key: cord-0286400-ybw1sbmn
authors: Jeong, Gi Uk; Yoon, Gun Young; Moon, Hyun Woo; Lee, Wooseong; Hwang, Insu; Kim, Hae Soo; Kim, Kyun-Do; Kim, Chonsaeng; Ahn, Dae-Gyun; Kim, Bum-Tae; Kim, Seong-Jun; Kwon, Young-Chan
title: Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern
date: 2021-10-01
journal: bioRxiv
DOI: 10.1101/2021.09.30.462687
sha: 6249c93ad331662512e8b1a46728a7615870f6f2
doc_id: 286400
cord_uid: ybw1sbmn

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha’s small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic. IMPORTANCE The global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.

(B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade 23 G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in 24 therapeutic or vaccine effectiveness. However, the physiological and virological understanding of 25 these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque 26 size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta 27 was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by 28 measuring infectivity and half-life after prolonged incubation at physiological temperature, was 29 correlated with plaque size in all variants except alpha. However, despite its relatively high thermal 30 stability, alpha's small plaque size resulted in lower replication rates and fewer progeny viruses. Our The global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have 39 resulted in the emergence of different variants. Many of these variants have become more virulent and 40 infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological 41 understanding of these variants remains poor. Here, we directly compared the plaque size, stability, 42 and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at 43 physiological temperatures. Our observations may help to characterize each variant in terms of their 44 interactions with host factors and responses to environmental conditions. We also believe that our 45 evaluations will improve understanding of the emergence of new variants and contribute to controlling 46 their spread. 47 4 OBSERVATION 49

Several novel variants of SARS-CoV-2 have been identified since the COVID-19 pandemic began in 50 December 2019. Although the expected mutation rate for SARS-CoV-2 is estimated to be 51 approximately 2.4 x 10 -3 per site per year (1), significantly more mutations and deletions across 52 thousands of variants, all of which may alter their pathogenic potential, have already been reported (2). 53

The World Health Organization identifies novel SARS-CoV-2 variants of concern (VOCs) based on 54 their potential impact on public health, according to evidence of enhanced transmissibility, increased 55 severity, reduced antibody neutralization arising from former infection or vaccination, detection 56 evasion ability, or decreased treatment or vaccine efficacy (3). 57

There are currently only four circulating SARS-CoV-2 VOCs: alpha (B.1.1.7, clade GRY), beta 58 (B.1.351, clade GH), gamma (P.1, clade GR), and delta (B.1.617.2, clade G). They share D614G 59 mutation, conferring increased infectivity, likely due to changes affecting the receptor binding and 60 fusion (4-7). N501Y mutation is also shared by alpha, beta, and gamma, increasing their receptor-61 binding affinity and subsequent cellular entry (8). However, the combination of mutations could result 62 in greater conformational changes and distinctive modifications (9). For example, VOCs exhibit 63 differential receptor-binding affinity. Alpha requires the most force to be detached from the receptor, 64 followed by beta/gamma and delta (10). 65

Here, we noted that each of these variants presented with different plaque sizes (Fig. 1a) . The mean 66 plaque size of beta was largest, followed by those of gamma, delta, and alpha ( Fig. 1b) . While there 67 are numerous determinants of plaque size, we hypothesized that changes in the receptor-binding 68 affinity, thermal stability, and replication rate of these viruses were likely factors. Based on the 69 previously reported receptor-binding affinity data described above, we examined the thermal stability 70 and replication rate of each of these VOCs. We first assessed the thermal stability of these variants in 71 culture media incubated at different temperatures over an eight-hour period. Their infectivity was then 72 5 measured using a focus-forming assay. Owing to the short incubation time following viral infection, 73 this assay allowed for more precise assessments of virion infectivity, as it is less affected by other 74 factors associated with viral replication. 75

Of the four VOCs, the beta variant was the most stable at 4, 24, and 37 °C. We then evaluated the 76 relative stability by measuring changes in their infectivity following prolonged incubation (2, 4, 8, 12, 77 and 24 h) in solutions at physiological temperatures using the focus-forming assay. As expected, beta 78 exhibited the highest thermal stability (Fig. 1d) , with a half-life approximately twice that of gamma or 79 delta (Fig. 1e) . These results indicated a correlation between thermal stability and plaque size in all 80 variants except alpha. Interestingly, despite the small plaque size of alpha, its half-life was relatively 81 long, suggesting an alternative mechanism. 82 Next, we examined the viral replication rates of these VOCs. Vero E6 cells were infected with VOCs 83 at the same multiplicity of infection (MOI: 0.1). Plaque forming assay and qRT-PCR were then used 84 to assess infectious viral particle numbers and viral RNA concentrations, respectively. Alpha had fewer 85 infectious viral particles than the other variants, although there were no significant differences in viral 86 RNA copy number (Fig. 1f, 1g) . Moreover, the intracellular viral RNA concentrations of alpha and 87 gamma were significantly lower than those of the other VOCs (Fig. 1h) . These results indicate that the 88 viral replication rate of alpha is likely low, contributing to its small plaque size. 89

It is important to analyze these VOCs in terms of both their clinical pathology and virology, as this 90 will help us better understand their increased virulence. For example, plaque size may be associated 91 with contagiousness or viral transmission. Here, we noted that most of our VOCs presented with 92 changes in plaque size. In addition, these changes were largely mirrored by changes in virion stability 93 at physiological temperatures and the concentration of infectious viral particles. Correlation analyses 94 suggested that there is a strong link between plaque size and thermal stability in these VOCs and that 95 the relatively large plaque size of beta may account for its increased thermal resistance. This increased 6 stability may contribute to its pathobiology and transmission, requiring further studies of the viral titer 97 and case fatality rate in humans. Conversely, although alpha presented with increased thermal stability, 98 this did not translate to increased plaque size. This reduced plaque size may be explained by the 99 reduced number of infectious particles produced by alpha under these conditions. Further studies will 100 be required to identify additional determinants of plaque size, including functional mutations, 101 interactions with host factors, and environmental composition. In addition, variants of interest and 102 other variants should also be investigated to effectively control their spread. Viral titer determination Plaque-and focus-forming assays were performed as previously reported 112 with some modifications (11). In brief, for the plaque assay, the virus was serially diluted in EMEM 113 supplemented with 2% FBS. The cell culture medium was removed from Vero E6 cells (1 × 10 5 per 114 24-well) one day prior to the assay, before the inoculum was transferred onto triplicate cell monolayers. 115

These cells were incubated at 37 °C for 1 h, and the inoculum was discarded before the infected cells 116

were overlaid with 1.8% carboxymethyl cellulose in MEM. Samples were incubated at 37 °C for 4 d 117 before they were fixed and stained using 0.05% crystal violet in 1% formaldehyde. Plaque counts and 118 size evaluations were completed using an ImmunoSpot analyzer (C.T.L). 119

For the focus-forming assay, Vero E6 cells (2 × 10 4 per 96-well) were infected with diluted inoculum 120 for 1 h and then placed in fresh medium for an additional 8 h, after which they were washed and fixed. 

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Cells were then stained with the N-specific antibody (Cat# 40143-R001, Sino Biological) and 122 secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (Cat# 170-6515, Bio-Rad). The 123 signal was then developed using an insoluble TMB substrate (Promega), and the number of infected 124 cells was counted using an ImmunoSpot analyzer. Intra-and extracellular viral RNA was quantified 125 using quantitative RT-PCR (QuantStudio 3, Applied Biosystems), which was completed using the one-126 step Prime script III qRT-PCR mix (Takara). Viral RNA was detected using a 2019-nCoV-N1 probe 127 (Cat#10006770, Integrated DNA Technologies). 128