tetano
Editor, Senior Moderator
Nat Protoc
. 2023 Oct 13.
doi: 10.1038/s41596-023-00897-6. Online ahead of print. Generation of quality-controled SARS-CoV-2 variant stocks
Maren de Vries[SUP] 1 [/SUP], Grace O Ciabattoni[SUP] 1 [/SUP], Bruno A Rodriguez-Rodriguez[SUP] 1 [/SUP], Keaton M Crosse[SUP] 1 [/SUP], Dominick Papandrea[SUP] 2 [/SUP], Marie I Samanovic[SUP] 3 4 [/SUP], Dacia Dimartino[SUP] 5 [/SUP], Christian Marier[SUP] 5 [/SUP], Mark J Mulligan[SUP] 3 4 [/SUP], Adriana Heguy[SUP] 5 6 [/SUP], Ludovic Desvignes[SUP] 2 3 [/SUP], Ralf Duerr[SUP] 1 3 4 [/SUP], Meike Dittmann[SUP] 7 [/SUP]
Affiliations
One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research-for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5-8 d are required when virus is isolated from clinical swab material, and another 6-7 d is needed for sucrose-purifying the stocks.
. 2023 Oct 13.
doi: 10.1038/s41596-023-00897-6. Online ahead of print. Generation of quality-controled SARS-CoV-2 variant stocks
Maren de Vries[SUP] 1 [/SUP], Grace O Ciabattoni[SUP] 1 [/SUP], Bruno A Rodriguez-Rodriguez[SUP] 1 [/SUP], Keaton M Crosse[SUP] 1 [/SUP], Dominick Papandrea[SUP] 2 [/SUP], Marie I Samanovic[SUP] 3 4 [/SUP], Dacia Dimartino[SUP] 5 [/SUP], Christian Marier[SUP] 5 [/SUP], Mark J Mulligan[SUP] 3 4 [/SUP], Adriana Heguy[SUP] 5 6 [/SUP], Ludovic Desvignes[SUP] 2 3 [/SUP], Ralf Duerr[SUP] 1 3 4 [/SUP], Meike Dittmann[SUP] 7 [/SUP]
Affiliations
- PMID: 37833423
- DOI: 10.1038/s41596-023-00897-6
One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research-for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5-8 d are required when virus is isolated from clinical swab material, and another 6-7 d is needed for sucrose-purifying the stocks.