A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon

Abstract

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.

Figure 1. Expression of firefly luciferase and GFP reporter genes by a propagation-incompetent VSVΔG replicon.

(a) Genome maps of authentic VSV and recombinant VSV*ΔG(Luc) vector. VSV*ΔG(Luc) lacks the glycoprotein (G) gene and drives expression of both enhanced green fluorescent protein (GFP) and firefly luciferase (Luc). (b) Cell lines derived from different species were infected with 4 ffu/cell of VSV*ΔG(Luc) (1^st cycle). Sixteen hours post infection, the undiluted cell culture supernatant was passed to fresh cells (2^nd cycle). GFP expression in live cells was monitored 6 h post infection using an inverse fluorescence microscope. Bar, 100 µm. (c) Firefly luciferase activity in BHK-21 cell lysates at different times post infection with 4 ffu/cell of VSV*ΔG(Luc). (d) Firefly luciferase levels in BHK-21 cell lysates 5 h post infection by VSV*ΔG(Luc) with the indicated multiplicities of infection (m.o.i.).

Figure 2. Determination of mammalian and avian type I IFN bioactivity.

(a) NHDF (human fibroblasts), (b) DF-1 (chicken fibroblasts), and (c) PK-15 (porcine kidney) cells in 96-well plates were incubated for the indicated times with fourfold serial dilutions of type I IFN from the homologous species and subsequently infected with VSV*ΔG(Luc) (5 ffu/cell). Five hours post infection, the cells were lysed and firefly luciferase activity was recorded. Reference cells did not receive any type I IFN and demonstrated no antiviral activity. The axes of abscissas indicate the activities of the type I IFNs as determined with a conventional CPE-based IFN bioassay.

Figure 3. Validation of the VSV*ΔG(Luc) replicon bioassay.

Serial twofold dilutions of IFN-β standard (starting with 100 pg/ml) and of two different test samples containing type IFN of unknown activity (starting with dilution 1/100) were prepared and incubated with NHDF for 20 h at 37°C. (a) Five hours post infection with VSV*ΔG(Luc) (m.o.i. = 5), the cells were lysed and firefly luciferase activity recorded. The antiviral activity relative to infected but non-treated cells was calculated. (b) Fourty-eight hours post infection with VSV (m.o.i. = 1), the cells were fixed with formalin containing 0.1% crystal violet, washed with water, and dried. The dye was dissolved in 70% ethanol before absorbance at 595 nm was recorded. The relative antiviral activity (%) was calculated.

Figure 4. Type I IFNs act in a species-specific manner.

(a) Cells derived from different species were treated for 20 h with the indicated type I IFNs (5 units/well) and subsequently infected with VSV*ΔG(Luc). Expression of GFP was monitored 6 h post infection by fluorescence microscopy. Bar, 30 µm. (b) NHDF were incubated for 20 h with serial fourfold dilutions of human, porcine and murine type I IFNs. The cells were subsequently infected with VSV*ΔG(Luc) for 5 h and firefly luciferase activity was recorded in the cell lysates.

Figure 5. Analysis of type I IFN stability.

(a) Human IFN-β and porcine IFN-α (each at 800 units/ml) were heated for 30 min at the indicated temperatures and thereafter diluted 1∶10 with medium containing 5% FBS. The heat-treated IFN preparations were incubated for 20 h with NHDF and PK-15 cells, respectively (8 units per 5×10⁴ cells). Luciferase reporter activity was determined in cell lysates 5 hours post infection with VSV*ΔG(Luc). (b) Human IFN-β and (c) human IFN-λ were incubated for 30 min at 20°C with either 0.1 M HCl, 0.1 M NaOH, or H₂O, and then adjusted to neutral pH. NHDF and Calu-3 cells were incubated for 20 h with fourfold serial dilutions of HCl/NaOH-treated IFN-β and IFN-λ, respectively, and subsequently infected with VSV*ΔG(Luc). Firefly luciferase activity was determined in cell lysates 5 h post infection.