VLP-Based Model for Study of Airborne Viral Pathogens

Abstract

The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution, as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus like particles (VLP). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling study of BSL3 and BSL4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins including those of newly discovered pathogens and mutant variants; (v) the ability to introduce viral sequences to develop nucleic acid amplification assays.

Figure 1:. Western blot analysis for presence of SARS-CoV-2 spike in VLP and inactivated SARS-CoV-2.

For this analysis, equivalent amounts of total protein were used.

Figure 2:. Real-time quantitative fluorescent LAMP analysis to detect plasmid.

(a) Amplification plots of solutions containing 3.1 × 10⁹, 3.1 × 10⁷, 3.1 × 10⁶, 3.1 × 10⁵, 3.1 × 10⁴, and 3.1 × 10³ gene copies (left to right). The control experiment, which contains no template, is shown as closed circles. (b) Standard curves for the Cq values versus gene copy number. Plasmid pNL4–3.Luc.R-E-. was used for these experiments.

Figure 3:. Temperature and sensitivity limits for detection by LAMP.

(a) LAMP colorimetric assay for different temperatures (no template control on the left and added plasmid on the right). (b) Colorimetric LAMP assay. (c) SYBR Green stained agarose gel of LAMP reaction products. M=Marker, 1=NTC, 2=50 copies, 3=25 copies, 4=12.5 copies, 5= 6.75 copies.

Figure 4:. Real-time quantitative fluorescent Reverse-Transciption LAMP.

For these experiments, heat treatment corresponded to incubation of VLP at 95 °C for 10 minutes before analysis and detergent treatment corresponded to VLP dilution into a buffer containing 12.5 mM TCEP, 5 mM EDTA, and 0.002% Triton X100 at pH 8.0.

Figure 5:. Detection of VLP by RT-LAMP.

(a) Colorimetric RT-LAMP assay. (b) SYBR Green stained agarose gel of LAMP reaction products. M=Marker, 1=NTC, 2=300 copies, 3=30 copies, 4=3 copies, 5= 0.3 copies.

Figure 6:

Experimental setup for aerosolized VLP.

Figure 7:. Detection of aerosolized VLP on a MCE filter.

(a) Colorimetric RT-LAMP assay. (b) SYBR Green stained agarose gel of LAMP reaction products. M=Marker, 1=NTC, 2=Filter Extract, 3=Filter Extract 1/10 dilution, 4=Filter Extract 1/100 dilution.