Detection of influenza A(H3N2) viruses with polymerase acidic subunit substitutions after and prior to baloxavir marboxil treatment during the 2022-2023 influenza season in Japan.

Baloxavir marboxil (baloxavir), approved as an anti-influenza drug in Japan in March 2018, can induce reduced therapeutic effectiveness due to PA protein substitutions. We assessed PA substitutions in clinical samples from influenza-infected children and adults pre- and post-baloxavir treatment, examining their impact on fever and symptom duration. During the 2022-2023 influenza season, the predominant circulating influenza subtype detected by cycling-probe RT-PCR was A(H3N2) (n = 234), with a minor circulation of A(H1N1)pdm09 (n = 10). Of the 234 influenza A(H3N2) viruses collected prior to baloxavir treatment, 2 (0.8%) viruses carry PA/I38T substitution. One virus was collected from a toddler and one from an adult, indicating the presence of viruses with reduced susceptibility to baloxavir, without prior exposure to the drug. Of the 54 paired influenza A(H3N2) viruses collected following baloxavir treatment, 8 (14.8%) viruses carried E23 K/G, or I38 M/T substitutions in PA. Variant calling through next-generation sequencing (NGS) showed varying proportions (6-100 %), a polymorphism and a mixture of PA/E23 K/G, and I38 M/T substitutions in the clinical samples. These eight viruses were obtained from children aged 7-14 years, with a median fever duration of 16.7 h and a median symptom duration of 93.7 h, which were similar to those of the wild type. However, the delayed viral clearance associated with the emergence of PA substitutions was observed. No substitutions conferring resistance to neuraminidase inhibitors were detected in 37 paired samples collected before and following oseltamivir treatment. These findings underscore the need for ongoing antiviral surveillance, informing public health strategies and clinical antiviral recommendations for seasonal influenza.

Molecular Epidemiology of Respiratory Syncytial Virus during 2019-2022 and Surviving Genotypes after the COVID-19 Pandemic in Japan.

To evaluate the changes in respiratory syncytial virus (RSV) collected between 2019 and 2022, we analyzed RSV-A and RSV-B strains from various prefectures in Japan before and after the COVID-19 pandemic. RT-PCR-positive samples collected from children with rapid test positivity at outpatient clinics in 11 prefectures in Japan were sequenced for the ectodomain of the G gene to determine the genotype. Time-aware phylogeographic analyses were performed using the second hypervariable region (HVR) of the G gene from 2012 to 2022. Of 967 samples, 739 (76.4%) were found to be RSV-positive using RT-PCR. RSV peaked in September 2019 but was not detected in 2020, except in Okinawa. Nationwide epidemics occurred with peaks in July 2021 and 2022. The genotype remained the same, ON1 for RSV-A and BA9 for RSV-B during 2019-2022. Phylogeographic analysis of HVR revealed that at least seven clusters of RSV-A had circulated previously but decreased to two clusters after the pandemic, whereas RSV-B had a single monophyletic cluster over the 10 years. Both RSV-A and RSV-B were transferred from Okinawa into other prefectures after the pandemic. The RSV epidemic was suppressed due to pandemic restrictions; however, pre-pandemic genotypes spread nationwide after the pandemic.

Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.

In Japan, major mumps outbreaks still occur every 4-5 years because of low mumps vaccine coverage (30-40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan's regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

Duration of fever and symptoms in influenza-infected children treated with baloxavir marboxil during the 2019-2020 season in Japan and detection of influenza virus with the PA E23K substitution.

Data on the clinical effectiveness of the novel anti-influenza drug baloxavir marboxil (baloxavir) in children remain limited. We conducted an observational study to compare the duration of fever and symptoms between baloxavir- and oseltamivir-treated children infected with influenza A and B. In total, 159 outpatients with influenza A(H1N1)pdm09 or B/Victoria-lineage infections, aged <19 years, during the 2019-2020 influenza season in Japan were enrolled and assessed the duration of fever and symptoms using the Kaplan-Meier method and a multivariate Cox proportional hazard regression model. Polymerase acidic (PA) variants were examined before and after baloxavir treatment. In the multivariable analysis, the duration of fever and symptoms was unaltered between the A(H1N1)pdm09 (n = 116) and B/Victoria-lineage (n = 43) groups. Conversely, the fever duration was marginally longer in the oseltamivir-treated group (n = 59) than in the baloxavir group (n = 100) (hazard ratio (HR) = 0.67, p = 0.05); however, the duration of symptoms was unaltered between the two groups (HR = 0.74, p = 0.11). No patient presented PA reduced susceptibility marker(s) before baloxavir treatment in the analyzed groups. The PA/E23K variant was detected in one case (1.5%, 1/66) of A(H1N1)pdm09 after baloxavir treatment. One case (2.0%, 1/50) of A(H1N1)pdm09 with an N295S substitution in neuraminidase was detected following oseltamivir treatment. These results suggested that the duration of fever was likely to be shorter with baloxavir than with oseltamivir, but the difference between influenza A (H1N1)pdm09 and B/Victoria-lineage was unclear. It is important to continue evaluating the clinical effectiveness of baloxavir and monitoring its drug susceptibility to the influenza virus.

Development of cycling probe based real-time PCR methodology for influenza A viruses possessing the PA/I38T amino acid substitution associated with reduced baloxavir susceptibility.

Baloxavir marboxil has been used for influenza treatment since March 2018 in Japan. After baloxavir treatment, the most frequently detected substitution is Ile38Thr in polymerase acidic protein (PA/I38T), and this substitution reduces baloxavir susceptibility in influenza A viruses. To rapidly investigate the frequency of PA/I38T in influenza A (H1N1)pdm09 and A (H3N2) viruses in clinical samples, we established a rapid real-time system to detect single nucleotide polymorphisms in PA, using cycling probe real-time PCR. We designed two sets of probes that were labeled with either 6-carboxyfluorescein (FAM) or 6-carboxy-X-rhodamine (ROX) to identify PA/I38 (wild type strain) or PA/I38T, respectively. The established cycling probe real-time PCR system showed a dynamic linear range of 101 to 106 copies with high sensitivity in plasmid DNA controls. This real-time PCR system discriminated between PA/I38T and wild type viruses well. During the 2018/19 season, 377 influenza A-positive clinical samples were collected in Japan before antiviral treatment. Using our cycling probe real-time PCR system, we detected no (0/129, 0.0%) influenza A (H1N1)pdm09 viruses with PA/I38T substitutions and four A (H3N2) (4/229, 1.7%) with PA/I38T substitution prior to treatment. In addition, we found PA/I38T variant in siblings who did not received baloxavir treatment during an infection caused by A (H3N2) that afflicted the entire family. Although human-to-human transmission of PA/I38T variant may have occurred in a closed environment, the prevalence of this variant in influenza A viruses was still limited. Our cycling probe-PCR system is thus useful for antiviral surveillance of influenza A viruses possessing PA/I38T.

Impact of thirteen-valent pneumococcal conjugate vaccine on nasopharyngeal carriage in healthy children under 24 months in Okinawa, Japan.

In November 2013, a 13-valent pneumococcal conjugate vaccine (PCV13) for all infants aged younger than 5 years was incorporated into the Japan national immunization program. An annual survey of nasopharyngeal carriage rates was performed on healthy infants aged 2-24 months from Okinawa, Japan to evaluate the effect of PCV13 on pneumococcal colonization. Of 756 evaluable infants, 203 pneumococcal strains were detected in 193 infants. The overall nasopharyngeal carriage rate was 25.5%, which was not different from our previously reported isolation rate before the introduction of PCV13. The main serotypes of the Streptococcus pneumoniae strains are 15A (18.2%), non-typeable (14.8%), and 15B (11.8%). The carriage rates of pneumococcal strains with 7-valent pneumococcal conjugate vaccine serotypes and PCV13 serotypes were 3.0% and 9.9%, respectively. These values were significantly lower than we reported before the introduction of PCV13. However, the carriage rates of non-PCV13 serotypes have increased. Multivariate logistic regression analysis suggested that siblings and day care attendance are risk factors for pneumococcal carriage.

Molecular epidemiology of human metapneumovirus from 2009 to 2011 in Okinawa, Japan.

To clarify the molecular epidemiology of human metapneumovirus (HMPV) in Okinawa Prefecture, located in a subtropical region of Japan, we performed genetic analysis of the F gene in HMPV from patients with acute respiratory infection from January 2009 to December 2011. HMPV was detected in 18 of 485 throat swabs (3.7%). Phylogenetic analysis showed that 17 strains belonged to subgroup A2 and 1 strain belonged to subgroup B1. We did not observe seasonal prevalence of HMPV during the investigation period. A high level of sequence identity was observed in the strains belonging to subgroup A2 (>95%), and no amino acid substitution was found compared with other strains detected in Japan and other countries. The pairwise distance values among the present strains belonging to subgroup A2 were short. Our results suggest that the predominant HMPV strains belonging to A2 are highly homologous and seasonal epidemics were not seen in Okinawa during the investigation period.