Thread by @Ayjchan

Some people are super confused about RaTG13, one of the most closely related viruses to SARS-CoV-2, because of how sparse/disjointed its descriptions have been in interviews and papers. I'll try and get the facts straight here as best as I can - based on the data available to me.

The Sunday Times recently confirmed that RaTG13 is actually a sample called RaBtCoV/4991 from this 2016 paper published by the WIV/Shi Zhengli's group. It was renamed and it's not a big deal. Sometimes new naming criteria are implemented. ncbi.nlm.nih.gov/pmc/articles/P…

In this paper, it's pretty buried, but the source of all the bat CoVs sampled is an "abandoned mineshaft in Mojiang County, Yunnan Province, China". The authors cite this other study by another group including the Chinese Academy of Medical Sciences. wwwnc.cdc.gov/eid/article/20…

That article reads: "In June 2012, in Mojiang Hani Autonomous County, Yunnan Province, China, severe pneumonia without a known cause was diagnosed in 3 persons who had been working in an abandoned mine; all 3 patients died. "

Linked to one medical thesis and one PhD thesis supervised by George Gao (Chinese CDC director) - both claiming that the deaths in the cave are viral + unresolved (unknown pathogen). Multiple labs across China went to that cave after the miner incident to collect viruses.

Back to the 2016 paper, what does it say about how RaTG13 was collected and characterized? "Bat fecal swabs were collected in August and September of 2012 and in April and July of 2013" from the cave in Mojiang, Yunnan. 4 trips. The WIV didn't visit again after they found RaTG13.

"The bat species were first identified based on morphology. PCR amplification and sequencing of cytochrome b (Cytb) or NADH dehydrogenase subunit 1 (ND1) from DNA extracted from the fecal swabs was used to confirm the bat species."

Based on this, I assume that samples were not pooled. They had to assign the bat species based on the animal they sampled from. They confirmed the species genetically.

What does the paper actually say about RaTG13/4991? "Two sequences (HiBtCoV/3740-2 and RaBtCoV/4991) were homologous to betacoronaviruses, all other 150 sequences were homologous to alphacoronaviruses."

"One of them (RaBtCoV/4991) was detected in a R. affinis sample and was related to SL-CoV. The conserved 440-bp RdRp fragment of RaBtCoV/4991 had 89% nt identity and 95% aa identity with SL-CoV Rs672."

Key: SL-CoV = SARS-like coronavirus

Ok, so out of 150 sequences, only 1 was related to SARS with 95% amino acid identity to a SARS-like coronavirus. Totally uninteresting. Especially because 6 miners contracted an unresolved virus-driven pneumonia after moving bat guano in this cave.

"RaBtCoV/4991 showed more divergence from human SARS-CoV than other bat SL-CoVs and could be considered as a new strain of this virus lineage"

Still not interesting. Not relevant to preventing outbreaks at al.

Paper goes on to amplify full-length spike genes from 8 non-SARS-related CoVs to see how similar they are to well-characterized spikes.

Notably, this other beta CoV: HiBtCoV/3740-2 remains uncharacterized (2 out of 150 sequences). If the sample hasn't disintegrated yet, would we be able to get its metagenomic data publicly released?

The Sunday Times interviewed Daszak who said: "It was just one of the 16,000 bats we sampled. It was a faecal sample, we put it in a tube, put it in liquid nitrogen, took it back to the lab. We sequenced a short fragment." This is correct.

But he added "But it did no more work on it until the Covid-19 outbreak because it had not been a close match to Sars." but was most likely misinformed because the amplicon data shows the WIV sequencing RaTG13 in 2017 and 2018.

Sunday Times: after sequencing the full genome for RaTG13 the lab’s sample of the virus disintegrated, (Daszak) said. “I think they tried to culture it but they were unable to, so that sample, I think, has gone.”

Ok, sample gone. No more independent verification. What does the 2020 paper from the WIV/Shi's lab that describes RaTG13 say?

"bat coronavirus (BatCoV RaTG13)—which was previously detected in Rhinolophus affinis from Yunnan province—showed high sequence identity to 2019-nCoV"

No citation of the 2016 paper, which also didn't mention the miners' deaths.

"We carried out full-length sequencing on this RNA sample (GISAID accession number EPI_ISL_402131). Simplot analysis showed that 2019-nCoV was highly similar throughout the genome to RaTG13 (Fig. 1c), with an overall genome sequence identity of 96.2%."

The reason for this series of tweets is multiple people asked me to look at episode 623 of This Week in Virology #TWiV where Daszak called RaTG13's genome a consensus sequence. This is not wrong. The RNA isolated from the bat fecal/anal sample was sequenced. The reads assembled.

It does not mean that multiple samples from different bats were combined. But it does mean that it will take work to figure out if the RaTG13 sequences truly came from 1 virus or multiple viruses.

Problem is, now you have dozens upon dozens of papers using RaTG13 to evaluate the evolution and origins of SARS-CoV-2. Coupled with the Guangdong pangolin CoV papers that are confused/disorganized about their source of pangolin samples and data.

Same Nature paper: "The S genes of 2019-nCoV and RaTG13 are longer than other SARSr-CoVs" and performs a careful analysis of the S1 portion of the spike, stopping just short of the infamous S1/S2 furin cleavage site.

Again, super important feature of the virus, not interesting.

Ultimately, they conclude: "The close phylogenetic relationship to RaTG13 provides evidence that 2019-nCoV may have originated in bats."

We cannot stop evolutionary biology researchers from moving forward on SARS2 research, but I would highly caution the use of RaTG13, RmYN02, and the Guangdong pangolin CoV in analysis. I personally don't understand if these genomes are chimeric.

This, to me, is not very surprising. A SARS-like CoV came from bats, where the 2002-2004 SARS likely originated from. The surprising part is how the scientists reacted to a totally new strain of SARS from a cave where people died from a SARS-like illness.

Now EcoHealth wants to sample bats in SE Asia despite all of the closest SARS2 relatives hailing from Yunnan, China, and none of 300+ pangolins in Malaysia testing positive for CoVs.
economist.com/science-and-te…

I'm very curious about this. Why sample so far away from the site of the outbreak (Wuhan, China)? Did finding the bat CoVs that could've recombined into SARS1 help at all in preventing another outbreak? Once you find more CoVs related to SARS2, what will you do?

Seriously? "To justify the costs of prevention, a year's worth of these preventive strategies would only need to reduce the likelihood of another pandemic like COVID-19 in the next year by about 27%" science.sciencemag.org/content/369/65…

In their supplementary: "annual cost estimates over the decade as $120 to $340 million per year and enter the midpoint as $230 million per year" for USAID’s PREDICT, DARPA’s PREEMPT, and USAID EPT programs. science.sciencemag.org/content/sci/su…