scholarly journals Establishment of Canine RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus: Its Application for H5N1 Vaccine Production

2007 ◽  
Vol 82 (3) ◽  
pp. 1605-1609 ◽  
Author(s):  
Shin Murakami ◽  
Taisuke Horimoto ◽  
Shinya Yamada ◽  
Satoshi Kakugawa ◽  
Hideo Goto ◽  
...  
Keyword(s):  
Cell Line ◽  
Influenza A ◽  
Reverse Genetics ◽  
Current System ◽  
Influenza Pandemic ◽  
Promoter Sequence ◽  
Vero Cells ◽  
Vaccine Production ◽  
Polymerase I ◽  
H5n1 Vaccine

ABSTRACT In the event of a new influenza pandemic, vaccines whose antigenicities match those of circulating strains must be rapidly produced. Here, we established an alternative reverse genetics system for influenza virus using the canine polymerase I (PolI) promoter sequence that works efficiently in the Madin-Darby canine kidney cell line, a cell line approved for human vaccine production. Using this system, we were able to generate H5N1 vaccine seed viruses more efficiently than can be achieved with the current system that uses the human PolI promoter in African green monkey Vero cells, thus improving pandemic vaccine production.

scholarly journals Establishment of Vero cell RNA polymerase I-driven reverse genetics for Influenza A virus and its application for pandemic (H1N1) 2009 influenza virus vaccine production

2013 ◽  
Vol 94 (6) ◽  
pp. 1230-1235 ◽  
Author(s):  
Min-Suk Song ◽  
Yun Hee Baek ◽  
Philippe Noriel Q. Pascua ◽  
Hyeok-il Kwon ◽  
Su-Jin Park ◽  
...  
Keyword(s):  
Vero Cell ◽  
Influenza A ◽  
Reverse Genetics ◽  
Vero Cells ◽  
Influenza Virus Vaccine ◽  
Influenza Viruses ◽  
Vaccine Production ◽  
Influenza A Viruses ◽  
Alternative Means ◽  
Polymerase I

The constant threat of newly emerging influenza viruses with pandemic potential requires the need for prompt vaccine production. Here, we utilized the Vero cell polymerase I (PolI) promoter, rather than the commonly used human PolI promoter, in an established reverse-genetics system to rescue viable influenza viruses in Vero cells, an approved cell line for human vaccine production. The Vero PolI promoter was more efficient in Vero cells and demonstrated enhanced transcription levels and virus rescue rates commensurate with that of the human RNA PolI promoter in 293T cells. These results appeared to be associated with more efficient generation of A(H1N1)pdm09- and H5N1-derived vaccine seed viruses in Vero cells, whilst the rescue rates in 293T cells were comparable. Our study provides an alternative means for improving vaccine preparation by using a novel reverse-genetics system for generating influenza A viruses.

scholarly journals Rescue of Influenza A Virus from Recombinant DNA

1999 ◽  
Vol 73 (11) ◽  
pp. 9679-9682 ◽  
Author(s):  
Ervin Fodor ◽  
Louise Devenish ◽  
Othmar G. Engelhardt ◽  
Peter Palese ◽  
George G. Brownlee ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Recombinant Dna ◽  
Vero Cells ◽  
Influenza Viruses ◽  
Polymerase I ◽  
Negative Sense ◽  
Expression Plasmids ◽  
Delta Virus

ABSTRACT We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.

scholarly journals Human RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus in Canine Cells

2010 ◽  
Vol 84 (7) ◽  
pp. 3721-3725 ◽  
Author(s):  
Pirada Suphaphiphat ◽  
Bjoern Keiner ◽  
Heidi Trusheim ◽  
Stefania Crotta ◽  
Annunziata Barbara Tuccino ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Rna Polymerase ◽  
Cell Line ◽  
Influenza A ◽  
Reverse Genetics ◽  
Rna Polymerase I ◽  
Influenza A Viruses ◽  
Pol I ◽  
Influenza Vaccine Production ◽  
Polymerase I

ABSTRACT We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a nonendogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.

scholarly journals Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells

2005 ◽  
Vol 79 (21) ◽  
pp. 13811-13816 ◽  
Author(s):  
Pascale Massin ◽  
Pierre Rodrigues ◽  
Monica Marasescu ◽  
Sylvie van der Werf ◽  
Nadia Naffakh
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Promoter Sequence ◽  
Influenza Viruses ◽  
Rna Polymerase I ◽  
Influenza A Viruses ◽  
Virus Research ◽  
Influenza Vaccine Production ◽  
Polymerase I

ABSTRACT Reverse genetics techniques to rescue influenza viruses have thus far been based on the use of a human polymerase I (PolI) promoter to direct the synthesis of the eight viral RNAs. They can only be used on cells from primate origin due to the species specificity of the PolI promoter. Here we report the cloning of the chicken PolI promoter sequence and the generation of recombinant influenza virus upon transfection of bidirectional PolI/PolII plasmids in avian cells. Potential contributions of this new reverse genetics system in the fields of influenza virus research and influenza vaccine production are discussed.

scholarly journals Growth Determinants for H5N1 Influenza Vaccine Seed Viruses in MDCK Cells

2008 ◽  
Vol 82 (21) ◽  
pp. 10502-10509 ◽  
Author(s):  
Shin Murakami ◽  
Taisuke Horimoto ◽  
Le Quynh Mai ◽  
Chairul A. Nidom ◽  
Hualan Chen ◽  
...  
Keyword(s):  
Cell Line ◽  
Influenza A ◽  
Mdck Cells ◽  
Influenza Pandemic ◽  
Influenza Viruses ◽  
Vaccine Production ◽  
Efficient Manner ◽  
Short Supply ◽  
Promising Alternative ◽  
H5n1 Influenza

ABSTRACT H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.

Reactogenicity and safety of AS03B-adjuvanted H5N1 influenza vaccine in children: an open-label, one-way, crossover trial

2018 ◽  
Vol 11 (4) ◽  
pp. 359-364
Author(s):  
Patricia Izurieta ◽  
Pope Kosalaraksa ◽  
Louise Frenette ◽  
Mamadou Dramé ◽  
Bruce L. Innis ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Pandemic ◽  
The United States ◽  
World Health ◽  
Open Label ◽  
Eligibility Criteria ◽  
Clinical Trial Registration ◽  
Post Vaccination ◽  
H5n1 Influenza ◽  
H5n1 Vaccine

AbstractBackgroundHuman cases of highly pathogenic avian-origin influenza A/H5N1 infection continue to be reported to the World Health Organization, and recent outbreaks of human cases of other zoonotic influenza strains highlight the continued need for strategies to mitigate influenza pandemic potential.MethodsA Phase II–III randomized, placebo-controlled, observer-blind trial was conducted to assess the immunogenicity, reactogenicity, and safety of two 1.9 μg hemagglutinin doses of AS03B-adjuvanted H5N1 (AS03B-H5N1; A/Indonesia) vaccine in children (6 months to <18 years old) of Thailand, the United States, and Canada (Year 1, published elsewhere). After database lock in Year 1, the trial was unblinded, and children who had been randomized to receive placebo and continued to fulfill the eligibility criteria were invited to participate in an open-label, one-way, crossover safety extension phase, in which they received AS03B-H5N1 vaccine. Here we report the safety analysis in Year 2.ResultsA total of 155 children were vaccinated in Year 2. The most frequent solicited adverse event (AE) during 7 days post vaccination was injection site pain. Irritability or fussiness was reported in about one-third of younger children (aged <6 years) during 7 days post vaccination and was the most common solicited general AE in this age group. Postvaccination temperature (≥38°C) was reported in 4 (5.1%) children. The most common solicited general AEs in older children (aged ≥6 years) were muscle aches, headache, and fatigue. The AS03B-H5N1 vaccine had a clinically acceptable safety profile up to 385 days post vaccination.ConclusionsSafety in the crossover phase was acceptable and consistent with that observed in vaccine recipients in the randomized, blinded phase of the study.Clinical trial registrationClinicalTrials.gov: NCT01310413.

scholarly journals A reverse-genetics system for Influenza A virus using T7 RNA polymerase

2007 ◽  
Vol 88 (4) ◽  
pp. 1281-1287 ◽  
Author(s):  
Emmie de Wit ◽  
Monique I. J. Spronken ◽  
Gaby Vervaet ◽  
Guus F. Rimmelzwaan ◽  
Albert D. M. E. Osterhaus ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Influenza Virus A ◽  
Sense Orientation ◽  
Polymerase I ◽  
Rna Polymerase Promoter

The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.

scholarly journals Unidirectional RNA polymerase I–polymerase II transcription system for the generation of influenza A virus from eight plasmids

2000 ◽  
Vol 81 (12) ◽  
pp. 2843-2847 ◽  
Author(s):  
Erich Hoffmann ◽  
Robert G. Webster
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
National Academy Of Sciences ◽  
Transcription System ◽  
Pol I ◽  
Positive Sense ◽  
Polymerase I ◽  
Academy Of Sciences ◽  
Negative Sense

Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and positive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108–6113, 2000). Here we report the establishment of a different transcription system for the expression of virus-like RNAs, allowing the intracellular synthesis of noncapped positive-sense cRNA and 5′-capped mRNA from one template. Cotransfection of eight RNA pol I–pol II tandem promoter plasmids containing the cDNA of A/WSN/33 (H1N1) resulted in the generation of infectious influenza A virus, albeit with a lower yield than the bidirectional system. Our approach of producing either vRNA and mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids should be useful for the establishment or optimization of reverse genetics systems for other RNA viruses.

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