Evidence for ‘live’ RaTG13 at WIV
Part 3

In two previous threads, I have presented evidence the genome of RaTG13, the closest relative of SARS2, was not generated from a fecal swab as claimed. In this thread I examine whether RaTG13 was generated from a mislabelled oral swab
2/ This might be a plausible explanation for the anomalies in the RaTG13 dataset. The WIV were known to take oral swabs as well as fecal/anal swabs and fecal pellets in their sampling expeditions

3/ Oral swabs were taken from Mojiang, Yunnan in 2014 (from EH_WIV NIAID grant R01Al110964, Accomplishments page 8), the area from where RaTG13 appears to have ultimately originated
4/ A copper mine in Mojiang, Yunnan was where RaTG13 (then labelled RaBtCov/4991) was originally sampled, described in the article below by Zheng-li Shi h/t @Rossana38510044 @TheSeeker268 @MonaRahalkar

5/ The copper mine is ~1800 km distant from Wuhan, and currently sarbecoviruses related to RaTG13 and SARS2 have not been detected in natural reservoirs close to Wuhan
6/ Bacterial rRNAs can provide a phylogenetic fingerprint that can identify origin of a sample. In Thread #2 I applied this approach to show that the bacterial taxa present in the RaTG13 sample were inconsistent with fecal material

7/ A NGS dataset generated by EcoHealth Alliance from an Ebola containing oral swab from the insectivorous bat Miniopterus nimbae from Zaire (SRA SRR14127641) was used as a comparison to the RaTG13 dataset
8/ The core oral microbiome should show similarities across different species of insectivorous bats. This is because the microbiomes in related groups of animals typically have common core bacterial taxa

9/ A Metaxa2 analysis was conducted on the RaTG13 and oral swab datasets. Metaxa2 identifies eukaryotic, mitochondrial and bacterial rRNA sequences in NGS datasets, and assigns taxonomic affiliation to them

10/ When the Metaxa2 analysis was conducted on the oral swab dataset, a number of bacterial taxa which are characteristic of the oral microbiome are revealed. These are lacking in the RaTG13 dataset. I will go these one by one
11/ Pasteurellaceae spp. are mostly commensals living on mucosal surfaces, particularly in the upper respiratory tract. They dominate the oral swab dataset, comprising 51.3 % of bacterial rRNAs, in contrast to only 0.03 % in the RaTG13 dataset

12/ Haemophilus spp. are members of the Pasteurellaceae family and are characteristic of the upper respiratory tract. They comprise 4.4 % of bacterial rRNAs in the oral swab dataset, but only 0.01 % in the RaTG13 dataset

13/ Members of the Gemella genus are characteristic of the oral microbiome in humans. They comprise 0.3 % of bacterial rRNAs in the oral swab dataset, but are completely absent in the RaTG13 dataset

14/ 18.1 % of bacterial rRNAs in the RaTG13 dataset belong to Enterobacteriaceae spp. These are characteristic of the gut, or of cell culture contamination, as discussed in Thread #2
15/ In contrast, only 2.4 % of bacterial rRNAs belong to Enterobacteriaceae spp. in the oral swab dataset. This is expected as gut bacteria are not present in the oral microbiome. Their abundance in the RaTG13 sample therefore is inconsistent with it being an oral swab sample
16/ Lactococcus spp. are abundant in the RaTG13 dataset (64 % of bacterial rRNAs), but are rare in the oral swab dataset (0.06 % of bacterial rRNAs)
17/ This is consistent with the observation that Lactococcus spp. are lactic acid bacteria, which are not expected in high abundance in the oral cavity
18/ An interesting observation is the high number of arthropod nuclear rRNA sequences in the oral swab dataset (0.4 % of eukaryotic SSU rRNA sequences). This is consistent with the insectivorous diet of Miniopterus nimbae, the bat species which the oral swab was sampled from
19/ In contrast, the RaTG13 dataset only 0.02 % of eukaryotic rRNAs belong to arthopods, indicating a further inconsistency with the oral swab dataset
20/ Interestingly, in a WIV anal swab dataset (ENA SRR11085736), only 0.01 % of eukaryotic rRNAs are arthopod. This is an indication that arthopod rRNA largely does not survive the digestion process in Rhinolophus spp.
21/ In Thread #1 I showed that 87.5 % of reads in the RaTG13 dataset map to the Rhinolophus ferrumequinum genome. As a comparison, I wanted to see how many reads in the oral swab dataset would map to the Miniopterus natalensis genome (GCF_001595765.1)
22/ M.natalensis is the most closely related bat genome to M.nimbae available. Using BBMap, only 27.9 % of filtered reads map to the M.natalensis genome, which is considerably less than the 87.5 % mapping efficiency of RaTG13 to the Rf genome
23/ A weakness with the analysis is that only forward reads are available, whereas forward and reverse read pairs were used for RaTG13 mapping to the Rf genome. Mapping forward reads only would presumably lead to a higher mapping % than paired reads, as there is less constraint
24/ In addition, the phylogenetic distance of M.nimbae to M.natalensis, may not be comparable to that of R,affinis to R.ferrumequinum, affecting relative mapping efficiency. Lastly, the sample extraction method is not clear from the SRA oral swab sample webpage
25/ It would be very helpful if someone from EcoHealth Alliance @EcoHealthNYC could clarify if reverse reads are available for the dataset, and what the sample extraction methodology was
@EcoHealthNYC 26/ To conclude this thread, the identity of the bacterial taxa present in the RaTG13 sample are inconsistent with bacteria associated with the oral cavity, indicating that RaTG13 is unlikely to be derived from an oral swab
@EcoHealthNYC 27/ In my next thread I will identify the bat species used to generate the RaTG13 genome sequence. Stay tuned !

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More from @stevenemassey

18 Nov
Evidence for ‘live’ RaTG13 at WIV, Part 2

In this thread I dissect the microbial taxa present in the RaTG13 dataset and show they are inconsistent with a fecal swab sample
2/ Using Metaxa2, only 1.8 % of the reads in the RaTG13 dataset (GSA CRR122287) correspond to small subunit rRNA sequences. This contrasts with 20.7 % present in a Rhinolophus sp. anal swab sample from the WIV (NCBI MN611522)
3/ This implies the RaTG13 sample underwent rRNA depletion, in contrast to the anal swab sample. This is an optional step when using the TruSeq library preparation kit, indicated as being used on the RaTG13 GSA webpage
Read 21 tweets
16 Nov
Evidence for ‘live’ RaTG13 at WIV
Part 1

Over the course of several threads, I will present evidence that the genome of RaTG13, the closest relative of SARS2, was not generated from a fecal swab as claimed but a ‘live’ isolate.
2/ Here, I will show that most reads in the RaTG13 dataset belong to a bat transcriptome. This is inconsistent with a bat fecal swab, where only a minority of reads are expected to belong to the animal being sampled, the rest belonging to bacteria
3/ 87.5 % of RaTG13 paired reads mapped to the Rhinolophus ferrumequinum genome (the closest genome to R.affinis available) using BBMap. This mapping rate is higher than to the genomes of other species known to be in cell culture at the WIV. Image
Read 12 tweets

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