Development and applications of single-cycle infectious influenza A virus (sciIAV)

Abstract

The diverse host range, high transmissibility, and rapid evolution of influenza A viruses justify the importance of containing pathogenic viruses studied in the laboratory. Other than physically or mechanically changing influenza A virus containment procedures, modifying the virus to only replicate for a single round of infection similarly ensures safety and consequently decreases the level of biosafety containment required to study highly pathogenic members in the virus family. This biological containment is more ideal because it is less apt to computer, machine, or human error. With many necessary proteins that can be deleted, generation of single-cycle infectious influenza A viruses (sciIAV) can be achieved using a variety of approaches. Here, we review the recent burst in sciIAV generation and summarize the applications and findings on this important human pathogen using biocontained viral mimics.

Keywords: Influenza HA-expressing MDCK cells (MDCK-HA); Influenza vaccine; Reporter virus; Reverse genetics; Single-cycle virus; Viral vectors.

Figure 1. Generation of stable MDCK-HA cell lines and rescue of single-cycle infectious influenza A virus (sciIAV)

A) Schematic representation for the generation of stable MDCK-HA cells: Polymerase II (Pol II) driven plasmids encoding IAV HA (top) and hygromycin-B resistance (bottom) are co-transfected (ratio 3:1) into parental MDCK cells. After transfection, cells are seeded at low density (cloning dilution) and hygromycin-resistant clones are individually selected. HA-expressing MDCK clones are screened by HA-deficient sciIAV complementation using microscopic analysis of reporter gene (GFP) expression (top), and by HA protein expression using specific Abs by immunofluorescence (right). B) Plasmid-based reverse genetics to generate HA-deficient sciIAV: Ambisense plasmids encoding PB2, PB1, PA, NP, NA, M, NS as well as a Polymerase I (Pol I) driven plasmid encoding the reporter gene (GFP) flanked by the IAV HA NCR and packaging signals and a Pol II driven HA protein expression plasmid are co-transfected into co-cultures of 293T/MDCK-HA cells. Virus containing tissue culture supernatants are subsequently passaged onto MDCK-HA cells for the amplification of the sciIAV ΔHA. pA: polyadenylation signal. RBZ: Hepatitis Delta Virus ribozyme.

Figure 2. Characterization of sciIAV ΔHA

A) Schematic representation of the ΔHA/GFP vRNA: Top, wild-type IAV HA vRNA segment. Bottom, HA(45)GFP(80) vRNA. NCR (thin black lines) at each vRNA termini and the IAV HA packaging signals (Ψ) necessary for the incorporation of vRNA into virions. Nucleotide lengths of NCR, packaging signals and vRNAs are shown. B) Plaque morphology of sciIAV ΔHA in parental and MDCK-HA cells: Confluent parental and MDCK-HA cells were infected with ~25 PFU of the sciIAV ΔHA and, at 3 days post-infection, monolayers were fixed and stained with crystal violet. C) Electron micrographs of WT IAV (left) and sciIAV ΔHA (right). Scale bar, 100 nm. D) sciIAV ΔHA RNA composition: vRNAs of purified WT (left) and sciIAV ΔHA (right) virions are indicated. RNAs were separated by electrophoresis on polyacrylamide gels containing 7 M urea and silver stained. The positions of the IAV vRNA segments are indicated. The black arrow indicates 18S ribosomal RNA (18S rRNA). The green arrow indicates the GFP vRNA segment in the sciIAV ΔHA.

Figure 3. Generation of HA-pseudotyped sciIAV

sciIAV ΔHA can be pseudotyped with various IAV HA proteins by infection of various MDCK-HA cell lines. Infection of parental MDCK cells will result in VLPs released that do not contain HA on the surface. Infection of MDCK-HA cells will produce sciIAV complemented with the cell-derived IAV HA, allowing the generation of HA-pseudotyped sciIAV.

Figure 4. In vitro and in vivo applications of sciIAV

A) In vitro applications: sciIAV can be used for the screening of influenza neutralizing antibodies (NAbs) and the identification of antiviral compounds. Fluorescent/luminescent assays can also be conducted to investigate host-virus factors important for IAV replication as well as to investigate co-infection and reassortment mechanisms of IAV infection. Color code in 96-well plates indicate single (green and red) or double (yellow) infections with sciIAV expressing green or red fluorescent proteins. B) In vivo applications: sciIAV can be used as vaccines or vaccine vectors to provide protection against subsequent viral lethal challenges, as viral vectors (left) for in vivo delivery of miRNA or genes like GFP (center), and to study the immunological consequences of an IAV single round of infection (right). For more details, see text.