In essense, the authors have “translated” the RNA virus into the language of DNA (using reverse transcriptase), which enabled them to manipulate its genome with the help of existing genetic engineering tools. Having created 7 such cDNA provirus segments, the authors then stitched them together “seamlessly” (i.e. without introducing any new, even silent mutations, including new restrictase sites), after which they transcribed their construct back into RNA, which was then translated into virus particles in other cells.
Baric-1990
Just so you appreciate how long Ralph Baric has been at this game — he was designing recombinant coronaviruses way before there were any DNA sequencing machines or other modern tools of genetic engineering. Here is his paper on the creation of “temperature mutants” from mouse coronavirus from 1990:
The A59 strain of mouse hepatitis virus (MHV-A59) was used throughout the course of this study. Virus was propagated and cloned three times in the continuous murine astrocytoma cell line (DBT).
…
Various combinations of [temperature sensitive] mutants were mixed and inoculated onto cells at a multiplicity of infection of 10 each.
So Dr. Baric has been creating mutant viruses for over 30 years.
https://link.springer.com/content/pdf/10.1007%2F978-1-4684-5823-7_47.pdf
But in terms of the potential lab-based origin of the furin site, I am more inclined to hypothesize a specific insertion — as in the Beijing paper from October 2019 with chicken coronavirus. After that, the synthetic strain could have acquired new mutations by subsequent culturing in vitro or in vivo — like the MA15 murine strain in 2007, for example. Or maybe even using the same mouse model with humanized lung tissues and immune system that was created at UNC by Baric’s and other groups in 2018, in which they reported testing several viruses including MERS:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832359/
Recombinant rYN-S2/RRKR virus containing an S protein with the furin-S2′ site was generated by vaccinia recombination, as described previously [20,28]. Briefly, plasmid with the furin-S2′ site was generated using the Seamless Assembly kit (Invitrogen, Carlsbad, CA, USA) and transfected into CV-1 cells infected by vaccinia virus containing the genome of YN-ΔS-GPT. Furin-S2’ site was introduced into the YN cDNA by homologous recombination using the transient dominant selection system [25]. After screening and transcription in vitro, the full-length RNA of rYN-S2/RRKR was electroporated into BHK cells. After 48 h of incubation at 37 °C, cells and supernatant were repeatedly subjected to freezing and thawing three times and inoculated into 10-day-old SPF embryonated eggs. Serial passage was executed until the stable appearance of dwarf embryos and the time of death of embryos was recorded. The 50% embryo infectious dose (EID50) was determined by inoculating serial 10-fold dilutions of allantoic fluid into SPF chicken embryos [29]. The TCID50 was determined by inoculating serial 10-fold dilutions of allantoic fluid into CEK cells cultured in DMEM with 10% FBS in 24-well plates.
On the 4% Genome Difference between RaTG13 and Cov2
Some critics of the lab-made hypothesis claim that the observed ~4% genetic difference between RaTG13 and CoV2 is too high to have possibly occurred in a lab if RaTG13 itself was used as a backbone. Observed mutation rates for RNA viruses vary widely — from 10⁻⁶ to 10⁻⁴ nucleotides per replication in vitro, and in humans CoV2 seems to mutate at a rate of 25 mutations per year. Thus, the logic goes, it would take years, if not decades, for two strains to diverge by 4%. While that is a valid point, there are several issues with that line of reasoning.
First, in vitro mutation speeds (i.e. per unit of time) are much higher, as you can passage cells much more often than infect new animals. As SARS and MERS in vitro experiments showed, significant mutations might be observed after only a few passages. For example, the 2004 paper reported that only after 600 passages there already was a 2.1% difference in the genomic sequences of spike proteins between the original strain and its progeny:
Moreover, in the presence of some antiviral compounds, such as nucleoside analogs (e.g. ribavirin or remdesivir), mutation rates in RNA viruses can increase even further:
We obtained an estimate of the spontaneous mutation rate of ca. 10⁻⁴ substitutions per site or lower, a value within the typically accepted range for RNA viruses. A roughly threefold increase in mutation rate and a significant shift in mutation spectrum were observed in samples from patients undergoing 6 months of interferon plus ribavirin treatment. This result is consistent with the known in vitro mutagenic effect of ribavirin and suggests that the antiviral effect of ribavirin plus interferon treatment is at least partly exerted through lethal mutagenesis.
So if ancestral CoV2 was being lab-tested to assess how its mutagenesis might affect the efficacy of potential vaccines or antiviral drugs, it could have accumulated mutations at a much higher rate.
But possibly, the biggest problem with the 4% difference argument is that it relies on RaTG13 being exactly what WIV says it is. If we are to seriously consider the lab leak hypothesis, we must concede that it does not make sense to blindly trust the data released by the very lab suspected of the leak. If the leak did occur, as is the premise of the lab hypothesis, then the description of what RaTG13 is could be furthering the goal of covering up the leak.
Again, I am not claiming with certainty that is what is happening here. All I am saying is that this is what could have happened, and we need a lot more evidence before we can reach a definitive conclusion. One thing that could help rule out tampering with RaTG13 is having independent labs sequence the 2013 Yunnan samples that She Zhengli extracted RaTG13 from. WIV must still have them if they re-sequenced RaTG13 in 2020.
https://yurideigin.medium.com/lab-made-cov2-genealogy-through-the-lens-of-gain-of-function-research-f96dd7413748
On balance, the current chances against this are still higher than for the natural origins of CoV2. Moreover, even if CoV2 was indeed an unfortunate lab leak, the scientists themselves are not to blame, as they were working within the established international laws and guidelines on such research. Now, those who might be trying to cover up that leak, that’s a different story.
I don't know if I agree.
The people partaking in gain of function research which was suspended by WH should not get involved just becaue Fauci grants it.