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Updated: Dec 12, 2025
Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2
Published on: April 10, 2009
Transcriptome & viral growth analysis of SARS-CoV-2-infected Vero CCL-81 cells
Dimpal A Nyayanit1, Prasad Sarkale1, Shreekant Baradkar1
1Maximum Containment Laboratory, ICMR-National Institute of Virology, Pune, Maharashtra, India.
Background & Objectives:
The 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.
Methods:
Vero 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.
Results:
In 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 & Conclusions:
This 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.
Insights
This study tracked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene expression in host cells. High expression of ORF8 and ORF7b genes suggests their crucial role in SARS-CoV-2 replication.
Area of Science:
- Virology
- Molecular Biology
- Genomics
Background:
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the Coronaviridae family, possesses genes encoding essential structural, non-structural, and accessory proteins for viral replication.
- Understanding the transcription patterns of these SARS-CoV-2 genes within host cells is crucial for comprehending viral kinetics.
Purpose of the Study:
- To investigate the transcription patterns of SARS-CoV-2 genes in Vero CCL-81 cells throughout the infection process.
- To assess the expression levels of specific viral genes during SARS-CoV-2 replication.
Main Methods:
- Vero CCL-81 cells were infected with SARS-CoV-2 at a 0.1 multiplicity of infection.
- Viral gene expression was analyzed using next-generation sequencing, mapping reads to the SARS-CoV-2 Wuhan HU-1 reference sequence.
- Quantification of viral load was performed using 50% tissue culture infective dose (TCID50) and cycle threshold (Ct) values for E and RdRp-2 genes.
Main Results:
- Viral gene copy numbers for E and RdRp-2 remained relatively stable for the first six hours post-infection before increasing significantly by 12 hours post-infection.
- Viral progeny release, indicated by TCID50, was detected in the supernatant after 7 hours post-infection.
- Higher expression levels were observed for ORF8, ORF7a, and nucleocapsid transcripts.
Conclusions:
- The study provides insights into the growth kinetics of the SARS-CoV-2 replication cycle.
- Elevated expression of ORF8 and ORF7b gene transcripts suggests their critical involvement in viral replication.
- Further research is warranted to elucidate the specific roles of these highly expressed genes in the SARS-CoV-2 life cycle.
