Epidemiological and clinical characteristics of infections with seasonal human coronavirus and respiratory syncytial virus in hospitalized children immediately before the coronavirus disease 2019 pandemic.

INTRODUCTION: Seasonal human coronavirus (HCoV)-229E, -NL63, -OC43, and -HKU1 are seasonal coronaviruses that cause colds in humans. However, the clinical characteristics of pediatric inpatients infected with HCoVs are unclear. This study aimed to compare and clarify the epidemiological and clinical features of HCoVs and respiratory syncytial virus (RSV), which commonly causes severe respiratory infections in children. METHODS: Nasopharyngeal swabs were collected from all pediatric inpatients with respiratory symptoms at two secondary medical institutions in Fukushima, Japan. Eighteen respiratory viruses, including RSV and four HCoVs, were detected via reverse transcription-polymerase chain reaction. RESULTS: Of the 1757 specimens tested, viruses were detected in 1272 specimens (72.4%), with 789 single (44.9%) and 483 multiple virus detections (27.5%). RSV was detected in 639 patients (36.4%) with no difference in clinical characteristics between RSV-A and RSV-B. HCoV was detected in 84 patients (4.7%): OC43, NL63, HKU1, and 229E in 25 (1.4%), 26 (1.5%), 23 (1.3%), and 16 patients (0.9%), respectively. Patients with HCoV monoinfection (n = 35) had a significantly shorter period from onset to hospitalization (median [interquartile range] days, 2 [1-4.5] vs. 4 [2-5]), significantly shorter hospitalization stays (4 [3-5] vs. 5 [4-6]), and more cases of upper respiratory infections (37.1% vs. 3.9%) and croup (17.1% vs. 0.3%) but less cases of lower respiratory infection (54.3% vs. 94.8%) than patients with RSV monoinfection (n = 362). CONCLUSION: Seasonal HCoV-infected patients account for approximately 5% of children hospitalized for respiratory tract infections and have fewer lower respiratory infections and shorter hospital stays than RSV-infected patients.

Deficiency of AMPAR-Palmitoylation Aggravates Seizure Susceptibility.

Synaptic AMPAR expression controls the strength of excitatory synaptic transmission and plasticity. An excess of synaptic AMPARs leads to epilepsy in response to seizure-inducible stimulation. The appropriate regulation of AMPARs plays a crucial role in the maintenance of the excitatory/inhibitory synaptic balance; however, the detailed mechanisms underlying epilepsy remain unclear. Our previous studies have revealed that a key modification of AMPAR trafficking to and from postsynaptic membranes is the reversible, posttranslational S-palmitoylation at the C-termini of receptors. To clarify the role of palmitoylation-dependent regulation of AMPARs in vivo, we generated GluA1 palmitoylation-deficient (Cys811 to Ser substitution) knock-in mice. These mutant male mice showed elevated seizure susceptibility and seizure-induced neuronal activity without impairments in synaptic transmission, gross brain structure, or behavior at the basal level. Disruption of the palmitoylation site was accompanied by upregulated GluA1 phosphorylation at Ser831, but not at Ser845, in the hippocampus and increased GluA1 protein expression in the cortex. Furthermore, GluA1 palmitoylation suppressed excessive spine enlargement above a certain size after LTP. Our findings indicate that an abnormality in GluA1 palmitoylation can lead to hyperexcitability in the cerebrum, which negatively affects the maintenance of network stability, resulting in epileptic seizures.SIGNIFICANCE STATEMENT AMPARs predominantly mediate excitatory synaptic transmission. AMPARs are regulated in a posttranslational, palmitoylation-dependent manner in excitatory synapses of the mammalian brain. Reversible palmitoylation dynamically controls synaptic expression and intracellular trafficking of the receptors. Here, we generated GluA1 palmitoylation-deficient knock-in mice to clarify the role of AMPAR palmitoylation in vivo We showed that an abnormality in GluA1 palmitoylation led to hyperexcitability, resulting in epileptic seizure. This is the first identification of a specific palmitoylated protein critical for the seizure-suppressing process. Our data also provide insight into how predicted receptors such as AMPARs can effectively preserve network stability in the brain. Furthermore, these findings help to define novel key targets for developing anti-epileptic drugs.

Comparison of mutations in human parainfluenza viruses during passage in primary human bronchial/tracheal epithelial air-liquid interface cultures and cell lines.

Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1-HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis. IMPORTANCE: Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses.

IMPORTANCE: Since the pandemic of coronavirus diseases 2019, the use of real-time PCR assay has become widespread among people who were not familiar with it in virus detection. As a result, whether a high real-time PCR value in one time test indicates virus transmissibly became a complicated social problem, regardless of the difference in assays and/or amplification conditions, the time and number of diagnostic test during the time course of infection. In addition, the multiple positives in the test of respiratory viruses further add to the confusion in the interpretation of the infection. To address this issue, we performed virus isolation using pediatric SARI (severe acute respiratory infections) specimens on air-liquid interface culture of human bronchial/tracheal epithelial cell culture. The result of this study can be a strong evidence that the specimens showing positivity for multiple agents in real-time PCR tests possibly contain infectious viruses.

Molecular Epidemiology of Human Metapneumovirus in East Japan before and after COVID-19, 2017-2022.

Human metapneumovirus (hMPV) is genetically classified into two major subgroups, A and B, based on attachment glycoprotein (G protein) gene sequences. The A2 subgroup is further separated into three subdivisions, A2a, A2b (A2b1), and A2c (A2b2). Subgroup A2c viruses carrying 180- or 111-nucleotide duplications in the G gene (A2c 180nt-dup or A2c 111nt-dup ) have been reported in Japan and Spain. The coronavirus disease 2019 (COVID-19) pandemic disrupted the epidemiological kinetics of other respiratory viruses, including hMPV. In this study, we analyzed the sequences of hMPV isolates in Tokyo and Fukushima obtained from 2017 to 2022, i.e., before and after the COVID-19 pandemic. Subgroup A hMPV strains were detected from 2017 to 2019, and most cases were A2c 111nt-dup, suggesting ongoing transmission of this clade, consistent with global transmission dynamics. Subgroup B viruses, but not subgroup A viruses, were detected in 2022 after the COVID-19 peak. Phylogenetic analysis showed that the subgroup B viruses were closely related to strains detected in Yokohama from 2013 to 2016, and strains detected in Fukushima in 2019, suggesting the reappearance of local endemic viruses in East Japan.

Development of a duplex real-time RT-PCR assay for the detection and identification of two subgroups of human metapneumovirus in a single tube.

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. Many genetic diagnostic assays have been developed, but most detect hMPV regardless of the subgroup. In this study, we developed a real-time RT-PCR assay that can detect and identify the two major subgroups of hMPV (A and B) in one tube. Primers and probes were designed based on the sequences of recent clinical isolates in Japan. The assay showed comparable analytical sensitivity to a previously reported real-time RT-PCR assay and specific reactions to hMPV subgroups. The assay also showed no cross-reactivity to clinical isolates of 19 species of other respiratory viruses. In a validation assay using post-diagnosed clinical specimens, 98% (167/170) positivity was confirmed for the duplex assay, and the three specimens not detected were of low copy number. The duplex assay also successfully distinguished the two major subgroups for all 12 clinical specimens, for which the subgroup had already been determined by genomic sequencing analysis. The duplex assay described here will contribute to the rapid and accurate identification and surveillance of hMPV infections.

Molecular Diversity of Human Respiratory Syncytial Virus before and during the COVID-19 Pandemic in Two Neighboring Japanese Cities.

Human respiratory syncytial viruses (HRSVs) are divided into subgroups A and B, which are further divided based on the nucleotide sequence of the second hypervariable region (HVR) of the attachment glycoprotein (G) gene. Understanding the molecular diversity of HRSV before and during the coronavirus disease 2019 (COVID-19) pandemic can provide insights into the effects of the pandemic on HRSV dissemination and guide vaccine development. Here, we analyzed HRSVs isolated in Fukushima Prefecture from September 2017 to December 2021. Specimens from pediatric patients were collected at two medical institutions in neighboring cities. A phylogenetic tree based on the second HVR nucleotide sequences was constructed using the Bayesian Markov chain Monte Carlo method. HRSV-A (ON1 genotype) and HRSV-B (BA9 genotype) were detected in 183 and 108 specimens, respectively. There were differences in the number of HRSV strains within clusters prevalent at the same time between the two hospitals. The genetic characteristics of HRSVs in 2021 after the COVID-19 outbreak were similar to those in 2019. HRSVs within a cluster may circulate within a region for several years, causing an epidemic cycle. Our findings add to the existing knowledge of the molecular epidemiology of HRSV in Japan. IMPORTANCE Understanding the molecular diversity of human respiratory syncytial viruses during pandemics caused by different viruses can provide insights that can guide public health decisions and vaccine development.

Thirteen Nearly Complete Genome Sequences of Human Bocavirus 1 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 13 genomic sequences of human bocavirus 1 isolated from pediatric inpatients in Fukushima, Japan, using an air-liquid interface culture of human bronchial tracheal epithelial cells. This work suggests the endemic circulation of a human bocavirus variant with a unique amino acid signature in Fukushima.

Practical Validation of United States Centers for Disease Control and Prevention Assays for the Detection of Human Respiratory Syncytial Virus in Pediatric Inpatients in Japan.

The World Health Organization initiated a global surveillance system for respiratory syncytial virus (RSV) in 2015, and the pilot surveillance is ongoing. The real-time RT-PCR RSV assays (Pan-RSV and duplex assays) developed by the United States Centers for Disease Control and Prevention are applied as the standard assays. To introduce these as standard assays in Japan, their practicality was evaluated using 2261 specimens obtained from pediatric inpatients in Japan, which were collected from 2018 to 2021. Although the Pan-RSV and duplex assays had similar analytical sensitivities, they yielded 630 (27.9%) and 786 (34.8%) RSV-positive specimens, respectively (p < 0.001). Although sequencing analysis showed mismatches in the reverse primer used in the Pan-RSV assay, these mismatches did not affect its analytical sensitivity. The analysis of read numbers of RSV isolates from air−liquid interface culture of human bronchial/tracheal epithelial cells showed that the duplex assay had a greater number of reads than did the Pan-RSV assay. Therefore, the duplex assay has superior detection performance compared with the Pan-RSV assay, but the two assays have similar analytical sensitivities.

Ten Nearly Complete Genome Sequences of Human Orthorubulavirus 4 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 10 nearly complete genomic sequences of human orthorubulavirus 4, also called human parainfluenza virus 4 (HPIV4), isolated from pediatric inpatients with respiratory infections in Fukushima, Japan, by using an air-liquid interface culture of human bronchial and tracheal epithelial cells.

Nearly Complete Genome Sequences of 12 Types of Human Rhinoviruses Isolated from Pediatric Inpatients in Fukushima, Japan.

We reported nearly complete genomic sequences of 12 serotypes of human rhinoviruses (HRVs) isolated from pediatric inpatients in Fukushima, Japan using an air-liquid interface culture of human bronchial tracheal epithelial cells. We found that various serotypes of HRV circulated locally and simultaneously from 2018 to 2021.

Changes in virus detection in hospitalized children before and after the severe acute respiratory syndrome coronavirus 2 pandemic.

The impact of strengthening preventive measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the prevalence of respiratory viruses in children was examined. After the SARS-CoV-2 pandemic, the rate of multiple virus detection among hospitalized children decreased. Immediately after the SARS-CoV-2 pandemic, respiratory syncytial and parainfluenza viruses were rarely detected and subsequently reemerged. Human metapneumovirus and influenza virus were not consistently detected. Non-enveloped viruses (bocavirus, rhinovirus, and adenovirus) were detected to some extent even after the pandemic. Epidemic-suppressed infectious diseases may reemerge as susceptibility accumulates in the population and should continue to be monitored.