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 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 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 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 Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine

2012 ◽  
Vol 93 (10) ◽  
pp. 2204-2214 ◽  
Author(s):  
Lindomar Pena ◽  
Amy L. Vincent ◽  
Crystal L. Loving ◽  
Jamie N. Henningson ◽  
Kelly M. Lager ◽  
...  
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Viruses ◽  
Dependent Manner ◽  
Lung Pathology ◽  
Influenza A Viruses ◽  
Virus Shedding ◽  
The Impact ◽  
Strain Dependent

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

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 Characterization of a porcine intestinal epithelial cell line for influenza virus production

2012 ◽  
Vol 93 (9) ◽  
pp. 2008-2016 ◽  
Author(s):  
Zhi Sun ◽  
Victor C. Huber ◽  
Kara McCormick ◽  
Radhey S. Kaushik ◽  
Adrianus C. M. Boon ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Epithelial Cell Line ◽  
Influenza B ◽  
Specific Cell ◽  
Influenza A Viruses

We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log10 TCID50 ml−1. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin–Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 ml−1, respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.

scholarly journals Role of Quail in the Interspecies Transmission of H9 Influenza A Viruses: Molecular Changes on HA That Correspond to Adaptation from Ducks to Chickens

2003 ◽  
Vol 77 (5) ◽  
pp. 3148-3156 ◽  
Author(s):  
Daniel R. Perez ◽  
Wilina Lim ◽  
Jon P. Seiler ◽  
Guan Yi ◽  
Malik Peiris ◽  
...  
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Viruses ◽  
Interspecies Transmission ◽  
Influenza A Viruses ◽  
Species Barrier ◽  
Ha Gene ◽  
Novel Variants ◽  
Efficient Replication

ABSTRACT H9 influenza viruses have become endemic in land-based domestic poultry in Asia and have sporadically crossed to pigs and humans. To understand the molecular determinants of their adaptation to land-based birds, we tested the replication and transmission of several 1970s duck H9 viruses in chickens and quail. Quail were more susceptible than chickens to these viruses, and generation of recombinant H9 viruses by reverse genetics showed that changes in the HA gene are sufficient to initiate efficient replication and transmission in quail. Seven amino acid positions on the HA molecule corresponded to adaptation to land-based birds. In quail H9 viruses, the pattern of amino acids at these seven positions is intermediate between those of duck and chicken viruses; this fact may explain the susceptibility of quail to duck H9 viruses. Our findings suggest that quail provide an environment in which the adaptation of influenza viruses from ducks generates novel variants that can cross the species barrier.

scholarly journals Interpretation of responses and protective levels of antibody against attenuated influenza A viruses using single radial haemolysis

1984 ◽  
Vol 93 (2) ◽  
pp. 301-312 ◽  
Author(s):  
R. Al-Khayatt ◽  
R. Jennings ◽  
C. W. Potter
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Haemagglutination Inhibition ◽  
Influenza Virus Vaccine ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
H1n1 Vaccine ◽  
Antibody Titres ◽  
Attenuated Viruses ◽  
Virus Strains

SummaryAntibody determinations against H3N2 and H1N1 type A influenza viruses were carried out on paired sera obtained from volunteers taking part in influenza virus vaccine studies, using both the haemagglutination-inhibition (HI) and single radial haemolysis (SRH) test. Good correlation between the HI and SRH test was found for both H3N2 and H1N1 antibody and the zone area increases corresponding to significant SRH antibody rises determined for both virus strains. In both H3N2 and H1N1 vaccine studies, intranasal infection of the volunteers with live attenuated viruses was involved and by the measurement of HI and SRH antibodies prior to and following infection, levels of antibody equating with protection against the infecting viruses could be estimated. For the HI test the antibody titres associated with 50% protection were 42 for H1N1, and 44 for H3N2 viruses; for the SRH test, 50% protection was associated with zone areas of 20·0–25·0 mm2for both H1N1 and H3N2 viruses.

scholarly journals Generation of High-Yielding Influenza A Viruses in African Green Monkey Kidney (Vero) Cells by Reverse Genetics

2004 ◽  
Vol 78 (4) ◽  
pp. 1851-1857 ◽  
Author(s):  
Hiroichi Ozaki ◽  
Elena A. Govorkova ◽  
Chenghong Li ◽  
Xiaoping Xiong ◽  
Robert G. Webster ◽  
...  
Keyword(s):  
Growth Kinetics ◽  
Influenza A ◽  
Reverse Genetics ◽  
Vero Cells ◽  
African Green Monkey ◽  
Monkey Kidney ◽  
Influenza A Viruses ◽  
Poor Growth ◽  
African Green Monkey Kidney ◽  
Green Monkey

ABSTRACT Influenza A viruses are the cause of annual epidemics of human disease with occasional outbreaks of pandemic proportions. The zoonotic nature of the disease and the vast viral reservoirs in the aquatic birds of the world mean that influenza will not easily be eradicated and that vaccines will continue to be needed. Recent technological advances in reverse genetics methods and limitations of the conventional production of vaccines by using eggs have led to a push to develop cell-based strategies to produce influenza vaccine. Although cell-based systems are being developed, barriers remain that need to be overcome if the potential of these systems is to be fully realized. These barriers include, but are not limited to, potentially poor reproducibility of viral rescue with reverse genetics systems and poor growth kinetics and yields. In this study we present a modified A/Puerto Rico/8/34 (PR8) influenza virus master strain that has improved viral rescue and growth properties in the African green monkey kidney cell line, Vero. The improved properties were mediated by the substitution of the PR8 NS gene for that of a Vero-adapted reassortant virus. The Vero growth kinetics of viruses with H1N1, H3N2, H6N1, and H9N2 hemagglutinin and neuraminidase combinations rescued on the new master strain were significantly enhanced in comparison to those of viruses with the same combinations rescued on the standard PR8 master strain. These improvements pave the way for the reproducible generation of high-yielding human and animal influenza vaccines by reverse genetics methods. Such a means of production has particular relevance to epidemic and pandemic use.

scholarly journals Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals

2020 ◽  
Vol 94 (16) ◽  
Author(s):  
Allen Zheng ◽  
Weina Sun ◽  
Xiaoli Xiong ◽  
Alec W. Freyn ◽  
Julia Peukes ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Virus Vaccine ◽  
Influenza Viruses ◽  
Virus Infections ◽  
Influenza A Viruses ◽  
Virus Surface ◽  
Virus Challenge ◽  
Packaging Signals

ABSTRACT Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responses—in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5′ and 3′ terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated “rewired” viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signals—thereby increasing the NA content on viral particles—is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine. IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection.

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