Epidemiologic, clinical, and genetic characteristics of influenza C virus infections among outpatients and inpatients in Sendai, Japan from 2006 to 2020.

BACKGROUND: Influenza C virus is a pathogen that causes acute respiratory illness in children. The clinical information about this virus is limited because of the small number of isolated viruses compared to influenza A or B viruses. METHODS: A total of 60 influenza C viruses were isolated by clinical tests using cell culture methods conducted in one hospital and one clinic during the 15 years from 2006 to 2020. These 60 cases were retrospectively analyzed by comparing outpatients and inpatients. Moreover, isolated viruses were analyzed for genomic changes during the study period. RESULTS: All were younger than 7 years, and 73% of inpatients (19 out of 26) were under 2 years of age. A significant difference was found in the frequency of pneumonia, accounting for 45% and 4% of inpatients and outpatients, respectively. Most of the viruses isolated from 2006 to 2012 belonged to the S/A sublineage of the C/Sao Paulo lineage, but three sublineage viruses, including the S/A sublineage with K190N mutation, S/V sublineage, and C/Kanagawa lineage, have cocirculated since 2014. Moreover, S/A sublineage viruses were undergoing reassortment since 2014, suggesting significant changes in the virus, both antigenically and genetically. Of the 10 strains from patients with pneumonia, 7 were in the S/A sublineage, which had circulated from 2006 to 2012. CONCLUSION: Infants under 2 years of age were more likely to be hospitalized with pneumonia. The genomic changes that occurred in 2014 were suggested to affect the ability of the virus to spread.

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.

Fluctuations in Antibody Titers against Enterovirus D68 in Pediatric Sera Collected in a Community before, during, and after a Possible Outbreak.

We previously reported a hospital-based epidemiological study on enterovirus (EV)-D68 infection among children during the autumn of 2015, which indirectly inferred an outbreak in Sendai, Japan. In this study, stocked sera of children (aged 0-6 years; without symptoms of infectious diseases) in the Sendai community collected during 4 periods (1 year before, 6 months before, immediately after, and 1 year after the possible outbreak period) were analyzed using the neutralization antibody titer assay to determine community children's immunity levels against EV-D68 infection. The immunity levels were confirmed to have increased during the possible outbreak period and to have gradually waned over 1 year without another outbreak. These results provide background information supporting the results of our previous hospital-based surveillance study.

Efficient isolation of human metapneumovirus using MNT-1, a human malignant melanoma cell line with early and distinct cytopathic effects.

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC-MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT-1, obvious syncytium formation occurs shortly after inoculation with HMPV-positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT-1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation-based surveillance. MNT-1 has the potential to facilitate clinical and epidemiological studies of HMPV.

Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71.

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.

Comparison of virus isolation using the Vero E6 cell line with real-time RT-PCR assay for the detection of human metapneumovirus.

BACKGROUND: The use of cell culture for the diagnosis of human metapneumovirus (hMPV) infection is uncommon at present and molecular method such as reverse-transcription PCR (RT-PCR) has been widely and most commonly used as the preferred test. We aimed to compare the results of virus isolation using Vero E6 cells with real-time RT-PCR for the detection of hMPV, since such a comparison data is not available. METHODS: Between December 2007 and July 2008, we obtained 224 nasopharyngeal swab specimens from patients with acute respiratory infection and tested by the two methods. RESULTS: Forty-three (19.2%) were found positive by cell culture and 62 (27.7%) by real-time RT-PCR. Cell cultures were positive for 42 of 62 specimens found positive by real-time RT-PCR (67.7% sensitivity) and for 1 of 162 specimens found negative by real-time RT-PCR (99.4% specificity), respectively. The sensitivity of the cell culture was 76.2-87.5% (mean 81.8%) when specimens were collected within 3 days after the onset of symptoms, and the sensitivity decreased to 50% or less thereafter. Among specimens collected within 3 days after symptom onset, all of the real-time RT-PCR positive specimens having a viral load of more than 1.25x105 copies/ml were found positive by cell culture. CONCLUSIONS: Cell culture using Vero E6 cell line has 81.8% sensitivity compared with the real-time RT-PCR method, when specimens are collected within 3 days after the onset of symptoms. Thus, this method is a useful method for epidemiological and virological research even in facilities with minimal laboratory resources.

Evaluation of a new rapid antigen test using immunochromatography for detection of human metapneumovirus in comparison with real-time PCR assay.

A new rapid human metapneumovirus (hMPV) detection kit using immunochromatography (SAS hMPV test) was compared to real-time PCR for 224 nasal swab specimens, 96.4% of which were obtained from children of <15 years of age. The overall sensitivity and specificity were 82.3% and 93.8%, respectively, suggesting that this test is useful for pediatricians to diagnose hMPV infection in a clinical setting.

Clinical features of influenza C virus infection in children.

BACKGROUND: Seroepidemiological studies have revealed that influenza C virus is widely distributed globally. However, because the isolation of this virus is difficult, there have been few reports on its clinical features. METHODS: Between December 1990 and November 2004, 84,946 respiratory-tract specimens were obtained from patients < or = 15 years old. On the basis of the results of isolation of virus, we examined the clinical data on children infected with influenza C virus. RESULTS: Of 170 children infected with influenza C virus, 157 (92.4%) were < 6 years old. Fever (frequency, 90.0%), cough (frequency, 74.1%), and rhinorrhea (frequency, 61.8%) were the most frequent symptoms. The mean duration of fever was 2.88 days (standard deviation, 1.66 days). Of the 170 children, 29 were hospitalized, and 21 (72.4%) of these 29 had lower-respiratory-tract illness such as pneumonia, bronchitis, and bronchiolitis. The rate of hospital admission was significantly higher in children < 2 years old than in children 2-5 years old (30.4% vs. 11.9%; P = .0043). CONCLUSIONS: Influenza C virus is a significant cause of upper-respiratory-tract illness in children < 6 years old, and the risk of complications with lower-respiratory-tract illness is particularly high in children < 2 years old.