The Impact of SARS-CoV-2 Multiple Spike Protein Mutations on COVID-19 Outcomes
Authors: Gunadi  , Hakim Mohamad Saifudin , Wibawa Hendra , Marcellus  , Trisnawati Ika , Supriyati Endah , Afiahayati  , Khair Riat El , Iskandar Kristy , Siswanto  , Irene  , Anggorowati Nungki , Daniwijaya Edwin Widyanto , Nugrahaningsih Dwi Aris Agung , Puspadewi Yunika , Simanjaya Susan , Puspitarani Dyah Ayu , Hanifin Hana Fauzyyah , Setiawan Alvina Alexandra , Tania Irene , Amalia Cita Shafira , Artayasa I Putu Aditio , Rachman Haries , Mulyawan Herdiyanto , Ananda Nur Rahmi , Arguni Eggi , Nuryastuti Titik , Wibawa Tri 
Published: July 2021

Abstract
Background: Recent studies focusing on the association of SARS-CoV-2 variants of concern (VOC) on COVID-19 outcomes have been reported. However, studies of the impact of multiple mutations within the spike (S) protein of SARS-CoV-2 on COVID-19 illness are limited. This study determined the association between multiple mutations within the S protein, prognosis factors, and the disease outcomes of SARS-CoV-2 infection. Methods: We included 51 COVID-19 patients from Yogyakarta and Central Java, Indonesia. Whole genome sequences of SARS-CoV-2 were determined by the Illumina MiSeq next-generation sequencer, followed by the phylogenetic analysis of 170 full-genomes of SARS-CoV-2 from different regions. We analyzed characteristics of COVID-19 patients and multiple mutations in association with different outcomes.Results: Among 51 patients, the clinical manifestations of COVID-19 were as follows: without any symptoms (13.7%), mild (47%), moderate (19.6%), severe (4%), critical (2%), and died (13.7%). The age of hospitalized patients (53.4 ± 18 years) was higher than non-hospitalized patients (34.6 ± 19) (p=0.001). A significant association between diabetes, hypertension, and anticoagulant and the hospitalization of patients was noted with p-value of 0.039 (OR=4.47 [95% CI=1.07-18.58]), 0.001 (OR=17 [95% CI=2-144]), and 0.02 (OR=27.97 [95% CI=1.54-507.13]), respectively; whereas a strong association between patients’ age, diabetes, anticoagulant, and steroid with the mortality of patients was revealed with p-value of 0.016 (OR=8.44 [95% CI=1.5-47.49]), 0.019 (OR=8.5 [95% CI=1.43-50.66]), 0.001 (46.8 [95% CI=4.63-472.77]), and 0.009 (OR=15.75 [95% CI=2-123.86]), respectively. All viruses contained the D614G variant, except one case. Accordingly, the samples were classified as the following clade: L (2%), GH (84.3%), GR (11.7%), and O (2%). Besides the D614G, the most common variants in the S protein were L5F (18.8%), V213A (18.8%), and S689R (8.3%). There was no significant association between multiple S protein variants with either hospitalization or mortality of COVID-19 (p=0.11 and 0.69, respectively). Multivariate analysis showed that hypertension and anticoagulant were the strong factors affecting the hospitalization and mortality of patients with COVID-19 with a p-value of 0.009 (OR=17.06 [95% CI=2.02-144.36]) and 0.001 (OR=46.8 (95% CI=4.63-472.77), respectively. Interestingly, the multiple S protein variants almost reached a significant level affecting the hospitalization of patients (p=0.07). Phylogenetic analysis showed that although most of the viruses from this study belonged to clade GH, none were detected as the variant of concern (VOC) and the variant of interest (VOI) of SARS-CoV-2.Conclusions: Here, we show for the first time the association between SARS-CoV-2 mutations within the S protein besides the VOC with the COVID-19 outcomes. Our findings suggest that multiple mutations in the S protein might affect the severity of COVID-19. Our study further suggests the importance of genomic surveillance to monitor SARS-CoV-2 variants, particularly those that might influence the outcomes of COVID-19 patients.