Differential expression of miRNAs in enterovirus 71-infected cells.

BACKGROUND: Enterovirus 71 (EV71) is one of the major etiological pathogens of hand, foot and mouth disease (HFMD) and can cause severe cerebral and pulmonary complications and even fatality. MicroRNAs (miRNAs), a class of small non-coding RNA molecules, play an important role in post-transcriptional regulation of gene expression and thereby influencing various physiological and pathological processes. Increasing evidence suggests that miRNAs act as key effector molecules in the complicated pathogen-host interactions. However, the roles of miRNAs in EV71 infection and pathogenesis are not well understood. METHODS: To identify special miRNAs involved in EV71 infection, a microarray assay was performed to study the expression pattern of miRNAs in EV71-infected human rhabdomyosarcoma cells (RD cells) and uninfected RD cells. We further predicted the putative target genes for the dysregulated miRNAs using the online bioinformatic algorithms (TargetScan, miRanda and PicTar) and carried out functional annotation including GO enrichment and KEGG pathway analysis for miRNA predicted targets. Then, the results of microarray were further confirmed by quantitative RT-PCR. RESULTS: Totally, 45 differentially expressed miRNAs ware identified by microarray, among which 36 miRNAs were up-regulated and 9 were down-regulated. 7166 predicted target genes for the dysregulated miRNAs were revealed by using TargetScan in conjunction with miRanda and PicTar. The GO annotation suggested that predicted targets of miRNAs were enriched into the category of signal transduction, regulation of transcription, metabolic process, protein phosphorylation, apoptotic process and immune response. KEGG pathway analysis suggested that these predicted target genes were involved in many important pathways, mainly including endocytosis and focal adhesion, MAPK signaling pathway, hypertrophic cardiomyopathy, melanogenesis and ErbB signaling pathway. The expression levels of 8 most differentially up-regulated miRNAs and 3 most differentially down-regulated miRNAs were confirmed by qRT-PCR. The expressions of hsa-miR-4530, hsa-miR-4492, hsa-miR-6125, hsa-miR-494-3p, hsa-miR-638, hsa-miR-6743-5p, hsa-miR-4459 and hsa-miR-4443 detected by qRT-PCR were consistent with the microarray data. CONCLUSION: These results might extend our understanding to the regulatory mechanism of miRNAs underlying the pathogenesis of EV71 infection, thus strengthening the preventative and therapeutic strategies of HFMD caused by EV71.

The highly conserved 5' untranslated region as an effective target towards the inhibition of Enterovirus 71 replication by unmodified and appropriate 2'-modified siRNAs.

BACKGROUND: Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71. METHODS: Unmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 2'-modified (2'-O-methylation or 2'-fluoro modification) siRNAs were designed to target highly conserved 5' untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs. RESULTS: Transfection of rhabdomyosarcoma (RD) cells with siRNAs targeting the EV71 genomic 5' UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2'-modified siRNAs exhibited similar RNA interference (RNAi) activity with dramatically increased serum stability in comparison with unmodified counterparts. CONCLUSION: Sequences were identified within the highly conserved 5' UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2'-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.

[Association between hand-foot-and-mouth disease in Xi'an and enterovirus 71].

OBJECTIVE: To isolate the prevalent strain of enterovirus 71 (EV71) in Xi'an area in 2008, and compare the concordance of viral isolation, reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent technique in detecting EV71, find the fast and effective method for detection, and analyze the differences between the EV71 strains isolated from Xi'an and Fuyang, Anhui. METHOD: Virus isolation and RT-PCR were carried out on vesicle fluid and throat swab specimens that were collected from the patients with hand-foot-and-mouth disease, RD and HEp-2 cell lines were used for viral isolation. The virus was identified by using immunofluorescence technique. Nucleotide sequencing was performed on positive product of RT-PCR, and compared with EV71 isolated from Fuyang in 2008, then submitted to Genbank. RESULT: Among the 56 samples of throat swab inoculated on RD and HEp-2 cells, the positive rates were 5.4% (3/56) and 1.8% (1/56), respectively. Among the 56 samples of vesicle fluid inoculated on RD and HEp-2 cells, the positive rates were 12.5% ( 7/56 ) and 5.4% (3/56), respectively. Cytopathic effect of RD and HEp-2 cells appeared on days 7 and 10, respectively. The positive rates of RT-PCR on throat swab and vesicle fluid samples were 21.4% (12/56) and 33.9% (19/56), respectively. Cytopathic effect was found in cell culture for 14 cases and immunofluorescence, showed that 9 of them were infected with EV71. The authors obtained the EV71 strain prevalent in Xi'an during 2008. The nucleotide sequence was submitted to the NCBI Genbank and gained the accession number EU812461. CONCLUSION: The EV71 in Xi'an prevalent during 2008 may have a weaker epithelial tropism. Comparison of the EV71 strain isolated from Xi'an with EU703812, EU703813 and EU703814 isolated from Fuyang, Anhui showed that the homology was 97%-98%. RT-PCR is an important method for rapid detection of EV71.