key: cord-1036083-59zhfh1o
authors: Dimonte, Salvatore; Babakir-Mina, Muhammed; Hama-Soor, Taib; Ali, Salar
title: Genetic Variation and Evolution of the 2019 Novel Coronavirus
date: 2021-01-06
journal: Public Health Genomics
DOI: 10.1159/000513530
sha: 1079c66c22989c7720bbe45cbcaf18173d3abc5a
doc_id: 1036083
cord_uid: 59zhfh1o

INTRODUCTION: SARS-CoV-2 is a new type of coronavirus causing a pandemic severe acute respiratory syndrome (SARS-2). Coronaviruses are very diverting genetically and mutate so often periodically. The natural selection of viral mutations may cause host infection selectivity and infectivity. METHODS: This study was aimed to indicate the diversity between human and animal coronaviruses through finding the rate of mutation in each of the spike, nucleocapsid, envelope, and membrane proteins. RESULTS: The mutation rate is abundant in all 4 structural proteins. The most number of statistically significant amino acid mutations were found in spike receptor-binding domain (RBD) which may be because it is responsible for a corresponding receptor binding in a broad range of hosts and host selectivity to infect. Among 17 previously known amino acids which are important for binding of spike to angiotensin-converting enzyme 2 (ACE2) receptor, all of them are conservative among human coronaviruses, but only 3 of them significantly are mutated in animal coronaviruses. A single amino acid aspartate-454, that causes dissociation of the RBD of the spike and ACE2, and F486 which gives the strength of binding with ACE2 remain intact in all coronaviruses. DISCUSSION/CONCLUSION: Observations of this study provided evidence of the genetic diversity and rapid evolution of SARS-CoV-2 as well as other human and animal coronaviruses.

Coronavirus disease is the severe acute respiratory disease in humans that is caused by the emergence of a new type of a coronavirus. The outbreak of the disease was first identified in Wuhan, China, in December 2019. Wuhan city became an epicenter of the disease, and then the outbreak spread outside of China and caused the emergence of the disease in 213 countries and territories. The total number of infected cases (25, 658, 983) and death (855,186) has been recorded globally until September 1, 2020. The World Health Organization called it as a public health emergency of worldwide concern (https://www. worldometers.info/coronavirus/). The estimated mean time of incubation period of SARS-CoV-2 is 5.1 days. In general, all 3 emerged coronaviruses have relatively similar mean time of incubation period around 5 days. More precisely it is 5.1 in SARS-CoV-2 (ranged from 4.5 to 5.8 days) [1] , 5 days in SARS (ranged from 2 to 14 days) [2] , and 7 days in MERS (ranged from 2 to 14 days) [3] .

Isolate was used as a reference strain for the definition of mutations. Multiple sequence alignments of sequences were performed using T-Coffee program (https://www.ebi.ac.uk/Tools/msa/tcoffee/) and were manually edited with the BioEdit software. The T-coffee program was used to mitigate the pitfalls of progressive alignment methods and because it is suitable for small alignments. In the case of amino acid insertion or deletion, these events were not considered.

All amino acid viral sequences of the spike, nucleocapsid, membrane, and envelope proteins were screened in both animal and human viruses, and the frequency of mutations was calculated and statistically compared using the χ 2 test (based on a 2 × 2 contingency table containing the number of isolates from animal and human viruses and the number of isolates with and without mutations) [16, 17] . Fisher exact tests were used to determine whether the differences in frequency between the 2 groups of samples (animal vs. human) were statistically significant.

The Benjamini-Hochberg method was used to identify results that were statistically significant in multiple hypothesis testing. A strong false discovery rate of 0.001 was used to determine statistical significance [18] .

The pandemic COVID-19 is the third known outbreak to cause respiratory illness in the current century which has been caused by zoonotic coronaviruses, and it is thought to have jumped from bats or/and pangolin hosts to humans [14, 15, [19] [20] [21] . The disease spread mostly everywhere globally very rapidly mainly due to traveling. Several mutations have been observed in the virus genome during the rapid dissemination of the disease across different continents. Antigenic drift may occur as a result of accumulation of several mutations in the virus during seasonal outbreaks which helps the virus to survive due to natural selection process like it is common among influenza viruses [14] . Tracking mutations in the genome of the virus is necessary especially in spike protein which directly has a role in binding and infecting the cell. Vaccine development is mostly dependent on the spike protein, and this is aimed to find antibody against spike or any other surface proteins to neutralize viruses [22, 23] . Therefore, in this study, the occurrence of mutations in different animal and human coronaviruses and finding genetic diversity among human coronaviruses and/ with animal coronaviruses were focused. For this purpose, over 50 protein sequences of each of the spike (S), nucleocapsid (N), envelope (E), and membrane (M) proteins from the online database GenBank was collected. The sum of SARS viruses' amino acid changes is listed in Table 1 . The sequences were processed for each of them separately in both human and animal viruses as follows. F4S  E340S  L387I  A2S  A68D  R131F  Y178V  S2A  I84H  Q163D Q242S  F307L  Y360V  V5L  E340G  N388A  D3N  V70S  R131I  K180R  D3S  Y87W  G164Q  G243S  A308V  K361G  E7N  F342S  D389G  T7S  Y71F  P132A  L181R  N4G  R88N  T165G  Q244S  S310N  F363R  L12S  A344G  D389S  T9D  R72Q  L133I  G182Q  N4V  R89E  L167I  T245R  A311S  T366D  I13V  R346L  D389T  T9P  I73P  L134E  A183R  G5K  A90Q  K169D  V246T  A311V  E367Q  V14L  F347I  L390G  L13V  I73V  E135G  S184Y  G5N  R93W  F171N  T247P  S312A  P368E  N15T  F347Y  F392K  K14V  W75T  E135S  Q185L  N8D  G96M  F171V  S250K  S312H  K370R  S16A  A348P  F392T  K15E  I76G  S136A  R186G  N8G  G96P  Y172F  A252R  F314C  K373S  L18Y  A348Q  N394D  L17F  T77F  S136V  R186N  Q9K  D98K  E174P  E253K  F314L  K374R  A22G S349T R385Q  L28I  I358H  K417C  F26A  M84L  V143T  G192R  G44V  P106S  S184T  R259Y  M322T Q390K  V29I  S359P  I418C  F26I  C86V  I144L  F193W  L45G  R107A  R185A  Q260C  E323K  T391E  V29L  V362P  D467N  L27F  L87F  L145K  A195F  L45R  R107N  S187R  Q260W E323R  T393D  T30S  A363F  T470Y  F28L  L87I  R146S  A195V  P46K  Y109H  N192A  A264P  V324E  L394V  L31R  D364R  Y473L  L29T  V88T  H148T  S197A  P46S  E118A  N192S  T265P  V324L  L395T  I33V  Y365F  T500C  T30F  G89C  R150L  S197V  N47R  E118H  S193P  K266P  P326G  P396D  L34Q  Y365L  T500G  T30I  G89L  R150Y  R198K  N48G  G120D  S197G  A267E  S327D  A397N  A36F  S366E  G504Y  W31F  L90V  I151C  Y199Q  T49N  L121A  S197R  A267G  T329L  A397Q  L37I  V367T  Y505F  W31L  M91L  I151F  Y199S  T49P  P122N  T198S  N269S  W330E A398L  L39A  L368I  Q506G  I32L  M91S  I151V  R200K  A50L  P122R  P199R  T271D  W330H D399E  C40A  L368V  P507E  C33V  W92F  A152E  R200S  W52L  Y123W G200S  Q272A  L331I  L400F  A41D  Y369N  P507S  L34V  S94G  H154F  I201H  T54A  A125D  S201N  A273C  Y333F  F403E  Y42A  N370S  Y508L  F37Y  S94M  H154Q  I201V  T54Q  N126R  A208G  A273V  G335F  S404P  Y42T  S371G  R509Y  A38G  F96W  H155K  G202D  A55P  K127Q  A211S  R276P  G335Y  Q406V  N45L  A372L  V512D  Y39H  A98N  H155W N203D  T57K  I130V  G214S  G278T  A336K  L407I  V47W  S373W  V512L  A40Y  F103Y  G157A  N203T  T57R  I131F  G215E  P279K  A336T  Q408N  N48Y  S375N  L513V  N41K  A104K  R158K  Y204G  Q58A  T135A  A217D  T282K  I337M  Q409W  V49S  T376K  S514Y  N41T  A104R  R158T  K205E  Q58V  E136K  A218I  Q283E  I337T  S410G  S50W  K378F  F515V  R42Y  T106C  C159V  K205S  H59K  L139D  A218L  G284K  K338V  S413A  L51I  C379S  E516Q  N43S  M109W D160E  L206A  H59T  L139K  L219I  Q289A  L339V  A414L  K53I  Y380V  E516T  R44A  S111A  D160Q  N207E  G60D  N140T  L219V  Q289D D340P  A414V  F56K  G381D  R44K  T116S  I161P  N207V  G60N  T141K  L221A  E290K  D341K  D415G  Y57G  G381S  L46I  N117D  I161V  D209N  E62P  K143R  L222A  L291M  K342D  S416E  V58A  V382I  I48G  I118A  D163H  D209V  D63F  D144S  L223A  I292V  K342S  Q418E  V58T  V382L  I49L  L119I  D163Q  S212T  L64P  H145N L223V  R293E  K347D  A419L  Y59A  P384Y  I49V  L119V  E167D  S213D  L64W  I146L  L224K  Q294E  K347E  A419N  S60F  T385G  L51M  L120G  E167F  S213E  F66V  I146Q  D225K  T296I  D348N  L65F  T385L  I52L  N121S  I168V  S214G  Q70N  T148V  D225Q T296V  Q349F  L65N  T385S  F53I  N121T  T169F  D215E  Q70S  A152D  R226A  D297K  Q349Y  S68D  K386P  L56C  V122I  L16H  D215S  G71A  N153K  R226I  Y298A  I351K  D72K  L57F  P123L  A171C  N216K  I74T  N154F  N228K  K299G  L352I  V60L  P123S  S173A  N216S  N75G  A155D  E231G  W301F L353C  T61N  L124V  S173P  I217V  T76E  A155P  S232I  W301V N354D  T61V  H125T  R174D  L220H  N77G  A156Q  K233Q  W301Y K355E  L62I  T127Q  T175R  V221L  S79K  I157Y  M234Q P302T  K355S  C64V  T127R  L176R  V221Y  S79N  V158P  K237Q  Q303A  H356C  F65G  I128Q  L176T  Q222T  P80K  Q160R  G238K  I304M  H356Q  L67F  L129C  S177N  Q222V  D81S  L161F  Q239S  A305L  I357V  L67I  T130N  Y178I  D82Q  P162S  Q241K  Q306N A359G Sum of the mutations found and observed in a statistically significant manner (p ≤ 0.001) for each viral protein analyzed in the text. In the table were listed the changes, related to envelope (ENV), spike (nAb and RDB epitopes crucial and enough to bind ACE2 receptor), membrane, and nucleocapsid viral proteins. The ENV protein is responsible for protection of the interior parts of the virus and has a role in viral assembly during viral replication. The spike protein helps in viral attachment to its corresponding receptor and mediates fusion of the cell and viral membrane. The surface membrane is the most abundant structural protein and defines the shape of the viral envelope. The nucleocapsid is the pretentious structure inside the box of the coronaviruses: N structural protein and CoV RNA genome make up the nucleocapsid. Envelope The entire envelope protein sequences, derived from 51 animal viruses and 55 human viruses, were analyzed ( Fig. 1 ). In Figure 1 , frequencies of SARS-CoV-2 envelope amino acid signatures were shown, using the isolate NC_045512.2 COVID-19/Wuhan as a reference.

In envelope virus sequences, among the 76 residues, 365 mutations were observed; 47, in a statistically significant manner (p ≤ 0.001). In animal viruses' sequences, 333 mutations were observed; 47 of them (F4S, V5L, E7N, L12S, I13V, V14L, N15T, S16A, L18Y, A22G, A22L, V24L, V25A, V25F, F26V, L27Y, L28I, V29I, V29L, T30S, L31R, I33V, L34Q, A36F, MUT, L37I, L39A, C40A, A41D, Y42A, Y42T, N45L, V47W, N48Y, V49S, S50W, L51I, K53I, F56K, Y57G, V58A, V58T, Y59A, S60F, L65F, L65N, S68D, and D72K) in a statistically significant manner (p ≤ 0.001) (Fig. 1a, b) . In envelope human virus sequences, 94 mutations were observed and just 1, L28I, was observed in a statistically significance manner (p ≤ 0.001) (3 isolates; 5.4%) (Fig. 1a) .

The entire spike protein sequences, derived from 51 animal viruses and 52 human viruses, were analyzed. This protein has several domains (1,273 amino acid residues): N-terminal domain, RBD, receptor-binding motif, subdomain-1, subdomain-2, fusion peptide, heptad repeat 1, heptad repeat 2, transmembrane region, and intracellular domain. The SARS-CoV-2 spike RBD bound to the cell receptor ACE2, and here this crucial region (domain located between amino acid residues 336-516) was analyzed [24] .

In Figure 2 , frequencies of SARS-CoV-2 spike RBD amino acid signatures were shown, using the isolate NC_045512.2 COVID-19/Wuhan as a reference.

In spike viruses' sequences, among the 1,273 residues, 9,782 mutations were observed; 982, in a statistically significant manner (p ≤ 0.001). In animal sequences, 920 mutations were observed, and specifically in the RBD region, 83 of them (E340S, E340G, F342S, A344G, R346L, F347I, F347Y, A348P, A348Q, S349T, A352G, W353F, W353Y, N354K, N354Y, K356S, I358H, S359P, V362P,  A363F, D364R, Y365F, Y365L, S366E, V367T, L368I,  L368V, Y369N, N370S, S371G, A372L, S373W, S375N,  T376K, K378F, C379S, Y380V, G381D, G381S, V382I,  V382L, P384Y, T385G, T385L, T385S, K386P, L387I,  N388A, D389G, D389S, D389T, L390G, F392K, F392T,  N394D, N394S, Y396F, A397L, V401R, D405T, T415A,  G416T, K417C, I418C, D467N, T470Y, Y473L, T500C,  T500G, G504Y, Y505F, Q506G, P507E, P507S, Y508L,  R509Y , V512D, V512L, L513V, S514Y, F515V, E516Q, and E516T) in a statistically significant manner (p ≤ 0.001) (Fig. 2a, b) . In spike human virus sequences, 222 mutations were observed, and specifically in the RBD region, 18 of them (E340S, F347I, W353F, K356S, V362P, Y369N, Y380V,   G381D, T385L, K386P, D389T, D467N, T470Y, Y473L,  G504Y , P507S, Y508L, and V512D) in a statistically significant manner (p ≤ 0.001) (Fig. 2a, b) .

To best study this domain, the geographical localization of sequences was analyzed. Homogeneity continental sources were observed: just the D405T animal virus mutation (10 Chinese isolates) shows a specific country (and continent, Asia).

The entire membrane protein sequences, derived from 51 animal viruses and 55 human viruses, were analyzed (Fig. 3 ). In Figure 3 , frequencies of SARS-CoV-2 membrane amino acid signatures were shown, using the isolate NC_045512.2 COVID-19/Wuhan as a reference. Frequency, % V70S  Y71F  R72Q  I73P  I73V  W75T  I76G  T77F  T77G  G78F  G79A  G79V  I80A  I80F  A83L  M84L  C86V  L87F  L87I  V88T  G89C  G89L  L90V  M91L  M91S  W92F  S94G  S94M  F96W  A98N  F103Y  A104K  A104R  T106C  M109W  S111A  T116S  N117D  I118A  L119I  L119V  L120G  N121S  N121T  V122I  P123L  P123S  L124V  H125T  T127Q  T127R  I128Q  L129C R131F  R131I  P132A  L133I  L134E  E135G  E135S  S136A  S136V  E137P  L138M  V139G  G141S  A142L  A142P  V143I  V143T  I144L  L145K  R146S  H148T  R150L  R150Y  I151C  I151F  I151V  A152E  H154F  H154Q  H155K  H155W  G157A  R158K  R158T  C159V  D160E  D160Q  I161P  I161V  D163H  D163Q  E167D  E167F  I168V  T169F  L16H  A171C  S173A  S173P  R174D  T175R  L176R  L176T  S177N K180R  L181R  G182Q  A183R  S184Y  Q185L  R186G  R186N  V187A  V187D  A188Q  G189S  D190G  S191K  S191T  G192R  F193W  A195F  A195V  S197A  S197V  R198K  Y199Q  Y199S  R200K  R200S  I201H  I201V  G202D  N203D  N203T  Y204G  K205E  K205S  L206A  N207E  N207V  D209N  D209V  S212T  S213D  S213E  S214G  D215E  D215S  N216K  N216S  I217V  L220H  V221L  V221Y  Q222T In membrane viruses sequences, among the 222 residues, 851 mutations were observed; 219, in a statistically significance manner (p ≤ 0.001). In animal membrane sequences, 794 mutations were observed, and specifically 219 of them (A2S, D3N, T7S, T9D, T9P, L13V, K14V,  K15E, L17F, E18R, Q19N, W20Y, L22F, V23F, G25N,  G25T, F26A, F26I, L27F, F28L, L29T, T30F, T30I, W31F,  W31L, I32L, C33V, L34V, F37Y, A38G, Y39H, A40Y,  N41K, N41T, R42Y, N43S, R44A, R44K, L46I, I48G, I49L,  I49V, L51M, I52L, F53I, L56C, L57F, V60L, T61N, T61V,  L62I, C64V, F65G, L67F, L67I, A68D, V70S, Y71F, R72Q,  I73P, I73V, W75T, I76G, T77F, T77G I151C, I151F, I151V, A152E, H154F, H154Q, H155K,  H155W, G157A, R158K, R158T, C159V, D160E, D160Q,  I161P, I161V, D163H, D163Q, E167D, E167F, I168V,  T169F , L16H, A171C, S173A, S173P, R174D, T175R, L176R, L176T, S177N, Y178I, Y178V, K180R, L181R, G182Q, A183R, S184Y, Q185L, R186G, R186N, V187A, V187D, A188Q, G189S, D190G, S191K, S191T, G192R, F193W, A195F, A195V, S197A, S197V , R198K, Y199Q,  Y199S, R200K, R200S, I201H, I201V, G202D, N203D,  N203T, Y204G, K205E, K205S, L206A, N207E, N207V,  D209N, D209V, S212T, S213D, S213E, S214G, D215E,  D215S, N216K, N216S, I217V, L220H, V221L, V221Y, Q222T, and Q222V) in a statistically significant manner (p ≤ 0.001) (Fig. 3a-d) .

In human membrane virus sequences, 262 mutations were observed and specifically 35 of them (A2S, D3N,  L22F, F26I, F28L, T30F, L34V, F37Y, A38G, N43S, L51M,  F53I, V60L, L62I, L67F, L87I, A98N, M109W, V122I,  L133I, V143T, R150Y, H155K, R158T, I168V, S173P, L181R, A195V, S197V, R198K, Y199S, R200K, I201V, S212T, and S214G) in a statistically significant manner (p ≤ 0.001) (Fig. 3a-d) .

The entire nucleocapsid protein sequences, derived from 51 animal viruses and 54 human viruses, were analyzed (Fig. 4) . In Figure 4 , frequencies of SARS-CoV-2 nucleocapsid amino acid signatures were shown, using the isolate NC_045512.2 COVID-19/Wuhan as a reference.

In nucleocapsid virus sequences, among the 419 residues, 1,885 mutations were observed; 317, in a statistically significance manner (p ≤ 0.001).

In animal nucleocapsid sequences, 613 mutations were observed and specifically 38 of them (D82Q, Y87W, A90Q, D98K, E118H, I130V, I131F, V158P, Q160R, L161F, G179R, S183N, T198S, A208G, A264P, A273C, L291M, W301F, F307L, A308V, G316F, M317G, E323R, S327D, W330E, K347E, I351K, K355E, K370R, A381P, and Q418E) in a statistically significant manner (p ≤ 0.001) (Fig. 4a-f ).

In human nucleocapsid sequences, 1,628 mutations were observed and specifically 317 of them (S2A, D3S,  N4G, N4V, G5K, G5N, N8D, N8G, Q9K, R10T, N11P,  F17L, S21K, R41P, Q43K, Q43R, G44V, L45G, L45R,  P46K, P46S, N47R, N48G, T49N, T49P, A50L, W52L,  T54A, T54Q, A55P, T57K, T57R, Q58A, Q58V, H59K,  H59T, G60D, G60N, E62P, D63F, L64P, L64W, F66V,  Q70N, Q70S, G71A, I74T, N75G, T76E, N77G, S79K,  S79N, P80K, D81S, D82Q, I84H, Y87W, R88N, R89E,  A90Q, R93W, G96M, G96P, D98K, K100G, K100Q,  M101R, L104Q, L104V, S105P, P106D, P106S, R107A,  R107N, Y109H, E118A, E118H, G120D, L121A, P122N,  P122R, Y123W, A125D, N126R, K127Q, I130V, I131F,  T135A, E136K, L139D, L139K, N140T, T141K, K143R,  D144S, H145N, I146L, I146Q, T148V, A152D, N153K,  N154F, A155D, A155P, A156Q, I157Y, V158P, Q160R,  L161F, P162S, Q163D, G164Q, T165G, L167I, K169D,  F171N , F171V, Y172F, E174P, E174V, S176N, G179R, Q181G, Q181R, A182R, S183N, S184T, R185A, S187R, N192A, N192S, S193P, S197G, S197R, T198S, P199R, I84H  Y87W  R88N  R89E  A90Q  R93W  G96M  G96P  D98K  K100G  K100Q  M101R  L104Q  L104V  S105P  P106D  P106S  R107A  R107N  Y109H  E118A  E118H  G120D  L121A  P122N  P122R  Y123W  A125D  N126R  K127Q  I130V  I131F  T135A  E136K  L139D  L139K  N140T  T141K  K143R  D144S  H145N  I146L  I146Q  T148V  A152D  N153K  N154F  A155D  A155P  A156Q  I157Y   c   Frequency, %   0   10   20   30   40   50   60   70   80   V158P  Q160R  L161F  P162S  Q163D  G164Q  T165G  L167I  K169D  F171N  F171V  Y172F  E174P  E174V  S176N  G179R  Q181G  Q181R  A182R  S183N  S184T  R185A  S187R  N192A  N192S  S193P  S197G  S197R  T198S  P199R  G200S  S201N  A208G  A211S  G214S  G215E  A217D  A218I  A218L  L219I  L219V  L221A  L222A  L223A  L223V  L224K  D225K  D225Q  R226A  R226I  N228K  E231G K237Q  G238K  Q239S  Q241K  Q242S  G243S  Q244S  T245R  V246T  T247P  S250K  A252R  E253K  A254M  S255A  K256D  K256H  K257R  P258R  R259E  R259Y  Q260C  Q260W  A264P  T265P  K266P  A267E  A267G  N269S  T271D  Q272A  A273C  A273V  R276P  G278T  P279K  T282K  Q283E  G284K  Q289A  Q289D  E290K  L291M  I292V  R293E  Q294E  T296I  T296V  D297K A273C, A273V, R276P, G278T, P279K, T282K, Q283E,  G284K, Q289A, Q289D, E290K, L291M, I292V, R293E,  Q294E, T296I, T296V, D297K, Y298A, K299G, W301F,  W301V, W301Y, P302T, Q303A, I304M, A305L, Q306N,  F307L, A308V, S310N, A311S, A311V, S312A, S312H,  F314C, F314L, F315L, G316F, M317G, S318G, I320V,  G321T, M322P, M322T, E323K, E323R, V324E, V324L,  P326G, S327D, T329L, W330E, W330H, L331I, Y333F,  G335F, G335Y, A336K, A336T, I337M, I337T, K338V,  L339V, D340P, D341K, K342D, K342S, K347D, K347E,  D348N, Q349F, Q349Y, I351K, L352I, L353C, N354D,  K355E, K355S, H356C, H356Q, I357V, A359G, Y360V,  K361G, F363R, T366D, E367Q, P368E, K370R, K373S,  K374R, A376S, D377E, D377S, E378R, A381P, L382A,  R385Q, Q390K, T391E, T393D, L394V, L395T, P396D,  A397N, A397Q, A398L, D399E, L400F, F403E, S404P,   Q406V, L407I, Q408N, Q409W, S410G, S413A, A414L,  A414V, D415G, S416E , Q418E, A419L, and A419N) in a statistically significant manner (p ≤ 0.001) (Fig. 4a-f ).

Different variants of coronaviruses have been identified infecting human and animals. The differences in genome structure and sequences make the coronaviruses to be different in severity of infection and host selectivity. Coronaviruses continuously change their structures via mutation, deletion, and/or insertion mutations. The most genomic variation part in SARS-CoV and SARS-CoV-2 is the RBD in the S protein [13, 14] and some locations in S protein sequence might be related to positive selection W301V I351K  L352I  L353C  N354D  K355E  K355S  H356C  H356Q  I357V  A359G  Y360V  K361G  F363R  T366D  E367Q  P368E  K370R  K373S  K374R  A376S  D377E  D377S  E378R  A381P  L382A  R385Q  Q390K  T391E  T393D  L394V  L395T  P396D  A397N  A397Q  A398L  D399E  L400F  F403E  S404P  Q406V  L407I  Q408N  Q409W  S410G  S413A  A414L  A414V  D415G  S416E  Q418E  A419L [15] . Due to the several changes in the genome sequences of SARS-CoV-2 isolates, it is necessary to find the location of mutations and to understand the role of these mutations in the pathogenicity of SARS-CoV-2. This is significant to understand the viral infection mechanisms and pave a way to find drug and vaccine to protect people from the next stage of the pandemic.

The surface envelope is the smallest but abundant structural protein. This protein participates to create envelope which is the important part of the virus, and it is responsible for protection of the interior parts of the virus and has a role in viral assembly during viral replication [25, 26] . Due to the envelope's roles in host cell infectivity, it is necessary to investigate the mutations in this protein in different animal and human coronaviruses, including SARS-CoV-2.

For this purpose, the full amino acid sequences of several envelope proteins were retrieved from GenBank and used in this study. Fifty-one animal viruses and 55 human viruses' envelope proteins were taken and analyzed (Fig. 1) .

In envelope viruses' sequences, among the 76 residues, 365 mutations were observed and 47 in a statistically significance manner (p ≤ 0.001).

In animal viruses' sequences, 333 mutations were observed; 47, of them in a statistically significant manner (p ≤ 0.001) (Fig. 1) . In envelope human virus sequences, 94 mutations were observed and just 1, L28I, was observed in a statistically significant manner (p ≤ 0.001) (3 isolates; 5.4%) (Fig. 1a) . These results show that there was high diversity seen in animal viruses than human viruses because animal viruses isolated in different species of animals, but for human viruses, only 1 host participated in the diversity. The rate of significant amino acid mutations (14.1%) is higher in animal coronaviruses than in human coronaviruses (0.9%). This means that human coronavirus preserves its envelope amino acids with the least number of amino acid mutations, and majority of its amino acids are conservative.

The surface spike glycoprotein is the most important part of the virus, and it is responsible in natural host selection to initiate infection. This trimmer protein helps in viral attachment to its corresponding receptor and mediates fusion of the cell and viral membrane [27] [28] [29] . The S protein has 2 main domains: S1 which is responsible to bind the host cell receptor, and S2 is responsible for the fusion of membrane of both viral and host cellular membranes [12] . Due to the variability in sequences of the RBD SARS-CoV-1 and SARS-CoV-2 [13, 14] , it is necessary to investigate the mutations in this protein in different animal and human coronaviruses including SARS-CoV-2.

For this purpose, the full amino acid sequences of several spike proteins were retrieved from GenBank and used in this study. Fifty-one animal viruses and 52 human viruses spike proteins were taken and analyzed. Due to the important role of RBD of the spike, only this domain was analyzed in this study. More accurately, amino acid residues 336-516 in SARS-CoV-2, which are crucial and enough to bind ACE2 receptor [24] , were analyzed.

In general, in spike virus's sequences, among the 1,273 residues, 9,782 mutations were observed; 982, in a statistically significant manner (p ≤ 0.001). This large number of mutations indicates the wide variation between different host coronaviruses. Of which, 982 mutations are significant which means it is found in most of the virus's spikes of most of the coronaviruses. The same investigation was made in animal and human coronaviruses. In animal coronaviruses, 920 mutations were observed, and specifically in the RBD region, 83 of them in a statistically significant manner (p ≤ 0.001) (Fig. 2) . The lower rate of significant number of mutations (8.1%) in human coronaviruses indicates the close relation between human coronaviruses more than in animal coronaviruses (9%). The variation in sequences is much less among human coronaviruses than animal coronaviruses. In spike human virus sequences, 222 mutations were observed, and specifically in the RBD region, 18 of them (E340S, F347I,  W353F, K356S, V362P, Y369N, Y380V, G381D, T385L,  K386P, D389T, D467N, T470Y, Y473L, G504Y, In RBD of SARS-CoV-2, many amino acids have a direct contact with ACE2 receptor and have a role in direct spike and corresponding receptor binding [24] . These  amino acids are K417, G446, Y449, Y453, L455, F456,  A475, F486, N487, Y489, Q493, G496, Q498, T500, N501,  G502 , and Y505. In our study, it is revealed that K417, T500, and Y505 amino acids are missing in most of animal coronaviruses and the differences are significant. The mentions amino acids are substituted to K417C, T500C, and Y505F in animal coronaviruses. According to ding [24] , these 3 amino acids are part of 17 (17.6%) amino acids which are important in the binding of spike protein of SARS-CoV-2 to human ACE2 receptor. Therefore, significant substitution mutation in animal coronaviruses of these amino acids may have a role in host specific infectivity and are unable to infect human cells.

It is revealed in a different study that 193 amino acid residues (318-510) of RBD of SARS-CoV-1 spike protein are enough to bind to ACE2 receptor and among them, one-point mutation (aspartate-454) can abort the binding interaction between S1 domain of spike and ACE2 receptor [30] . In our study, this amino acid is not among significant animal and human coronaviruses mutations, and amino acids found previously are not required to have contact with ACE2 receptor [30] . Therefore, this amino acid may be very conservative in all coronaviruses and it has a big role in spike receptor binding but not host selectivity. F486 in RBD of the spike protein gives strength for RBD binding of spike protein of SARS-CoV-2 to the ACE2 receptor [31] . This mutation is also not present among significant amino acid mutations in animal and human coronaviruses; therefore, it may be one of the conservative amino acids.

Finally, a recent observation underlied that RDB plays a fundamental role as a damping element of the massive viral particle's motion prior to cell recognition, while also facilitating viral attachment, fusion, and entry [32] .

The surface membrane protein participates to create an outer layer of the virus which is the important part of the virus and it is responsible for giving a shape to the virus and protection of the interior parts of the virus. This membrane has a role in pathogenesis and virus entry into the cell. After binding of spike to the ACE2 receptor, it moves closer to its corresponding host cell membrane and mediates fusion of the cell viral membranes [6-9, 24, 31] . Due to its important role in pathogenesis, it is necessary to investigate the mutations in this protein in different animal and human coronaviruses, including SARS-CoV-2.

For this purpose, the full amino acid sequences of several membrane proteins were retrieved from GenBank and used in this study. Fifty-one animal viruses and 55 human viruses' membrane proteins were taken and analyzed.

In Figure 3 , frequencies of SARS-CoV-2 membrane amino acid signatures were shown, using the isolate NC_045512.2 COVID-19/Wuhan as a reference. In membrane viruses' sequences, among the 222 residues, 851 mutations were observed; 219, in a statistically significance manner (p ≤ 0.001). In animal membrane sequences, 794 mutations were observed, and specifically, 219 of them in a statistically significant manner (p ≤ 0.001). In animal viruses, rate of significant amino acid mutations is very high (27.5%) which means the diversity among them is very significant. This may have been related to the diversity of animal hosts ranging from birds, land animals, and sea animals where the virus lives and propagates because the virus needs to adapt themselves in new hosts and enjoinments to survive. On the other hand, the significant mutation rate (13.3%) is less in human hosts because there is no diversity between human hosts as seen in animals. In human membrane virus sequences, 262 mutations were observed, and specifically, 35 of them in a statistically significant manner (p ≤ 0.001).

Nucleocapsid is the pretentious structure inside the box of the coronaviruses and it combines with the nucleic acid, RNA. The function of nucleocapsid is to hang viral nucleic acid around itself by packaging the genomic viral genome into long, flexible, helical ribonucleoprotein complexes, the nucleocapsid. The nucleocapsid protects the RNA of the virus and ensures its replication and transmission [33, 34] . Due to large number of diversity between human and animal coronaviruses, it is necessary to investigate the rate of mutations of nucleocapsid between animal and human coronaviruses. For this purpose, the entire nucleocapsid protein sequences, derived from 51 animal viruses and 54 human viruses, were analyzed (Fig. 4) .

In nucleocapsid virus's sequences, among the 419 residues, 1,885 mutations were observed; 317, in a statistically significance manner (p ≤ 0.001). In animal nucleocapsid sequences, 613 mutations were observed, and specifically, 38 of them in a statistically significant manner (p ≤ 0.001). In human nucleocapsid sequences, 1,628 mutations were observed, and specifically, 317 of them in a statistically significant manner (p ≤ 0.001). The number of significant amino acid mutations is lower in animal vi-12 DOI: 10.1159/000513530 ruses at a rate of 6.1% than in human viruses, 19.4%. In all previously mentioned structural proteins, envelope, membrane, and spike, the rate of significant mutations was higher than in that of human viruses, whereas it is opposite in nucleocapsid protein.

A connection between conserved viral structure and possible target medicine treatment is mandatory. As recently reported, thousands of compounds including approved drugs and drugs in the clinical trial are available in the literature, and some anti-COVID-19 candidates based on computer-aided drug design can be followed up [35] .

Here, in general, in all human structural proteins, significant amino acid mutation(s) was observed. The highest significant mutation (19.4%) was observed in nucleocapsid protein, whereas the lowest rate was in envelope protein (0.9%). This means that the structure of envelope and envelope protein is very stable and the amino acids are very conservative while it is opposite in nucleocapsid. The rate of significant amino acid mutations in spike protein (8.1%) is less than that in nuleocapsid (19.4%). In spite of the existing less-significant amino acid mutations in spike protein, the mutations in spike protein still have a crucial role in the viruses' host selectivity and infectivity because it is directly related to receptor binding and indicating the type of cell and host to infect. Therefore, the least mutation in RBD of spike may make a catastrophic effect on the human hosts than other structural proteins of the coronaviruses.

The structural proteins, spike, envelope, nucleocapsid, and membrane proteins are very genetically diverse among human and animal coronaviruses. The highest rate of significant mutation was found in nucleocapsid rather than in spike protein, but most of mutations in spike proteins are in the RBD part of S1 of the spike. The least rate of mutation was in envelope protein which means envelope protein amino acid residues are more stable and conservative. A single amino acid which dissociates spike binding with ACE2 receptor remains intact in all human and animal coronaviruses. In addition, among 17 amino acids that have a direct contact with ACE2 receptor, only 3 of them are significantly mutated and substituted in animal coronaviruses.

Large number of significant mutations were recorded in animal coronaviruses than in human coronaviruses. The highest rate of significant amino acid substitution was found in nucleocapsid viral protein, but it is the low-est in envelope protein which means envelope protein is more stable and less diverse. In spike protein, the highest number of significant amino acid mutations was found in RBD. Three out of 17 binding amino acids in RBD are significantly mutated in animal coronaviruses. A single amino acid of RBD, aspartate-454, which is essential for the binding of spike protein with ACE2 cell receptor remains intact in all human and animal coronaviruses.

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We acknowledge the president of Sulaimani Polytechnic University and especially Assistant Professor Dr. Alan Faraydoon Ali for his great help and academic support to performing this research. Also, we would like to appreciate Mam Humanitarian Foundation for its financial and logistic support.

No ethical approval was required, as it is not applicable to our research. According to the study design, neither medical treatments nor procedures involving humans or animals were performed.

The authors have no conflicts of interest to declare.

This work was supported by Mam Humanitarian Foundation.