Abstract
Enterovirus 71 (EV71) belongs to the genus Enterovirus within the family Picornaviridae. In the past three decades, there are several reported EV71 outbreaks in the world, including Brazil, Bulgaria, Hungary, Japan, Malaysia, Singapore, Taiwan, and USA. Most patients infected with EV71 only exhibited mild clinical symptoms, such as hand-foot-and-mouth diseases (HFMD) and pharyngitis. However, acute meningitis, encephalitis, encephalomyelitis, polio-like syndrome, and even fatal cases might be caused by EV71 infection in some severe cases. Although the neurological damage is a critical cause for severe clinical syndromes, the underlying molecular mechanism that regulates the EV71-triggered neuronal cell death remains unclear. Herein, we show that Cdk5, a critical signaling effector of various neurotoxic insults in the brain, is activated by EV71 infection of neuronal cells. EV71-induced neuronal apoptosis could be effectively repressed by blocking either Cdk5 kinase activity or its protein expression. Moreover, EV71-induced Cdk5 activation was modulated by c-Abl. The suppression of c-Abl kinase activity by STI571 notably repressed both the Cdk5 activation and neuronal apoptosis in cells infected with EV71. Although EV71 also induces apoptosis in non-neuronal cells, it did not affect Abl and Cdk5 activities in several non-neuronal cell lines. Intriguingly, coxsackievirus A16 (CA16), a genetically closely related serotype to EV71 that usually does not induce severe neurological disorders, could only weakly stimulate Abl, but not Cdk5 kinase activity. Taken together, our data suggests a serotype- and cell type-specific mechanism, by which EV71 induces Abl kinase activity, which in turn triggers Cdk5-signaling for neuronal apoptosis. Despite the close genetic relationship between EV71 and CA16, 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 specimens remain unclassifiable due to the limited number of serotype-specific antibodies against enterovirus surface proteins. Array-based assay is able to simultaneously detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex RT-PCR (MRT-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 glass slides. Using this enterovirus microarray to study 144 clinical specimens of HFMD or suspected cases, 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%. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.