Jyh-Yuan Yang

Combining Multiplex Reverse Transcription-PCR and a Diagnostic Microarray To Detect and Differentiate Enterovirus 71 and Coxsackievirus A16

ABSTRACT Cluster A enteroviruses, including enterovirus 71 (EV71) and coxsackievirus A16 (CA16), are known to cause hand-foot-and-mouth disease (HFMD). Despite the close genetic relationship between these two viruses, EV71 is generally known to be a more perpetuating pathogen involved in severe clinical manifestations and deaths. While the serotyping of enteroviruses is mostly done by conventional immunological methods, many clinical isolates remain unclassifiable due to the limited number of antibodies against enterovirus surface proteins. Array-based assays are able to detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex reverse transcription-PCR to try to develop a method of sensitively and accurately detecting and differentiating EV71 and CA16. In an effort to design serotype-specific probes for detection of the virus, we first did an elaborate bioinformatic analysis of the sequence database derived from different enterovirus serotypes. We then constructed a microarray using 60-mer degenerate oligonucleotide probes covalently bound to array slides. Using this enterovirus microarray to study 144 clinical specimens from patients infected with HFMD or suspected to have HFMD, we found that it had a diagnostic accuracy of 92.0% for EV71 and 95.8% for CA16. Diagnostic accuracy for other enteroviruses (non-EV71 or -CA16) was 92.0%. All specimens were analyzed in parallel by real-time PCR and subsequently confirmed by neutralization tests. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.

Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan

Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.

Sentinel Hospital Surveillance for Rotavirus Diarrhea in Taiwan, 2001–2003

We examined the epidemiological profile of rotavirus infection among children hospitalized for diarrhea in Taiwan, to assess the burden of this disease. From 1 April 2001 through 31 March 2003, children <5 years old with gastroenteritis admitted to 4 sentinel hospitals were enrolled in a surveillance study and had stool specimens tested for the presence of rotavirus, enteric adenovirus, and the bacterial pathogens for which routine screening is performed. For 52% of patients, a recognized enteric pathogen was identified, including rotavirus (43% of patients), bacteria (11%), enteric adenovirus (2.5%), and a mixture of pathogens (3.9%). Rotavirus was detected year-round, but great month-to-month variability made it difficult to identify a distinct seasonal pattern. Rotavirus disease was most common among children 7-23 months old, but the rate of rotavirus detection varied little between the youngest and oldest age groups. The novel strain P[8]G9 was detected most commonly (37% of strains), followed by strains P[8]G1 (31%), P[4]G2 (10%), P[8]G3 (9.3%), and P[8]G4 (3.7%). Rotavirus infection is the most important cause of diarrhea among hospitalized children in Taiwan, and a rotavirus vaccination program for young children might significantly reduce this problem.

Detection of SARS-associated Coronavirus in Throat Wash and Saliva in Early Diagnosis

Early detection of SARS-CoV in throat wash and saliva suggests that these specimens are ideal for SARS diagnosis.

Sensitive and Quantitative Detection of Severe Acute Respiratory Syndrome Coronavirus Infection by Real-Time Nested Polymerase Chain Reaction

Abstract A quantitative, real-time, nested polymerase chain reaction (PCR) method, combining the high sensitivity of nested PCR with time-saving real-time instrumentation, was developed for large-scale screening for severe acute coronavirus (SARS) coronavirus. Forty-six clinical specimens were analyzed by this method, and results were compared with those obtained by conventional, single-round, real-time reverse-transcriptase PCR (RT-PCR) performed in parallel. Of the 17 positive results, 2 identified by our method were not detected by single-round, real-time RT-PCR, which suggests that real-time nested PCR has the potential for increased sensitivity, leading to earlier detection of SARS.

Genetic diversity and C2-like subgenogroup strains of enterovirus 71, Taiwan, 2008

BackgroundHuman enterovirus 71 (EV-71) is known of having caused numerous outbreaks of hand-foot-mouth disease, and other clinical manifestations globally. In 2008, 989 EV-71 strains were isolated in Taiwan.ResultsIn this study, the genetic and antigenic properties of these strains were analyzed and the genetic diversity of EV-71 subgenogroups surfacing in Taiwan was depicted, which includes 3 previously reported subgenogroups of C5, B5, and C4, and one C2-like subgenogroup. Based on the phylogenetic analyses using their complete genome nucleotide sequences and neutralization tests, the C2-like subgenogroup forms a genetically distinct cluster from other subgenogroups, and the antisera show a maximum of 128-fold decrease of neutralization titer against this subgenogroup. In addition, the subgenogroup C4 isolates of 2008 were found quite similar genetically to the Chinese strains that caused outbreaks in recent years and thus they should be carefully watched.ConclusionsOther than to be the first report describing the existence of C2-like subgenogroup of EV-71 in Taiwan, this article also foresees a potential of subgenogroup C4 outbreaks in Taiwan in the near future.

Molecular Epidemiology of HIV-1 Infection and Full-Length Genomic Analysis of Circulating Recombinant Form 07_BC Strains from Injection Drug Users in Taiwan

BACKGROUND Previously, we reported that there was an outbreak of human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) in Taiwan in 2004. The objectives of the present study were to conduct a molecular epidemiological analysis and to characterize the full-length genome of the Taiwanese CRF07_BC. METHODS Three hundred and fifty-eight patients with HIV-1/AIDS from hospitals and 133 HIV-1-infected inmates from detention centers were recruited. DNA sequencing and phylogenetic analysis were conducted to determine subtypes and evolutionary relationships. Recombination breakpoints of 2 full-length CRF07_BC strains were elucidated using a bootscanning method. RESULTS Of 206 HIV-1-infected patients who received a diagnosis in 2004, 44.7% were infected with subtype B, 53.4% with CRF07_BC, and 1.5% with CRF01_AE. Ninety-eight percent (109/111) of IDUs were infected with CRF07_BC. Deletions of 7-11 amino acids in both p6(gag) and p6(pol) proteins were noted among the Taiwanese CRF07_BC strains. The CRF_07BC strains belonged to 2 phylogenetic clusters, and the first cluster contained only CRF07_BC strains from the southern part of Taiwan. CONCLUSIONS The Taiwanese CRF07_BC strains had 97% full-length sequence homology with the prototype from mainland China. CRF07_BC was first introduced into the southern region in 2002 and then spread to other regions in Taiwan in 2004.

The effect of prior compressive deformation of austenite on toughness property in an ultra-low carbon bainitic steel

For the purpose of investigating the trend of toughness versus variation of microstructural constituents for an experimental ultra-low carbon bainitic steel, the experiments (with and without prior compressive deformation) have been carried out on a Gleeble 1500 machine. The Charpy impact specimens were prepared from the samples treated by the Gleeble machine. The Charpy impact absorbed energy for toughness was measured, and the corresponding fractographs, optical metallographs and transmission electron micrographs have been examined. The result shows that the prior compressive deformation of austenite promotes the formation of intragranular non-parallel plates of acicular ferrite but stifles the formation of sheaf-like parallel plates of bainitic ferrite. Furthermore, the study emphasizes that the microstructure containing a high volume fraction of acicular ferrite possesses better toughness and strength than the microstructure containing mainly bainite.

Ultraviolet Electroluminescence From n-ZnO–SiO

Ultraviolet (UV) light-emitting diodes composed of n-ZnO:Al-SiO2-ZnO nanocomposite/p-GaN:Mg heterojunction were fabricated on the (0002) Al2O3 substrate. A SiO2 layer embedded with ZnO nanodots was prepared on the p-type GaN using spin-on coating of SiO2 nanoparticles together with atomic layer deposition (ALD). An n-type Al-doped ZnO layer was deposited also by ALD. The SiO2-ZnO nanocomposite layer accomplishes a role of the current blocking layer and also causes, by its low refractive index, the increase in the light extraction efficiency from n-ZnO. Significant UV electroluminescence from n-ZnO was achieved at a low forward-bias current of 1.8 mA. Strong UV emission arising from impact ionization in GaN, ZnO, and GaN:Mg states was also observed at reverse breakdown bias.

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We previously reported the detection of genotype P[19] rotavirus strains from children hospitalized with acute dehydrating diarrhea during a 5‐year surveillance period in Taiwan. The characterization of five P[19] strains (0.4% of all typed), including three G3P[19], a novel G5P[19], and a unique G9P[19] genotype is described in this study. Phylogenetic analysis of the VP4, VP7, VP6, and NSP4 genes was performed, which demonstrated novel lineages for respective genotypes of the VP4 and the VP7 genes. The sequence similarities of the P[19] VP4 gene among Taiwanese human strains was higher (nt, 91.5–96.2%; aa, 93.7–97.6%) than to other P[19] strains (nt, 83.5–86.6%; aa, 89.4–94.1%) from different regions of the world. The VP7 gene of the three G3P[19] Taiwanese strains shared up to 93.4% nt and 97.5% aa identity to each other but had lower similarity to reference strain sequences available in GenBank (nt, <90.1%; aa, <95.6%). Similarly, the VP7 gene of the novel G5P[19] strain was only moderately related to the VP7 gene of reference G5 strains (nt, 82.2–87.3%; aa, 87.0–93.1%), while the VP7 gene of the single G9P[19] strain was genetically distinct from other known human and animal G9 rotavirus strains (nt, ≤92.0%; aa, ≤95.7%). Together, these findings suggest that the Taiwanese P[19] strains originated by independent interspecies transmission events. Synchronized surveillance of human and animal rotaviruses in Taiwan should identify possible hosts of these uncommon human rotavirus strains. J. Med. Virol. 83:1279–1287, 2011.