A conventional PCR-based method to detect the E2 gene of the rubella virus for epidemiological analysis.

To eliminate the rubella virus (RV), genetic characterization is vital for its detection, identification of endemic transmission, and diagnosis of imported cases. The 739-nucleotide region in the E1 gene has primarily been used for genotyping for epidemiological analysis. However, in the 2018-2019 RV outbreak, identical sequences were observed in patients who were not epidemiologically linked. Additionally, the 739 nt sequences from the outbreak in Tokyo in 2018-2019 were identical to RV identified in China in 2019. This suggests that this region may be insufficient to identify the detected RV strains as endemic or imported. In 62.4% of the specimens, the E1 gene sequences of the 1E RV genotype were identical. Additionally, the observed discordance of sequences from the mainly detected identical sequence in the 739-nt sequence of the E1 gene were one (31.0%), two (3.5%), three (2.6%), and four (0.23%). Moreover, a comparison of the complete structural protein-coding region suggests that the E2 gene is more diverse than the E1 and the capsid gene. Thus, conventional polymerase chain reaction (PCR) primers were developed to detect the E2 gene and improve epidemiological analysis. A comparison of the sequences identified during the RV outbreak in Tokyo revealed genetic differences in the sequences (15 of the 18 specimens). These results suggest that additional information could be obtained by simultaneously analyzing the E2 and the E1 region. The identified sequences can potentially aid in evaluating the RV strains detected during epidemiological analysis.

Genetic Characteristics of the Virus Detected in the First Mpox Imported Case in Tokyo, Japan.

Mpox, caused by the mpox virus (MPXV), produces symptoms similar to those of smallpox when transmitted to humans. Since 1970, this disease has been endemic, particularly in Africa. However, since May 2022, the number of patients without a history of travel to endemic areas has increased rapidly globally. Under these circumstances, in July 2022, two different real-time PCR methods were used on specimens brought to the Tokyo Metropolitan Institute of Public Health. MPXV was detected in the skin samples, and it was inferred that the virus was a West African strain. Furthermore, a more detailed analysis of the genetic characteristics of the detected MPXV using next-generation sequencing revealed that the MPXV detected in Tokyo was strain B.1, which corresponds to the same strain that is prevalent in Europe and the USA. This suggests that mpox reported for the first time in Japan was imported and related to outbreaks in Europe and the USA. Therefore, it is necessary to continue monitoring outbreaks in Japan in conjunction with global epidemics.

Molecular and Epidemiological Analysis of Respiratory Syncytial Virus Detected in Tokyo, Japan in 2021 Season.

During the COVID-19 pandemic in 2021, Japan experienced an outbreak of respiratory syncytial virus (RSV) infection. A total of 51 RSV cases were detected in infant specimens, including 38 rhinorrhea and 13 nasopharyngeal swabs, collected at the Tokyo Metropolitan Institute of Public Health. Of the 51 cases, 12 were RSV-A and 39 were RSV-B. The G protein gene sequence of RSV-A belonged to the ON1 genotype, whereas RSV-B belonged to the BA9 genotype; thus, different types of RSV were detected during the same period, suggesting that the unusual 2021 RSV season was not due to a single strain or genotype. Of all RSV-positive cases, the proportion of patients aged ≥2 years was 56.8% in 2021, higher than the 31.2% reported in the past 5 years. This indicates that infants aged <1 year who were originally susceptible to RSV infection were less likely to be infected with RSV because of the COVID-19 control measures. The 2021 epidemic peaked in the 28th week, 9 weeks earlier than the average from 2016 to 2020. Therefore, it seems necessary to accumulate and analyze further data, such as factors that led to the outbreak and the characteristics of the detected viruses in 2021.

Shedding of Rubella Virus among Infants with Congenital Rubella Syndrome Born in Tokyo, Japan, 2013-2014.

Rubella is usually a mild illness, with febrile rash being its main symptom. However, serious consequences of rubella infection can result when the infection occurs during the early stages of pregnancy. After the occurrence of a rubella outbreak in Japan that was observed from 2012 to 2013, 45 infants were reportedly born with congenital rubella syndrome (CRS). We prospectively followed the 15 CRS cases reported in Tokyo to determine the virus shedding periods by using nested reverse transcriptase-polymerase chain reaction to detect rubella virus genes. Throast swabs were used for virus detection. The virus shedding period was measured from birth until the time when the sample last tested positive followed by 2 consecutive negative samples. Kaplan-Meier method was used to estimate the proportion of cases remaining positive for rubella virus genes over time. The proportion of CRS cases shedding virus dropped steadily after birth, dropping to 33.8% at 6 months and 16.9% at 12 months. Our findings also suggested that the earlier the mother's onset of rubella during pregnancy, the longer the infant remained positive. Based on our findings, we believe that infants with CRS should be monitored for rubella virus shedding until 1 year of age.

[An autochthonous outbreak of dengue type 1 in Tokyo, Japan 2014].

OBJECTIVES: An outbreak of autochthonous dengue fever was reported in August 2014, with cases suspected mainly from Yoyogi Park in Tokyo. This is the first epidemic of dengue fever in Japan since 1945. METHODS: From August to October 2014, the following measures were taken to control the outbreak: 1) risk communication and information sharing; 2) active case finding; 3) vector surveillance in affected sites; and 4) laboratory testing. We also reviewed the surveillance data as reported to the National Epidemiological Surveillance of Infectious Diseases during the 44 epidemiological weeks. results: An official dengue fever call center was set up temporarily for the general public and 3,005 calls were received. The Tokyo Metropolitan Government issued 39 press releases regarding patients and nine related to dengue virus (DENV) detection and vector control activities for the media. Confirmed autochthonous dengue fever cases were reported between the 35th and 44th epidemiological weeks. Out of 160 cases of outbreak, 108 (67.5%) confirmed cases were reported in Tokyo. The estimated illness onset dates were between August 9 and October 7, and estimated dates of infections were between August 3 and October 3, 2014. The data suggest that the infective mosquitoes had already been present in Yoyogi Park at the end of July 2014. During the weekly vector surveillance at Yoyogi Park, a total of 1,152 adult mosquitoes, of which 856 (73.3%) were Aedes mosquitoes, were collected over 11 weeks by a light trap with dry ice. DENV was detected from adult Aedes mosquito samples collected on the 2nd, 9th, and 16th of September, 2014. Serum samples from 240 suspected cases were examined at the Tokyo Metropolitan Institute of Public Health, and 78 were positive for the DENV NS1 antigen, DENV-specific IgM antibody, or DENV nucleic acid with reverse transcriptase polymerase chain reaction (RT-PCR) (NS1: 66 cases; IgM: 50 cases; PCR: 57 cases). Genetic analysis of DENV-positive serum and mosquito samples found all to be categorized as DENV-serotype 1 (gene type I). Phylogenetic analysis of the envelope protein genome sequence from patients and mosquitoes in Tokyo revealed more than 99% similarity with each other and with the strain from the first outbreak-associated patient in Saitama. CONCLUSION: Measures important for control of infectious disease epidemic were learned during this recent indigenous dengue outbreak in Tokyo. It also highlighted the importance of preparedness for epidemics of indigenous or imported infectious diseases, especially in light of the fact that Tokyo is in preparation for the Olympic and Paralympic Games in 2020.

The 2011 measles outbreak in Tokyo. An analysis of surveillance data.

OBJECTIVES: The study was conducted with the intention of establishing a strategy to eliminate measles on the basis of an analysis of the epidemiological profile of measles cases reported in Tokyo during the year 2011. METHODS: We investigated measles cases reported to the Tokyo Metropolitan Government in 2011, recorded as part of the National Epidemiological Surveillance of Infectious Diseases. Factors analyzed included age, vaccination status for each patient, cases for which records were discarded after laboratory confirmation, genotype of the measles virus and relationships between dates of specimen collection and results of polymerase chain reaction (PCR) and IgM antibody tests. RESULTS: A total of 178 measles cases were reported in Tokyo during 2011, and the majority of cases (128, 71.9%) were reported during the peak period from epiweeks 13 to 24. The largest age group reported was one to four years of age (40, 22.5%) followed by groups of 20-29 and 30-39 years of age (both 34, 19.1%). Most cases were sporadic, with only six outbreaks occurring. Even then, the numbers of cases for each outbreak was less than five. More than half of the patients in all age groups, except for the 1-4-year-old group, had not been vaccinated or did not have a record of vaccination. Genotypes D4 and D9 of measles virus were detected in most cases. However, genotype D5, which had been circulating in Japan before 2008, was not detected. CONCLUSION: Imported viruses were the cause of measles cases reported in Tokyo during 2011. The disease control was better than that in 2007 and 2008 because of the swift and appropriate responses to the occurrences. It is also possible that there has been an increase in the proportion of people with immunity to measles. Increasing the rate of immunization, performing effective surveillance, and confirming suspicious measles cases by using molecular methods are important for achieving the elimination of measles.

Rapid typing of influenza viruses using super high-speed quantitative real-time PCR.

The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2h per run. Detection assays were performed with super high-speed RT-PCR (SHRT-PCR) developed according to a newly designed heating system. The new method uses a high-speed reaction (18s/cycle; 40 cycles in less than 20min) for typing influenza viruses. The detection limit of SHRT-PCR was 1 copy/reaction and 10(-1) plaque-forming unit/reaction for viruses in culture supernatants during 20min. Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. Twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between SHRT-PCR and immunochromatography exhibited 100% agreement for both positive and negative results. The rapid reaction time and high sensitivity of SHRT-PCR makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks.

[Development of effective detection method for Coxiella burnetii in mayonnaise by real-time PCR and investigation of C. burnetii contamination in commercial mayonnaise in Tokyo].

A PCR method for the effective detection of Coxiella burnetii in commercially available mayonnaise was developed. Sample preparations were isolated from 50 g portions of each mayonnaise product by four successive extraction steps in phosphate buffer with 2.0 M NaCl. These extracts were then centrifuged at 20,000 x g for 60 min. DNA was isolated from the solution containing the precipitate with a commercial kit, and amplified quantitatively using real-time PCR that targeted the com1 region of C. burnetii. The recoveries of C. burnetii from 2 kinds of commercial mayonnaise specimens, with a baseline control of 1 x 10(7) particles of the Nine Mile phase II strain, were 85.0 +/- 6.0% and 72.0 +/- 0.4%, respectively. The determination limit of this method was 500 C. burnetii particles per 50 g of mayonnaise. The DNA specimens isolated from 50 different commercial mayonnaise samples sold in Tokyo using this method were amplified using both nested PCR and real-time PCR. No contamination by C. burnetii was detected in any of the mayonnaise samples.

Phylogenetic analysis of envelope glycoprotein (E1) gene of rubella viruses prevalent in Japan in 2004.

We performed a molecular epidemiological study on the envelope glycoprotein gene (E1 gene) obtained by PCR amplification from specimens of 17 rubella patients in certain areas (Gunma, Saitama, and Kagoshima prefectures, and Tokyo metropolitan area) in Japan in 2004. In these sequences of partially amplified DNAs (283 bases) within the E1 gene, no nucleotide substitution was observed. They were classified into genotype 1D of clade 1 in the constructed phylogenetic tree. One amino acid substitution was found between the amino acid sequence predicted from these DNAs and those of Japanese strains [To-336 vaccine strain (To-336 vac) and its wild progenitor (To-336 wt)]. The results suggest that the rubella viruses (RV) prevalent in certain areas of Japan in 2004 were highly homologous and were closely related with Japanese vaccine strain.

Multiple viral infections and genomic divergence among noroviruses during an outbreak of acute gastroenteritis.

An epidemic outbreak of both norovirus (NV) and astrovirus (ASV) occurred on a research ship surveying Tokyo Bay, causing acute gastroenteritis in 26 of its 37 crew members. The presence of viral pathogens in fecal specimens was analyzed, and noroviruses were identified by reverse transcription-PCR in 18 (48.6%) of these specimens. In addition, astroviruses were identified in 14 (37.8%) of the fecal samples from the affected crew members, and multiple viral infections of both NV and ASV were observed in 6 cases. The genogrouping of the NV-positive samples was then examined by dot blot hybridization, and it was determined that all of the isolates were from genogroup II (GII). No bacterial pathogens were subsequently isolated from fecal specimens. Furthermore, a variety of NV strains were identified by sequencing and single-stranded conformational polymorphism (SSCP) analyses of PCR products from the fecal samples. One recombinant NV isolate, Minato/14, was identified as a recombinant NV strain of GII/6 and GII/1. The other NV isolates from this outbreak were classified into three NV genotypes (GII/1 [Minato/10], GII/4 [Minato/33], and GII/5 [Minato/6]). Furthermore, ASVs in positive samples were determined to belong to serotypes 1 and 2 by sequencing analysis. Our findings thus indicate that coinfections with NV and ASV, including a number of NV genotypes, persisted during an outbreak of gastroenteritis in a closed environment.