Invest Clin 62(4): 371 - 377, 2021 https://doi.org/10.22209/IC.v62n4a07

Corresponding author: Flor H Pujol. Laboratorio de Virología Molecular, Instituto Venezolano de Investigaciones

Científicas, CMBC, Caracas ,Venezuela .Tel/Fax: +58-2125041623. E-mail: fhpujol@gmail.com

A simple method for detection of mutations

in amino acid 452 of the Spike protein

of SARS-CoV-2 using restriction enzyme

analysis.

Rossana C. Jaspe1, Yoneira Sulbaran1, Mariana Hidalgo2, Carmen L. Loureiro1,

Zoila C. Moros3, Domingo J. Garzaro1, Héctor R. Rangel1 and Flor H. Pujol1

1Laboratorio de Virología Molecular, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

2Laboratorio de Inmunoparasitología, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

3Laboratorio de Biología de Virus, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

Key words: COVID-19; SARS-CoV-2; Delta Variant of Concern; RFLP; rapid screening;

mutation L452R.

Abstract. Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI),

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

Mutations in the amino acid 452 of the Spike protein are particularly important

and associated with some of these variants: L452R, present in Delta VOC, and

L452Q, present in Lambda VOI. These mutations have been associated with

both increased infectivity and evasion of protective immune response. A search

on GISAID to detect the number of sequences harboring the L452R mutation

and the frequency of Delta VOC among them, showed that since August 2021,

most of these sequences belong to the Delta VOC. Restriction enzyme analysis

is proposed as a rapid method to detect L452R. A small amplicon from the

Spike gene was digested with MspI. A 100% concordance was observed between

digestion and sequencing results. The mutation L452Q can also be detected by

restriction analysis, allowing the identification of putative Lambda VOIs. The

proposed methodology, which allows screening of a great number of samples,

could provide a faster information on the prevalence of Delta VOC cases.

372 Jaspe et al..

Investigación Clínica 62(4): 2021

Un método simple para la detección de mutaciones en el

aminoácido 452 de la proteína de la Espiga del SARS-CoV-2,

usando análisis de enzimas de restricción.

Invest Clin 2021; 62 (4): 371-377

Palabras clave: COVID-19; SARS-CoV-2; Delta Variant of Concern; RFLP; detección

rápida; mutación L452R.

Resumen. Las variantes de preocupación o interés del SARS-CoV-2 (VOC

o VOI, por sus siglas en inglés), el coronavirus responsable de la COVID-19,

han surgido en varios países. Las mutaciones en el aminoácido 452 de la pro-

teína de la Espiga son particularmente importantes y están asociadas con al-

gunas de estas variantes: L452R, presente en la VOC Delta, y L452Q, presente

en la VOI Lambda. Estas mutaciones se han asociado con un aumento de la

infectividad y la evasión de respuesta inmunitaria protectora. Una búsqueda

en GISAID para detectar el número de secuencias que albergan la mutación

L452R y la frecuencia de la VOC Delta entre ellas, mostró que desde agosto de

2021, la mayoría de estas secuencias pertenecen a la VOC Delta. Se propone

el análisis de enzimas de restricción como un método rápido para detectar

L452R. Se digirió un pequeño amplicón de un fragmento del gen de la espiga

con MspI. Se observó una concordancia del 100% entre la identificación de

la mutación a través de la digestión y los resultados de la secuenciación. La

mutación L452Q también se puede detectar mediante análisis de restricción,

lo que permite la identificación de posibles VOI Lambda. La metodología

propuesta, que permite el cribado de un gran número de muestras, podría

contribuir a proporcionar más información sobre la prevalencia y a detectar

rápidamente los casos de la VOC Delta.

Received: 05-09-2021 Accepted: 26-10-2021

INTRODUCTION

The COVID-19 pandemic is caused by

an emerging coronavirus, SARS-CoV-2, and

has caused more than 200 million cases

and more than 4 million deaths worldwide.

This virus belongs to the family Coronaviri-

dae. The tremendous number of replication

events that this virus has experienced, in

addition to an elevated frequency of recom-

bination, and the probable action of host

deaminases on the viral genome (1), has al-

lowed the emergence of many mutations in

the viral genome (2).

Different variants (lineages of viruses

sharing particular types of mutations) have

emerged since the end of 2020. Some of

these variants have been defined as of Inter-

est (VOI) or Concern (VOC) by WHO, asso-

ciated with more transmissibility, or partial

resistance to protective immunity, among

other characteristics. The variants with con-

firmed increased capacities are named VOC

(3-7). There are at present four VOCs: vari-

ant Alpha which emerged in the UK, vari-

ant Beta in South Africa, variant Gamma in

Brazil and variant Delta in India. Genomic

surveillance is recommended for monitoring

Rapid detection of SARS-CoV-2 mutations in amino acid 452 373

Vol. 62(4): 371 - 377, 2021

the introduction of SARS-CoV-2 Variants of

Concern (VOCs) in each country (6,7).

Multiple mutations have emerged at

amino acid 452 of the Spike protein, par-

ticularly L452R and L452Q. L452R was first

described in variants from California US at

the end of 2020 (Epsilon Variant), and has

now been found in several lineages (8), no-

tably including Delta VOC (3). In the Fall of

2021, Delta VOC is predominating in many

countries, despite high vaccination cover-

ages (9-11). The Delta variant has been as-

sociated with higher viral load compared to

previous SARS-CoV-2 isolates (12), being

at least two-times more transmissible than

the original isolate first detected in Wu-

han (13), and possibly associated with an

increased severity (14). Delta VOC is now

predominating in many countries and is

thought to predominate in more countries

with time.

Thus, rapid detection of Delta VOC cas-

es can be particularly useful in a clinical set-

ting or for epidemiological purposes. L452R,

a characteristic mutation of Delta VOC, can

be detected by partial or complete genome

sequencing and also by real-time PCR with

commercial probes. Here we propose an al-

ternative method for rapid detection of this

mutation by restriction enzyme analysis,

which can also be applied to the L452Q al-

lele, present in other variants like Lambda

VOI (15).

MATERIALS AND METHODS

Sequences available at GISAID on Sep-

tember 25, 2021, were analyzed for the

presence of L452R, at https://www.gisaid.

org/phylodynamics/global/nextstrain/ and

https://www.epicov.org/. The number of se-

quences belonging to the Delta VOC among

the ones harboring this mutation was also

estimated.

A restriction enzyme analysis was devel-

oped to detect the presence of L452R mu-

tation. RNA from clinical samples positive

by qRT-PCR (classified upon sequencing as

wild-type, WT, or harboring L452R muta-

tion) was amplified with primers 76.1L and

76.8R as previously described (16). Five µL

of the amplicon were digested with 1 unit

of MspI for 1 hour at 37°C and then load-

ed 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 Macrogen Sequencing Service

(Macrogen, Korea). This study was approved

by the Bioethical Committee of IVIC.

RESULTS

All sequences available at GISAID on

September 25, 2021 were analyzed for the

presence of L452R. At GISAID a total of

1,269,918 sequences of SARS-CoV-2 har-

boring the mutation L452R were avail-

able in samples collected between January

1 2021 and August 31 2021. Fig. 1 shows

the frequency of Delta VOC sequences for

each month. Since June 2021, 98% of the

sequences harboring the L452R mutation

correspond to Delta VOC. Before this date,

Delta VOC accounted only for 44% of the

sequences harboring the mutation L452R,

while 36% of them were grouped in lineages

B.1.427 or B.1.429, known as Epsilon vari-

ant (8).

Fig. 2 shows the expected restriction

pattern of Wild Type (WT) samples and iso-

lates harboring mutations L452R or L452Q,

by using two restriction enzymes: MspI for

L452R and BsrI for L452Q: each enzyme

yields an additional restriction site in the

respective mutated sample. Fig. 2C shows

the digestion of the PCR-amplified product

with the MspI enzyme of two samples with

the L452R mutation and two WT samples. A

total of 56 samples were analyzed for their

restriction pattern with MspI enzyme, and

compared to the presence or not of the mu-

tation L452R in their sequence. A 100% con-

cordance was observed in the detection of

the L452R mutation between the two meth-

ods (Table I).

374 Jaspe et al..

Investigación Clínica 62(4): 2021

DISCUSSION

Delta VOC is becoming the predomi-

nant lineage of SARS-CoV-2 in many coun-

tries (8,17). The presence of one of its most

important mutation, L452R, does not nec-

essarily mean the presence of a Delta VOC

isolate, but, as shown in Fig. 1, this associa-

tion is at present very high, because of the

predominance of the Delta VOC in many

countries. This mutation is not the only one

responsible for the particularly high fitness

phenotype of Delta VOC: P681R, for exam-

ple, has been associated with a more fuso-

genic phenotype, conferring more pathoge-

nicity to virus harboring this mutation in

experimental animals (9, 18).

On the other hand, the presence of L452Q

does not mean either the identification of the

Lambda VOI, but in regions where this variant

circulates, it could be strongly correlated. Thus

a rapid 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 samples.

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

sequencing. It also allows analyzing samples

with suspicion of Delta VOC in a short time,

without the need for commercial kits, be-

fore getting sequencing results. On the oth-

Fig. 1. Number of sequences with L452R mutation available at GISAID from January to August, according to

the month of collection. The total number of sequences harboring the mutation L452R is shown for

each month (bars). The number of these sequences belonging to lineages B.1.617.2 is shown in dark

blue: the percent number is shown for each bar.

TABLE I

CONCORDANCE BETWEEN RESTRICTION

ENZYME ANALYSIS AND SEQUENCING

RESULTS.

Sequence analysis

Restriction analysis

L452 R452

Concordance

L452 33 0

100%

R452 0 23

A total of 56 samples were analyzed: 23 Delta, with

L452R, and 33 non-Delta, Variants Alpha, Gamma, and

Mu, and other lineages.

Rapid detection of SARS-CoV-2 mutations in amino acid 452 375

Vol. 62(4): 371 - 377, 2021

Fig. 2. Restriction analysis of amplicons with L452R or L452Q mutations. A. Sequence of the amplified

product showing the restriction sites which discriminate Wild-type (WT) or mutant (L452R or

L452Q) viruses. The use of these two enzymes generates a restriction pattern characteristic for each

situation (WT, L452R and L452Q). The restriction sites are underlined. The numbers in the align-

ment indicate the bp position in the PCR-amplified product. Nucleotides 79-81 code for the amino

acid L452 (CTG), R452 (CGG), or Q452 (CAG). B. Expected digestion pattern with MspI enzyme

and similar enzymes (restriction site CCGG) and with BsrI and similar enzymes (restriction site

ACTGG). With MspI digestion, WT amplicon generates a product with two close bands of 140-155,

while L452R mutated amplicon generates 3 bands: two close bands of 71-78 and one of 144 bp. With

BsrI digestion, WT amplicon generates an undigested product of 293 bp, while L452Q mutated am-

plicon generates two bands of 78 and 215 bp. C. Agarose gel electrophoresis of PCR-amplified pro-

ducts digested with MspI. The PCR-amplified products digested with the MspI enzyme were run with

molecular weight markers (1Kb plus DNA ladder): smaller bands are signaled (100, 200, and 300 bp).

376 Jaspe et al..

Investigación Clínica 62(4): 2021

er hand, once the presence of the variants

is confirmed by whole genome sequencing,

this method can be used for the rapid esti-

mation of their prevalence in different geo-

graphical regions. We previously reported a

restriction analysis to detect another impor-

tant mutations: E484K or E484Q in the RBD

of the Spike protein (16). This method was

particularly useful during the dissemination

of Gamma VOC in Venezuela (Jaspe, RC,

personal communication). We have already

experienced in our laboratory the usefulness

of these restriction analysis, which can be

also combined for detecting several VOCs.

ACKNOWLEDGEMENTS

This study was supported by Ministerio

del Poder Popular de Ciencia, Tecnología e

Innovación of Venezuela. We wish to thank

Dr. Ferdinando Liprandi for his suggestion

to performing this study and for critical

reading of the manuscript.

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