Dataset Information

Molecular analysis of virulent determinants of enterovirus 71.

ABSTRACT: Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5'-NTR and 3'-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (Gly(P710)/Gln(P710)/Arg(P710) and Glu(P729)) on the DE and EF loop of VP1, one (Lys(P930)) on the surface of protease 2A and four nucleotides on three positions (G(P272), U(P488) and A(P700)/U(P700)) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the A(P700)/U(P700) was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR.

SUBMITTER: Li R

PROVIDER: S-EPMC3198388 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Molecular analysis of virulent determinants of enterovirus 71.

Li Renqing R Zou Qinghua Q Chen Lijuan L Zhang Herun H Wang Yumei Y

PloS one 20111019 10

Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5'-NTR and 3'-NTR in 25 SC-EV71 strains (strains from severe cases) and ...[more]

PMID: 22039449

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Project description:We have previously shown that Heparin (Hep) significantly inhibited Enterovirus 71 (EV71) infection and binding in both Vero and a human neural cell line, SK-N-SH, in vitro. Therefore, in this study we intended to gain insight into the cellular and molecular mechanisms of action of Hep against clinical EV71 infection in SK-N-SH cells. Instead of stating a long list of gene functions and pathways, we tried to select for EV71-induced genes that were exclusively affected by antiviral activity of Hep through a multi-level comparison and characterization. Overall, our microarray analysis may suggest that Hep might exert its anti-EV71 activity in SK-N-SH cells, at least in part, through the following mechanisms: i. Reduction of the down-regulation effect of EV71 on TOX that would lead to an increased population of effective, mature T-cells and NK-cells; ii. Reduction of the up-regulation effect of EV71 on GIP, that in turn, would reduce recruiting different GPCRs, leading to a reduced viral infection in host cells; iii. Partially neutralization of the EV71-mediated apoptosis through expression of CARP2 that acts as an anti-apoptosis ubiquitin protein ligase (E3). EV71 is a neurovirological virus that can sometimes cause severe and fatal CNS complications in infected patients. Since still there is no approved drug for prophylaxis of EV71-casued disease, discovering a molecular drug target(s) for EV71 infection would be crucial. This was the first study to report direct assessment of mechanisms of action of antivirals against EV71 infection. SK-N-SH cells were infected with a clinical EV71 isolate followed by treatment with 125 µg/mL of Hep. At 72 hours post infection, antiviral activity and cytotoxicity of Hep at 125 µg/mL in 12-well plates were carried out at the same time as RNA extraction. This way, we could ensure that we would assess transcript profiles of the host cells under the same condition and time as assessment of antiviral activity and cytotoxicity for the same replicates. Changes in expression profiles of the host cells were comparatively assessed under four conditions: cell control (neither infection nor treatment, designated CC), treated only with Hep (compound control, designated Cyto), EV71-infected cells treated with Hep (treatment, designated H), and infected with EV71 without treatment with Hep (virus control, designated V). All experiments were applied in triplicate, and totally twelve GeneChip® Human Gene 1.0 ST arrays were purchased from Affymetrix and processed. Then, the following five contrasts were made: Hep vs. CC; VC vs. CC; Cyto vs CC; Hep vs. VC; and Hep vs. Cyto. For each contrast, only samples from the two target groups were included. The statistical parameters of ANOVAs, p values, multiple test corrections, and fold changes were calculated within each contrast. Then, a multi-level selection and analysis procedure was employed in order to attribute changes in the gene transcription level to antiviral activity of Hep.

Project description:We have previously shown that Heparin (Hep) significantly inhibited Enterovirus 71 (EV71) infection and binding in both Vero and a human neural cell line, SK-N-SH, in vitro. Therefore, in this study we intended to gain insight into the cellular and molecular mechanisms of action of Hep against clinical EV71 infection in neural cells. Instead of stating a long list of gene functions and pathways, we tried to select for EV71-induced genes that were exclusively affected by antiviral activity of Hep through a multi-level comparison and characterization. Overall, the findings of this study suggest several molecular targets by which Hep might exert its antiviral activity against clinical EV71 infection in neural cells. Mostly, Hep appeared to modulate induction effect of EV71 on several genes in a way that may benefit the host cell and inhibit the viral infection. The most important genes where expression patterns were significantly changed by Hep include genes related to; (i) cell growth, (ii) DNA repair and replication, (iii) cytoplasmic microfilaments and related apoptosis, (iv) regulation of energy metabolism and mitochondrion-related apoptosis, (v) cytoplasmic viral transport, (vi) transmembrane proteins and the related signaling pathways, (vii) phosphoribosyltransferase, (viii) transcription factors, (ix) G protein-coupled receptors and G protein-coupled receptors-interacting proteins, (x) remodeling actin filament assembly, (xi) chemokin receptors, and (xii) immunosuppression. In addition, Hep sometimes appeared to be harmful for cells, like the case in that Hep likely suppresses regulation of Metallothionein 1E. Furthermore, it should not be ignored that the Hep-caused affects seen in this microarray analysis may partially be attributed to a significant inhibitory effect of Hep on EV71 entry into the cells. In conclusion, we propose that our microarray findings may suggest new directions for further studies on molecular targets of anti-EV71 activity of Hep. EV71 is a neurovirological virus that can cause severe and fatal CNS complications in infected patients. There is no approved drug for prophylaxis of EV71-casued disease and discovering a molecular drug target(s) for EV71 infection would be beneficial. The microarray analysis reported here was a large-scale microarray pilot study and thus further confirmatory experiments such as real time RT PCR and Western blotting would be warranted in order to confirm the mode of action of Hep implied here.

Dataset Information

Molecular analysis of virulent determinants of enterovirus 71.

Publications

Molecular analysis of virulent determinants of enterovirus 71.

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