This Week in the New Normal #47

[Comment rescued & relocated by Admin]

The Virus has never been isolated! See also my reply on el zalfo below this reply.

To understand why it is important to isolate certain substances and how to do that I want you to watch the following movie from minute 3.20 till 11.00. Dr Cowan describes how you can test if caffeine in coffee beans causes high blood pressure. Here is the link .

According to Zhang, who was the first to publish the complete RNA strand of the coronavirus, the total length of the RNA strand of the coronavirus is 29903 nt long (nt = nucleotides).

The WHO refers to a study by Corman for the primers that can be used for a PCR test. Here is the link for the WHO document (at the bottom of page 3). The primers for the corona PCR test are no longer than 21 nt.

Let’s see if Corman got the primers directly form the virus. Corman’s article states in the Abstract ( link ):

The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur.
We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.

And in the Discussion they report it again:

The present report describes the establishment of a diagnostic workflow for detection of an emerging virus in the absence of physical sources of viral genomic nucleic acid.

The researchers did not have the complete RNA strand of the coronavirus at their disposal to develop the primers, so they did not know what the complete RNA strand of the coronavirus looked like. To take the coffee example of Dr Cowan , they did not have access to the isolated caffeine from the coffee beans. If they did not know what the full RNA strand of the coronavirus looked like, then they cannot say with certainty that the primers they developed are unique to the coronavirus. How were they able to develop the primers?

On page 3 under Results it says:
Before public release of virus sequences from cases of 2019-nCoV, we relied on social media reports announcing detection of a SARS-like virus. We thus assumed that a SARS-related CoV is involved in the outbreak. We downloaded all complete and partial (if>400 nt) SARS-related virus sequences available in GenBank by 1 January 2020.

So primers were developed based on social media reports suggesting that it was a SARS-like virus, and on the researchers’ suspicions that it might be a SARS-like virus. Despite the fact that they did not have the specific RNA strand at their disposal, they nevertheless developed primers with the SARS related virus sequences available until then (01-01-2020).

Corman refers to Zhang for the complete RNA strand, see ref.2 of Corman’s research ( here the RNA strand entry in the database and here the study that led to the complete RNA strand). Whether and how Corman used Zhang’s entire strand is not mentioned by Corman in his article. For the sake of convenience, let’s assume that Corman had this entire RNA strand from Zhang at his disposal for developing the primers for the PCR test, even though Corman himself writes that this was not the case!! Then he could have ‘cut’ a number of unique pieces of RNA from that complete RNA strand for the PCR test. Now it gets a little more technical, but this is perhaps the most important thing to understand.

Zhang and his colleagues modeled the entire RNA strand, not isolated as we will see. Zhang took a bronchoalveolar lavage fluid (BALF) from 1 patient. This is fluid from the lower respiratory tract, see here . Then they use the BALF to read a lot of RNA strands of different lengths. The sample (BALF) consists of the fluid/mucus from the lower respiratory tract. That sample contains not only the virus, but probably much more genetic material from the patient himself and/or other microorganisms. Zhang then uses this mixture of genetic material to model the entire RNA strand.

On page 270, under RNA library construction and sequencing, they write:
Total RNA was extracted from the BALF sample using the RNeasy Plus Universal Mini kit (Qiagen) following the manufacturer’s instructions.

On the same page under Data processing and identification of the viral agent they write:
Sequencing reads were first adaptor and quality trimmed using the Trimmomatic program. The remaining 56,565,928 reads were assembled de novo using both Megahit (v.1.1.3) and Trinity (v.2.5.1).
Megahit generated a total of 384,096 assembled contigs (size range of 200–30,474 nt), whereas Trinity generated 1,329,960 contigs with a size range of 201–11,760 nt.

Okay, here’s the explanation of what that all means.

The pieces of RNA are taken directly from the sample (the BLAF). It is not described anywhere whether and how they isolate only the RNA pieces from the coronavirus out of the sample.

Trimming the dataset is the ‘cleaning’ of the dataset, see here from minute 1.06. The remaining 56 million reads were loaded into a computer and two software programs calculated / modelled possible connections between these small pieces of RNA, or were assembled de novo . (Here are the links to the description of the 2 software programs, Megahit and Trinity .)

Here is a definition of assembled de novo:
De novo assembly is a method for constructing genomes from a large number of (short- or long-) DNA fragments, with no a priori knowledge of the correct sequence or order of those fragments .

A contig (from contiguous) is a set of overlapping DNA segments that together represent a consensus region of DNA or RNA, see here .

Zhang uses the moisture from the sample to model the entire RNA strand, without first separating the virus particles from the rest of the genetic material.

Then with one computer program he uses the 56565928 pieces of RNA to calculate 384096 longer pieces of RNA with different lengths. And with another computer program he uses the 56565928 pieces of RNA to calculate 1329960 longer pieces of RNA with different lengths. The final RNA strand is then selected from this, after comparison with a meat mouse virus. And the reference RNA strand itself exists only as a computer model, and has never been isolated directly from a bat or any of its fluids.

Furthermore, what would happen if we repeated the calculation, would we get another RNA strand? A mutant? This control is not performed by Zhang.

The full RNA strand is a fictitious strand that only exists in a computer program, this is why Corman did not have a physical sample of the virus or of some virus material to his disposal. There are several studies in which the RNA strand is calculated / modeled only with the help of a computer program. The coronavirus has never been isolated directly from a human or any of its fluids! So what do they show with the PCR tests, if the complete RNA strand was not available at the time of developing the primers or if a computer model of the RNA strand is used. Also, a number of primers used are not unique, and do not only belong to the coronavirus.

I’m not saying that the people who got sick didn’t get sick, quite the contrary. But there is no scientific evidence that this is due to a transmissible virus.