Genetic Reassortment in a Child Coinfected with Two Influenza B Viruses, B/Yamagata Lineage and B/Victoria-Lineage Strains.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Longitudinal antigenic and seroepidemiological analyses of parechovirus A1 in Yamagata, Japan.

To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and PeVA1/Yamagata.JPN/2021-4785, isolated in 2021, using immune sera and 207 and 237 human serum specimens collected in 2021 and 1976, respectively. Although rabbit immune sera showed the highest neutralization antibody (NT-Ab) titers against the immunized viruses at 1:12 800-1:102 400, they were cross-reactive at 1:400-1:800. All 62 Yamagata isolates obtained between 2001 and 2021 (Yamagata strains), belonging to phylogenetic lineage 1B, reacted more strongly (mostly 4-64 times) to antiserum against PeVA1/Yamagata.JPN/2021-4785 than to antiserum against Harris, belonging to phylogenetic lineage 1 A. Human serum specimens obtained in 2021 showed higher NT-Ab titers against PeVA1/Yamagata.JPN/2021-4785, whereas those obtained in 1976 had similar NT-Ab titers against both strains. These findings suggested that Yamagata strains and Harris were antigenically cross-reactive, although there were differences. There are still high NT-Abs titers present against Harris in 2021 in particular, indicating that PeVA1 has been in circulation with high immunity in the population. In conclusion, this study suggested that PeVA1 has been endemically perpetuated with only minor antigenic changes as well as with high immunity over several decades in the community.

Measles Outbreak Response Activity in Japan, and a Discussion for a Possible Strategy of Outbreak Response Using Cycle Threshold Values of Real-Time Reverse Transcription PCR for Measles Virus in Measles Elimination Settings.

Measles is a highly contagious, but vaccine-preventable disease caused by the measles virus (MeV). Although the administration of two doses of measles vaccines is the most effective strategy to prevent and eliminate measles, MeV continues to spread worldwide, even in 2022. In measles-eliminated countries, preparedness and response to measles outbreaks originating from imported cases are required to maintain elimination status. Under these circumstances, real-time reverse transcription (RT) PCR for MeV could provide a diagnostic method capable of strengthening the subnational capacity for outbreak responses. Real-time RT-PCR can detect MeV RNA from patients with measles at the initial symptomatic stage, which can enable rapid public health responses aimed at detecting their contacts and common sources of infection. Furthermore, low cycle threshold (Ct) values (i.e., high viral load) of throat swabs indicate high infectiousness in patients with measles. The high basic reproduction number of measles suggests that patients with high infectiousness can easily become super-spreaders. This opinion proposes a possible strategy of rapid and intensive responses to counter measles outbreaks caused by super-spreader candidates showing low Ct values in throat swabs. Our strategy would make it possible to effectively prevent further measles transmission, thereby leading to the early termination of measles outbreaks.

Recombinant parechovirus A3 possibly causes various clinical manifestations, including myalgia; findings in Yamagata, Japan in 2019.

BACKGROUND: Parechovirus A3 was first reported in 2004 and has been recognized as a causative agent of mild and severe infections in children. Since we first reported an outbreak of adult parechovirus A3-associated myalgia in Yamagata, Japan in 2008, this disease has since been recognized across Japan, but has not yet been reported from other countries. AIM: We analysed 19 cases of parechovirus A3 infections identified in Yamagata in 2019 to further clarify the epidemiology of this disease. METHODS: We performed phylogenetic analyses of parechovirus A3 isolates and analysed the clinical manifestations and the genomic clusters. RESULTS: There were two clusters, with cluster 2019B replacing 2019 A around October/November. Phylogenetic analysis revealed that 2019B cluster strains and Australian recombinant strains, which appeared between 2012 and 2013, were grouped in one cluster at non-structural protein regions, suggesting that the ancestor to these regions of 2019B cluster strains were Australian recombinant lineage strains. The strains from both clusters caused various infections in children including myalgia. These findings strongly support that parechovirus A3 strains cause myalgia and other paediatric infections irrespective of the virus strains involved, including recombinant strains.　　. CONCLUSIONS: We have reported repeatedly sporadic cases of myalgia and here showed that recombinant strains also cause myalgia. We hope our experiences will help better understand these infections and possibly result in detection of more cases in the world.

A seroepidemiologic study of a measles outbreak, Yamagata Prefecture, Japan, 2017: The estimation of spreaders using serological assays in a measles elimination setting.

INTRODUCTION: In regions where the endemic measles virus has been eliminated, early detection of contagious patients is important for preventing the spread of measles and sustaining elimination. To investigate whether serological assays can be used for the estimation of highly infectious patients with measles, we performed a seroepidemiologic study of a measles outbreak in Yamagata Prefecture, Japan, in 2017. METHODS: We tested plaque reduction neutralization (PRN), IgG avidity, and gelatin particle agglutination (PA) assays in 31 patients with measles, subdivided into two super-spreaders, three spreaders, and 26 non-spreaders. Simultaneously, these results were compared with the cycle threshold (Ct) of a semi-quantitative real-time reverse transcription PCR for the measles virus from throat swab specimens. RESULTS: In the PRN assay, one super-spreader and two spreaders lacked protective antibodies. The IgG avidity assay showed that two super-spreaders and one spreader had low avidity. The PA assay indicated that two super-spreaders and two spreaders lacked protective antibodies. Comparison of the results of the three serological assays and Ct revealed that patients whose antibody titers were judged as low in the IgG avidity and PA assays showed low Ct (i.e., high viral load), whereas non-spreaders tended to show low viral load. CONCLUSIONS: Our preliminary seroepidemiologic analysis of a population of 31 patients with measles suggests that PA and IgG avidity assays may be used for the identification of super-spreader/spreader candidates. However, further investigations are necessary to validate the robustness of these serological assays in detecting contagious measles cases.

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.

Epidemiology of Coronavirus Disease 2019 in Yamagata Prefecture, Japan, January-May 2020: the Importance of Retrospective Contact Tracing.

Public health interventions have played an important role in controlling coronavirus disease 2019 (COVID-19), which is a rapidly spreading infectious disease. To contribute to future COVID-19 countermeasures, we aimed to verify the results of the countermeasures employed by public health centers (PHCs) against the first wave of COVID-19 in Yamagata Prefecture, Japan (Yamagata). Between January and May 2020, 1,253 patients suspected of SARS-CoV-2 infection were invited for testing. Simultaneously, based on retrospective contact tracings, PHCs investigated the infection sources and transmission routes of laboratory-confirmed COVID-19 cases and tested 928 contacts. Consequently, 69 cases were confirmed between March 31 and May 4, 58 of whom were from among the contacts (84.1%; 95% confidence interval [CI] 75.5-92.7). The spread of infection was triggered in cases harboring epidemiological links outside Yamagata. Subsequently, the number of cases rapidly increased. However, PHCs identified epidemiological links in 61 (88.4%; 95% CI 80.8-96.0) of the 69 cases, and transmission chains up to the fifth generation. Finally, the spread of infection ended after approximately one month. Our results indicate that the identification of infection sources and active case finding from contacts based on retrospective contact tracing was likely to be an effective strategy in ending the first wave of COVID-19 in Yamagata.

Proposal for the Recognition of a New Disease Concept from Japan: Parechovirus A3-Associated Myalgia.

Parechovirus A3 (PeVA3) was first reported in 2004 and has been recognized as a causative agent of mild and severe infectious diseases in children. We first reported an outbreak of PeVA3-associated myalgia (PeVA3-M) in Yamagata, Japan, in 2008. We have repeatedly observed PeVA3-M cases in 2011, 2014, and 2016, and identified the first child case in 2014. Reports of PeVA3-M have increased since 2014, indicating that the recognition of PeVA3-M has spread across Japan. The findings showed that PeVA3-M commonly occurs among adults aged 30-40 years, particularly in males. Elevation of creatinine phosphokinase, C-reactive protein, and myoglobin, as well as magnetic resonance imaging findings, suggest inflammation of the muscles and/or fascia of the four limbs. Patients recover within 1-2 weeks without any sequelae. A longitudinal molecular epidemiological study in Yamagata revealed that PeVA3 strains cause a variety of diseases, ranging from mild to severe, including PeVA3-M, in subjects ranging from neonates to adults, irrespective of their genetic cluster. As PeVA3-M has not yet been reported abroad, more widespread recognition of PeVA3-M as an emerging disease is important. We hope this review will help clinicians and researchers in understanding PeVA3-M and therefore advance related research in Japan as well as around the world.

Longitudinal epidemiology of human coronavirus OC43 in Yamagata, Japan, 2010-2017: Two groups based on spike gene appear one after another.

Human coronavirus OC43 (HCoV-OC43) is divided into genotypes A to H based on genetic recombination including the spike (S) gene. To investigate the longitudinal transition of the phylogenetic feature of the HCoV-OC43 S gene in a community, phylogenetic analysis of the S1 region of the S gene was conducted using 208 strains detected in Yamagata during 2010 to 2017 with reference strains of the genotype. The S1 sequences were divisible into four groups: A to D. All Yamagata strains belonged to either group B or group D. In group B, 46 (90.2%) out of 51 Yamagata strains were clustered with those of genotype E reference strains (cluster E). In group D, 28 (17.8%) and 122 (77.7%) out of 157 Yamagata strains were clustered, respectively, with genotype F and genotype G reference strains. In cluster G, 28 strains formed a distinct cluster. Monthly distributions of HCoV-OC43 in Yamagata in 2010 to 2017 revealed that group B and group D appeared one after another. In group B, the cluster E strains were prevalent recurrently. In conclusion, epidemics of HCoV-OC43 in Yamagata, Japan might be attributable to two genetically different groups: group B showed a recurrent epidemic of strains belonging to a single phylogenetic cluster and group D showed epidemic strains belonging to multiple clusters.

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.

Seroprevalence of parechovirus A1, A3 and A4 antibodies in Yamagata, Japan, between 1976 and 2017.

Introduction. Although new parechovirus A (PeVA) types, including parechovirus A3 (PeVA3) and PeVA4, have been reported in this century, there have not yet been any seroepidemiological studies on PeVA over a period of several decades.Hypothesis/Gap Statement. The authors hypothesize that PeVA3 and PeVA4 emerged recently.Aims. The aim was to clarify changes in the seroprevalence of PeVA1, PeVA3 and PeVA4.Methodology. Neutralizing antibodies (NT Abs) were measured among residents in Yamagata, Japan in 1976, 1983, 1985, 1990, 1999 and 2017.Results. The total NT Ab-positive rate for PeVA1 was between 90.7 and 100 % for all years analysed, with that for PeVA3 increasing from 39.6 % in 1976 to 69.6 % in 2017, and that for PeVA4 decreasing from 93.9 % in 1976 to 49.1 % in 2017. The distribution of NT Ab titres for PeVA1, PeVA3 and PeVA4 among those aged less than 20 years old was as follows: those ≥1 : 32 for PeVA1 were between 68.0-89.2 % for all years analysed; those ≥1 : 32 for PeVA3 was 15.4 % in 1976, 44.3-54.9 % in 1983-1990 and 64.8-68.0 % in 1999-2017; and those ≥1 : 32 for PeVA4 were between 49.1-67.2 % in 1976-1990, 41.3 % in 1999 and 23.8 % in 2017.Conclusions. Our findings in this seroepidemiological study over four decades suggested that PeVA1 has been stably endemic, while PeVA3 appeared around 1970s and has spread since then as an emerging disease, and occasional PeVA4 infections were common in 1970s and 1980s but have been decreasing for several decades in our community.

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.

Phylogenetic and antigenic analyses of coxsackievirus A6 isolates in Yamagata, Japan between 2001 and 2017.

Although coxsackievirus A6 (CV-A6) is generally recognized as a causative agent of herpangina in children, CV-A6 infections globally emerged as a new and major cause of epidemic hand-foot-and-mouth-diseases (HFMDs) around 2008. To clarify the longitudinal epidemiology of CV-A6, we carried out sequence and phylogenetic analyses for the VP1 and partially for the VP4-3D regions as well as antigenic analysis using 115 CV-A6 isolates and 105 human sera in Yamagata, Japan between 2001 and 2017. Phylogenetic analysis revealed that CV-A6 isolates were clearly divided into two clusters; strains in circulation between 2001 and 2008 and those between 2010 and 2017. Neutralizing antibody titers of two rabbit antisera, which were immunized with Yamagata isolates in 2001 and 2015, respectively, against 28 Yamagata representative strains as well as the prototype Gdula strain were 1:2560-1:5120 and 1:160-1:640, respectively. The neutralizing antibody titers among residents in Yamagata against the above two strains were similar. Our analyses revealed that there were cross-antigenicities among all analyzed CV-A6 strains, although the newly emerged strains were introduced into Yamagata around 2010 and replaced the previous ones. With regard to control measures, these findings suggest that we can prevent CV-A6 infections through the development of a vaccine that effectively induces neutralizing antibodies against CV-A6, irrespective of genetic cluster.

Parechovirus A3 (PeV-A3)-associated myalgia/myositis occurs irrespective of its genetic cluster: a longitudinal molecular epidemiology of PeV-A3 in Yamagata, Japan between 2003 and 2016.

No longitudinal molecular epidemiology of parechovirus A3 (PeV-A3) over a decade is available and PeV-A3-associated myalgia/myositis has been reported only in Japan. Thus, we aimed to clarify the longitudinal molecular epidemiology of PeV-A3 with a major focus on the strains detected from PeV-A3-associated myalgia/myositis cases. We performed sequence and phylogenetic analysis for the VP1 region of PeV-A3 strains in Yamagata, Japan, between 2003 and 2016. The phylogenetic analysis indicated that PeV-A3 strains caused PeV-A3-associated myalgia/myositis as well as a variety of infectious diseases, ranging from mild to severe, in subjects ranging from neonates to adults, irrespective of genetic cluster or variations. PeV-A3 strains are causative agents of a variety of human diseases, irrespective of their genetic cluster. Furthermore, we consider that PeV-A3-associated myalgia/myositis may occur, not only in Japan, but also in other countries, as closely related PeV-A3 strains have been circulating around the world.

The Largest Measles Outbreak, Including 38 Modified Measles and 22 Typical Measles Cases in Its Elimination Era in Yamagata, Japan, 2017.

The incidence of modified measles (M-Me), characterized by milder symptoms than those of typical measles (T-Me), has been increasing in Japan. However, the outbreak dominated by M-Me cases has not been thoroughly investigated worldwide. The largest importation-related outbreak of measles with genotype D8 occurred in Yamagata Prefecture, Japan, from March to April 2017. This phenomenon was observed after Japan had achieved measles elimination in 2015. We confirmed 60 cases by detecting the genome of the measles virus (MeV). Among the cases, 38 were M-Me and 22 were T-Me. Thirty-nine (65.0%) patients were 20-39 years of age. Three out of 7 primary cases produced 50 transmissions, of which each patient caused 9-25 transmissions. These patients were 22-31 years old and were not vaccinated. Moreover, they developed T-Me and kept contact with the public during their symptomatic periods. Considering that M-Me is generally caused by vaccine failure, some individuals in Japan may have insufficient immunity for MeV. Accordingly, additional doses of measles vaccine may be necessary in preventing measles importation and endemicity among individuals aged 20-39 years. Furthermore, to accurately and promptly diagnose individuals with measles, particularly those who can be considered as primary cases, efforts must be exerted to detect all measles cases using epidemiological and genetic approaches in countries where measles elimination had been achieved.

Serum leptin levels during the periparturient period in cows.

Serum leptin concentrations were measured in antenatal and postnatal cows housed at two different locations. The mean serum leptin concentration was 9.2 +/- 0.6 ng/m l (n=22) in one group, and was slightly lower in the other (7.4 +/- 0.4 ng/ml, n=54), probably because of the different nutritional conditions between the two groups. There was no consistent variation in relation to the menstrual cycle and the periparturient period in both groups. Moreover, serum leptin concentrations during the periparturient period were independent of the number of delivery and the incidence of mastitis and milk fever. These results are quite different from those in rodents and human, suggesting the different regulatory mechanism of circulating leptin concentration in cows.