A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA

Abstract

The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.

FIG. 1.

Replication and expression of HABCAT influenza B virus model RNAs. CAT protein synthesized from two constructs containing an influenza B vRNA-like CAT reporter gene was quantified. pPRGCAT and pPRFCAT contain the CAT gene in a negative-sense orientation flanked by the influenza B virus HA gene noncoding regions between a human RNA polymerase I promoter and an HDV ribozyme terminator. One microgram of each construct was cotransfected into 5 × 10⁵ 293T cells with 0.5 μg of pCIPB1, 0.5 μg of pCIPB2, 0.5 μg of pCIPA, and 1 μg of pCINP. The pCI plasmids express B/Panama/45/90 polymerase and NP proteins. pPRGCAT was also cotransfected with plasmids expressing the A/Victoria/3/75 PB1, PB2, PA, and NP proteins or with pcDNA3-based plasmids expressing the same proteins from the B/Ann Arbor/66 virus. Transfections were performed in duplicate using the FuGENE 6 transfection reagent (Roche). After 48 h cells were lysed and cell lysates were used in a CAT enzyme-linked immunosorbent assay (Roche).

FIG. 2.

(A) Nucleotide and amino acid sequences of B⁺WNP, B⁺E116G and B⁺HA2X rescued virus NA. B⁺WNP and B⁺HA2X contain the E116 sequence and B⁺E116G contains the 116G sequence. The bold lettering in the E116 nucleotide sequence highlights the codon coding for glutamic acid at amino acid position 116. The bold lettering in the 116G sequence highlights single base mutations (introduced into the pPRNA plasmid via site-directed mutagenesis) within the NA gene of B⁺E116G rescued virus. (B) Multiple-step growth curve of B⁺WNP (•), B⁺E116G (□), and B⁺HA2X (▴) rescued viruses in MDCK cells. Eight 3.5-cm² wells of confluent MDCK cells (approximately 10⁶ cells) were infected with each virus at a multiplicity of infection of 0.001 for 1 h at 34°C. Cells were washed, and serum-free Dulbecco's modified Eagle's medium containing 2.5 μg of trypsin/ml was added to each well, followed by incubation at 34°C. Cell supernatant from one well was harvested after each of the following time points: 12, 24, 36, 48, 60, 72, 84, and 99 h postinfection (p.i).

FIG. 3.

Inhibition of B⁺WNP (○) and B⁺E116G (▪) rescued virus NA activity by zanamivir via the adaptation of the NA assay of Potier et al. (21).