Replication-incompetent influenza A viruses that stably express a foreign gene

Abstract

A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.

A Replication-Incompetent PB2-Knockout Influenza A Virus Vaccine Vector

“…AX4 cells, which are an MDCK-derived cell line with enhanced expression of human-type receptors for influenza virus and were produced by stable transfection of a plasmid expressing the human ␣-2,6-sialyltransferase (8), were maintained in 5% NCS-MEM supplemented with puromycin (2 g/ml). AX4/ PB2 cells (AX4 cells stably expressing the PB2 protein derived from A/Puerto Rico/8/34 [H1N1, PR8], established by transduction with a retroviral vector [16]) were maintained in 5% NCS-MEM supplemented with puromycin (2 g/ml) and blasticidin (10 g/ml). All cells were maintained in a humidified incubator at 37°C in 5% CO 2 .…”

“…Recently, our group genetically engineered PB2-knockout (PB2-KO) influenza viruses that are able to harbor a reporter gene, such as the green fluorescent protein (GFP) gene, between the packaging signals (the noncoding and coding 120 bases at both the 5= and 3= ends) of the PB2 gene (16). Growth of such PB2-KO viruses is restricted to a cell line stably expressing the PB2 protein and yields high virus titers (Ͼ10 8 PFU/ml).…”

“…Growth of such PB2-KO viruses is restricted to a cell line stably expressing the PB2 protein and yields high virus titers (Ͼ10 8 PFU/ml). The HA and NA genes of a heterologous influenza virus could be accommodated in the PB2-KO virus (16). Furthermore, the recombinant PB2/reporter gene was stably incorporated into progeny PB2-KO virions and was retained through sequential passages.…”

Competitive Incorporation of Homologous Gene Segments of Influenza A Virus into Virions

“…By using these tandem reporter plasmids instead of an intact PB2 vRNA-expressing plasmid, we generated PB2-knockout viruses by means of reverse genetics. We then counted the GFP-and/or DsRed-positive plaques formed by the transfectant viruses in PB2 protein-expressing MDCK cells that were established by using a retrovirus vector as described previously (16). Like the NA-knockout viruses, these PB2-knockout viruses mainly formed plaques expressing only either GFP (75.6%) or DsRed (23.8%); only a limited portion (0.6%) of the plaques expressed both GFP and DsRed (Fig.…”

An Eight-Segment Swine Influenza Virus Harboring H1 and H3 Hemagglutinins Is Attenuated and Protective against H1N1 and H3N2 Subtypes in Pigs

“…Such a packaging signal, which is necessary for viral genome segment-specific packaging, was believed to reside in both the 3= and 5= ends of the noncoding regions (NCRs) and the adjacent terminal coding region of each RNA segment (26)(27)(28)(29)(30)(31)(32)(33)(34). It was demonstrated that foreign genes or influenza virus genes from different types and subtypes could be efficiently cloned between the packaging signals of different influenza virus genes and expressed in the form of proteins (34)(35)(36) (35,37,38). These approaches resulted in the generation of replication-deficient recombinant influenza virus.…”