Improved dual promotor-driven reverse genetics system for influenza viruses

Mostafa, Ahmed; Kanrai, Pumaree; Ziebuhr, John; Pleschka, Stephan

doi:10.1016/j.jviromet.2013.07.021

Cited by 25 publications

(44 citation statements)

References 24 publications

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“…Further plasmids used were (i) pPol-I-PB2, -PB1, -PA, -HA, -NP, -NA, -M and -NS, expressing the vRNAs of the respective FPV segments (Wagner et al, 2000), (ii) pBD-NS and pPol-I-NS-MA expressing the NS vRNA of GD and MA (Ma et al, 2010), (iii) the pPol-I-NS-GD/MA and -NS-MA/GD plasmids (encoding the N-terminal sequence of the NS GD and the C-terminal sequence of the NS MA segment or, vice versa), (iv) pHW2000-PB2, -PB1, -PA, -HA, -NP, -NA, -M and -NS containing the viral genome sequences of SC35 (H7N7) or A/Thailand/1(KAN-1)/ 2004 (H5N1, KAN-1), with the latter encoding an HA with a monobasic ('low pathogenicity') cleavage site as previously described (Auewarakul et al, 2007;Mostafa et al, 2015;Schmier et al, 2015) and (v) pMPccdB-PB2, -PB1, -PA, -HA, -NP, -NA, -M and -NS containing the viral genome sequences of A/Giessen/6/2009 (H1N1pdm09) (Mostafa et al, 2013). Mutations in pcDNA3.1-NS1-MA, pPol-I-NS-MA, pHW2000-NS1-SC35, -NS1-H5N1 and -NS1-H1N1 were generated and confirmed as previously described using specific oligonucleotide primers (sequences available upon request).…”

Section: Methodsmentioning

confidence: 99%

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Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems

Kanrai

Mostafa

Madhugiri

et al. 2016

Journal of General Virology

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Reassortment of their segmented genomes allows influenza A viruses (IAV) to gain new characteristics, which potentially enable them to cross the species barrier and infect new hosts. increased virus titres, (ii) larger plaque sizes and (iii) increased levels and faster kinetics of viral mRNA and protein accumulation in mammalian cells. Interestingly, the NS1 substitutions did not affect viral growth characteristics in avian cells. Furthermore, we show that an FPV mutant with N74 in the NS1 (already possessing S3+K41) is able to replicate and cause disease in mice, demonstrating a key role of NS1 in the adaptation of avian IAV to mammalian hosts. Our data suggest that (i) adaptation to mammalian hosts does not necessarily compromise replication in the natural (avian) host and (ii) very few genetic changes may pave the way for zoonotic transmission. The study reinforces the need for close surveillance and characterization of circulating avian IAV to identify genetic signatures that indicate a potential risk for efficient transmission of avian strains to mammalian hosts.

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Section: Methodsmentioning

confidence: 99%

“…Transfection was performed for 8 h as described previously (Mostafa et al, 2013). The cells were harvested 24 h post-transfection, pelleted and subjected to protein extraction as previously described .…”

mentioning

confidence: 99%

Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems

Kanrai

Mostafa

Madhugiri

et al. 2016

Journal of General Virology

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“…The plasmids carrying the eight gene segments of the high-yield PR8 (H1N1) virus were kindly supplied by Richard Webby at St Jude Children's Research Hospital, TN, USA (Hoffmann et al, 2000). and A/Anhui/1/2013 (Anhui, H7N9) were cloned into pMPccd B as described previously (Mostafa et al, 2013).…”

Section: Methodsmentioning

confidence: 99%

“…Using the eight-plasmid reverse genetics system employing either pHW2000 or our own previously developed vector pMPccd B, reassortant and recombinant viruses were generated as described previously (Hoffmann et al, 2000(Hoffmann et al, , 2001Mostafa et al, 2013). Briefly, 8 mg plasmid DNA (1 mg per plasmid) was transfected into a co-culture of 293T/MDCK-II cells with Trans-IT2020 (Mirus) (2 ml per 1 mg plasmid DNA to be transfected).…”

Section: Methodsmentioning

confidence: 99%

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The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs

Mostafa

Kanrai

Ziebuhr

et al. 2016

Journal of General Virology

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Influenza vaccine strains (IVSs) contain the haemagglutinin (HA) and neuraminidase (NA) genome segments of relevant circulating strains in the genetic background of influenza A/ PR/8/1934 virus (PR8). Previous work has shown that the nature of the PB1 segment may be a limiting factor for the efficient production of IVSs. Here, we showed that the PB1 segment (PB1 Gi ) from the 2009 pandemic influenza A virus (IAV) A/Giessen/06/2009 (Gi wt, H1N1pdm) may help to resolve (some of) these limitations. We produced a set of recombinant PR8-derived viruses that contained (i) the HA and NA segments from representative IAV strains (H3N2, H5N1, H7N9, H9N2); (ii) the PB1 segment from PR8 or Gi wt, respectively; and (iii) the remaining five genome segments from PR8. Viruses containing the PB1 Gi segment, together with the heterologous HA/NA segments and five PR8 segments (5+2+1), replicated to higher titres compared with their 6+2 counterparts containing six PR8 segments and the equivalent heterologous HA/NA segments. Compared with PB1 PR8 -containing IVSs, viruses with the PB1 Gi segment replicated to higher or similar titres in both cell culture and embryonated eggs, most profoundly IVSs of the H5N1 and H7N9 subtype, which are known to grow poorly in these systems. IVSs containing either the PB1 Gi or the cognate PB1 segment of the respective specific HA/NA donor strain showed enhanced or similar virus replication levels. This study suggests that substitution of PB1 PR8 with the PB1 Gi segment may greatly improve the large-scale production of PR8-derived IVSs, especially of those known to replicate poorly in vitro. INTRODUCTIONInfluenza viruses belong to the family Orthomyxoviridae and are classified into three genera termed Influenza virus A, B and C. Among these, influenza A viruses (IAVs) pose the greatest threat to human and animal health. They contain eight segments of negative-sense ssRNA (Ritchey et al., 1976;Shaw & Palese, 2013). The eight genomic segments encode at least 10 viral proteins, comprising the three subunits of the viral RNA polymerase complex [polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA)], the surface glycoproteins [haemagglutinin (HA) and neuraminidase (NA)], the nucleoprotein (NP), the matrix protein (M1), the ion channel protein (M2), the non-structural protein 1 (NS1) and the nuclear export protein (NEP). IAVs are genetically diverse, with new strains rapidly emerging by point mutations introduced by the low-fidelity viral RNA replicase (potentially resulting in antigenic drift if changes occur in the HA and/or NA coding sequences and affect important antigenic epitopes) or by genetic reassortment of genomic segments (potentially leading to antigenic shift if antigenically distant HA and NA segments are incorporated). Minor and major antigenic changes in the HA and NA proteins of circulating IAV are important driving forces for the emergence of new strains causing epidemics or even major pandemics (Neumann et al., 2009;Peng et al., 2014;Smith...

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Reverse Genetics of Influenza Virus

Lee

2014

Methods in Molecular Biology

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Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an "infectious clone," and is one of the most powerful genetic tools in modern virology. Since its development in 1999, plasmid-based reverse genetics has been effectively applied to numerous aspects of influenza studies which include revolutionizing the production of seasonal and pandemic influenza vaccine seed strains. Although continual improvement in reverse genetics system is being made in different laboratories for the efficient rescue of the influenza virus, the basic concept of synthesizing viral RNA using RNA polymerase I remains the same. Coupled with in vitro mutagenesis, reverse genetics can be applied widely to accelerate progress in understanding the influenza virus life cycle, the generation of customized vaccine seed strains, development of live-attenuated vaccines, and the use of influenza virus as vaccine and gene delivery vectors.

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Improved dual promotor-driven reverse genetics system for influenza viruses

Cited by 25 publications

References 24 publications

Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems

Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems

The PB1 segment of an influenza A virus H1N1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs

Reverse Genetics of Influenza Virus

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