Chit Laa Poh

The largest outbreak of hand; foot and mouth disease in Singapore in 2008: The role of enterovirus 71 and coxsackievirus A strains

BACKGROUND During 2008, Singapore experienced its largest ever outbreak of hand, foot and mouth disease (HFMD), resulting in 29686 cases, including four cases of encephalitis and one fatality. METHODS A total of 51 clinical specimens from 43 patients with suspected HFMD at the National University Hospital, Singapore were collected for virus isolation and identification by reverse transcription polymerase chain reaction (RT-PCR) and sequencing. RESULTS Enteroviruses were identified in 34 samples (66.7%), with 11 samples (21.6%) being positive for enterovirus 71 (EV71). Other non-EV71 enteroviruses (including coxsackievirus A4, A6, A10, and A16) were identified in 23 samples (45.1%). The most prevalent virus serotypes were CA6, CA10, and EV71. CA6 and CA10 accounted for 35.3% of all HFMD cases, which may explain the high transmissibility and low fatality that characterized this unprecedented epidemic associated with relatively mild disease. Phylogenetic analyses of 10 circulating EV71 strains indicated that they belonged to two subgenogroups, i.e., B5 (80%) and C2 (20%). The VP1 sequences of the 2008 EV71 strains also exhibited continuous mutations during the outbreak, reflecting the relatively high mutation rate of the EV71 capsid protein, which may have implications for future vaccine development. CONCLUSIONS A safe and effective vaccine against EV71 is certainly warranted in view of its potential neurovirulence and its role in HFMD epidemics of recurring frequency with resultant fatalities in Asia, as well as other parts of the world.

Direct Detection of Enterovirus 71 (EV71) in Clinical Specimens from a Hand, Foot, and Mouth Disease Outbreak in Singapore by Reverse Transcription-PCR with Universal Enterovirus and EV71-Specific Primers

ABSTRACT A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.

Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor

ABSTRACT Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor.

Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000).

Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non‐structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence.

Novel β-Lactamase Genes from Two Environmental Isolates of Vibrio harveyi

ABSTRACT Two ampicillin-resistant (Ampr) isolates ofVibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel β-lactamase genes from W3B (blaVHW-1) and HB3 (blaVHH-1) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed for the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced forblaVHH-1. At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homology to any other β-lactamases in the databases. The deduced proteins were found to be class A β-lactamases bearing low levels of homology (<50%) to other β-lactamases of the same class. The highest level of identity was obtained with β-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employingblaVHW-1 as a gene probe demonstrated that thebla gene was not located in the plasmid. A total of nine ampicillin-resistant V. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis ofNotI-digested genomic DNA. Despite a high level of intrastrain genetic diversity, theblaVHW-1 probe hybridized only to an 80- or 160-kb NotI genomic fragment in different isolates.

Cloning and sequences of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putida P35X

Pseudomonas putida P35X (NCIB 9869) metabolises phenol and cresols via a chromosomally encoded meta-cleavage pathway. A 13.4-kb fragment of the chromosome involved in encoding phenol catabolism was cloned and characterized. Deletion analysis and nucleotide sequencing of a 6589-bp region, in conjunction with enzyme assays, were used to identify the phhKLMNOP genes encoding the phenol hydroxylase, the phhB gene encoding catechol 2,3-dioxygenase (EC 1.13.11.2) and the phhQ gene that encodes a small ferredoxin-like protein. The genes are organised in an operon-like structure, in the order phhKLMNOPQB, and the deduced amino-acid sequences share high homology (68.3-99.7%) with those of the plasmid-encoded genes dmpKLMNOPQB of Pseudomonas sp. strain CF600. Genetic evidence is presented that the difference in the growth substrate ranges of Pseudomonas P35X and CF600 are due to the effector activation specificities of the regulators of these systems, rather than the substrate specificities of the catabolic enzymes.

Genome fingerprinting of Salmonella typhi by pulsed-field gel electrophoresis for subtyping common phage types.

The genomic DNA of 39 strains of Salmonella typhi isolated from local residents and patients who had visited countries in the Asian region was analysed for restriction fragment length polymorphisms (RFLP). Pulsed-field gel electrophoretic (PFGE) analysis of Xba I- and Spe I-generated genomic restriction fragments established 22 PFGE types whereas phage typing differentiated the 39 isolates into 9 distinct phage types. This study showed that PFGE is more discriminatory than phage typing as it is capable of subtyping S. typhi strains of the same phage types. Genetic relatedness among the isolates was determined. Seven major clusters were identified at SABs of > 0.80 and the remaining 13 isolates were distributed into minor clusters which were related at SABs of less than 0.80. In conclusion, PFGE analysis in conjunction with distance matrix analysis served as a useful tool for delineating common S. typhi phage types of diverse origins from different geographical locales and separated in time.

Inhibition of Gene Expression and Growth by Antisense Peptide Nucleic Acids in a Multiresistant β-Lactamase-Producing Klebsiella pneumoniae Strain

ABSTRACT Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of β-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 μM and 40 μM, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (104 CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.

Insights into environmental bioremediation by microorganisms through functional genomics and proteomics

Environmental pollutants in the soil are a major concern worldwide. Bioremediation mediated by microorganisms is a highly promising technology that is environmentally friendly, safe, and effective. However, incomplete biological information regarding the cellular responses in many microbial communities restricts progress in the site‐specific mineralization process. The application of proteomics in environmental bioremediation research provides a global view of the protein compositions of the microbial cells and offers a promising approach to address the molecular mechanisms of bioremediation. With the combination of proteomics, functional genomics provide an insight into global metabolic and regulatory networks that can enhance the understanding of gene functions. This article deals with the applications of functional genomics and proteomics to dissect the cellular responses to environmental stimuli, such as stress response, induction and expressions of regulatory proteins/enzymes in response to aromatic hydrocarbons and heavy metals. An understanding of the growth conditions governing the expression of the proteome (for example, enzymes and regulatory proteins of aromatic hydrocarbon degradation, energy generation pathways, transport and stress‐related proteins) in a specific environment is essential for developing rational strategies for successful bioremediation.

Rapid detection of Enterovirus 71 by real-time TaqMan RT-PCR

BACKGROUND Enterovirus 71 (EV71) is the main etiological agent of Hand, Foot and Mouth Disease (HFMD) and has been associated with neurological complications which resulted in fatalities during recent outbreaks in Asia Pacific region. OBJECTIVE Develop a real-time TaqMan RT-PCR for rapid detection of EV71. STUDY DESIGN Specific primers and probe were designed based on highly conserved VP1 region of EV71. The sensitivity of the real-time RT-PCR was evaluated with 67 clinical specimens collected from pediatric patients with suspected HFMD. RESULTS Our real-time TaqMan RT-PCR showed 100% specificity in detecting EV71 and showed an analytical sensitivity of 5 viral copies. High sensitivity was also achieved in detecting EV71 directly from clinical specimens. CONCLUSIONS Real-time TaqMan RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.