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.

Increased risk of rhinovirus infection in children during the coronavirus disease-19 pandemic.

BACKGROUND: Coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in Japan in January 2020 and has spread throughout the country. Previous studies have reported that viral interference among influenza virus, rhinovirus, and other respiratory viruses can affect viral infections at the host and population level. METHODS: To investigate the impact of COVID-19 on influenza and other respiratory virus infections, we analyzed clinical specimens collected from 2244 patients in Japan with respiratory diseases between January 2018 and September 2020. RESULTS: The frequency of influenza and other respiratory viruses (coxsackievirus A and B; echovirus; enterovirus; human coronavirus 229E, HKU1, NL63, and OC43; human metapneumovirus; human parainfluenza virus 1, 2, 3, and 4; human parechovirus; human respiratory syncytial virus; human adenovirus; human bocavirus; human parvovirus B19; herpes simplex virus type 1; and varicella-zoster virus) was appreciably reduced among all patients during the COVID-19 pandemic except for that of rhinovirus in children younger than 10 years, which was appreciably increased. COVID-19 has not spread among this age group, suggesting an increased risk of rhinovirus infection in children. CONCLUSIONS: Rhinovirus infections should be continuously monitored to understand their increased risk during the COVID-19 pandemic and viral interference with SARS-CoV-2.

Recent Molecular Evolution of Human Metapneumovirus (HMPV): Subdivision of HMPV A2b Strains.

Human metapneumovirus (HMPV) is a major etiological agent of acute respiratory infections in humans. HMPV has been circulating worldwide for more than six decades and is currently divided into five agreed-upon subtypes: A1, A2a, A2b, B1, and B2. Recently, the novel HMPV subtypes A2c, A2b1, and A2b2 have been proposed. However, the phylogenetic and evolutionary relationships between these recently proposed HMPV subtypes are unclear. Here, we report a genome-wide phylogenetic and evolutionary analysis of 161 HMPV strains, including unique HMPV subtype A2b strains with a 180- or 111-nucleotide duplication in the G gene (nt-dup). Our data demonstrate that the HMPV A2b subtype contains two distinct subtypes, A2b1 and A2b2, and that the HMPV subtypes A2c and A2b2 may be different names for the same subtype. HMPV A2b strains with a nt-dup also belong to subtype A2b2. Molecular evolutionary analyses indicate that subtypes A2b1 and A2b2 diverged from subtype A2b around a decade after the subtype A2 was divided into the subtypes A2a and A2b. These data support the A2b1 and A2b2 subtypes proposed in 2012 and are essential for the unified classification of HMPV subtype A2 strains, which is important for future HMPV surveillance and epidemiological studies.

Nationwide Molecular Epidemiology of Measles Virus in Japan Between 2008 and 2017.

Genotyping evidence that supports the interruption of endemic measles virus (MV) transmission is one of the essential criteria to be verified in achieving measles elimination. In Japan since 2014, MV genotype analyses have been performed for most of the measles cases in prefectural public health institutes nationwide. With this strong molecular epidemiological data, Japan was verified to have eliminated measles in March, 2015. However, even in the postelimination era, sporadic cases and small outbreaks of measles have been detected repeatedly in Japan. This study investigated the nationwide molecular epidemiology of MV between 2008 and 2017. The 891 strains in the total period between 2008 and 2017 belonged to seven genotypes (D5, D4, D9, H1, G3, B3, and D8) and 124 different MV sequence variants, based on the 450-nucleotide sequence region of the N gene (N450). The 311 MV strains in the postelimination era between 2015 and 2017 were classified into 1, 7, 8, and 32 different N450 sequence variants in D9, H1, B3, and D8 genotypes, respectively. Analysis of the detection period of the individual N450 sequence variants showed that the majority of MV strains were detected only for a short period. However, MV strains, MVs/Osaka.JPN/29.15/ [D8] and MVi/Hulu Langat.MYS/26.11/ [D8], which are named strains designated by World Health Organization (WHO), have been detected in many cases over 2 or 3 years between 2015 and 2017. The WHO-named strains have circulated worldwide, causing outbreaks in many countries. Epidemiological investigation revealed repeated importation of these WHO-named strains into Japan. To demonstrate the elimination status (interruption of endemic transmission) in situations with repeated importation of the same strains is challenging. Nevertheless, the detailed sequence analysis of individual MV strains and chronological analysis of these strains provided sufficient evidence to show that Japan has still maintained its measles elimination status in 2017.

Predominant Detection of the Subgroup A2b Human Metapneumovirus Strain with a 111-Nucleotide Duplication in the G gene in Yokohama City, Japan in 2018.

Human metapneumovirus (HMPV) has been a major causative agent of acute respiratory infections in humans. Recently, two types of variant A2b subtype HMPV strains possessing a 111- or 180-nucleotide duplication (nt-dup) in the G gene (HMPV A2b180nt-dup and HMPV A2b111nt-dup, respectively) were detected in Japan, Spain, Vietnam, and China. Our surveillance for infectious agents in Yokohama City, Japan revealed that the HMPV A2b111nt-dup strain became predominant in Yokohama City in 2018. In contrast, no classic HMPV A2b strain was detected after 2017. These data indicate a beneficial role of the 111nt-dup in the G gene for the transmission of HMPV.

Genetic and antigenic characterisation of influenza A(H3N2) viruses isolated in Yokohama during the 2016/17 and 2017/18 influenza seasons.

BACKGROUND: Influenza A(H3N2) virus rapidly evolves to evade human immune responses, resulting in changes in the antigenicity of haemagglutinin (HA). Therefore, continuous genetic and antigenic analyses of A(H3N2) virus are necessary to detect antigenic mutants as quickly as possible. AIM: We attempted to phylogenetically and antigenically capture the epidemic trend of A(H3N2) virus infection in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons. METHODS: We determined the HA sequences of A(H3N2) viruses detected in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons to identify amino acid substitutions and the loss or gain of potential N-glycosylation sites in HA, both of which potentially affect the antigenicity of HA. We also examined the antigenicity of isolates using ferret antisera obtained from experimentally infected ferrets. RESULTS: Influenza A(H3N2) viruses belonging to six clades (clades 3C.2A1, 3C.2A1a, 3C.2A1b, 3C.2A2, 3C.2A3 and 3C.2A4) were detected during the 2016/17 influenza season, whereas viruses belonging to two clades (clades 3C.2A1b and 3C.2A2) dominated during the 2017/18 influenza season. The isolates in clades 3C.2A1a and 3C.2A3 lost one N-linked glycosylation site in HA relative to other clades. Antigenic analysis revealed antigenic differences among clades, especially clade 3C.2A2 and 3C.2A4 viruses, which showed distinct antigenic differences from each other and from other clades in the antigenic map. CONCLUSION: Multiple clades, some of which differed antigenically from others, co-circulated in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons.

A novel 111-nucleotide duplication in the G gene of human metapneumovirus.

In 2017, novel human metapneumovirus (HMPV) A2b subgroup strains with a 111-nucleotide duplication in the G gene was detected by the present team. These strains were related to previously identified HMPV A2b strains with a 180-nucleotide duplication; however, they appeared to be different strains, produced by an independent duplication event. The recent evolution of HMPV suggests that careful monitoring of this virus is required.

Molecular Epidemiology of Rubella Virus Strains Detected Around the Time of the 2012-2013 Epidemic in Japan.

A nationwide rubella epidemic occurred from 2012 to 2013 in Japan, resulting in around 17,000 rubella cases and the birth of 45 infants with congenital rubella syndrome. The aim of this study was to genetically characterize the rubella viruses (RVs) circulating around the time of the epidemic in Japan. In total, 221 RV strains detected from 14 prefectures in Japan between 2010 and 2014 were sequenced in the 739 nucleotide-window region within the E1 gene. The virus strains were chronologically and geographically characterized into groups based on phylogenetic analysis. Among the 221 strains analyzed, 192 (87%), 26 (12%), and 3 (1%) strains were classified into genotypes 2B, 1E, and 1J, respectively. The majority (n = 184) of the genotype 2B strains belonged to lineage 2B-L1 and shared nucleotide homology with the strains detected in Southeast and East Asian countries. Phylogenetic analyses demonstrated that at least six distinct clusters of RV strains (clusters 1-6) induced outbreaks in Japan between 2010 and 2014. Among them, strains from clusters 3, 4, and 6 circulated almost simultaneously during 2012-2013. The cluster 3 strains circulated locally, whereas strains from cluster 4 spread nationwide. The findings suggest that RVs were introduced into Japan many times from neighboring countries. The 2012-2013 epidemic was a complex of outbreaks induced by at least three clusters of RV strains.

180-Nucleotide Duplication in the G Gene of Human metapneumovirus A2b Subgroup Strains Circulating in Yokohama City, Japan, since 2014.

Human metapneumovirus (HMPV), a member of the family Paramyxoviridae, was first isolated in 2001. Seroepidemiological studies have shown that HMPV has been a major etiological agent of acute respiratory infections in humans for more than 50 years. Molecular epidemiological, genetic, and antigenetic evolutionary studies of HMPV will strengthen our understanding of the epidemic behavior of the virus and provide valuable insight for the control of HMPV and the development of vaccines and antiviral drugs against HMPV infection. In this study, the nucleotide sequence of and genetic variations in the G gene were analyzed in HMPV strains prevalent in Yokohama City, in the Kanto area, Japan, between January 2013 and June 2016. As a part of the National Epidemiological Surveillance of Infectious Diseases, Japan, 1308 clinical specimens (throat swabs, nasal swabs, nasal secretions, and nasal aspirate fluids) collected at 24 hospitals or clinics in Yokohama City were screened for 15 major respiratory viruses with a multiplex reverse transcription-PCR assay. HMPV was detected in 91 specimens, accounting for 7.0% of the total specimens, and the nucleotide sequences of the G genes of 84 HMPV strains were determined. Among these 84 strains, 6, 43, 10, and 25 strains were classified into subgroups A2a, A2b, B1, and B2, respectively. Approximately half the HMPV A2b subgroup strains detected since 2014 had a 180-nucleotide duplication (180nt-dup) in the G gene and clustered on a phylogenic tree with four classical 180nt-dup-lacking HMPV A2b strains prevalent between 2014 and 2015. The 180nt-dup causes a 60-amino-acid duplication (60aa-dup) in the G protein, creating 23-25 additional potential acceptor sites for O-linked sugars. Our data suggest that 180nt-dup occurred between 2011 and 2013 and that HMPV A2b strains with 180nt-dup (A2b180nt-dup HMPV) became major epidemic strains within 3 years. The detailed mechanism by which the A2b180nt-dup HMPV strains gained an advantage that allowed their efficient spread in the community and the effects of 60aa-dup on HMPV virulence must be clarified.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens.

BACKGROUND: An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. OBJECTIVES: To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. STUDY DESIGN: The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. RESULTS: The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. CONCLUSIONS: The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination.

Effect of Th1/Th2 cytokine pretreatment on RSV-induced gene expression in airway epithelial cells.

BACKGROUND: Respiratory syncytial virus (RSV) infection in infants with Th2 predisposition is thought to increase the risk of allergic sensitization, recurrent wheezing, and bronchial asthma during childhood. We attempted to clarify the molecular mechanisms by which Th1/Th2 predisposition in the host alters RSV infection and facilitates airway inflammation. METHODS: A549 human airway epithelial cells were inoculated with live or UV-treated RSV after pretreatment with either a combination of tumor necrosis factor (TNF)-α and interferon-γ (Th1-primed) or a combination of TNF-α and interleukin-4 (Th2-primed) for 48 h. The gene and protein expression profiles of RSV-infected A549 cells were examined. RESULTS: GeneChip analysis indicated that, at 96 h after inoculation with RSV, the expression of 62 genes was specifically enhanced (more than 2-fold by normalized data) in Th2-primed cells compared to that in unprimed or Th1-primed cells. An increase in mRNA and protein levels of monocyte chemoattractant protein (MCP)-1/CCL2 among those 62 genes was confirmed by real-time PCR and cytometric bead assay, respectively. RSV replication was markedly diminished in Th1-primed airway epithelial cells but not in Th2-primed cells, which was presumably caused at least in part by the early induction of antiviral genes. CONCLUSIONS: These results suggest that Th1/Th2 predisposition in the host prior to RSV infection critically regulates inflammatory reactions in the airways through alteration of gene expression, and that MCP-1/CCL2 plays an important role in the pathogenesis of severe RSV infection and the subsequent development of asthma in Th2-predisposed hosts.

[An epidemiological study of respiratory syncytial virus infection in Yokohama, Japan for five years].

The epidemiologic features of respiratory syncytial virus (RSV) infection were investigated by detecting the virus in throat swab specimens from patients with acute respiratory symptoms attending the sentinel surveillance clinics in Yokohama City in 5 seasons from July 1998 to June 2003. Throughout the 5 seasons, RSV was found from 181 in 2683 specimens tested (6.7%) by virus isolation in cell culture or genome detection using nested RT-PCR, and this detection rate followed that of influenza virus (infl.v.) (441/2683; 16.4%), while the proportion of RSV isolates in a season fluctuated from 12 to 22% of all causative viruses identified. Analysis of monthly detected number of strains revealed that the peak of RSV isolation was present in December which preceded that of infl.v. by 2 months. Moreover, RSV strains were isolated sporadically during late spring to early autumn (from May to September) when infl.v. was scarcely detected. Among 181 RSV strains, 172 could be subgrouped; 104 were identified as subgroup A, while 68 were B. Subgroup A were detected more frequently throughout the 5 seasons (57%), though the proportion varied seasonally and subgroup B exceeded both in 2000/2001 and 2002/2003 seasons (61% and 70%, respectively). Clinical characteristics of RSV-infected patients were compared with those infected with infl.v. Age distribution of cases revealed that RSV detected predominantly (79%) from lower age groups (less than 5 years) compared to infl. v (41%). As for the proportion of cases showing clinical symptoms of lower respiratory inflammation predominated in RSV-infected irrespective of age groups.

Isolation of influenza A H1N2 viruses from an outbreak in Yokohama City during the 2001-2002 influenza season in Japan.

Emergence of Influenza A H1N2 viruses was documented worldwide during the 2001-2002 influenza season. In Japan, H1N2 viruses were isolated from two students of a junior high school in an influenza outbreak in Yokohama City, February 2001. Genetic and antigenic analyses demonstrated that the H1N2 viruses isolated in Japan shared common features with those isolated in other countries.

[Study of a respiratory syncytial virus diagnostic test kit based on immunochromatography].

We carried out clinical and basic studies of the Directigen Lateral Flow RSV (Becton, Dickinson and Company, USA), a rapid test kit that detects respiratory syncytial virus (hereinafter referred to as "RSV") antigens based on immunochromatography. For the clinical study, 103 nasopharyngeal aspirates from patients with acute respiratory infections were used to evaluate the kit. Compared to the cell culture method, the Directigen Lateral Flow RSV showed a sensitivity of 100% (16/16) and a specificity of 94.3% (82/87), and an agreement rate of 95.1% (98/103). When compared to conventional testing kits, we found that the total agreement rate with the Directigen RS (Nippon Becton Dickinson and Company) was 88.3% (91/103) and with RSV TestPack (Dainabot Co., Ltd.) was 91.3% (94/103). The detection limit of the Directigen Lateral Flow RSV was 2 x 10(3) PFU/ml for both RSV subgroups A and B. In the crossreactivity test, only RSV was found positive. No other microorganisms were crossreactive. We also studied storage stability of nasopharyngeal aspirates and found that stability was not affected by storage at room and refrigerator temperatures for 14 days. Taken all together, the Directigen Lateral Flow RSV is useful for the diagnosis of RSV infection in a clinical setting because its performance is equivalent to conventional testing kits and is easy to use.