A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells.
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ABSTRACT:
SUBMITTER: Funnell SGP
PROVIDER: S-EPMC8211640 | biostudies-literature |
REPOSITORIES: biostudies-literature
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| EMPIAR-10753 | biostudies-other
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| S-EPMC7303940 | biostudies-literature
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| EMPIAR-11417 | biostudies-other
Project description:BACKGROUND:Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV). It is an enveloped, single-stranded, plus-sense RNA virus with a genome of approximately 30 kb. The structural proteins E, M and N of SARS-CoV play important roles during host cell entry and viral morphogenesis and release. Therefore, we have studied whether expression of these structural proteins can be down-regulated using an antisense technique. METHODS:Vero E6 cells were transfected with plasmid constructs containing exons of the SARS-CoV structural protein E, M or N genes or their exons in frame with the reporter protein EGFP. The transfected cell cultures were treated with antisense phosphorothioated oligonucleotides (antisense PS-ODN, 20mer) or a control oligonucleotide by addition to the culture medium. RESULTS:Among a total of 26 antisense PS-ODNs targeting E, M and N genes, we obtained six antisense PS-ODNs which could sequence-specifically reduce target genes expression by over 90% at the concentration of 50 microM in the cell culture medium tested by RT-PCR. The antisense effect was further proved by down-regulating the expression of the fusion proteins containing the structural proteins E, M or N in frame with the reporter protein EGFP. In Vero E6 cells, the antisense effect was dependent on the concentrations of the antisense PS-ODNs in a range of 0-10 microM or 0-30 microM. CONCLUSIONS:The antisense PS-ODNs are effective in downregulation of SARS. The findings indicate that antisense knockdown of SARS could be a useful strategy for treatment of SARS, and could also be suitable for studies of the pathological function of SARS genes in a cellular model system.
| S-EPMC7166999 | biostudies-literature
Project description:BackgroundThe coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter.ObjectiveThe objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level.MethodsIn this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients.FindingsThe infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles.Main conclusionsTherefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.
| S-EPMC7874846 | biostudies-literature
Project description:We conducted a high-throughput drug repositioning screen using the LOPAC®1280 and the ReFRAME drug libraries to identify existing drugs that harbor antiviral activity against SARS-CoV-2, in a Vero E6 cell-based assay. We additionally performed RNA sequencing on control and SARS-CoV-2 infected Vero E6 cells to study the biological changes after SARS-CoV-2 infection and to elucidate the potential mechanisms underlying the positive hits identified from our high-throughput screen. Vero E6 cells were either mock-infected or infected with SARS-CoV-2 USA-WA1/2020 (MOI = 0.3) with three replicates. Cells were harvested 24 hours after infection, and total RNA was extracted using the Qiagen® RNeasy® Plus Mini Kit. The quality of the extracted RNA was assessed with the Agilent® 2100 Bioanalyzer. Libraries were prepared from total RNA following ribosome RNA depletion using standard protocol according to Illumina®. Total RNA sequencing was then performed on the Illumina® NextSeq system; 150bp paired-end runs were performed and 100 million raw reads per sample were generated.
2020-07-08 | GSE153940 | GEO
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, and scientists around the world are currently studying the virus intensively in order to fight against the on-going pandemic of the virus. To do so, SARS-CoV-2 is typically grown in the lab to generate viral stocks for various kinds of experimental investigations. However, accumulating evidence suggests that such viruses often undergo cell culture adaptation. Here, we systematically explored cell culture adaptation of two SARS-CoV-2 variants, namely the B.1.36.16 variant and the AY.30 variant, a sub lineage of the B.1.617.2 (Delta) variant, propagated in three different cell lines, including Vero E6, Vero E6/TMPRSS2, and Calu-3 cells. Our analyses detected numerous potential cell culture adaptation changes scattering across the entire virus genome, many of which could be found in naturally circulating isolates. Notable ones included mutations around the spike glycoprotein's multibasic cleavage site, and the Omicron-defining H655Y mutation on the spike glycoprotein, as well as mutations in the nucleocapsid protein's linker region, all of which were found to be Vero E6-specific. Our analyses also identified deletion mutations on the non-structural protein 1 and membrane glycoprotein as potential Calu-3-specific adaptation changes. S848C mutation on the non-structural protein 3, located to the protein's papain-like protease domain, was also identified as a potential adaptation change, found in viruses propagated in all three cell lines. Our results highlight SARS-CoV-2 high adaptability, emphasize the need to deep-sequence cultured viral samples when used in intricate and sensitive biological experiments, and illustrate the power of experimental evolutionary study in shedding lights on the virus evolutionary landscape.
| S-EPMC10078795 | biostudies-literature
Project description:RNAseq was used to identify host and viral transcriptome changes in SARS-CoV-2 infection in Vero E6 cells at 8hpi.
2020-11-21 | GSE161881 | GEO
Project description:Helium ion microscopy (HIM) offers the opportunity to obtain direct views of biological samples such as cellular structures, virus particles, and microbial interactions. Imaging with the HIM combines sub-nanometer resolution, large depth of field, and high surface sensitivity. Due to its charge compensation capability, the HIM can image insulating biological samples without additional conductive coatings. Here, we present an exploratory HIM study of SARS-CoV-2 infected Vero E6 cells, in which several areas of interaction between cells and virus particles, as well as among virus particles, were imaged. The HIM pictures show the three-dimensional appearance of SARS-CoV-2 and the surface of Vero E6 cells at a multiplicity of infection of approximately 1 with great morphological detail. The absence of a conductive coating allows for a distinction between virus particles bound to the cell membrane and virus particles lying on top of the membrane. After prolonged imaging, it was found that ion-induced deposition of hydrocarbons from the vacuum renders the sample sufficiently conductive to allow for imaging even without charge compensation. The presented images demonstrate the potential of the HIM in bioimaging, especially for the imaging of interactions between viruses and their host organisms.
| S-EPMC7871036 | biostudies-literature
Project description:Background & objectivesThe genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the family Coronaviridae, encodes for structural, non-structural, and accessory proteins, which are required for replication of the virus. These proteins are encoded by different genes present on the SARS-CoV-2 genome. The expression pattern of these genes in the host cells needs to be assessed. This study was undertaken to understand the transcription pattern of the SARS-CoV-2 genes in the Vero CCL-81 cells during the course of infection.MethodsVero CCL-81 cells were infected with the SARS-CoV-2 virus inoculum having a 0.1 multiplicity of infection. The supernatants and cell pellets were harvested after centrifugation at different time points, post-infection. The 50% tissue culture infective dose (TCID50)and cycle threshold (Ct) values of the E and the RdRp-2 genes were calculated. Next-generation sequencing of the harvested sample was carried out to observe the expression pattern of the virus by mapping to the SARS-CoV-2 Wuhan HU-1 reference sequence. The expressions were in terms of the reads per kilobase million (RPKM) values.ResultsIn the inital six hours post-infection, the copy numbers of E and RdRp-2 genes were approximately constant, which raised 10 log-fold and continued to increase till the 12 h post-infection (hpi). The TCID50 was observed in the supernatant after 7 hpi, indicating the release of the viral progeny. ORF8 and ORF7a, along with the nucleocapsid transcript, were found to express at higher levels.Interpretation & conclusionsThis study was a step towards understanding the growth kinetics of the SARS-CoV-2 replication cycle. The findings indicated that ORF8 and ORF7b gene transcripts were expressed in higher amounts indicating their essential role in viral replication. Future studies need to be conducted to explore their role in the SARS-CoV-2 replication.
| S-EPMC7853258 | biostudies-literature