Coronavirus pseudovirus packaging system, packaging method therefor, and application of coronavirus pseudovirus in evaluating disinfection efficacy
A packaging system for a coronavirus pseudovirus, including a vesicular stomatitis virus (VSV) vector in which Fluc and EGFP dual-reporter genes replace a GP gene, and packaging cell that expresses a coronavirus spike protein. The packaging system may quickly package pseudoviruses by using a one-step packaging method, and may be used in the research of coronaviruses such as COVID-19 (SARS-CoV-2), SARS (SARS-CoV) and MERS, and other viruses. The packaging system and thereby pseudovirus method may also be used to evaluate the efficacy of disinfectants by means of virus contamination distribution models, setting up scenarios, and sampling and testing steps.
1 . A coronavirus pseudovirus packaging system, comprising a modified vesicular stomatitis virus (VSV) and a packaging cell that expresses a coronavirus spike protein; wherein the coronavirus is SARS, MERS, or COVID-19 virus, and the amino acid sequence of the spike protein is set forth in SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7; the modified vesicular stomatitis virus VSV is defined as a replication-defective virus with structural gene replaced by Fluc and EGFP dual-reporter genes, the modified vesicular stomatitis virus VSV is named as dVSVΔG-Fluc-EGFP, a gene coding GP in the genetic material of the VSV is replaced by Fluc reporter gene, the EGFP reporter gene is integrated between Fluc and VSV polymerase L gene, and the gene sequence of the dVSVΔG-Fluc-EGFP is set forth in SEQ ID NO: 3.
2 . The coronavirus pseudovirus packaging system according to claim 1 , wherein the packaging cell is selected from 293, 293T, 293sus, HEK293, HEK293T, HEK293FT, BHK, or Vero, and the packaging cell transiently or stably or inductively expresses the coronavirus spike protein, the transient expression is realized by transfecting the cell with an eukaryotic expression vector, the stable expression is realized by transducing the cell with a lentiviral vector system, and the induced expression is realized by transducing the cell with a tetracycline-regulated tet-on/off vector system.
3 . A one-step packaging method for a pseudovirus packaging system, wherein the pseudovirus packaging system comprises the coronavirus pseudovirus packaging system according to claim 1 , expression of the coronavirus spike protein is mediated by a transient expression plasmid or a stable expression plasmid or a stable and inducible expression lentivirus vector, dVSVΔG-Fluc-EGFP and the packaging cell that expresses the coronavirus spike protein are mixed in one step, and supernatant is collected after a certain time to obtain the coronavirus pseudovirus.
4 . The one-step packaging method for the pseudovirus packaging system according to claim 3 , comprising the following steps:
(1) adding dVSVΔG-Fluc-EGFP to 293T cell that transiently or stably or inductively expresses VSV envelope protein GP, collecting supernatant after 24 h to obtain the amplified VSV replication-defective virus, and measuring its titer; and
(2) passaging the packaging cell 293T that transiently or stably or inductively expresses the coronavirus spike protein into a 60 mm dish, adding dVSVΔG-Fluc-EGFP, wherein multiplicity of infection MOI is 0.1 to 5, culturing in an incubator at 32° C. to 37° C., harvesting pseudovirus supernatant after 24 h, then treating with anti-VSV neutralizing antibody for 2 h, and filtering with 0.22 μm filter membrane to obtain coronavirus pseudovirus.
5 . A coronavirus pseudovirus packaged by the coronavirus pseudovirus packaging system according to claim 1 as a biological indicator to replace a wild-type coronavirus for detection and evaluation of efficacy of a biological and chemical substance and a physical treatment method for inhibiting and disinfecting coronavirus, wherein the substance and the method for inhibiting and disinfecting coronavirus comprise an anti-coronavirus neutralizing antibodies, macromolecule and small-molecule drugs, physical virucidal disinfection methods, and chemical virucidal disinfectants.
6 . A method of evaluating efficacy of a virucidal disinfectant using a coronavirus pseudovirus, comprising the following steps:
(1) construction of virus-contaminated environment simulating distribution of a target virus under a virucidal disinfectant evaluation scene, including the existence of medium, temperature, humidity and gas disturbance, through analysis of virus contamination distribution models;
diluting the packaged coronavirus pseudovirus, uniformly smearing the diluted coronavirus pseudovirus on a medium, and setting environmental parameters of evaluation scenarios;
(2) determination of concentration of coronavirus pseudovirus before virucidal disinfection treatment based on the evaluation requirements, performing standard virus characteristic detection before treatment with sampling the coronavirus pseudovirus at different positions and different points; and
(3) sampling and determination during and after virucidal disinfection treatment uniformly spraying or smearing the virucidal disinfectant on the medium;
based on the evaluation requirements, selecting the positions and points selected in the step (2), sampling the coronavirus pseudovirus at different times, and detecting titer activity of the coronavirus pseudovirus.
7 . The method according to claim 6 , wherein the virus-contaminated environment in the step (1) comprises a logistics environment, a home environment, a public place environment, and a school environment.
8 . The method according to claim 6 , wherein multiple experimental groups are constructed in the step (1) to avoid excessive errors, and the step (3) comprises observing expression of fluorescent protein and luciferase after 293T-hACE2 is infected by the pseudovirus for measurement and calculation of infection capacity and bioactivity titer (TCID50 method, unit: PFU/ml) of the pseudovirus as well as detection of copy number of the pseudovirus nucleic acid (PCR method).
9 . The method according to claim 6 , wherein the virucidal disinfectant (peroxides, quaternary ammonium salts, chlorine-containing compounds, and alcohols) and the physical treatment method in the step (3) comprises combinations of one or more of ozone, peroxyacetic acid, hydrogen peroxide, chlorine dioxide, oxydol, sodium dichloroisocyanurate, ultraviolet light, negative ions, irradiation, or the like.
10 . The method according to claim 6 , wherein the medium in the step (1) and step (3) comprises one or more of a plastic, a foam, a bookbinding paperboard, a boxboard, a textile, and a metal foil.