Seroprevalence of coxsackievirus A21 neutralizing antibodies in Yamagata, Japan, between 1976 and 2019; coxsackievirus A21 has rarely affected young children.

Although coxsackievirus A21 (CV-A21) has been associated with an acute respiratory infection (ARI) as well as poliomyelitis-like paralysis, reports of CV-A21 detection have been quite limited both globally and in Japan. CV-A21 strains were isolated from five sporadic pediatric cases with ARI in 2019 in Yamagata, Japan. Neutralizing antibodies (NT Abs) were then measured against CV-A21 using sera collected in 1976, 1985, 1999, 2009, and 2019 in Yamagata, to clarify the longitudinal epidemiology of CV-A21. The total Ab-positive rate in each year was 15.2% (35/233), 10.7% (30/281), 14.3% (28/196), 3.1% (7/236), and 1.3% (3/226), respectively. Ab-positive rates generally increased with age, especially between 1976 and 1999. Among the total Ab-positive cases, the Ab titers were relatively low; 50 cases belonged to the 1:8-1:16, 40 to 1:32-1:64, 12 to 1:128-1:256, and 1 to 1:1024< groups, respectively. No Ab-positive cases under the age of 10 were observed in any of the years analyzed. In conclusion, this study and previous works suggested that CV-A21 is a unique enterovirus, which is not transmitted readily among young children but causes sporadic ARI cases mainly among those ≥15 years of age in the community.

Isolation of Human Coronaviruses OC43, HKU1, NL63, and 229E in Yamagata, Japan, Using Primary Human Airway Epithelium Cells Cultured by Employing an Air-Liquid Interface Culture.

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.

Longitudinal Epidemiology of Viral Infectious Diseases Combining Virus Isolation, Antigenic Analysis, and Phylogenetic Analysis as Well as Seroepidemiology in Yamagata, Japan, between 1999 and 2018.

We introduced a microplate method for virus isolation in the Department of Microbiology, Yamagata Prefectural Institute of Public Health (YPIPH) in 1999 in Yamagata, Japan. We have since carried out longitudinal epidemiological studies on viral infectious diseases, particularly respiratory viruses, combining traditional technologies such as virus isolation and serological techniques and newly developed molecular methods. Here, we provide an overview of our activities at YPIPH between 1999 and 2018. During the study period, we observed emerging and re-merging diseases such as those caused by echovirus type 13, enterovirus D68, parechovirus-A3 (PeV-A3), and Saffold virus. With regard to PeV-A3, we proposed a new disease concept, "PeV-A3-associated myalgia/myositis." We also revealed the longitudinal epidemiologies of several viruses such as enterovirus A71 and coxsackievirus A16. To perform longitudinal epidemiological studies at any time in Yamagata, we established a system for stocking clinical specimens, viral isolates, complementary DNAs, and serum specimens. We have also pursued collaboration works with virology laboratories across Japan. We hope our experiences, findings, and research materials will further contribute to the development of countermeasures against viral infectious diseases and improvement in public health strategies in Yamagata, Japan, Asia, and around the world.