Infectious viruses from transfected SARS-CoV-2 genomic RNA

Abstract

SARS-CoV-2 emerged at the end of 2019, and like other novel pathogens causing severe symptoms, WHO recommended heightened biosafety measures for laboratories working with the virus. The positive-stranded genomic RNA of coronaviruses has been known to be infectious since the 1970s, and overall, all experiments with the possibility of SARS-CoV-2 propagation are carried out in higher containment level laboratories. However, as SARS-CoV-2 RNA has been routinely handled in BSL-2 laboratories, the question of the true nature of RNA infectiousness has risen along with discussion of appropriate biosafety measures. Here, we studied the ability of native SARS-CoV-2 genomic RNA to produce infectious viruses when transfected into permissive cells and discussed the biosafety control measures related to these assays. In transfection assays large quantities of genomic vRNA of SARS-CoV-2 was required for a successful production of infectious viruses. However, the quantity of vRNA alone was not the only factor, and especially when the transfected RNA was derived from infected cells, even small amounts of genomic vRNA was enough for an infection. Virus replication was found to start rapidly after transfection, and infectious viruses were detected in the cell culture media at 24 h post-transfection. In addition, silica membrane-based kits were shown to be as good as traditional TRI-reagent based methods in extracting high-quality, 30 kb-long genomic vRNA. Taken together, our data indicates that all transfection experiments with samples containing genomic SARS-CoV-2 RNA should be categorized as a propagative work and the work should be conducted only in a higher containment BSL-3 laboratory.

Keywords: SARS-CoV-2; biosafety; genomic RNA; infectivity; transfection.

FIGURE 1

(A) Flowchart of the RNA transfection experiments and infectivity analysis. VeroE6-TMPRSS2 (VE6-T2) cells were infected with a low MOI of SARS-CoV-2 and vRNA was harvested either from the culture media or from infected cells. After RNA transfection to VE6-T2 cells, cytopathic effect (CPE) as an indication of infection was monitored, and media and cell samples were collected during 3–4 days of incubation. The infectious viruses in the media samples were titrated with cultivation in VE6-T2 cells in an endpoint dilution assay, or vRNA was quantified from the media or cell samples with RT-qPCR with SARS-CoV-2 E gene specific assay (negative control wells). (B) Gel analysis of the RNA on a native agarose gel. RNA samples (1 µg/well) were run on a 0.8% native agarose gel. RNA samples in the native state can form secondary structures that may run faster or differently than linear DNA ladder. Therefore, the size of vRNA or rRNA may appear lower than DNA ladder in the gel. M, GeneRuler 1 kb DNA ladder (Thermo Fisher; the highest band is 10 kb); TRI, RNA extracted with TRIsure reagent; RNeasy, RNA extracted with RNeasy Mini kit. The red arrow indicates the genomic vRNA of SARS-CoV-2, and blue arrows indicate 28S and 18S rRNA, respectively.

FIGURE 2

Correlation of the transfected RNA amount to the production of infectious viruses. Different amounts of SARS-CoV-2 vRNA (concentrated genomic vRNA or total cellular RNA from virus-infected cells) were transfected into VeroE6-TMPRSS2 cells and the formation of CPE by infectious viruses was monitored. The presence or lack of infectious viruses was confirmed by an endpoint dilution assay. The graph shows the percentage of wells with an infection after transfection (both CPE and TCID₅₀ titer positive). The means and standard errors of the means have been calculated from the following number of experiments (the total number of replicates in parenthesis): 500 ng, 1 (3); 200 ng, 10 (63); 100 ng, 5 (39); 50 ng, 2 (15); 20 ng, 3 (30); 2 ng, 3 (30); 200 ng kit, 7 (58); Cell. RNA, TRIsure, 5 (46); Cell. RNA, RNeasy, 2 (20). Cell. RNA, total cellular RNA from virus-infected cells; TRIsure, RNA extracted with TRIsure reagent; RNeasy, RNA extracted with RNeasy Mini kit. p < 0.05 (*), p < 0.001 (***), not significant (ns).

FIGURE 3

Daily follow-up of the appearance of infectious viruses into media post-transfection. Supernatant samples were collected daily from transfected wells and analyzed with an endpoint dilution assay for the presence of infectious viruses. The figure presents the TCID₅₀/ml results from daily samples only from wells in which infectious viruses were observed at 72 h timepoint. The means and standard errors of the means have been calculated from the following number of experiments (the total number of replicates in parenthesis): 500 ng TRIsure, 1 (3); 200 ng TRIsure, 3 (13); 200 ng RNeasy, 4 (14). The results for total cellular RNA transfection are from a single positive well for both TRIsure and RNeasy extracted RNA. TRIsure, RNA extracted with TRIsure reagent; RNeasy, RNA extracted with RNeasy Mini kit.