Monitoring Influenza C and D Viruses in Patients With Respiratory Diseases in Japan, January 2018 to March 2023.

BACKGROUND: Influenza viruses can cause zoonotic infections that pose public health risks. Surveillance of influenza A and B viruses is conducted globally; however, information on influenza C and D viruses is limited. Longitudinal monitoring of influenza C virus in humans has been conducted in several countries, but there has been no long-term monitoring of influenza D virus in humans. The public health risks associated with the influenza D virus therefore remain unknown. METHODS: We established a duplex real-time RT-PCR to detect influenza C and D viruses and analyzed respiratory specimens collected from 2144 patients in Japan with respiratory diseases between January 2018 and March 2023. We isolated viruses and conducted hemagglutination inhibition tests to examine antigenicity and focus reduction assays to determine susceptibility to the cap-dependent endonuclease inhibitor baloxavir marboxil. RESULTS: We detected three influenza C viruses belonging to the C/Kanagawa- or C/Sao Paulo-lineages, which recently circulated globally. None of the specimens was positive for the influenza D virus. The C/Yokohama/1/2022 strain, isolated from the specimen with the highest viral RNA load and belonging to the C/Kanagawa-lineage, showed similar antigenicity to the reference C/Kanagawa-lineage strain and was susceptible to baloxavir. CONCLUSIONS: Our duplex real-time RT-PCR is useful for the simultaneous detection of influenza C and D viruses from the same specimen. Adding the influenza D virus to the monitoring of the influenza C virus would help in assessing the public health risks posed by this virus.

Antiviral susceptibility of SARS-CoV-2 and influenza viruses from 3 co-infected pediatric patients.

In Japan, influenza activity was low throughout the COVID-19 pandemic until the 2022-23 season, when the first influenza outbreak occurred since the 2020-21 season. In our influenza surveillance during the COVID-19 pandemic, co-infection with SARS-CoV-2 and influenza virus had not been detected; however, in January 2024, we identified three pediatric outpatients co-infected with these viruses: one with SARS-CoV-2 Omicron EG.5 sublineage HK.3 and influenza A(H3N2) and two with SARS-CoV-2 Omicron BA.2.86 sublineage JN.1.5 and influenza A(H1N1)pdm09. We evaluated the susceptibility of SARS-CoV-2 against RNA-dependent RNA polymerase inhibitors (remdesivir and molnupiravir) and 3C-like protease inhibitors (nirmatrelvir and ensitrelvir), and that of influenza viruses against neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir) and the cap-dependent endonuclease inhibitor baloxavir. All viruses tested were susceptible to these antiviral drugs and did not possess amino acid substitutions associated with reduced antiviral susceptibility. The patients were treated with anti-influenza drugs and did not develop severe symptoms despite the co-infection. Since SARS-CoV-2 and influenza viruses continue to evolve, continuous monitoring of their circulation remains essential to assess public health measures and support clinical management.

Lung ultrasound in the assessment of interstitial lung disease in patients with connective tissue disease: Performance in comparison with high-resolution computed tomography.

OBJECTIVE: To investigate clinical relevance of performing lung ultrasound (LUS) in patients with connective tissue disease (CTD)-associated interstitial lung disease (ILD) in comparison with high-resolution computed tomography (HRCT). METHODS: This single-centre study enrolled eligible patients with CTD-ILD from the prospective LUS registry. Total B-lines were detected by assessment at 14 sites via LUS. Forced vital capacity, diffusing lung capacity for carbon monoxide (DLCO), DLCO/alveolar volume, 6-minute walking distance, and the ILD-GAP index were used as ILD prognostic parameters. Correlations were examined using single and multiple regression analyses. RESULTS: Sixty-seven patients were enrolled, including 29 with idiopathic inflammatory myopathy or anti-synthetase syndrome, 25 with systemic sclerosis (SSc), 10 with rheumatoid arthritis, and 3 with mixed connective tissue disease. The total number of B-lines correlated with ILD extent on HRCT in patients with CTD-ILD (r = 0.66; P < 0.001), particularly in patients with SSc-ILD (r = 0.78; P < 0.001). Total B-lines and ILD extent on HRCT showed comparable correlations with prognostic parameters, while multiple regression analysis revealed the limited benefit of performing LUS in addition to HRCT in predicting correlations with prognostic factors. CONCLUSIONS: LUS serves as an alternative tool for assessing the severity and prognosis of patients with CTD-ILD.

Effect of medium-chain triglycerides supplements and walking on health-related quality of life in sedentary, healthy middle-aged, and older adults with low BMIs: a randomized, double-blind, placebo-controlled, parallel-group trial.

Introduction: To extend individuals' healthy life expectancies, the improvement of subjective health and quality of life (QOL) has been increasingly prioritized, alongside the improvement of their physical functioning. Reports have indicated that intake of medium-chain triglycerides (MCTs) benefits the physical health of older individuals requiring nursing care, and athletes, and healthy individuals. But there are few studies investigating the effects of MCTs on subjective health and QOL. The present study sought to evaluate the combined effects of 12-week MCTs supplements and moderate-intensity walking exercise on the subjective health and QOL of middle-aged and older adults aged 60-74 with low BMIs (< 24 kg/m2) and who had no exercise habits. Methods: A placebo-controlled, double-blind, parallel-group trial was conducted. Three MCTs supplement groups with different doses and fatty acid compositions were compared with a control group. The study used the SF-36v2 questionnaire to assess subjective health and health-related QOL (HRQOL). Results: The result showed significant improvements in the scores on subscales of the physical QOL, such as Physical functioning and General health, and summary scores on the mental QOL, compared to the control. Conclusion: It is estimated that the combination of continuous intake of MCTs and walking exercise may affect HRQOL and improve subjective physical and mental health in sedentary, healthy, middle-aged and older adults. Clinical trial registration: https://rctportal.niph.go.jp/s/detail/um?trial_id=UMIN000046861, UMIN000046861.

Antigenic drift and subtype interference shape A(H3N2) epidemic dynamics in the United States.

Influenza viruses continually evolve new antigenic variants, through mutations in epitopes of their major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Antigenic drift potentiates the reinfection of previously infected individuals, but the contribution of this process to variability in annual epidemics is not well understood. Here we link influenza A(H3N2) virus evolution to regional epidemic dynamics in the United States during 1997-2019. We integrate phenotypic measures of HA antigenic drift and sequence-based measures of HA and NA fitness to infer antigenic and genetic distances between viruses circulating in successive seasons. We estimate the magnitude, severity, timing, transmission rate, age-specific patterns, and subtype dominance of each regional outbreak and find that genetic distance based on broad sets of epitope sites is the strongest evolutionary predictor of A(H3N2) virus epidemiology. Increased HA and NA epitope distance between seasons correlates with larger, more intense epidemics, higher transmission, greater A(H3N2) subtype dominance, and a greater proportion of cases in adults relative to children, consistent with increased population susceptibility. Based on random forest models, A(H1N1) incidence impacts A(H3N2) epidemics to a greater extent than viral evolution, suggesting that subtype interference is a major driver of influenza A virus infection dynamics, presumably via heterosubtypic cross-immunity.

Antiviral efficacy against and replicative fitness of an XBB.1.9.1 clinical isolate.

The emergence and spread of new SARS-CoV-2 variants with mutations in the spike protein, such as the XBB.1.5 and XBB.1.9.1 sublineages, raise concerns about the efficacy of current COVID-19 vaccines and therapeutic monoclonal antibodies (mAbs). In this study, none of the mAbs we tested neutralized XBB.1.9.1 or XBB.1.5, even at the highest concentration used. We also found that the bivalent mRNA vaccine could enhance humoral immunity against XBB.1.9.1, but that XBB.1.9.1 and XBB.1.5 still evaded humoral immunity induced by vaccination or infection. Moreover, the susceptibility of XBB.1.9.1 to remdesivir, molnupiravir, nirmatrelvir, and ensitrelvir was similar to that of the ancestral strain and the XBB.1.5 isolate in vitro. Finally, we found the replicative fitness of XBB.1.9.1 to be similar to that of XBB.1.5 in hamsters. Our results suggest that XBB.1.9.1 and XBB.1.5 have similar antigenicity and replicative ability, and that the currently available COVID-19 antivirals remain effective against XBB.1.9.1.

Magnetic dynamics and nonreciprocal excitation in uniform hedgehog order in icosahedral 1/1 approximant crystal.

The hedgehog state in the icosahedral quasicrystal (QC) has attracted great interest as the theoretical discovery of topological magnetic texture in aperiodic systems. The revealed magnetic dynamics exhibits nonreciprocal excitation in the vast extent of the reciprocal lattice [Formula: see text]-energy [Formula: see text] space, whose emergence mechanism remains unresolved. Here, we analyze the dynamical as well as static structure of the hedgehog order in the 1/1 approximant crystal (AC) composed of the cubic lattice with spatial inversion symmetry. We find that the dispersion of the magnetic excitation energy exhibits nonreciprocal feature along the N-P-[Formula: see text] line in the [Formula: see text] space. The dynamical structure factor exhibits highly structured intensities where high intensities appear in the high-energy branches along the [Formula: see text]-H line and the P-[Formula: see text]-N line in the [Formula: see text] space. The nonreciprocity in the 1/1 AC and also in the QC is understood to be ascribed to inversion symmetry breaking by the hedgehog ordering. The sharp contrast on the emergence regime of nonreciprocal magnetic excitation between the QC and the 1/1 AC indicates that the emergence in the vast [Formula: see text]-[Formula: see text] regime in the QC is attributed to the QC lattice structure.

A community cluster of influenza A(H3N2) virus infection with reduced susceptibility to baloxavir due to a PA E199G substitution in Japan, February to March 2023.

A community cluster of influenza A(H3N2) caused by viruses with an E199G substitution in PA was detected in Nara, Japan, between February and March 2023. The three patients with these mutant viruses had not received antiviral treatment before specimen collection but patients in the same hospital had. The sequences of the mutant viruses were closely related, suggesting clonal spread in Nara. They showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear.

A Randomized, Double-Blind, Controlled Trial Assessing If Medium-Chain Triglycerides in Combination with Moderate-Intensity Exercise Increase Muscle Strength in Healthy Middle-Aged and Older Adults.

An adequate nutritional intake is recommended for the prevention of physical frailty and sarcopenia. In particular, medium-chain fatty acids (MCFAs) are reportedly important for muscle strength in nursing home residents. However, the effects of MCFAs on healthy adults at risk for frailty remain unknown. Hence, a randomized, placebo-controlled study was conducted to investigate the effects of 12 weeks of medium-chain triglycerides (MCTs) intake and walking on muscle mass and function in healthy, sedentary, middle-aged and older adults with a low body mass index. Three MCT intake groups with different amounts of octanoic and decanoic acid intake were compared with a control group. After 12 weeks, knee extension strength increased in all groups, with the increases in all MCT intake groups being significantly higher than those in the control group (p < 0.05). Grip strength significantly increased from baseline in the MCT 6 g/day intake group (p < 0.05). The combination of aerobic exercise and MCT intake may be effective in preventing decline in muscle strength and promoting increase in muscle strength as they can improve muscle energy production, thereby contributing to the maintenance of good health for middle-aged and older adults at high risk for frailty and sarcopenia.

Assessment of the frequency of SARS-CoV-2 Omicron variant escape from RNA-dependent RNA polymerase inhibitors and 3C-like protease inhibitors.

The emergence and spread of antiviral-resistant SARS-CoV-2 is of great concern. In this study, we evaluated the propensity of Omicron variants to escape from RNA-dependent RNA polymerase (RdRP) inhibitors and 3C-like protease (3CLpro) inhibitors. SARS-CoV-2 Delta and Omicron variants were serially passaged in vitro in the presence of RdRP inhibitors (remdesivir and molnupiravir) and 3CLpro inhibitors (nirmatrelvir and lufotrelvir) to detect SARS-CoV-2 escape mutants. After five passages with 3CLpro inhibitors, mutant viruses that escaped from 3CLpro inhibitors emerged; however, in the presence of RdRP inhibitors all variants disappeared within 2-4 passages. Our findings suggest that the frequency of SARS-CoV-2 mutant escape from RdRP inhibitors is lower than that from 3CLpro inhibitors. We also found that Delta variants were more likely to acquire amino acid substitutions associated with resistance to 3CLpro inhibitors under the selective pressure of this drug compared with Omicron variants.

Saturation time of exposure interval for cross-neutralization response to SARS-CoV-2: Implications for vaccine dose interval.

Evaluating the serum cross-neutralization responses after breakthrough infection with various SARS-CoV-2 variants provides valuable insight for developing variant-proof COVID-19 booster vaccines. However, fairly comparing the impact of breakthrough infections with distinct epidemic timing on cross-neutralization responses, influenced by the exposure interval between vaccination and infection, is challenging. To compare the impact of pre-Omicron to Omicron breakthrough infection, we estimated the effects on cross-neutralizing responses by the exposure interval using Bayesian hierarchical modeling. The saturation time required to generate saturated cross-neutralization responses differed by variant, with variants more antigenically distant from the ancestral strain requiring longer intervals of 2-4 months. The breadths of saturated cross-neutralization responses to Omicron lineages were comparable in pre-Omicron and Omicron breakthrough infections. Our results highlight the importance of vaccine dosage intervals of 4 months or longer, regardless of the antigenicity of the exposed antigen, to maximize the breadth of serum cross-neutralization covering SARS-CoV-2 Omicron lineages.

Molecular Scale Structure and Kinetics of Layer-by-Layer Peptide Self-Organization at Atomically Flat Solid Surfaces.

Understanding the mechanisms of self-organization of short peptides into two- and three-dimensional architectures are of great interest in the formation of crystalline biomolecular systems and their practical applications. Since the assembly is a dynamic process, the study of structural development is challenging at the submolecular dimensions continuously across an adequate time scale in the natural biological environment, in addition to the complexities stemming from the labile molecular structures of short peptides. Self-organization of solid binding peptides on surfaces offers prospects to overcome these challenges. Here we use a graphite binding dodecapeptide, GrBP5, and record its self-organization process of the first two layers on highly oriented pyrolytic graphite surface in an aqueous solution by using frequency modulation atomic force microscopy in situ. The observations suggest that the first layer forms homogeneously, generating self-organized crystals with a lattice structure in contact with the underlying graphite. The second layer formation is mostly heterogeneous, triggered by the crystalline defects on the first layer, growing row-by-row establishing nominally diverse biomolecular self-organized structures with transient crystalline phases. The assembly is highly dependent on the peptide concentration, with the nucleation being high in high molecular concentrations, e.g., >100 µM, while the domain size is small, with an increase in the growth rate that gradually slows down. Self-assembled peptide crystals are composed of either singlets or doublets establishing P1 and P2 oblique lattices, respectively, each commensurate with the underlying graphite lattice with chiral crystal relations. This work provides insights into the surface behavior of short peptides on solids and offers quantitative guidance toward elucidating molecular mechanisms of self-assembly helping in the scientific understanding and construction of coherent bio/nano hybrid interfaces.

Characterization of SARS-CoV-2 Omicron BA.2.75 clinical isolates.

The prevalence of the Omicron subvariant BA.2.75 rapidly increased in India and Nepal during the summer of 2022, and spread globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs is higher than that of BA.2 and BA.5. Of note, BA.2.75 causes focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which is not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicates better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 in a hamster model and should be closely monitored.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Use of the particle agglutination/particle agglutination inhibition test for antigenic analysis of SARS-CoV-2.

Background: The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell-based assays using special equipment such as plate reader or ELISPOT analyzer. Methods: Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus-infected hamster antisera in addition to the beads and virus (PAI test). Results: The PA titer was positively correlated with the plaque-forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods. Conclusions: The PAI test is an easy and rapid method to analyze the antigenicity of SARS-CoV-2.

Impact of Reinfection with SARS-CoV-2 Omicron Variants in Previously Infected Hamsters.

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCE The emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

Antiviral Susceptibilities of Distinct Lineages of Influenza C and D Viruses.

The emergence and spread of antiviral-resistant influenza viruses are of great concern. To minimize the public health risk, it is important to monitor antiviral susceptibilities of influenza viruses. Analyses of the antiviral susceptibilities of influenza A and B viruses have been conducted globally; however, those of influenza C and D viruses are limited. Here, we determined the susceptibilities of influenza C viruses representing all six lineages (C/Taylor, C/Yamagata, C/Sao Paulo, C/Aichi, C/Kanagawa, and C/Mississippi) and influenza D viruses representing four lineages (D/OK, D/660, D/Yama2016, and D/Yama2019) to RNA polymerase inhibitors (baloxavir and favipiravir) by using a focus reduction assay. All viruses tested were susceptible to both drugs. We then performed a genetic analysis to check for amino acid substitutions associated with baloxavir and favipiravir resistance and found that none of the viruses tested possessed these substitutions. Use of the focus reduction assay with the genotypic assay has proven valuable for monitoring the antiviral susceptibilities of influenza C and D viruses as well as influenza A and B viruses. Antiviral susceptibility monitoring of all influenza virus types should continue in order to assess the public health risks posed by these viruses.

The potential of a universal influenza virus-like particle vaccine expressing a chimeric cytokine.

The efficacy of the current influenza vaccines is frequently reduced because of antigenic drift, a trade-off of developing improved vaccines with broad cross-protective activity against influenza A viruses. In this study, we have successfully constructed a chimeric cytokine (CC) comprising the M2 protein, influenza A neuraminidase stalk, and interleukin-12. We produced virus-like particles (VLPs) containing CC and influenza hemagglutinin (HA) proteins using a baculovirus system in Eri silkworm pupae. The protective efficacy of the CCHA-VLP vaccine was evaluated in mice. The CCFkH5HA-VLP vaccine increased the survival rates of BALB/c mice, infected with a lethal dose of PRH1 and HKH5 viruses, to 80% and 100%, respectively. The results suggested that CCHA-VLP successfully induced potent cross-reactive protective immunity against infection with homologous and heterologous subtypes of the influenza A virus. This is the first study to design a CC-containing HA-VLP vaccine and validate its protective efficacy.

Mapping Nanomechanical Properties of Basal Surfaces in Metastatic Intestinal 3D Living Organoids with High-Speed Scanning Ion Conductance Microscopy.

Studying mechanobiology is increasing of scientific interests in life science and nanotechnology since its impact on cell activities (e.g., adhesion, migration), physiology, and pathology. The role of apical surface (AS) and basal surface (BS) of cells played in mechanobiology is significant. The mechanical mapping and analysis of cells mainly focus on AS while little is known about BS. Here, high-speed scanning ion conductance microscope as a powerful tool is utilized to simultaneously reveal morphologies and local elastic modulus (E) of BS of genotype-defined metastatic intestinal organoids. A simple method is developed to prepare organoid samples allowing for long-term BS imaging. The multiple nano/microstructures, i.e., ridge-like, stress-fiber, and E distributions on BS are dynamically revealed. The statistic E analysis shows softness of BS derived from eight types of organoids following a ranking: malignant tumor cells > benign tumor cells > normal cells. Moreover, the correlation factor between morphology and E is demonstrated depending on cell types. This work as first example reveals the subcellular morphologies and E distributions of BS of cells. The results would provide a clue for correlating genotype of 3D cells to malignant phenotype reflected by E and offering a promising strategy for early-stage diagnosis of cancer.