Structure of human Aichi virus and implications for receptor binding.

Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries1,2. The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life3,4. There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the ß-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' ß-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.

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.

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.

Investigation of toilet activities in elderly patients with dementia from the viewpoint of motivation and self-awareness.

Toilet activities of the elderly patients with dementia were observed focusing on care conditions and investigated based on Hull's drive reduction theory (behavior = drive × habit × incentive) and our self-awareness model (consisting of theory of mind, self-evaluation, and self-consciousness) to evaluate the association between self-awareness and toilet activities in patients with dementia and to explain the time when and the reason why a series of toilet activities as habit once acquired become unfeasible. If theory of mind is lost, awareness of one's desire and intention becomes vague, and toilet activities begin to collapse. Furthermore, if incentive disappears, one's intention hardly arises and toilet activities further collapse. If self-evaluation is lost, time sense fades, future goals based on the present time cannot exist, and behavior loses directivity. As a result, toilet activities collapse, and with a decrease in drive toilet activities cease.

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.

Impact of controlled pericardial drainage on critical cardiac tamponade with acute type A aortic dissection.

BACKGROUND: Cardiac tamponade is associated with fatal outcomes for patients with acute type A aortic dissection, and the presence of cardiac tamponade should prompt urgent aortic repair. However, treatment of the patient with critical cardiac tamponade who cannot survive until surgery remains unclear. We analyzed our experience of controlled pericardial drainage (CPD) managing critical cardiac tamponade. METHODS AND RESULTS: Between September 2003 and May 2011, 175 patients with acute type A aortic dissection were treated surgically, including 43 (24.6%) who presented with cardiac tamponade on arrival. Eighteen patients, who did not respond to intravenous volume resuscitation, underwent CPD in the emergency department. An 8F pigtail drainage catheter was inserted percutaneously, and drainage volume was controlled by means of several cycles of intermittent drainage to maintain blood pressure at ≈90 mm Hg. After CPD, all of the patients were transferred to the operating room, and immediate aortic repair was performed. Systolic blood pressure before CPD was 64.3 ± 8.2 mm Hg and elevated significantly in all of the cases after CPD. Systolic blood pressure after CPD was 94.8 ± 10.5 mm Hg, and increase in systolic pressure was 30.5 ± 11.7 mm Hg. Total volume of aspirated pericardial effusion was 40.1 ± 30.6 mL, and 10 patients required only ≤30-mL aspiration volume. All of the patients underwent aortic repair successfully. In-hospital mortality was 16.7%; however, there was no complications or mortality related to CPD. CONCLUSIONS: Preoperative pericardial drainage with control of volume is a safe and effective procedure for acute type A aortic dissection complicated by critical cardiac tamponade. In our patient population, timely controlled pericardial drainage is warranted.

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.

Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71.

Human enterovirus species A (HEV-A) consists of at least 16 members of different serotypes that are known to be the causative agents of hand, foot, and mouth disease (HFMD), herpangina, and other diseases, such as respiratory disease and polio-like flaccid paralysis. Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of HFMD. CVA5, CVA6, CVA10, and CVA12 mainly cause herpangina or are occasionally involved with sporadic cases of HFMD. We have previously shown that human scavenger receptor class B, member 2 (SCARB2) is a cellular receptor for EV71 and CVA16. Using a large number of clinical isolates of HEV-A, we explored whether all clinical isolates of EV71 and other serotypes of HEV-A infected cells via SCARB2. We tested this possibility by infecting L-SCARB2 cells, which are L929 cells expressing human SCARB2, by infecting human RD cells that had been treated with small interfering RNAs for SCARB2 and by directly binding the viruses to a soluble SCARB2 protein. We showed that all 162 clinical isolates of EV71 propagated in L-SCARB2 cells, suggesting that SCARB2 is the critical receptor common to all EV71 strains. In addition, CVA7, CVA14, and CVA16, which are most closely related to each other, also utilized SCARB2 for infection. EV71, CVA14, and CVA16 are highly associated with HFMD, and EV71 and CVA7 are occasionally associated with neurological diseases, suggesting that SCARB2 plays important roles in the development of these diseases. In contrast, another group of viruses, such as CVA2, CVA3, CVA4, CVA5, CVA6, CVA8, CVA10, and CVA12, which are relatively distant from the EV71 group, is associated mainly with herpangina. None of these clinical isolates infected via the SCARB2-dependent pathway. HEV-A viruses can be divided into at least two groups depending on the use of SCARB2, and the receptor usage plays an important role in developing the specific diseases for each group.

Detection of human parechoviruses from clinical stool samples in Aichi, Japan.

Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5' untranslated region (5'UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema, "hand, foot, and mouth disease," aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.

Molecular identification of enteroviruses including two new types (EV-98 and EV-107) isolated from Japanese travellers from Asian countries.

Of 58 enterovirus strains isolated from Japanese travellers returning from Asian countries, eight were non-serotypable with existing antisera. By sequencing a part of the VP1 region, six of these strains were typed as echovirus 9, enterovirus (EV)-73, EV-79 or EV-97. The nucleotide identity of the VP1 region of isolate T92-1499 to all enterovirus prototypes was <70 %. The VP1 sequence of isolate TN94-0349 was closely related to coxsackievirus (CV)-A9 (73.3 % nucleotide identity), but the virus could not be neutralized with a serum raised against the prototype CV-A9 strain. On the basis of complete molecular comparisons, T92-1499 and TN94-0349 were identified as EV-98 and EV-107, respectively, by the ICTV Picornavirus Study Group. Serum neutralization tests of Japanese individuals revealed a seroprevalence rate of 11 % for EV-73, and even lower seroprevalence rates, 1.0-3.8 %, were found for the other new enteroviruses, suggesting that prior circulation of these viruses in Japan was unlikely.

Congenital quadricuspid aortic valve associated with severe regurgitation.

A 56-year-old man was referred because of severe aortic regurgitation. He had a quadricuspid aortic valve with a small accessory cusp between the right coronary and noncoronary cusps. The ostium of the right coronary artery was deviated toward the accessory cusp commissure. Aortic valve replacement was performed with a bioprosthesis. The resected cusps showed fibrotic thickening with calcification and fenestration.

Abdominal aortic aneurysm associated with horseshoe kidney.

The optimal approach to abdominal aortic aneurysm with horseshoe kidney is still debated. We describe a successful abdominal aortic aneurysm repair through a left retroperitoneal approach in a 77-year-old woman with a horseshoe kidney.

Acute dissection of the innominate artery: a case of report.

A 61-year-old man developed a ruptured innominate artery associated with localized acute dissection. The innominate artery was reconstructed with a bifurcated prosthetic graft without brain complication.