The Upregulation of a Novel Long Noncoding RNA AK097647 Promotes Enterovirus 71 Replication and Decreases IFN-λ1 Secretion.

BACKGROUND: Enterovirus 71 (EV71) infects millions of children every year in China and has become a challenge to public health. However, there is no effective treatment for EV71 infection. Long noncoding RNAs (lncRNAs) have been found to play various roles in virus replication and infection. OBJECTIVE: We aimed to explore the role of a novel long noncoding RNA AK097647 (lncRNA-AK097647) during EV71 infection. METHODS: To assess the role of lncRNA-AK097647 during EV71 infection, siRNAs were used to silence lncRNA-K097647 expression. RT-qPCR assay and Western blotting were applied to measure the mRNA and protein levels of EV71 VP1 and the phosphorylation of NF-κB. ELISA was used to detect the level of IFN-λ1 expression. RESULTS: The novel lncRNA-AK097647 was upregulated in human rhabdomyosarcoma cells and the blood of hand, foot, and mouth disease patients infected with EV71, as demonstrated by RT-qPCR. Interestingly, RNAi-mediated knockdown of lncRNA-AK097647 dramatically increased the level of IFN-λ1 expression, resulting in the suppression of EV71 replication. In contrast, overexpression of lncRNA-AK097647 decreased the level of IFN-λ1 expression and resulted in increased EV71 replication. In addition, we found that lncRNA-AK097647 could inhibit the phosphorylation of NF-κB. CONCLUSION: These results suggest a novel mechanism by which EV71 evades the IFN-mediated host antiviral response by increasing lncRNA-AK097647 expression.

Practical application of Six Sigma management in analytical biochemistry processes in clinical settings.

BACKGROUND: Six Sigma methodology with a zero-defect goal has long been applied in commercial settings and was utilized in this study to assure/improve the quality of various analytes. METHODS: Daily internal quality control (QC) and external quality assessment data were collected and analyzed by calculating the sigma (σ) values for 19 analytes based on the coefficient of variation, bias, and total error allowable. Standardized QC sigma charts were established with these parameters. Quality goal index (QGI) analysis and root cause analysis (RCA) were used to discover potential problems for the analytes. RESULTS: Five analytes with σ ≥ 6 achieved world-class performance, and only the Westgard rule (13s ) with one control measurement at two QC material levels (N2) per QC event and a run size of 1000 patient samples between QC events (R1000) was needed for QC. In contrast, more control rules (22s /R4s /41s ) along with high N values and low R values were needed for quality assurance for five analytes with 4 ≤ σ < 6. However, the sigma levels of nine analytes were σ < 4 at one or more QC levels, and a more rigorous QC procedure (13s /22s /R4s /41s /8x with N4 and R45) was implemented. The combination of QGI analysis and RCA further revealed inaccuracy or imprecision problems for these analytes with σ < 4 and discovered five aspects of potential causes considered for quality improvement. CONCLUSIONS: Six Sigma methodology is an effective tool for evaluating the performance of biochemical analytes and is conducive to quality assurance and improvement.

Hsa-let-7c-5p augments enterovirus 71 replication through viral subversion of cell signaling in rhabdomyosarcoma cells.

BACKGROUND: Human enterovirus 71 (EV71) causes severe hand, foot and mouse disease, accompanied by neurological complications. During the interaction between EV71 and the host, the virus subverts host cell machinery for its own replication. However, the roles of microRNAs (miRNAs) in this process remain obscure. RESULTS: In this study, we found that the miRNA hsa-let-7c-5p was significantly upregulated in EV71-infected rhabdomyosarcoma cells. The overexpression of hsa-let-7c-5p promoted replication of the virus, and the hsa-let-7c-5p inhibitor suppressed viral replication. Furthermore, hsa-let-7c-5p targeted mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) and inhibited its expression. Interestingly, downregulation of MAP4K4 expression led to an increase in EV71 replication. In addition, MAP4K4 knockdown or transfection with the hsa-let-7c-5p mimic led to activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas the hsa-let-7c-5p inhibitor inhibited activation of this pathway. Moreover, EV71 infection promoted JNK pathway activation to facilitate viral replication. CONCLUSIONS: Our data suggested that hsa-let-7c-5p facilitated EV71 replication by inhibiting MAP4K4 expression, which might be related to subversion of the JNK pathway by the virus. These results may shed light on a novel mechanism underlying the defense of EV71 against cellular responses. In addition, these findings may facilitate the development of new antiviral strategies for use in future therapies.

Combination of intratypic and intertypic recombinant events in EV71: a novel evidence for the "triple-recombinant" strains of genotype A viruses in Mainland China from 2008 to 2010.

The first Enterovirus 71 (EV71) strain isolated in 1969 was classified as genotype A. It is interesting that the genotype A disappeared nearly 40 years until its re-emergence in mainland China in 2008-2010. Few studies on genetic characterization of the re-emerged genotype A viruses have been reported. In this study, a series of analyses were performed on molecular epidemiology and genome recombination of genotype A viruses in China. Phylogenetic analysis indicated that except for 17 reported genotype A strains and 3 orphan strains (C0, C3 and B5), almost all EV71 strains in mainland China were belonging to subgenotype C4 during 1987-2011. The subgenotype C4 was further divided into 3 clades C4a1, C4a2, and C4b. The genotype A viruses co-circulated with the predominant clade C4a2 and the re-emerged clade C4b both in eastern and central China in 2008-2009. Moreover, comprehensive recombination analysis showed that the genotype A viruses were "triple-recombinant" by combination of intratypic and intertypic recombination. Intertypic recombination between the oldest C4b strain (SHZH98) and Coxsackievirus A5 (CVA5) and intratypic recombination between the SHZH98 and C1 strains both with one junction in 5'-UTR were observed for some specific C4a2 strains and the re-emerged C4b strain, respectively. And intratypic recombination between the re-emerged C4b strain and the specific C4a2 strains with one junction in 5'-UTR was observed for the Chinese genotype A viruses. Taken together, these results provided potential explanations for the genesis of Chinese genotype A viruses which were significant for preventing and controlling outbreaks.

RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.

Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

miR-27a suppresses EV71 replication by directly targeting EGFR.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has broken out several times and was accompanied by neurological disease. microRNAs, a class of small non-coding RNAs that are approximately 20 nucleotides long, play important roles in the regulation of various biological processes, including antiviral defense. However, the roles of miRNAs in EV71 replication and pathogenesis are not well understood. In this study, we found that the expression of miR-27a was significantly decreased in EV71-infected cells. Interestingly, the over-expression of miR-27a could inhibit EV71 replication, as measured by virus titration, qPCR, and Western blotting. We identified EGFR mRNA is a bona fide target of miR-27a by computational analysis and luciferase reporter assays. Furthermore, miR-27a could decrease EGFR expression, as measured by qPCR and Western blotting. Moreover, the inhibition of EGFR expression by miR-27a decreased the phosphorylation of Akt and ERK, which facilitate EV71 replication. These results suggest that miR-27a may have antiviral activity against EV71 by inhibiting EGFR.

A novel finding for enterovirus virulence from the capsid protein VP1 of EV71 circulating in mainland China.

Enterovirus 71 (EV71) is a neurotropic virus that causes various clinical manifestations in young children, ranging from asymptomatic to fatal. Different pathotypes of EV71 notably differ in virulence. Several virulence determinants of EV71 have been predicted. However, these reported virulence determinants could not be used to identify the EV71 strains of subgenotype C4, which mainly circulate in China. In this study, VP1 sequences of 37 EV71 strains from severe cases (SC-EV71) and 192 EV71 strains from mild cases (MC-EV71) in mainland China were analyzed to determine the potential virulence determinants in the capsid protein VP1 of EV71. Although most SC-EV71 strains belonged to subgenotype C4a, no specific genetic lineages in C4a were correlated with EV71 virulence. Interestingly, amino acid substitutions at nine positions (H22Q, P27S, N31S/D, E98K, E145G/Q, D164E, T240A/S, V249I, and A289T) were detected by aligning the VP1 sequences of the SC-EV71 and MC-EV71 strains. Moreover, both the constituent ratios of the conservative or mutated residues in the MC-EV71 and SC-EV71 strains and the changes in the VP1 3D structure resulting from these mutations confirmed that the conservative residues (22H, 249V, and 289A) and the mutated residues (27S, 31S/D, 98K, 145G/Q, 164E, and 240A/S) might be potential virulence determinants in VP1 of EV71. Furthermore, these results led to the hypothesis that VP1 acts as a sandwich switch for viral particle stabilization and cellular receptors attachment, and specific mutations in this protein can convert mild cases into severe cases. These findings highlight new opportunities for diagnostic and therapeutic interventions.