Generation and Functional Analysis of Defective Viral Genomes during SARS-CoV-2 Infection.

Defective viral genomes (DVGs) have been identified in many RNA viruses as a major factor influencing antiviral immune response and viral pathogenesis. However, the generation and function of DVGs in SARS-CoV-2 infection are less known. In this study, we elucidated DVG generation in SARS-CoV-2 and its relationship with host antiviral immune response. We observed DVGs ubiquitously from transcriptome sequencing (RNA-seq) data sets of in vitro infections and autopsy lung tissues of COVID-19 patients. Four genomic hot spots were identified for DVG recombination, and RNA secondary structures were suggested to mediate DVG formation. Functionally, bulk and single-cell RNA-seq analysis indicated the interferon (IFN) stimulation of SARS-CoV-2 DVGs. We further applied our criteria to the next-generation sequencing (NGS) data set from a published cohort study and observed a significantly higher amount and frequency of DVG in symptomatic patients than those in asymptomatic patients. Finally, we observed exceptionally diverse DVG populations in one immunosuppressive patient up to 140 days after the first positive test of COVID-19, suggesting for the first time an association between DVGs and persistent viral infections in SARS-CoV-2. Together, our findings strongly suggest a critical role of DVGs in modulating host IFN responses and symptom development, calling for further inquiry into the mechanisms of DVG generation and into how DVGs modulate host responses and infection outcome during SARS-CoV-2 infection. IMPORTANCE Defective viral genomes (DVGs) are generated ubiquitously in many RNA viruses, including SARS-CoV-2. Their interference activity to full-length viruses and IFN stimulation provide the potential for them to be used in novel antiviral therapies and vaccine development. SARS-CoV-2 DVGs are generated through the recombination of two discontinuous genomic fragments by viral polymerase complex, and this recombination is also one of the major mechanisms for the emergence of new coronaviruses. Focusing on the generation and function of SARS-CoV-2 DVGs, these studies identify new hot spots for nonhomologous recombination and strongly suggest that the secondary structures within viral genomes mediate the recombination. Furthermore, these studies provide the first evidence for IFN stimulation activity of de novo DVGs during natural SARS-CoV-2 infection. These findings set up the foundation for further mechanism studies of SARS-CoV-2 recombination and provide evidence to harness the immunostimulatory potential of DVGs in the development of a vaccine and antivirals for SARS-CoV-2.

Preparation of noninfectious scRNAseq samples from SARS-CoV-2-infected epithelial cells.

Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, there are only 190 BSL3 laboratory facilities registered with the U.S. Federal Select Agent Program, as of 2020, and only a subset of these are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility-thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. Briefly, we infected African green monkey kidney epithelial cells (Vero-E6) with SARS-CoV-2 for 96 hours, trypsin-dissociated the cells, and inactivated them with methanol-acetone in a single-cell suspension. Fixed cells were tested for the presence of infectious SARS-CoV-2 virions using the Tissue Culture Infectious Dose Assay (TCID50), and also tested for viability using flow cytometry. We then tested the dissociation and methanol-acetone inactivation method on primary human lung epithelial cells that had been differentiated on an air-liquid interface. Finally, we performed scRNAseq quality control analysis on the resulting cell populations to evaluate the effects of our virus inactivation and sample preparation protocol on the quality of the cDNA produced. We found that methanol-acetone inactivated SARS-CoV-2, fixed the lung epithelial cells, and could be used to obtain noninfectious, high-quality cDNA libraries. This methodology makes investigating SARS-CoV-2, and related high-containment RNA viruses at a single-cell level more accessible to an expanded community of researchers.