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

Murakami, Shin; Horimoto, Taisuke; Yamada, S.; Kakugawa, Satoshi; Goto, Hideo; Kawaoka, Yoshihiro

doi:10.1128/jvi.01876-07

Cited by 42 publications

(62 citation statements)

References 20 publications

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“…Since PA is one of the components of the viral replication complex, we evaluated viral polymerase activity in human A549 and avian DF-1 cells by using a luciferase-based mini-replicon assay, as previously described (24,25). A549 or DF-1 cells were transfected with viral protein expression plasmids for NP, PB1, PB2, and PA or its mutants (i.e., PA-A100V, PA-R356K, PA-N409S, PA-A100V/ R356K/N409S, PA-S37A, PA-8Mut, or PA-11Mut), a plasmid ex- …”

Section: Resultsmentioning

confidence: 99%

“…A minigenome assay based on the dual-luciferase system was performed as previously reported (24,25). Briefly, A549 and DF-1 cells, incubated at 37 and 39°C, respectively, were transfected with viral protein expression plasmids for NP, PB1, PB2, and PA or its mutants (0.2 g of each), with a plasmid expressing a reporter vRNA encoding the firefly luciferase gene under the control of the human or chicken RNA polymerase I promoter [pPolI/NP(0)Fluc(0) or pPolIGG-NP(0)Fluc(0), respectively; 0.2 g of each], and pRL-null (Promega, 0.2 g), which expresses Renilla luciferase, as an transfection control.…”

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confidence: 99%

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Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

Yamayoshi

Yamada

Fukuyama

et al. 2014

J Virol

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108

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Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals. (1). To date, 143 confirmed cases of A(H7N9) virus infection and 45 associated deaths have been reported. This represents a case-fatality rate of ca. 31.5%; however, this rate may actually be lower because the number of mild cases is unknown (2). Phylogenetic analysis has revealed that the hemagglutinin (HA) and neuraminidase (NA) genes of the A(H7N9) viruses share the greatest sequence identities with Eurasian avian A(H7N3) viruses and N9 viruses of different HA subtypes, respectively (3-6). The other six viral genes are highly related to A(H9N2) viruses that have circulated in poultry in China (1, 3-7). These findings indicate that A(H7N9) viruses are reassortant viruses with genes from several avian isolates. IMPORTANCE Novel avian-origin influenzaWe (8) and others (9-14) recently reported that prototype A(H7N9) viruses replicate efficiently in mammalian cells, mice, and ferrets, and transmit among subsets of pairs of ferrets. A(H7N9) viruses isolated from humans possess several amino acid changes already known to facilitate infection of mammals, including leucine at position 226 of HA (H3 HA numbering), which confers increased binding to human-type receptors (15), and lysine at position 627 (PB2-627K) (16,17) and asparagine at position 701 (PB2-701N) (18,19) in the polymerase protein PB2, which increase the polymerase activity in mammalian cells. Notably, the PB2-627K and PB2-701N markers have been detected in almost all human, but not avian or environmental, A(H7N9) isolates (6).In addition to the known mammalian-adapting amino acid changes in the PB2 and HA proteins, additional amino acid changes in A(H7N9) proteins may have enabled these viruses to infect mammals. We therefore undertook a comprehensive analysis of A(H7N9) sequences and identified several amino acids in the polymerase PA protein that are typically found in human (but not avian) influenza A viruses or are characteristic of A(H7N9) viruses (i.e., not commonly detected among human or avian influenza viruses). Here, we evaluated these distinguishing amino acid changes in vivo and in vitro. Cells. Madin-Darby canine kidney ...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

Yamayoshi

Yamada

Fukuyama

et al. 2014

J Virol

Self Cite

108

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“…It has been reported that although an RG system driven by the human RNA pol I promoter supports influenza virus rescue in primate cells, it does not support rescue in canine cells due to the host species specificity of RNA pol I (6,9,17). Therefore, an alternative 8-plasmid RG system that uses the canine RNA pol I promoter sequence was developed for influenza virus rescue in MDCK cells (9,17).…”

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confidence: 99%

“…This virus-inducible reporter plasmid system allows us to measure reporter gene activity as a surrogate for viral RNA synthesis under the control of the canine pol I promoter. For comparison, we also tested a similar virus-inducible reporter plasmid under the control of the human pol I promoter, which has been reported to be transcriptionally inactive in MDCK cells (9).…”

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

Suphaphiphat

Keiner

Trusheim

et al. 2010

J Virol

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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.

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“…The DNA plasmids serve as intracellular templates for both the synthesis of viral genome segments and the viral proteins that then assemble into infectious viruses that are capable of replication. This technique allows the recovery of infectious viruses directly from cells that are approved for the production of human vaccines (13,14) . Moreover, in the case of genetic engineering through classical cloning procedures, because proteins are denatured and removed during the initial cloning steps and the subsequent preparation of plasmids, plasmid-driven transfection represents an effi cient purifi cation step for eliminating potential adventitious agents that may be present in human infl uenza virus isolates.…”

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confidence: 99%

Exploring synergies between academia and vaccine manufacturers: a pilot study on how to rapidly produce vaccines to combat emerging pathogens

Strecker

Uhlendorff

Diederich

et al. 2012

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background: In spring 2009, a new swine-origin infl uenza A (H1N1) virus emerged in Mexico. During the following weeks the virus spread worldwide, prompting the World Health Organization to declare the fi rst infl uenza pandemic of the 21st century. Sustained human-to-human transmission and severe disease progression observed in some patients urged public health authorities to respond rapidly to the disease outbreak and vaccine manufacturers to develop pandemic infl uenza vaccines for mass distribution. With the onset of the pandemic we began to explore the potential of academic/ industrial collaboration to accelerate the production of vaccines during an outbreak of an emerging virus by combining the use of an academic BSL-4 laboratory with the expertise of a commercial vaccine manufacturer. Methods and results: To obtain virus seed stocks used for the production of a vaccine to combat the pandemic H1N1 2009 infl uenza virus (H1N1pdm), we followed various strategies: (i) optimization of cell culture conditions for growth of wild-type H1N1pdm isolates; (ii) classical reassortment of H1N1pdm and standard infl uenza vaccine donor strain PR8; and (iii) generation of corresponding reassortant viruses

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

Cited by 42 publications

References 20 publications

Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

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

Exploring synergies between academia and vaccine manufacturers: a pilot study on how to rapidly produce vaccines to combat emerging pathogens

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