COVID-19 Research: Using Vero E6 Cells In Vitro Studies Might Not Model Key Aspects Of SARS-CoV-2 Viral Life Cycle As Virus Mutates Fast To Adapt

COVID-19 Research: Scientists from Erasmus Medical Center-Netherlands and the University of Illinois at Urbana-Champaign-USA have found that the SARS-CoV-2 rapidly adapts to Vero E6 cell culture propagation that is often used in in vitro studies, hence not proper representing the actual viral life cycle as would be in a typical human host setting and can sometimes also lead to misleading laboratory study results.


 
In the study abstract, the research team said, “Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein that facilitates serine protease-mediated entry into human airway cells. We report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases-prevents MBCS mutations. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.01.22.427802v1
 
While the COVID-19 pandemic continues to wreak havoc across the globe, with almost 2.16 deaths caused by it so far including more than 425,200 Americans and 100,400 British citizens perishing, scientists race to further understand the virus and how it infects the body. This way, safe and effective therapeutics and other potential preventive measures can be developed to bring surging case-loads across the globe under control.
 
The pandemic which is caused by the SARS-CoV-2 virus has now taken a new turn with the emergence of various new variants, some more infectious and some more deadly. Among one variant that is far more infectious and is fast becoming dominant is the B.1.1.7 variant that originated in the United Kingdom. This new variants through increased transmissibility, has contributed to surging cases worldwide. To date, there have been nearly more than 100.3 million cases confirmed globally.
 
The study team from Netherlands and the United States found that SARS-CoV-2 rapidly adapts to Vero E6 cell culture propagation and that this can be prevented by using cell lines with an active serine protease-mediated entry pathway.
 
The researchers also noted that propagating SARS-Cov-2 on the human airway cell line Calu-3, which expresses serine proteases, can help prevent multibasic cleavage site (MBCs) mutation.
 
Similar to any other virus, the SARS-CoV-2 tends to mutate, causing the emergence of new variants.
 
Studies and COVID-19 vaccine development commenced many months ago, and a few vaccine candidates have already been approved for roll out in many countries, including the UK, the US, and Canada.
 
But the question of whether or not new variants pose a threat to the current vaccines’ efficacy is still unclear. The continual emergence of new strains, the lack of targe ted antivirals, and the adaptive capacity of the virus show that further research into SARS-CoV-2’s viral evolution and its implications on vaccination efforts is crucial.
 
The SARS-CoV-2 laboratory studies pave the way for scientists to explore the virus’s structure and how it can affect the body. The first step in most laboratory studies is in vitro virus propagation to get highly concentrated virus stocks.
 
Even despite recent advances in physiologically relevant in vitro cell culture systems, techniques to propagate clinical isolates have not changed to produce progeny viruses after inoculation of these cells with a specimen containing the virus.
 
The most popular cell line in virology is the Vero cell line, which was obtained from the kidney of an African green monkey.
 
However this cell line and its derivatives contain deletions of genes, which work in the antiviral interferon response. Some of the mutations are mostly found in transformed cell lines and open the door for virus replication, producing a high titer virus.
 
Researchers previously reported that the SARS-CoV-2 MBCs in cell culture have shown that in vitro propagation systems may sometimes fail to show important aspects of the virus life cycle.
 
As mutations impact the results and relevance of laboratory experiments with SARS-CoV-2, it is imperative to know why these happen in order to prevent them.
 
Past studies have shown that SARS-CoV-2 MBCs boost serine protease-mediated entry.
 
Importantly however it must be noted that Vero E6 cells, which are commonly used in the laboratory to grow virus stocks, lack this entry pathway, making the virus use another entry point ie the endosomal cathepsins.
 
The study team believes that mutations in the MBCs could be averted in cells with an active serine protease-mediated entry pathway.
 
The researchers state that virus propagation methods use transformed cell lines to grow viruses from clinical specimens, making some viruses rapidly adapt to cell culture conditions, a process helped by high viral mutation rates.
 
As expected when the researchers propagated SARS-CoV-2 in Vero E6 cells, the virus mutated or deleted the multibasic cleavage site (MBCs) in the spike protein that allows the serine protease to enter human airway cells.
 
The study team also noted that propagating SARS-CoV-2 on the human airway cell line Calu-3, which expresses serine proteases, prevented MBCs mutation.
 
The team also showed that the ectopic expression of the serine protease TMPRSS2 in Vero E6 cells prevented MBCs mutations.
 
Interestingly when the study team used a human airway organoid-based culture system for viral propagation, the same results came out.
 
The study team recommends that further studies, to develop more in-depth knowledge of this area of viral biology, should be done to help formulate methods to prevent cell culture adaptation.
 
The team also recommends that a 2D airway organoid-based cell culture model can be used for SARS-CoV-2 propagation if in the future new variants emerge that are not genetically stable on Calu-3 cells.
 
The study team also warns that the study also shows that deep-sequencing rather than consensus sequencing of viral stocks is critical for obtaining relevant and reproducible results in SARS-CoV-2 studies.
 
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