菅義偉は米国から2度と帰ってこないでください!――査読前論文:新型コロナ米国変異株、日本人の6割が持つ白血球型で免疫効果低下の可能性『An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity』

プレプリント=査読前論文ですが、こういうのが公表されました→An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity(※フルバージョン)

今年2月から3ヶ月連続で第1巻第2巻 第3巻と改訂版が出されたブルーバックス新書の『アメリカ版 新・大学生物学の教科書』を手はじめに、医学部生が使うような分子生物学や免疫学の教科書で勉強しはじめたところで(生物は中学までで高校の理科は物理と化学だけど化学が得意だったせいか難しくても面白い)、機械翻訳で流し読みしても「?」だらけとはいえ、いま製剤化されているワクチンでは効き目があるとは思えず、抗原定量検査というザルザルな空港検疫で素通りされて日本に入ってきたら、どうなるんでしょう?

松戸市議会議員DELIさんのYouTubeチャンネルPlanet Rockにコロラド先生こと牧田寛さんが特別出演されてた時に、カリフォルニア州で最凶のハイブリッドな変異株が発生したって話をされてた記憶が朧気にあるのですが、これのことだったのかな〜と・・・

新たな変異株の出現でワクチン開発が後手に回っているイタチごっこな状況で、SARS-CoV-2対策でワクチン頼み以外は何もしないという今の菅政権や日本政府の態度は非常に悪手だし、それよりも早いとこ全国民が無料で何度もPCR検査を受診できる体制を作って地道にコツコツ叩いていく他ないでしょう。早期に発見して隔離と治療を行えば不治の病ではないのですし。

ところで、カリフォルニア州のあるアメリカといえば、どこぞのソーリが訪米するんでしたっけ?
いい機会です、ヘンテコリンな変異株を日本に持ち込まれたら非常に迷惑ですので、
菅義偉は入国禁止にしましょう!(もちろんお付きの方々全員まとめて)

新型コロナ米国変異株、日本人の6割が持つ白血球型で免疫効果低下の可能性【マイナビニュース:TECH+ 2021年4月14日】

東京大学医科学研究所は、米国カリフォルニア州で見つかっている新型コロナウイルスの変異株(B.1.427およびB.1.429)が日本人の免疫から逃避する可能性を明らかにした。

同成果は、東京大学医科学研究所 附属感染症国際研究センター システムウイルス学分野の佐藤佳 准教授の研究室が主催する新型コロナ研究コンソーシアム「The G2P-Japan」によるもので、佐藤准教授のほか、ヒトレトロウイルス学共同研究センター(熊本大学/鹿児島大学)の本園千尋 講師、同 上野貴将 教授、東京大学医科学研究所 附属感染症国際研究センター システムウイルス学分野の木村出海 博士課程3年、同・瓜生慧也 博士課程2年、東海大学医学部 基礎医学系分子生命科学の中川草 講師らが参加している。詳細は、生物学のプレプリントリポジトリ「bioRχiv」にて公開された

それによると、同コンソーシアムでは新型コロナのスパイクタンパク質の一部が、CTLやマクロファージが直接細胞を攻撃する免疫反応である細胞性免疫のうち、日本人の6割が持つとされる白血球の型「HLA-A24」によって強く認識ことを免疫学実験によって実証したほか、B.1.427系統ではY453F、B.1.429系統ではL452Rというスパイクタンパク質のアミノ酸変異があり、これらの変異はいずれもHLA-A24による細胞性免疫から逃避、つまり免疫細胞の働きを抑制するなど、免疫の効果を弱めることを実証したという。

さらに、これらの変異が、新型コロナウイルスの感染と複製効率に与える影響を、ウイルス学実験で検討した結果、L452R変異は、ウイルスの感染力を増強させることも判明したという。

今回の結果を受けてコンソーシアムでは、L452R変異は、日本人に多いHLA-A24による免疫から逃避するだけでなく、ウイルスの感染力を増強しうる変異であることから、日本人あるいは日本社会にとって、他の変異株よりも危険な変異株である可能性が示唆されるとしており、この変異株へのリスク対応のためにも、流行株のサーベイランスや空港での水際対策の強化の必要性が考えられるとしている。

 

An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity【bioRxiv Posted April 05, 2021.】

Summary

During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic.

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Introduction

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since an unusual outbreak in Wuhan, Hubei province, China, in December 2019 (Wu et al., 2020; Zhou et al., 2020), SARS-CoV-2 has rapidly spread all over the world, and as of March 2021, SARS-CoV-2 is an ongoing pandemic: more than one hundred million cases of infections have been reported worldwide, and more than two million people died of COVID-19 (WHO, 2020a).

During the current pandemic, a variety of SARS-CoV-2 mutants have emerged and some of them have dominantly spread [reviewed in (Plante et al., 2021)]. A well-studied SARS-CoV-2 mutant harbors D614G substitution in the spike (S) protein. Recent studies have revealed that the D614G mutation increases the SARS-CoV-2 binding affinity to ACE2, the SARS-CoV-2 receptor (Ozono et al., 2021; Yurkovetskiy et al., 2020; Zhou et al., 2021), infectivity (Ozono et al., 2021; Yurkovetskiy et al., 2020; Zhou et al., 2021), fitness (Hou et al., 2020; Plante et al., 2020; Zhou et al., 2021), and transmissibility in the human population (Volz et al., 2021). However, there is no evidence suggesting that the D614G variant is associated with viral pathogenicity and lethality (Hou et al., 2020; Korber et al., 2020; Plante et al., 2020). Additionally, since the fall of 2020, new SARS-CoV-2 variants such as B.1.1.7 (also known as a variant of concern 202012/01 or 20I/501Y.V1), B.1.351 (also known as 20H/501Y.V2), and P.1 (also known as 501Y.V3) lineages emerged in the UK, South Africa, and Brazil, respectively, and have rapidly spread worldwide (CDC, 2020). At the end of 2020, another lineage, B.1.427/429 (also known as CAL.20C), has been predominant particularly in California state, the USA (Deng et al., 2021; Zhang et al., 2021). Moreover, cross-species viral infection can accelerate the emergence of diversified viruses [reviewed in (Banerjee et al., 2021; Parrish et al., 2008)]. In the case of SARS-CoV-2, a variety of mammals such as nonhuman primates (Chandrashekar et al., 2020; Munster et al., 2020; Yu et al., 2020) and carnivores (Halfmann et al., 2020; Kim et al., 2020; Shi et al., 2020) are prone to its infection (Damas et al., 2020; Martinez-Hernandez et al., 2020; OIE, 2021). Strikingly, the emergence of a SARS-CoV-2 variant, B.1.298, has likely to be associated with the outbreak in farmed minks in Denmark (Koopmans, 2021; WHO, 2020b), and phylogenetic analysis has provided evidence of mink-to-human transmission of SARS-CoV-2 within Danish mink farms (Oude Munnink et al., 2021). Because newly emerging variants can potentially change viral infectivity, transmissibility and pathogenicity, deep monitoring of the SARS-CoV-2 strains circulating globally and locally and evaluating the effects of mutations detected on virological characteristics are urgent and crucial.

The emergence of mutated viruses is mainly due to error-prone viral replication, and the spread of emerged variants is attributed to the escape from immune selective pressures [reviewed in (Duffy et al., 2008)]. In fact, several SARS-CoV-2 mutants can be resistant to the neutralization mediated by the antibodies from COVID-19 patients (Baum et al., 2020; Chen et al., 2021; Liu et al., 2021c; McCarthy et al., 2021; Weisblum et al., 2020) as well as those from vaccinated individuals (Liu et al., 2021b). Although the B1.1.7 variant is sensitive to convalescent and vaccinated sera (Collier et al., 2021; Garcia-Beltran et al., 2021; Shen et al., 2021; Supasa et al., 2021; Wang et al., 2021), the B.1.351 and P.1 variants are relatively resistant to anti-SARS-CoV-2 humoral immunity (Garcia-Beltran et al., 2021; Hoffmann et al., 2021a; Wang et al., 2021).

In addition to the humoral immunity mediated by neutralizing antibodies, another protection system against pathogens is the cellular immunity mediated by cytotoxic T lymphocytes (CTLs) [reviewed in (Fryer et al., 2012; Leslie et al., 2004)]. CTLs recognize the nonself epitopes that are presented on virus-infected cells via human leukocyte antigen (HLA) class I molecules, and therefore, the CTL-mediated antiviral immunity is HLA-restricted [reviewed in (La Gruta et al., 2018)]. Recent studies have reported the HLA-restricted SARS-CoV-2-derived epitopes that can be recognized by human CTLs (Kared et al., 2021; Kiyotani et al., 2020; Nelde et al., 2021; Schulien et al., 2021; Wilson et al., 2021). More importantly, Bert et al. have recently reported that the functionality of virus-specific cellular immunity is inversely correlated to the COVID-19 severity (Le Bert et al., 2021). Therefore, it is conceivable to assume that the HLA-restricted CTLs play crucial roles in controlling SARS-CoV-2 infection and COVID-19 disorders. However, comparing to humoral immune responses, it remains unclear whether the SARS-CoV-2 variants can potentially escape from cellular immunity.

In this study, we investigate the possibility for the emergence of the SARS-CoV-2 mutants that can escape from the HLA-restricted cellular immunity. We demonstrate that at least two naturally occurring substitutions in the receptor binding motif (RBM; residues 438-506) of the SARS-CoV-2 S protein, L452R and Y453F, which were identified in the two major variants, B.1.427/429 (L452R) and B1.1.298 (Y453F), can be resistant to the cellular immunity in the context of HLA-A*24:02, an allele of HLA-I. More intriguingly, the L452R and Y453F mutants increase the binding affinity to ACE2, and the experiments using pseudoviruses show that the L452R substitution increases viral infectivity. Furthermore, we artificially generate the SARS-CoV-2 harboring these point mutations by reverse genetics and demonstrate that the L452R mutants enhance viral replication capacity.

Results

Evasion from the HLA-A24-restricted CTL responses by acquiring mutations in the RBM of SARS-CoV-2 S protein

We set out to address the possibility of the emergence of the naturally occurring mutants that can potentially confer the resistance to antigen recognition by HLA-restricted cellular immunity. A bioinformatic study has suggested that the 9-mer peptide in the RBM, NYNYLYRLF (we designate this peptide “NF9”), which spans 448-456 in the S protein, can be the potential epitope presented by HLA-A24 (Kiyotani et al., 2020), an HLA-I allele widely distributed all over the world and particularly predominant in East and Southeast Asian area (Table S1). Additionally, three immunological analyses using COVID-19 convalescents have shown that the NF9 peptide is an immunodominant epitope presented by HLA-A*24:02 (Gao et al., 2021; Hu et al., 2020; Kared et al., 2021). To verify these observations, we obtained the peripheral blood mononuclear cells (PBMCs) from nine COVID-19 convalescents with HLA-A*24:02 and stimulated these cells with the NF9 peptide. As shown in Figure 1A, a fraction of CD8+ T cells upregulated two activation markers, CD25 and CD137, in response to the stimulation with NF9. In the nine samples of COVID-19 convalescents with HLA-A*24:02, the percentage of the CD25+CD137+ cells in the presence of the NF9 peptide (5.3% in median) was significantly higher than that in the absence of the NF9 peptide (0.49% in median) (Figure 1B; P=0.016 by Wilcoxon signed-rank test). Additionally, the stimulation with the NF9 peptide did not upregulate CD25 and CD137 in the CD8+ T cells of three seronegative samples with HLA-A*24:02 and the percentage of the CD25+CD137+ cells in seronegative samples (0.93% in median) was significantly lower than that in COVID-19 convalescent samples (Figure 1B; P=0.011 by Mann-Whitney U test). Consistent with previous reports (Gao et al., 2021; Hu et al., 2020; Kared et al., 2021; Kiyotani et al., 2020), our data suggest that the NF9 peptide is an immunodominant HLA-A*24:02-restricted epitope recognized by the CD8+ T cells of COVID-19 convalescents in our cohort.

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〔Figure 1. Escape of the two naturally occurring SARS-CoV-2 mutations from the S RBM-specific CD8+ T cells.
(A and B) Detection of the HLA-A24-restricted NF9-specific CTLs. The HLA-A*24:02-positive CTL lines of 3 seronegative donors and 9 COVID-19 convalescents were stimulated with or without 1 μM NF9 peptide (NYNYLYRLF, residues 448-456 of the SARS-CoV-2 S protein). Representative FACS plots showing the surface expression of CD25 and CD137 in the CD8+ T cell subset (i.e., CD3+CD8+ cells) of a seronegative donor (left) and a COVID-19 convalescent donor #1 (right) (A) and the median of the percentage of CD25+CD137+ cells in CD8+ T cells (B) are shown. In B, the COVID-19 convalescent samples >3 SD of the median of NF9-stimulated seronegative samples are indicated with red asterisks.
(C and D) Multifunctionality of the HLA-A24-restricted NF9-specific CTLs. The HLA-A*24:02-positive CTL lines of 6 COVID-19 convalescents were stimulated with or without 10 nM NF9 peptide. Representative FACS plots showing the intracellular expression of IFN-γ, TNF-α and IL-2 in the CD8+ T cell subset of a COVID-19 convalescent (donor #1) (C) and the pie charts showing the proportion of cytokine positive cells in each convalescent sample (D) are shown.
(E and F) Potential killing activity of the HLA-A24-restricted NF9-specific CTLs. The HLA-A*24:02-positive CTL lines of 6 COVID-19 convalescents were stimulated with the C1R-A2402 cells pulsed with or without 10 nM NF9 peptide. Representative FACS plots showing the surface expression of CD107a in the CD8+ T cell subset of a COVID-19 convalescent (donor #1) (E) and the median of the percentage of CD107a+ cells in CD8+ T cells (F) are shown.
(G) Distribution of the L452R and Y453F mutants during the current pandemic. The top 5 countries where the variants harboring the L452R (top) and Y453F (bottom) mutations are shown. The raw data are summarized in Table S2.
(H and I) Mutations escaped from the HLA-A24-restricted NF9-specific CTLs. The HLA-A*24:02-positive CTL lines of 5 COVID-19 convalescents were stimulated with 1 nM NF9 peptide or its derivatives: NF9-L452R (NYNYRYRLF) and NF9-Y453F (NYNYLFRLF). Representative FACS plots showing the intracellular expression of IFN-γ in the CD8+ T cell subset of a COVID-19 convalescent (donor #1) (H) and the mean of the percentage of IFN-γ+ cells in CD8+ T cells (I) are shown.
In A, E and H, the numbers in the FACS plot represent the percentage of gated cells in CD8+ T cells. In C, the number represents the percentage of the cells in each quadrant.
In B, a statistically significant difference (*, P<0.05) between SARS-CoV-2 seronegative and COVID-19 convalescent samples is determined by Mann-Whitney U test, and a statistically significant difference (*, P<0.05) between with and without NF9 peptide in COVID-19 convalescent samples is determined by Wilcoxon signed-rank test. In F, each symbol of the COVID-19 convalescent data represents the mean of technical triplicate. Statistically significant differences (*, P<0.05) between with and without NF9 peptide in COVID-19 convalescent samples are determined by Wilcoxon signed-rank test. In I, the assay was performed in triplicate, and the means are shown with SD. Statistically significant differences (*, P<0.05) versus “no are determined by ANOVA with multiple comparisons by Bonferroni correction. See also Figure S1 and Tables S1 and S2.〕 We next assessed the profile of cytokine production by the NF9 stimulation. As shown in Figure 1C, the stimulation with the NF9 peptide induced the production of IFN-γ, TNF-α and IL-2 in the CD8+ T cells of a COVID-19 convalescent. The analysis using six COVID-19 convalescent samples showed that CD8+ T cells produce multiple cytokines in response to the NF9 stimulation (Figure 1D), demonstrating the multifunctional nature of the NF9-specific CD8+ T cells of COVID-19 convalescents. Moreover, the cytotoxic potential of the NF9-specific CD8+ T cells was assessed by staining with surface CD107a, a degranulation marker (Figure 1E). As shown in Figure 1F, the percentage of CD107a+ cells in the CD8+ T cells with the NF9 peptide (12.9% in median) was significantly higher than that without the NF9 peptide (0.83% in median) (; P=0.031 by Wilcoxon signed-rank test), suggesting the cytotoxic potential of the NF9-specific CD8+ T cells. To assess the presence of naturally occurring variants harboring mutations in this region (residues 448-456 in the S protein), we analyzed the diversity of SARS-CoV-2 during the current pandemic. We downloaded 750,243 viral genome sequences from the global initiative on sharing all influenza data (GISAID) database (https://www.gisaid.org; as of March 15, 2021). The L452R substitution was most frequent among the sequences analyzed (5,677 sequence), and 1,380 sequences reported contained the Y453F substitution (Table 1). Notably, the B.1.427/429 and B.1.1.298 lineages (CDC, 2020) in the PANGO lineages (https://cov-lineages.org/index.html) mainly harbor the L452R and Y453F mutations, respectively (Figure 1G and Table S2). To address the possibility that the naturally occurring mutations in the NF9 region, L452R and Y453F, evade the NF9-specific CD8+ T cells of HLA-A24-positive COVID-19 convalescents, two NF9 derivatives containing either L452R or Y453F substitution (NF9-L452R and NF9-Y453F) were prepared and used for the stimulation experiments. As shown in Figure S1, parental NF9 induced IFN-γ expression in a dose-dependent manner. In contrast, the induction level of IFN-γ expression by the NF9-Y453F derivative was significantly lower than that by parental NF9, and more intriguingly, the NF9-L452R derivative did not induce IFN-γ expression even at the highest concentration tested (10 nM) (Figure S1). In the five HLA-A24-positive COVID-19 convalescent samples, parental NF9 peptide significantly induced IFN-γ expression, while the NF9-L452R and NF9-Y453F derivatives did not (Figures 1H and 1I). Altogether, these results suggest that the NF9 peptide, which is derived from the RBM of SARS-CoV-2 S protein, is an immunodominant epitope of HLA-A24, and two naturally occurring mutants, L452R and Y453F, evade the HLA-A24-restricted cellular immunity. Augmentation of the binding affinity to ACE2 by the L452 and Y453 mutations

We next addressed whether the mutations of interest affect the efficacy of virus infection. Structural analyses have shown that the Y453 and N501 residues in the RBM are located on the interface between the SARS-CoV-2 RBM and human ACE2 and directly contribute to the binding to human ACE2, while the L452 residue is not on the RBM-ACE2 interface (Lan et al., 2020; Wang et al., 2020; Zhao et al., 2020) (Figure 2A). To directly assess the effect of these mutations in the RBM on the binding affinity to ACE2, we prepared the yeasts expressing parental SARS-CoV-2 receptor binding domain RBD (residues 336-528) and its derivatives (L452R, Y453F and N501Y) and performed in vitro binding assay using the yeast surface display of the RBD and soluble ACE2 protein. Consistent with recent studies including ours (Supasa et al., 2021; Zahradník et al., 2021b), the N501Y mutation, which is a common mutation in the B1.1.7, B1.351 and P.1 variants [reviewed in (Plante et al., 2021)] as well as the Y453F mutation (Bayarri-Olmos et al., 2021; Zahradník et al., 2021b) significantly increased the binding affinity to human ACE2 (Figures 2B and 2C; RBD parental KD = 2.05 ± 0.26 nM; RBD N501Y KD = 0.59 ± 0.03 nM; and RBD Y453F KD = 0.51 ± 0.06 nM). We also found that the L452R mutant significantly increased the binding affinity to human ACE2 (Figures 2B and 2C; RBD L452R KD = 1.20 ± 0.06 nM). Intriguingly, the L452R mutations increased the surface expression, which reflects protein stability (Traxlmayr and Obinger, 2012), while the Y453F and N501Y mutations decreased (Figure 2D).

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〔Figure 2. Increase of the binding affinity to ACE2 and viral infectivity by the L452 mutation.
(A) Location of the NF9 peptide (residues 448-456) in the cocrystal structure of the SARS-CoV-2 S and human ACE2 proteins (PDB: 6M17) (Yan et al., 2020). An overview (left), the enlarged view of the boxed area in the left panel (middle) and the view of the middle panel rotated 180° on the y-axis (right) are shown. The residues 448-456 of SARS-CoV-2 S (corresponding to the NF9 peptide) are shown in black. (B-D) Binding affinity of SARS-CoV-2 S RBD to ACE2 by yeast surface display. The percentage of the binding of the SARS-CoV-2 S RBD expressing on yeast to soluble ACE2 (B) and the KD values (C) are shown. (D) The level of stable expression of the SARS-CoV-2 RBD on yeast (x-axis) and the binding affinity to ACE2 (y-axis) compared to parental RBD. In B, the fitting curve of parental RBD is shown in all panels as black lines.
(E) Pseudovirus assay. The HIV-based reporter virus pseudotyped with the parental SARS-CoV-2 S or its derivatives (L452R, Y453F and N501Y) were inoculated into the 293 cells transiently expressing human ACE2 and TMPRSS2 at 4 different doses (1, 3, 5 and 10 ng p24 antigens). The percentages of the infectivity compared to the virus pseudotyped with parental S (10 ng p24 antigen) are shown.
(F) Gain of electrostatics complementarity by the L452R substitution. (Left) The surface structure of the SARS-CoV-2 S and ACE2 (PDB: 6M17) (Yan et al., 2020). The residue 452 of the SARS-CoV-2 S and the negatively charged patch on ACE2 (residues E35, E37 and D38) are indicated by black and red. The boxed area is enlarged in the upper right panel. (Right) Coulombic surface coloring at the structures of the SARS-CoV-2 S and ACE2 (PDB: 6M17) (Yan et al., 2020) (top) and a model of the L452R substitution (bottom). The black line indicates the border between SARS-CoV-2 S and ACE2.
In B and E, these assays were performed in quadruplicate.
In C, statistically significant differences (*, P<0.05) versus parental S are determined by Mann-Whitney U test. In E, statistically significant differences (*, P<0.05) versus parental S at the same dose are determined by ANOVA with multiple comparisons by Bonferroni correction.〕 Increase of pseudovirus infectivity by the L452R mutation

To directly analyze the effect of the mutations of interest on viral infectivity, we prepared the HIV-1-based reporter virus pseudotyped with the SARS-CoV-2 S protein and its mutants and the 293 cells transiently expressing human ACE2 and TMPRSS2. As shown in Figure 2E, although the N501Y mutation faintly affected viral infectivity in this assay, the L452R mutations significantly increased viral infectivity compared to parental S protein. In contrast to the yeast display assay (Figures 2B and 2C), the infectivity of the Y453F mutant was significantly lower than that of parental S protein (Figure 2E). Altogether, these findings suggest that the L452R substitution increases the binding affinity of the SARS-CoV-2 RBD to human ACE2, protein stability, and viral infectivity. Although the L452 residue is not directly located at the binding interface (Figure 2A), structural analysis and in silico mutagenesis suggested that the L452R substitution can cause a gain of electrostatics complementarity (Selzer et al., 2000) (Figure 2F). Because the residue 452 is located close proximity to the negatively charged patch of ACE2 residues (E35, E37, D38), the increase of viral infectivity by the L452R substitution can be attributed to the increase in the electrostatic interaction with ACE2.

Promotion of SARS-CoV-2 replication in cell cultures by the L452 mutation

To investigate the effect of the mutations in the RBM on viral replication, we artificially generated the recombinant SARS-CoV-2 viruses that harbor the mutations of interest as well as parental recombinant virus by a reverse genetics system (Torii et al., 2021). The nucleotide similarity of SARS-CoV-2 strain WK-521 (GISAID ID: EPI_ISL_408667) (Matsuyama et al., 2020), the backbone of the artificially generated recombinant SARS-CoV-2, to strain Wuhan-Hu-1 (GenBank: NC_045512.2) (Wu et al., 2020) is 99.91% (27 nucleotides difference) and the sequences encoding the S protein between these two strains are identical, indicating that the strain WK-521 is a SARS-CoV-2 prototype. We verified the insertions of the targeted mutations in the generated viruses by direct sequencing (Figure 3A) and performed virus replication assay using these recombinant viruses. As shown in Figure 3B, we revealed that the growth of the L452R mutant in VeroE6/TMPRSS2 cells was significantly higher than that of parental virus. Together with the findings in the binding assay (Figures 2B-2D) and the assay using pseudoviruses (Figure 2E), our results suggest that the L452R mutation potentially increase viral replication.

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〔Figure 3. Enhancement of viral replication by the L452 mutation.
(A) Chromatograms of the mutated regions of the SARS-CoV-2 viruses artificially generated by reverse genetics. The chromatograms of the nucleotide positions 22,913-22,924 (left) and 23,060-23,068 (right) of parental SARS-CoV-2, the L452R, Y453F and N501Y mutants are shown.
(B) Growth kinetics of parental SARS-CoV-2 and the SARS-CoV-2 mutants. Parental SARS-CoV-2, the L452R, Y453F and N501Y mutants (100 pfu) were inoculated into VeroE6/TMPRSS2 cells (top) and 293-ACE2 cells (middle) and the copy number of viral RNA in the culture supernatant was quantified by real-time RT-PCR. (Left) The growth curve of the viruses inoculated. The result of parental virus is shown in all panels as a black line. (Right) The amount of viral RNA in the culture supernatant at 72 h postinfection. The assays were performed in quadruplicate (VeroE6/TMPRSS2 cells) or triplicate (293-ACE2 cells), and statistically significant differences (*, P<0.05) versus parental S are determined by Student’s t test. (Bottom) Representative figures of the blight fields of 293-ACE2 cells infected with the viruses indicated at 72 h post infection are also shown. Bars, 200 μm.〕 Dynamics of the spread of the RBM mutants during the current pandemic

We finally assessed the epidemic dynamics of the naturally occurring variants containing the substitutions in L452 and Y453. As shown in Figure 4A and Table S3, the L452R mutants were mainly found (3,967 sequences) in the B.1.427/B.1.429 lineage that forms a single clade (Deng et al., 2021). Although the L452R mutant was first detected the B.1.39 lineage in Denmark on March 17, 2020 (GISAID ID: EPI_ISL_429311) (Table 1), this variant did not spread. The oldest sequence that contains the L452R mutation in the B.1.427/B.1.429 lineage was isolated in Quintana Roo state, Mexico, on July 6, 2020 (GISAID ID: EPI_ISL_942929) (Table 1), and the L452R-harboring mutants have been first detected in California state, the USA, on September 28, 2020 (GISAID ID: EPI_ISL_730092 and EPI_ISL_730345) (Figure 4B). The B.1.427/B.1.429 lineage harboring the L452R mutation has started expanding in California state, the USA, at the beginning of November, 2020 (Figure 4B, top). In 2021, this lineage has expanded throughout the USA, and currently, is one of the most predominant lineages in the country (Figure 4B, bottom and Table S4).

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〔Figure 4. Epidemic dynamics of the B.1.298 and B.1.427/429 lineages during the current pandemic.
The PANGO lineages harboring the L452R (A and B) and Y453F (C and D) and their epidemic dynamics are summarized.
(A and C) Distribution of the L452R and Y453F mutants during the current pandemic. The top 5 PANGO lineages (https://cov-lineages.org/index.html) that harbor the L452R (A) and Y453F (C) mutations are shown. The raw data are summarized in Table S3.
(B and D) Epidemic dynamics of the L452R-harboring B.1.427/429 lineage in California state, the USA (B, top) and the USA (B, bottom) and the Y453F-harboring B.1.298 lineage in Denmark (D). The numbers of the sequences harboring mutation per day (left y-axis, bars) and the numbers of total sequences per day (right y-axis, dots), from January 22, 2020 to March 6, 2021, are summarized. Note that a L452R variant isolated from gorilla and three Y453F variants isolated from cats are not included.
See also Figure S2 and Tables S3 and S4.〕

For the Y453F mutation, 1,274 out of the 1,380 mutated sequences belong to the B.1.1.298 lineage, which has been exclusively detected in Denmark (Table S3). The oldest sequence that contains the Y453F mutation in the B.1.1.298 lineage was isolated from a human in Denmark on April 20, 2020 (GISAID ID: EPI_ISL_714253) (Figure 4C). Intriguingly, the B.1.1.298 variants containing either Y453 or F453 are detected not only in humans but also in minks (Figure 4D).

The phylogenetic analysis of the whole genome sequences of the B.1.1.298 lineage SARS-CoV-2 suggested multiple SARS-CoV-2 transmissions between humans to minks (Figure S2). Additionally, the three sequences that contain the Y453F mutation were isolated from cats in Denmark: the two sequences out of them (GISAID ID: EPI_ISL_683164 and EPI_ISL_683166) made a single clade, while the other one (GISAID ID: EPI_ISL_683165) had a distinct origin (Figure S2). These results suggest that this SARS-CoV-2 variant has transmitted from humans to cats multiple times and some of them may spread among Danish cat population. However, the epidemic of a fraction of the B.1.1.298 lineage containing the Y453F mutation in Denmark peaked during October to November, 2020, and subsequently, gradually reduced (Figure 4D). The variant containing the Y453F mutation was last collected in Denmark on January 18, 2021 (GISAID ID: EPI_ISL_925998) and it has not been reported worldwide since then (Figure 4D).

 

・・・『Discussion』以下まだまだ続きますけど長いので省略・・・