A human H5N1 influenza virus expressing bioluminescence for evaluating viral infection and identifying therapeutic interventions

Abstract

A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in the United States dairy cattle was first reported on March 2024, followed by a zoonotic cattle-to-human virus transmission to a dairy farm worker in Texas. To facilitate real-time tracking of HPAIV H5N1, we generated a recombinant nanoluciferase (Nluc)-expressing H5N1 virus, HPhTX NSs-Nluc, by introducing an Nluc reporter into the non-structural gene of human A/Texas/37/2024 H5N1 (HPhTX). HPhTX NSs-Nluc replicated with kinetics and plaque morphology comparable to wild-type virus in vitro. In vivo and ex vivo, HPhTX NSs-Nluc allowed tracking viral infection in the living animals and their necropsied organs using in vivo imaging systems (IVISs). Treatment with baloxavir effectively inhibited HPhTX NSs-Nluc replication, comparable to wild-type virus, validating its applicability for high-throughput screening of potential antiviral therapeutics. These results demonstrate that HPhTX NSs-Nluc is a robust tool for studying H5N1 pathogenesis and assessing antiviral efficacy against HPAIV H5N1.

Keywords: Bioengineering; Methodology in biological sciences; Optical imaging; Virology.

Graphical abstract

Figure 1

In vitro characterization of HPhTX NSs-Nluc (A and B) Schematic representation of HPhTX NSs (A) and HPhTX NSs-Nluc (B) viral segments. The viral NS1 and NEP ORFs and PTV-1 2A sequences are represented in white, black, and gray, respectively. The Nluc ORF is represented in blue. (C) Multicycle growth kinetics of HPhTX, HPhTX NSs, and HPhTX NSs-Nluc as determined by plaque assay in MDCK cells (top); and Nluc activity from same cell culture supernatants (bottom). Data represent means and SD for triplicates. (D) Plaque phenotype of HPhTX, HPhTX NSs, and HPhTX NSs-Nluc in MDCK cells. Viral plaques were evaluated at 48 and 72 hpi via staining with Nluc substrate (left) and NP immunostaining (right). (E) Quantification of viral plaque sizes from (D). Data are represented as mean ± SD. A two-way repeated measure ANOVA with Geisser-Greenhouse correction. Post hoc multiple comparisons performed using Šídák or Bonferroni method to compare groups within each time point. The significant differences are indicated (ns, non-significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 2

HPhTX NSs-Nluc inhibits IFNβ promoter activation MDCK IFNβ-GFP/IFNβ-FFluc cells were mock infected or infected (MOI 1) with HPhTX, HPhTX NSs, HPhTX NSs-Nluc, or LPhTXdNS1 (control). At 12 hpi, IFNβ promoter activation was analyzed by GFP (A) and FFluc (B) expression. The scale bar was set for 100 μm. Nluc expression was evaluated in the cell culture supernatants of infected cells (C). Data are represented as mean ± SD. A Welch’s one-way ANOVA with Geisser-Greenhouse correction. Post hoc multiple comparisons performed using Dunnett’s method to compare groups within each time point. The significant differences are indicated (ns, non-significant, ∗∗∗∗p < 0.0001).

Figure 3

Stability of HPhTX NSs-Nluc in vitro MDCK cells were infected with serial passages of HPhTX NSs-Nluc. (A) Nluc expression in cell culture supernatants was evaluated for each of the viral passages. (B) Plaque assays of HPhTX NSs-Nluc collected at passages 1 (P1), 5 (P5), and 10 (P10) stained with Nluc substrate (left) or immunostained with the NP MAb HT103 (right). (C) Average plaque sizes of P1, P5, and P10. The average plaque sizes were determined from 10 plaques for each virus. (D) Next-generation sequencing data covering the NSs-Nluc segment across P1, P5, and P10. (E) Non-reference allele frequency. The passaged samples were compared to the NSs-Nluc reference sequence to identify variants. A single-nucleotide variant (SNV) allele frequency from P1, P5, and P10 is shown with circles. One variant HPhTX NSs-Nluc (nucleotide 1,538) was at high frequency in all three samples. Variants <25% frequency are not shown. Allele frequencies are provided in Table 2. The red line indicates 25% allele frequency. (F) Growth kinetics of HPhTX NSs-Nluc P0, P1, P5, and P10 in MDCK cells infected at MOI of 0.0001. Monolayers of MDCK cells were infected with the indicated passages of HPhTX NSs-Nluc (triplicates), and cell culture supernatants were collected at 12, 24, 48, and 72 hpi. Viral titers were calculated by standard plaque assay. (G) Nluc values in cell culture supernatants from cells infected in (E). Data are represented as mean ± SD. A two-way ANOVA with Geisser-Greenhouse correction. Post hoc multiple comparisons performed using Dunnett’s method to compare groups within each time point. The significant differences are indicated (ns, non-significant).

Figure 4

Pathogenicity of HPhTX NSs-Nluc in C57BL/6J mice Female 6-week-old C57BL/6J mice (n = 5) were inoculated with 10,10²,10³, and 10⁴ PFU of HPhTX NSs (A) or HPhTX NSs-Nluc (B) and monitored daily for 14 days for body weight (left) and survival (right). Data are represented as mean ± SD. Mice that lost 25% or greater of initial weight were humanely sacrificed. The MLD₅₀ was calculated by Reed and Muench method. Data represent the means and SD of the results for individual mice.

Figure 5

In vivo Nluc expression in HPhTX NSs-Nluc-infected C57BL/6J mice Female 6-week-old C57BL/6J mice infected in Figure 4 were monitored for Nluc expression at 1, 2, 4, 6, and 8 DPI using IVIS. Radiance, defined as the number of photons per s per square cm per steradian (p s⁻¹ cm⁻² sr⁻¹), is shown on the heatmap at the bottom.

Figure 6

In vivo and ex vivo imaging of Nluc in C57BL/6J mice infected with HPhTX NSs-Nluc (A) In vivo imaging of female 6-week-old C57BL/6J mice infected with the indicated doses of HPhTX NSs-Nluc at 2 and 4 DPI. (B) Ex vivo imaging of the lungs and brains of infected mice in (A) at 2 and 4 DPI. Radiance, defined as the number of photons per s per square cm per steradian (p s⁻¹ cm⁻² sr⁻¹), is shown on each of the indicated heatmaps.

Figure 7

Viral titers and Nluc expression in the NT, lung, and brain homogenates of HPhTX NSs-Nluc-infected C57BL/6J mice (A) Viral titers in the different tissues and DPI are represented as log₁₀ PFU/mL. (B) Nluc expression in tissue homogenates collected from the same NT, lungs, and brain tissues of mice infected with HPhTX NSs and HPhTX NSs-Nluc. The limit of detection (LOD) is indicated with a dashed line. Data are represented as mean ± SD. A mixed-effects ANOVA followed by Dunnett’s multiple comparisons test (ns, non-significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 8

Antiviral activity of BXA against HPhTX, HPhTX NSs, and HPhTX NSs-Nluc in vitro The IC₅₀ of BXA against HPhTX, HPhTX NSs, and HPhTX NSs-Nluc was determined by microneutralization assay (A) or Nluc activity (B). The percent neutralization is calculated using sigmoidal dose-response curves. Dotted line indicates 50% of viral inhibition. Data are represented as mean ± SD.

Figure 9

Antiviral activity of BXA against HPhTX NSs-Nluc in vivo Female 6-week-old C57BL/6J mice (n = 5/group) were mock treated/mock infected, mock treated/infected, or treated/infected. Mice treated with BXA received 15 mg/kg of BXA twice daily by oral gavage. Mice were challenged with 10² PFU of HPhTX NSs-Nluc virus at 6 h post treatment. (A and B) Mice were monitored for body weight changes (A) and survival (B) for 14 days. (C) Nluc expression in same groups of mice was monitored by IVIS on 2, 4, 6, and 8 DPI. Data are represented as mean ± SD. Radiance, defined as the number of photons per s per square cm per steradian (p s⁻¹ cm⁻² sr⁻¹), is shown on the heatmap at the bottom.

Figure 10

In vivo and ex vivo detection of Nluc expression and viral loads in HPhTX NSs-Nluc-infected mice following treatment with BXA (A) In vivo imaging of the entire mice and ex vivo imaging of lungs and brains at 6 DPI. Mice were challenged with 10² PFU of HPhTX NSs-Nluc virus at 6 h post treatment. Radiance, defined as the number of photons per s per square cm per steradian (p s⁻¹ cm⁻² sr⁻¹), is shown on the heat maps. (B and C) Viral titers (PFU/mL) (B) and Nluc expression (C) from the supernatants of homogenized NT, lung, and brain tissues from mice in (A). The LOD is indicated with a dashed line. Data are represented as mean ± SD. A mixed-effects ANOVA followed by Dunnett’s multiple comparisons test (ns, non-significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).