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ABSTRACT Helicobacter pylori vacuolating cytotoxin A (VacA) is an intracellular-acting protein exotoxin that induces mitochondrial dysfunction and energy depletion within host cells. Although exposure to VacA results in mitochondrial dysfunction, one recent study revealed that, following limited exposure to VacA, mitochondrial function and cellular ATP levels were restored in a time-dependent manner. Studies performed to address the mechanism by which host cells detect and respond to intracellular VacA identified the adenosine monophosphate (AMP)-activated protein kinase (AMPK) as a sensor of toxin-dependent alterations in cellular energy status. Activation of AMPK in response to VacA was demonstrated to orchestrate alterations in mitochondrial dynamics which resulted in restoration of mitochondrial function. Specifically, upregulation of dynamin-related protein 1 (Drp-1)-dependent mitochondrial fission resulted in reversible fragmentation of filamentous mitochondria and time-dependent reduction in mitochondrial-associated VacA, suggesting that fragmentation is important for removal of VacA from mitochondria. Cells with reduced levels of Drp-1 were more susceptible to VacA-dependent cell death, suggesting that mitochondrial dynamics is important for maintaining cell viability through the reduction in mitochondrial-associated toxin. Collectively, these studies support a model that cellular recovery and survival in response to VacA-dependent mitochondrial dysfunction is linked to host cell modulation of mitochondrial dynamics. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies. IMPORTANCE Persistent human gastric infection with Helicobacter pylori is the single most important risk factor for development of gastric malignancy, which is one of the leading causes of cancer-related deaths worldwide. An important virulence factor for Hp colonization and severity of gastric disease is the protein exotoxin VacA, which is secreted by the bacterium and modulates functional properties of gastric cells. VacA acts by damaging mitochondria, which impairs host cell metabolism through impairment of energy production. Here, we demonstrate that intoxicated cells have the capacity to detect VacA-mediated damage, and orchestrate the repair of mitochondrial function, thereby restoring cellular health and vitality. This study provides new insights into cellular recognition and responses to intracellular-acting toxin modulation of host cell function, which could be relevant for the growing list of pathogenic microbes and viruses identified that target mitochondria as part of their virulence strategies.

Abstract Background Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. Results In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. Conclusion The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.

Exosomes are nanosized extracellular vesicles secreted by various cell types, including those of the immune system, such as natural killer (NK) cells. They play a role in intercellular communication by transporting signal molecules between the cells. Recent studies have reported that NK cell-derived exosomes (NK-exo) contain cytotoxic proteins-induced cell death. However, the characteristics and potential functions of NK-exo, especially for the liver cancer are poorly understood. In this study, we investigated the anti-tumor effects of NK-exo in the primary liver cancer, hepatocellular carcinoma (HCC), using the orthotopic and subcutaneous tumor model. We found that NK-exo expressed both typical exosomal markers (e.g. CD63, CD81, and Alix) and cytotoxic proteins (e.g. perforin, granzyme B, FasL, and TRAIL). NK-exo were selectively taken up by HCC cells (e.g. Hep3B, HepG2, and Huh 7). Interestingly, Hep3B cells induced the highest cytotoxicity compared with HepG2 and Huh7 cells, and substantially enhanced the apoptosis by NK-exo. Furthermore, we demonstrated that NK-exo inhibited the phosphorylation of serine/threonine protein kinases (e.g. AKT and ERK1/2), and enhanced the activation of specific apoptosis markers (e.g. caspase-3, -7, -8, -9, and PARP) in Hep3B cells. NK-exo also exhibit the active targeting ability and potent therapeutic effects in both orthotopic and subcutaneous HCC mouse models. Overall, these results suggest that NK-exo indicate strong anti-tumor effects in HCC, which are mediated by novel regulatory mechanisms involved in serine/threonine kinase pathway-associated cell proliferation and caspase activation pathway-associated apoptosis.

A 14-day-old female baby was admitted to the neonatal Intensive Care Unit (ICU) with complaints of fever for one week, along with reduced intake of feeds and weight loss. Routine investigations, blood culture, Cerebrospinal Fluid (CSF) routine analysis, and CSF culture were performed. Both cultures grew non typhoidal Salmonella enterica subspecies enterica. Magnetic Resonance Imaging (MRI) brain with contrast revealed leptomeningeal enhancement and basal exudates, both suggestive of meningitis, as well as ventriculitis and arachnoiditis. The baby was treated with intravenous Ceftriaxone and Meropenem. Follow-up CSF analysis showed improvement, and the cultures were sterile.

Abstract Cisplatin is effective against many types of carcinoma. However, a high rate of renal damage is a clinical problem. Thus, there is a need to establish a method to prevent it. Although various compounds have been reported to be effective against cisplatin‐induced renal injury, there are no examples of their clinical application. Therefore, we attempted to search for prophylactic agents with a high potential for clinical application. We used Cascade Eye to identify genes that are altered during cisplatin‐induced renal injury, Library of Integrated Network‐based Cellular Signatures (LINCS) to identify drugs that inhibit changes in gene expression, and a large database of spontaneous adverse drug reaction reports to identify drugs that could prevent cisplatin‐induced kidney injury in clinical practice. In total, 10 candidate drugs were identified. Using the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS), we identified drugs that reduce cisplatin‐induced kidney injury. Fenofibrate was selected as a candidate drug to prevent cisplatin‐induced kidney injury based on the FAERS analysis. A model was used to evaluate the efficacy of fenofibrate against cisplatin‐induced renal injury. Studies using HK2 cells and mouse models showed that fenofibrate significantly inhibited cisplatin‐induced renal injury but did not inhibit the antitumor effect of cisplatin. Fenofibrate is a candidate prophylactic drug with high clinical applicability for cisplatin‐induced renal injury. Analysis of data from multiple big databases will improve the search for novel prophylactic drugs with high clinical applicability. For the practical application of these findings, evaluation in prospective controlled trials is necessary.

Previous studies have suggested that change-related cortical responses are phenomena similar to the onset response and could be applied to the loudness dependence of auditory evoked potential (LDAEP) paradigm. In the present study, we examined the relationship between LDAEP and the change-related response using electroencephalography findings in 50 healthy subjects. There were five conditions (55, 65, 75, 85, and 95 dB) for LDAEP and five similar conditions (abrupt sound pressure increase from 70 to 75, 80, 85, 90, and 95 dB) for the change-related response. Both the onset and abrupt sound pressure increase evoked a triphasic response with peaks at approximately 50 (P50), 100 (N100), and 200 (P200) ms. We calculated the peak-to-peak amplitudes for P50/N100 and N100/P200. Medians and slopes for P50/N100 and N100/P200 amplitudes were calculated and compared between the two measures. Results revealed a significant correlation for both the slope and median for P50/N100 (r = 0.36, 0.37, p = 1.0 × 10-2, 7.9 × 10-3), N100/P200 (r = 0.40, 0.34, p = 4.0 × 10-3, 1.6 × 10-2), and P50/N100/P200 (r = 0.36, 0.35, p = 1.0 × 10-2, 1.3 × 10-2). These results suggested that the change-related response and LDAEP shared generation mechanisms at least partially.

Since Mongolian gerbils are broadly susceptible to hepatitis E virus (HEV), including genotypes 1, 4, 5, and 8 (HEV-1, HEV-5, HEV-5, and HEV-8) and rat HEV, they are a useful small animal model for HEV. However, we have observed that the subtypes HEV-3k and HEV-3ra in genotype 3 HEV (HEV-3) were not infected efficiently in the gerbils. A small-animal model for HEV-3 is also needed since HEV-3 is responsible for major zoonotic HEV infections. To investigate whether gerbils can be used as animal models for other subtypes of HEV-3, we injected gerbils with five HEV-3 subtypes (HEV-3b, -3e, -3f, -3k, and -3ra) and compared the infectivity of the subtypes. We detected viral RNA in the gerbils' feces. High titers of anti-HEV IgG antibodies in serum were induced in all HEV-3b/ch-, HEV-3f-, and HEV-3e-injected gerbils. Especially, the HEV-3e-injected animals released high levels of viruses into their feces for an extended period. The virus replication was limited in the HEV-3b/wb-injected and HEV-3k-injected groups. Although viral RNA was detected in HEV-3ra-injected gerbils, the copy numbers in fecal specimens were low; no antibodies were detected in the sera. These results indicate that although HEV-3's infectivity in gerbils depends on the subtype and strain, Mongolian gerbils have potential as a small-animal model for HEV-3. A further comparison of HEV-3e with different genotype strains (HEV-4i and HEV-5) and different genera (rat HEV) revealed different ALT elevations among the strains, and liver damage occurred in HEV-4i- and HEV-5-infected but not HEV-3e- or rat HEV-infected gerbils, demonstrating variable pathogenicity across HEVs from different genera and genotypes in Mongolian gerbils. HEV-4i- and HEV-5-infected Mongolian gerbils might be candidate animal models to examine HEV's pathogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected or isolated from domestic cats. It is unclear whether cats play an important role in the SARS-CoV-2 transmission cycle. In this study, we examined the susceptibility of cats to SARS-CoV-2, including wild type and variants, by animal experiments. Cats inoculated with wild type, gamma, and delta variants secreted a large amount of SARS-CoV-2 for 1 week after the inoculation from nasal, oropharyngeal, and rectal routes. Only 100 TCID 50 of virus could infect cats and replicate well without severe clinical symptoms. In addition, one cat inoculated with wild type showed persistent virus secretion in feces for over 28 days post-inoculation (dpi). The titer of virus-neutralizing (VN) antibodies against SARS-CoV-2 increased from 11 dpi, reaching a peak at 14 dpi. However, the omicron variant could not replicate well in cat tissues and induced a lower titer of VN antibodies. It is concluded that cats were highly susceptible to SARS-CoV-2 infection, but not to the Omicron Variant, which caused the attenuated pathogenicity., (© 2024. The Author(s).)

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 10 9 to 3.4 × 10 11 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection., Importance: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material., Competing Interests: The authors declare no conflict of interest.

Bacterial pathogens utilize the factors of their hosts to infect them, but which factors they exploit remain poorly defined. Here, we show that a pathogenic Salmonella enterica serovar Typhimurium (STm) exploits host polyamines for the functional expression of virulence factors. An STm mutant strain lacking principal genes required for polyamine synthesis and transport exhibited impaired infectivity in mice. A polyamine uptake-impaired strain of STm was unable to inject effectors of the type 3 secretion system into host cells due to a failure of needle assembly. STm infection stimulated host polyamine production by increasing arginase expression. The decline in polyamine levels caused by difluoromethylornithine, which inhibits host polyamine production, attenuated STm colonization, whereas polyamine supplementation augmented STm pathogenesis. Our work reveals that host polyamines are a key factor promoting STm infection, and therefore a promising therapeutic target for bacterial infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Miki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human T cell leukemia/T-lymphotropic virus type 1 (HTLV-1) infection occurs by cell-to-cell transmission and can induce fatal adult T cell leukemia. Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of 10 vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 10 8 HTLV-1-producing cells. In contrast, the 2 vaccinated macaques without NAb induction and 10 unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8 + cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development., Competing Interests: Declaration of interests T.M. is an inventor on Patent Cooperation Treaty (PCT) application for SeV-NVP-EnvF vaccine., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro . DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate., Competing Interests: D.T., K.Ma., A.U., Y.W., and M.I are employees of VLP Therapeutics, Inc.; J.F.S. is an employee of and holds stocks in VLP Therapeutics, Inc. W.A. is a board member and employee of and holds stocks in VLP Therapeutics, Inc. W.A. is an inventor on a related vaccine patent. The remaining authors declare no competing interest.

Hyperuricemia (HUA) is a symptom of high blood uric acid (UA) levels, which causes disorders such as gout and renal urinary calculus. Prolonged HUA is often associated with hypertension, atherosclerosis, diabetes mellitus, and chronic kidney disease. Studies have shown that gut microbiota (GM) affect these chronic diseases. This study aimed to determine the relationship between HUA and GM. The microbiome of 224 men and 254 women aged 40 years was analyzed through next-generation sequencing and machine learning. We obtained GM data through 16S rRNA-based sequencing of the fecal samples, finding that alpha-diversity by Shannon index was significantly low in the HUA group. Linear discriminant effect size analysis detected a high abundance of the genera Collinsella and Faecalibacterium in the HUA and non-HUA groups. Based on light gradient boosting machine learning, we propose that HUA can be predicted with high AUC using four clinical characteristics and the relative abundance of nine bacterial genera, including Collinsella and Dorea. In addition, analysis of causal relationships using a direct linear non-Gaussian acyclic model indicated a positive effect of the relative abundance of the genus Collinsella on blood UA levels. Our results suggest abundant Collinsella in the gut can increase blood UA levels., (© 2024. The Author(s).)

Interstitial fluid flow is a feature of many solid tumors. In vitro experiments have shown that such fluid flow can direct tumor cell movement upstream or downstream depending on the balance between the competing mechanisms of tensotaxis (cell migration up stress gradients) and autologous chemotaxis (downstream cell movement in response to flow-induced gradients of self-secreted chemoattractants). In this work we develop a probabilistic-continuum, two-phase model for cell migration in response to interstitial flow. We use a kinetic description for the cell velocity probability density function, and model the flow-dependent mechanical and chemical stimuli as forcing terms that bias cell migration upstream and downstream. Using velocity-space averaging, we reformulate the model as a system of continuum equations for the spatiotemporal evolution of the cell volume fraction and flux in response to forcing terms that depend on the local direction and magnitude of the mechanochemical cues. We specialize our model to describe a one-dimensional cell layer subject to fluid flow. Using a combination of numerical simulations and asymptotic analysis, we delineate the parameter regime where transitions from downstream to upstream cell migration occur. As has been observed experimentally, the model predicts downstream-oriented chemotactic migration at low cell volume fractions, and upstream-oriented tensotactic migration at larger volume fractions. We show that the locus of the critical volume fraction, at which the system transitions from downstream to upstream migration, is dominated by the ratio of the rate of chemokine secretion and advection. Our model also predicts that, because the tensotactic stimulus depends strongly on the cell volume fraction, upstream, tensotaxis-dominated migration occurs only transiently when the cells are initially seeded, and transitions to downstream, chemotaxis-dominated migration occur at later times due to the dispersive effect of cell diffusion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The human intestinal microbiota that comprise over 1,000 species thrive in dark and anaerobic environments. They are recognized for the production of diverse low-molecular-weight metabolites crucial to human health and diseases. Carotenoids, low-molecular-weight pigments known for their antioxidative activity, are delivered to humans through oral intake. However, it remains unclear whether human intestinal bacteria biosynthesize carotenoids as part of the in-situ microbiota. In this study, we investigated carotenoid synthesis genes in various human gut and probiotic bacteria. As a result, novel candidates, the crtM and crtN genes, were identified in the carbon monoxide-utilizing gut anaerobe Eubacterium limosum and the lactic acid bacterium Leuconostoc mesenteroides subsp. mesenteroides . These gene candidates were isolated, introduced into Escherichia coli , which synthesized a carotenoid substrate, and cultured aerobically. Structural analysis of the resulting carotenoids revealed that the crtM and crtN gene candidates of E. limosum and L. mesenteroides mediate the production of 4,4'-diaponeurosporene through 15- cis -4,4'-diapophytoene. Evaluation of the crtE -homologous genes in these bacteria indicated their non-functionality for C 40 -carotenoid production. E. limosum and L. mesenteroides , along with the known carotenogenic lactic acid bacterium Lactiplantibacillus plantarum , were observed to produce no carotenoids under strictly anaerobic conditions. The two lactic acid bacteria synthesized detectable levels of 4,4'-diaponeurosporene under semi-aerobic conditions. The findings highlight that the obligate anaerobe E. limosum retains aerobically functional C 30 -carotenoid biosynthesis genes, potentially with no immediate self-utility, suggesting an evolutionary direction in carotenoid biosynthesis. (229 words)., Competing Interests: All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this research article., (© 2024 The Author(s).)

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 + Ifnar flox/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 + Ifnar flox/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/β 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kurosu, Sakai, Ami, Shimojima, Yoshikawa, Fukushi, Nagata, Suzuki, Ebihara and Saijo.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sakai, Mura, Kuwabara, Kagimoto, Sakurai, Morimoto, Park, Shimojima, Nagata, Ami, Yoshikawa, Iwata-Yoshikawa, Fukushi, Watanabe, Kurosu, Okutani, Kimura, Imaoka, Saijo, Morikawa, Suzuki and Maeda.)

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/β and γ receptor knockout mice (IFN-α/β/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/β/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-1β, 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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kurosu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: We performed our standard air leak, leak percentage, and cuff leak percentage tests in pediatric patients intubated with microcuff pediatric tracheal tubes (MPTTs) just before extubation. We examined the association between test findings and the subsequent occurrence of post-extubation laryngeal edema (PLE)., Design: Prospective, single-center, observational study., Setting: PICU (June 1, 2020 to May 31, 2021)., Patients: Pediatric patients intubated and scheduled for extubation during the day shift in the PICU., Interventions: Multiple pre-extubation leak tests were performed on each patient immediately before extubation. In our center, the standard leak test is positive if a leak is audible at 30 cm H 2 O applied pressure with the MPTT cuff deflated. Two other tests were calculated in the pressure control-assist control ventilator mode using the following formulas: leak percentage with deflated cuff = (inspiratory tidal volume [V t ]-expiratory V t ) × 100/inspiratory V t ; cuff leak percentage = (expiratory V t with inflated cuff-expiratory V t with deflated cuff) × 100/expiratory V t with inflated cuff., Measurements and Main Results: The diagnostic criteria for PLE was made by at least two healthcare professionals and included upper airway stricture with stridor-requiring nebulized epinephrine. Eighty-five pediatric patients (< 15 yr) who had been intubated for at least 12 hours using the MPTT were included. Positive rates for the standard leak, leak percentage (cutoff 10%), and cuff leak percentage (cutoff 10%) tests were 0.27, 0.20, and 0.64, respectively. The standard leak, leak percentage, and cuff leak tests showed sensitivities of 0.36, 0.27, and 0.55, respectively; and specificities of 0.74, 0.81, and 0.35, respectively. PLE occurred in 11 of 85 patients (13%), and there were no instances of needing reintubation., Conclusions: The pre-extubation leak tests in current practice for intubated pediatric patients in the PICU all lack diagnostic accuracy for PLE., Competing Interests: Dr. Fujii disclosed work for hire. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

Human T-cell leukemia virus type 1 (HTLV-1) induces chronic asymptomatic latent infection with a substantial proviral load but without significant viral replication in vivo . Cumulative studies have indicated involvement of CD8-positive (CD8 + ) cells, including virus-specific CD8 + T cells in the control of HTLV-1 replication. However, whether HTLV-1 expression from latently infected cells in vivo occurs in the absence of CD8 + cells remains unclear. Here, we examined the impact of CD8 + cell depletion by monoclonal anti-CD8 antibody administration on proviral load in HTLV-1-infected cynomolgus macaques. Five cynomolgus macaques were infected with HTLV-1 by inoculation with HTLV-1-producing cells. Administration of monoclonal anti-CD8 antibody in the chronic phase resulted in complete depletion of peripheral CD8 + T cells for approximately 2 months. All five macaques showed an increase in proviral load following CD8 + cell depletion, which peaked just before the reappearance of peripheral CD8 + T cells. Tax-specific CD8 + T-cell responses were detected in these recovered CD8 + T cells. Importantly, anti-HTLV-1 antibodies also increased after CD8 + cell depletion, indicating HTLV-1 antigen expression. These results provide evidence indicating that HTLV-1 can proliferate from the latent phase in the absence of CD8 + cells and suggest that CD8 + cells are responsible for the control of HTLV-1 replication. IMPORTANCE HTLV-1 can cause serious diseases such as adult T-cell leukemia (ATL) in humans after chronic asymptomatic latent infection with substantial proviral load. Proviruses are detectable in peripheral lymphocytes in HTLV-1 carriers, and the association of a higher proviral load with a higher risk of disease progression has been observed. However, neither substantial viral structural protein expression nor viral replication was detectable in vivo . Cumulative studies have indicated involvement of CD8 + cells, including virus-specific CD8 + T cells in the control of HTLV-1 replication. In the present study, we showed that CD8 + cell depletion by monoclonal anti-CD8 antibody administration results in HTLV-1 expression and an increase in proviral load in HTLV-1-infected cynomolgus macaques. Our results indicate that HTLV-1 can proliferate in the absence of CD8 + cells, suggesting that CD8 + cells are responsible for the control of HTLV-1 replication. This study provides insights into the mechanism of virus-host immune interaction in latent HTLV-1 infection., Competing Interests: The authors declare no conflict of interest.

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 10 7 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 10 8 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 10 4 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.

Polyamine intake has been reported to help extend the lifespan of animals. Fermented foods contain high concentrations of polyamines, produced by fermenting bacteria. Therefore, the bacteria, isolated from fermented foods that produce large amounts of polyamines, are potentially used as a source of polyamines for humans. In this study, the strain Levilactobacillus brevis FB215, which has the ability to accumulate approximately 200 µM of putrescine in the culture supernatant, was isolated from fermented foods, specifically the Blue Stilton cheese. Furthermore, L. brevis FB215 synthesized putrescine from agmatine and ornithine, which are known polyamine precursors. When cultured in the extract of Sakekasu, a byproduct obtained during the brewing of Japanese rice wine containing high levels of both agmatine and ornithine, L. brevis FB215 grew to OD 600 = 1.7 after 83 h of cultivation and accumulated high concentrations (~1 mM) of putrescine in the culture supernatant. The fermentation product also did not contain histamine or tyramine. The Sakekasu-derived ingredient fermented by the food-derived lactic acid bacteria developed in this study could contribute to increasing polyamine intake in humans.

The live-attenuated oral polio vaccine has long been used as the standard for polio prevention, but in order to minimize the emergence of pathogenic revertants, the inactivated polio vaccine (IPV), which is administered intramuscularly or subcutaneously, is being increasingly demanded worldwide. However, there is a global shortage of IPV, and its cost is an obstacle in developing countries. Therefore, dose-sparing with intradermal administration of IPV has been investigated. In this study, rats were immunized by intradermal (ID) and intramuscular (IM) administration of Sabin-derived inactivated polio vaccine (sIPV) produced in Japan, and the immune responses were evaluated. The results showed that one-fifth (1/5)-dose of ID administration yielded neutralizing antibody titers comparable to the full-dose IM administration, whereas 1/5-dose of IM administration was less effective than the full dose. Furthermore, a vertical puncture-type ID injection device (Immucise) that was originally developed for humans was modified for rats, resulting in successful and stable ID administration into the thin skin of rats. Based on these results, the ID administration of sIPV using Immucise in clinical use is expected to offer benefits such as reduced amounts of vaccine per dose, cost-effectiveness, and thereby the feasibility of vaccination for more people., Competing Interests: Declaration of competing interest Eriko Itoh, Sakiko Shimizu, and Yoichiro Iwase are permanent employees of TERUMO CORPORATION, Japan. Yasushi Ami is a reappointed researcher of NIID, and Yuichi Someya is a permanent researcher of NIID. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837C RNA , T2885C RNA , and T2837C/T2885C RNA , replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836T RNA , accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Introduction: Although outbreaks of parainfluenza virus type 3 (PIV-3) have been reported in children, to our knowledge none have been reported in a nursery school. As the symptoms of PIV-3 infection are similar to those of COVID-19 infection, accurate diagnosis of PIV-3 and other respiratory viruses is important during the COVID-19 pandemic., Aims: We experienced an outbreak of upper respiratory symptoms at a nursery school in Miyagi Prefecture, Japan, from 29/5/2021 to 13/6/2021 and aimed to determine the causative organism(s)., Methods: A multiplex polymerase chain reaction (PCR) assay which enabled rapid detection of a variety of causative microorganisms of respiratory tract infections was used to analyse 13 nasopharyngeal swabs collected during the outbreak. Infection Prevention and control measures were implemented to prevent further spread of infection., Results: All 13 samples were positive for PIV-3 infection. 2 of the 13 samples were also positive for rhinovirus/enterovirus and 1 sample was also positive rhinovirus/enterovirus and coronavirus NL 63. No samples were positive for SARS-CoV-2., Discussion: Children in school settings are especially vulnerable to respiratory viral infections, including COVID-19. Children under two years are unable to wear masks reliably, and the COVID-19 vaccine was approved only for older children. Multiplex PCR assays can be used for the rapid diagnosis of respiratory infections., Conclusion: We identified an outbreak of PIV-3 in a nursery school during the COVID-19 pandemic. The investigation of the outbreak highlighted that it was important not to overlook other respiratory infections including PIV-3 during the COVID-19 pandemic. The multiplex PCR assay provided rapid and accurate diagnosis of the causative organisms in the outbreak and helped to direct appropriate interventions to control the outbreak., (© 2022 The Authors.)

Although cell culture systems for hepatitis E virus (HEV) have been established by using cell lines such as PLC/PRF/5 and A549, small-animal models for this virus are limited. Since Mongolia gerbils are susceptible to genotype 1, 3 and 4 HEV (HEV-1, HEV-3 and HEV4), we intraperitoneally inoculated Mongolia gerbils with HEV-5, HEV-7, HEV-8, rabbit HEV or rat HEV in addition to the above three genotypes to investigate the infectivity and to assess whether Mongolia gerbil is an appropriate animal model for HEV infection. The results indicated that (i) HEV-5 and rat HEV were effectively replicated in the Mongolia gerbils in the same manner as HEV-4: large amounts of the viral RNA were detected in the feces and livers, and high titers of the serum anti-HEV IgG antibodies were induced in all animals. The feces were shown to contain HEV that is infectious to naïve gerbils. Furthermore, HEV-4, HEV-5 and rat HEV were successfully transmitted to the gerbils by oral inoculation. (ii) Although the viral RNA and serum anti-HEV IgG antibodies were detected in all animals inoculated with HEV-1 and HEV-8, both titers were low. The viral RNA was detected in the feces collected from two of three HEV-3-inoculated, and one of three HEV-7-inoculated gerbils, but the titers were low. The serum antibody titers were also low. The viruses excreted into the feces of HEV-1-, HEV-3-, HEV-7- and HEV-8-inoculated gerbils failed to infect naïve Mongolia gerbils. (iii) No infection sign was observed in the rabbit HEV-inoculated gerbils. These results demonstrated that Mongolia gerbils are broadly susceptible to HEV, and their degree of sensitivity was dependent on the genotype. Mongolia gerbils were observed to be susceptible to not only HEVs belonging to HEV-A but also to rat HEV belonging to HEV-C1, and thus Mongolia gerbil could be useful as a small-animal model for cross-protection experiments between HEV-A and HEV-C1. Mongolia gerbils may also be useful for the evaluation of the efficacy of vaccines against HEV.

Human parechoviruses (HPeVs) are being increasingly recognized as pathogens that cause mild-to-life-threatening diseases in children and adults. Recently, nucleic acid detection has become the mainstream method for pathogen detection. However, virus isolation is important for virus detection and further virologic characterization studies, and securing human pathogenic virus bioresources. We recently explored conventional cell lines suitable for human sapovirus isolation and accidentally identified a human duodenal cell line, HuTu80, that supported efficient growth of human parechovirus type 3 (HPeV-3) with clear cytopathic effects (CPE). Subsequently, we confirmed that all representative prototype HPeV type 1-6 strains were propagated efficiently in HuTu80 cells with clear CPE within 4 days. Another human ileocecal cell line, HCT-8 (HRT-18), also supports HPeV propagation except for HPeV-3. Titer values in HuTu80 and HCT-8 reached approximately 6.83-8.83 and 6.50-8.17 log 10 50% tissue culture infectious dose/50 μL, respectively, when inoculated with multiplicity of infection of 0.0025. Previously reported cell lines likely support HPeV types 1-6 with different efficiency, especially for HPeV-3. In summary, HuTu80 can be used as an additional cell line for HPeV isolation, propagation with a clear CPE to produce a high titer value and for the virus neutralization assays.

Rat hepatitis E virus (rat HEV) was first identified in wild rats and was classified as the species Orthohepevirus &nbsp; C in the genera Orthohepevirus , which is genetically different from the genotypes HEV-1 to HEV-8, which are classified as the species Orthohepevirus &nbsp; A . Although recent reports suggest that rat HEV transmits to humans and causes hepatitis, the infectivity of rat HEV to non-human primates such as cynomolgus and rhesus monkeys remains controversial. To investigate whether rat HEV infects non-human primates, we inoculated one cynomolgus monkey and five rhesus monkeys with a V-105 strain of rat HEV via an intravenous injection. Although no significant elevation of alanine aminotransferase (ALT) was observed, rat HEV RNA was detected in fecal specimens, and seroconversion was observed in all six monkeys. The partial nucleotide sequences of the rat HEV recovered from the rat HEV-infected monkeys were identical to those of the V-105 strain, indicating that the infection was caused by the rat HEV. The rat HEV recovered from the cynomolgus and rhesus monkeys successfully infected both nude and Sprague-Dawley rats. The entire rat HEV genome recovered from nude rats was identical to that of the V-105 strain, suggesting that the rat HEV replicates in monkeys and infectious viruses were released into the fecal specimens. These results demonstrated that cynomolgus and rhesus monkeys are susceptible to rat HEV, and they indicate the possibility of a zoonotic infection of rat HEV. Cynomolgus and rhesus monkeys might be useful as animal models for vaccine development.

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced, viral-antigen-specific CD8 + T cell responses. Our results indicate that CD8 + T cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8 + T cell responses to contribute to COVID-19 containment., Competing Interests: H.I., K.K., R.S., and T.M. are the inventors on Patent Cooperation Treaty (PCT) application for SeV-NME vaccine. The authors declare no other competing interests., (© 2022 The Author(s).)

Rabbit hepatitis E virus (HEV) has been detected among rabbits and recently isolated from immunocompromised patients, suggesting zoonotic transmission. In this study, HEV infection among feral rabbits (Oryctolagus cuniculus) was assessed by detection of anti-HEV antibodies and HEV RNA. The prevalence of anti-HEV antibodies in sera was of 33 % (20/60) and HEV RNA was detected from only one of fecal swabs (1.7 %, 1/58). Furthermore, one naïve rabbit was intravenously inoculated with the suspension of the HEV-positive fecal specimen, exhibiting persistent HEV shedding in feces, intermittent viremia, seroconversion to anti-HEV IgM and IgG, and high alanine aminotransferase (ALT) values, indicating persistent HEV infection. The isolate JP-59 had a length of 7,282 bp excluding a poly (A) tail and possessed the characteristic 93 bp-insertion in ORF1. Phylogenetic analysis indicated that JP-59 formed a cluster with other rabbit HEV isolates from rabbits and human origin. The JP-59 shared the nucleotide sequence identities less than 87 % with other rabbit HEVs, suggesting that a novel rabbit HEV strain was circulating in Japan., (Copyright © 2021 Elsevier B.V. All rights reserved.)