key: cord-0267732-ak85v5pe authors: Desingu, Perumal Arumugam; Nagarajan, K. title: SARS-CoV-2 originated from SARS-CoV-1-related Bat-CoVs through Pan-CoVs rather than from SARS-CoV-2-related Bat-CoVs date: 2021-09-07 journal: bioRxiv DOI: 10.1101/2021.09.06.459210 sha: 3b7005a9bd338db872e66aab0ebc37f2f13f21ad doc_id: 267732 cord_uid: ak85v5pe The emergence of the novel SARS-CoV-2 in 2019 sparked a dispute concerning its origin. Here, we report that the SARS-CoV-2 originated through pangolin-coronavirus (Pan-CoVs) from the SARS-CoV-related-bat-coronaviruses (SARS-CoV-1-rB-CoVs) rather than from SARS-CoV-2-related-bat-coronaviruses (SARS-CoV-2-rB-CoVs), in contrast to the previous thought. Further, our analyses strongly suggest that the Pan-CoVs evolved from the SARS-CoV-1-rB-CoVs without recombination. Further, our results suggest that the SARS-CoV-1-rB-CoVs’ perhaps jumped into the pangolin, which forced the viruses to mutate and adapt to the new host, and resulted in the origin of Pan-CoVs. Surprisingly, the Pan-CoVs formed an evolutionary intermediate between SARS-CoV-2 and SARS-CoV-2-rB-CoVs at the spike gene. Our findings also suggest that the Pan-CoV/GX and Pan-CoV/Guangdong lineages recombined to form the SARS-CoV-2 spike gene. We also found evidence that the SARS-CoV-2-rB-CoVs spike gene evolved via recombination between Pan-CoV/Guangdong and SARS-CoV-1-rB-CoVs. Overall, our findings suggest that the SARS-CoV-2 emerged from SARS-CoV-1-rB-CoVs through host jumping. Except for RaTG13, all SARS-CoV-2-rB-CoVs had a lot of genetic variabilities when 218 compared to SARS-CoV-2 at the entire spike (S) gene levels (5) (6) (7) 14) . In our phylogenetic 219 analysis, the Pan-CoVs formed an evolutionary intermediate between the SARS-CoV-2 and 220 SARS-CoV-2-rB-CoVs in the spike gene ( Figure 5A ). On the other hand, in NBGMD 221 analysis, the SARS-CoV-2-rB-CoVs displayed almost equal genetic diversity with CoV-2 and SARS-CoV-1/SARS-CoV-1-rB-CoVs ( Figure 5B) . However, the SARS-CoV-2- showed considerable evolution in Pan-CoVs when compared to SARS-CoV-1/SARS-CoV-1-257 rB-CoVs. A virus that jumps from natural host to another must overcome the following host 258 genetic barriers such as host surface receptors, host antiviral proteins for viral replication, 259 virus protein translation, virus assembly, release, and the host's innate and adaptive immune 260 protection (35) (36) (37) (38) (39) . Viruses used to mutate and bypass all these host barriers to accomplish in 261 the new species through host jump (35) (36) (37) 40) . In majority of the time, accidental virus 262 introduction to an unnatural host does not result in effective infection (35) (36) (37) 40, 41) . 263 However, in rare cases/continuous/forced exposure to an unnatural host, the viruses begin to 264 adapt to the new host through virus mutations to tackle the host selection pressure and 265 prolong their lifecycle (35) (36) (37) 41) . Viruses must then continue to evolve to increase their 266 adaptability. Finally, it becomes highly adapted to new species to produce a large viral load 267 that can be transmitted within the new hosts (35, 37, 38, 40) . Furthermore coronaviruses in 268 general, the quasispecies evolutionary processes have been well described (39, (42) (43) (44) (45) (46) (47) (48) (49) . To 269 support this, the ongoing SARS-CoV-2 outbreak in humans displayed widespread 270 8 mutation/deletions and evolution in the critical genes of ORF1a (46, [50] [51] [52] [53] [54] ; spike gene 271 (46, 50, 51, 55, 56) ; ORF3 (50, [57] [58] [59] ; and ORF8 (33,50,60-65) those viral proteins regulates 272 the virus transmission, better adaptability to host and evasion of the host immunity. 273 Furthermore, the presence of selection pressure for the evolution of the SARS-CoV-2 has 274 been highly characterized (10, 52, 66, 67) . More importantly, among the Pan-CoV/GX and 275 Pan-CoV/Guangdong lineages, Pan-CoV/Guangdong lineage displayed high sequence 276 identity with SARS-CoV-2 and was isolated from the clinically ill pangolins (8, 9) . Therefore, 277 collectively it can be inferred that the SARS-CoV-1/SARS-CoV-1-rB-CoVs were jumped (Figure 7) . This notion is further supported by the fact that pangolins were unwell 284 and deceased as a result of the Pan-CoV/Guangdong lineage (8, 9) . In addition, huge genetic diversity was documented between the SARS-CoV-2 and SARS- CoV-2-rB-CoVs in the spike gene (5-7,14) which is responsible for critical host range, 287 transmissibility, and virus entry. Furthermore, the occurrence of widespread recombination 288 events in the spike gene of SARS-CoV-2-rB-CoVs has been extensively studied for this 289 gene's evolution (8,10-13). As per our findings in this study, revealed that the progenitor 290 SARS-CoV-2/RaTG13-like virus is recombinant of the Pan-CoV/Guangdong and Pan-291 CoV/GX lineages (Figure 7) . In this perspective, our study implies that a recombinant of the 292 Pan-CoV/Guangdong and Pan-CoV/GX lineages may have entered humans and begun 293 evolving in these genes for improved adaptation (Figure 7) . Due to the mild illness or short- CoV-1-rB-CoVs in the part of pp1a and S1-RBD (Figure 7) . Surprisingly, Our findings 301 found evidence that the Bat/China/RpYN06 virus evolved by recombination of Pan-302 CoV/Guangdong lineages and bat-SL-CoVZC45 virus, which is the next one to bat/RaTG13 303 virus in terms of overall genome similarity to SARS-CoV-2 (Figure 7) . 304 In conclusion, the SARS-CoV-1/SARS-CoV-1-rB-CoVs were perhaps jumped into the Pan-CoV/GX and Pan-CoV/Guangdong lineages in the S1-NTD. Similarly, progenitor 314 SARS-CoV-2-rB-CoVs-like viruses were evolved through recombination of Pan-CoVs and 315 SARS-CoV-1-rB-CoVs in the part of pp1ab and S1-RBD. Further, the progenitor SARS-316 CoV-2-like virus and SARS-CoV-2-rB-CoVs-like virus's gave raise to SARS-CoV-2 and 317 SARS-CoV-2-rB-CoVs respectively through host jump mediated evolution. This study 318 demonstrates that the SARS-CoV-2 outbreak emerged through host jump mediated virus 319 evolution (Figure 7) . The close monitoring and large-scale surveillance of SARS-CoV-320 2/SARS-CoV-2-rB-CoVs in different domestic and wild species are warranted to predict their 321 potential to evolve as a new virus that may cause the next pandemic outbreak, and also get 322 prepared with the vaccines and antivirals. Joining method used to infer evolutionary history, bootstrap tests (1000 replicates), the 329 Maximum Composite Likelihood method used to compute evolutionary distances, and 330 gamma distribution (shape parameter = 5) used to compute the evolutionary distances being 331 employed to model the rate variation among sites. For evolutionary comparisons, differences 332 in composition bias among sequences were analyzed, and all ambiguous sites were discarded 333 in the analysis for each sequence pair. The NBGMD were measured using MEGA7 tool and the Kimura 2-parameter model, with a 336 gamma distribution (shape parameter=5) used as a model to measure rate variation among 337 sites, a bootstrap test (1000 replicates) was used to estimate the Standard error and all 338 ambiguous sites for each sequence pair were removed from the analysis. Above the diagonal, 339 standard error estimates were displayed. SimPlot 3.5.1 was used to calculate the percent identity between the query and reference 343 sequences. MEGA7 was used to align the nucleotide sequences before they were exported to 344 SimPlot 3.5.1 for further analysis. We used the Kimura two-parameter method to measure the 345 identity between the query and reference sequences, using the 500 base pair of the window at 346 a 50 base pair step. Manipulation of autophagy by SARS-CoV-2 proteins. Autophagy, 1-3. CoVs, SARS-CoV-2, and related viruses using the nucleotide sequences of E, M, protein 6, 581 protein 7a, and protein 7b. SARS-CoV-2 is set as a query sequence. Window : 500 bp, Step: 50 bp, GapStrip: On, Kimura (2-parameter), T/t: 2.0 Position 9,500 9,000 8,500 8,000 7,500 7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 Position 820 800 780 760 740 720 700 680 660 640 620 600 580 560 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 Position 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Position 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Position 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Position 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Position 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Position 29,000 28,000 27,000 26,000 25,000 24,000 23,000 22,000 21,000 20,000 19,000 18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 377 (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications 378 for virus origins and receptor binding A new coronavirus associated with human respiratory disease in China 383 (2020) A Novel Coronavirus from Patients with Pneumonia in China 1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45ar Bat/Cambodia** Bat/Cambodia/RShSTT200, and Bat/Cambodia/RShSTT182 MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS ASARS-CoV-1* DQ071615.1_Bat_SARS/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1_BtRs-BetaCoV/YN2013 /BtRs-BetaCoV/HuB2013 Bat-SARS/HKU@ JX993988.1_Bat/Cp/Yunnan2011 Bat/China/RsYN09 MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS Severe acute respiratory syndrome coronavirus 2 MN994468.1 /SARS-CoV SARS-CoV-1* DQ071615.1/Bat/SARS/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 /BtRs-BetaCoV/HuB2013 Bat-SARS/HKU@ JX993988.1/Bat/Cp/Yunnan2011 Step: 30 bp, GapStrip: On, Kimura Guangdong##-Pangolin/Guangdong/A22-2, Pangolin/Guangdong/A22-2, MT121216.1/Pangolin/MP789 , and Pangolin/Guangdong RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS /PCoV GX-P5E MT040334.1 /PCoV GX-P1E MT040333.1 /PCoV GX-P4L 1MT072864.1 /PCoV GX-P2V MT040335.1/PCoV GX-P5L SARS-CoV-1* DQ071615.1/Bat/SARS/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 MN996532.2/Bat/RaTG13 Bat/Cambodia** bat/Yunnan/RmYN02 MW251308.1_Bat/RacCS203 Step: 30 bp, GapStrip: On, Kimura SARS-CoV-1-rB-CoVs) CoVs At complete genome levels: evolutionary intermediate strain Between SARS-CoV-1 and SARS-CoV-2 At Spike gene levels: evolutionary intermediate strain Between SARS-CoV-2 and SARS-CoV-2-rB-CoVs CoVs Bat -SARS -CoVs (SARS-CoV-1-rB-CoVs) Bat-SL-CoVZC45-like virus SARS-CoV-1* DQ071615.1_Bat_SAR_Rp3 MK211375.1_BtRs-BetaCoV/YN2018A KJ473816.1_BtRs-BetaCoV/YN2013 Step: 50 bp, GapStrip: On, Kimura DQ071615.1_Bat_SAR_Rp3 MK211375.1_BtRs-BetaCoV/YN2018A KJ473816.1_BtRs-BetaCoV/YN2013 Step: 50 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS DQ071615.1/Bat/SARS/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 T/t: 2.0 SARS-CoV-1* DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 MW703458.1_Bat/PrC31 Bat-SL-CoV*** Pan-CoV/Guangdong## Bat/China$ KJ473814.1/BtRs-BetaCoV/HuB2013 Bat-SARS/HKU@ JX993988.1/Bat/Cp/Yunnan2011 Step: 50 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS SARS-CoV-1* DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Bat/China$$ DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 MW703458.1_Bat/PrC31 Bat-SL-CoV*** Pan-CoV/GX# Pan-CoV/Guangdong## Bat/China$ KJ473814.1/BtRs-BetaCoV/HuB2013 Bat-SARS/HKU@ JX993988.1/Bat/Cp/Yunnan2011 Step: 50 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS DQ071615.1_Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Step: 20 bp, GapStrip: On, Kimura SARS-CoV-1* DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Step: 20 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 SARS-CoV-1* DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Step: 20 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS DQ071615.1/Bat/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Bat/China$$ SARS-CoV-1* DQ071615.1/Bat/p3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Step: 30 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS DQ071615.1_Bat_SARS/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Bat/Cambodia** bat/Yunnan/RmYN02 MW251308.1_Bat/RacCS203 Step: 30 bp, GapStrip: On, Kimura SARS-CoV-1* DQ071615.1_Bat_SARS/Rp3 MK211375.1/BtRs-BetaCoV/YN2018A KJ473816.1/BtRs-BetaCoV/YN2013 Bat/Cambodia** bat/Yunnan/RmYN02 MW251308.1_Bat/RacCS203 Step: 30 bp, GapStrip: On, Kimura MG772934.1/bat-SL-CoVZXC21, and MG772933.1/bat-SL-CoVZC45 Pan-CoV/GX# -MT072864.1/PCoV GX-P2V, MT040335.1/PCoV GX-P5L, MT040334.1/PCoV GX-P1E, MT040336.1/PCoV GX-P5E, and MT040333.1/PCoV GX-P4L Pan-CoV/Guangdong##-Pangolin/Guangdong/A22-2 RsYN04 Bat-SARS/HKU@ -DQ022305.2 Bat SARS/HKU3-1, GQ153547.1 Bat SARS/HKU3-12, and GQ153543.1 Bat SARS SimPlot -Query: Pan-CoV/GX# Window : 500 bp, Step: 50 bp, GapStrip: On, Kimura (2-parameter), T/t: 2.0 Position 9,500 9,000 8,500 8,000 7,500 7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000 Position 9,500 9,000 8,500 8,000 7,500 7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000