Genetic comparison of human parainfluenza virus type 3 detected in respiratory samples from patients with encephalopathy and airway inflammation in Aichi Prefecture, Japan.

Human parainfluenza virus type 3 (HPIV-3, Human respirovirus 3) is the second most frequently detected virus after human respiratory syncytial virus (HRSV) in lower respiratory tract infections in children. HPIV-3, like its close relative respiratory viruses, HRSV and influenza virus, may cause encephalopathy, but the relevance of HPIV-3 as a pathogenic factor in encephalopathy is unknown. We attempted to detect HPIV-1 through 4, HRSV, and human metapneumovirus (HMPV) in 136 patients with encephalitis/encephalopathy, or suspected encephalitis/encephalopathy during a 6-year period from 2014 to 2019. As a result, HPIV-3 was detected most frequently in 6 patients, followed by HRSV in 3. The HPIV-3 strains detected were closely related to those detected in a patient with respiratory disease at the same period. Although HPIV-3 is less recognized than HRSV as a triggering virus of encephalopathy, our results suggest that HPIV-3 is at least as important as HRSV. Surveillance of the causative virus of encephalopathy, including HPIV-3, would help to clarify the actual status of encephalopathy, the cause of which is currently reported in less than half of cases in Japan.

Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.

In Japan, major mumps outbreaks still occur every 4-5 years because of low mumps vaccine coverage (30-40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan's regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

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.

The Association Between Documentation of Koplik Spots and Laboratory Diagnosis of Measles and Other Rash Diseases in a National Measles Surveillance Program in Japan.

Koplik spots are considered a disease-specific sign for measles, although comprehensive virological studies have not been conducted to date. In Japan, a national survey of 3023 measles and measles-suspected cases was conducted between 2009 and 2014 using polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) to detect various rash/fever-associated viruses. Koplik spots were observed in 717 of 3023 cases (23.7%). Among these, the measles virus was detected in 202 cases (28.2%), while the rubella virus was detected in 125 cases (17.4%). Other viruses were detected in 51 cases having the spots (7.1%). In some of the cases with spots, two or three viruses, such as the rubella virus, parvovirus, and human herpesvirus type 6 were also detected. The sensitivity and specificity of Koplik spots as a diagnostic marker for measles were 48 and 80%, respectively. The results suggested that Koplik spots might appear not only in measles but also in other viral infections, such as rubella, as a clinical sign.

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.

Description of the Pathogenic Features of Streptococcus pyogenes Isolates from Invasive and Non-Invasive Diseases in Aichi, Japan.

We identified hypervirulent Streptococcus pyogenes in 27 and 420 isolates from patients with invasive and non-invasive diseases, respectively, in Aichi Prefecture, Japan, between 2003 and 2012, in an attempt to understand why the prevalence of streptococcal toxic shock syndrome (STSS) suddenly increased in this location during 2011. Hypervirulent strains belong to the emm1 genotype, with a mutation in the covR/S genes that regulate many other genes, encoding virulence determinants and resulting in the absence of the proteinase streptococcal exotoxin B and the production of virulence factors such as the superantigen streptococcal exotoxin A, the nuclease streptococcal DNase, the cytotoxin NAD-glycohydrolase, and the hemolysin streptolysin O. We found 1 strain from invasive disease and 1 from non-invasive disease with traits similar to those of hypervirulent strains, except that the sda1 gene was absent. We also found 1 non-emm1 strain with phenotypic and genetic traits identical to those of the emm1 hypervirulent strains except that it did not belong to emm1 genotype, from non-invasive diseases cases in 2011. These findings suggested that hypervirulent and hypervirulent-like strains from invasive and non-invasive disease cases could have at least partially contributed to the sudden increase in the number of patients with STSS in Aichi during 2011.

Molecular epidemiology of enteric viruses in patients with acute gastroenteritis in Aichi prefecture, Japan, 2008/09-2013/14.

Acute gastroenteritis is a critical infectious disease that affects infants and young children throughout the world, including Japan. This retrospective study was conducted from September 2008 to August 2014 (six seasons: 2008/09-2013/14) to investigate the incidence of enteric viruses responsible for 1,871 cases of acute gastroenteritis in Aichi prefecture, Japan. Of the 1,871 cases, 1,100 enteric viruses were detected in 978 samples, of which strains from norovirus (NoV) genogroup II (60.9%) were the most commonly detected, followed by strains of rotavirus A (RVA) (23.2%), adenovirus (AdV) type 41 (8.2%), sapovirus (SaV) (3.6%), human astrovirus (HAstV) (2.8%), and NoV genogroup I (1.3%). Sequencing of the NoV genogroup II (GII) strains revealed that GII.4 was the most common genotype, although four different GII.4 variants were also identified. The most common G-genotype of RVA was G1 (63.9%), followed by G3 (27.1%), G2 (4.7%) and G9 (4.3%). Three genogroups of SaV strains were found: GI (80.0%), GII (15.0%), and GV (5.0%). HAstV strains were genotyped as HAstV-1 (80.6%), HAstV-8 (16.1%), and HAstV-3 (3.2%). These results show that NoV GII was the leading cause of sporadic acute viral gastroenteritis, although a variety of enteric viruses were detected during the six-season surveillance period.

Case-based surveillance enhanced with measles virus detection/genotyping is essential to maintain measles elimination in Aichi Prefecture, Japan.

BACKGROUND: Japan was verified as having achieved measles elimination by the Measles Regional Verification Commission in the Western Pacific Region in March 2015. Verification of measles elimination implies the absence of continuous endemic transmission. After the last epidemic in 2007 with an estimated 18,000 cases, Japan introduced nationwide case-based measles surveillance in January 2008. Laboratory diagnosis for all suspected measles cases is essentially required by law, and virus detection tests are mostly performed by municipal public health institutes. Despite relatively high vaccination coverage and vigorous response to every case by the local health center staff, outbreak of measles is repeatedly observed in Aichi Prefecture, Japan. METHODS: Measles virus N and H gene detection by nested double RT-PCR was performed with all specimens collected from suspected cases and transferred to our institute. Genotyping and further molecular epidemiological analyses were performed with the direct nucleotide sequence data of appropriate PCR products. RESULTS: Between 2010 and 2014, specimens from 389 patients suspected for measles were tested in our institute. Genotypes D9, D8, H1 and B3 were detected. Further molecular epidemiological analyses were helpful to establish links between patients, and sometimes useful to discriminate one outbreak from another. All virus-positive cases, including 49 cases involved in three outbreaks without any obvious epidemiological link with importation, were considered as import-related based on the nucleotide sequence information. Chain of transmission in the latest outbreak in 2014 terminated after the third generations, much earlier than the 2010-11 outbreak (6th generations). CONCLUSION: Since 2010, almost all measles cases reported in Aichi Prefecture are either import or import-related, based primarily on genotypes and nucleotide sequences of measles virus detected. In addition, genotyping and molecular epidemiological analyses are indispensable to prove the interruption of endemic transmission when the importations of measles are repeatedly observed.

Reduced Tyk2 gene expression in ß-cells due to natural mutation determines susceptibility to virus-induced diabetes.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic ß-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by ß-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic ß-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections.

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus.

Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

Detection and genotyping of rubella virus from exanthematous patients suspected of having measles using reverse transcription-PCR.

Between July 2012 and March 2013, a total of 133 clinical specimens from 47 patients suspected of having measles were collected for virological surveillance in Aichi Prefecture, Japan. Facing the rubella epidemic, the reverse transcription (RT)-PCR protocol for measles virus (MeV) was modified to simultaneously detect rubella virus (RUBV) in these clinical specimens. As a result, 30 specimens from 15 patients were positive for RUBV and 8 specimens from 3 patients were positive for MeV. The RUBV genotype analysis for the samples from 13 patients revealed 12 samples as 2B and 1 sample as 1E. The results provided additional evidence for the difficulty in the diagnosis of exanthematous diseases based on clinical manifestations alone and the necessity of virological diagnosis to maintain the accuracy of case-based surveillance. Furthermore, the results indicated that the modified RT-PCR protocol could be useful as a routine procedure to simultaneously detect MeV and RUBV in clinical specimens of patients suspected of having exanthematous disease caused by these viruses.

Molecular detection and nucleotide sequence analysis of a new Aichi virus closely related to canine kobuvirus in sewage samples.

Between 2001 and 2005, 207 raw sewage samples were collected at the inflow of a sewage treatment plant in Aichi Prefecture, Japan. Of the 207 sewage samples, 137 (66.2 %) were found to be positive for amplification of Aichi virus (AiV) nucleotide using reverse transcription (RT)-PCR with 10 forward and 10 reverse primers in the 3D region corresponding to the nucleotide sequence of all kobuviruses. AiV genotype A sequences were detected in all 137 samples. New sequences of AiV were detected in nine samples, exhibiting 83 % similarity with AiV A846/88, but 95 % similarity with canine kobuvirus (CKV) US-PC0082 in this region. The nucleotide sequences from the VP3 region to the 3' untranslated region (UTR) of sewage sample Y12/2004 were determined. The number of nucleotides in each region was the same as that of CKV. The similarity of the nucleotide (amino acid) identity of a complete VP1 region was 90.5 % (94.8 %) between Y12/2004 and CKV US-PC0082. The phylogenic analyses based on the nucleotide and the deduced amino acid sequences of VP1 and 3D showed that Y12/2004 was independent from AiV, but closely related to CKV. These results suggested that CKV is present in Aichi Prefecture, Japan.

Hyperferritinemia in neonatal and infantile human parechovirus-3 infection in comparison with other infectious diseases.

Human parechovirus-3 (HPeV-3) has been associated with severe clinical manifestations in neonates and infants in the form of sepsis or hemophagocytic lymphohistiocytosis (HLH)-like illness. To clarify the clinical features of HPeV-3 infection, we compared clinical signs and laboratory findings among enteroviruses (EVs), HPeV-3, and other infections. Participants were 26 febrile infants in whom EVs (n = 20) or HPeV-3 (n = 6) were isolated from throat swab or fecal specimens. Clinical and laboratory data were compared among EVs, HPeV-3, respiratory syncytial virus (RSV) infection (n = 15), and bacterial meningitis (n = 8) groups. Apnea was frequently seen in the HPeV-3 group although there were no significant differences in other clinical symptoms. Leukocyte count was significantly lower in the HPeV-3 group than in the EV and RSV group. Platelet count was significantly lower in the HPeV-3 group than in the RSV group. Serum ferritin levels in the HPeV-3 group (mean, 2437 ng/ml) and EV group (mean, 552 ng/ml) were significantly higher than in the RSV group (mean 237 ng/ml; P = 0.008 and P = 0.002, respectively). The frequency of patients with clearly high ferritin levels ≥1000 ng/ml was comparatively higher in the HPeV-3 group (4/6) than the EV group (3/20) (P = 0.03). In the HPeV-3 group, ferritin levels were high on Days 4-5. Elevated ferritin levels, decreased leukocyte and platelet counts could offer diagnostic clues to HPeV-3 infection in infant. These laboratory findings might be associated with aberrant immune response to HPeV-3, which could contribute to the development of sepsis or HLH-like illness in neonates.

Development of a reverse transcription-quantitative PCR system for detection and genotyping of aichi viruses in clinical and environmental samples.

Aichi viruses (AiVs) have been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. In the present study, we developed a TaqMan minor groove binder (MGB)-based reverse transcription-quantitative PCR (RT-qPCR) system that is able to quantify AiVs and differentiate between genotypes A and B. This system consists of two assays, an AiV universal assay utilizing a universal primer pair and a universal probe and a duplex genotype-specific assay utilizing the same primer pair and two genotype-specific probes. The primers and probes were designed based on multiple alignments of the 21 available AiV genome sequences containing the capsid gene. Using a 10-fold dilution of plasmid DNA containing the target sequences, it was confirmed that both assays allow detection and quantification of AiVs with a quantitative range of 1.0 × 10(1) to 1.0 × 10(7) copies/reaction, and the genotype-specific assay reacts specifically to each genotype. To validate the newly developed assays, 30 clinical stool specimens were subsequently examined with the assays, and the AiV RNA loads were determined to be 1.4 × 10(4) to 6.6 × 10(9) copies/g stool. We also examined 12 influent and 12 effluent wastewater samples collected monthly for a 1-year period to validate the applicability of the assays for detection of AiVs in environmental samples. The AiV RNA concentrations in influent and effluent wastewater were determined to be up to 2.2 × 10(7) and 1.8 × 10(4) copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples.

Phenotypic and genetic analyses of Campylobacter jejuni Lior serotype 76 isolated from chicken meat and clinical specimens.

The aim of this study was to examine the link between Campylobacter jejuni isolates obtained from chicken meat (n = 7) and gastroenteritis patients (n = 744). In total, 751 isolates were subjected to Lior serotyping. All the isolates from chicken meats were serotyped as Lior serotype 76 (LIO76). Among 23 of the identified LIO76 strains, 13 strains (6 from chicken meat and 7 from clinical specimens) were indistinguishable by Penner serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis. These strains were isolated in 2 different Japanese prefectures in 2004-2005, suggesting that chicken meat is an etiological agent of Campylobacter gastroenteritis and that a diffuse outbreak occurred during this time. Therefore, a continuous surveillance program should be established in Japan in order to prevent Campylobacter gastroenteritis, especially large-scale food-borne outbreaks.

A foodborne outbreak of sapovirus linked to catered box lunches in Japan.

Sapovirus (SaV) is a common cause of acute viral gastroenteritis worldwide, and SaV outbreaks have become more frequent in recent years. In January 2010, an outbreak of acute gastroenteritis due to SaV occurred in Aichi, Gifu and Mie Prefectures, Japan. The illness was strongly associated with eating a delivered box lunch prepared by one catering company. In total, 655 (17.1 %) of 3827 individuals developed gastroenteritic symptoms. SaV was detected in seven of the nine people who became ill and in seven of the 52 food handlers at the catering company, but all the tested samples were negative for norovirus and enteropathogenic bacteria. Sequence analysis of RT-PCR products indicated that the nucleotide sequences of SaV strains from the people who became ill and the food handlers were identical. The detected SaV strains were genogrouped as SaV genotype I.2. This was the largest foodborne outbreak of sapovirus in Japan.