Two Candidates re SARS-2 Origin.
Zhiyan-Le, 2021-04-29.
https://zhiyanleback.blogspot.com/p/two-candidates-re-sars-2-origin.html
https://sites.google.com/site/zhiyanleback/2021-1/z20210429-two-origins

Key Words: Science and research communications, SARS-2, Covid-19.


There are two possible candidates regarding the SARS-2 origin. One is bat RaTG13, as the WHO-China joint report said. The other is WIV1 series (Wuhan Institute of Virology, No.1 virus. Totally, it has 8 bat samples, plus WIV16). The following are key info/data suggesting that RaTG13 is too good to be true as the SARS-2 origin and that WIV1 is the real origin.

Samples for this essay:
 

Samples, Raw Data Souce: NIH GenBank.
Name	GenBank ID	Descriptions
WH-01	NC_045512.1	Basic sample, Wuhan patient.
	URL:	https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.1
WIV1	KF367457.1	Bat SARS-like coronavirus WIV1, lab product by PRC WIV.
	URL:	https://www.ncbi.nlm.nih.gov/nucleotide/KF367457.1
RaTG13	MN996532.1	Bat coronavirus RaTG13, said collected in PRC Yunnan, 2013.
	URL:	https://www.ncbi.nlm.nih.gov/nuccore/MN996532.1
Bat-12	NC_028824.1	Bat coronavirus, collected in PRC Yunnan, 2012.
	URL:	https://www.ncbi.nlm.nih.gov/nuccore/971746735
Bat-14	NC_030886.1	Bat coronavirus, collected in PRC Yunnan, 2014.
	URL:	https://www.ncbi.nlm.nih.gov/nuccore/NC_030886.1

Note: If RaTG13 were really a natural bat sample, its nCov should behave as the same, or at least very close to, how the other two natural bat samples do.

And here is the alignment on single-gene level:
 

Global Align re Samples (Raw data and Method: NIH BLAST, by 2021-02)
Query x Subject	S-Gene		Complete Sequence
	Identities	Gaps	Identities	Gaps
RaTG13 x NC-045512	98%	0%	96%	0%
RaTG13 x WIV1	88%	2%	78%	3%
RaTG13 x Bat-2012	57%	19%	55%	16%
RaTG13 x Bat-2014	61%	13%	56%	12%
NC-045512 x Bat-2012	57%	19%	55%	16%
NC-045512 x Bat-2014	61%	13%	56%	12%
WIV1 x NC-045512	87%	2%	78%	3%

Taking WH-01(NC_045512) as the basic sample, RaTG13 has highest identities, and much higher than that of two natural bat sample (2012 and 2014). Question: in the same location (PRC, Yunnan Province) and in just one year, how come in nature that the nCov in RaTG13 could be mutated so close to a patient sample which will be coming over 6.5 years later? However, a lab can do it in 10 days.


And here are similarities on Codon-level:
 

S-Gene: Codon-Leveled Similarities to NC-045512
	NC-045512	WIV1	RaTG13	Bat-2012	Bat-2014
Length by Codon	1273	1260	1256	1269	1132
Matching Score		78	693	66	94
Matching Ratio		0.0619	0.5518	0.0520	0.0830
ATG	14	17	14	21	29
TAA	0	0	0	0	0
TAG	0	0	0	0	0
TGA	0	0	0	0	0

Again, the similarity score of RaTG13 goes much, much higher than that of natural bat samples and, for the codon ATG, RaTG13 has it exactly as the same as what the Wuhan patient (NC_045512) has. Is this possible in natural mutations? Obviously not.


The RaTG13 sample has two versions: one is issued at 2020-03-24 and the new version is issued at 2020-11-24. And here is their aligned result by NIH Blast:
 

Alignment of RaTG13: V.1 (2020-03-24) x V.2 (2020-11-24)

v.1 (2020-03-24): https://www.ncbi.nlm.nih.gov/nuccore/MN996532.1

v.2 (2020-11-24): https://www.ncbi.nlm.nih.gov/nuccore/MN996532.2

Query Length: 29855 (v.1)

Subject Length: 29855 (v.2)

Identities: 29834/29870 (99%)

Query  1                     CTTTCCAGGTAACAAACCAACGAACTCTCGATCTCTTGTAGATCT  45

|||||||||||||||||||||||||||||||||||||||||||||

Sbjct  1      ATTAAAGGTTTATACCTTTCCAGGTAACAAACCAACGAACTCTCGATCTCTTGTAGATCT  60

Query  2086   GCTAACAAATATCTTTGGCACTGTCTATGAGCAACTCAAACCTGTTCTTGATTGGCTCGA  2145

|||||||||||||||||||||||| |||||| ||||||||||||||||||||||||||||

Sbjct  2101   GCTAACAAATATCTTTGGCACTGTTTATGAGAAACTCAAACCTGTTCTTGATTGGCTCGA  2160

Query  6886   TTCATTTAATTACCTGAAGTCACCTAATTTTTTTACATTGATTAATATTATAATTTGGTT  6945

||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||

Sbjct  6901   TTCATTTAATTACCTGAAGTCACCTAATTTTTTTAAATTGATTAATATTATAATTTGGTT  6960

Query  7066   TACTAATGTCACTACAGCAATCTACTGTACTGGTTCTATACCTTGTGGTGTTTGTCTTAG  7125

|||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||

Sbjct  7081   TACTAATGTCACTACAGCAATCTACTGTACTGGTTCTATACCTTGTAGTGTTTGTCTTAG  7140

Query  29806  GTGATTTTAATAGCTTCTTAGGAGAATGACAAAAAAAAAAAAAAAAAAAA  29855

|||||||||||||||||||||||||||||||||||

Sbjct  29821  GTGATTTTAATAGCTTCTTAGGAGAATGACAAAAA                 29855

That is blasted on single-gene level. It shows a 99% identity with a few allele changes and a 15-bp gap (ATTAAAGGTTTATAC). To summarize:
 

RaTG13 v.1(Ref) and v.2 (Alt): Codon Changes
	BC	Hidden Codons							BC	Hidden Codon Values							BC-Gain	Total Gain
		I	II	III	IV	V	VI			I	II	III	IV	V	VI	SUM
Ref	CTA	GCA	CGT	TGC	ACA	TGG	TAC		29	37	28	58	5	59	50	266	32	-13
Alt	TTA	AGC	TCG	TAG	CAT	CGG	CTA		61	10	55	51	20	27	29	253
Ref	GCA	TGA	GTC	CTG	AGA	CTT	CAG		37	57	46	31	9	32	19	231	-4	24
Alt	GAA	GTC	GTC	AGT	CAG	TCA	GTC		33	46	46	12	19	53	46	255
Ref	TAC	TCG	TGA	AGT	CTC	AGG	ACT		50	55	57	12	30	11	8	223	-1	18
Alt	TAA	TCG	GTC	CTG	AGA	CTT	CAG		49	55	46	31	9	32	19	241
Ref	TGG	CTA	CTA	ATC	GCG	ATG	CTA		59	29	29	14	39	15	29	214	-8	141
Alt	TAG	TCG	TAC	TCA	GAG	TCC	TGA		51	55	50	53	35	54	57	355
Ref	CGC	TAC	ATG	TGC	AGG	ACA	CGT		26	50	15	58	11	5	28	193	5	37
Alt	CTG	GCA	TAC	CAT	GTG	CAA	CGT		31	37	50	20	47	17	28	230

In the above, BC refers to Basic Codon, of which each has its own 6 hidden codons. Hidden codon value refers to energy level corresponding to each codon.

It shows that RaTG13 version 2 gained 207 units on gene-energy-level. Question is: How could the change and its energy gain naturally happen as the WIV leaders said that they have genomic sequence without live-sample regarding the bat RaTG13 nCov?

Regarding the 15-bp gap (ATTAAAGGTTTATAC), here is a comparison:
 

Assesion	Name	Genomic Data
NC_045512.1	WH01-1	CGGTGACGCATACAA AACATTCCCACCATACCTTCCCAGGTAACAAACCAACCAACTTTCGATCTCTTGT
NC_045512.2	WH01-2	ATTAAAGGTTTATAC CTTCCCAGGTAACAAACCAACCAACTTTCGATCTCTTGTAGATCTGTTCTCTAAA
MT511081.1	Poland+	ATTAAAGGTTTATAC CTTTCCAGGTAACAAACCAACCAACTTTCGATCTCTTGTAGATCTGTTCTCTAAA
MN996532.1	RaTG13-1	CTTTCCAGGTAACAAACCAACGAACTCTCGATCTCTTGTAGATCTGTTCTCTAAA
MN996532.2	RaTG13-2	ATTAAAGGTTTATAC CTTTCCAGGTAACAAACCAACGAACTCTCGATCTCTTGTAGATCTGTTCTCTAAA

The genomic data comes from each sample (Poland+, NIH Blast-Search result: hundreds 100% identical samples re patients around the world. Taking the first one, Poland.).

Question: where does the 15-bp gap come from? How does it go exactly the same 15-bp gap come to both RaTG13 (with no live-sample) and patients around the world in almost the same time around 2020 autumn?

There is only one convincing answer to the above questions, that is, RaTG13 nCov is a lab product made for similarities or identities as close as possible to that of patient samples so as to cover up the real SARS-2 origin, such as WIV1.

Regarding the changes for RaTG13 gene data, here is the relevant info by USRTK:

RATG13: ALTERED DATASETS RAISE MORE QUESTIONS ABOUT RELIABILITY OF KEY STUDIES ON CORONAVIRUS ORIGINS.
Posted on December 29, 2020.
NO PEER REVIEW FOR ADDENDUM TO PROMINENT CORONAVIRUS ORIGINS STUDY?
Posted on December 18, 2020
URL: https://usrtk.org/tag/ratg13/ 

It says that, when updating RaTG13 genomic sequence data, the provider, PRC-WIV, did not give a clear reason, nor did they go through peer-review and other normal processes.


Speaking of WIV1, its identities (87% to basic sample) should be good enough as the SARS-2 origin. The provider PRC-WIV said that it is a bat-nCov sample. However, in international meetings, it was reviewed and approved as a lab-product. For example:
 

The University of North Carolina, Institutional Biosafety Committee Meeting Minutes,  Jan.09,2019 3:30 PM.  Burnett-Womack 9001
60350	Infectious clones of bat SARS-like coronaviruses WIV1-CoV and SHC-014 (including reporter-expressing variants) or expressing WIV1 or SHC014 Spike genes - 2019. Renewal
APPROVED	Summary: The aim of this study is to generate reverse genetic infectious clones of bat SARS- like coronaviruses WIV1-CoV and SHC-014, which are genetically similar to. Additionally, to determine if the Spike proteins from these viruses are sufficient to confer infectivity, the Spike genes from the bat viruses will be introduced into the genome background. Replication of recombinant viruses will be monitored through viral passage in cells and infectious of mice. Committee Comments: The proposed containment and safety procedures are adequate for the experimental design. Community Comments: None. III-D, BSL-3, plasmids, mice.
60351	Ralph Baric Transposon mutagenesis of WIV16-CoV to identify genetically flexible regions of CoV genomes
APPROVED	Summary: The aim of this experiment is to generate a transposon mutant library spanning the WIVI6-CoV genome. The virus library will be screened in cell culture for viral fitness via passage in cell lines. Additionally, the screen will be interrogated for genes responsible for interferon antagonism or RNA replication fidelity. Committee Comments: The proposed containment and safety procedures are adequate for the experimental design. Community Comments: None III-D, BSL-3, plasmids
Link ::  https://2f7nhsvfj5dyz0312njuuj14-wpengine.netdna-ssl.com/wp-content/uploads/2020/08/For-Production-to-Requestor-IBC-Meeting-Minutes.pdf

In the above re BP genes frequency and distribution on Codon level, WIV1 has the smallest distance from patient sample (NC_045512), while RaTG13 has three times bigger distance than that of WIV1 and obviously different from that of natural samples. See below:
 

Global Codon BP Freq.Distribution (raw data:NIH GenBank, by 2021-02)
	NC_045512	WIV1	RaTG13	Bat-12	Bat-14
A	3038.33	2884.67	2975.33	2342.33	2552.33
C	1868.00	2020.67	1836.67	1462.67	2135.67
G	1983.67	2098.00	1948.67	1936.00	2419.00
T	3267.00	3099.67	3190.33	3250.00	2946.00
total:	10157	10103	9951	8991	10053
avrg:	2539.25	2525.75	2487.75	2247.75	2513.25
total gap w NC_045512		-54	-206	-1166	-104
avrg gap w NC_045512		-14	-52	-292	-26

In more detailed terms, WIV1 and RaTG13 behave the same way:
 

SARS-2: Global Codon Frequency & Distribution (raw data: NIH GenBank, by 2021-02-22)
	NC_045512			WIV1			RaTG13			Bat-12			Bat-14
	1st	2nd	3rd	1st	2nd	3rd	1st	2nd	3rd	1st	2nd	3rd	1st	2nd	3rd
A	3075	2903	3137	3061	2855	2738	2828	3087	3011	2316	2091	2620	2754	2547	2356
C	2132	1610	1862	2051	2135	1876	1599	1838	2073	1555	1387	1446	2000	2285	2122
G	1525	1909	2517	2389	1629	2276	1921	2466	1459	1692	1852	2264	2729	2014	2514
T	3425	3735	2641	2602	3484	3213	3603	2560	3408	3428	3661	2661	2570	3207	3061
total:	10157			10103			9951			8991			10053
avrg:	2539.25			2525.75			2487.75			2247.75			2513.25
align w NC_045512:				0.99468			0.97972			0.88520			0.98976

In the above, 1st-2nd-3rd refer to three base genes. In terms of Codons frequency and distribution, which in fact the virus and ACE2 run and interact with each other, WIV1 has the highest score, 0.99468, to that of patient sample (NC_45512), while RaTG13 has clearly lower ration and obviously different from natural bat samples.

In sum, between the two candidates of SARS-2 origin, WIV1 is the real one; RaTG13 is not the SARS-2 origin, nor is it a natural sample.

In March 2016, PNAS (publication by US National Academy of Science) published an article with editor’s notes about the WIV1. Please see:
This Issue, by PNAS March 15, 2016 113 (11) 2793-2795;
https://www.pnas.org/content/113/11/2793 



it warned that WIV1 is a lab nCov product, which can directly jump to humans and cause huge pandemic with global economic loses, can even change existing living styles. Now the warning is a big reality that the US and the whole world are facing.
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