Dataset Information

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.

SUBMITTER: Singh S

PROVIDER: S-EPMC120643 | biostudies-literature | 2002 Aug

REPOSITORIES: biostudies-literature

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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.

Singh Sunita S Chow Vincent T K VT Phoon M C MC Chan K P KP Poh Chit Laa CL

Journal of clinical microbiology 20020801 8

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 di ...[more]

PMID: 12149336

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Project description:Enterovirus 71 (EV71) and coxsackieviruses (CV) are the major causative agents of hand, foot and mouth disease (HFMD). There is not currently a vaccine available against HFMD, even though a newly developed formalin-inactivated EV71 (FI-EV71) vaccine has been tested in clinical trial and has shown efficacy against EV71. We have designed and genetically engineered a recombinant adenovirus Ad-EVVLP with the EV71 P1 and 3CD genes inserted into the E1/E3-deleted adenoviral genome. Ad-EVVLP were produced in HEK-293A cells. In addition to Ad-EVVLP particles, virus-like particles (VLPs) formed from the physical association of EV71 capsid proteins, VP0, VP1, and VP3 expressed from P1 gene products. They were digested by 3CD protease and confirmed to be produced by Ad-EVVLP-producing cells, as determined using transmission electron microscopy and western blotting. Mouse immunogenicity studies showed that Ad-EVVLP-immunized antisera neutralized the EV71 B4 and C2 genotypes. Activation of VLP-specific CD4+ and CD8+/IFN-? T cells associated with Th1/Th2-balanced IFN-?, IL-17, IL-4, and IL-13 was induced; in contrast, FI-EV71 induced only Th2-mediated neutralizing antibody against EV71 and low VLP-specific CD4+ and CD8+ T cell responses. The antiviral immunity against EV71 was clearly demonstrated in mice vaccinated with Ad-EVVLP in a hSCARB2 transgenic (hSCARB2-Tg) mouse challenge model. Ad-EVVLP-vaccinated mice were 100% protected and demonstrated reduced viral load in both the CNS and muscle tissues. Ad-EVVLP successfully induced anti-CVA16 immunities. Although antisera had no neutralizing activity against CVA16, the 3C-specific CD4+ and CD8+/IFN-? T cells were identified, which could mediate protection against CVA16 challenge. FI-EV71 did not induce 3C-mediated immunity and had no efficacy against the CVA16 challenge. These results suggest that Ad-EVVLP can enhance neutralizing antibody and protective cellular immune responses to prevent EV71 infection and cellular immune responses against CV infection.

Dataset Information

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.

Publications

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.

Similar Datasets

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