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Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid.

Zhang X, Curtiss R - Virol. J. (2015)

Bottom Line: A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid.The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid.In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA. xiangmin.zhang@wayne.edu.

ABSTRACT

Background: The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse PolI-driven plasmid for efficient production of influenza virus.

Results: A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 10(5)-10(6) 50% tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin-Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method.

Conclusions: An all-in-one plasmid and a 3-plasmid murine PolI-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.

No MeSH data available.


Related in: MedlinePlus

Illustration of the bidirectional vector. A cytomegalovirus (CMV) promoter and Simian virus 40 (SV40) polyadenylation signal (pA) direct the synthesis of mRNA. The mouse RNA PolI promoter (MPI) and terminator (MTI) compose the vRNA transcription unit. Between the two BsmBI sites, there is a prokaryotic green fluorescent protein (GFP) gene cassette. The MPI and MTI sequences adjacent to GFP cassette are shown in blue or purple. Substitution of the GFP cassette with the influenza virus gene resulted in the loss of green fluorescent protein expression in the bacterial host
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Fig1: Illustration of the bidirectional vector. A cytomegalovirus (CMV) promoter and Simian virus 40 (SV40) polyadenylation signal (pA) direct the synthesis of mRNA. The mouse RNA PolI promoter (MPI) and terminator (MTI) compose the vRNA transcription unit. Between the two BsmBI sites, there is a prokaryotic green fluorescent protein (GFP) gene cassette. The MPI and MTI sequences adjacent to GFP cassette are shown in blue or purple. Substitution of the GFP cassette with the influenza virus gene resulted in the loss of green fluorescent protein expression in the bacterial host

Mentions: Using the truncated mouse PolI promoter (MPI, 250 bp), plasmid pYA4924 was constructed to transcribe negative sense EGFP RNA flanked by the 5′ and 3′ non-translating regions of influenza virus M segment (vRNA-like). EGFP expression was observed in BHK-21 cells only when pYA4924 has been cotransfected with plasmids encoding influenza polymerase and NP (Data not shown), suggesting that the cloned MPI is functional in hamster cells. By combining MPI with the SV40 polyadenylation sequence (SV40 pA, 131 bp), truncated mouse PolI terminator (MTI, 41 bp), and functional truncated CMV promoter (188 bp), a bidirectional CMV/MPI-driven transcription vector, pYA4963, was constructed. Two BsmBI sites were designed between MPI and MTI to allow precise insertion of influenza cDNA. A BamHI site was designed between the two BsmBI sites. By inserting a prokaryotic GFP-expression cassette at the BamHI site, an easy-to-use bidirectional vector pYA4964 was constructed (Fig. 1). Substitution of the GFP cassette with influenza cDNA resulted in loss of green fluorescence in bacterial host cells. The GFP-based selection method facilitated construction of the mouse PolI-driven 8-plasmid system that included plasmids pYA4965 (PB1), pYA4966 (PB2), pYA4967 (PA), pYA4968 (NP), pYA4969 (HA), pYA4970 (NA), pYA4971 (M), and pYA4972 (NS) (Table 1).Fig. 1


Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid.

Zhang X, Curtiss R - Virol. J. (2015)

Illustration of the bidirectional vector. A cytomegalovirus (CMV) promoter and Simian virus 40 (SV40) polyadenylation signal (pA) direct the synthesis of mRNA. The mouse RNA PolI promoter (MPI) and terminator (MTI) compose the vRNA transcription unit. Between the two BsmBI sites, there is a prokaryotic green fluorescent protein (GFP) gene cassette. The MPI and MTI sequences adjacent to GFP cassette are shown in blue or purple. Substitution of the GFP cassette with the influenza virus gene resulted in the loss of green fluorescent protein expression in the bacterial host
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495709&req=5

Fig1: Illustration of the bidirectional vector. A cytomegalovirus (CMV) promoter and Simian virus 40 (SV40) polyadenylation signal (pA) direct the synthesis of mRNA. The mouse RNA PolI promoter (MPI) and terminator (MTI) compose the vRNA transcription unit. Between the two BsmBI sites, there is a prokaryotic green fluorescent protein (GFP) gene cassette. The MPI and MTI sequences adjacent to GFP cassette are shown in blue or purple. Substitution of the GFP cassette with the influenza virus gene resulted in the loss of green fluorescent protein expression in the bacterial host
Mentions: Using the truncated mouse PolI promoter (MPI, 250 bp), plasmid pYA4924 was constructed to transcribe negative sense EGFP RNA flanked by the 5′ and 3′ non-translating regions of influenza virus M segment (vRNA-like). EGFP expression was observed in BHK-21 cells only when pYA4924 has been cotransfected with plasmids encoding influenza polymerase and NP (Data not shown), suggesting that the cloned MPI is functional in hamster cells. By combining MPI with the SV40 polyadenylation sequence (SV40 pA, 131 bp), truncated mouse PolI terminator (MTI, 41 bp), and functional truncated CMV promoter (188 bp), a bidirectional CMV/MPI-driven transcription vector, pYA4963, was constructed. Two BsmBI sites were designed between MPI and MTI to allow precise insertion of influenza cDNA. A BamHI site was designed between the two BsmBI sites. By inserting a prokaryotic GFP-expression cassette at the BamHI site, an easy-to-use bidirectional vector pYA4964 was constructed (Fig. 1). Substitution of the GFP cassette with influenza cDNA resulted in loss of green fluorescence in bacterial host cells. The GFP-based selection method facilitated construction of the mouse PolI-driven 8-plasmid system that included plasmids pYA4965 (PB1), pYA4966 (PB2), pYA4967 (PA), pYA4968 (NP), pYA4969 (HA), pYA4970 (NA), pYA4971 (M), and pYA4972 (NS) (Table 1).Fig. 1

Bottom Line: A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid.The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid.In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA. xiangmin.zhang@wayne.edu.

ABSTRACT

Background: The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse PolI-driven plasmid for efficient production of influenza virus.

Results: A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 10(5)-10(6) 50% tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin-Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method.

Conclusions: An all-in-one plasmid and a 3-plasmid murine PolI-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.

No MeSH data available.


Related in: MedlinePlus