Jurnal Riset Biologi dan Aplikasinya, Volume 3, Issue 2, September 2021 S-D614G Mutation Reveals the Euro-America and East-Asia Origin SARS-CoV-2 Virus Spread in Indonesia Nina Bunga Anggraini1, Dwi Listyorini1,2* 1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang 2Department of Biotechnology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang Jln. Semarang 5, Malang, East Java 65145, Indonesia *Corresponding Author: e-mail: listyorini.aljabari@um.ac.id Article History ABSTRACT Received : 30 April 2021 COVID-19 is a pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The first case was found in the city of Wuhan, Hubei province, China. The first case in Indonesia was reported in March 2020 and currently there are 0.5 million cases with a death rate of 3.1%. This rapid increase in cases is thought to due to presence of the mutant strain S-D614G, which causes a faster rate of infection and spread. The purpose of this study was to determine the presence of S-D614G mutations in Indonesian samples in order to find the origin of COVID-19 which was spread in Indonesia based on the Spike gene sequences and the RdRp genes from 25 countries, and one control sequence China/Wuhan-Hu-1 obtained from the NCBI and GISAID databases. Mutation analysis was carried out through multiple alignments using BioEdit software. Phylogenetic tree reconstruction using MEGA6 software with the Neighbor Joining method. This study found mutation of S-D614G in one Indonesian sample, namely the Indonesian/SBY9 sample along with 23 samples from Europe, America, and Africa. The phylogenetic tree reconstruction confirmed these findings; the mutated samples were closely related to samples from these continents, while the non- mutated Indonesian samples were closely related to sample from East Asia. These findings indicate that the origin of the SARS-CoV-2 virus in Indonesia possibly came from the East Asia cluster and the European-American cluster. Revised : 5 July 2021 Approved : 25 August 2021 Published : 30 September 2021 Keywords COVID-19; SARS-CoV-2 virus; S- D614G mutation; Spike gene and RdRp gene; phylogenetics How to cite: Anggraini, N.B., & Listyorini, D. (2021). S-D614G Mutation Reveals the Euro-America and East-Asia Origin SARS-CoV-2 Virus Spread in Indonesia. Jurnal Riset Biologi dan Aplikasinya, 3(2):45-53. DOI: 10.26740/jrba.v3n2.p45-53 INTRODUCTION COVID-19 is a pandemic caused by the Severe acute respiratory syndrome coronavirus (SARS- CoV-2) (Decaro & Lorusso, 2020; Gralinski & Menachery, 2020). This virus attacks human respiratory system (Rhotan et al., 2020). Early 2020 this virus shocked the world with the surge of infected patients. The origin of this case began the discovery of pneumonia cluster cases with unclear etiology from Wuhan City, Hubei Province, China, according to the information released by World Health Organization (WHO) on December 31, 2019 (Shreen et al., 2020). That case continued to develop until there are reports of fatality and cases reported outside China (Safrizal et al., 2020). As of November 19, 2020 there were 56,564,475 cases worldwide with the number of deaths reaching 1,354,858 (Worldometer, 2020). The global COVID-19 outbreak is a pandemic that is very important to be taken seriously. Based on the predictive value set by the CDC around 20-60% of the population worldwide will be infected by this virus (Baud et al., 2020). Coronavirus was confirmed for the first time in Indonesia at March 2020 (Indonesian Ministry of Health, 2020). A patient was confirmed positive for Jurnal Riset Biologi dan Aplikasinya https://journal.unesa.ac.id/index.php/risetbiologi Jurnal Riset Biologi dan Aplikasinya, 3(2): 45-53, September 2021 | 46 COVID-19 after attending an event in Jakarta where the patient had been in contact with a foreigner, the patient complained of fever, cough and blown (WHO, 2020). As of November 2020, there were 478,720 cases of COVID-19 in Indonesia and the death rate reached 15,530 or 3,2% of confirmed cases (Worldmeter.info, 2020; Satgas penanganan COVID-19, 2020). Corona virus is transmitted by aerosol droplet or nasopharyngeal droplets of patient (Mao et al., 2020). Aerosol or nasopharyngeal droplets are transmitted when people come into direct contact with patient (Safrizal et al., 2020; Huang et al., 2020). Several efforts to prevent the transmission rate of COVID-19 have been carried out, such as physical distancing, sels-quarantine, early detection, and basic protection in the form of wearing masks when traveling and lockdowns area (WHO, 2020; Susilo et al., 2020). SARS-CoV-2 virus is included in the subgenus Sarbecovirus, genus Betacoronavirus, and the Coronaviridae family (Khot & Nadkar, 2020) which has incubation symptoms similar to SARS-CoV 2016, there are fever, dry cough, blown, and various other symptoms different from each individual (Safrizal et al., 2020; Gorbalenya et al., 2020; Jin et al., 2020). The incubation period for this virus is between 5-14 days (Jin et al., 2020). The recovery rate from the COVID-19 reached 36% (Mahase et al., 2020), even so this virus is an obstacle to human activities in various fields such as economy, development and education (Schleicher, 2020; Wren-Lewis, 2020). SARS-CoV-2 virus has spread throughout the world along with the mobilization of its host cell (Zheng et al., 2020). Differences in climatic conditions, environment and host cell antibodies will make the virus adapt or even mutate (Sanjuán & Domingo-Calap, 2016). Mutations that have an impact on the spread of SARS-CoV-2 are mutations in the 614th amino acid position of Spike protein or called S-D614G mutation, that is substitution of the aspartate to glycine (Laha et al., 2020; Hu et al., 2020). This mutation causes the virus become more pathogenic and spread faster (Korber et al., 2020). Research on the Spike protein is mostly carried out due to the important role of this protein in inserting the genetic material of the SARS-CoV-2 virus into host cells through the ACE2 receptor (Shang et al., 2020; Tay et al., 2020). According to Biswas & Majumder (2020) there are two type of SARS-CoV-2, there are type (O) and type (A2a). Type (O) is a type that does not have mutations in the Spike protein. Type A2a is a variant of SARS- CoV-2 that has a mutation in the amino acid S- D614G. The crucial process after the insertion of the virus into host cell is the replication facilitated by the RdRp gene (Lung et al., 2020). This gene produces the RNA polymerase enzyme which is needed in the replication process (Zhu et al., 2020). The length of this gene is from 182 to 881 base pairs, that will be translated into protein with 60 to 269 amino acids long as orf1ab polyprotein (Parikesit & Nurdiansyah, 2020). Phylogenetic tree reconstruction will provide information on the relationship, similarity based on genes and the origin of SARS-CoV-2 in Indonesia (Turista et al., 2020). Information about the relationship and origin of SARS-CoV-2 in Indonesia can be a source to find out the type of SARS-CoV-2 in Indonesia. Therefore, the right steps can be found to overcome the COVID-19 pandemic in Indonesia. Hence, more in-depth research is needed the distribution, types of viruses and mutations that occur to be used as the basis for appropriate prevention and treatment efforts in the future. This research aimed to determine the presence of S- D614G mutations in Indonesian samples in order to find the origin of COVID-19 which was spread in Indonesia based on the Spike gene sequences and the RdRp genes from 25 countries, and one control sequence China/Wuhan-Hu-1 obtained from the NCBI and GISAID databases MATERIALS AND METHODS This research was included in a qualitative descriptive study conducted in the Biotechnology room, O4 Building, Laboratorium Sentral Mineral dan Material Maju, Universitas Negeri Malang in June to August 2020. The samples of this study were 50 SARS-CoV-2 Spike and RdRp genes sequences from 25 countries obtained through data mining from the National Center for Biotechnology Information (NCBI) database and the Global Initative on Sharing All Influenza Data (GISAID). The control sequences used were RNA sequences of Spike and RdRp SARS-CoV-2 genes from Wuhan China (Wuhan-Hu-1). The tools used are BioEdit for alignment sequences process and MEGA6 software for phylogenetic tree reconstruction using neighbour joining method. Data analysis was by observing the 1842th base to determine the presence of base substitution from Adenine (A) to Guanine (G). Analysis of the relationship and origin of the SARS- 47 | Anggraini & Listyorini; S-D614G Mutation Reveals the Euro-America and East-Asia CoV-2 viruses based on the formed phylogenetic tree. RESULTS AND DISCUSSION Multiple alignment result of Spike gene using BioEdit software showed there was an 1842th base change in some samples from base A to G (Table 1). The multiple alignment result of Spike gene sequences using BioEdit software showed that there was mutation on 1842th base in several samples of the Spike gene, from base A to G (Table 1). This base substitution result in a change in the amino acid aspartate (GAT) to glycine (GGT) (Eaaswarkhanth et al., 2020; Hu et al., 2020). Table.1 Mutations A>G cause a change in the amino acid aspartate (S-D614) to glycine (S-G614). No Sample Origin Accession Number A-G 1 Indonesia /Jakarta 1 EPI_ISL_467370 A 2 Indonesia/Jakarta 2 EPI_ISL_437192 A 3 Indonesia/Jakarta 3 EPI_ISL_437191 A 4 Indonesia/Jakarta 4 EPI_ISL_437190 A 5 Indonesia/Jakarta 5 EPI_ISL_437189 A 6 Indonesia/Jakarta 6 EPI_ISL_435283 A 7 Indonesia/Jakarta 7 EPI_ISL_435282 A 8 Indonesia/Jakarta 8 EPI_ISL_435281 A 9 Indonesia/Surabaya 9 EPI_ISL_437188 G 10 Indonesia/Surabaya 10 EPI_ISL_437187 A 11 Indonesia/Manado 11 EPI_ISL_467375 A 12 Indonesia/Samarinda 12 EPI_ISL_467374 A 13 Indonesia/Pasuruan 13 EPI_ISL_458081 A 14 China 1 MT510727 A 15 China/Guangzhou 2 MT568634 A 16 China/Guangzhou 3 MT568638 A 17 China/Guangzhou 4 MT568641 A 18 South Korea 1 MT039890 A 19 South Korea 2 MT304474 A 20 South Korea 3 MT304475 A 21 South Korea 4 MT304476 A 22 Japan 1 LC556320 G 23 Japan 2 LC549340 G 24 Taiwan MT374104 A 25 Hongkong MT114412 A 26 Malaysia MT372481 A 27 Australia/Northern terrritory MT641648 A 28 Australia/Victoria MT451009 G 29 India MT415321 G 30 Bangladesh MT762396 G 31 Saudi Arabia MT755885 G 32 Italy 1 MT531537 G 33 Italy/Lazio 2 MT527178 G 34 Italy/Lazio 3 MT527184 G 35 Spain MT359866 G 36 Netherlands MT705206 G 37 Germany MT358643 G 38 France MT470150 A 39 USA/Virginia MT929083 G 40 USA/North America MT764170 G 41 Brazil MT738101 G 42 Colombia MT470219 G 43 South Africa MT324062 G 44 Egypt 1 MT627391 G 45 Egypt 2 MT510690 G 46 Marocco MT731292 G 47 Guam MT459985 A 48 Jamaica MT507793 A 49 Serbia 1 MT569470 G 50 Serbia 2 MT569471 G 51 China/Wuhan-Hu-1 NC_045512 A Jurnal Riset Biologi dan Aplikasinya, 3(2): 45-53, September 2021 | 48 Total samples that had mutations were 23 of the 50 samples used. The result of the alignment of 13 samples from Indonesia found 1 sample Indonesia/JKT9 that had mutation. Other samples that had mutations included 3 samples from Italy, two samples from America, Egypt, Japan and Serbia. One sample from Spain, Netherlands, South Africa, Morocco, Germany, France, Brazil, Colombia, India, Saudi Arabia, Bangladesh. The remaining 27 samples did not change base or were identical to the control sequence from China/Wuhan-Hu-1. Most of the samples that had mutations came from countries in Europe (Eaaswarkhanth et al. 2020) and America (Gudbjartsson et al., 2020). Mutations in viral RNA have a high rate and affect the process of spread and virulence (Hu et al., 2020; Becerra-Flores & Cardozo, 2020). In the case of the S-D614G mutation, the substitution of aspartate to glycine causes the S-G614G strain virus to be more pathogenic and have higher transmission rate than the S-D614G strain (Becerra-Flores & Cardozo, 2020). This is due to the presence of single nucleotide deletion (delC) at a known variant site (rs35074065) in the cis-eQTL of TMPRSS2 which facilitates SARS-CoV-2 to be more easily inserting host cells (Bhattacharyya et al., 2020). The Spike gene in the S-G614 strain divides faster due to the presence of new serine protease (elastase-2) cleavage site located near the S1-S2 junction of the Spike gene (Bhattacharyya et al., 2020), this strain is also more stable than S- D614 so it is strongly suspected to have a more efficient transmission (Zhang et al., 2020). Until November 19, 2020 Indonesia was in the 19th position among countries with high cases of COVID-19 in the world with a total of 478,720 cases (Worldmeter, 2020; WHO, 2020; CSIS, 2020). East Java Province is the second highest number of COVID-19 cases in Indonesia after DKI Jakarta with total of 56,070 cases and highest fatality with a total of 4008 death (Satgas Penanganan COVID-19, 2020) although a mutant SARS-CoV-2 was found from East Java. It is suggested that there were some samples with mutation which were not yet uploaded to both NCBI GenBank and GISAID within the research sampling period. The discovery of the S- G614 variant virus from the city of Surabaya could be one of the causes of COVID-19 cases in Indonesia increase due to the high transmission rate of this strain (Bhattacharyya et al., 2020). In addition, countries in Europe and America are in the row of countries with the most cases in the world (Bhattacharyya et al., 2020; Worldometer., 2020) accompanied by many samples that have mutations in the amino acid 614 Spike gene on both continents. Phylogenetic tree reconstruction based on the Spike gene form two large cluster, there are cluster A and cluster G. Most of the SARS-CoV-2 Spike gene sequences from Indonesia were in cluster A, but the Indonesian/SBY9 samples were in cluster G which was in one clade with Italy/LZ3 sample. The bootstrap value (54) revealed that the Indonesian/SBY9 sample has a close relationship to Italy/LZ3 sample. These results can be an indication that the Indonesian/SBY9 patient was in a close contact with a COVID-19 patient from Italy or the related patient was suspected of having a history of travel to Italy. This can occur due to the spread of the SARS-CoV-2 virus through aerosols or nasopharyngeal droplets which are transmitted when people have direct contact with infected persons (Safrizal et al., 2020; Huang et al., 2020). The other samples from Indonesia were in cluster A. The sample Indonesia/JKT3 was in one branch with South Korea3 with a bootsrap value of 79. However, the Indonesia/JKT3 sample formed different branch along with Asian countries in which several small clades were formed. The samples of Indonesia/MND were in one branch with samples from China/GHZ2 and France with a bootsrap value of 85 and were in one clade with Jamaica and Taiwan. The sample of Indonesia/JKT6 was in one branch with South Korea2 with a bootsrap value of 80 and formed a small clade with the samples of Indonesia/JKT1 and Indonesia/SBY10. The samples of Indonesia/JKT7 and Indonesia/PSR13 were in one branch with a bootsrap value of 88 and formed a small clade with Indonesia/SMR12 and Australia/NT. The sample Indonesia/JKT5 was in one branch with China/GHZ3 with a bootsrap value of 76 and formed a small clade with China1 and China/Wuhan-Hu-1. The Indonesian/JKT2 sample was in one branch with the China/GHZ4 sample with a bootsrap value of 74 and formed a small clade with Indonesia/JKT4 and South Korea4. The sample Indonesia/JKT8 was in one branch with South Korea1 with bootsrap value of 87 and s in the same clade as Hongkong, Malaysia and Guam (Figure 1). The results of the phylogenetic tree reconstruction also proved that the mutant samples formed separate clades from the non-mutant samples. Generally, samples of the Spike gene in 49 | Anggraini & Listyorini; S-D614G Mutation Reveals the Euro-America and East-Asia cluster G (type A2a) were suggested came from European and American countries with type A2a since this type was commonly found in Europe and America which bear a non-synonymous S-D614G Spike gene mutation which making it easier for the virus to enter the lung cells of the host (Biswas et al., 2020; Gudbjartsson, et al., 2020). This is the reason why type A2a has the advantage of infecting and surviving which results in faster spread across geographic areas (Biswas et al., 2020). One of the causes of mutations in viruses is the adaptation of a virus to geographic environmental conditions and the condition of host cell antibodies (Sanjuán & Domingo-Calap, 2016). Although many samples of Indonesia were found in cluster A, Indonesian samples were not grouped in the same branch. This could be an indication that there are variations in the Indonesian Spike gene sequence. Most of the samples of Indonesian Spike genes are closely related to the samples from East Asia countries, especially China and South Korea. It can be assumed that the patient has had contact with foreigners from or had a history of travel to those two countries. Figure 1. Phylogenetic tree reconstruction of Spike gene sequences using the Neighbour Joining method G G A G (28) Guam (32) Malaysia (35) Hongkong (9) Indonesia/JKT 8 (22) S outh Korea 1 (5) Indonesia/JKT 4 (43) S outh Korea 4 (3) Indonesia/JKT 2 (48) China/GHZ 4 (6) Indonesia/JKT 5 (47) China/GHZ 3 (21) China 1 (1) China/Wuhan-Hu-1 (8) Indonesia/JKT 7 (17) Indonesia/PS R 13 (16) Indonesia/S MR 12 (27) Australia/NT (2) Indonesia/JKT 1 (14) Indonesia/S BY 10 (7) Indonesia/JKT 6 (41) S outh Korea 2 (36) Jamaica (38) Taiwan (15) Indonesia/MND 11 (25) France (46) China/GHZ 2 (4) Indonesia/JKT 3 (42) S outh Korea 3 (10) S pain (11) Netherlands (19) Egypt 1 (20) Morocco (23) Germany (24) Italy 1 (29) Brazil (31) India (33) S audi Arabia (34) Japan 1 (39) Egypt 2 (40) Japan 2 (44) S erbia 1 (45) S erbia 2 (49) Italy/LZ 2 (12) US A/VRG (26) US A/NA (18) S outh Africa (30) Colombia (13) Indonesia/S BY 9 (50) Italy/LZ 3 (37) Bangladesh (51) Australia/VCT (52) (53) (54) (55) (56) (57) (58) (59) (60) (61) (62) (63) (64) (65) (66) (67) (68) (69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) (91) (92) (93) (94) (95) (96) (97) (98) (99) (100) Jurnal Riset Biologi dan Aplikasinya, 3(2): 45-53, September 2021 | 50 (23) Germany (45) S erbia 2 (49) Italy/LZ 2 (33) S audi Arabia (24) Italy 1 (10) S pain (19) Egypt 1 (30) Colombia (13) Indonesia/S BY 9 (39) Egypt 2 (18) S outh Africa (40) Japan 2 (31) India (20) Morocco (11) Netherlands (26) US A (34) Japan 1 (44) S erbia 1 (51) Australia/VCT (12) US A/VRG (29) Brazil (37) Bangladesh (50) Italy/LZ 3 (1) China/Wuhan-Hu-1 (2) Indonesia/JKT 1 (4) Indonesia/JKT 3 (5) Indonesia/JKT 4 (7) Indonesia/JKT 6 (9) Indonesia/JKT 8 (14) Indonesia/S BY 10 (15) Indonesia/MND 11 (16) Indonesia/S MR 12 (17) Indonesia/PS R 13 (27) Australia/NT (35) Hongkong (22) S outh Korea 1 (38) Taiwan (47) China/GHZ 3 (6) Indonesia/JKT 5 (3) Indonesia/JKT 2 (8) Indonesia/JKT 7 (46) China/GHZ 2 (25) France (36) Jamaica (41) S outh Korea 2 (42) S outh Korea 3 (43) S outh Korea 4 (21) China 1 (48) China/GHZ 4 (28) Guam (32) Malaysia (52) (53) (54) (55) (56) (57) (58) (59) (60) (61) (62) (63) (64) (65) (66) (67) (68) (69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) (91) (92) (93) (94) (95) (96) (97) (98) (99) (100) Figure 2. Phylogenetic tree reconstruction of RdRp gene sequences using the Neighbour Joining method The result of phylogenetic tree reconstruction using the RdRp gene showed that Indonesian samples were in one large clade but separated in three different small clade (Figure 2). The non- mutant Indonesian samples on this phylogenetic tree are in the adjacent clade, namely clade A and clade B. In clade A the samples Indonesia/JKT2, Indonesia/JKT5 and Indonesia/JKT7 are in one small clade with China/GHZ3 with bootsrap value 61, and formed a larger clade A with South Korea1 and Taiwan with bootsrap value 66. Samples of Australia/NT, Hongkong and China-Wuhan-Hu-1 form clade B together with Indonesian samples including Indonesia/MND11, Indonesia/PSR13, Indonesia/SBY10, Indonesia/JKT8, Indonesia/JKT6, Indonesia/JKT4, Indonesia/JKT4, Indonesia/JKT3 and Indonesia/JKT1 with a bootsrap value of 68. In this clade, the Indonesian sample appears to form new sub-branches with varied bootstrapping values, thus indicating that there are variations between the RdRp gene sequences in the Indonesian sample. As well as having close similarities to Hongkong, Australia/NT and China-Wuhan-Hu-1 (Figure 2, Clade B). Clade A Clade B Clade G 51 | Anggraini & Listyorini; S-D614G Mutation Reveals the Euro-America and East-Asia In clade G, the sample Indonesia/SBY9 is in the same branch with Egypt2 with a value of 84 and is in a small clade with Colombia and Egypt1. The small clade was in one clade G together with samples from Spain, Italy/LZ2, Serbia and Germany with a value of 99. Samples in clade G were samples that had the S-D614G mutation or mutant strains. The results of phylogenetic tree reconstruction using Spike and RdRp genes were found to be similar (Figure 1 & 2) where 12 samples of Indonesia are closely related to East Asian countries, while one sample Indonesia/SBY9, is closely related to countries from the continents of Europe, Africa and America. This indicates that the origins of the SARS-CoV-2 virus in Indonesia came from East Asia, the continent of Europe, America and Africa either through direct patient contact with foreigners from these countries or a history of travel to these countries. The Spike gene and the RdRp gene are two crucial genes in the process of viral infection into host cells. The Spike gene acts as an intermediary for fusion between the virus and the cellular membrane of the host cell through endocytosis (Zhang et al., 2020; Tay et al., 2020), and the RdRp gene activates the RNA polymerase enzyme for the virus replication process in the host cell (Lung et al., 2020). High variations through this research can cause obstacles for scientists to determine drugs based on the anti-sense silencing RNA (SiRNA) method (Parikesit & Nurdiansyah, 2020). Transmission of the SARS-CoV-2 virus through aerosols or nasopharyngeal droplets which is transmitted when people make direct contact with patient (Safrizal et al., 2020; Huang et al., 2020) accompanied by high mobilization and interaction between humans in their daily lives will also be an obstacle as well in reducing cases of COVID-19. Therefore, vaccines, physical distancing (Aminnejad & Alikhani, 2020), wearing masks (Howard et al., 2021) and diligently washing hands are still the most appropriate steps to take for prevention of each individual in preventing the spread and transmission of COVID-19 (Bloomfield et al., 2020). CONCLUSION This study found a mutation of the Spike gene at amino acid14 in Indonesian/SBY9 sample originating from the city of Surabaya. 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