When a new pathogen that causes a global epidemic in humans, one key question is where it comes from. This is especially important for a zoonotic infectious disease that jumps from animals to humans. Knowing the origin of such a pathogen is critical to develop means to block further transmission and to develop vaccines. Discovery of the origin of a newly human pathogen is a sophisticated process that requires extensive and vigorous scientific validations and generally takes many years, such as the cases for HIV-1, SARS and MERS. Unfortunately, before the natural sources of new pathogens are clearly defined, conspiracy theories that the new pathogens are man-made often surface as the source. However, in all cases, such theories have been debunked in history

Gao, Feng

Gao, Shou-Jiang

Li, Xiaojun

Liu, Shuying

Sang, Yongming

Xiao, Chuan

English

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Tennessee State University Digital Scholarship @ Tennessee State University Agricultural and Environmental Sciences Faculty Research Department of Agricultural and Environmental Sciences 2-14-2020 HIV-1 did not contribute to the 2019-nCoV genome Chuan Xiao University of Texas at El Paso Xiaojun Li Duke University Shuying Liu University of Massachusetts Medical School Yongming Sang Tennessee State University Shou-Jiang Gao University of Pittsburgh See next page for additional authors Follow this and additional works at: https://digitalscholarship.tnstate.edu/agricultural-and-environmental-sciences-faculty  Part of the Immunology and Infectious Disease Commons Recommended Citation Chuan Xiao, Xiaojun Li, Shuying Liu, Yongming Sang, Shou-Jiang Gao & Feng Gao (2020) HIV-1 did not contribute to the 2019-nCoV genome, Emerging Microbes & Infections, 9:1, 378-381, DOI: 10.1080/22221751.2020.1727299 This Article is brought to you for free and open access by the Department of Agricultural and Environmental Sciences at Digital Scholarship @ Tennessee State University. It has been accepted for inclusion in Agricultural and Environmental Sciences Faculty Research by an authorized administrator of Digital Scholarship @ Tennessee State University. For more information, please contact XGE@Tnstate.edu. Authors Chuan Xiao, Xiaojun Li, Shuying Liu, Yongming Sang, Shou-Jiang Gao, and Feng Gao This article is available at Digital Scholarship @ Tennessee State University: https://digitalscholarship.tnstate.edu/agricultural-and-environmental-sciences-faculty/161 Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=temi20Emerging Microbes & InfectionsISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/temi20HIV-1 did not contribute to the 2019-nCoV genomeChuan Xiao, Xiaojun Li, Shuying Liu, Yongming Sang, Shou-Jiang Gao & FengGaoTo cite this article: Chuan Xiao, Xiaojun Li, Shuying Liu, Yongming Sang, Shou-Jiang Gao & FengGao (2020) HIV-1 did not contribute to the 2019-nCoV genome, Emerging Microbes & Infections,9:1, 378-381, DOI: 10.1080/22221751.2020.1727299To link to this article:  https://doi.org/10.1080/22221751.2020.1727299© 2020 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup, on behalf of Shanghai ShangyixunCultural Communication Co., LtdPublished online: 14 Feb 2020.Submit your article to this journal Article views: 31998View related articles View Crossmark dataCiting articles: 22 View citing articles COMMENTHIV-1 did not contribute to the 2019-nCoV genomeChuan Xiaoa, Xiaojun Li b, Shuying Liuc, Yongming Sangd, Shou-Jiang Gaoe and Feng Gao b,faDepartment of Chemistry and Biochemistry, The University of Texas at El Paso, El Paso, TX, USA; bDepartment of Medicine, Duke UniversityMedical Center, Durham, NC, USA; cNA BioTech Corp, M2D2 Incubator, University of Massachusetts Medical School, Worcester, MA, USA;dDepartment of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN, USA; eUPMC Hillman Cancer Center,Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA; fNational Engineering Laboratory forAIDS Vaccine, School of Life Sciences, Jilin University, Changchun, People’s Republic of ChinaARTICLE HISTORY Received 4 February 2020; Accepted 4 February 2020When a new pathogen that causes a global epidemic inhumans, one key question is where it comes from. Thisis especially important for a zoonotic infectious diseasethat jumps from animals to humans. Knowing the ori-gin of such a pathogen is critical to develop means toblock further transmission and to develop vaccines.Discovery of the origin of a newly human pathogenis a sophisticated process that requires extensive andvigorous scientific validations and generally takesmany years, such as the cases for HIV-1 [1], SARS[2] and MERS [3]. Unfortunately, before the naturalsources of new pathogens are clearly defined, conspi-racy theories that the new pathogens are man-madeoften surface as the source. However, in all cases,such theories have been debunked in history.Infection from an emerging pathogenic coronaviruswas first reported in December 2019 in China. Ithas now affected over 42,000 people and causedover 1,000 deaths in 25 countries (https://2019ncov.Chinacdc.Cn/2019-Ncov). The complete genome ofthis new virus was quickly sequenced and made publicon January 12, only about 2 weeks after the diseasewas first observed [4]. It was named as 2019-nCoVthe following day by the World Health Organization(WHO). Phylogenetic analysis shows that 2019-nCoV is a new member of coronaviruses that infecthumans. It is genetically homogenous but distinctfrom coronaviruses that cause SARS and MERS[5,6]. However, it shares a high level of genetic simi-larity (96.3%) with a bat coronavirus RaTG13 whichwas obtained from bat in Yunnan in 2013, suggestingthat RaTG13-like viruses are most likely the reservoir,but not the immediate sources of the current 2019-nCoV viruses [7].Lack of the definite origin of 2019-nCoV has ledto speculation that 2019-nCoV might be derivedfrom genetic manipulation or even for the purposeof use as a bioweapon. This notion has been fullydebunked in the media. A recent informally pre-sented report, however, showed that 2019-nCoVhad four insertions in the spike glycoprotein genethat is critical for the virus to enter the target cellswhen compared to other coronaviruses [8]. It wasclaimed that these inserts were either identical orsimilar to the motifs in the highly variable (V)regions (V1, V4 and V5) in the envelope glyco-protein or in the Gag protein of some unique HIV-1 strains from three different countries (Thailand,Kenya and India). Together with the structure mod-elling analysis, the authors speculated that thesemotif insertions sharing similarity with HIV-1 pro-teins could provide an enhanced affinity towardshost cell receptors and increase the range of hostcells of 2019-nCoV. This study implies that 2019-nCoV might be generated by gaining gene fragmentsfrom the HIV-1 genome.Current report conducted careful examination ofthe sequences of 2019-nCoV, other CoV viruses andHIV-1 as well as GenBank database. Our resultsdemonstrated no evidence that the sequences of thesefour inserts are HIV-1 specific or the 2019-nCoVviruses obtain these insertions from HIV-1. First, theresults of blast search of these motifs against GenBankshows that the top 100 identical or highly homologoushits are all from host genes of mammalian, insects, bac-terial and others. There are only a few hits on corona-viruses, but none of them are HIV-1 related. Blastagainst viral sequence database also showed theseinsertion sequences widely exist in all kinds of virusesfrom bacteriophage, influenza, to giant eukaryoticviruses (Table 1). More hits were found for corona-viruses and a few also hit on HIV-1 sequences thanthe search against the entire database (Table 1). How-ever, while the 100%match between the insertion 1 and© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., LtdThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricteduse, distribution, and reproduction in any medium, provided the original work is properly cited.CONTACT Chuan Xiao cxiao@utep.edu Department of Chemistry and Biochemistry, The University of Texas at El Paso, 500 W. University Ave, El Paso,TX 79968, USA; Feng Gao fgao@duke.edu Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; National EngineeringLaboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, People’s Republic of ChinaEmerging Microbes & Infections2020, VOL. 9https://doi.org/10.1080/22221751.2020.17272992 sequences and the HIV sequences were found in 19entries, the matches between the insertion 3 and 4sequences and HIV-1 sequences were rather poor(from 42% to 88%). Moreover, the insertion 4 sequenceambiguously hit multiple different genes (gag, pol andenv) in the HIV-1 genome, suggesting that similarities(as low as 42%) between them are too low to be reliable.Search these four insertion sequences against HIV-1Sequence Database (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html)yielded similar results. Sequences that completelymatch the insertion 3 and 4 sequences were notfound in any HIV-1 sequences. This clearly showsthat these insertioin sequences are widely present inliving organisms including viruses, but not HIV-1specific. All these regions in HIV-1 envelope glyco-protein are highly variable with many large insertionsand deletions, indicating that they are not essentialfor biological functions of HIV-1 envelope glyco-protein. The detection of completely matchedsequences of 1 and 2 insertions in only a few HIV-1strains demonstrated that four insertions are veryrare or not present among tens of thousands of naturalHIV-1 sequences. This also explains why four insertionhomolog sequences could only be independently foundin different HIV-1 genomes [8]. Because of their poorTable 1. Blast search results of four insertion sequences against sequence databases.Database Gene source Insertion 1 TNGTKR Insertion 2 HKNNKS Insertion 3 RSYLTPGDSSSG Insertion 4 QTNSPRRAWhole database CoV 2 (2) 0 3 (3) 2 (2)HIV-1 0 0 0 0Prokaryotic 27 (27) 3 (3) 74 (0) 66 (1)Eukaryotic 71 (71) 97 (97) 23 (0) 32 (1)Only viral database CoV 3 (3) 3 (3) 5 (3) 3 (2)HIV-1 18 (18) 1 (1) 4 (0)* 6 (0)**Other Eukaryotic viruses 49 (2) 66 (8) 69 (0) 62 (0)Prokaryotic viruses 29 (13) 30 (1) 21 (0) 28 (0)Unclassified virus 1 (1) 0 1 (0) 1 (0)Top 100 hits are analyzed and the numbers of 100% matches are shown in parentheses. * Similarity at 67%; ** Random hits in Gag, Pro and Env sequenceswith similarity between 42% and 88%.Figure 1. Sequence and structure analysis of 2019-nCoV and bat coronaviruses. (A) Phylogenetic tree analysis of the spike genesequences. (B) Sequence alignment of suspected insertion sites between the 2019-nCoV and bat coronavirus sequences. The del-etions in the alignment are shown as dashes. The numbers of insertions are indicated at the top of the alignment. (C) Structurecomparison of the four insertions in the CoV spike protein and HIV-1 gp120. 2019-nCoV structure was modelled using I-TASSERserver with default parameters. Only relevant domains with residues 1 to 708 (exclude residues from 305 to 603) were presentedas ribbon diagram. The four insertions were labelled and coloured in red, blue, green and magenta, respectively. HIV-1 gp120 struc-ture (PDB 1GC1) is presented as ribbon diagram. V4, V5, V1/V2 and LE loops were labelled and coloured in red, blue, green, andblack, respectively.Emerging Microbes & Infections 379identities to and rareness in the HIV-1 sequences,HIV-1 could not be the source for those insertionsequences in the 2019-nCoV genome.Second, these insertions are present not only in2019-nCoV viruses but also in three betaCoVsequences from bats: two (ZC45 and ZXC21) fromZhejiang deposited in GenBank in 2018 and RaTG13from Yunnan obtained in 2013 [8]. The RaTG13 ismuch more similar to 2019-nCoV than both ZC45and ZXC21 (Figure 1A). The similarity of the spikeprotein between RaTG13 and 2019-nCoV is 97.7%.In the RaTG13 genome, two inserts are identical(HKNNKS and RSYLTPGDSSSG) to those in 2019-nCoV, one has one T→ I substitution (TNGIKR),and the fourth one misses the C-terminal 4 aminoacids (QTNS----) (Figure 1B). ZC45 and ZXC21 aremore divergent from 2019-nCoV than RaTG13, butboth also contain similar insertions at three insertionsites, except insertion 4 (Figure 1B). Furthermore,many other CoV viruses have similar insertions butwith different sequences at the insertion 1 position.These results clearly show that three out of four ofthese inserts naturally exist in three bat CoV virusesbefore 2019-nCoV was identified. This undoubtedlyrefutes the possibility that 2019-nCoV is generatedthrough obtaining gene fragments from the HIV-1genome. Instead, it is much more likely that 2019-nCoV originated from RaTG13-like CoV viruses.Third, insertions 1 and 2 in 2019-nCoV have 6-AAmotifs identical to those in V4 and V5 of certain HIV-1gp120 isolates, which are structurally close to eachother but separated by a LE loop (Figure 1C) [9]. How-ever, insertion 3 located between insertions 1 and 2 in2019-nCoV has sequences similar (with deletions) tothose in the V1 region of HIV-1 gp120. V1 is faraway from V4 and V5 on the opposite side of gp120,which should not interact with V4/V5 in gp120 (Figure1C) but is now inserted between V4 and V5 in themodelled the 2019-nCoV spike protein structure [10].Insertion 4 was found in Gag protein of HIV-1 thatis not associated with viral entry. This insertion islocated too far to be considered to form the same struc-tural unit with the other three insertions in the 2019-nCoV spike protein (Figure 1C). We do not see anyselection benefit or rationale for 2019-nCoV to obtainand mix structurally unrelated parts of HIV-1 to gener-ate a unique structure for its enhanced receptor bind-ing as indicated by the authors [8].How the three bat CoV viruses obtain those insertsremains unknown. For any virus to obtain additionalinsert sequences from other organisms, it requiresthat it has direct interactions with other organisms,most likely through homologous or non-homologousrecombination [11]. For bat CoV viruses to gain thegene fragments from HIV-1, it will require both virusesto co-infect the same cells. Because the host cells for batCoV viruses and HIV-1 are different, the chance forboth to exchange genetic materials is negligible. Onthe contrary, these motifs are widely present in variousmammalian cells and so it will be more likely for batCoV viruses to gain those motifs from the genomes oftheir infected cells if recombination indeed occurs. How-ever, extensive studies of more CoV viruses in wild anddomestic animals are warranted to address this question.Identification of the origins of these insertedsequences in three bat CoV viruses and the new epi-demic 2019-nCoV strain will be important for us tounderstand how CoV viruses jump from animals tohumans and adapt in the latter. Current data showedthat RaTG13 is most closely related to 2019-nCoV[7]. However, the genetic difference between them istoo high for RaTG13 to serve as the immediate ances-tor of 2019-nCoV. Other viruses that are more closelyrelated to 2019-nCoV in intermediate animals like civetfor SARS and camel for MERS [3,12] are remained tobe identified. More studies are necessary to identifythe real source of 2019-nCoV. This may take a longtime to identify the origin of 2019-nCoV by screeninga large number of wild and domestic animals. In anycase, reducing or eliminating direct contacts withwild animals will be critical to control the new epi-demic infection diseases in the future.The advances in bioinformatics analysis tools arewidely used to easily and rapidly analyse newly obtainedsequences. However, great care is required for compre-hensive and thorough analysis to fully understand thereal biological implications of the new genomic infor-mation. Biased, partial and incorrect analysis candangerously lead to conclusions that fuel conspiraciesand harm the process of true scientific discoveries andthe effort to control the damage to public health.AcknowledgmentsWe greatly appreciate Youyu He (Shanghai Center for Bioin-formation Technology) in helping us to blast the insertionsequences against the viral sequence database.Disclosure statementNo potential conflict of interest was reported by the author(s).ORCIDXiaojun Li http://orcid.org/0000-0002-5780-0880Feng Gao http://orcid.org/0000-0001-8903-0203References[1] Gao F, Bailes E, Robertson DL, et al. Origin of HIV-1 inthe chimpanzee Pan troglodytes troglodytes. Nature.1999;397(6718):436–441.[2] Li W, Shi Z, Yu M, et al. Bats are natural reservoirs ofSARS-like coronaviruses. Science (New York, NY).2005 Oct 28:310(5748):676–679.380 C. Xiao et al.[3] Azhar EI, El-Kafrawy SA, Farraj SA, et al. Evidence forcamel-to-human transmission of MERS coronavirus.N Engl J Med. 2014 Jun 26;370(26):2499–2505.[4] Wu F, Zhao S, Yu B, et al. A new coronavirus associ-ated with human respiratory disease in China.Nature. 2020. doi:10.1038/s41586-020-2008-3[5] Zhu N, Zhang D, Wang W, et al. A Novel coronavirusfrom patients with Pneumonia in China, 2019. N EnglJ Med. 2020. doi:10.1056/NEJMoa2001017[6] Lu R, Zhao X, Li J, et al. Genomic characterisation andepidemiology of 2019 novel coronavirus: implicationsfor virus origins and receptor binding. Lancet. 2020.doi:10.1016/S0140-6736(20)30251-8[7] Zhou P, Yang XL, Wang XG, et al. A pneumonia out-break associated with a new coronavirus of probablebat origin. Nature. 2020. doi:10.1038/s41586-020-2012-7.[8] Prashant Pradhan, Ashutosh Kumar Pandey, AkhileshMishra, et al. Uncanny similarity of unique inserts inthe 2019-nCoV spike protein to HIV-1 gp120 andGag. bioRxiv. 2020. doi:10.1101/2020.01.30.927871.[9] Kwong PD, Wyatt R, Robinson J, et al. Structure of anHIV gp120 envelope glycoprotein in complex with theCD4 receptor and a neutralizing human antibody.Nature. 1998 Jun 18;393(6686):648–659.[10] Roy A, Kucukural A, Zhang Y. I-TASSER: a unifiedplatform for automated protein structure and functionprediction. Nat Protoc. 2010 Apr;5(4):725–738.[11] Holmes EC. The Evolution and Emergence of RNAviruses. New York: Oxford University Press; 2009.[12] Guan Y, Zheng BJ, He YQ, et al. Isolation and charac-terization of viruses related to the SARS coronavirusfrom animals in southern China. Science (New York,NY). 2003 Oct 10;302(5643):276–278.Emerging Microbes & Infections 381

HIV-1 did not contribute to the 2019-nCoV genome

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University of Massachusetts Medical School eScholarship@UMMS Coronavirus COVID-19 Publications by UMMS Authors 2020-02-14 HIV-1 did not contribute to the 2019-nCoV genome Chuan Xiao University of Texas at El Paso Et al. Let us know how access to this document benefits you. Follow this and additional works at: https://escholarship.umassmed.edu/covid19  Part of the Bioinformatics Commons, Computational Biology Commons, Genomics Commons, Immunology and Infectious Disease Commons, Infectious Disease Commons, Structural Biology Commons, Virology Commons, and the Virus Diseases Commons Repository Citation Xiao C, Li X, Liu S, Sang Y, Gao S, Gao F. (2020). HIV-1 did not contribute to the 2019-nCoV genome. Coronavirus COVID-19 Publications by UMMS Authors. https://doi.org/10.1080/22221751.2020.1727299. Retrieved from https://escholarship.umassmed.edu/covid19/2 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Coronavirus COVID-19 Publications by UMMS Authors by an authorized administrator of eScholarship@UMMS. For more information, please contact Lisa.Palmer@umassmed.edu. COMMENTHIV-1 did not contribute to the 2019-nCoV genomeChuan Xiaoa, Xiaojun Li b, Shuying Liuc, Yongming Sangd, Shou-Jiang Gaoe and Feng Gao b,faDepartment of Chemistry and Biochemistry, The University of Texas at El Paso, El Paso, TX, USA; bDepartment of Medicine, Duke UniversityMedical Center, Durham, NC, USA; cNA BioTech Corp, M2D2 Incubator, University of Massachusetts Medical School, Worcester, MA, USA;dDepartment of Agricultural and Environmental Sciences, Tennessee State University, Nashville, TN, USA; eUPMC Hillman Cancer Center,Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA; fNational Engineering Laboratory forAIDS Vaccine, School of Life Sciences, Jilin University, Changchun, People’s Republic of ChinaARTICLE HISTORY Received 4 February 2020; Accepted 4 February 2020When a new pathogen that causes a global epidemic inhumans, one key question is where it comes from. Thisis especially important for a zoonotic infectious diseasethat jumps from animals to humans. Knowing the ori-gin of such a pathogen is critical to develop means toblock further transmission and to develop vaccines.Discovery of the origin of a newly human pathogenis a sophisticated process that requires extensive andvigorous scientiﬁc validations and generally takesmany years, such as the cases for HIV-1 [1], SARS[2] and MERS [3]. Unfortunately, before the naturalsources of new pathogens are clearly deﬁned, conspi-racy theories that the new pathogens are man-madeoften surface as the source. However, in all cases,such theories have been debunked in history.Infection from an emerging pathogenic coronaviruswas ﬁrst reported in December 2019 in China. Ithas now aﬀected over 42,000 people and causedover 1,000 deaths in 25 countries (https://2019ncov.Chinacdc.Cn/2019-Ncov). The complete genome ofthis new virus was quickly sequenced and made publicon January 12, only about 2 weeks after the diseasewas ﬁrst observed [4]. It was named as 2019-nCoVthe following day by the World Health Organization(WHO). Phylogenetic analysis shows that 2019-nCoV is a new member of coronaviruses that infecthumans. It is genetically homogenous but distinctfrom coronaviruses that cause SARS and MERS[5,6]. However, it shares a high level of genetic simi-larity (96.3%) with a bat coronavirus RaTG13 whichwas obtained from bat in Yunnan in 2013, suggestingthat RaTG13-like viruses are most likely the reservoir,but not the immediate sources of the current 2019-nCoV viruses [7].Lack of the deﬁnite origin of 2019-nCoV has ledto speculation that 2019-nCoV might be derivedfrom genetic manipulation or even for the purposeof use as a bioweapon. This notion has been fullydebunked in the media. A recent informally pre-sented report, however, showed that 2019-nCoVhad four insertions in the spike glycoprotein genethat is critical for the virus to enter the target cellswhen compared to other coronaviruses [8]. It wasclaimed that these inserts were either identical orsimilar to the motifs in the highly variable (V)regions (V1, V4 and V5) in the envelope glyco-protein or in the Gag protein of some unique HIV-1 strains from three diﬀerent countries (Thailand,Kenya and India). Together with the structure mod-elling analysis, the authors speculated that thesemotif insertions sharing similarity with HIV-1 pro-teins could provide an enhanced aﬃnity towardshost cell receptors and increase the range of hostcells of 2019-nCoV. This study implies that 2019-nCoV might be generated by gaining gene fragmentsfrom the HIV-1 genome.Current report conducted careful examination ofthe sequences of 2019-nCoV, other CoV viruses andHIV-1 as well as GenBank database. Our resultsdemonstrated no evidence that the sequences of thesefour inserts are HIV-1 speciﬁc or the 2019-nCoVviruses obtain these insertions from HIV-1. First, theresults of blast search of these motifs against GenBankshows that the top 100 identical or highly homologoushits are all from host genes of mammalian, insects, bac-terial and others. There are only a few hits on corona-viruses, but none of them are HIV-1 related. Blastagainst viral sequence database also showed theseinsertion sequences widely exist in all kinds of virusesfrom bacteriophage, inﬂuenza, to giant eukaryoticviruses (Table 1). More hits were found for corona-viruses and a few also hit on HIV-1 sequences thanthe search against the entire database (Table 1). How-ever, while the 100%match between the insertion 1 and© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., LtdThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricteduse, distribution, and reproduction in any medium, provided the original work is properly cited.CONTACT Chuan Xiao cxiao@utep.edu Department of Chemistry and Biochemistry, The University of Texas at El Paso, 500 W. University Ave, El Paso,TX 79968, USA; Feng Gao fgao@duke.edu Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; National EngineeringLaboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, People’s Republic of ChinaEmerging Microbes & Infections2020, VOL. 9https://doi.org/10.1080/22221751.2020.17272992 sequences and the HIV sequences were found in 19entries, the matches between the insertion 3 and 4sequences and HIV-1 sequences were rather poor(from 42% to 88%). Moreover, the insertion 4 sequenceambiguously hit multiple diﬀerent genes (gag, pol andenv) in the HIV-1 genome, suggesting that similarities(as low as 42%) between them are too low to be reliable.Search these four insertion sequences against HIV-1Sequence Database (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html)yielded similar results. Sequences that completelymatch the insertion 3 and 4 sequences were notfound in any HIV-1 sequences. This clearly showsthat these insertioin sequences are widely present inliving organisms including viruses, but not HIV-1speciﬁc. All these regions in HIV-1 envelope glyco-protein are highly variable with many large insertionsand deletions, indicating that they are not essentialfor biological functions of HIV-1 envelope glyco-protein. The detection of completely matchedsequences of 1 and 2 insertions in only a few HIV-1strains demonstrated that four insertions are veryrare or not present among tens of thousands of naturalHIV-1 sequences. This also explains why four insertionhomolog sequences could only be independently foundin diﬀerent HIV-1 genomes [8]. Because of their poorTable 1. Blast search results of four insertion sequences against sequence databases.Database Gene source Insertion 1 TNGTKR Insertion 2 HKNNKS Insertion 3 RSYLTPGDSSSG Insertion 4 QTNSPRRAWhole database CoV 2 (2) 0 3 (3) 2 (2)HIV-1 0 0 0 0Prokaryotic 27 (27) 3 (3) 74 (0) 66 (1)Eukaryotic 71 (71) 97 (97) 23 (0) 32 (1)Only viral database CoV 3 (3) 3 (3) 5 (3) 3 (2)HIV-1 18 (18) 1 (1) 4 (0)* 6 (0)**Other Eukaryotic viruses 49 (2) 66 (8) 69 (0) 62 (0)Prokaryotic viruses 29 (13) 30 (1) 21 (0) 28 (0)Unclassiﬁed virus 1 (1) 0 1 (0) 1 (0)Top 100 hits are analyzed and the numbers of 100% matches are shown in parentheses. * Similarity at 67%; ** Random hits in Gag, Pro and Env sequenceswith similarity between 42% and 88%.Figure 1. Sequence and structure analysis of 2019-nCoV and bat coronaviruses. (A) Phylogenetic tree analysis of the spike genesequences. (B) Sequence alignment of suspected insertion sites between the 2019-nCoV and bat coronavirus sequences. The del-etions in the alignment are shown as dashes. The numbers of insertions are indicated at the top of the alignment. (C) Structurecomparison of the four insertions in the CoV spike protein and HIV-1 gp120. 2019-nCoV structure was modelled using I-TASSERserver with default parameters. Only relevant domains with residues 1 to 708 (exclude residues from 305 to 603) were presentedas ribbon diagram. The four insertions were labelled and coloured in red, blue, green and magenta, respectively. HIV-1 gp120 struc-ture (PDB 1GC1) is presented as ribbon diagram. V4, V5, V1/V2 and LE loops were labelled and coloured in red, blue, green, andblack, respectively.Emerging Microbes & Infections 379identities to and rareness in the HIV-1 sequences,HIV-1 could not be the source for those insertionsequences in the 2019-nCoV genome.Second, these insertions are present not only in2019-nCoV viruses but also in three betaCoVsequences from bats: two (ZC45 and ZXC21) fromZhejiang deposited in GenBank in 2018 and RaTG13from Yunnan obtained in 2013 [8]. The RaTG13 ismuch more similar to 2019-nCoV than both ZC45and ZXC21 (Figure 1A). The similarity of the spikeprotein between RaTG13 and 2019-nCoV is 97.7%.In the RaTG13 genome, two inserts are identical(HKNNKS and RSYLTPGDSSSG) to those in 2019-nCoV, one has one T→ I substitution (TNGIKR),and the fourth one misses the C-terminal 4 aminoacids (QTNS----) (Figure 1B). ZC45 and ZXC21 aremore divergent from 2019-nCoV than RaTG13, butboth also contain similar insertions at three insertionsites, except insertion 4 (Figure 1B). Furthermore,many other CoV viruses have similar insertions butwith diﬀerent sequences at the insertion 1 position.These results clearly show that three out of four ofthese inserts naturally exist in three bat CoV virusesbefore 2019-nCoV was identiﬁed. This undoubtedlyrefutes the possibility that 2019-nCoV is generatedthrough obtaining gene fragments from the HIV-1genome. Instead, it is much more likely that 2019-nCoV originated from RaTG13-like CoV viruses.Third, insertions 1 and 2 in 2019-nCoV have 6-AAmotifs identical to those in V4 and V5 of certain HIV-1gp120 isolates, which are structurally close to eachother but separated by a LE loop (Figure 1C) [9]. How-ever, insertion 3 located between insertions 1 and 2 in2019-nCoV has sequences similar (with deletions) tothose in the V1 region of HIV-1 gp120. V1 is faraway from V4 and V5 on the opposite side of gp120,which should not interact with V4/V5 in gp120 (Figure1C) but is now inserted between V4 and V5 in themodelled the 2019-nCoV spike protein structure [10].Insertion 4 was found in Gag protein of HIV-1 thatis not associated with viral entry. This insertion islocated too far to be considered to form the same struc-tural unit with the other three insertions in the 2019-nCoV spike protein (Figure 1C). We do not see anyselection beneﬁt or rationale for 2019-nCoV to obtainand mix structurally unrelated parts of HIV-1 to gener-ate a unique structure for its enhanced receptor bind-ing as indicated by the authors [8].How the three bat CoV viruses obtain those insertsremains unknown. For any virus to obtain additionalinsert sequences from other organisms, it requiresthat it has direct interactions with other organisms,most likely through homologous or non-homologousrecombination [11]. For bat CoV viruses to gain thegene fragments from HIV-1, it will require both virusesto co-infect the same cells. Because the host cells for batCoV viruses and HIV-1 are diﬀerent, the chance forboth to exchange genetic materials is negligible. Onthe contrary, these motifs are widely present in variousmammalian cells and so it will be more likely for batCoV viruses to gain those motifs from the genomes oftheir infected cells if recombination indeed occurs. How-ever, extensive studies of more CoV viruses in wild anddomestic animals are warranted to address this question.Identiﬁcation of the origins of these insertedsequences in three bat CoV viruses and the new epi-demic 2019-nCoV strain will be important for us tounderstand how CoV viruses jump from animals tohumans and adapt in the latter. Current data showedthat RaTG13 is most closely related to 2019-nCoV[7]. However, the genetic diﬀerence between them istoo high for RaTG13 to serve as the immediate ances-tor of 2019-nCoV. Other viruses that are more closelyrelated to 2019-nCoV in intermediate animals like civetfor SARS and camel for MERS [3,12] are remained tobe identiﬁed. More studies are necessary to identifythe real source of 2019-nCoV. This may take a longtime to identify the origin of 2019-nCoV by screeninga large number of wild and domestic animals. In anycase, reducing or eliminating direct contacts withwild animals will be critical to control the new epi-demic infection diseases in the future.The advances in bioinformatics analysis tools arewidely used to easily and rapidly analyse newly obtainedsequences. However, great care is required for compre-hensive and thorough analysis to fully understand thereal biological implications of the new genomic infor-mation. Biased, partial and incorrect analysis candangerously lead to conclusions that fuel conspiraciesand harm the process of true scientiﬁc discoveries andthe eﬀort to control the damage to public health.AcknowledgmentsWe greatly appreciate Youyu He (Shanghai Center for Bioin-formation Technology) in helping us to blast the insertionsequences against the viral sequence database.Disclosure statementNo potential conﬂict of interest was reported by the author(s).ORCIDXiaojun Li http://orcid.org/0000-0002-5780-0880Feng Gao http://orcid.org/0000-0001-8903-0203References[1] Gao F, Bailes E, Robertson DL, et al. Origin of HIV-1 inthe chimpanzee Pan troglodytes troglodytes. Nature.1999;397(6718):436–441.[2] Li W, Shi Z, Yu M, et al. Bats are natural reservoirs ofSARS-like coronaviruses. Science (New York, NY).2005 Oct 28:310(5748):676–679.380 C. Xiao et al.[3] Azhar EI, El-Kafrawy SA, Farraj SA, et al. Evidence forcamel-to-human transmission of MERS coronavirus.N Engl J Med. 2014 Jun 26;370(26):2499–2505.[4] Wu F, Zhao S, Yu B, et al. A new coronavirus associ-ated with human respiratory disease in China.Nature. 2020. doi:10.1038/s41586-020-2008-3[5] Zhu N, Zhang D, Wang W, et al. A Novel coronavirusfrom patients with Pneumonia in China, 2019. N EnglJ Med. 2020. doi:10.1056/NEJMoa2001017[6] Lu R, Zhao X, Li J, et al. Genomic characterisation andepidemiology of 2019 novel coronavirus: implicationsfor virus origins and receptor binding. Lancet. 2020.doi:10.1016/S0140-6736(20)30251-8[7] Zhou P, Yang XL, Wang XG, et al. A pneumonia out-break associated with a new coronavirus of probablebat origin. Nature. 2020. doi:10.1038/s41586-020-2012-7.[8] Prashant Pradhan, Ashutosh Kumar Pandey, AkhileshMishra, et al. Uncanny similarity of unique inserts inthe 2019-nCoV spike protein to HIV-1 gp120 andGag. bioRxiv. 2020. doi:10.1101/2020.01.30.927871.[9] Kwong PD, Wyatt R, Robinson J, et al. Structure of anHIV gp120 envelope glycoprotein in complex with theCD4 receptor and a neutralizing human antibody.Nature. 1998 Jun 18;393(6686):648–659.[10] Roy A, Kucukural A, Zhang Y. I-TASSER: a uniﬁedplatform for automated protein structure and functionprediction. Nat Protoc. 2010 Apr;5(4):725–738.[11] Holmes EC. The Evolution and Emergence of RNAviruses. New York: Oxford University Press; 2009.[12] Guan Y, Zheng BJ, He YQ, et al. Isolation and charac-terization of viruses related to the SARS coronavirusfrom animals in southern China. Science (New York,NY). 2003 Oct 10;302(5643):276–278.Emerging Microbes & Infections 381

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HIV-1 did not contribute to the 2019-nCoV genome

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