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. 2002 Aug 20;99(17):11411-6.
doi: 10.1073/pnas.172393399. Epub 2002 Aug 9.

Rescue of influenza B virus from eight plasmids

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Rescue of influenza B virus from eight plasmids

Erich Hoffmann et al. Proc Natl Acad Sci U S A. .

Abstract

Influenza B virus causes a significant amount of morbidity and mortality, yet the systems to produce high yield inactivated vaccines for these viruses have lagged behind the development of those for influenza A virus. We have established a plasmid-only reverse genetics system for the generation of recombinant influenza B virus that facilitates the generation of vaccine viruses without the need for time consuming coinfection and selection procedures currently required to produce reassortants. We cloned the eight viral cDNAs of influenza B/Yamanashi/166/98, which yields relatively high titers in embryonated chicken eggs, between RNA polymerase I and RNA polymerase II transcription units. Virus was detected as early as 3 days after transfection of cocultured COS7 and Madin-Darby canine kidney cells and achieved levels of 10(6)-10(7) plaque-forming units per ml of cell supernatant 6 days after transfection. The full-length sequence of the recombinant virus after passage into embryonated chicken eggs was identical to that of the input plasmids. To improve the utility of the eight-plasmid system for generating 6 + 2 reassortants from recently circulating influenza B strains, we optimized the reverse transcriptase-PCR for cloning of the hemagglutinin (HA) and neuraminidase (NA) segments. The six internal genes of B/Yamanashi/166/98 were used as the backbone to generate 6 + 2 reassortants including the HA and NA gene segments from B/Victoria/504/2000, B/Hong Kong/330/2001, and B/Hawaii/10/2001. Our results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza B virus.

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Figures

Fig 1.
Fig 1.
Eight-plasmid bidirectional transcription system for the generation of influenza B virus. The eight plasmids contain cDNAs representing the eight gene segments of influenza B virus. In each of the plasmids, the cDNAs are flanked by pol I (pI) promoter/terminator (tI) sequences. The RNA pol I transcription unit is flanked by pol II promoter (pII)/polyadenylation (aII) sequences. After transfection of these constructs into COS7 cells, cellular pol I synthesize virus-like noncapped negative sense vRNAs and pol II capped mRNAs encoding the viral proteins. After translation of PB1, PB2, PA, and NP proteins, the eight negative sense vRNAs are transcribed and replicated. Ultimately, the viral ribonucleoproteins (vRNPs) and structural proteins derived from cellular transcription or viral amplification (dashed rectangle) are assembled into new virus particles.
Fig 2.
Fig 2.
Kinetics of virus generation after transfection. COS7 or cocultured COS7-MDCK cells were transfected with eight plasmids representing the eight segments of B/Yamanashi/166/98. The virus yield of the supernatant was determined at different times after transfection by plaque assay on MDCK cells.
Fig 3.
Fig 3.
Genetic characterization of recombinant influenza B viruses. RNA was isolated from allantoic fluid of chicken eggs either infected with wt B/Yamanashi/166/98 (1) or with supernatant of transfected COS7-MDCK cells with eight plasmids (2). RT-PCR was performed by using PA-specific primers. The PCR products were sequenced. The nucleotides in the electropherogram that differ between recombinant virus and wt virus are encircled.
Fig 4.
Fig 4.
RT-PCR for amplification of the HA and NA segments. RT-PCR was performed with the primer pair Bm-NAb-1/Bm-NAb-1557R. The PCR products were subjected to gel electrophoresis on a 1% agarose gel. RNA isolated from the following virus isolates were used: 1, B/Lee/40; 2, B/Ann Arbor/1/94; 3, B/Yamanashi/166/98; 4, B/Johannesburg/5/99; 5, B/Victoria/504/2000; 6, B/Sichuan/317/2001; 7, B/Shizuoka/15/2001; 8, B/Hawaii/10/2001; 9, B/Hong Kong/330/2001; –, no RNA.
Fig 5.
Fig 5.
Growth of 6 + 2 reassortants in eggs. Embryonated chicken eggs were inoculated with wt or recombinant virus. The virus titer was determined by titration on MDCK cells .

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