Dr. Sam Bailey gives a number of references in her YouTube video on PCR tests stating the virus has never been purified as in actually separated from the infected cells. I'm curious if this is actually true, and if so is this trivially true since viruses can't exactly thrive without other cells. Has this been done for viruses like influenza and Sars 1, I think it must have been as it would be the only direct and explicit way of specifying said virus

The actual statement and references at 5.50 mark

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At 6:12 she says (approximately):

Hence it cannot be concluded with any confidence that the so-called Covid-19 PCR test was calibrated to a brand new viral pathogen.


It is essentially irrelevant whether the virus was purified, and the reason it wasn't in the cases that the Youtube video mentioned is that it simply was not necessary or useful to add a purification step there.

I'll quote a pagagraph from a random electron microscopy paper about SARS-CoV2 I found here:

To avoid artefacts associated with virus concentration or purification, we aimed to image SARS-CoV-2 virions from the supernatant of infected cells without further concentrating or purifying the virus. Vero E6 cells were infected with SARS-CoV-2 (isolate Germany/BavPat1/2020)24. At 48 h after infection, the supernatant was clarified, inactivated by fixation with formaldehyde and stored at −80 °C.

Supernatant is essentially everything outside the cells in this case, the liquid in which the cells were grown, but not the cells themselves. This is arguably a purification step already, if a rather coarse one.

They made some pictures of single viruses in this paper, and they explain that they didn't purify the virus any further. You can take a look at the pictures there, so it is obvious that purification isn't necessary to take these images. It doesn't matter if you have some other things present because you're taking images and can simply ignore those parts of the images that don't show your virus.

Any purification steps could introduce more problems, and there is obviously enough virus around that they can just take the pictures they wanted. So purification would be harmful in this case, and that is why it is not done.

And if someone is trying to argue that you don't know that it's actually SARS-CoV2, that is not really true. Because those cells were intentionally infected by SARS-CoV2, so where would another random virus that looks like a Coronavirus come from?

But the whole argument is a giant distraction and not relevant at all. We know how PCR works, and it is incredibly specific. It's probably the most specific biological test we have. If you perform a PCR targeted on a specific sequence, you know that that sequence is present in your sample. You obviously have to make sure that you're choosing the target well, and that your target isn't present in other viruses. But that is exactly what Drosten and his team did in that first paper.

PCR is the gold standard in this case, not any purification or microscopy. It tells us exactly what we want to know, is an RNA with this particular new sequence present in the sample.

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    Good answer, I'm dubious about a few things thaugh. Like how do you know it was infected with sarcs cov 2 and only Sars cov 2 if you don't have the isolated PURIFIED and completely sequenced Sars cov 2 to begin with. Also, as per the sub question it is emportant to establish whether Sars or hiv for example have been purified ever. As I understand this could be dangerous but still. Im still uncertain as to how they actually sequence DNA if a specific virus and is that full sequence required for unambiguous classification – Matko Jan 18 at 15:40
  • @Matko In the linked paper they got the virus from a central repository (european-virus-archive.com/virus/human-2019-ncov-isolate). You can see on that page that the virus was fully sequenced, which does tell you exactly which sequences are in there and that no other viruses are present (as you would detect those other sequences mixed in). – Mad Scientist Jan 18 at 15:44
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    But that's what I'm asking essentially, how was that original isolate sequenced when, doesn't it have to be fully purified to be sequenced correctly – Matko Jan 18 at 15:51
  • They actually note a bit later in the paper "The low concentration of particles in supernatant makes high-resolution structure determination difficult. We therefore concentrated the virus by pelleting through a sucrose cushion. Concentrated virions deviated from the spherical morphology (Extended Data Fig. 5), but the overall features were preserved." – Fizz Jan 18 at 17:20
  • So meaning they have better resolution in a non purified case? – Matko Jan 18 at 19:21

The whole you-didn't-image-this-in-a-highly-purified-sample is a red herring with regard to determining whether a (suspected) virus is a "a brand new viral pathogen" or not. Nobody does that kind of novelty determination based on imaging the virus... simply because lots of viruses look alike even under electron microscopy. Imaging is not a useful way to tell apart viruses from similar "genres", e.g. various coronaviruses one from another. I didn't expect there to be a paper about this, but (amusingly) there is (a short) one that's somewhat relevant, starting with:

Caution in Identifying Coronaviruses by Electron Microscopy

We are concerned about the erroneous identification of coronavirus directly in tissues by authors using electron microscopy. Several recent articles have been published that purport to have identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly in tissue. Most describe particles that resemble, but do not have the appearance of, coronaviruses. [...]

(I'm not going to delve into who is right in that little spat, just saying that identification by imaging is much more problematic, and has been superseded by molecular methods by and large.)

Furthermore, while the video lambast at length PCR tests as a diagnostic tool, it's completely silent on how suspected new viruses are actually sequenced in the first place. There is a general rant against molecular biology in the final minutes of the video, but this is still unspecific. That rant is basically akin to: we can never make/understand computers with billions of transistors because of their complexity.

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N.B.: that's a quote from a 1966 position paper of Macfarlane Burnet. The same author in a 1970s book "predicted that scientific progress would end soon", according to Wikipedia. His beef/prognostication generally was with DNA/RNA-based identification (which wasn't actually possible during his active career).

Basically, Dr. Bailey doesn't (explicitly) outline a single method by which she would agree that something is a new pathogen. So to a large extent, the whole video is an argument that we can never know this.

And while Burnett may have railed against molecular methods, the micrographs he used in his own papers on influenza don't seem to ever show the purified influenza virus alone, but commonly show it among cells. E.g., these are images from Burnett's 1957 article in Sci. Am.

enter image description here enter image description here

So really, this purification before micrographs is a Bailey-made-up standard that Burnett himself probably would have not considered seriously.

Now to save this answer from triviality, the way by which suspected new viruses are sequenced is a bit different than what (later) PCR diagnostic tests do... because the latter use highly specific primers, which aren't known (what they should be) when sequencing an unknown virus in the first place. An umbrella term for such methods is metagenomic sequencing.

If you look at actual (NEJM) paper for SARS-CoV-2 discovery, they mention using Illumnia and nanopore sequencing specifically. They don't bother citing any publication for these methods, presumably because they are considered (already) well known, although nanopore sequencing is actually kinda new.

The NEJM paper on SARS-CoV-2 identification doesn't detail much purification steps (it's a brief report) but does mention that they excluded known respiratory pathogens with standard kit for these (details are in a supplement to the paper). Simplifying, the logic of the paper is: these patients have these symptoms of a respiratory disease, they test negative for (a whole lot of) known pathogens that can cause these symptoms, and looky we found a novel virus sequence in their throats; ergo, that must be the cause of their disease. I'm simplifying, because they also cultured the new pathogen etc. and used control samples of the culture without the pathogen:

Extraction of nucleic acids from clinical samples (including uninfected cultures that served as negative controls) was performed with a High Pure Viral Nucleic Acid Kit, as described by the manufacturer (Roche)

If you read the mfg description

The High Pure Viral Nucleic Acid Kit is intended for general laboratory use and efficiently purifies viral nucleic acids from serum, plasma, or whole blood using sample volumes of up to 200 μL. The purified viral nucleic acid is eluted in nuclease-free water, and is suitable for direct use in PCR and RT-PCR.

An earlier (2015) paper on nanopore sequencing for example (which compares it to Illumnia) also does mention some purification steps

cDNA were purified using AMPure XP beads [...]

An even earlier 2013 review on viral identification goes into some nitty gritty details on some purification steps, e.g.

Pathogen enrichment or host depletion before microarray and deep sequencing analyses hence becomes critical to maximize sensitivity for identification of novel agents in clinical samples. For viruses, capsid purification procedures involving repeated freeze/thaw cycles, filtration, ultracentrifugation, and prenuclease digestion have been developed to enrich host tissues or body fluids for infectious particles. Strategies to deplete the sample of background host DNA can also be implemented, including the use of methylation-specific DNAse to selectively degrade host genomes, removal of host ribosomal RNA, and/or removal of the most abundant host sequences by duplex-specific nuclease (DSN) normalization. Another complementary approach is to perform target enrichment using biotinylated probes to enrich NGS libraries for sequences corresponding to pathogens, akin to now well-established techniques that have been developed in the cancer field. [...]

Which steps are relevant (for which method) may a good question on Biology SE, but Dr. Bailey seems completely ignorant of any of this, even on a high level, at least in the video. Basically, Dr. Bailey's rant is that you can't purify some RNA or resultant cDNA (which isn't present in control cell cultures), you need to "purify the [whole] virus". This is nonsense from the point of view of practitioners in field of new virus discovery.

As far as Covid-19 is concerned largely similar approach is taken in Shi et al.'s Nature paper: metagenomic sequencing, followed by a general confirmation with electron microscopy.

I'm gonna close this by saying that sequencing is still a fast moving field, technologically speaking. Methods that were used e.g. in 2003 to identify the SARS virus are only relevant nowadays as a general idea... but even that paper mentions:

nucleic acids were purified from the supernatant. Random amplification was performed with 15 different PCRs under low-stringency conditions. We had previously shown that this method is able to detect unknown pathogens growing in cell culture (unpublished data). To detect RNA viruses, an initial reverse-transcription step was included.

Basically someone from outside the field who rants that the field is invalid to begin with because of its complexity and also complains that some (almost certainly not needed) whole-virus purification steps aren't performed to their idea-of-a-standard is basically (almost) as laughable as the average guy on the street complaining that some mathematician isn't writing proofs according to Trump's speeches.

What gives a high degree of confidence in the sequencing results regarding SARS-CoV-2 is also the fact that the sequencing process (and I'm not talking about PCR testing with specific kits but basically a replication of the initial analysis/experiments be it with various sequencing technologies) for has been done for more than 90,000 samples, worldwide. Basically that's an experiment/analysis replicated 90,000+ times. This wasn't done because the initial results were deemed not credible, but mainly because viruses mutate/evolve and establishing lineages and keeping track of mutations which may affect is important for epidemiologist too. (See e.g. an early mutation that enhanced transmissibility being identified; or lineages of concern because e.g. they have some antigenic mutations, meaning they "work around" some antibodies hosts may have against other lineages.)

Purified whole-virus samples are actually used in some studies. For example there's one such study on mouse hepatitis virus (which also a coronavirus), but the goal there was to measure the variation in particle size and likewise for their wall thickness and spike lengths. For something like that, it's convenient to have a bunch viral particles in close proximity so you can easily measure a set of them from a few images.

Perhaps with all that methods stuff, I didn't make some basic science clear enough. You can recreate a virus' capsule (thus the "whole virus") solely from its nucleic acid, by injecting the latter into an appropriate host cell. The virus will replicate inside the cell and eject from the cell new, encapsulated viral particles. (More technically, that viral capsule is called a capsid.) Some viruses--including coronaviruses-- are additionally coted with a lipid layer, which is also obtained in roughly the same way, although a bit later in the cell-egress process. (Viruses which cause the cell the to burst [aka lysis] don't get this additional envelope.)

Such "capsule recreation" was done e.g. with Covid-19's virus (SARS-CoV-2). Not only that, but the whole nucleic acid of the virus was created "from scratch" in that (last) experiment, simply following the "recipe" given by its published sequence. (This kind of tech has existed for about two decades.) This is why the capsule is irrelevant to the identity of the virus.

Not only that, but as an aside, the same virus can have different envelopes, e.g.

Hepatitis A virus (HAV) spreads by both the fecal-oral route (by ingestion of food contaminated with fecal matter) and the blood route (such as by use of contaminated needles). HAV can have both naked and quasi-enveloped capsids. We know that HAV particles isolated from fecal matter are naked, while particles circulating through the blood are protected by a quasi-envelope that prevents detection of these particles by the host immune system.

  • Comments are not for extended discussion; this conversation has been moved to chat. – Oddthinking Jan 19 at 15:52
  • Sorry but all you did above was make a case for there never being anything properly identified as a virus. Sounds like you’re a bit invested in the field in some capacity (like you put time into thinking it was all legitimate) and now you have no idea how to disprove the suspicion that there’s a bunch of BS at it’s core. Anyway you’re not alone in feeling a bit duped – froimovi Apr 24 at 14:32

Here is a press release with isolated SARS-NCoV-2 including a photo.

Lab that first isolated coronavirus in Australia shares how it was done

“A nasopharyngeal swab and sputum taken on presentation tested positive for SARS-CoV-2,” Catton and colleagues wrote. “Inoculation with material from the initial nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles displaying morphology characteristic of the family Coronaviridae."

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    I'm sure it wasn't purified to Dr. Bailey's standards beforehand, since it's a Mar 9, 2020 publication. (It's also not the first such image, although it does claim to be "the first to grow coronavirus outside of China"). The Park et al. paper (S. Korea) was actually published before that though (Feb 24). It's included in the screenshot table in the question. – Fizz Jan 27 at 16:35

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