Ultra-rapid inactivation of SARS-CoV-2 with 266 nm lasers

SARS-CoV-2/Wuhan/WIV04/2019 (SARS-CoV-2 WIV04) and SARS-CoV-2/630–1 (SARS-CoV-2 delta) were included in our experiments to verify the efficacy of the laser inactivation. Sindbis virus (SINV) with a similar molecular structure (also an enveloped RNA virus) but a different host cell was used to confirm that the surrounding environment can affect viral sensitivity to laser irradiation. Pseudorabies virus (PRV) with DNA as genetic material was used for comparison with RNA viruses since the difference in mechanisms between UV-damaged DNA and RNA, i.e. i.e. one of the disinfection mechanisms by UV irradiation for DNA is the formation of TT dimers, while for RNA it is the formation of UU dimers. Human enterovirus 71 (EV71) and porcine parvovirus (PPV) with non-enveloped RNA/DNA were used to test for any interactions between the viral envelope and UV photons that may affect viral susceptibility.

Cell lines and culture

The cell lines used in the experiments included Vero E6, BHK, PK15, ST and RD cells from the National Virus Resource Center of the Wuhan Institute of Virology, Chinese Academy of Sciences, which were cultured in essential medium minimum (MEM, Gibco™, Cat No: 42360032) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10099-141) and 100 U/mL penicillin and streptomycin each (Gibco, 15140-122) at 37°C in 5% CO solution2 incubator. The viruses and cells used in our experiments are shown in Table 1.

Table 1 Viruses and cells used in the study.

Viral proliferation

SARS-CoV-2 WIV04 and SARS-CoV-2 delta were propagated in Vero E6 cells. Vero E6 cells were seeded in a T-75 cell culture flask overnight and viruses were inoculated into the culture at a multiplicity of infection (MOI) = 0.1 when the cells were 80% confluent. The infected cell culture flask was placed in a 37°C incubator for virus adsorption for 1 h, and fresh medium of MEM + 2% FBS was replaced. Supernatants from infected cells were collected in a 15 ml centrifuge tube two days later. Cell fragments were discarded after centrifugation at 3000 rpm at 4°C for 10 min, and viruses were obtained. The viruses obtained were separated and frozen in a refrigerator at -80°C for later use.23.

Additionally, SINV was propagated in BHK cells at 0.01 MOI, EV71 was propagated in RD cells at 0.01 MOI, PRV was propagated in PK15 cells at 0.1 MOI, and PPV was propagated in ST cells at 0.1 MOI, and they were harvested at 24 h, 24 h, 48 h and 72 h respectively. The media they suspended was still MEM + 2% FBS. They were harvested in the same way as SARS-CoV-2. All experiments involving SARS-CoV-2 experiments were performed in a Biosafety 3 (P3) laboratory, and the SINV, PRV, EV71, and PPV experiments were performed in a Biosafety 2 (P2) laboratory. All viruses were obtained from the National Virus Resource Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

Virus titration

Vero E6, BHK, RD, ST and PK15 cells were seeded in 6-well plates overnight. The cell inoculation density was 104 per well. A strain of virus stored in the refrigerator at -80°C was sampled. After virus thawing, MEM + 2% FBS was used to perform a ten-fold gradient dilution, with a total of 9 dilutions from 10−1 at 10−9, and 6 to 8 repetitions were made for each dilution. Three days after inoculation, the cytopathic effect (CPE) was noted and the Reed-Muench formula was used to calculate the 50% tissue culture infectious dose (TCID50)24. Virus inoculum titer started at ~6 Lg TCID50/0.1ml25. The lower titer limit was 1 Lg TCID50/0.1 ml (completely inactivated).

Laser

An in-house built pulsed laser with a 10 ps pulse width, 200 kHz repetition rate and adjustable output power was used. The beam diameter is approximately 5.16 mm at the facet of the laser head and the beam divergence is approximately 18 mrad. The wavelength tested was 266 nm, which is close to the maximum of RNA absorption at around 260 nm15. The laser spectrum is shown in Fig. 2 recorded by HR4000CG-UV-NIR (Ocean optics).

Figure 2

Inactivation of SARS-CoV-2 by UV irradiation

A 0.1 ml droplet of viral inoculum was placed in a defined well of a 6-well plate. Next, the laser head was positioned about 10 cm above the well containing the virus. The laser spot size was 0.785 cm2 or 0.601cm2 (fully covering the surface of the viral inoculum droplet in the well). After exposure for the designated time (controlled using a laser shutter, GCI-7102 M Daheng Optics Ltd.), each virus inoculum was subjected to virus titration. The number of repetitions performed in our experiments is three.

Data analysis

UV rate constant k is equipped with η= 1 e−kDwhere the inactivation efficiency η is the decreasing titer percentage and D is the UV exposure dose. The k values ​​are developed using the first 2 points for SARS-CoV-2 WIV04 and the first 3 points for other viruses. This is because the titer for the third dose from small to large in SARS-CoV-2 WIV04 reaches the lower limit (1 Lg TCID50/0.1 ml), whereas in the other viruses this appears for the 4th dose. Once kis obtained, the dose required for the indicated inactivation efficiency, for example 90%, 99%, 99.9% and 99.99%, can also be calculated using η= 1e−kD. The time required for the indicated inactivation efficiency is calculated using D =Iyou.

The absorptions and reflections from the MEM medium and the 6-well plate are included in Supplementary Material 1. The percentage of energy absorbed in the MEM medium is 39.69% in our experimental analysis. The absorbed laser dose Dabsorbed could be calculated as Dabsorbed= 39.69% Dirradiated where Dirradiated is the irradiated laser dose. UV rate constant kabsorbed can be easily calculated as kabsorbed= kirradiated/39.69%. For simplicity, we didn’t do the conversion and only used Dirradiated and kirradiated asDandkin the following analysis.

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