Sains Malaysiana 53(8)(2024): 1767-1776

http://doi.org/10.17576/jsm-2024-5308-04

 

Genome Sequencing and Phylogenetic Analysis of SARS-CoV-2 Isolated from Patients with COVID-19 in Hospital Canselor Tuanku Muhriz (HCTM), Malaysia

(Penjujukan Genom dan Analisis Filogenetik SARS-CoV-2 Dipencilkan daripada Pesakit COVID-19 di Hospital Canselor Tuanku Muhriz (HCTM), Malaysia)

 

KON KEN WONG1, NOOR ZETTI ZAINOL RASHID1, UMI KALSOM ALI1, NUR HAZLIN CHONG2, SHAIRAH ABDUL RAZAK2, NAJMA KORI3, PETRICK PERIYASAMY3 & ALFIZAH HANAFIAH1

 

1Department of Medical Microbiology & Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia

2Department of Biological Sciences and Biotechnology, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

3Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia

 

Diserahkan: 18 Mac 2024/Diterima: 26 Jun 2024

 

Abstract

SARS-CoV-2 infection has reached pandemic status in numerous countries worldwide, including Malaysia. Monitoring the genetic diversity of SARS-CoV-2 is essential for identifying the emergence and prevalence of novel variants in different geographical areas. From May to October 2021, this research endeavor analyzed the SARS-CoV-2 genome of 40 COVID-19 patients diagnosed using real-time RT-PCR at the local hospital HCTM-UKM. The process involved extracting RNA from these patients, which was subsequently subjected to whole genome sequencing. Our discovery underscores the primary strains responsible for the fourth wave of the COVID-19 pandemic in Malaysia in May 2021 were the Beta variant, primarily associated with the B.1.351 lineage. However, by October 2021, the Delta variant had become predominant and was categorized within the AV.59 and AV.79 lineages. Genomic epidemiological analysis showed about 19 amino acid mutations in the spike protein that were prevalent during the Beta variant outbreak. Among these, the N501Y mutation is particularly noteworthy as it significantly enhances the virus's ability to bind to the ACE2 receptor. Additionally, 32 amino acid mutations were identified during the Delta variant outbreak, with the T478K mutation being linked to increased viral infectivity and affecting the virus's affinity for human cells. Throughout our research, we consistently noted the presence of Spike D614G mutations in all the strains we collected which is known to reduce S1 shedding and increase infectivity. These findings could contribute significantly to our understanding of specific variants due to their clinical implications and rapid spread within the community.

 

Keywords: COVID-19; genome sequencing; phylogenetic; spike protein mutations; variant

 

Abstrak

Jangkitan SARS-CoV-2 telah mencapai status pandemik di hampir semua negara di seluruh dunia, termasuk Malaysia. Pemantauan kepelbagaian genetik SARS-CoV-2 adalah penting untuk mengenal pasti penemuan dan prevalen varian baharu di kawasan geografi yang berbeza. Dari Mei hingga Oktober 2021, kami menganalisis genom SARS-CoV-2 bagi 40 pesakit COVID-19 dengan menggunakan kaedahrealtime RT-PCR’ di hospital tempatan HCTM-UKM. Proses ini melibatkan pengekstrakan RNA daripada sampel pesakit ini dan dikaji dengan teknik jujukan lengkap genom. Penemuan utama kami adalah strain SARS-CoV-2 yang berleluasa semasa pandemik COVID-19 gelombang keempat di Malaysia pada Mei 2021 adalah varian Beta dengan garis keturunan B.1.351. Namun demikian, varian Delta telah menjadi dominan pada Oktober 2021, dan dikategorikan dalam garis keturunan AV.59 dan AV.79. Analisis epidemiologi genom menunjukkan kira-kira 19 mutasi asid amino dalam protein spike yang mendominasi semasa wabak varian Beta. Antara ini, mutasi N501Y adalah perlu diperhatikan kerana ia akan meningkatkan keupayaan virus untuk berikatan dengan reseptor ACE2 secara signifikan. Tambahan pula, sebanyak 32 mutasi asid amino dikenal pasti semasa wabak varian Delta dengan mutasi T478K dikaitkan dengan peningkatan jangkitan virus dan mempengaruhi keupayaan virus untuk berikatan dengan sel manusia. Sepanjang penyelidikan kami, mutasi Spike D614G dijumpai dalam semua strain yang kami kumpulkan. Mutasi ini dapat mengurangkan pembuangan S1 dan meningkatkan jangkitan virus. Dapatan kajian ini boleh memberi sumbangan yang signifikan kepada pemahaman kita mengenai varian tertentu disebabkan implikasi klinikal dan penyebaran yang pesat dalam komuniti.

 

Kata kunci: COVID-19; filogenetik; mutasi protein Spike; penjujukan genom; varian

 

RUJUKAN

Aleem, A., Akbar Samad, A.B. & Slenker, A.K. 2022. Emerging variants of SARS-CoV-2 and novel therapeutics against coronavirus (COVID-19). StatPearls Publishing 54(1): 524-540.

Capobianchi, M.R., Rueca, M., Messina, F., Giombini, E., Carletti, F., Colavita, F., Castilletti, C., Lalle, E., Bordi, L., Vairo, F., Nicastri, E., Ippolito, G., Gruber, CEM. & Bartolini, B. 2020. Molecular characterization of SARS-CoV-2 from the first case of COVID-19 in Italy. Clinical Microbiology and Infection 26(7): 954-956.

Centers for Disease Control and Prevention (CDC). 2022. About Variants of the Virus that Causes COVID-19.  https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html#concern (Accessed on 6 October 2022).

Chan, J.F., Yuan, S., Kok, K.H., To, K.K., Chu, H., Yang, J., Xing, F., Liu, J., Yip, C.C., Poon, R.W., Tsoi, H.W., Lo, S.K., Chan, K.H., Poon, V.K., Chan, W.M., Ip, J.D., Cai, J.P., Cheng, V.C., Chen, H., Hui, C.K. & Yuen, K.Y. 2020. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster. Lancet 395(10223): 514-523.

CMMID Repository. 2022. Estimates of Severity and Transmissibility of Novel Sars-Cov-2 Variant 501y.V2 in South Africa. https://cmmid.github.io/topics/covid19/sa-novel-variant.html [30 Dec 2022].

COVID-19 pandemic in Malaysia. 2022. https://en.wikipedia.org/wiki/COVID-19_pandemic_in_Malaysia (Accessed on 12 October 2022).

Cui, J., Li, F. & Shi, Z.L. 2019. Origin and evolution of pathogenic coronaviruses. Nature Reviews Microbiology 17: 181-192.

Deng, X., Garcia-Knight, M.A., Khalid, M.M., Servellita, V., Wang, C., Morris, M.K., Sotomayor-González, A., Glasner, D.R., Reyes, K.R., Gliwa, A.S., Reddy, N.P., Martin, C.S.S., Federman, S., Cheng, J., Balcerek, J., Taylor, J., Streithorst, J.A., Miller, S., Kumar, G.R., Sreekumar, B., Chen, P.Y., Schulze-Gahmen, U., Taha, T.Y., Hayashi, J., Simoneau, C.R., McMahon, S., Lidsky, P.V., Xiao, Y., Hemarajata, P., Green, N.M., Espinosa, A., Kath, C., Haw, M., Bell, J., Hacker, J.K., Hanson, C., Wadford, D.A., Anaya, C., Ferguson, D., Lareau, L.F., Frankino, P.A., Shivram, H., Wyman, S.K., Ott,  M., Andino, R. & Chiu, C.Y. 2021. Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation. MedRxiv doi: http://doi.org/10.1101/2021.03.07.21252647. Preprint.

Di Giacomo, S., Mercatelli, D., Rakhimov, A. & Giorgi, F.M. 2021. Preliminary report on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike mutation T478K. Journal of Medical Virology 93(9): 5638-5643.

Dowell, S.F., Simmerman, J.M., Erdman, D.D, Wu, J.S., Chaovavanich, A., Javadi, M., Yang, J.Y., Anderson, L.J., Tong, S. & Ho, M.S. 2004. Severe acute respiratory syndrome coronavirus on hospital surfaces. Clinical Infectious Diseases 39: 652-657.

Drosten, C., Günther, S., Preiser, W., van der Werf, S., Brodt, H.R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R.A., Berger, A., Burguière, A.M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J.C., Müller, S., Rickerts, V., Stürmer, M., Vieth, S., Klenk, H.D., Osterhaus, A.D., Schmitz, H. & Doerr, H.W. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. The New England Journal of Medicine 348(20): 1967-1976.

Forster, P., Forster, L., Renfrew, C. & Forster, M. 2020. Explaining phylogenetic network analysis of SARS-CoV-2 genomes. Proceedings of the National Academy of Sciences 117(23): 12524-12525.

Gao, G.F. 2018. From “A”IV to “Z”IKV: Attacks from emerging and re-emerging pathogens. Cell  172: 1157-1159.

Guruprasad, K. 2022. Mutations in human SARS-CoV-2 spike proteins, potential drug binding and epitope sites for COVID-19 therapeutics development. Current Research in Structural Biology  4: 41-50.

Korber, B., Fischer, W.M., Gnanakaran, S., Yoon, H., Theiler, J., Abfalterer, W., Foley, B., Giorgi, E., Bhattacharya, T., Parker, M., Partridge, D., Evans, C., Freeman, T., de Silva, T., LaBranche, C. & Montefiori, D. 2020a. Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. BioRxiv doi: https://doi.org/10.1101/2020.04.29.069054 Preprint.

Korber, B., Fischer, W.M., Gnanakaran, S., Yoon, H., Theiler, J., Abfalterer, W., Hengartner, N., Giorgi, E.E., Bhattacharya, T., Foley, B., Hastie, K.M., Parker, M.D., Partridge, D.G., Evans, C.M., Freeman, T.M., de Silva, T.I., Sheffield COVID-19 Genomics Group; McDanal, C., Perez, L.G., Tang, H., Moon-Walker, A., Whelan, S.P., LaBranche, C.C., Saphire, E.O. & Montefiori, D.C. 2020b. Tracking changes in SARS-CoV-2 spike: Evidence that D614G increases infectivity of the COVID-19 virus. Cell 182(4): 812-827.

Ksiazek, T.G., Erdman, D., Goldsmith, C.S., Zaki, S.R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J.A., Lim, W., Rollin, P.E., Dowell, S.F., Ling, A.E., Humphrey, C.D., Shieh, W.J., Guarner, J., Paddock, C.D., Rota, P., Fields, B., DeRisi, J., Yang, J.Y., Cox, N., Hughes, J.M., LeDuc, J.W., Bellini, W.J., Anderson, L.J. & SARS Working Group. 2003. A novel coronavirus associated with severe acute respiratory syndrome. The New England Journal of Medicine 348(20): 1953-1966.

Otter, J.A., Donskey, C., Yezli, S., Douthwaite, S., Goldenberg, S.D. & Weber, D.J. 2016. Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: The possible role of dry surface contamination. Journal of Hospital Infection 92: 235-250.

Park, D., Huh, H.J., Kim, Y.J., Son, D.S., Jeon, H.J., Im, E.H., Kim, J.W., Lee, N.Y., Kang, E.S., Kang, C.I., Chung, D.R., Ahn, J.H., Peck, K.R., Choi, S.S., Kim, Y.J., Ki, C.S. & Park, W.Y. 2016. Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus. Cold Spring Harbor Molecular Case Studies 2(6): a001214.

Ramanathan, M., Ferguson, I.D., Miao, W. & Khavari, P.A. 2021. SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity. The Lancet Infectious Diseases 21(8): 1070. 

Science. 2022. Delta variant triggers dangerous new phase in the pandemic. https://www.sciencemag.org/news/2021/06/deltavariant-triggers-dangerous-new-phase-pandemic (Accessed on 31 December 2022).

Su, S., Wong, G., Shi, W., Liu, J., Lai, A.C.K., Zhou, J., Liu, W., Bi, Y. & Gao, G.F. 2016. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol. 24(6): 490-502.

Weis, S.R. & Leibowitz, J.L. 2011. Coronavirus pathogenesis. Advances in Virus Research 81: 85-164.

Wise, J. 2021. COVID-19: The E484K mutation and the risks it poses. British Medical Journal 372: n359.

World Health Organization (WHO) 2020. Coronavirus Disease (COVID-2019) Situation Reports- 39. Geneva: WHO. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports (Accessed on 1 October 2022).

Wu, Y., Guo, C., Tang, L., Hong, Z., Zhou, J., Dong, X., Yin, H., Xiao, Q., Tang, Y., Qu, X., Kuang, L., Fang, X., Mishra, N., Lu, J., Shan, H., Jiang, G. & Huang, X. 2020. Prolonged presence of SARS-CoV-2 viral RNA in faecal samples. Lancet Gastroenterol. Hepatol. 5(5): 434-435.

Xu, D., Zhang, Z. & Wang, F.S. 2004. SARS-associated coronavirus quasispecies in individual patients. The New England Journal of Medicine 350(13): 1366-1367.

Zaki, A.M., van Boheemen, S., Bestebroer, T.M., Osterhaus, A.D. & Fouchier, R.A. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. The New England Journal of Medicine 367: 1814-1820.

Zhang, L., Jackson, C.B., Mou, H., Ojha, A., Rangarajan, E.S., Izard, T., Farzan, M. & Choe, H. 2020. The D614G mutation in the SARS-CoV-2 spike  protein reduces S1 shedding and increases infectivity. BioRxivhttp://doi.org/10.1101/2020.06.12.148726  Preprint.

Zhong, N.S., Zheng, B.J., Li, Y.M., Poon, X.Z.H., Chan, K.H., Li, P.H., Tan, S.Y., Chang, Q., Xie, J.P., Liu, X.Q., Xu, J., Li, D.X., Yuen, K.Y., Peiris & Guan, Y. 2003. Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China. Lancet 362(9393): 1353-1358. 

 

*Pengarang untuk surat-menyurat; email: alfizah@ppukm.ukm.edu.my

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

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