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.

Genotypic diversity of Streptococcus suis and the S. suis-like bacterium Streptococcus ruminantium in ruminants.

Although Streptococcus suis has attracted public attention as a major swine and human pathogen, this bacterium has also been isolated from other animals, including ruminants. However, recent taxonomic studies revealed the existence of other species that were previously identified as S. suis, and some of these isolates were reclassified as the novel species Streptococcus ruminantium. In Japan, biochemically identified S. suis is frequently isolated from diseased ruminants; however, such isolates have not yet been identified accurately, and their aetiological importance in ruminants is unclear. Therefore, to understand the importance of S. suis and S. suis-like bacteria in ruminants, we reclassified S. suis isolates from ruminants according to the updated classification and investigated their genetic diversity. Although both S. suis and S. ruminantium were isolated from healthy and diseased ruminants, most of the isolates from diseased animals were S. ruminantium, implying that S. ruminantium is more likely to be associated with ruminant disease than S. suis. However, the ruminant S. suis and S. ruminantium isolates from diseased animals were classified into diverse genotypes rather than belonging to certain clonal groups. Genome sequence analysis of 20 S. ruminantium isolates provided information about the antibiotic resistance, potential virulence, and serological diversity of this species. We further developed an S. ruminantium-specific PCR assay to aid in the identification of this bacterium. The information obtained and the method established in this study will contribute to the accurate diagnosis of ruminant streptococcal infections.

Detection of Saffold viruses from children with acute respiratory infections in Yamagata, Japan, between 2008 and 2015.

Although Saffold virus (SAFV) was reported as a novel human cardiovirus in 2007, no causative association between SAFV and clinical disease has been proven and the longitudinal epidemiology of SAFVs is not available. To establish the relationship between SAFVs and acute respiratory infections (ARIs) and to clarify the longitudinal epidemiology of SAFVs, 7258 nasopharyngeal specimens were collected from children with ARIs in Yamagata, Japan between 2008 and 2015. The specimens were inoculated on a microplate including six cell lines as part of routine surveillance, and molecular screening was performed for SAFVs using a reverse transcription (RT)-PCR method. Throughout the study period, 95 (1.3%) SAFV genotype 2 (SAFV2), and 28 (0.4%) SAFV3 were detected, mainly between September and November. There were two outbreaks of SAFV2 in 2009 and 2013, and one outbreak of SAFV3 in 2012 and the positive rates during these outbreaks were 12.1% (53/439), 11% (35/319), and 4.4% (20/453), respectively. Sixty-three SAFV2 and 28 SAFV3 strains were detected as a single virus from children with ARIs such as pharyngitis, herpangina, and tonsillitis. These results suggested that SAFV2 and SAFV3 are possible causative agents of ARIs among children and their infections occur mainly in the autumn season in Japan.

Clinical characteristics of children infected with enterovirus D68 in an outpatient clinic and the association with bronchial asthma.

BACKGROUND: All reports of increases in severe respiratory disease associated with human enterovirus D68 (EV-D68) are from hospital settings. However, there are few reports describing clinical characteristics in less severely affected populations. METHODS: We conducted a retrospective observational study from January 2010 to December 2015 in Yamagata, Japan. Using regional passive surveillance, 5794 respiratory specimens were collected from children who initially presented to an outpatient clinic with acute respiratory symptoms. The collected samples were tested for EV-D68 by reverse transcription PCR. RESULTS: EV-D68 was detected in 79 specimens mainly during the two epidemic periods in August-October 2010 and August-October 2015, when detection rates were 10.2% (31 of 304 specimens) and 16.3% (46 of 282 specimens), respectively. Among the 69 EV-D68-positive children, excluding those with viral coinfection, 39 (57%) had upper respiratory tract infections, 23 (33%) bronchiolitis or asthma attack, 5 (7%) bronchitis, 1 (1%) meningitis and 1 (1%) acute flaccid paralysis. In 23 children with wheezing, retraction was observed in 10 (43%), and six (26%) were diagnosed with asthma exacerbation. Six children required hospital admission, five (83%) because of asthma exacerbation. A history of asthma or wheezing was the most significant risk factor for the development of wheezing (odds ratio, 8.23; 95% CI, 2.65-25.50; p < .001). CONCLUSIONS: The low rate of hospitalization (9%, 6 of 69) indicates that most cases with EV-D68 infection were managed as outpatients. A history of asthma or wheezing was a potential risk factor for wheezing, resulting in hospitalization due to a severe asthma attack.

Seroepidemiology of human parechovirus types 1, 3, and 6 in Yamagata, Japan, in 2014.

To clarify the seroepidemiology of human parechovirus type 1 (HPeV1), 3 and 6, neutralizing antibodies (NT Abs) were measured in 214 serum specimens collected in 2014 in Yamagata, Japan. The seroprevalence against HPeV1 was 100% in all age groups, while that against HPeV3 and HPeV6 was 79.4% and 66.8%, respectively, overall. The geometric mean titers of NT Abs against HPeV1, 3 and 6 were 755.2, 255.0 and 55.9, respectively, overall. Our findings indicate that HPeV1 is the most prevalent HPeV circulating in Yamagata, followed by HPeV3 and HPeV6.

Chronological changes of mumps virus genotypes in Japan between 1999-2013.

BACKGROUND: The molecular epidemiology of mumps virus (MuV) has been carried out worldwide based on genotyping proposed by the World Health Organisation. However, longitudinal molecular epidemiological studies of MuV are still limited. METHODS: This study carried out genotyping of MuVs isolated in Yamagata prefecture, which is located in northern Japan, between 1999-2013, using standard nomenclature based on the sequence analysis of the entire 316 nucleotides of the small hydrophobic (SH) gene. RESULTS: During this 15-year period, 249 MuVs were isolated, with the majority of them belonging to genotype G. Phylogenetic analysis revealed that genotype G strains were divided into two distinct clusters 1 and 2, consisting of 178 and 47 strains, respectively. The cluster 1 strains were isolated every year since 2001, except for 2012. The cluster 2 strains first appeared in 2011 and were dominant in 2011 and 2012. The epidemic pattern of genotype G strains observed in Yamagata was similar to those in Kanagawa and Hyogo prefectures located in eastern and western Japan, respectively. Only one L, three H and one F genotype strains were isolated in 2001, 2004 and 2010, respectively. Almost every year several genotype B strains related to Japanese vaccine strains were isolated. CONCLUSIONS: These data demonstrated that the genotype G strains have been endemically perpetuating as the major type over a wide area of Japan since 2001, although the genotype G strains that emerged after 2011 differed from the earlier strains.

Detection of the human coronavirus 229E, HKU1, NL63, and OC43 between 2010 and 2013 in Yamagata, Japan.

The available literature on human coronaviruses (HCoVs) in Japan is limited to epidemiological studies conducted over a maximum of 1 year. We conducted a 4-year study of HCoVs by analyzing 4,342 respiratory specimens obtained in Yamagata, Japan, between January 2010 and December 2013. A pan-coronavirus reverse transcription-PCR screening assay was performed, and all HCoV-positive specimens were subsequently confirmed by sequencing of the PCR products. We detected in 332 (7.6%) HCoV strains during the study period, comprising 133 (3.1%) HCoV-NL63, 83 (1.9%) HCoV-HKU1, 78 (1.8%) HCoV-OC43, and 38 (0.9%) HCoV-229E strains. HCoV detection per year ranged from 3.5% to 9.7%. HCoVs were detected mainly in winter, with January (28.5%) and February (25.3%) 2011 and December 2012 (14.6%) being the only months in which HCoV-NL63 detection per month exceeded 10.0%. HCoV-HKU1 displayed clear biennial peaks in January (18.3%) and February (10.7%) 2010 and in February (18.8%) and March (14.7%) 2012. The peak detection of HCoV-OC43 was 13.6% in November 2010, while that of HCoV-229E was 10.8% in March 2013. Our results indicated that there may be annual variations in the circulation of individual HCoV strains. Further long-term surveillance is necessary to clarify HCoV prevalence and circulation patterns in Japan.

Molecular epidemiology of enterovirus 71 strains isolated from children in Yamagata, Japan, between 1990 and 2013.

Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.

Molecular evidence for the presence of new Babesia species in feral raccoons (Procyon lotor) in Hokkaido, Japan.

We recently reported that feral raccoons (Procyon lotor) with splenomegaly native to Japan were carriers of a Babesia microti-like parasite identical to that found in the United States, which was likely introduced to Japan from North America via raccoons imported as pets. Thus, we attempted extensive molecular survey for piroplasma infections of feral raccoon with normal spleen in Hokkaido, Japan using nested PCR that target broadly to 18S ribosomal RNA gene (SSU-rDNA) of all the parasites in the genus Babesia, Theileria, Cytauxzoon and B. microti group. Of the 348 raccoon samples analyzed, 9 gave positive signals. Cloning and phylogenetic analysis on SSU-rDNA sequences revealed that six of nine positives were found to be infected with Babesia and the remaining three with previously unreported Sarcocystis. Babesia sequences were further separated into two distantly related groups, those that reside in a novel phylogenetic group were consisted solely of four parasites found in this study, while those which included one identical sequence found in the three of our specimens were assembled together with both Babesia parasites of tick's in Japan and of raccoon's in U.S. These results may indicate that not only a B. microti-like parasite but also at least two yet undescribed Babesia species are being established in their new life cycles in the feral raccoon populations in Japan.

Epidemiology, histopathology, and muscle distribution of Trichinella T9 in feral raccoons (Procyon lotor) and wildlife of Japan.

The prevalences of Trichinella T9 in trapped raccoons (Procyon lotor) and several other potential mammalian reservoirs in Hokkaido, Wakayama, and Nagasaki Prefectures were investigated. Muscle samples were collected from 2003 to 2006 from 1,080 raccoons, 113 raccoon dogs including 2 species (Nyctereutes procyonoides albus and N. p. viverrinus), 41 wild boars (Sus scrofa leucomystax), 14 martens (Martes melampus), 10 badgers (Meles meles), 5 Siberian weasels (Martes sibirica coreana), 7 mink (Mustela vison), and 1 red fox (Vulpes vulpes japonica). The samples were digested, and the prevalence and mean intensity of infection with the Trichinella muscle larvae were determined. The prevalence and intensity of the muscle larvae were 0.9% and 93.3 larvae/g (range 0.4-201.8) in raccoons, and 1.6% and 61.6 larvae/g in raccoon dogs, respectively. The infected animals were captured in different areas in Hokkaido Prefecture. These results confirmed that raccoons, which have been introduced from North America since the 1970s, are involved in the sylvatic cycle of Trichinella in Japan. In raccoons, the muscle density of Trichinella T9 larvae was highest in the tongue, and larvae were not found in the heart muscle or diaphragm. This is the first report of Trichinella T9 infection of feral raccoons in Japan.