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Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells

BACKGROUND: Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK)...

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Autores principales: Wang, Zhaoti, Duke, Gregory M
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2241602/
https://www.ncbi.nlm.nih.gov/pubmed/17956624
http://dx.doi.org/10.1186/1743-422X-4-102
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author Wang, Zhaoti
Duke, Gregory M
author_facet Wang, Zhaoti
Duke, Gregory M
author_sort Wang, Zhaoti
collection PubMed
description BACKGROUND: Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells. RESULTS: The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 10(6 )to 10(7 )plaque forming units per ml by 5 to 7 days post-transfection. CONCLUSION: This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.
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spelling pubmed-22416022008-02-13 Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells Wang, Zhaoti Duke, Gregory M Virol J Methodology BACKGROUND: Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells. RESULTS: The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 10(6 )to 10(7 )plaque forming units per ml by 5 to 7 days post-transfection. CONCLUSION: This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness. BioMed Central 2007-10-23 /pmc/articles/PMC2241602/ /pubmed/17956624 http://dx.doi.org/10.1186/1743-422X-4-102 Text en Copyright © 2007 Wang and Duke; 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
Wang, Zhaoti
Duke, Gregory M
Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
title Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
title_full Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
title_fullStr Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
title_full_unstemmed Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
title_short Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
title_sort cloning of the canine rna polymerase i promoter and establishment of reverse genetics for influenza a and b in mdck cells
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2241602/
https://www.ncbi.nlm.nih.gov/pubmed/17956624
http://dx.doi.org/10.1186/1743-422X-4-102
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