key: cord-0855192-0qdqb2em
authors: Abe, K.; Kabe, Y.; Uchiyama, S.; Iwasaki W., Y.; Ishizu, H.; Uwamino, Y.; Takenouchi, T.; Uno, S.; Ishii, M.; Murata, M.; Hasegawa, N.; Saya, H.; Kitagawa, Y.; Fukunaga, K.; Amagai, M.; Siomi, H.; Suematsu, M.; Kosaki, K.
title: Severity of COVID-19 is inversely correlated with increased number counts of non-synonymous mutations in Tokyo
date: 2020-11-24
journal: nan
DOI: 10.1101/2020.11.24.20235952
sha: 064bb5d22975fa857ef22e848544c716da13e51a
doc_id: 855192
cord_uid: 0qdqb2em

Background: SARS-CoV-2 genome accumulates point mutations in a constant manner. Whether the accumulation of point mutations is correlated with milder manifestations of COVID-19 remains unknown. Methods: We performed SARS-CoV-2 genome sequencing in 90 patients with COVID-19 infection treated at a tertiary medical center in Tokyo between March and August 2020. The possible association between disease severity and viral haplotype was then assessed by counting the number of mutations in addition to performing phylogenic tree analysis, comparative amino acid sequence analysis among {beta}-coronaviruses, and mathematical prediction of the functional relevance of amino acid substitutions. Results: The number of non-synonymous mutations was inversely correlated with COVID-19 severity, as defined by requiring oxygen supplementation. Phylogenic tree analysis identified two predominant groups which were separated by a set of 6 single nucleotide substitutions, including four leading to non-synonymous amino acid substitutions. Among those four, Pro108Ser in 3 chymotrypsin-like protease (3CLpro) and Pro151Leu in nucleocapsid protein occurred at conserved locations and were predicted to be deleterious. Patients with Pro108Ser in 3CLpro and Pro151Leu in nucleocapsid protein had a lower odds ratio for developing hypoxia requiring supplemental oxygen (odds ratio of 0.24 [95% confidence interval of 0.07-0.88, P-value = 0.032]) after adjustments for age and sex, compared with patients lacking this haplotype in Clade 20B. Conclusion: Viral genome sequencing in 90 patients treated in the Tokyo Metropolitan area showed that the accumulation of point mutations, including Pro108Ser in 3CLpro and Pro151Leu in nucleocapsid protein, was inversely correlated with COVID-19 severity. Further in vitro research is awaited.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is causing the coronavirus disease 2019 (COVID-19) pandemic, is spreading around the world. As an RNA virus with limited fidelity for genome replication, the SARS-CoV-2 viral genome accumulates mutations in a constant manner at an average of two nucleotides per months (GISAIDs: http://www.gisaid.org/), with the exception of large rearrangements such as a deletion spanning 382 nucleotides. 1, 2 Although the 382-nucleotide deletion was associated with a milder clinical course, whether the accumulation of point mutations is associated with a better prognosis remains unknown.

Keio University Hospital, which has a catchment that includes the Tokyo Metropolitan area and surrounding prefectures, has been performing whole viral genome sequencing of SARS-CoV-2 in COVID-19 patients since March 2020 to characterize healthcare-associated infections rapidly and effectively and to prevent the spread of infection. 3 Molecular viral genome sequencing studies have indicated that the number of mutations is indeed increasing. 4, 5 According to a Japanese governmental report, the daily number of newly identified COVID-19 patients in Tokyo plateaued during the time period when restrictions were imposed on foreign entry to Japan, while the number of seriously ill patients has been decreasing since May in All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020. 11.24.20235952 doi: medRxiv preprint Tokyo, Japan (https://www.mhlw.go.jp/stf/covid-19/kokunainohasseijoukyou.html). Hence, the relative fraction of seriously ill patients has been decreasing. Actually, the mortality rates in Japan appear to be lower than those in Western countries (https://covid19.who.int/). 6 We hypothesized that the accumulation of mutations may have contributed to the decrease in clinical virulence.

From March 17 to August 31, 2020, 187 patients with a reverse transcription polymerase chain reaction (RT-PCR)-positive result who had been diagnosed as having COVID-19 at Keio University Hospital between March 17th and Augst 31th, 2020, were enrolled in the present study. Of these 187 patients, 134 COVID-19 patients underwent whole viral genome sequencing. After the exclusion of 44 patients in whom only partial genome sequences were obtained because of insufficient PCR amplification, 90 patients were included in the present study (Supplementary Figure 1) . Thirty-two of these 90 patients were previously reported in an article describing their viral genome sequencing results. 3 The medical records of all the patients were reviewed, and data were obtained All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint regarding both the clinical characteristics of the eligible patients and the treatments that they received. PCR data obtained from samples collected from the nasopharynx, sputum or saliva were also collected. The present study protocol was approved by the ethics committee of the Keio University School of Medicine (approval number: 20200062) and was conducted in accordance with the Declaration of Helsinki.

The definitions of healthcare-associated SARS-CoV-2 transmission are shown in the Supplementary Table 1 . We first divided the subjects into healthcare workers or patients and then subdivided the non-healthcare workers into those with "community onset" and those with "hospital onset" to detect nosocomial infection (Supplementary Figure 2 ). 7 Next, we classified the patients according to the grade of disease severity based on their symptoms or conditions as outlined in the clinical management guidelines from the World Health Organization 8 and the Ministry of Health, Labour, and Welfare in Japan (https://www.mhlw.go.jp/content/000650160.pdf). In some COVID-19 patients with mild or moderate symptoms, the presence of pneumonia could not be determined because they did not receive either chest X-ray or computed tomography examinations. Therefore, we All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint classified disease severity as follows: "Mild to Moderate," patients did not require oxygen administration; "Severe," patients required oxygen supplementation but did not require a ventilator; and "Critical," patients who developed sepsis or acute respiratory distress syndrome and required a ventilator (Supplementary Table 2 ). 8

The whole viral genome sequences were determined as previously reported (Takenouchi et al.) . 3 PCR-based amplification was performed using the Artic ncov-2019 primers, version 3 (https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-201 9/V3/nCoV-2019.tsv) in two multiplex reactions according to the globally accepted "nCoV-2019 sequencing protocol" (https://www.protocols.io/view/ncov-2019-sequencingprotocol-bbmuik6w). 3 The sequencing library for amplicon sequencing was prepared using the Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Paired end sequencing was performed on the MiSeq platform (Illumina, CA). The bioinformatic pipeline used in this All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint study, "Mutation calling pipeline for amplicon-based sequencing of the SARS-CoV-2 viral genome," is available at https://cmg.med.keio.ac.jp/sars-cov-2/.

All point mutations including non-synonymous and synonymous mutations were annotated by the ANNOVAR software and were assessed using VarSifter 

The three-dimensional (3D) structure was visualized using PyMol v2.4.

(https://pymol.org/2/) based on publicly available SARS-CoV-2 protein structure coordinate for the dimer of 3CL pro (protein data bank ID "6LU7") and the tetramer of All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint nucleocapsid protein (protein data bank ID "6VYO"). We then used the DynaMut server (http://biosig.unimelb.edu.au/dynamut/) to infer the effect of non-synonymous mutations on the 3CL pro and nucleocapsid protein 3D structures in terms of molecular stabilities, and flexibilities. 9 We also estimated the differences of free energy change (ΔΔG) and vibrational entropy change (ΔΔSVib) accompanied by the mutation using DynaMut, 10 which implements ENCoM reports on the impact on protein stability and flexibility accompanied by mutations in the wild-type structure. Structural changes, such as changes in cavity volume, packing density, and accessible surface area, are correlated with Δ Δ G, thereby Δ Δ G can be used as an indicator of the impact of a mutation on protein stability. 11 A Δ Δ G value of less than zero indicates that the mutation causes destabilization, while a Δ Δ G above zero indicates protein stabilization.

The main parameter of this study was the grade of severity. Comparisons of categorical variables between two groups were assessed using the Fisher exact test. A Student t-test was used to compare abnormally distributed quantitative variables between two groups, and a Jonckheere-Terpstra test was used to analyze the tendency among three All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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groups. An exact logistic regression analysis was used to examine the relationship between the Clade 20B-T and the need for supplemental oxygen. The following covariates were considered for inclusion in the multivariate model: age group (<65 years or ≧ 65 years), sex, and infection group (Clade 20B-T or 20B-nonT). Statistical analyses were performed using R (version 3.6.2), and all the statistical tests were two-sided. A P-value <0.05 was considered significant. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint (frameshift mutations, non-sense mutations). The number counts of non-synonymous mutations among the strains varied from 2 to 12 (mean, 7.5 [SD], 2.4]), compared with the Wuhan reference strain. The functional relevance of the non-synonymous mutations was predicted using the computer algorithm PROVEAN, the calculations of which are not dependent on sequence conservation among animals.

The clinical characteristics of the 90 patients whose complete viral genome sequences were obtained, are shown in the Supplementary Table 4 . Nineteen patients (23.3%) required supplemental oxygen, and 8 (8.9%) developed acute respiratory distress syndrome; five of the eight patients died.

The number counts of thr non-synonymous mutations in SARS-CoV-2 found in COVID-19 patients who did and did not require supplemental oxygen were compared.

The number counts of non-synonymous mutations was significantly higher among the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint COVID-19 patients who did not require supplemental oxygen ( 

We tested whether any of the phylogenic clade containing any of the non-synonymous mutations might contribute to a milder clinical course. The overall genetic diversity was relatively low, presumably because of effective international border restrictions and successful quarantine efforts. A divergent tree analysis of the complete viral genome sequences from the 90 patients and classification according to the internationally recommended nomenclature (GISAIDs: http://www.gisaid.org/) showed that most (i.e., 87) patients had strains derived from Clade 20B (Figure 2a) . 12 The All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint remaining 5 patients belonged to Clade 19A, which lacks the Asp614Gly mutation in the Spike protein. Because the Asp614Gly mutation in the spike protein is known to be functionally relevant, 13 the 5 patients infected with Clade 19A viruses were excluded from further study.

Patients from Clade 20B (N = 85) were additionally divided into two subgroups by defining a subgroup as patients who had strains with no more than 5 nucleotide differences within the subgroup. The first subgroup, which we arbitrarily referred to as preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint cumulative total number and frequency curve showed that the relative fraction of Clade 20B-T increased during the time frame of this study (since May 2020) (Figure 2b-c) .

Mapping of the suspected geographic locations where infection in each patient likely occurred indicated that patients with the Clade 20B-T strain and those with the Clade 20B-nonT strain become infected in various regions in the Tokyo metropolitan area and its neighboring prefectures (Figure 2d ). This observation, together with a lack of patients with strains belonging to other clades (except for the 5 patients with Clade 19A who belonged to the same cluster) did not prove but suggested that Clade 20B and its variation Clade 20B-T were the predominant strains during the observation period.

A comparison of the clinical characteristics between patients with the Clade 20B-T viral strain (N = 48) and those with other strains (N = 37) is shown in Table 1 . Age, sex, symptoms at admission, and outcome did not differ significantly between the two main groups, but the use of oxygen supplementation was significantly lower among the patients with Clade 20B-T viral strain, compared with those with other strains (Fisher exact test: 12.5% vs. 32.4%, P value = 0.033). An exact logistic regression analysis showed that patients with the Clade All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

to Clade 20B-T.

We further aimed to decipher which of the 4 non-synonymous mutations that characterize the Clade 20B-T haplotype may contribute to a milder clinical course through a molecular evolutionary analysis. The conservation of the amino acid residues around the non-synonymous mutations observed in Clade 20B-T was evaluated using the software MEGA (https://www.megasoftware.net/) and the amino acid sequences of known β -coronaviruses. Amino acid residues at and around the Pro108Ser mutation in the 3CL pro protein (nonstructural polyprotein 5; NSP5), and those at and around the Pro151Leu mutation in the nucleocapsid protein were highly conserved, whereas amino acid residues at and around the Ser543Pro mutation in the papain-like protease (PL pro , NSP3) protein and those at and around the Ala423Val mutation in RNA-dependent RNA polymerase All rights reserved. No reuse allowed without permission.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint 4a). [14] [15] [16] The modeling showed that the Pro108Ser substitution in 3CL pro could not induce significant structural changes at or around residue 108 or intramolecular interactions between the residue at 108 and surrounding residues (Figure 4b) 

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The number count of non-synonymous mutations was inversely correlated with the severity of COVID-19 disease in a cohort of 90 patients whose viral genome sequences had been completely sequenced. Patients with a viral haplotype containing both the 3CL pro Pro108Ser mutation and the nucleocapsid protein Pro151Leu mutation tended to have a milder disease course than those with a viral haplotype lacking these two mutations.

Our observation that a mutation of 3CL pro , Pro108Ser, was associated with disease severity strongly supports the notion that inhibitors for 3CL pro , the main protease that cleaves viral polyproteins into functional proteins, 18 could be a promising antiviral agent that may be effective against SARS-CoV, 19,20 as well as SARS-Cov-2. 21 Further enzymatic analysis of the Pro108Ser mutation in 3CL pro is needed.

The other observation that the Pro151Leu mutation in nucleocapsid protein, which enters the host cells along with the viral RNA, may be associated with disease severity is also intriguing in that nucleocapsid protein is responsible for promoting viral replication and processing the assembly and release of viral particles. 22 The computer-based protein structure predicted that the mutant 3CL pro and the mutant nucleocapsid proteins are less flexible, with no significant changes in protein structure or stabilities. However, their interactions and dominance are not known. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020. 11.24.20235952 doi: medRxiv preprint In general, RNA viruses continue to survive and proliferate by constantly changing their forms and adapting to various environments, but quasispecies with a high replicability seem to be unfavorable in terms of long-term survival because of a high sensitivity to environmental influences (i.e., survival of the fittest). 23 On the other hand, quasispecies with a low replicability may have reduced infectivity and pathogenicity but actually have long-term survival advantages because they are less likely to be subjected to natural selection (i.e., survival of the flattest). 24, 25 Coronaviruses, including SARS-CoV or SARS-CoV-2, are well known to have a low fidelity in replicating their genome. 26, 27 Of the proteins involved in the replication of the viral genome, proteases, including 3CL pro , are critical for viral replication. 28 Therefore, the Pro108Ser mutation in 3CL pro may lead to a situation in which the virus is less susceptible to natural selection and long-term survival is more favorable, reducing viral infectivity and virulence.

The present study had several limitations. First, the decrease in the fraction of critically ill patients might be accounted for by the seasonality of vulnerability to viruses, in general. 29 One report has suggested that SARS-CoV-2 is more stable and has a higher persistence at the same temperature than SARS-CoV. 30 However, the lack of such seasonal trends in the SARS-CoV-2 pandemic observed in other countries suggests that a All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint seasonal bias is unlikely. Second, drawing a decisive conclusion based on a relatively small number of subjects at a single center could be premature in terms of the relative virulence of the two subclades in Tokyo. However, the near absence of haplotype groups other than Clade 20B-T or 20B-nonT in our cohort of 87 patients, whose suspected locations of infection were scattered across the city (Figure 2d ), strongly suggest that these two groups represent the predominant or almost exclusive strains in Tokyo. Currently, the number of whole viral genome sequences determined in Japan and deposited in international databases is not sufficient to conclude that viral strains with a Clade 20B-nonT haplotype carrying the 3CL Pro Pro108Ser mutation and the nucleocapsid protein Pro151Leu mutation are predominant in Japan. Nevertheless, recent data deposited from a tertiary medical center in Nagoya, Japan's third largest city located 500 km away from Tokyo, support our notion. Among the 27 viral strains collected in Nagoya between March and August of 2020, 19 strains belonged to the Clade 20B-T haplotype and 8 belonged to the Clade 20B-nonT haplotype (Supplementary Figure 3) . This distribution essentially recapitulates our observations in Tokyo.

In conclusion, viral genome sequencing in 90 patients living in the Tokyo Metropolitan area showed that the accumulation of point mutations, including Pro108Ser All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint in 3CL pro and Pro151Leu in nucleocapsid protein, was inversely correlated with COVID-19 severity. Further in vitro research is awaited.

KA contributed to writing of the report and data analysis. YK, SU, TM and MN contributed to review and editing of the report and data analysis. YI, HI, TT and HS contributed to sequencing and analysis. YU, SU and NH contributed to public health intelligence and case identification. MI and KF contributed to clinical data and clinical care. HS, YK and MA contributed to writing and editing of the report. MM contributed to diagnostics and laboratory management. MS and KK had the idea for the study and contributed to diagnostics, formal analysis, and writing and editing of the report.

Authors have no conflicts of interests.

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The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint Wuhan-strain type and mutants.

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The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint Figure 2 : Phylogenic tree analysis, temporal trends, and spatial distribution around Keio University Hospital (purple dot in Fig. 2d) showed consistent increase of a strain with a unique haplotype.

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Acknowledgements Table. We have uploaded the full nucleotide sequences of our cohort to the GISAID database.

We thank all the patients and healthcare workers who have fought against COVID-19. This work was supported by the Keio Donner Project and is devoted to the late Professor Shibasaburo Kitasato, the founder of Keio University School of Medicine. We also thank the members of Center for Medical Genetics in Keio University School of Medicine, and SUNTORY Co., Ltd.All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this this version posted November 24, 2020. ; https://doi.org/10.1101/2020.11.24.20235952 doi: medRxiv preprint