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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

Analysis of gene expression in BHK-21, CHO, and MDCK cells. The cells were transfected with reporter plasmids carrying a CMV-driven mCherry cassette. The large pYA4732 plasmid showed significantly lower mCherry expression in CHO cells than the small pYA4731 plasmid. Cells were also cotransfected with pYA4732 and pYA4924. The EGFP expression indicated that vRNA-like EGFP RNA was generated from pYA4924 and converted into mRNA for EGFP synthesis in the presence of the influenza NP and polymerase provided by pYA4732. Compared with BHK-21 and CHO cells, very few MDCK cells expressed EGFP. Expression of EGFP and mCherry were recorded from the same field
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Fig4: Analysis of gene expression in BHK-21, CHO, and MDCK cells. The cells were transfected with reporter plasmids carrying a CMV-driven mCherry cassette. The large pYA4732 plasmid showed significantly lower mCherry expression in CHO cells than the small pYA4731 plasmid. Cells were also cotransfected with pYA4732 and pYA4924. The EGFP expression indicated that vRNA-like EGFP RNA was generated from pYA4924 and converted into mRNA for EGFP synthesis in the presence of the influenza NP and polymerase provided by pYA4732. Compared with BHK-21 and CHO cells, very few MDCK cells expressed EGFP. Expression of EGFP and mCherry were recorded from the same field

Mentions: The BHK-21, CHO, and MDCK cells in 80–90 % confluence were transfected with a 6.1-kb plasmid pYA4731 (CMV-mCherry) or a 25.3-kb plasmid pYA4732 (CMV-mCherry) [19]. Both plasmids resulted in similar levels of mCherry expression in BHK-21 and in MDCK cells (Fig. 4). The CHO cells showed preferential uptake of the smaller plasmid. The three types of cells were cotransfected with pYA4924 and pYA4732 which encodes influenza viral polymerase and NP. The BHK-21 and CHO cells showed efficient EGFP expression, indicating that the vRNA-like molecules were converted into mRNA. In contrast, only numerous MDCK cells expressed EGFP, which suggests that the mouse PolI promoter is poorly active in canine cells.Fig. 4


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

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

Analysis of gene expression in BHK-21, CHO, and MDCK cells. The cells were transfected with reporter plasmids carrying a CMV-driven mCherry cassette. The large pYA4732 plasmid showed significantly lower mCherry expression in CHO cells than the small pYA4731 plasmid. Cells were also cotransfected with pYA4732 and pYA4924. The EGFP expression indicated that vRNA-like EGFP RNA was generated from pYA4924 and converted into mRNA for EGFP synthesis in the presence of the influenza NP and polymerase provided by pYA4732. Compared with BHK-21 and CHO cells, very few MDCK cells expressed EGFP. Expression of EGFP and mCherry were recorded from the same field
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Analysis of gene expression in BHK-21, CHO, and MDCK cells. The cells were transfected with reporter plasmids carrying a CMV-driven mCherry cassette. The large pYA4732 plasmid showed significantly lower mCherry expression in CHO cells than the small pYA4731 plasmid. Cells were also cotransfected with pYA4732 and pYA4924. The EGFP expression indicated that vRNA-like EGFP RNA was generated from pYA4924 and converted into mRNA for EGFP synthesis in the presence of the influenza NP and polymerase provided by pYA4732. Compared with BHK-21 and CHO cells, very few MDCK cells expressed EGFP. Expression of EGFP and mCherry were recorded from the same field
Mentions: The BHK-21, CHO, and MDCK cells in 80–90 % confluence were transfected with a 6.1-kb plasmid pYA4731 (CMV-mCherry) or a 25.3-kb plasmid pYA4732 (CMV-mCherry) [19]. Both plasmids resulted in similar levels of mCherry expression in BHK-21 and in MDCK cells (Fig. 4). The CHO cells showed preferential uptake of the smaller plasmid. The three types of cells were cotransfected with pYA4924 and pYA4732 which encodes influenza viral polymerase and NP. The BHK-21 and CHO cells showed efficient EGFP expression, indicating that the vRNA-like molecules were converted into mRNA. In contrast, only numerous MDCK cells expressed EGFP, which suggests that the mouse PolI promoter is poorly active in canine cells.Fig. 4

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