Invest Clin 62(Suppl. 2): 18 - 26, 2021 https://doi.org/10.22209/IC.v62s2a02

Corresponding author: Flor H Pujol. Laboratorio de Virología Molecular, Centro de Microbiología y Biología

Celular, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. Tel/Fax: +58-2125041623.

E-mail: fhpujol@gmail.com

Importance of mutations in amino acid 484

of the Spike protein of SARS-CoV-2: rapid

detection by restriction enzyme analysis

Rossana C Jaspe

Yoneira Sulbarn

, Carmen L Loureiro

, Pierina D´Angelo

Lieska Rodríguez

, Domingo J Garzaro

, Héctor R Rangel

and Flor H Pujol

Laboratorio de Virología Molecular, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

Instituto Nacional de Higiene “Rafael Rangel”, Caracas, Venezuela.

Key words: COVID-19; SARS-CoV-2; Variants of Concern (VOC); RFLP; rapid screening;

mutations.

Abstract. Variants of Concern of SARS-CoV-2 (VOCs), the new coronavirus

responsible for COVID-19, have emerged in several countries. Mutations in the

amino acid 484 of the Spike protein are particularly important and associated

with some of these variants: E484K or E484Q. These mutations have been as-

sociated with evasion to neutralizing antibodies. Restriction enzyme analysis is

proposed as a rapid method to detect these mutations. A search on GISAID was

performed in April 2021 to detect the frequency of these two mutations in the

sequence available and their association with other lineages. E484K, present in

some VOCs, has emerged in several other lineages and is frequently found in re-

cent viral isolates. A small amplicon from the Spike gene was digested with two

enzymes: HpyAV, and MseI. The use of these two enzymes allows the detection of

mutations at position 484, and to differentiate between these three conditions:

non-mutated, and the presence of E484K or E484Q. A 100% correlation was

observed with sequencing results. The proposed methodology, which allows for

the screening of a great number of samples, will probably help to provide more

information on the prevalence and epidemiology of these mutations worldwide,

to select the candidates for whole-genome sequencing.

Rapid detection of SARS-CoV-2 mutations 19

Vol. 62(Suppl. 2): 18 - 26, 2021

Importancia de las mutaciones en el amino acido 484

de la Espiga del SARS-CoV-2: identificación rápida

por análisis con enzimas de restricción

Invest Clin 2021; 62 (Suppl. 2): 18-26

Palabras clave: COVID-19; SARS-CoV-2; Variantes de preocupación (VOC); RFLP;

detección rápida; mutaciones.

Resumen. En varios países han surgido variantes de preocupación del

SARS-CoV-2 (VOC), el nuevo coronavirus responsable de la COVID-19. Las mu-

taciones en el aminoácido 484 de la proteína de la Espiga (S) son particular-

mente importantes y están asociadas con algunas de estas variantes: E484K o

E484Q. Estas mutaciones han sido asociadas a evasión de la respuesta de anti-

cuerpos neutralizantes. Se propone el análisis con enzimas de restricción como

un método rápido para detectar estas mutaciones. En abril de 2021 se realizó

una búsqueda en GISAID para detectar la frecuencia de estas dos mutaciones en

la secuencia disponible y su asociación con otros linajes. La mutación E484K,

presente en algunas VOCs, ha surgido en varios otros linajes y se encuentra con

mayor frecuencia en aislados virales recientes. Se generó un producto ampli-

ficado de un fragmento pequeño del gen S, que fue digerido con dos enzimas:

HpyAV y MseI. El uso de estas dos enzimas permite detectar la mutación en la

posición 484 y diferenciar entre estas 3 condiciones: no mutado y la presencia

de E484K o E484Q. Se observó una correlación del 100% con los resultados de

la secuenciación. La metodología propuesta, que permite el cribado de un gran

número de muestras, probablemente ayudará a proporcionar más información

sobre la prevalencia y epidemiología de estas mutaciones en todo el mundo,

para seleccionar los candidatos para la secuenciación del genoma completo.

Received: 25-06-2021 Accepted: 10-07-2021

INTRODUCTION

In March 2020, the COVID-19 pandemic

was declared, caused by an emerging corona-

virus, SARS-CoV-2. One year and a half later,

this infection has caused almost 200 million

cases and almost 3 million deaths worldwide.

This virus belongs to the family Coronaviri-

dae, order Nidovirales. These viruses possess

an envelope that surrounds a helicoidal nu-

cleocapsid which packs a large continuous

RNA genome of almost 30,000 nt, which

codes for 4 structural proteins (nucleocapsid

or N, spike or S, matrix or M and envelope

or E), 16 non-structural protein and 8 addi-

tional accessory ones (Fig. 1). This viral order

is unique among the RNA viruses since the

viruses belonging to this order encode for an

exonuclease, which enables proof-reading ca-

pacity to the replication machinery, limiting

mutational events. However, the tremendous

number of replication events that this virus

has undergone, in addition to an elevated

frequency of recombination, and to the prob-

able action of host deaminases on the viral

genome (1), has allowed the emergence of

many point mutations and frequent deletions

in the viral genome (2).

20 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

Different variants (groups of viruses

sharing particular types of mutations) have

emerged at the end of 2020. Some of these

variants have been defined of Interest (VOI)

or Concern (VOC) by WHO: VOI harbor mu-

tations that may confer to these groups of vi-

ruses more transmissibility, or partial resis-

tance to protective immunity or treatment,

among others. When some of these charac-

teristics are demonstrated for a particular

VOI, this VOI is named VOC (3). The vari-

ant B1.1.7 (VOC α), for which an increase in

incidence after its emergence was observed

in the UK, variant B.1.351 (VOC β), with an

increase in prevalence in South Africa, vari-

ants B.1.1.28.1 or P.1 (VOC γ) and B.1.1.28.2

or P.2 (VOI), which are now predominant in

Brazil, and variants B.1.617.1 (VOI θ) and

B.1.617.2 (VOC δ) are examples of these

variants. The first name corresponds to the

lineage according to Pango lineages classi-

fication (https://cov-lineages.org/lineages.

html) and the Greek letter corresponds to

WHO classification (4-7). Genomic surveil-

lance has been proposed for monitoring the

introduction of SARS-CoV-2 Variants of Con-

cern (VOCs) in different countries (8,9).

Some of the mutations harbored by these

variants are of public health concern for two

main reasons: mutation N501Y (tyrosine sub-

stituting an asparagine), for example, might

lead to increased transmissibility of the vi-

ruses harboring this mutation, and mutation

E484K (lysine substituting a glutamic acid)

has been frequently associated with reinfec-

tion cases, and might reduce the neutraliz-

ing activity of antibodies produced by vacci-

nation (7,10). Both mutations are located in

the Receptor Binding Domain (RBD) of the

S protein (Fig. 1). Amino acid 484 of S is of

particular interest, since at least two impor-

tant mutations have emerged in this position:

E484K and E484Q. E484K is found in VOCs β

and γ, and sometimes in VOC α, while E484Q

is found in VOI κ.

A rapid method for detecting those mu-

tations might be useful for screening large

quantities of samples, in settings where se-

quencing facilities are limited. This would be

very useful for rapid and massive screening

of these variants. In this study, we propose a

simple restriction enzyme digestion analysis

for the detection of these two mutations in

the SARS-CoV-2 Spike: E484K and E484Q.

METHODOLOGY

Analysis of sequences available at GISAID

for E484K and E484Q mutations

Sequences available at GISAID in

April 2021 were analyzed for the presence

of E484K, E484Q and N501Y, at https://

Fig. 1. SARS-CoV-2 virion structure, genome. Structural proteins of SARS-CoV-2 are shown in the virion on

the left side of the figure. The genome of almost 30,000 nt is shown on the right side, with the diffe-

rent Open Reading Frames (ORFs) and the proteins produced. RBD is shown inside the S protein, as

well as the small PCR-amplified product used for restriction analysis.

Rapid detection of SARS-CoV-2 mutations 21

Vol. 62(Suppl. 2): 18 - 26, 2021

www.gisaid.org/phylodynamics/global/next-

strain/ and https://www.epicov.org/.

RT-PCR

This study was approved by the Bioethi-

cal Committee of IVIC. We previously detect-

ed in Venezuela, the circulation of variants of

the B.1.1.28.1 lineage (P1-like) and samples

harboring the E484K mutation without the

N501Y one, by sequencing part of the Spike

gene in Venezuelan isolates (Jaspe, RC, in

preparation). RNA from clinical samples

positive by qRT-PCR (classified as wild-type,

WT, or harboring E484K) was amplified with

primers 76.1L (5´-CCAGATGATTTTACAG-

GCTGCG-3´) and 76.8R (5´-GTTGCTG-

GTGCATGTAGAAGTTC-3´) (Fig. 1), using

SuperScript III One-Step RT-PCR System

with Platinum Taq High Fidelity DNA Poly-

merase (Thermo Fisher Scientific), and the

following PCR conditions: an incubation at

55°C for 30 min, followed by 94°C/3min

and 40 cycles of 94°C/15 sec, 55°C/30 sec

and 68°C/30 sec, with a final extension of

68°C for 7 min. Superscript II One-Step PCR

System with Platinum Taq DNA Polymerase

(Thermo Fisher Scientific), was also used

successfully.

Restriction analysis

Five µl of the amplicon were digested

with 1 unit of HpyAV or MseI for 1 hour at

37°C and then loaded in a 3% agarose gel

electrophoresis for band visualization with

Ethidium bromide. Restriction results were

compared with the sequence obtained by

sending PCR purified fragments to Macro-

gen Sequencing Service (Macrogen, Korea).

RESULTS

A total of 1,224,815 sequences of SARS-

CoV-2 were available on April 24 at GISAID.

Fig. 2 shows the frequency of sequences

harboring the N501Y, E484K, or both muta-

tions. However, this frequency does not re-

flect the true prevalence of these mutations

worldwide, since a strong bias exists, which

has been accentuating over the last months.

There are strong differences in genomic se-

quencing capacities between developed and

developing countries, and even between de-

veloped countries (Table I).

For example, UK is the country that has

provided the highest number of sequences

to GISAID, more than the USA, even if the

total number of COVID-19 cases is 7.4 times

lower than the ones in the USA. VOC of the

lineage B.1.1.7, which generally lacks the

E484K mutations, emerged in the UK and

is also frequent in the USA. In contrast, the

other two VOCs, which harbor E484K in ad-

dition to N501Y, emerged in Brazil and South

Africa, countries with robust sequencing ca-

pacities but not comparable to the countries

previously mentioned (Table I). This is the

main reason for the higher abundancy of

N501Y mutation, compared to E484K one

TABLE I

NUMBER OF SEQUENCES HARBORING E484K OR N501Y MUTATIONS IN SELECTED COUNTRIES.

Country COVID-19 cases Total sequences N501Y E484K

UK 4,403,170 382,862 212,371 1,398

USA 32,788,341 331,109 57,661 16,443

Germany 3,277,661 64,503 37,412 1,668

Brazil 14,308,215 5,827 1,063 1,872

South Africa 1,574,370 5,075 1,974 2,020

Total COVID-19 cases and sequences available at GISAID on April 24, 2021. The number

of sequences with the N501Y mutation is shown for comparison.

22 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

(Fig. 2). E484Q mutation is less commonly

found in SARS-CoV-2 isolates (4966 in total

until April 24, 2021). Even if the frequency

of the E484K mutation appears relatively

low in the sequences available at GISAID,

this mutation has been found associated

with many lineages (Table II).

Fig. 3 shows the restriction pattern of

Wild Type (WT) samples and isolates harbor-

ing mutations E484K. Digestion with HpyAV

of the 293 bp amplicon of the Spike partial

genomic region yielded two bands of 170 and

123 bp for samples with E484, while the di-

gestion site is abolished with K484 or Q484.

Digestion with MseI yielded a fragment of 183

and 110 for WT samples and Q484, and a frag-

ment of 174, 110, and 9 bp (this last one not

observed in the gel) for samples with K484.

The presence of E484Q mutation generates

a hybrid pattern between the two other situ-

ations: no digestion with HpyAV (as E484K)

and fragments of 183 and 110 as WT samples

when digested with MseI (Fig. 3A). The 9 bp

difference of the product between the WT and

E484K mutated samples was easily differen-

tiated in 3% agarose gels (Fig. 3B). NuSieve

agarose or polyacrylamide gel electrophore-

sis can also be used for more discrimination,

particularly of bands of 174 and 183 bp. How-

ever, this was not necessary in our hands.

A total of 40 samples (12 WT and 28

with E484K) were analyzed for their restric-

tion pattern and compared to the presence

or not of the mutation in their sequence.

A 100% correlation was observed in the de-

tection of the mutations between the two

methods (data not shown). No sample was

available harboring the E484Q mutation.

However, the analysis of the sequence with

the E484Q mutation allows inferring a dis-

tinct restriction pattern compared to WT

and E484K (Fig. 3B).

DISCUSSION

SARS-CoV-2 variants are emerging and

spreading rapidly in several parts of the

world (3). Mutations E484K and E484Q

have been associated with many of the VOCs

and may appear in other variants already un-

known. Indeed, a survey of sequences avail-

able at GIASID showed that these mutations,

particularly E484K, have been found in se-

quences for many lineages, suggesting that

this mutation is emerging frequently (Jaspe,

R.C. et al., in preparation) (11). As stated

Fig. 2. Frequency of sequences in GISAID (until April 2021) harboring N501Y, E484K mutations, or both.

The frequency of N501Y isolates is shown for comparison.

Rapid detection of SARS-CoV-2 mutations 23

Vol. 62(Suppl. 2): 18 - 26, 2021

before, the E484K mutation has been found

in cases of reinfection and may represent a

mutation that emerged to escape the pres-

ence of neutralizing antibodies (11,12). The

frequency of variants or mutations cannot

be estimated with the sequences available at

GISAID, since huge disparities exist between

the sequencing capacities among countries.

The presence of any of these mutations

does not necessarily represent that a VOC is

circulating in a specific country. Their pres-

ence does not necessarily mean that this

isolate will gain the phenotypic advantages

provided by these mutations in VOCs. Many

other mutations present in these VOCs (point

mutations and deletions) might be contribut-

ing to the enhanced transmissibility of these

VOCs (13). However, the specific detection

of these two mutations, which play a key role

in determining the phenotype of the isolate,

might be of particular interest. Thus a rap-

id method for identifying those mutations

should be very useful, particularly in settings

where massive whole genome sequencing is

not available. Rapid methodologies might be

used for the rapid screening of several sam-

ples. The whole protocol can be run in a day.

The proposed methodology allows ana-

lyzing a great number of samples to select

samples that may harbor mutations of con-

cern, before proceeding to whole genome se-

quencing. On the other hand, once the pres-

ence of the variants is confirmed by whole

genome sequencing, this method can be

used for the rapid estimation of their preva-

lence in different geographical regions.

TABLE II

LINEAGES WHERE E484K MUTATION CAN BE FOUND

Mutation Lineages where some or all sequences harbor this mutation

E484K Total lineages: 127

Lineages with less than

10 sequences with the

mutation (n=99)

A A.23 B B.1.1.1 B.1.1.10 B.1.1.101 B.1.1.115 B.1.1.16 B.1.1.161

B.1.1.214 B.1.1.216 B.1.1.220 B.1.1.241 B.1.1.25 B.1.1.270 B.1.1.273

B.1.1.297 B.1.1.316 B.1.1.317 B.1.1.322 B.1.1.34 B.1.1.348 B.1.1.351

B.1.1.365 B.1.1.38 B.1.1.393 B.1.1.398 B.1.1.406 B.1.1.412 B.1.1.434

B.1.1.461 B.1.1.482 B.1.1.486 B.1.1.51 B.1.1.515 B.1.1.519 B.1.1.57

B.1.110 B.1.111 B.1.131 B.1.134 B.1.139 B.1.160.26 B.1.177.10

B.1.177.12 B.1.177.31 B.1.177.4 B.1.177.40 B.1.177.44 B.1.177.51

B.1.177.62 B.1.177.77 B.1.177.85 B.1.177.86 B.1.214.3 B.1.218 B.1.220

B.1.221 B.1.234 B.1.237 B.1.241 B.1.243 B.1.258 B.1.265 B.1.279

B.1.281 B.1.311 B.1.313 B.1.314 B.1.349 B.1.36 B.1.36.10 B.1.361

B.1.369 B.1.384 B.1.395 B.1.396 B.1.409 B.1.411 B.1.416 B.1.429

B.1.441 B.1.486 B.1.526.1 B.1.526.2 B.1.527 B.1.538 B.1.555 B.1.564

B.1.573 B.1.577 B.1.595 B.1.609 B.1.612 B.1.620 C.11 C.22 L.3 P.1.1

Lineages with 10-100

sequences (n=18)

A.23.1 B.1.1 B.1.1.28 B.1.1.306 B.1.1.318 B.1.1.345 B.1.110.3 B.1.160

B.1.177 B.1.177.88 B.1.243.1 B.1.375 B.1.400 B.1.596 B.1.618 N.9 P.3 R.2

Lineages with more

than 100 sequences

(n=10)

B.1.2 (104) B.1.619 (167) B.1.1.207 (190) B.1.1.7* (378) B.1 (403) P.2

(1.896) R.1 (2.175) P. 1 (4.192) B.1.526 (6.011) B.1.351 (7.595)

The list of lineages was assessed in the GISAID. The lineages in bold refer to VOCs. The

number of lineages harboring the mutation is shown under parenthesis. This analysis includes

fewer sequences than the ones included in Fig. 2, since only sequences with high coverage

were analyzed. *Some isolates belonging to the B.1.1.7 have acquired the E484K mutation.

24 Cepeda et al.

Investigación Clínica 62(Suppl. 2): 2021

Fig. 3. Restriction analysis of amplicons with E484K or E484Q mutations. A. Sequence of the amplified pro-

duct showing the restriction sites which discriminate Wild-type (WT) or mutant (E484K or E484Q)

viruses. Predicted size of the restriction fragments produced by digestion with each enzyme and vi-

sible on agarose gel. The use of these two enzymes generates a restriction pattern characteristic for

each situation (WT, E484K and E484Q). The arrow indicates the position in bp of the restriction site.

The numbers in the alignment indicate the bp position in the PCR-amplified product (see Fig. 1).

Nucleotides 175-177 codes for the amino acid E484 (GAA), K484 (AAA), or Q484 (CAA). B. Agarose

gel electrophoresis of digested PCR-amplified products. The PCR-amplified products digested with

the enzyme (HpyAV or MseI) were run with molecular weight markers (1Kb plus DNA ladder): smaller

bands are signaled (100, 200, and 300 bp). *Samples with E484Q mutations were not available for

analysis but the predicted restriction pattern is shown.

Rapid detection of SARS-CoV-2 mutations 25

Vol. 62(Suppl. 2): 18 - 26, 2021

ACKNOWLEDGEMENTS

This study was supported by the Minis-

terio del Poder Popular de Ciencia, Tecnolo-

gía e Innovación of Venezuela.

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