Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Feb 17;11(2):e0149470.
doi: 10.1371/journal.pone.0149470. eCollection 2016.

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Haibing Liu  1 , Yanyan Qin  2 , Zhenzhen Kong  2 , Qixiang Shao  2 , Zhaoliang Su  2 , Shengjun Wang  1 , Jianguo Chen  1
Affiliations

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Haibing Liu et al. PLoS One. .

Abstract

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2Apro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID50) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT-PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2Apro region of the EV71 genome exerted antiviral effects in vitro.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Transfection efficiency of siRNA in RD cells.
(A) Cellular distribution of BLOCK-iT Fluorescent Oligo in transfected RD cells. RD cells were transfected with different concentrations of BLOCK-iT Fluorescent Oligo and 2 μl of Lipofectamine 2000. At 24 h after transfection, the cells were observed under a fluorescence microscope. (B) RD cells transfected with BLOCK-iT Fluorescent Oligo were quantified with flow cytometry. The cells were assayed in three independent experiments.
Fig 2
Fig 2. siRNAs protect cells against EV71-induced cytopathic effects.
Morphological changes in RD cells were observed after infection. Cells were transfected with each siRNA at a final concentration of 60 nM and then infected with strain EV71-2006-52-9 at an MOI of 0.01. Micrographs were taken at 48 h postinfection under an inverted microscope. The tests were performed in three independent experiments.
Fig 3
Fig 3. Cell viability assay.
Viability was determined with a MTT assay at 48 h postinfection. The cell viability of each group is expressed relative to the normal RD cell control, which was defined as 100% survival. The data shown represent the means ± SD of three independent experiments (**p < 0.01).
Fig 4
Fig 4. Viral titer assay.
Virus titers were determined in term of TCID50. RD cells pretreated with siRNAs were infected with strain EV71-2006-52-9 at an MOI of 0.01. At 48 h postinfection, the supernatants were collected to detect the progeny viral titers. Data are the means ± SD of three independent experiments (**p < 0.01).
Fig 5
Fig 5. RNAi inhibits viral RNA.
RD cells were treated with each siRNA and then infected with strain EV71-2006-52-9 at an MOI of 0.01. At 24 h postinfection, the viral RNA was extracted and analyzed with real-time RT–PCR. The data shown represent the means ± SD of three independent experiments (**p < 0.01).
Fig 6
Fig 6. RNAi inhibits viral protein.
RD cells transfected with siRNA were infected with strain EV71-2006-52-9 at an MOI of 0.01. At 36 h postinfection, total protein was extracted and analyzed with western blotting. β-Actin was used as the internal loading control. The protein measurements were made in three independent experiments.
Fig 7
Fig 7. RNAi inhibits the replication of different EV71 strains.
RD cells transfected with siRNA were infected with strain EV71-2008-43-16 at an MOI of 0.01. Total protein was extracted at 36 h after infection, and VP1 expression was analyzed with western blotting. β-Actin was used as the internal loading control. Protein measurements were made in three independent experiments.
Fig 8
Fig 8. Interferon pathway was not activated by siRNA.
The levels of IFN-α (A) and IFN-β (B) were determined by measuring the absorbance at 490 nm. The data shown represent the means ± SD of three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Shih SR, Stollar V, Li ML. Host Factors in Enterovirus 71 Replication. Journal of Virology. 2011;85(19):9658–66. 10.1128/jvi.05063-11 - DOI - PMC - PubMed
    1. Yip CCY, Lau SKP, Woo PCY, Yuen K-Y. Human enterovirus 71 epidemics: what's next? Emerging Health Threats Journal. 2013;6(0). 10.3402/ehtj.v6i0.19780 - DOI - PMC - PubMed
    1. Wang Y, Zou G, Xia A, Wang X, Cai J, Gao Q, et al. Enterovirus 71 infection in children with hand, foot, and mouth disease in Shanghai, China: epidemiology, clinical feature and diagnosis. Virology Journal. 2015;12(1):83 10.1186/s12985-015-0308-2 - DOI - PMC - PubMed
    1. Wang S-M, Lei H-Y, Liu C-C. Cytokine Immunopathogenesis of Enterovirus 71 Brain Stem Encephalitis. Clinical and Developmental Immunology. 2012;2012:1–8. 10.1155/2012/876241 - DOI - PMC - PubMed
    1. Tu Y-F, Lin C-H, Lee H-T, Yan J-J, Sze C-I, Chou Y-P, et al. Elevated cerebrospinal fluid endothelin 1 associated with neurogenic pulmonary edema in children with enterovirus 71 encephalitis. International Journal of Infectious Diseases. 2015;34:105–11. 10.1016/j.ijid.2015.03.017 - DOI - PubMed

Grants and funding

The authors have no support or funding to report.

LinkOut - more resources