Masahiro Miyoshi

Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription-polymerase chain reaction

Conventional reverse transcription‐polymerase chain reaction (RT‐PCR) to detect norovirus (NV) is a complex of multi‐step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT‐PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1‐ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT‐PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT‐PCR. Two hundred fifty‐one of the 255 specimens that were negative by the conventional RT‐PCR were also negative by the TaqMan RT‐PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT‐PCR end‐point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT‐PCR is useful for the detection of NV in clinical specimens. J. Med. Virol. 80:913–920, 2008.

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.

Import-Associated Measles Outbreak Including Hospital- and Clinic-Based Transmission in the Non-Endemic Hokkaido District, Japan, 2014

Accepted July 15, 2015. DOI: 10.7883/yoken.JJID.2015.237 *Corresponding author: Mailing address: Hokkaido Institute of Public Health, North 19 West 12, Kita-ku, Sapporo 060-0819, Japan. Tel: +81-11-747-2764, Fax: +81-11-747-2757, E-mail: miyo@iph.pref.hokkaido.jp Table 1. Laboratory diagnoses of suspected cases of measles performed in commercial laboratories and prefectural or municipal Public Health Institutes in Hokkaido district, Japan, 2014

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.

Genetic characterization of hemagglutinin protein of measles viruses in Hokkaido district, Japan, 2006-2015: Characterization of MeV-H in Hokkaido

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.

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.

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.

Usefulness of the rapid determination system of viral genome sequences in human stool specimens.

The rapid determination system of viral genome sequences (the RDV method) consists of detecting and determining the nucleotide sequences of viral genomes without using specific primers. To evaluate the usefulness of the RDV method, the detection of human norovirus (NV) genomes in stool specimens was investigated. In addition, the effect of nuclease treatment of the process was examined. A total of 23 human stool specimens were used, all of which were collected from patients with acute viral gastroenteritis, and were shown to contain NV genomes and also determined the cDNA copy numbers by the real-time reverse transcriptase-polymerase chain reaction. NV genomes were detected by the RDV method with nuclease treatment in nine specimens containing cDNA copies ranging between 6.2×10(9) and 9.8×10(11)/g stool. In contrast, NV genome was found by the method in 15 specimens without nuclease treatment and the number of NV cDNA copies ranged between 1.2×10(6) and 9.8×10(11)/g stool. These results suggest that the RDV method has potential for detecting viral genomes in stool specimens. The procedure without a step of nuclease treatment appears to be sensitive.

Corrigendum to “Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens” [J. Clin. Virol. 80 (2016) 98–101]

orrigendum to “Evaluation of sensitivity of TaqMan RT-PCR for ubella virus detection in clinical specimens” J. Clin. Virol. 80 (2016) 98–101] iyoko Okamotoa, Yoshio Moria,∗, Rika Komagomeb, Hideki Naganob, Masahiro Miyoshib, otohiko Okanob, Yoko Aokic, Atsushi Ogurad, Chiemi Hottad, Tomoko Ogawad, iwako Saikusae, Hiroe Kodamaf, Yoshihiro Yasuig, Hiroko Minagawag, Takako Kuratah, aiki Kanbayashih, Tetsuo Kaseh, Sachiko Murata i, Komei Shirabe i, Mitsuhiro Hamasaki j, akashi Katok, Noriyuki Otsukia, Masafumi Sakataa, Katsuhiro Komasea, Makoto Takedaa