So, Flo Debarre et al’s raccoon dog analysis that has caused such a media frenzy has been released and what does it show ?
Not much 🧵
2/ Essentially, it confirms Gao et al’s preprint analysis that there was nuc acid from animals in addition to humans in the samples 👇 (no surprise there)
It adds some species specific info
3/ The analysis is crude, and taxonomic attribution method naïve
They rely on seq assembly, which miss a lot of info
They use numbers of assembled contigs as a metric for quantity of species specific material
This is semi-quantitative at best (due to the vagaries of assembly)
4/ Even then, they fail to see if the species specific material correlates with numbers of SARS2 reads, which is inexplicable
5/ They apparently identify a potentially new subspecies of raccoon dog – this would worth following up for forensic purposes, but bafflingly they fail to do so
6/ But they also seem to identify new subspecies of masked palm civet, hoary bamboo rat, Malayan porcupine and Amur hedgehog. This could be interesting, or more likely indicates problems with their assembly. Oddly, they fail to follow this up and validate their assembly qualities
7/ Bizarrely, they seem unable to differentiate between DNA and RNA (hint, trying mapping reads to annotated mito genome or nuc genome). This has importance as it can affect estimates of relative species proportions
8/ Tellingly, none of the stalls with raccoon dog nuc acid have a human SARS2 case linked to it (which puzzlingly they fail to mention)
(figure on left from our HSM Zoonosis critique, on right from Debarre et al)
9/ This saga is a case study in the perils of making grandiose claims without having completed the analysis, and of the hubris to embark into a new subject area without specialists (metagenomic) to scrutinize and suggest robust analyses
Some further insights into the SARS2 spike sequences found in Pseudomonas aeruginosa datasets, recorded as being sampled in 2019 🧵
2/ Complete SARS2 spike gene sequences were found in contigs generated from Pseudomonas aeruginosa cultures sampled in 2019, by @iximeno
The spike sequence displayed codon optimization and lacked the furin cleavage site
3/ The spike sequences are found in four contigs 👇, inserted into the pcDNA3.1 plasmid h/t @raqueltobes , with a t-PA (tissue plasminogen activator) leader h/t @Daoyu15
Differential gene expression analysis of the controversial RaTG13 dataset reveals strong similarity to the RaTG15 dataset, also described as generated from a Rhinolophus affinis 'rectal swab' from the Mojiang Mine
This indicates they have a common, undefined source 🧵
2/ The source of the RaTG13 dataset has been a key puzzle of the C19 Origin debate
RaTG13 was sequenced by the Wuhan Institute of Virology prepandemic in 2017/2018 and remains the closest related CoV backbone to SARS2
3/ While the sample is described as being generated from a Rhinolophus affinis 'fecal swab', numerous investigators have noted this is inconsistent with the low % of bacteria present in the NGS dataset
The Zhang group of Fudan University have identified and validated two A-B intermediate SARS2 genomes from the early pandemic
This provides a key to understanding the origin of COVID19 🧵
2/ In their new paper, the Zhang group sequence 343 new SARS2 genomes from the early pandemic (sampled up to Oct 2020). The genomes were obtained from COVID19 patients in the Shanghai Public Health Center academic.oup.com/ve/advance-art…
3/ Importantly, they identify two SARS2 genomes intermediate between lineage A and lineage B
These were validated using two methods, RT-PCR (Sanger sequencing), and Next Generation Sequencing (NGS). @jbloom_lab verified the sequencing depth on one (high)
Was Baric aware of the work on the human α-ENaC furin cleavage site (FCS) at the University of North Carolina (UNC) ? 🧵
In a striking coincidence, the human α-ENaC FCS is exactly the same as that of SARS2, as first noted by Anand et al in 2020 elifesciences.org/articles/58603
2/ Furin cleavage of human α-ENaC has been studied by M.Jackson Stutts, who is based at the UNC School of Medicine
Ralph Baric is also based at UNC School of Medicine, and also has an interest in lung disease. Was he aware of this work ?
A potential explanation for the alanine (A684) in the SARS2 FCS ?
Alanine mutational scanning is used to systematically mutate residues in functional sites, to determine if they ablate function, so indicating their importance 🧵
2/ In the recent FOIA-ed DEFUSE documents released by @emilyakopp and @USRightToKnow there is a section that proposes to 'ablate' 'human-specific cleavage sites' introduced into SARSr-CoV spike proteins
3/ This would present a method of tweaking the efficiency of the FCS, and identifying key residues. There is a possibility that overly-efficient cleavage might result in excessive shedding of the S1 domain (reducing ACE2 binding rates)