Genetic characterization of a novel recombinant echovirus 30 strain causing a regional epidemic of aseptic meningitis in Hokkaido, Japan, 2017.

A regional epidemic of aseptic meningitis caused by echovirus 30 (E30) occurred in Hokkaido, Japan, during the period of August-December 2017. To investigate their phylogenetic relationship to other human enteroviruses, we determined the complete genomic nucleotide sequences of isolates from this outbreak. Phylogenetic analysis of the viral capsid protein 1 gene showed that the strains were most closely related to E30 strains detected in Germany, France, and Russia in 2013. In contrast, the region encoding the viral protease and the RNA-dependent RNA polymerase had a close phylogenetic relationship to non-E30 enteroviruses detected in the United Kingdom and Switzerland in 2015-2017, suggesting that a recombination event had occurred.

Genetic characterization of hemagglutinin protein of measles viruses in Hokkaido district, Japan, 2006-2015.

Strains of measles virus of genotypes D5, H1, D4, D8, and B3 were detected among epidemic, endemic, imported and import-associated cases in Hokkaido district, Japan, during 2006-2015. In the present study, their antigenic features were evaluated by determining the complete nucleotide sequences of their hemagglutinin proteins, which are a major target for neutralizing antibodies, and their amino acid sequences deduced. It was found that the hemagglutinin proteins of these strains had several novel amino acid changes in some functional regions. Although these strains have not caused further infections thus far, these antigenic changes should continue to be monitored to maintain their elimination status.

Genomic characterization of echovirus 6 causing aseptic meningitis in Hokkaido, Japan: a novel cluster in the nonstructural protein coding region of human enterovirus B.

We determined four complete nucleotide sequences of echovirus 6 (E6) isolated from an epidemic of aseptic meningitis (AM) in Hokkaido, Japan, in 2011. Phylogenetic analysis of the genes encoding viral capsid protein 1 revealed that the strains were closely related to E6 strains isolated in China in recent years, but they were distantly related to E6 strains isolated from patients with AM in Osaka Prefecture, Japan, in 2011. The genes encoding the viral protease and RNA-dependent RNA polymerase (3CD) were closely related to those of several non-E6 strains of the species Human enterovirus B isolated in China, South Korea, and Australia from 1999 to 2010, resulting in a novel cluster in the phylogenetic tree. These results suggest that the incidence of AM in Japan in 2011 was caused by at least two lineages of E6 strains, and a lineage of the 3CD gene was interspersed among different serotypic strains isolated in Western Pacific countries.

Detection and molecular characterization of hepatitis E virus in clinical, environmental and putative animal sources.

Putative animal reservoirs and environmental samples were studied to investigate potential routes of transmission for indigenous hepatitis E virus (HEV) infection in Hokkaido, Japan. A total of 468 liver samples and 954 environmental samples were collected from 2003 to 2011 for this study. Four swine livers (1 %) were positive for HEV RNA; two strains belonged to genotype 3 and the other two strains were genotype 4. Genotype 3 HEV was detected in a sewage sample and a seawater sample. HEV strains derived from swine liver, seawater and raw sewage samples shared 93-100 % sequence similarity with human HEV strains.

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan.

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.

Outbreak of aseptic meningitis caused by echovirus 30 in Kushiro, Japan in 2017.

BACKGROUND: Echovirus 30 (E30) is one of the most common causative agents for aseptic meningitis. OBJECTIVES: In the autumn of 2017, there was an outbreak caused by E30 in Kushiro, Hokkaido, Japan. The aim of this study was to characterize this outbreak. STUDY DESIGN: Fifty-nine patients were admitted to the Department of Pediatrics, Kushiro Red Cross Hospital (KRCH) with clinical diagnosis of aseptic meningitis. Among those, 36 patients were finally diagnosed as E30-associated aseptic meningitis by the detection of viral RNA using reverse transcription-polymerase chain reaction (RT-PCR) and/or the evidence of more than four-fold rise in neutralizing antibody (NA) titers in the convalescent phase relative to those in the acute phase. We investigated these 36 confirmed cases. RESULTS: The median age was 6 years (range: 6 months-14 years). The positive signs and symptoms were as follows: fever (100%), headache (94%), vomiting (92%), jolt accentuation (77%), neck stiffness (74%), Kernig sign (29%), and abdominal pain (28%). The median cerebrospinal fluid (CSF) white cell count, neutrophil count, and lymphocyte count were 222/µL (range: 3-1434/µL), 144/µL (range: 1-1269/µL), and 85/µL (range: 2-354/µL), respectively. Although the detected viral genes demonstrated same cluster, they were different from E30 strains observed in Japan between 2010 and 2014. CONCLUSION: We mainly showed clinical and virological features of the E30-associated aseptic meningitis outbreak that occurred in Kushiro. To prevent further spread of E30 infection, continuous surveillance of enterovirus (EV) circulation and standard precautions are considered essential.

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.

Hepatitis E outbreak at a nursing home for aged people in Hokkaido, Japan, between February and March 2016.

BACKGROUND: Infection with hepatitis E virus (HEV) genotypes 3 and 4 are usually asymptomatic but can occasionally result in life-threatening acute hepatitis. To date, only sporadic cases together with a few outbreaks have been documented. Seroprevalence studies with assays for the detection of HEV IgG antibodies, suggest that HEV is more prevalent than previously thought, even in non-endemic regions. OBJECTIVES: The aim of this study was to characterize an outbreak of hepatitis E (HE) in a nursing home for aged people between February and March 2016. STUDY DESIGN: After the identification of two cases living in the same nursing home, the presence of antibodies against HEV and HEV RNA were examined in serum samples collected from the other residents and staff members to identify any additional cases. An epidemiological investigation was also carried out. RESULTS: Only 4 patients showed mild symptoms such as anorexia, abdominal pain and fatigue. Among the 125 persons tested, 28 residents and one dietitian were confirmed positive for anti-HEV IgA or IgM antibodies, and/or HEV RNA. Eight samples had only IgG antibodies. Finally, 22 cases were notified with HE on the basis of the presence of IgA antibodies. All HEV isolates obtained were 99.8-100% identical and belonged to genotype 3. CONCLUSION: HEV infections seem to be under-reported or underestimated possibly due to cases being generally asymptomatic. Testing for the presence of both anti-HEV antibodies and HEV RNA would be beneficial for both the comprehensive diagnosis of HE infections and the prevention of further infections.

Amoebal endosymbiont Neochlamydia protects host amoebae against Legionella pneumophila infection by preventing Legionella entry.

Acanthamoeba isolated from environmental soil harbors the obligate intracellular symbiont Neochlamydia, which has a critical role in host amoebal defense against Legionella pneumophila infection. Here, by using morphological analysis with confocal laser scanning fluorescence microscopy and transmission electron microscopy, proteome analyses with two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry (LC/MS), and transcriptome analysis with DNA microarray, we explored the mechanism by which the Neochlamydia affected this defense. We observed that when rare uptake did occur, the symbiotic amoebae allowed Legionella to grow normally. However, the symbiotic amoebae had severely reduced uptake of Legionella when compared with the aposymbiotic amoebae. Also, in contrast to amoebae carrying the endosymbiont, the actin cytoskeleton was significantly disrupted by Legionella infection in aposymbiotic amoebae. Furthermore, despite Legionella exposure, there was little change in Neochlamydia gene expression. Taken together, we concluded that the endosymbiont, Neochlamydia prevents Legionella entry to the host amoeba, resulting in the host defense against Legionella infection.

Detection of Measles Virus Genotypes B3, D4, D5, D8, and H1 in the Surveillance System in Hokkaido, Japan, 2006-2015, the Last Decade toward the Elimination.

Measles is an acute and highly contagious disease caused by measles virus (MeV). The government of Japan, following the last epidemic in 2007 and 2008, which was caused by genotype D5 strains, introduced a catch-up-vaccination program for teenagers during Japan fiscal years 2008-2012 and a mandatory case-based reporting system for the nationwide elimination. Furthermore, laboratory confirmation of measles cases by genotyping of isolates has been performed to clarify the source of infection and support the interruption of measles cases. Owing to these preventive measures, the number of measles cases has been steadily decreasing after the last epidemic. In March 2015, Japan was internationally verified as having achieved measles elimination by the World Health Organization Regional Office for the Western Pacific. The continuous elimination of measles and high levels of vaccination coverage for MeV have been maintained nationally. However, imported or import-associated cases of measles have sporadically occurred during this time. After the last nationwide epidemic, 17 imported or import-associated measles cases (MeV strains identified as genotypes H1, D4, D8, and B3) were reported in Hokkaido, the northern islands of Japan. In this study, we present the occurrence of measles and surveillance activities in Hokkaido during 2006-2015.

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.

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.

[Relationship between ABO histo-blood group type and an outbreak of norovirus gastroenteritis among primary and junior high school students: results of questionnaire-based study].

Noroviruses are common causative agents of epidemic gastroenteritis in humans. Recent studies showed that human susceptibility to noroviruses was associated with ABO histo-blood group type. It was also observed that various degrees of susceptibility were exhibited by different norovirus strains. In January 2003, an outbreak of acute gastroenteritis including 661 affected primary and junior high school students occurred through lunch bread contaminated with norovirus in Hokkaido, Japan. To clarify the relationship between ABO histo-blood group type and the norovirus infection, we performed a written questionnaire to schoolchildren about the consumption of the bread, onset of symptoms and person-to-person transmission in their household. Questionnaires were returned from 722 schoolchildren (response rate, 65.8%), of whom 55.3% suffered gastroenteritis. As a result of this survey, it was found that schoolchildren with blood group type A (71.1%, 133/187) were more susceptible to the norovirus infection, whereas, schoolchildren with blood group type AB (55.3%, 26/47) were less affected (P (Z0) < 0.025). In addition, the presumptive prevalence rate of person-to-person transmission in each household indicated that schoolchildren with blood group type AB (19.2%, 5/26) had a lower risk of infection than those with blood group type A or O [A : 41.4%, 55/133 O : 39.5%, 49/124 (unknown for one case) ] [P (Z0) < 0.025]. Our findings suggested that persons with blood group type AB were less affected by the norovirus infection in this outbreak.

Mutations associated with floral organ number in rice.

How floral organ number is specified is an interesting subject and has been intensively studied in Arabidopsis thaliana. In rice (Oryza sativa L.), mutations associated with floral organ number have been identified. In three mutants of rice, floral organ number 1 (fon1) and the two alleles, floral organ number 2-1 (fon2-1) and floral organ number 2-2 (fon2-2), the floral organs were increased in number centripetally. Lodicules, homologous to petals, were rarely affected, and stamens were frequently increased from six to seven or eight. Of all the floral organs the number of pistils was the most frequently increased. Among the mutants, fon1 showed a different spectrum of organ number from fon2 -1 and fon2 -2. Lodicules were the most frequently affected in fon1, but pistils of more than half of fon1 flowers were unaffected; in contrast, the pistils of most flowers were increased in fon2 -1 and fon2-2. Homeotic conversion of organ identity was also detected at a low frequency in ectopically formed lodicules and stamens. Lodicules and stamens were partially converted into anthers and stigmas, respectively. Concomitant with the increased number of floral organs, each mutant had an enlarged apical meristem. Although meristem size was comparable among the three mutants and wild type in the early phase of flower development, a significant difference became apparent after the lemma primordium had differentiated. In these mutants, the size of the shoot apical meristem in the embryo and in the vegetative phase was not affected, and no phenotypic abnormalities were detected. These results do not coincide with those for Arabidopsis in which clavatal affects the sizes of both shoot and floral meristems, leading to abnormal phyllotaxis, inflorescence fasciation and increased floral organs. Accordingly, it is considered that FON1 and FON2 function exclusively in the regulation of the floral meristem, not of the vegetative meristem.

The immediate early gene of canine herpesvirus is transcribed through early and late phases.

The immediate early (IE) gene of canine herpesvirus (CHV), homologue of the infected cell protein 4 (ICP4) gene of herpes simplex virus 1, is transcribed as a 4.9kb mRNA during IE phase. The IE gene was further transcribed as a 4.8kb mRNA through early (E) and late (L) phases of productive infection. Transcription of the 4.8kb mRNA initiated from downstream of the TATA box in an intron which was spliced out during IE phase. The reverse transcription-polymerase chain reaction revealed that the IE promoter was turned off during L phase at a permissive temperature. We, thus, propose to redesignate the IE gene of CHV as CICP4 gene.

Epidemiology and laboratory diagnoses of rubella in Hokkaido district during the Nationwide Outbreak in Japan, 2011-2013.

We report the epidemiology and laboratory diagnostic results of rubella cases from 2011 to 2013 in Hokkaido district, Japan. A total of 150 cases were officially reported as rubella; 102 (68%) involved males and 48 (32%) involved females. The highest proportion of cases were notified in 40-49-year-old age group among males and the 20-29-years-old age group among females. Forty-six cases (25 males and 21 females) had not been vaccinated, and 17 had been vaccinated, whereas 87 had the unknown vaccination status. Eighty-three cases (55.3%) showed the 3 typical principal rubella symptoms (fever, rash, and lymphadenopathy). Seven, 11, 92, and 40 cases were identified in the northern, eastern, central, and southern areas of Hokkaido district, respectively. In the central and southern areas of Hokkaido district, endemic rubella transmissions were indicated by both the epidemiological survey and molecular analyses. However, these outbreaks terminated spontaneously and did not expand to other areas of Hokkaido district. Fortunately, no congenital rubella syndrome (CRS) cases were reported during this observation period. However, to control virus transmission, prevent CRS, and maintain the routine vaccination program, the immediate introduction of an immunization strategy is required for susceptible individuals, particularly young adults.

Recent progress toward measles elimination in Hokkaido, Japan, during 2011-2012.

Laboratory diagnoses for measles were performed in a total of 97 cases in Hokkaido, Japan, during 2011-2012. Two patients were confirmed to be positive for measles virus (MV), both of whom lived in the Iburi district of Hokkaido. Molecular analysis of the nucleotide sequences of the nucleoprotein (N) gene revealed that these 2 strains had high homology with each other and belonged to the genotype D8. The onset interval of these cases and epidemiological data suggested that MV transmission had occurred between them and then terminated. Phylogenetic analysis of the N gene revealed that the strains identified in Hokkaido were classified into a cluster that contained many genotype D8 strains that were detected within a large area of Japan. Eventually, 9 cases were officially reported as measles. However, other than the abovementioned 2 cases, no genetic information regarding MV was obtained. In future, further active surveillance combined with the genetic investigation should be required in all suspected measles cases to verify the elimination status.

Oscillating thrombi in bilateral extracranial internal carotid arteries demonstrated on ultrasonography: two autopsy cases of cardioembolic stroke.

We herein report two autopsy cases of severe cardioembolic stroke with oscillating thrombi in the bilateral extracranial internal carotid arteries (ICAs) demonstrated on carotid ultrasonography performed on admission. An autopsy study of case 1 conducted on the third hospital day revealed no thrombi, while that of case 2 conducted on the 42nd hospital day revealed red thrombi in the extracranial ICAs. Our postmortem studies confirm that oscillating thrombi may be seen in the region of blood stasis caused by occlusion of the distal portion of the ICA, thus reflecting a pre-state of thrombus formation.