Influenza A virus infection is a zoonosis. The natural reservoirs of the viruses are migratory water birds, particularly ducks. Through a process of reassortment of the genomes of avian and human influenza viruses, novel virus strains may arise to which humans are not immune. Such a virus can spread rapidly throughout populations as a pandemic, usually with serious consequences. Effective preparedness for future influenza pandemics depends on robust global surveillance of avian, swine, mammalian, and human influenza and continuous updating of our Library of Influenza Viruses from these hosts to aid diagnosis and vaccine production. Enhancing and refining strategies for controlling seasonal influenza, including the development of more potent vaccines and therapeutic medicines, that can be seamlessly implemented when a new influenza virus emerges is crucial. This review, which draws on more than half a century of our research on the ecology of influenza viruses, describes how pandemic influenza viruses arise and outlines strategies for preparedness against future pandemics.

Nontuberculous mycobacteria occasionally harbour clustered tRNA genes, referred to as a tRNA array unit, which is considered a putative antidefense system within their genomes. However, the precise genomic location of these tRNA array units remains unclear. To address this, we sequenced the complete genomes of 5 Mycobacterium avium strains carrying a tRNA array unit using a hybrid assembly of long and short reads followed by manual curation. The assemblies indicated that each strain harbours 3 to 5 extrachromosomal elements. In all genomes, the tRNA array unit was found on a linear contig exceeding 300 kb. Pulse-field gel electrophoresis (PFGE) and sodium dodecyl sulphate-PFGE revealed that the strains harbour linear plasmids corresponding to these large contigs with protein-capped termini. These linear plasmids encode a hybrid type VII/type IV secretion system but lack relaxase genes, which are typically present in mycobacterial circular plasmids. Additionally, they contain approximately 415 bp inverted repeats at the termini. Sequences of related plasmids were identified exclusively in the genomes of M. avium isolates from Japan available in public databases, suggesting a possible Asian origin. This study provides the first experimental evidence that M. avium harbours giant invertron-type linear plasmids carrying a tRNA array unit.

The genus Mycobacterium, including Mycobacterium tuberculosis and over 200 nontuberculous mycobacteria (NTM), shows wide variability in clinical outcomes and drug susceptibility. Although culture-based identification remains the gold standard, slow mycobacterial growth delays diagnosis and treatment. In this study, we evaluated a novel culture-free method for subspecies-level identification directly from sputum. In this single-center prospective cohort study at Osaka Toneyama Medical Center, we analyzed 125 sputum samples from 115 patients with NTM pulmonary disease and 10 with non-NTM respiratory conditions. Samples were decontaminated using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) or succinic acid. We compared the reference culture method (mycobacterial culture plus whole-genome sequencing) and a culture-free direct target capture sequencing method. Core genome multi-locus sequence typing identified subspecies in both workflows, covering 186 mycobacterial species, including M. tuberculosis. The 115 NTM cohort specimens yielded 57 smear-positive and 93 culture-positive results. The identified subspecies included 48 Mycobacterium avium subsp. hominissuis, 22 Mycobacterium intracellulare subsp. intracellulare, 5 subsp. chimaera, 7 Mycobacterium abscessus subsp. abscessus, 5 subsp. massiliense, 1 M. tuberculosis, and 5 other NTM species. The culture-free method showed a high identification rate for smear-positive specimens (75.4%) but a low identification rate for smear-negative specimens (13.9%). NALC-NaOH pretreatment resulted in higher accuracy (90.5%) than did succinic acid pretreatment (66.7%). Thus, our culture-free subspecies-level identification method achieved high accuracy, especially in alkaline-treated smear-positive sputum samples, achieving rates above 90%. This method is recommended in clinical practice for patients who require rapid diagnosis and timely initiation of appropriate treatment, bypassing time-consuming culture steps.IMPORTANCEAccurate identification of Mycobacterium species and subspecies is crucial for effective treatment, as drug susceptibility and clinical outcomes vary significantly among them. However, conventional diagnosis relies on culture-based methods that can take several weeks, critically delaying appropriate therapy. This study validates a novel culture-free method using target capture sequencing for the comprehensive, subspecies-level identification of over 186 mycobacterial species directly from sputum specimens. Our findings revealed the high accuracy of this approach for smear-positive specimens, especially with alkaline pretreatment. This rapid method is applicable in clinical settings and enables timely and precise treatment decisions, greatly benefiting patients who require urgent intervention.

A rapidly growing acid-fast bacterium not consistent with existing species had previously been isolated from an immunocompetent patient with chronic and progressive pulmonary disease. Herein, we aimed to further characterize five clinically isolated strains based on genetic and phenotypic examinations and propose a novel species. The 16S rRNA gene sequence of the five strains had 99.6-99.7% similarity with that of Mycobacterium mucogenicum and clustered most closely to M. mucogenicum in a phylogenetic analysis based on single-copy marker genes. The average nucleotide identity values of the strains against M. mucogenicum and Mycobacterium phocaicum were ≤93.6% and ≤93.2%, respectively, indicating that they are separate species. The strains formed smooth colonies within 3-4 days and grew at temperatures between 24 and 42 °C, with optimal growth at 37 °C. The biochemical characteristics of the novel species were similar to those of M. mucogenicum and M. phocaicum. It predominantly contained C16:0 and C18:1 (n-9cis) fatty acid and molecular species of mycolic acid, such as C77:2 and C79:2 of α-mycolic acids, C77:0 and C79:0 of keto-mycolic acids and C58:1 and C60:1 of dicarboxy-mycolic acids. Based on the genomic analyses, we propose a novel species, named Mycobacterium toneyamaense, with the type strain LRC2408T (=JCM 37957T=KCTC 59531T).

Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disease caused by autoantibodies targeting granulocyte-macrophage colony-stimulating factor (GM-CSF). Although serum GM-CSF autoantibody levels are markedly increased in aPAP patients, total antibody titer does not correlate with disease severity. Here, we characterize 186 monoclonal anti-GM-CSF autoantibodies derived from GM-CSF-specific B cells in 28 aPAP patients with varying disease severity, including three longitudinal cohorts, to determine whether epitope specificity and affinity of the autoantibodies contribute to disease pathophysiology. We classify these antibodies into two groups based on their epitopes: class 1 (targeting A, BD, or D epitopes) and class 2 (targeting B or C epitopes). In class 1 antibodies, affinity strongly correlates with neutralization activity, whereas this relationship is weak or absent in class 2 antibodies. High-affinity class 1 antibodies are present at higher levels in patients with more severe disease and are sufficient to induce PAP symptoms in a humanized mouse model. Thus, these findings identify epitope specificity and affinity as key determinants of pathogenicity and provide a mechanistic framework for understanding why total serum autoantibody levels fail to reflect disease severity in aPAP.

Since the emergence of the SARS-CoV-2 Omicron variant, decreased morbidity and mortality relative to early strains have been widely reported. However, the virological characteristics of recent subvariants dominating the post-pandemic era remain to be fully elucidated. In the present study, the intrinsic pathogenicities of the Omicron subvariants JN.1, EG.5.1, and BA.2.86.1 were investigated using an immunologically naïve cynomolgus macaque model. All three variants exhibited robust replication in the upper respiratory tract, surpassing the viral titers observed with the early Wuhan strain. Omicron subvariant infection caused prolonged fever and sustained viral shedding in the nasal mucosa for at least seven days, indicating enhanced adaptation to upper airway tissues. Despite this shift in viral replication tropism, histological analysis showed that these variants retained the characteristics to induce lower respiratory tract disease. All infected macaques developed bronchopneumonia characterized by cellular exudates in the alveolar spaces. The histological scores were comparable to those observed with the early strain. These findings demonstrate that the pathogenicity of the Omicron subvariants has not been fully attenuated in the absence of pre-existing immunity, since they caused severe lung disease in immunologically naïve macaques. Furthermore, their enhanced propagation in the upper airway likely facilitates efficient transmission among humans. The continuous circulation of SARS-CoV-2 variants underscores the necessity of ongoing surveillance of viral gene variations and the maintenance of protective immunity in vulnerable populations.

The nontuberculous mycobacteria (NTM) isolated from gastric aspirate have demonstrated >85% strain concordance with those from the sputum, suggesting that they originate from the lungs rather than the environment. Gastric aspirate, although not yet internationally recognized, may be a useful supplementary specimen for diagnosing NTM pulmonary disease.

Bacterial species cultured from sputum change during treatment or observation for non-tuberculous mycobacterial pulmonary disease; however, strain-level changes remain unrecognized. Variable number tandem repeat typing is a standard technique for strain identification; nonetheless, its labor-intensive and time-consuming nature limits routine clinical use. Therefore, we aimed to elucidate species-subspecies and strain dynamics in non-tuberculous mycobacteria and develop a simple sequence-based strain-level determination method. We performed a single-center prospective cohort study of 112 patients with non-tuberculous mycobacterial pulmonary disease. Whole-genome sequencing was performed on two sputum samples collected at enrollment and at the end of follow-up, followed by variable number tandem repeat (VNTR) typing. We also developed a simple long-read sequencing-based digital VNTR (dVNTR) typing method and evaluated its efficacy. Our results demonstrate that core genome multi-locus sequencing typing revealed species/subspecies changes in 13 patients (11.6%); VNTR typing detected strain changes in 16 patients (14.3%) without species/subspecies changes. Overall, pathogen shifts occurred in 29 patients (shift [+] group, 25.9%), whereas 83 had no detectable pathogen shift (shift [-] group, 74.1%). Interestingly, macrolide and amikacin susceptibility changed in both groups, but resistance remained higher in shift (-) patients. dVNTR results aligned with those of conventional VNTR typing. In conclusion, since susceptibility factors remain unclear, routine species/subspecies identification and molecular typing, such as VNTR, are optimal for patient care. Core genome multi-locus sequencing typing with a dVNTR identified pathogen shifts, innovating non-tuberculous mycobacterial pulmonary disease management.Clinical TrialsThis study is registered with UMIN as UMIN 000056067. IMPORTANCE:Pulmonary non-tuberculous mycobacterial disease is a chronic infection in which the causative pathogens may change at the species, subspecies, or strain level over time. Accurate tracking of these changes is essential for optimizing treatment; however, conventional clinical practice lacks efficient methods for monitoring such dynamics. Our study revealed pathogen changes in approximately one-quarter of patients over 1.5 years, prompting the development of a novel surveillance system that integrates next-generation sequencing for both species-subspecies identification and strain-level molecular epidemiology. This innovation enables real-time monitoring of pathogen dynamics, allowing clinicians to promptly adjust treatment strategies and improve patient care through more informed decision-making.

It has been recognized that it is difficult to maintain the virus particle structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be associated with lower immunogenicity of inactivated vaccines against coronavirus disease 2019 (COVID-19). We have previously demonstrated that an intact structure of the virus particles is critical for influenza inactivated whole virus particle vaccine (WPV) to be immunogenically potent. Here, we tested 37, 35, 33, and 31 °C for the virus propagation temperatures and the timing of formaldehyde treatment of the virus before and after centrifugation-based purification to obtain virus particles with an intact structure. Virus particles cultured at 33 °C retained spike proteins on the surface the most abundantly. The pretreatment of the virus with formaldehyde prevented the dissociation of the spike proteins from the viral surface during the centrifugation-based purification. The immunogenicity of the prepared vaccines, intact WPV and non-intact WPV that had lost the spike proteins, was evaluated in a mouse model. A single dose of intact WPV effectively induced humoral immunity compared to non-intact WPV, as indicated by higher titers of neutralizing antibodies. After a virus challenge, the mice vaccinated with a single dose of inactivated intact WPV showed less severe weight loss and lower virus titers in the lungs compared to those vaccinated with non-intact WPV. These results demonstrate the importance of the structural integrity of WPV in inducing effective and protective immunity, and provide significant insight into the development of COVID-19 WPV for practical use.

Outbreaks of H5 highly pathogenic avian influenza A viruses (HPAIVs) in animals pose a threat to humans immunologically naïve to avian influenza viruses. However, annual vaccination, such as for seasonal influenza is not planned because the number of human patients infected with H5 HPAIVs is small, and the possibility of human-to-human transmission of H5 HPAIVs is low at present. However, various clades of H5 HPAIVs have emerged continuously. Therefore, a vaccine that confers long-term and cross-clade immunity is required. To examine the long-term effectiveness and cross-clade reactivity of an H5 influenza virus vaccine, cynomolgus macaques were infected with an H5N1 HPAIV 5 years after two subcutaneous vaccinations with inactivated H5N1 whole-virus particles (H5 clade classical/outlier), which showed higher immunogenicity than did split vaccines in our previous studies. Neutralization titers against the vaccine strain were maintained for 5 years, and a recall immune response was observed on challenge infection against the challenge strain (clade 1) and other H5N1 HPAIV strains (clades 2.2, 2.3.2.1, and 2.3.4.4b). Compared with unvaccinated macaques, viral titers were low, and the cytokine signaling pathways related to the pathogenesis of an influenza virus infection were not activated in the vaccinated macaques. Thus, a whole-virus particle vaccine induced long-term memory sufficient to prevent severe pneumonia caused by an H5N1 HPAIV in cynomolgus macaques.

We identified 2 novel species, Mycobacterium novusgordonae and M. shingordonae, from sputum specimens of pulmonary disease patients in Japan. Genetic and biochemical analyses revealed a close relationship with M. paragordonae. One M. shingordonae case-patient experienced severe progressive infection, highlighting the variation in pathogenicity of the M. gordonae clade species.

Aberrant immune responses to viral pathogens contribute to pathogenesis, but our understanding of pathological immune responses caused by viruses within the human virome, especially at a population scale, remains limited. We analyzed whole-genome sequencing datasets of 6,321 Japanese individuals, including patients with autoimmune diseases (psoriasis vulgaris, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), pulmonary alveolar proteinosis (PAP) or multiple sclerosis) and coronavirus disease 2019 (COVID-19), or healthy controls. We systematically quantified two constituents of the blood DNA virome, endogenous HHV-6 (eHHV-6) and anellovirus. Participants with eHHV-6B had higher risks of SLE and PAP; the former was validated in All of Us. eHHV-6B-positivity and high SLE disease activity index scores had strong correlations. Genome-wide association study and long-read sequencing mapped the integration of the HHV-6B genome to a locus on chromosome 22q. Epitope mapping and single-cell RNA sequencing revealed distinctive immune induction by eHHV-6B in patients with SLE. In addition, high anellovirus load correlated strongly with SLE, RA and COVID-19 status. Our analyses unveil relationships between the human virome and autoimmune and infectious diseases. Analysis of the blood DNA virome in patients with COVID-19 and autoimmune disease associates endogenous HHV-6 (eHHV-6) and high anellovirus load with increased disease risk, most notably for systemic lupus erythematosus. eHHV-6 carriers show a distinct immune response.

PURPOSE:The suitability of progressive pulmonary fibrosis (PPF) as a criterion for antifibrotic use remains uncertain. We aimed to evaluate the effectiveness of antifibrotics for patients with different criteria of progressive non-idiopathic pulmonary fibrosis interstitial lung disease (non-IPF ILD). MATERIAL AND METHODS:In this multicenter, retrospective cohort study, we estimated the effect of antifibrotic drugs in three cohorts of PF-ILD (progression within 24 months under standard non-antifibrotic therapy, as in the INBUILD trial), PPF (progression within 12 months based on ATS/ERS/JRS/ALAT guidelines), and PPF "despite management" (a subset of PPF with progression despite appropriate non-antifibrotic therapy). Analyses used the parametric G-formula, the time-varying Cox hazard model, and inverse probability weighting (IPW). RESULTS:Among 1754 patients with non-IPF ILD, 327, 567, and 326 patients were diagnosed with PF-ILD (134 antifibrotics, 193 non-antifibrotics), PPF (149 antifibrotics, 418 non-antifibrotics), and PPF "despite management" (115 antifibrotics, 211 non-antifibrotics), respectively. Using the parametric G-formula, antifibrotic therapy was associated with higher estimated survival in PF-ILD, with statistically significant differences during the first three years, and with a consistent survival advantage in the PPF "despite management" cohort. In contrast, no clear survival benefit was observed in the PPF cohort. These findings were consistent with the time-varying Cox hazard model and IPW analysis results. CONCLUSION:Our results demonstrate antifibrotic therapy was associated with higher estimated survival in patients with PF-ILD in a real-world setting, suggesting the importance of including "despite management" as a criterion for antifibrotic therapy eligibility in the PPF diagnosis.

ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating inflammatory responses. ARNAX activates cellular immunity via cross-presentation; hence, its practical application has been demonstrated in cancer immunotherapy. Given the importance of cellular immunity in virus infections, ARNAX is expected to be a more effective vaccine adjuvant for virus infections than alum, an adjuvant approved for human use that mainly enhances humoral immunity. In the present study, the trimeric recombinant spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was prepared as a vaccine antigen and formulated with ARNAX. When T-cell and neutralizing antibody responses were evaluated in immunized mice, antigen formulated with ARNAX generated significantly larger numbers of antigenspecific CD4+ and CD8+ T cells, as well as higher titers of neutralizing antibodies, compared to antigen alone or antigen formulated with alum. In experiments where immunized mice were challenged with a SARS-CoV-2 mouse-adapted virus derived from the ancestral strain, immunization with antigen formulated with ARNAX reduced virus titers in the lungs at 3 days post-infection to a much greater extent than did immunization with either antigen alone or that formulated with alum. These results show that ARNAX potently enhances the levels of both cellular and humoral immunity above those seen with alum, providing significantly greater viral clearing responses. Thus, ARNAX may act as a useful adjuvant for prophylactic vaccines, particularly for viral infectious diseases. IMPORTANCE:Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote strong cellular immunity. To prepare for the next pandemic of viral origin, the development of Th1-type adjuvants with low adverse reactions that induce cellular immunity is necessary. ARNAX is a TLR3 agonist consisting of DNA-RNA hybrid nucleic acid, which is expected to be an adjuvant that induces cellular immunity. The present study using a coronavirus disease 2019 mouse model demonstrated that ARNAX potently induces cellular immunity in addition to humoral immunity with minimal induction of inflammatory cytokines. Therefore, ARNAX has the potential to be used as a potent and welltolerated adjuvant for vaccines against pandemic viruses emerging in the future.

Leptospirosis a significant and life-threatening zoonosis with global reach. If diagnosis and treatment are delayed, the infection may lead to fatal Weil's disease. In the Philippines, a 21-year-old man was admitted to the hospital with leptospirosis-like symptoms, including fever, myalgia, headache, anuria, jaundice, hemorrhage, skin rash, and diarrhea. Although he was immediately treated with penicillin G, the patient died shortly after admission. The serological test result was negative for Leptospira; however, three leptospires were isolated from the patient through selective cultivation under varying conditions. Microscopic agglutination tests and a phylogenetic analysis of the flaB gene encoding the flagellin subunit protein revealed that the isolates belonged to three different serovars, suggesting that the patient was simultaneously infected with at least three distinct Leptospira serovars. In this case report, we highlight the potential risk of multiple infections with leptospires in humans, a critical consideration for diagnosis and treatment strategies.

Progressive Fibrosing Interstitial Lung Disease (PF-ILD) is a severe phenotype of Interstitial Lung Disease (ILD) with a poor prognosis, typically requiring prolonged clinical observation and multiple CT examinations for diagnosis. Such requirements delay early detection and treatment initiation. To enable earlier identification of PF-ILD, we propose ILD-Slider, a parameter-efficient and lightweight deep learning framework that enables accurate PF-ILD identification from a limited number of CT slices. ILD-Slider introduces anatomy-based position markers (PMs) to guide the selection of representative slices (RSs). A PM extractor, trained via a multi-class classification model, achieves high PM detection accuracy despite severe class imbalance by leveraging a peak slice mining (PSM)-based strategy. Using the PM extractor, we automatically select three, five, or nine RSs per case, substantially reducing computational cost while maintaining diagnostic accuracy. The selected RSs are then processed by a slice-level 3D Adapter (Slider) for PF-ILD identification. Experiments on 613 cases from The University of Osaka Hospital (UOH) and the National Hospital Organization Osaka Toneyama Medical Center (OTMC) demonstrate the effectiveness of ILD-Slider, achieving an AUPRC of 0.790 (AUROC 0.847) using only five automatically extracted RSs. ILD-Slider further validates the feasibility of diagnosing PF-ILD from non-contiguous slices, which is particularly valuable for real-world and public datasets where contiguous volumes are often unavailable. These results highlight ILD-Slider as a practical and efficient solution for early PF-ILD identification.

Combined pulmonary fibrosis and emphysema (CPFE) is characterized by emphysematous lesions in the upper lung field and pulmonary fibrosis in the lower lung field and is often associated with pulmonary hypertension and severe exercise-induced hypoxemia (EIH). We herein report a 62-year-old man with CPFE who presented with severe EIH despite relatively preserved lung volumes. Cardiopulmonary exercise testing suggested exercise-induced right-to-left shunt (EIS) through a patent foramen ovale (PFO). EIS was attributed to exercise-induced pulmonary hypertension. In this case report, we highlight the possibility of EIS using PFO for CPFE. We also discuss potential treatments including pharmaceutical interventions and PFO closures.

Background/Objectives: In preparation for a potential pandemic caused by the H5N1 highly pathogenic avian influenza (HPAI) virus, pre-pandemic vaccines against several viral clades have been developed and stocked worldwide. Although these vaccines are well tolerated, their immunogenicity and cross-reactivity with viruses of different clades can be improved. Methods: To address this aspect, we generated recombinant influenza vaccines against H5-subtype viruses using two different strains of highly attenuated vaccinia virus (VACV) vectors. Results: rLC16m8-mcl2.2 hemagglutinin (HA) and rLC16m8-mcl2.3.4 HA consisted of a recombinant LC16m8 vector encoding the HA protein from clade 2.2 or clade 2.3.4 viruses (respectively); rDIs-mcl2.2 HA consisted of a recombinant DIs vector encoding the HA protein from clade 2.2. A single dose of rLC16m8-mcl2.2 HA showed rapid (1 week after vaccination) and long-term protection (20 months post-vaccination) in mice against the HPAI H5N1 virus. Moreover, cynomolgus macaques immunized with rLC16m8-mcl2.2 HA exhibited long-term protection when challenged with a heterologous clade of the HPAI H5N1 virus. Although the DIs strain is unable to grow in most mammalian cells, rDIs-mcl2.2 HA also showed rapid and long-lasting effects against HPAI H5N1 virus infection. Notably, the protective efficacy of rDIs-mcl2.2 HA was comparable to that of rLC16m8-mcl2.2 HA. Furthermore, these vaccines protected animals previously immunized with VACVs from a lethal challenge with the HPAI H5N1 virus. Conclusions: These results suggest that both rLC16m8-mcl2.2 HA and rDIs-mcl2.2 HA are effective in preventing HPAI H5N1 virus infection, and rDIs-mcl2.2 HA is a promising vaccine candidate against H5 HA-subtype viruses.