VLP-based model for the 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 (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification 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 the study of biosafety level 3 and biosafety level 4 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; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays.

Importance: The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

Keywords: RT-LAMP; VLP; aerosols; airborne microorganisms; coronavirus; influenza; nucleic acid technology.

Fig 1

Western blot analysis for the presence of SARS-CoV-2 spike in VLP and inactivated SARS-CoV-2. For this experiment, monoclonal antibody CR-3022 (BEI Resources) was used with equivalent amounts of total protein in each lane. Heat-inactivated and gamma-irradiated SARS-CoV-2 were obtained from BEI Resources.

Fig 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 in 20 µL of assay mixture (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 in the reaction mix. Plasmid pNL4-3.Luc.R-E- was used for these experiments. The curves shown represent averages of triplicate experiments, and the error bars represent the estimated standard deviation.

Fig 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; and 5, 6.75 copies. Gene copies correspond to the number in 20 µL of assay mixture.

Fig 4

Real-time quantitative fluorescent reverse-transcription LAMP. For these experiments, heat treatment corresponded to the incubation of VLP at 95°C for 10 min before analysis, and detergent treatment corresponded to VLP dilution into a buffer containing 12.5 mM TCEP, 5 mM EDTA, and 0.002% Triton X-100 at pH 8.0. The curves shown represent averages of triplicate experiments.

Fig 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; and 5, 0.3 copies. Gene copies correspond to the number of copies in 20 µL of the assay mixture.

Fig 6

Experimental setup for aerosolized VLP. Aerosolization experiments were performed with a custom-built cabinet in a biosafety cabinet located in a BSL2 laboratory. Aerosolization of VLP is achieved using the BANG from CH Technologies in the MPA mode. Aerosolized droplets are collected using Millipore 13 mm 0.45 µm MCE filters.

Fig 7

Detection of aerosolized VLP on an MCE filter. VLPs were aerosolized as described in Materials and Methods. (a) Colorimetric RT-LAMP assay of filter extracts after 40 min at 65°C. (b) SYBR Green-stained agarose gel of RT-LAMP reaction products from filter extracts. M, marker; 1, NTC; 2, filter extract; 3, filter extract 1/10 dilution; and 4, filter extract 1/100 dilution.