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.

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.

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.

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 and 0.32, respectively, whereas those for users of books, commercial video sites, Facebook, and "personal blog or bulletin board system" were 3.34, 2.22, 2.36, and 4.81, respectively. Social media use among older or male participants was associated with lower vaccine uptake.

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.

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.

Lethal severe fever with thrombocytopenia syndrome virus infection causes systemic germinal centre failure and massive T cell apoptosis in cats.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is a fatal viral disease characterized by high fever, thrombocytopenia, leukopenia, and multi-organ haemorrhage. Disruption of the humoral immune response and decreased lymphocyte numbers are thought to contribute to the disease severity. These findings have been obtained through the analysis of peripheral blood leukocytes in human patients, whereas analysis of lymph nodes has been limited. Thus, in this study, we characterized the germinal centre response and apoptosis in the lymph nodes of cats with fatal SFTS, because SFTS in cats well mimics the pathology of human SFTS. Methods: Lymph node tissue sections collected during necropsy from seven fatal SFTS patients and five non-SFTS cases were used for histopathological analysis. Additionally, lymph node tissue sections collected from cats with experimental infection of SFTS virus (SFTSV) were also analysed. Results: In the lymphoid follicles of cats with SFTS, a drastic decrease in Bcl6- and Ki67-positive germinal centre B cells was observed. Together, the number of T cells in the follicles was also decreased in SFTS cases. In the paracortex, a marked increase in cleaved-caspase3 positivity was observed in T cells. These changes were independent of the number of local SFTS virus-positive cell. Furthermore, the analysis of cats with experimental SFTSV infection revealed that the intrafollicular Bcl6- and CD3-positive cell numbers in cats with low anti-SFTSV antibody production were significantly lower than those in cats with high anti-SFTSV antibody production. Discussion: These results suggest that dysfunction of the humoral response in severe SFTS was caused by the loss of germinal centre formation and massive apoptosis of T cells in the lymph nodes due to systemically circulating viruses.