Two-step evolution of HIV-1 budding system leading to pandemic in the human population.

The pandemic HIV-1, HIV-1 group M, emerged from a single spillover event of its ancestral lentivirus from a chimpanzee. During human-to-human spread worldwide, HIV-1 diversified into multiple subtypes. Here, our interdisciplinary investigation mainly sheds light on the evolutionary scenario of the viral budding system of HIV-1 subtype C (HIV-1C), a most successfully spread subtype. Of the two amino acid motifs for HIV-1 budding, the P(T/S)AP and YPxL motifs, HIV-1C loses the YPxL motif. Our data imply that HIV-1C might lose this motif to evade immune pressure. Additionally, the P(T/S)AP motif is duplicated dependently of the level of HIV-1 spread in the human population, and >20% of HIV-1C harbored the duplicated P(T/S)AP motif. We further show that the duplication of the P(T/S)AP motif is caused by the expansion of the CTG triplet repeat. Altogether, our results suggest that HIV-1 has experienced a two-step evolution of the viral budding process during human-to-human spread worldwide.

The incorporation of extracellular vesicle markers varies among vesicles with distinct surface charges.

Extracellular vesicles (EVs) are important mediators of intercellular communication. However, the methods available for distinguishing the heterogeneity of secreted EVs and isolating and purifying them are limited. This study introduced a HiBiT-tag to detect various EV markers, including CD63, CD9, Epidermal Growth Factor Receptor (EGFR), Flotilin1, and Syndecan-1, and investigated whether these marker-containing vesicles were capable of binding to differently charged column carriers. Four column carriers, Diethylaminoethyl (DEAE), Capto Adhere, Blue and Heparin, showed affinity for CD63 containing EVs, but their elution patterns varied. Furthermore, we observed that the elution patterns of the EV markers differed among vesicles with distinct surface charges when a DEAE column was used. This suggests that the incorporation of EV markers varied between these vesicles. The markers showed different subcellular localizations, indicating that the site of vesicle formation may contribute to the production of vesicles with varying charges and marker incorporation. These findings may have implications for the development of methods to purify homogeneous EVs, which could be useful in EV-mediated drug delivery systems.

Purification of hepatitis C virus core protein in non-denaturing condition.

Hepatitis C virus (HCV) is the major cause of chronic hepatitis and hepatocellular carcinoma. Among its structural proteins, the HCV core protein has been implicated in liver disease. Understanding the role of HCV core proteins in viral diseases is crucial to elucidating disease mechanisms and identifying potential drug targets. However, purification challenges hinder the comprehensive elucidation of the structure and biochemical properties of HCV core proteins. In this study, we successfully solubilized bacterially expressed core protein using a high-salt and detergent-containing buffer and bypassed the denaturing-refolding process. Size-exclusion chromatography revealed three distinct peaks in the HCV-infected cell lysate, with the bacterially expressed soluble core protein corresponding to its second peak. Using a combination of affinity, size exclusion, and multi-modal chromatography purification techniques, we achieved a purity of > 95% for the core protein. Analytical ultracentrifugation revealed monomer formation in the solution. Far UV Circular dichroism spectroscopy identified 25.53% alpha helices and 20.26% beta sheets. These findings strongly suggest that the purified core proteins retained one of the native structures observed in HCV-infected cells.

Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells.

IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.

N-linked glycosylation of flavivirus E protein contributes to viral particle formation.

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

HIV-1 Integrase Inhibitor, Dihydroobionin B, and the Investigation of Its Extraordinary Specific Rotation.

Dihydroobionin B (1), a chiral congener of known obionin B, was isolated from Pseudocoleophoma sp. KT4119, a freshwater fungus collected from a submerged wood block in Kochi Prefecture, Japan, in 2020. The planar structure of 1 was characterized by mass and NMR spectral analysis and confirmed by density functional theory (DFT)-based chemical shift calculations. Its absolute structure was determined by electronic circular dichroism spectral analysis. Notably, 1 exhibited an extraordinarily large specific rotation [[α]20D +1080 (c 0.056, CHCl3)], which was verified by DFT-based specific rotation calculations. However, these calculations indicated that the sign of the specific rotation based on static analysis was insufficient to determine the absolute configuration in this case. Furthermore, Pseudocoleophoma KT4119 produced coleophomapyrones A (2) and B (3) and coleophomaldehyde A (4). While this is the first report of 2 isolated from a natural source, it has also been prepared previously using a synthetic approach. Compound 1 potently inhibited HIV type 1 integrase (IC50 = 0.44 µM) without significant cytotoxicity. Finally, docking experiments were conducted to propose a plausible mechanism for the behavior of 1.

Protein Pull-down Assay Using HiBiT-tag-dependent Luciferase Activity Measurement.

Co-immunoprecipitation or pull-down assays are frequently used to analyze protein-protein interactions. In these experiments, western blotting is commonly used to detect prey proteins. However, sensitivity and quantification problems remain in this detection system. Recently, the HiBiT-tag-dependent NanoLuc luciferase system was developed as a highly sensitive detection system for small amounts of proteins. In this report, we introduce the method of using HiBiT technology for the detection of prey protein in a pull-down assay. Using this protocol, we demonstrate the formation of a ternary complex consisting of Japanese encephalitis virus NS4B and two host factors, namely valosin-containing protein, and nuclear protein localization protein 4, which is a critical biological event during flavivirus replication in cells.

Integrasone Derivatives Isolated from Lepteutypa sp. KT4162 and Their Anti-HIV-1 Integrase Activity.

Five integrasone derivatives, integrasone C (1), isointegrasone C (2), integrasone D1 (3), integrasone D2 (4), and integrasone E (5), were isolated from the culture broth of Lepteutypa sp. KT4162. Neither conventional NMR analyses nor DFT (density functional theory)-based computationally assisted chemical shift discussions were sufficient to elucidate the relative configuration of the 1,4-epoxydiol moiety. A combined analysis using the calculated nJCH values and HMBC spectra was helpful to establish the relative configuration. The absolute configurations of 1-5 were determined using DFT-based ECD (electronic circular dichroism) spectral analysis. Biological assays of these compounds revealed that 2 potently inhibits HIV-1 integrase without cytotoxicity.

Tracking of Human Parvovirus B19 Virus-Like Particles Using Short Peptide Tags Reveals a Membrane-Associated Extracellular Release of These Particles.

B19 virus (B19V) is a pathogenic human parvovirus that infects erythroid progenitor cells. Because there are limited in vitro culture systems to propagate this virus, little is known about the molecular mechanisms by which it propagates in cells. In this study, we introduced a HiBiT peptide tag into various loops of VP2 located on the surface of B19V particles and evaluated their ability to form virus-like particles (VLPs). Three independent sites were identified as permissive sites for peptide tag insertion without affecting VLP formation. When the HiBiT tag was introduced into B19V clones (pB19-M20) and transfected into a semipermissive erythroleukemia cell line (UT7/Epo-S1), HiBiT-dependent luciferase activities (HiBiT activities) increased depending on helicase activity of viral NS1. Furthermore, we used a GFP11 tag-split system to visualize VLPs in the GFP1-10-expressing live cells. Time-lapse imaging of green fluorescent protein (GFP)-labeled VLPs revealed that nuclear VLPs were translocated into the cytoplasm only after cell division, suggesting that the breakdown of the nuclear envelope during mitosis contributes to VLP nuclear export. Moreover, HiBiT activities of culture supernatants were dependent on the presence of a detergent, and the released VLPs were associated with extracellular vesicles, as observed under electron microscopy. Treatment with an antimitotic agent (nocodazole) enhanced the release of VLPs. These results suggest that the virions accumulated in the cytoplasm are constitutively released from the cell as membrane-coated vesicles. These properties are likely responsible for viral escape from host immune responses and enhance membrane fusion-mediated transmission. IMPORTANCE Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. This study provides evidence of mitosis-dependent nuclear export of B19V and extracellular vesicle-mediated virion release. Moreover, this study provides methods for modifying particle surfaces with various exogenous factors and contributes to the development of fine nanoparticles with novel valuable functions. The pB19-M20 plasmid expressing HiBiT-tagged VP2 is a novel tool to easily quantify VP2 expression. Furthermore, this system can be applied in high-throughput screening of reagents that affect VP2 expression, which might be associated with viral propagation.

A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection.

Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped ß-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown.

Safety and immunogenicity of parvovirus B19 virus-like particle vaccine lacking phospholipase A2 activity.

Parvovirus B19 (B19) belongs to the Erythroparvovirus genus and is known to cause the fifth disease in children. Primary infection of pregnant women is associated with a high risk of hydrops fetalis and stillbirth due to severe fetal anemia. Virus-like particle (VLP) vaccine candidates for B19 have been developed, although none have been approved so far. The B19 phospholipase A2 domain (B19 PLA2), located in the VP1 unique region, is believed to be associated with adverse inflammatory reactions, and previous effective attempts to improve this vaccine modality inserted a mutation to impair the PLA2 activity of VLPs. In this study, we designed VLPs with a deletion mutant of PLA2 (⊿PLA2 B19 VLP), devoid of PLA2 activity, and confirmed their immunogenicity and safe use in vivo. These results were supported by the lack of histological inflammatory reactions at the site of immunization or the production of IL-6 in ⊿PLA2 B19 VLP-immunized mice, that were observed in mice immunized with B19 VLPs. CD4+ T cells from mice vaccinated with VLPs and B19-seropositive human samples were not activated by B19 PLA2 stimulation, suggesting that the B19 PLA2 domain does not constitute a major CD4+ T cell epitope. Most importantly, the ⊿PLA2 B19 VLPs induced neutralizing antibodies against B19, in levels similar to those found in B19-seropositive human samples, indicating that they could be used as a safe and effective vaccine candidate against B19.

Surveillance of pollen-food allergy syndrome in elementary and junior high school children in Saitama, Japan.

BACKGROUND: Because few studies have epidemiologically evaluated pollen-food allergy syndrome (PFAS), relevant information about this disease is limited in children. OBJECTIVE: We wanted to clarify the epidemiological details of PFAS by creating a questionnaire which enables to distinguish class 2 food allergy from that of class 1. METHODS: We conducted a questionnaire survey for schoolchildren attending to public elementary and junior high schools. In this questionnaire, we asked about both the allergy to fruits and/or vegetables and allergic rhinitis (AR). PFAS was, then, defined as allergy for fruits and/or vegetable which occurred after the symptoms of AR appeared. RESULTS: A total of 2,346 children (median age, 10.6±2.5 years; 1,157 boys) were evaluated. The prevalence of PFAS was 6.9% among subjects. The mean ages in the onset of AR and PFAS were 4.59±2.76 and 7.38±3.17 years old, respectively. Various kinds of foods were shown to be causative, among which kiwifruits were the commonest. As high as approximately 30% of children with PFAS experienced systemic symptoms including cutaneous (21.8%) and respiratory symptoms (9.6%). Anaphylaxis was diagnosed in 5.8% children. CONCLUSION: Our results indicated that the prevalence of PFAS was getting higher and the mean age of onset was getting lower. These may be attributed to the increasing number of patients with AR and also to the lower age of onset of AR. We have to be careful to not only local but also systemic symptoms when examining children with PFAS.

Cellular ESCRT components are recruited to regulate the endocytic trafficking and RNA replication compartment assembly during classical swine fever virus infection.

As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.

Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication.

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). METHODS: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-ß, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. RESULTS: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-ß or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. CONCLUSION: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.

Fusion of parvovirus B19 receptor-binding domain and pneumococcal surface protein A induces protective immunity against parvovirus B19 and Streptococcus pneumoniae.

BACKGROUND: Parvovirus B19 (B19) is a well-known cause of fifth disease in children, but infection during pregnancy may cause hydrops fetalis and stillbirth. The receptor-binding domain (RBD) of the VP1 unique capsid plays a pivotal role in infection. Here, we aimed to improve the immunogenicity of an RBD-based vaccine by genetically fusing it with Streptococcus pneumoniae surface protein A (PspA). METHODS: Mice were intramuscularly injected with RBD-based vaccines. Antigen-specific antibodies and neutralizing activity against B19 were measured. Protective immunity against S. pneumoniae was evaluated by monitoring the survival of mice nasally challenged with bacteria and determining antigen-specific T cell activation in splenic cells. RESULTS: RBD alone failed to generate neutralizing antibodies against B19, but fusion with PspA induced higher levels of neutralizing IgG compared to B19 virus-like particles. Furthermore, a comparable level of PspA-specific IgG was induced by RBD-PspA and PspA alone, which was sufficient to protect mice against pneumococcal infection. Stimulation with PspA, but not RBD, induced cytokine production in splenic cells from mice immunized with RBD-PspA, suggesting that PspA-specific T cells supported immunoglobulin class switching of both RBD- and PspA-specific B cells. CONCLUSIONS: RBD-PspA should be an effective bivalent vaccine against B19 and S. pneumoniae infections.

Membrane-Associated Flavivirus Replication Complex-Its Organization and Regulation.

Flavivirus consists of a large number of arthropod-borne viruses, many of which cause life-threatening diseases in humans. A characteristic feature of flavivirus infection is to induce the rearrangement of intracellular membrane structure in the cytoplasm. This unique membranous structure called replication organelle is considered as a microenvironment that provides factors required for the activity of the flaviviral replication complex. The replication organelle serves as a place to coordinate viral RNA amplification, protein translation, and virion assembly and also to protect the viral replication complex from the cellular immune defense system. In this review, we summarize the current understanding of how the formation and function of membrane-associated flaviviral replication organelle are regulated by cellular factors.

[A PEDIATRIC CASE OF DIFFUSE PANBRONCHIOLITIS WHO PREDOMINANTLY SHOWED RESTRICTIVE PULMONARY DYSFUNCTION AND DRAMATICALLY RESPONDED TO MACROLIDE LOW-DOSE LONG-TERM THERAPY.]

A 12-year-old boy visited our hospital with complaints of chronic cough and dyspnea. Chest X-ray and CT revealed diffuse granular shadow in the bilateral lung fields and "Tree-in-bud appearance" in the peripheral airways, respectively. Sinusitis was present, and restrictive disorder was predominantly found in pulmonary function. The patient was diagnosed with DPB, and long-term therapy was started with low-dose clarithromycin (CAM), The patient showed a dramatic response to CAM, with improvements of both the clinical symptoms and pulmonary function within 1-2 months. According to the relevant literature, in adult patients with this disease, pulmonary dysfunction starts from an obstructive pattern; however, this is not the case in pediatric patients. It was therefore suggested that the mechanisms underlying the development of pulmonary dysfunction in cases of childhood onset differs from those with an adult onset.

Endoplasmic Reticulum-Associated Degradation Controls Virus Protein Homeostasis, Which Is Required for Flavivirus Propagation.

Many positive-stranded RNA viruses encode polyproteins from which viral proteins are generated by processing the polyproteins. This system produces an equal amount of each viral protein, though the required amounts for each protein are not the same. In this study, we found the extra membrane-anchored nonstructural (NS) proteins of Japanese encephalitis virus and dengue virus are rapidly and selectively degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. Our gene targeting study revealed that ERAD involving Derlin2 and SEL1L, but not Derlin1, is required for the viral genome replication. Derlin2 is predominantly localized in the convoluted membrane (CM) of the viral replication organelle, and viral NS proteins are degraded in the CM. Hence, these results suggest that viral protein homeostasis is regulated by Derlin2-mediated ERAD in the CM, and this process is critical for the propagation of these viruses. IMPORTANCE The results of this study reveal the cellular ERAD system controls the amount of each viral protein in virus-infected cells and that this "viral protein homeostasis" is critical for viral propagation. Furthermore, we clarified that the "convoluted membrane (CM)," which was previously considered a structure with unknown function, serves as a kind of waste dump where viral protein degradation occurs. We also found that the Derlin2/SEL1L/HRD1-specific pathway is involved in this process, whereas the Derlin1-mediated pathway is not. This novel ERAD-mediated fine-tuning system for the stoichiometries of polyprotein-derived viral proteins may represent a common feature among polyprotein-encoding viruses.

[A CASE OF VOCAL CORD DYSFUNCTION, WHO USED ADRENALIN AUTOINJECTOR (EPIPEN®) FREQUENTLY AFTER BEING DIAGNOSED AS ANAPHYLAXIS].

We experienced a case of vocal cord dysfunction (VCD) in a child to whom an adrenaline autoinjector (Epipen®) had been prescribed and frequently used following a diagnosis of exercise-induced anaphylaxis. An exercise test was performed to investigate her frequent episodes of anaphylaxis-like symptoms. A few minutes after starting the test, signs of dyspnea, such as throat tightness and stridor, appeared, although hypoxia was not present and her respiratory sounds were normal. Medications were not effective for treating her respiratory symptoms. Laryngoscopy performed at the test revealed bizarre vocal cord movement, which was diagnosed as VCD. The symptoms gradually diminished after the initiation of biofeedback therapy, including pursed lips breathing and abdominal breathing. Thereafter, she did not use an adrenaline autoinjector when symptoms appeared; instead, she would perform biofeedback therapy before using the adrenaline autoinjector. Thus, VCD should be included in the differential diagnosis of patients who show anaphylactic symptoms that are resistant to preventive therapy.