A Novel Fluorescent and Bioluminescent Bireporter Influenza A Virus To Evaluate Viral Infections

Abstract

Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select a fluorescent or a luciferase reporter gene, depending on the type of study. To circumvent this limitation, we engineered a novel recombinant replication-competent bireporter IAV (BIRFLU) expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed growth kinetics comparable to those of wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows monitoring of viral inhibition by fluorescence or bioluminescence, overcoming the limitations associated with the use of one reporter gene as a readout. In vivo, BIRFLU effectively infected mice, and both reporter genes were detected using in vivo imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections.IMPORTANCE Influenza A virus (IAV) causes a human respiratory disease that is associated with significant health and economic consequences. In recent years, the use of replication-competent IAV expressing an easily traceable fluorescent or luciferase reporter protein has significantly contributed to progress in influenza research. However, researchers have been forced to select a fluorescent or a luciferase reporter gene due to the restricted capacity of the influenza viral genome for including foreign sequences. To overcome this limitation, we generated, for the first time, a recombinant replication-competent bireporter IAV (BIRFLU) that stably expresses two reporter genes (one fluorescent and one luciferase) to track IAV infections in vitro and in vivo The combination of cutting-edge techniques from molecular biology, animal research, and imaging technologies brings researchers the unique opportunity to use this new generation of reporter-expressing IAV to study viral infection dynamics in both cultured cells and animal models of viral infection.

Keywords: Nano luciferase; Venus fluorescence; bioluminescence; fluorescence; in vivo expression technology; influenza; reporter genes.

FIG 1

Generation of a recombinant PR8 IAV expressing two reporter genes (BIRFLU). (A and B) Schematic representation of the modified recombinant PR8 IAV HA (A) and NS (B) viral segments. PR8 IAV HA and NS viral products are indicated by black (HA), dark gray (NS1), or light gray (NEP) boxes. Noncoding regions (NCR) are represented with black lines, and original or duplicated (HA viral segment) packaging signals are indicated with white or striped boxes, respectively. Nano luciferase (NLuc), Venus, and PTV-1 2A are indicated in blue, green, and red boxes, respectively. For the HA segment, the nucleotide sequences surrounding NLuc are included: the last 13 nucleotides of the packaging signal (white box) with the mutated ATG (green underlined), NLuc flanked by AgeI and NheI restriction sites (underlined), 2A (red), and the HA ORF (depicted with lines), including silent mutations (green underlined). Diagrams are not drawn to scale. (C) Schematic representation of the recombinant BIRFLU PR8 IAV. (D) Analysis of protein expression. MDCK cells (6 well plates, 10⁶ cells/well) were infected with the PR8 WT or BIRBLU at an MOI of 0.1 or mock infected (lane M). Protein expression was examined by Western blotting using specific antibodies for NS1, Venus, NLuc, and NP. Actin was used as a loading control. The numbers on the left indicate the molecular size of the protein markers (in kilodaltons).

FIG 2

Analysis of BIRFLU protein expression by direct fluorescence and immunofluorescence. MDCK cells (24-well plates, 2 × 10⁵ cells/well) were infected with PR8 WT or BIRFLU (MOI, 0.1) or mock infected. Infected cells were fixed at 18 h p.i. to directly visualize Venus expression and to visualize NP (A), NS1 (B), HA (C), and NLuc (D) using specific antibodies. Nuclei were stained with DAPI. Representative images are shown. Bars, 100 μm; magnification, ×20.

FIG 3

Growth kinetics and plaque morphology of BIRFLU. (A) Multicycle growth kinetics. Viral titers (in FFU per milliliter) in culture supernatants from MDCK cells (6-well plates, 10⁶ cells/well, triplicates) infected with PR8 WT and BIRFLU (MOI, 0.001) were determined by immunofocus assay at the indicated times postinfection. Data represent the means ± SD for triplicates. The dashed line denotes the limit of detection (200 FFU/ml). *, P < 0.05, using an unpaired two-tailed Student's t test. (B) NLuc expression. NLuc was evaluated in the same culture supernatants obtained from the experiment whose results are presented in panel A. RLU, relative light units. (C) Venus expression. MDCK cells (24-well-plate format, 2 × 10⁵ cells/well) infected (MOI, 0.001) with PR8 WT or BIRFLU were visualized at the indicated times (in hours) p.i. using a fluorescence microscope. Representative images are shown. Bars, 100 μm; magnification, ×20. (D) Plaque phenotype. Representative pictures of viral plaques in MDCK cells (6-well-plate format, 10⁶ cells/well) infected with PR8 WT and BIRFLU at 3 days p.i. are shown. Fluorescent Venus expression (top), NLuc immunostaining (middle), and crystal violet staining (bottom) are indicated. White arrows show the colocalization of Venus fluorescence (top), NLuc expression (middle), and virus lysis plaques stained with crystal violet (bottom).

FIG 4

A bireporter-based microneutralization assay for evaluating IAV NAbs. Two hundred PFU of BIRFLU was preincubated with 2-fold serial dilutions (starting concentration, 10 µg/ml) of HA-specific NAbs for PR8 (NAb PY102) or pH1N1 (NAb 29E3) for 1 h. Subsequently, MDCK cells (96-well plates, 2 × 10⁴ cells/well, triplicates) were infected with the antibody-virus mix. (A and B) Virus neutralization was determined by quantitating Venus (A) and NLuc (B) reporter expression at 48 h p.i. using a fluorescent microplate reader or a luminometer, respectively. (C and D) The percent neutralization (NC₅₀) was calculated using sigmoidal dose-response curves. Mock-infected cells were used as internal controls for basal levels of fluorescence (Venus) or luciferase (NLuc) expression. PR8 BIRFLU-infected cells in the absence of NAbs were used to determine maximum reporter expression. Data show the means ± SD of the results determined in triplicate.

FIG 5

A bireporter-based microneutralization assay for assessing IAV antivirals. MDCK cells (96-well plates, 2 × 10⁴ cells/well, triplicates) were infected with 200 PFU of BIRFLU and incubated with 2-fold serial dilutions (starting concentration, 100 µM) of ribavirin or amantadine. As an internal control, infected cells were not treated with antivirals. (A and B) Inhibition of viral replication was evaluated by quantitating Venus (A) and NLuc (B) reporter expression at 48 h p.i, using a fluorescent microplate reader or a luminometer, respectively. (C and D) The percent inhibition (IC₅₀) was calculated using sigmoidal dose-response curves. Percent viral inhibition was normalized to that with infection in the absence of antivirals. Data show the means ± SD of the results determined in triplicate.

FIG 6

Virulence of BIRFLU in mice. Five- to 7-week-old female BALB/c mice were intranasally inoculated with the indicated dose (10³, 10⁴, 10⁵, or 10⁶ PFU) of BIRFLU and monitored for 10 days for body weight loss and survival (not shown). Data represent the means ± SD of the results determined for individual mice.

FIG 7

In vivo kinetics of BIRFLU infection by real-time monitoring of NLuc expression. Five- to 7-week-old female BALB/c mice were mock infected (PBS) or intranasally inoculated with 10⁶ PFU of BIRFLU (n = 4). NLuc activity in the whole mouse was evaluated on the indicated day p.i. (2, 4, 6, 8, and 10). (Top) Representative images of the same mouse for each time point show the radiance (number of photons per second per square centimeter per steradian [p/sec/cm²/sr]). (Bottom) Bioluminescence values were quantified, and the total flux [in log₁₀ (number of photons per second) (p/s)] is presented.

FIG 8

In vivo bioluminescence and fluorescence correlation after BIRFLU infection. Five- to 7-week-old female BALB/c mice were mock infected (PBS) or infected intranasally with 10⁶ PFU of BIRFLU (n = 4). (A) NLuc activity in the whole mouse was determined at 2, 4, and 6 days p.i. Representative images of a single mouse for each time point show the radiance (number of photons per second per square centimeter per steradian [p/sec/cm²/sr]). (B) On the same days p.i., lungs from mock-infected and BIRFLU-infected mice were harvested to assess fluorescent Venus expression. Representative fluorescent pictures from whole lungs of the same mice used in the experiment whose results are presented in panel A are shown. (C and D) Bioluminescence (C) or Venus radiance (D) values were quantitated. (C) The average total flux [in log₁₀ (number of photons per second) (p/s)] is shown. (D) To quantify Venus expression, the mean values for the regions of interest were normalized to the lung autofluorescence in mock-infected mice at each time point, and fold changes in fluorescence were calculated. (E) Viral lung titers. After imaging, whole lungs from mice from the experiments whose results are shown in panel B were homogenized and used to infect MDCK cells and determine viral titers (in FFU per milliliter) by immunofocus assay. Bars represent the mean ± SD of lung virus titers.

FIG 9

Genetic stability of BIRFLU in vivo and in vitro. (A) In vivo genetic stability. To analyze the genetic stability of BIRFLU in vivo, viruses recovered from mouse lungs at 2 (n = 4) and 4 (n = 4) days p.i. (dpi) (Fig. 7) were evaluated for Venus (top) and NLuc (middle) expression. Viral plaques were determined by crystal violet staining (bottom). Representative images of BIRFLU obtained from one mouse are shown. To determine the percentage of reporter-expressing viruses, 160 plaques (40 plaques/mouse, 4 mice) for each day p.i. were evaluated for Venus (top), NLuc (bottom), and crystal violet (bottom) staining as indicated in the legend to Fig. 2. (B) In vitro genetic stability. To analyze the genetic stability in vitro, BIRFLU was passaged four times (P1 to P4) in MDCK cells and infectious virus-containing tissue culture supernatants were harvested and assessed for Venus (top) or NLuc (middle) expression, before crystal violet staining (bottom). The percentage of reporter-expressing viruses was determined from ∼70 to 100 viral plaques per passage. Representative images of BIRFLU obtained from each passage are shown.

FIG 10

Generation and characterization of mCherry-expressing BIRFLU. (A) Multicycle growth kinetics. Viral titers (in FFU per milliliter) from culture supernatants of MDCK cells (6-well plates, 10⁶ cells/well, triplicates) infected with PR8 WT or BIRFLU expressing mCherry (MOI, 0.001) were determined by immunofocus assay at the indicated times postinfection. Data represent the means ± SD for triplicates. The dashed line denotes the limit of detection (200 FFU/ml). *, P < 0.05, using an unpaired two-tailed Student's t test. (B and C) Reporter gene expression. NLuc activity was quantitated from the same culture supernatants (B), and mCherry expression was imaged using a fluorescence microscope (C). Representative images are shown. Bars, 100 μm; magnification, ×20. (D) Plaque phenotype. MDCK cells (6-well-plate format, 10⁶ cells/well) were infected with PR8 WT-mCherry and BIRFLU-mCherry, and viral plaques were evaluated at 3 days p.i. for mCherry fluorescence (top), NLuc immunostaining (middle), and crystal violet staining (bottom). White arrows indicate the colocalization of mCherry fluorescence (top), NLuc immunostaining (middle), and viral lysis plaques (bottom). (E) Analysis of protein expression. MDCK cells (6-well plates, 10⁶ cells/well) were infected with PR8 WT-mCherry or BIRFLU-mCherry at an MOI of 0.1 or mock infected (lane M). Protein expression was examined by Western blotting using specific antibodies for NS1, mCherry, NLuc, and NP. Actin was used as a loading control. Numbers on the left indicate the size of molecular markers for proteins (in kilodaltons).