high mortality rate in humans (>40%). Epidemiological studies on various fruit bat species, which are natural reservoirs of the virus, have shown that NiV is widely distributed throughout Southeast Asia. Therefore, there is an urgent need to develop effective NiV vaccines. In this study, we generated recombinant vaccinia viruses expressing the NiV glycoprotein (G) or fusion (F) protein using the LC16m8 strain, and examined their antigenicity and ability to induce immunity. Neutralizing antibodies against NiV were successfully induced in hamsters inoculated with LC16m8 expressing NiV G or F, and the antibody titers were higher than those induced by other vaccinia virus vectors previously reported to prevent lethal NiV infection. These findings indicate that the LC16m8-based vaccine format has superior features as a proliferative vaccine compared with other poxvirus-based vaccines. Moreover, the data collected over the course of antibody elevation during three rounds of vaccination in hamsters provide an important basis for the clinical use of vaccinia virus-based vaccines against NiV disease. Trial Registration: NCT05398796.

Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice.

Dengue is a major health problem in tropical and subtropical regions. Some patients develop a severe form of dengue, called dengue hemorrhagic fever, which can be fatal. Severe dengue is associated with a transient increase in vascular permeability. A cytokine storm is thought to be the cause of the vascular leakage. Although there are various research reports on the pathogenic mechanism, the complete pathological process remains poorly understood. We previously reported that dengue virus (DENV) type 3 P12/08 strain caused a lethal systemic infection and severe vascular leakage in interferon (IFN)-α/ß and γ receptor knockout mice (IFN-α/ß/γRKO mice), and that blockade of TNF-α signaling protected mice. Here, we performed transcriptome analysis of liver and small intestine samples collected chronologically from P12/08-infected IFN-α/ß/γRKO mice in the presence/absence of blockade of TNF-α signaling and evaluated the cytokine and effector-level events. Blockade of TNF-α signaling mainly protected the small intestine but not the liver. Infection induced the selective expansion of IL-17A-producing Vγ4 and Vγ6 T cell receptor (TCR) γδ T cells in the small intestine, and IL-17A, together with TNF-α, played a critical role in the transition to severe disease via the induction of inflammatory cytokines such as TNF-α, IL-1ß, and particularly the excess production of IL-6. Infection also induced the infiltration of neutrophils, as well as neutrophil collagenase/matrix metalloprotease 8 production. Blockade of IL-17A signaling reduced mortality and suppressed the expression of most of these cytokines, including TNF-α, indicating that IL-17A and TNF-α synergistically enhance cytokine expression. Blockade of IL-17A prevented nuclear translocation of NF-κB p65 in stroma-like cells and epithelial cells in the small intestine but only partially prevented recruitment of immune cells to the small intestine. This study provides an overall picture of the pathogenesis of infection in individual mice at the cytokine and effector levels.

Long-term survival from progressive multifocal leukoencephalopathy in living-donor liver transplant recipient with preformed donor-specific antibody.

Intensive immunosuppression has enabled liver transplantation even in recipients with preformed donor-specific antibodies (DSA), an independent risk factor for graft rejection. However, these recipients may also be at high risk of progressive multifocal encephalopathy (PML) due to the comorbid immunosuppressed status. A 58-year-old woman presented with self-limited focal-to-bilateral tonic-clonic seizures 9 months after liver transplantation. She was desensitized using rituximab and plasma exchange before transplantation and was subsequently treated with steroids, tacrolimus, and everolimus after transplantation for her preformed DSA. Neurological examination revealed mild acalculia and agraphia. Cranial MRI showed asymmetric, cortex-sparing white matter lesions that increased over a week in the left frontal, left parietal, and right parieto-occipital lobes. Polymerase chain reaction (PCR) of the cerebrospinal fluid for the JC supported the diagnosis of PML. Immune reconstitution by reducing the immunosuppressant dose stopped lesion expansion, and PCR of the cerebrospinal fluid for the JC virus became negative. Graft rejection occurred 2 months after immune reconstitution, requiring readjustment of immunosuppressants. Forty-eight months after PML onset, the patient lived at home without disabling deficits. Intensive immunosuppression may predispose recipients to PML after liver transplantation with preformed DSA. Early immune reconstitution and careful monitoring of graft rejection may help improve outcomes.

Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection.

Manipulating viral genomes is an essential technique in reverse genetics and recombinant vaccine development. A strategy for manipulating large viral genomes involves introducing their entire genome into bacterial artificial chromosomes and employing Escherichia coli genetic tools. For sequence manipulation on bacterial artificial chromosomes (bacterial artificial chromosomes recombineering), a well-established method that relies on the Escherichia coli strain GS1783, and the template plasmid, pEPKan-S, is often used. This method, known as markerless DNA manipulation, allows for the generation of a recombinant bacterial artificial chromosome that does not retain the selection markers used during recombination. Although this method is highly innovative, there remains room for improvement as the plasmid is currently only available for positive selection. Additionally, differentiating true recombinants from false negatives often proves time-consuming. Consequently, an improved method for bacterial artificial chromosomes recombineering, which utilizes fluorescent proteins, has been developed. This method's core comprises three plasmids containing the I-SceI recognition site, antibiotic resistance genes (ampicillin, kanamycin, and zeocin), and fluorescent genes (YPet, mOrange, and mScarlet). The success or failure of Red recombination can be confirmed via fluorescent signals. To validate this method, the Lassa virus genes were introduced into the bacterial artificial chromosomes, containing the entire genome of the vaccinia virus strain LC16m8. Consequently, the expression of fluorescent protein genes contributed to positive selection, such as blue-white screening and counter-selection during the first and second Red recombination.

Nationwide Laboratory Surveillance of Progressive Multifocal Leukoencephalopathy in Japan: Fiscal Years 2011-2020.

Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disease caused by JC virus (JCV), predominantly affecting patients with impaired cellular immunity. PML is a non-reportable disease with a few exceptions, making national surveillance difficult. In Japan, polymerase chain reaction (PCR) testing for JCV in the cerebrospinal fluid (CSF) is performed at the National Institute of Infectious Diseases to support PML diagnosis. To clarify the overall profile of PML in Japan, patient data provided at the time of CSF-JCV testing over 10 years (FY2011-2020) were analyzed. PCR testing for 1537 new suspected PML cases was conducted, and 288 (18.7%) patients tested positive for CSF-JCV. An analysis of the clinical information on all individuals tested revealed characteristics of PML cases, including the geographic distribution, age and sex patterns, and CSF-JCV-positivity rates among the study subjects for each type of underlying condition. During the last five years of the study period, a surveillance system utilizing ultrasensitive PCR testing and widespread clinical attention to PML led to the detection of CSF-JCV in the earlier stages of the disease. The results of this study will provide valuable information not only for PML diagnosis, but also for the treatment of PML-predisposing conditions.

Case-Control Study of Long COVID, Sapporo, Japan.

We conducted a cross-sectional survey among SARS-CoV-2-positive persons and negative controls in Sapporo, Japan, to clarify symptoms of long COVID. We collected responses from 8,018 participants, 3,694 case-patients and 3,672 controls. We calculated symptom prevalence for case-patients at 2-3, 4-6, 7-9, 10-12, and 13-18 months after illness onset. We used logistic regression, adjusted for age and sex, to estimate the odds ratio (OR) for each symptom and control reference. We calculated symptom prevalence by stratifying for disease severity, age, and sex. At 4-18 months from illness onset, ORs for anosmia, ageusia, dyspnea, alopecia, and brain fog were consistently >1, whereas ORs for common cold-like, gastrointestinal, and dermatologic symptoms were <1. Time trend ORs increased for diminished ability to concentrate, brain fog, sleep disturbance, eye symptoms, and tinnitus. Clinicians should focus on systemic, respiratory, and neuropsychiatric symptoms among long COVID patients.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Evaluation of a novel severe combined immunodeficiency mouse model for antiviral drug evaluation against Chandipura virus infection.

Chandipura virus (CHPV) is a negative-sense single-stranded RNA virus known to cause fatal encephalitis outbreaks in the Indian subcontinent. The virus displays tropism towards the pediatric population and holds significant public health concerns. Currently, there is no specific, effective therapy for CHPV encephalitis. In this study, we evaluated a novel C.B-17 severe combined immunodeficiency (SCID) mouse model which can be used for pre-clinical antiviral evaluation. Inoculation of CHPV developed a lethal infection in our model. Plaque assay and immunohistochemistry detected increased viral loads and antigens in various organs, including the brain, spinal cord, adrenal glands, and whole blood. We further conducted a proof-of-concept evaluation of favipiravir in the SCID mouse model. Favipiravir treatment improved survival with pre-symptomatic (days 5-14) and post-symptomatic (days 9-18) treatment. Reduced viral loads were observed in whole blood, kidney/adrenal gland, and brain tissue with favipiravir treatment. The findings in this study demonstrate the utility of SCID mouse for in vivo drug efficacy evaluation and the potential efficacy of favipiravir against CHPV infection.

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.

Amodiaquine derivatives as inhibitors of severe fever with thrombocytopenia syndrome virus (SFTSV) replication.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral infection caused by a bandavirus in the family of Phenuiviridae, commonly known as SFTS virus (SFTSV). We have previously isolated SFTSV from blood samples of SFTS patients and established an antiviral assay system to identify selective inhibitors of SFTSV in vitro. Using the assay system, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. However, due to its insufficient antiviral activity, 98 amodiaquine derivatives were newly synthesized and examined for their anti-SFTSV activity. Among the derivatives, some compounds showed selective inhibitory effect on SFTSV replication in vitro. The 50% effective concentration (EC50) and cytotoxic concentration (CC50) of the most active compound (C-90) were 2.6 ± 0.6 and >50 µM, respectively. This EC50 value was comparable to or slightly better than that of favipiravir (4.1 ± 0.6 µM). On the other hand, pharmacokinetic studies in vivo revealed that C-90 was poor in its oral bioavailability in mice. Therefore, we further designed and synthesized derivatives and obtained 2 compounds with selective anti-SFTSV activity in vitro and improved pharmacokinetics in vivo.

Japanese Encephalitis Virus Genotypes 1 and 3 Isolation in Kochi, Japan.

Japanese encephalitis virus (JEV) is a mosquito-borne virus belonging to the JEV serocomplex within the genus Flavivirus, family Flaviviridae. It has 5 genotypes, G1-G5, based on the envelope (E) protein nucleotide sequence. JEV G3 circulated in Japan until the early 1990s when it was replaced by G1. JEV G3 was isolated from swine serum samples (sw/Kochi/1/2004) in the Kochi Prefecture, western Japan, in 2004. In addition, the 2018 isolates from pigs and cows (sw/Kochi/492/2018 and bo/Kochi/211/2018) in the same prefecture were identified as G3. The nucleotide sequencing results of the sw/Kochi/492/2018 and bo/Kochi/211/2018 polyprotein region differed from those of the sw/Kochi/1/2004 strain described in our previous report. Seven JEV isolates were identified as G1 in the same geographical area as that in this study. This result indicates that both JEV G1 and G3 are present in the Kochi area.