Replication-competent fluorescent-expressing influenza B virus

Abstract

Influenza B viruses (IBVs) cause annual outbreaks of respiratory illness in humans and are increasingly recognized as a major cause of influenza-associated morbidity and mortality. Studying influenza viruses requires the use of secondary methodologies to identify virus-infected cells. To this end, replication-competent influenza A viruses (IAVs) expressing easily traceable fluorescent proteins have been recently developed. In contrast, similar approaches for IBV are mostly lacking. In this report, we describe the generation and characterization of replication-competent influenza B/Brisbane/60/2008 viruses expressing fluorescent mCherry or GFP fused to the C-terminal of the viral non-structural 1 (NS1) protein. Fluorescent-expressing IBVs display similar growth kinetics and plaque phenotype to wild-type IBV, while fluorescent protein expression allows for the easy identification of virus-infected cells. Without the need of secondary approaches to monitor viral infection, fluorescent-expressing IBVs represent an ideal approach to study the biology of IBV and an excellent platform for the rapid identification and characterization of antiviral therapeutics or neutralizing antibodies using high-throughput screening approaches. Lastly, fluorescent-expressing IBVs can be combined with the recently described reporter-expressing IAVs for the identification of novel therapeutics to combat these two important human respiratory pathogens.

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus

“…At 3 days p.i., cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After the overlays were removed, cells were permeabilized (0.5% Triton X-100 in PBS) for 15 min at room temperature and prepared for immunostaining as previously described (63,68), using the NP MAb HB-65 and vector kits (Vectastain ABC kit and DAB HRP substrate kit: Vector) following the manufacturer's specifications.…”

Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

“…To engineer the recombinant PR8 virus NS and M split segments (NSs and Ms, respectively), overlapping PCR and standard molecular biology techniques were used to introduce the modifications into the ambisense pDZ-NS or pDZ-M viral rescue plasmid (26). The modified plasmids, named pDZ-NSs and pDZ-Ms, respectively, contain the NS1 (NS segment) or M1 (M segment) open reading frames (ORFs) without stop codons or splice acceptor sites, followed by the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site (ATNFSLLKQAGDVEE NPGP) and the entire sequence of the NEP (NS segment) or M2 (M segment) ORF (27,29). The sequences of the modified pDZ-NSs and -Ms plasmids were confirmed by sequencing, and the plasmids were used for virus rescue.…”

“…At 3 days postinfection, the cells were fixed overnight with 4% paraformaldehyde (PFA) and the overlays were removed. Cells were then permeabilized (0.5% Triton X-100 in PBS) for 15 min at room temperature and prepared for immunostaining using NP MAb HB-65 and vector kits (Vectastain ABC vector kits and DAB HRP substrate kit; Vector) according to the manufacturer's specifications (27,29).…”

“…Antiviral assay. Antiviral-mediated inhibition of WT and recombinant PR8 viruses was evaluated as previously described (27,29). Briefly, confluent MDCK cells (24-well plate format, in triplicate) were infected (MOI, 0.001) for 1 h and incubated with infectious medium supplemented with 3-fold serial dilutions of oseltamivir (starting concentration, 10 M).…”

“…To that end, the overlapping regions in both segments of the viral genome were duplicated and the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site (33) was inserted between the NS1 and NEP genes (segment 8) or between the M1 and M2 genes (segment 7). This strategy ensured that the viral proteins (the NS1 and NEP proteins and the M1 and M2 proteins, respectively) would be translated as two separate, nonoverlapping, independent ORFs, similar to the strategy that we have previously described for the NS segment (27,29,34).…”