tetano
Editor, Senior Moderator
Indian J Med Res
. 2020 Jul 30.
doi: 10.4103/ijmr.IJMR_2257_20. Online ahead of print.
Transcriptome & viral growth analysis of SARS-CoV-2-infected Vero CCL-81 cells
Dimpal A Nyayanit[SUP] 1 [/SUP], Prasad Sarkale[SUP] 1 [/SUP], Shreekant Baradkar[SUP] 1 [/SUP], Savita Patil[SUP] 1 [/SUP], Pragya D Yadav[SUP] 1 [/SUP], Anita Shete-Aich[SUP] 1 [/SUP], Kaumudi Kalele[SUP] 1 [/SUP], Pranita Gawande[SUP] 1 [/SUP], Triparna Majumdar[SUP] 1 [/SUP], Rajlaxmi Jain[SUP] 1 [/SUP], Gajanan Sapkal[SUP] 2 [/SUP]
Affiliations
Abstract
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 (TCID 50") 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 TCID 50 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.
Keywords: India - next-generation sequencing - RT-PCR - replication cycle - SARS-CoV-2 - transcriptome.
. 2020 Jul 30.
doi: 10.4103/ijmr.IJMR_2257_20. Online ahead of print.
Transcriptome & viral growth analysis of SARS-CoV-2-infected Vero CCL-81 cells
Dimpal A Nyayanit[SUP] 1 [/SUP], Prasad Sarkale[SUP] 1 [/SUP], Shreekant Baradkar[SUP] 1 [/SUP], Savita Patil[SUP] 1 [/SUP], Pragya D Yadav[SUP] 1 [/SUP], Anita Shete-Aich[SUP] 1 [/SUP], Kaumudi Kalele[SUP] 1 [/SUP], Pranita Gawande[SUP] 1 [/SUP], Triparna Majumdar[SUP] 1 [/SUP], Rajlaxmi Jain[SUP] 1 [/SUP], Gajanan Sapkal[SUP] 2 [/SUP]
Affiliations
- PMID: 32773420
- DOI: 10.4103/ijmr.IJMR_2257_20
Abstract
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 (TCID 50") 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 TCID 50 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.
Keywords: India - next-generation sequencing - RT-PCR - replication cycle - SARS-CoV-2 - transcriptome.