Freedom Requires Live-Dead analysis: Subgenomic RNA

You need to pay attention to Wolfel et al and Kim et al.

A very elegant method for Live-Dead analysis.

But before we dive into that Mol-Bio hole, you must see the gravity of Liotti’s work.

jamanetwork.com/journals/jamai…
Liotti (&Wolfel) demonstrate that your infectious stage is above 1-2M RNA copies/ml and usually lasts 5 days. However non-infectious RNA can last 77 days. To be safe they suggest 10 days and 100,000 copies/ml as non-infectious.
So what is creating the long tail of qPCR positivity that is not infectious.

This is believed to be due to subGenomic RNA. Kim et al has the best review on this.

ncbi.nlm.nih.gov/pmc/articles/P…
Before we do the deep dive on Subgenomic RNA, Have a look at how Wolfel et al used this to track Infectious vs non-infectious (Live-Dead) SARs-CoV-2.

Wolfel
pubmed.ncbi.nlm.nih.gov/32235945/
The Virus makes 29Kb genomic RNA to be packaged. But it also makes 9 subgenomic RNAs that do not get packaged. These fragments have a leader sequence on their 5' end that can be targeted with an independent qPCR assays so one can measure total gRNA/SubgenomicRNA.
Wolfel exploits this to create a Delta CQ or the difference between live-dead qPCR signals. They then do a very thorough job tracking this with cell culture and Antibody studies and its a wealth of information.

This once again highlights the lack of infectability with late CQ
So how many people will tolerate an 11+ week quarantine. I hope zero but I'm worried they are boiling frogs and people are getting conditioned to Ivory Tower rulership.

Given there are published methods to estimate infectiousness, what should we do?
Live-Dead blind qPCR cannot be the sole data point to classify a case or steal someones liberty. Given the long persistence of qPCR signal seen in Liotti, much of the positivity rise we are seeing today may be seasonality + testing Vol + subgenomic RNA detection.
While cases are on the seasonal rise, excess deaths are not rising as fast as the spring. Flu is gone and likely reclassified as C19 given the endemic nature of its spread in hospitals.

The responsible thing to do for society is gRNA/subgRNA qPCR to understand infectiousness.
It's critical we push for this as the economic ruin of lockdown is immense. It is a 1st world privilege that starves the 3rd world to satiate a glutinous fear. A fear so irrational and ungrounded in trade-offs that we need to back up and think globally.

npr.org/sections/coron…
You may be tempted to make a blunt CQ cut off with the existing tests. The problem with this is that it's not a very sharp knife. Infectious vs non-infectious patients sit on both sides of any chosen cut off and this defaults into a position of caution. This can sharpen the knife
This also raises the question if the qPCR kits in the field that target ORF1a will do a better job at separating infectious vs non-infectious patients as there are no subgenomic RNAs that include ORF1a.
Just imagine if we had public CQ data to surf!!!
fda.gov/media/140922/d…
This is the nanopore coverage map of C19 (Kim et al).S and N qPCR primers are targeting regions have 50-300X more copy number than regions in ORF1a. Seeing as most of this coverage is from subgenomic RNA, these primers will remain positive longer and are not infectious.
One could qPCR ORF1a and N2 in different wavelengths with multiplex qPCR. This would provide a ratio of full length packaged gRNA vs fragmented sgRNA.

This company has an assay for ORF1a. You'll notice they miss some positives in their EUA! Dead +ves ?
fda.gov/media/140922/d…
A few other ORF1a assays are described in this primer Bake-off paper. Notice how each assay has a different LOD.
This is why making a centralized decision on CQ>35 is not wise. Its needs to be test specific.
ncbi.nlm.nih.gov/pmc/articles/P…
Some Bake off papers have signs of ORF1a assays having a log scale lower LOD. One has to be careful reading too deep into this as many LODs are measured with packaged virus where this sgRNA problem isn't as significant.
sciencedirect.com/science/articl…
This brings into picture the validation problem ORF1a primers have. If all True Positives are measured with qPCR primers that target sgRNA, primer pairs that measure the lower copy number gRNA (but more informative for infectiveness), will never get to market.
Instead, we have optimized for RNA sensitivity not clinical or infectiveness sensitivity.

We're digging in the wrong place.
This is an important reference to understand subgenomic RNA and its lack of packaging.
ncbi.nlm.nih.gov/pmc/articles/P…
This is a good read regarding strandedness of the RNAs and the nuclease resistance of the sgRNA.

medrxiv.org/content/10.110…
Recent Work from the CDC confirming sgRNA Vs gRNA ratio provides more resolution on infectiveness.
I would have expected the opposite ratio but as long as it provides additional information for understanding qPCR... let’s use it.

wwwnc.cdc.gov/eid/article/26…

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More from @Kevin_McKernan

13 Nov
What happens when you use RNA polymerase mutagens in your fight against a virus?

Life Finds a way. Mutations accelerate.
nejm.org/doi/full/10.10…
I’m just speculating on this. This is a great read on the types of polymerase errors this modified nucleotide tends to create.

Keep in mind the patient was on a lot of drug including HCQ so this one patient study won’t answer this. Image
From the supplement.
Poor soul had a rough fight. Image
Read 4 tweets
13 Nov
This genome was Crypto funded and Crypto peer reviewed.
Project published in 5 months.

Decentralized funding and peer review seemed crazy in 2018. If you recall Surgisphere, you might appreciate censorship resistant science.
osf.io/7d968/
@saifedean
@JamesTodaroMD
We presented on this Crypto Incentivized Blockchain recorded Review (CIBR) concept @TexasBitcoin in 2018. It foreshadows much the censorship and distortion of science seen in C19.

We propose as solution that introduces price signals and free markets.

Many people cringe at $ in peer review.
Meanwhile Journal publication prices continue to rise while digital content distribution costs fall.

The $ in peer review should go to the people doing the most valuable work (review). Free work generates errors.

medicinalgenomics.com/cryptocurrency…
Read 5 tweets
12 Nov
Inside and Outside of kids cloth mask worn all day at school.
Plated on PDA with no selection.

Central planning and it’s effect on evolution and anti-fragility?
If 7B people all wear 300um filters that collect microbes, the human microbiome will change. Is this Good or bad? ImageImage
In order for one to identify the microbes. ITS/16S sequencing or whole genome sequencing is required.

We describe that in these manuscripts.

f1000research.com/articles/5-2471

f1000research.com/articles/4-1422 Image
Cannabis microbiomes are relevant to the mask debate as this is the study of microbes you might inhale and thus we are particularly sensitized to fungi that can cause aspergillosis or fusariosis.

Microbes that synthesize mycotoxins are important as well. Image
Read 9 tweets
11 Nov
In Mass over 85-90% of the tests are CQ >30.
John conflates this with the 1:2000 FP rate specified in my tweet which he didn’t read because he was too busy verify GBD signatories.

CVs are seasonal. Its expected their viral loads go down in the summer.

frontiersin.org/articles/10.33…
Other CVs seasonality.
I’d love an explanation for how a virus does this and maintains constant CQ across the population.

I won’t hold my breath as I’m a GBD signatory and that makes me a conspiracy theorist that needs remote mocking as opposed to a normal scientific discussion
A normal person would ask a question on your thread.
“have you considered X?”

A toxic person will take your work out of context, mis-represent it and then use an associative GBR fallacy to mock you while barely scraping the surface of what you’ve said.

Not science. Politics
Read 8 tweets
11 Nov
Schrodinger’s CQ

I asked many labs if they could share CQ data. The Broad Institute responded immediately with: possible but requires some work.

Another lab became defensive, threatened to litigate and tweeted me “a horrible person” for probing

How did we get here? Lessons?
Lesson #1) everyone is stressed out and getting hit with questions over FPs and conflicts. I’m guilty of pointing out that testing labs have revenue interests and higher positive tests create more contact map testing.
Underscoring this COI doesn’t make friends. I will do better.
Lesson 2) Resistance to public CQ was mostly defended by demanding evidence they have a FP problem.

“Prove it before we show you.”

I was probably the 100th person since the NYTimes article that hit them up on this and I see why they were short.
Read 13 tweets
2 Nov
Are PFU/Cq curves perfect?
They are the best we have today.

The pioneering work that looked at this by Jaafar et al. Used Monkey cells or Vero cells.

Now let’s look at the work at the CDC culturing the virus.

wwwnc.cdc.gov/eid/article/26…
Vero cells were chosen as they were the most sensitive to forming plaques.
Aka the virus was very good at killing these cells.
But the CDC was thorough. They didn’t just look at Vero cells. They looked at many other cell lines including human cell lines.

Have a look at Figure 3.

Virus isn’t as virulent to other cell lines. I say virulent because they are measuring Viral titres as PFU.
Plaques are dead.
Read 5 tweets

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