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Producing infectious enterovirus type 71 in a rapid strategy

BACKGROUND: Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-str...

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Autores principales: Han, Jian-Feng, Cao, Rui-Yuan, Tian, Xue, Yu, Man, Qin, E-De, Qin, Cheng-Feng
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892457/
https://www.ncbi.nlm.nih.gov/pubmed/20525351
http://dx.doi.org/10.1186/1743-422X-7-116
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author Han, Jian-Feng
Cao, Rui-Yuan
Tian, Xue
Yu, Man
Qin, E-De
Qin, Cheng-Feng
author_facet Han, Jian-Feng
Cao, Rui-Yuan
Tian, Xue
Yu, Man
Qin, E-De
Qin, Cheng-Feng
author_sort Han, Jian-Feng
collection PubMed
description BACKGROUND: Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF), which is flanked by untranslated regions at both the 5' and 3' ends. RESULTS: A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA) assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice. CONCLUSIONS: Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method.
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spelling pubmed-28924572010-06-26 Producing infectious enterovirus type 71 in a rapid strategy Han, Jian-Feng Cao, Rui-Yuan Tian, Xue Yu, Man Qin, E-De Qin, Cheng-Feng Virol J Methodology BACKGROUND: Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF), which is flanked by untranslated regions at both the 5' and 3' ends. RESULTS: A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA) assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice. CONCLUSIONS: Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method. BioMed Central 2010-06-04 /pmc/articles/PMC2892457/ /pubmed/20525351 http://dx.doi.org/10.1186/1743-422X-7-116 Text en Copyright ©2010 Han et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology
Han, Jian-Feng
Cao, Rui-Yuan
Tian, Xue
Yu, Man
Qin, E-De
Qin, Cheng-Feng
Producing infectious enterovirus type 71 in a rapid strategy
title Producing infectious enterovirus type 71 in a rapid strategy
title_full Producing infectious enterovirus type 71 in a rapid strategy
title_fullStr Producing infectious enterovirus type 71 in a rapid strategy
title_full_unstemmed Producing infectious enterovirus type 71 in a rapid strategy
title_short Producing infectious enterovirus type 71 in a rapid strategy
title_sort producing infectious enterovirus type 71 in a rapid strategy
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892457/
https://www.ncbi.nlm.nih.gov/pubmed/20525351
http://dx.doi.org/10.1186/1743-422X-7-116
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