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Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease (HFMD). Several recent outbreaks of HFMD in East Asia were associated with neurological complications and numerous deaths. In 2000, an outbreak in Singapore afflicted thousands of children, resulting in four fatal cases from whom EV71 was isolated. The virus possesses four structural proteins VP1, VP2, VP3, and VP4, each of which is involved in forming the pentameric icosahedral structure of the virus. Here we report that the full-length VP1 structural protein of EV71 is capable of self-association. Dimerization of VP1 was tested using the yeast two-hybrid system, fluorescence resonance energy transfer (FRET) analysis, in vitro coupled transcription-translation binding assays, and mammalian cell transcription-translation experiments. Dimerization of various truncated versions of the VP1 protein was also studied by mutational analysis. Systematic deletions of parts of VP1 revealed that the region spanning amino acids 66-132 of VP1 contains the major dimerization domain. However, the region between amino acids 132 and 297 was indispensable, and contributed largely to increasing the strength of the interaction. This ability of EV71 VP1 to self-associate and to participate significantly in forming the characteristic icosahedral capsid strongly enhances the pathogenicity and stability of the virus to withstand the environment of the gastrointestinal tract.

Differential display (DD)-RT-PCR was employed to analyze mRNAs from ECV304 human endothelial-like cells undergoing apoptosis following infection with the virulent New Guinea C strain of dengue virus type 2 in order to elucidate the cellular gene responses to dengue viral infection at the transcriptional level. We isolated, sequenced, and identified 203 differentially expressed and overlapping cDNA fragments, all of which were of human origin except 1 that was of viral origin. Out of these, 78 were individual distinct clones comprising 46 and 32 expressed sequence tags (ESTs) that exhibited upregulated and downregulated trends, respectively. Of the 78 differentially expressed mRNAs, 16 did not match any characterized genes or ESTs. The remaining 62 mRNAs modified by dengue virus infection matched known genes, including those encoding components of the cell cycle (Anillin, CDC27), cytoskeleton (epsilon-tubulin), signal transduction (OPHN1, PPP2R2A, TIRAP), protein translation and modification (EIF3S10, IF2, TMEM1), transcriptional regulation (alpha-NAC, C20orf104, EGR1, ELP2), apoptotic cell death (RICK), membrane (BPAG1), and mitochondrial-related proteins. Semiquantitative RT-PCR and real-time RT-PCR authenticated further the altered expression patterns of selected genes of interest. These data demonstrate the feasibility of mRNA DD in providing insights into the complex responses of the transcriptional machinery of permissive and apoptotic human endothelial-like cells in the pathogenesis of dengue and/or its complications induced by the virulent dengue virus type 2.

Integrins are critical for initiating T‐cell activation events. The integrin‐binding motif Arg‐Gly‐Asp (RGD) was incorporated into the pcDNA 3.1 mammalian expression vector expressing the codon‐optimized extracellular domain of SARS coronavirus (SARS‐CoV) spike protein, and tested by immunizing C57BL/6 mice. Significant cell‐mediated immune responses were characterized by cytotoxic T‐lymphocyte 51Cr release assay and interferon‐gamma secretion ELISPOT assay against RMA‐S target cells presenting predicted MHC class I H2‐Kb epitopes, including those spanning residues 884–891 and 1116–1123 within the S2 subunit of SARS‐CoV spike protein. DNA vaccines incorporating the Spike‐RGD/His motif or the Spike‐His construct generated robust cell‐mediated immune responses. Moreover, the Spike‐His DNA vaccine construct generated a significant antibody response. Immunization with these DNA vaccine constructs elicited significant cellular and humoral immune responses. Additional T‐cell epitopes within the SARS‐CoV spike protein that may contribute to cell‐mediated immunity in vivo were also identified. J. Med. Virol. 81:1131–1139, 2009. © 2009 Wiley‐Liss, Inc.

During 1999-2000, a sustained mumps outbreak in the highly vaccinated population in Singapore was attributed to vaccine failure associated with the Rubini vaccine strain. To explain this phenomenon, the complete nucleotide and amino acid sequences of the hemagglutinin-neuraminidase (HN) gene of eight mumps virus isolates from patients with parotitis in Singapore were determined and compared with those of known vaccine strains. Phylogenetic trees constructed on the basis of HN nucleotide and amino acid sequences showed that the Singapore mumps virus isolates were more closely related to the Urabe strain and belonged to a different cluster from the Rubini and Jeryl-Lynn strains. The Rubini vaccine showed only approximately 93% nucleotide and approximately 96% amino acid sequence similarity to Urabe and Singapore isolates. Compared with the vaccine strains, six of the eight isolates lacked the extracellular glycosylation site at residues 400-402. Other significant amino acid disparities (e.g., at residue 354) may also affect the antigenic properties of the HN protein. These findings suggest that the evolution and adaptation of the currently circulating mumps virus strains in the community has led to the emergence of genetically distinct viral strains. The low vaccine efficacy of the Rubini strain represents a major reason for the recent mumps resurgence and failure of mumps immunization in Singapore.

The incidence of varicella in Singapore has been increasing since 1984. In 1991,17,930 cases were reported in a population of about 3 million. A serological survey completed in 1990 demonstrated that only 43% of the cohort had antibodies to varicella-zoster virus (VZV), indicating inadequate herd immunity. To exclude novel VZV strains, representative VZV isolates from 9 chicken pox and 4 zoster patients were characterised by restriction endonuclease analysis. DNAs were extracted from viral isolates propagated in MRC5 human embryo lung cells and were digested separately with Bg/ll, EcoRI, Pstl, Sall, and Xbal enzymes. The cleavage profiles of these VZV strains derived from both chicken pox and zoster lesions revealed no distinct differences. This observation implies that the current upsurge of chicken pox most likely stems from closely related VZV genotypes infecting a susceptible population with insufficient herd immunity. Comparison of the restriction fragments of the Singapore and the Dumas strains revealed polymorphisms of the Sal/I-D, SaI/l-E, and Xbal-l fragment lengths, which correlated with variable regions I, II, and Ill of the VZV genome, thereby representing geographically distinct genotypic variants of VZV. © 1993 Wiley-Liss, Inc.