Invest Clin 63(3): 262 - 274, 2022 https://doi.org/10.54817/IC.v63n3a05

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

lar, Instituto Venezolano de Investigaciones Científicas, Caracas, Miranda, Venezuela. E-mail: fhpujol@gmail.com

Sub-lineages of the Omicron variant

of SARS-CoV-2: characteristic mutations

and their relation to epidemiological

behavior.

José Luis Zambrano1, Rossana C Jaspe2, Mariana Hidalgo3, Carmen L Loureiro2,

Yoneira Sulbarán2, Zoila C Moros1, Domingo J Garzaro2, Esmeralda Vizzi4,

Héctor R Rangel2, Ferdinando Liprandi4, and Flor H Pujol2

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

Instituto Venezolano de Investigaciones Científicas, Caracas, Miranda, Venezuela.

2Laboratorio de Virología Molecular, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Caracas, Miranda, Venezuela.

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

Instituto Venezolano de Investigaciones Científicas, Caracas, Miranda, Venezuela.

4Laboratorio de Biología de Virus, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas, Caracas, Miranda, Venezuela.

Key words: COVID-19; SARS-CoV-2; Omicron Variant of Concern; mutations; tropism.

Abstract. By the end of 2021, the Omicron variant of SARS-CoV-2, the

coronavirus responsible for COVID-19, emerges, causing immediate concern,

due to the explosive increase in cases in South Africa and a large number of

mutations. This study describes the characteristic mutations of the Omicron

variant in the Spike protein, and the behavior of the successive epidemic waves

associated to the sub-lineages throughout the world. The mutations in the

Spike protein described are related to the virus ability to evade the protec-

tion elicited by current vaccines, as well as with possible reduced susceptibil-

ity to host proteases for priming of the fusion process, and how this might be

related to changes in tropism, a replication enhanced in nasal epithelial cells,

and reduced in pulmonary tissue; traits probably associated with the apparent

reduced severity of Omicron compared to other variants.

Omicron variant 263

Vol. 63(3): 262 - 274, 2022

Sub-linajes de la variante Ómicron del SARS-CoV-2: mutaciones

características y su relación con el comportamiento

epidemiológico.

Invest Clin 2022; 63 (3): 262 – 274

Palabras clave: COVID-19; SARS-CoV-2; variante de preocupación Ómicron;

mutaciones; tropismo.

Resumen. A finales de 2021 surge la variante Omicron del SARS-CoV-2, el

coronavirus responsable de la COVID-19, causando preocupación inmediata,

debido al aumento explosivo de casos en Suráfrica, y a su gran cantidad de

mutaciones. Este estudio describe las mutaciones características de la variante

Ómicron en la proteína de la Espiga (S) y el comportamiento de las sucesivas

olas epidémicas asociadas a la circulación de sus sub-linajes en todo el mundo.

Las mutaciones en la proteína S descritas están relacionadas con su capacidad

para evadir la protección provocada por las vacunas actuales, así como su posi-

ble susceptibilidad reducida a las proteasas del hospedero para la preparación

del proceso de fusión. Se infiere cómo esto podría estar relacionado con su

cambio en el tropismo, con una replicación mayor en las células epiteliales

nasales y menor en el tejido pulmonar, rasgos probablemente asociados a su

aparente menor gravedad en comparación con otras variantes.

Received: 10-07-2022 Accepted: 22-07-2022

INTRODUCTION

SARS-CoV-2 infection, responsible for

the COVID-19 pandemic, has caused more

than 550 million cases and more than 6

million deaths worldwide until June 2022.

This virus belongs to the family Coronaviri-

dae. This family comprises enveloped virus-

es, with a positive sense genome of around

30,000 nt. In the case of SARS-CoV-2, the

genome codes for four structural proteins

(nucleocapsid or N, spike or S, membrane

or M and envelope or E), 15 non-structural

proteins and eight accessory proteins. The S

protein contains two regions: S1, which in-

cludes the receptor-binding domain (RBD),

and S2, with the furin-cleavage site and the

fusion peptide. RBD, specifically the receptor

binding motif (RBM), is the region respon-

sible for the attachment to the angiotensin-

converting enzyme 2 (ACE2) cellular recep-

tor (Fig. 1)1,2. Two other putative receptors

(Asialoglycoprotein Receptor 1 - ASGR1- and

KREMEN1) have been described recently for

SARS-CoV-2, which are not used by the previ-

ous SARS-CoV. The virus appears to interact

with these two additional receptors through

the RBD and also with N-terminal domain of

the S1 region3,4. These new candidates add

to the list of other potential ligands that

may interact with SARS-CoV-25,6.

During these two years of a high rate

of replication, this virus has accumulated

several mutations, allowing for its clas-

sification in more than 2000 lineages by

June 20227-9. Some of these lineages were

denominated as variants by WHO10. These

variants (lineages of viruses sharing partic-

ular types of mutations) emerged since the

end of 2020, and were defined as Variants

Under Monitoring (VUM), Variants of Inter-

est (VOI), and Variants of Concern (VOC),

264 Zambrano et al.

Investigación Clínica 63(3): 2022

when any different phenotypic trait, such as

increased transmissibility or immune eva-

sion, among others, was suspected for the

two firsts and confirmed for the last10. Until

June 2022, five VOCs were described: Al-

pha variant, which emerged in the UK (lin-

eage B.1.1.7), Beta variant in South Africa

(B.1.351), Gamma variant in Brazil (P1),

Delta variant in India (B.1.617.2), and Omi-

cron variant, first identified in South Africa

(B.1.1529). The last one was included in

the list of VOCs very soon after its identi-

fication. By the end of June, only the Omi-

cron VOC and its sub-lineages were present

as a VOC10.

This variant caused immediate concern,

due to the explosive increase in cases in

South Africa11, and the large number of mu-

tations exhibited by this new lineage. In this

study, we describe the characteristic muta-

tions of the Omicron variant of SARS-CoV-2,

and the behavior of the epidemic waves as-

sociated with the dissemination of the differ-

ent sub-lineages throughout the world.

MATERIALS AND METHODS

Description of the mutations charac-

teristic of each sub-lineage of Omicron.

The identification of characteristic muta-

Fig. 1. Mutations of the SARS-CoV-2 Omicron VOC sub-lineages. Venn diagram showing common mutations

between the SARS-CoV-2 Omicron VOC sub-lineages.

Omicron variant 265

Vol. 63(3): 262 - 274, 2022

tions of the five sub-lineages of Omicron

was performed using the following data-

bases: SARS-CoV-2 lineages (Cov-lineages.

org), Outbreak.info and, GISAID EpiFlu™

(https://www.gisaid.org/). The Venn dia-

gram was made in Bioinformatics & Systems

Biology UGent/VIB (bioinformatics.psb.

ugent.be), and edited in Illustrator CC (ver.

23.0.1).

Analysis of the frequency of Omicron

VOC in the world from November 2021 to

May 2022. The number of sequences of Omi-

cron VOC available at the GISAID database

(https://www.gisaid.org/) on June 26, 2022

was compared to the total number of se-

quences foe each month, between November

2021 and May 2022.

Frequency of Omicron VOC sub-lineag-

es in the world. The number of sequences

reported for each Omicron VOC sub-lineage,

together with the date of the first and peak

detection, were analyzed from “SARS Cov2-

Lineages.” https://cov-lineages.org/lineage_

list.html9, and accessed on June 26, 2022.

Analysis of the epidemic waves caused

by the Omicron VOC in different coun-

tries. The peak number of cases caused by

the circulating Omicron VOC was analyzed

in selected countries, and compared with

the previous highest peak of cases occurring

in the same country, using the Worldometer

database (https://www.worldometers.info/

coronavirus/, accessed on July 1, 2022).

RESULTS

Key mutations of the Omicron variant

The Omicron variant exhibits a large

number of mutations in its genome, par-

ticularly in the S protein (Fig. 1). Five sub-

lineages of the Omicron variant have already

been identified, which exhibit some differ-

ences with the original B.1.1.529 lineage

and between them (Figs. 1 and 2). Addition-

ally, a recombinant BA.1/BA.2 sub-lineage

has also been described (Table 1).

The number of mutations exhibited by

the different sub-lineages of Omicron VOC

ranges from 22 for BA.3 to 57 in BA.4. Key

mutations of the S protein and present in al-

most all the sub-lineages are shown in Fig.

2. The S371L, K417N, and N440K muta-

tions, although not present in all the BA.1

isolates, are however also frequently found,

according to a search in GISAID database

(data not shown) In the case of BA.3, many

key mutations present in the other lineages

of Omicron, such as D405N, K417N, N440K,

T478K, E484A and N501Y, were found in

most sequences of lineage BA.3 available in

the GISAID database (data not shown).

Of particular relevance is the absence

of the deletion of the amino acids 69 and

70 in the S protein in the BA.2 sub-lineage,

which instead possesses a deletion of the

amino acids 25 to 279 (Fig. 1). Another im-

portant mutation acquired by the BA.4 and

Fig. 2. Key mutations in the different Omicron VOC sub-lineages.

266 Zambrano et al.

Investigación Clínica 63(3): 2022

BA.5 sub-lineages is the L452R mutation

(Fig. 2), a key mutation in the Delta VOC,

which was previously identified, particularly

in some Californian variants. The sub-lin-

eage BA.2.12.1 carries the mutation L452Q,

previously described in the C.37 VOI9.

Omicron dissemination and sub-lineages

Soon after its emergence, the Omicron

VOC displaced very rapidly the other lineag-

es circulating in the world, mainly the Delta

VOC (Fig. 3). At the end of June 2022, the

Omicron VOC was the only VOC recognized

by the WHO, although some Delta VOC iso-

lates were still described in the GISAID da-

tabase.

At the end of June 2022, the most fre-

quent lineages found in several parts of the

world were the sub-lineages BA.2.12.1, BA.4

and BA.5. These 3 lineages are expected to

circulate abundantly in the next months of

2022. Very few sequences of sub-lineage BA.3

were available at GISAID database (Table 1).

Table 2 shows some representative pat-

terns of the epidemic waves caused by the

Omicron VOC and its successive sub-linea-

ges. Generally, the Omicron VOC dissemi-

nation caused an epidemic wave of unprece-

dented magnitude, when compared to the

previous waves. Exceptions to this are India

and Iran, for which the ratio of Peak number

of cases with Omicron VOC/Peak number

of cases before Omicron VOC (PNO/PNBO)

was below 1 (Table 2). Additionally, this ratio

is highly variable between countries, being

generally lower in American countries, com-

pared to European ones, for example. The

second epidemic wave of Omicron VOC, due

to the dissemination of sub-lineages such as

BA.2, BA.4 and BA.5, was observed earlier in

many countries of Europe, while in America,

for example, some countries were suffering

an ongoing peak at the end of June 2022

(Table 2).

Several factors may have affected the

analysis. The testing capacity of each cou-

ntry, which ranged in July 1, 2022, from

five tests/million inhabitants in Algeria,

to 21,890,462 tests/million inhabitants in

Denmark (https://www.worldometers.info/

coronavirus/), may have limited more accu-

rate estimation of cases during the epidemic

waves, particularly during the Omicron VOC

one. With the highest testing capacity, Den-

mark exhibited a high PNO/PNBO of 11.54

(Table 2). The intensity of the Delta VOC

epidemic wave, which was particularly high

in some Asian countries, like India and Iran,

Table 1

Description of Omicron VOC sub-lineages.

Omicron main

lineage

Sub-lineages1Date of first

detection

Date of peak

detection2

Number of

sequences3

BA.1 50 (54) 2/9/21 4/1/22 2166293

BA.2 53 (108) 11/10/21428/3/22 1625851

BA.3 2 23/11/21 3/2/22 745

BA.4 1 (4) 10/1/22 25/5/22 13334

BA.5 2 (15) 5/1/22 30/5/22 27560

XE51 19/1/22 28/3/22 2438

Information from (9), accessed on June 26, 2022.

1 The effective number of sub lineages is shown, with the number of proposed ones indicated under parentheses.

2Date when the highest number of sequences were available was June 26, 2022. This date may change,

particularly for lineages BA.4 and BA.5, which are more actively disseminating in June 2022.

3The number of total sequences tentatively assigned to this lineage is shown.

4An earlier date of detection is reported for sub-lineage BA.2.3 (29/9/21).

5Recombinant BA.1/BA.2.

Omicron variant 267

Vol. 63(3): 262 - 274, 2022

led to a reduction of this ratio in these cou-

ntries, probably combined with saturated

testing capacities. PNO/PNBO is of parti-

cular interest in countries like South Korea

(83.58), Australia (59.61) and New Zealand

(unassigned since no peak in the number of

cases was observed before the Omicron wave

(Table 2). In Colombia and Venezuela, for

example, the PNO/PNBO was close to 1 (Ta-

ble 2). In addition to possible testing satura-

tion during the Omicron VOC wave in these

countries, the lower severity associated with

this variant, particularly in young persons,

may have reduced the demand for testing in

some groups.

DISCUSSION

Soon after its identification, the Omi-

cron variant was declared as a VOC, especia-

lly due to the huge number of mutations it

harbors11. Of the more than 30 mutations

present in the Omicron variant in the S pro-

tein, around half of them are located in the

RBD12. Some of these mutations can be used

detecting of this VOC by rapid methods13. Se-

ven produce a modest increase in the affinity

of the RBD for the ACE2 receptor: Q493R,

N501Y, S371L, S373P, S375F, Q498R, and

T478K12,14. It has been shown that Omicron

VOC can infect murine cell lines15. At least

two of the substitutions responsible for the

increase in affinity for the human ACE2 re-

ceptor are also responsible for the increa-

se in binding to mouse ACE2: Q493R and

Q498R15,16. Furthermore, the molecular

spectrum of Omicron-acquired mutations

was significantly different from the spectrum

for viruses that evolved in human patients.

However, it resembles the spectra associa-

ted with virus evolution in a mouse cellular

environment. These observations lead to the

suggestion that the progenitor of Omicron

might have jumped from humans to mice,

accumulating mutations conductive to in-

fecting that host, then jumping back into

humans, indicating an interspecies evolutio-

nary trajectory for the Omicron outbreak15.

Thus, this increased propensity for reverse

zoonosis makes this variant more likely than

previous variants to establish an animal re-

servoir of SARS-CoV-2.

Recent studies show that Omicron VOC

exhibit a replication enhanced in human

primary nasal epithelial cells, and reduced

in pulmonary cells17-20. This trait appears

to be related to the fact that, unlike other

SARSCoV-2 variants, Omicron is capable of

efficiently entering cells in a TMPRSS2-in-

dependent manner, via the endosomal rou-

te. This enables Omicron to infect a greater

number of cells in the respiratory epithe-

lium, allowing it to be more infectious at

lower exposure doses and resulting in en-

hanced transmissibility17. Syncytia forma-

tion depends on the fusogenic activity of the

S2 region of the S protein after cleavage by

the TMPRSS2. The Omicron also forms fewer

syncytia than other lineages20,21. The authors

suggest that this phenotypic trait may be

due to the presence of the mutation N969K.

The role of syncytia formation in the patho-

genic effect of SARS-CoV-2 remains a mat-

ter of debate22. Other substitutions, such as

N679K and H655Y, may be contributing to

Fig. 3. Frequency of Omicron VOC around the world

from November 2021 to May 2022. Green:

Omicron VOC. Red: other lineages, mainly

Delta VOC. From GISAID (https://www.gisaid.

org/accessed on June 26, 2022). In November

2021, only 3217 sequences of Omicron were

available out of 810820 total sequences.

268 Zambrano et al.

Investigación Clínica 63(3): 2022

this particular phenotype in Omicron VOC.

The N679K substitution is located in the

QTQTN motif upstream of the furin cleavage

site (QTQTK in the Omicron VOC). The clea-

vage site is known to play an important role

in viral pathogenesis. Its deletion attenuates

viral replication in respiratory cells in vitro

and attenuates disease in vivo22. The subs-

titution N679K in the QTQTN motif of the

Omicron VOC has been shown to reduce the

affinity of the protein for furin, resulting in

a reduced susceptibility to serine proteases

on the cell surface for entry23,24. The substi-

tution H655Y, in contrast, has been shown to

enhance viral replication and spike protein

cleavage25.

One of the most striking characteris-

tics of the Omicron VOC is its ability to eva-

de protective immunity induced by previous

infection or vaccination26,27. In fact, it is be-

lieved that the increase in transmissibility of

this variant is primarily due to its ability to

Table 2

Omicron VOC epidemic waves in selected countries.

Country Pop

(M hab)

Cases1

(M)

PNBO2PNO3PNO

/PNBO

SOP4

America

USA 334.9 89.4 (1) 310,438 (8/1/21) 908,909 (14/1/22) 2.93 140,047 (23/6/22)

Brazil 215.6 32.4 (3) 115,041 (23/6/21) 282,050 (3/2/22) 2.45 75,106 (30/6/22)

Argentina 45 9.4 (13) 41,080 (27/5/21) 139,853 (14/1/22) 3.40 8,314 (26/5/22)

Colombia 52 6.2 (18) 33,594 (26/6/21) 35,575 (15/1/22) 1.06 3,403 (28/6(22)

Mexico 131.6 6 (20) 28,953 (19/8/21) 60,552 (20/1/22) 2.09 32,216 (30/6/22)

Chile 19.4 4 (31) 9151 (9/4/22) 39,840 (10/2/22) 4.35 13,104 (17/5/22)

Venezuela 28.3 0.5 (86) 1,786 (4/4/21) 2,646 (30/1/22) 1.48 199 (24/6/22)

Africa

South Africa 60.8 4 (30) 26,645 (3/7/21) 37,875 (12/12/22) 1.42 10,017 (11/5/22)

Asia

India 1,407 43.5 (2) 414,433 (6/5/21) 347,254 (20/1/22) 0.88 19118 (30/6/22)

South Korea 51.3 18.4 (9) 7434 (17/12/21) 621,328 (17/3/22) 83.58 10,437 (29/6/22)

Iran 86.1 7.2 (17) 50,228 (17/8/21) 39,819 (7/2/22) 0.79 4,615 (5/4/22)

Hong Kong 7.6 1.2 (55) absent 79,876 (3/3/21) +++ 2,352 (30/6/22)

Europe

France 65.6 31.1 (4) 83,321 (7/11/20) 501,635 (25/1/22) 6.02 217,480 (29/3/22)

UK 68.6 22.7 (6) 83,088 (30/12/20) 275,618 (5/1/22) 3.32 109,320 (22/5/22)

Italy 60.3 18.6 (7) 41,386 (13/11/20) 228,740 (18/1/22) 5.53 100,823 (20/4/22)

Denmark 5.8 3 (39) 4,508 (18/2/20) 52,009 (10/2/22) 11.54 2,407 (28/6/22)

Oceania

Australia 26.1 8.2 (16) 2,528 (9/10/21) 150,702 (13/1/22) 59.61 67,379 (30/3/22)

New Zealand 5 1.3 (53) absent 24,106 (2/3/22) +++ 8,331 (28/6/22)

1Cases: Total number of cases in millions (M) until July 1, 2022 (position in the ranking of cases).

2PNBO: Peak number of cases before Omicron VOC (date). 3PNO: Peak number with Omicron VOC (date).

4SOP: Secondary Omicron VOC peak (date).

Omicron variant 269

Vol. 63(3): 262 - 274, 2022

evade previous protective immunity rather

than to of a higher affinity of its S protein

for the ACE2 receptor28. Several mutations

of Omicron VOC could impair neutralizing

antibodies (Nabs) with different specifici-

ties. Specifically, mutations K417N, G446S,

E484A, and Q493R induce escape of NAbs

in Group A-D, whose epitope overlaps with

the ACE2-binding motif. Group E (S309

site) and F (CR3022 site) NAbs, which of-

ten exhibit broad Sarbecovirus neutralizing

activity, are still somehow effective against

the Omicron VOC, but mutations G339D,

N440K, and S371L may induce resistance to

a subset of Nabs27.

The Omicron VOC also exhibits an inser-

tion of 3 amino acids (EPE) in the N-terminal

region of the S protein, at residue 214. This

insertion of EPE has not been observed in

other major variants of SARS-CoV-2 and may

induce a structural change, which might be

associated with a decrease in neutralizing

antibody binding ability to this region. This

genomic insertion has also been suggested to

be incorporated into SARS-CoV-2 by recombi-

nation with another coronavirus or even oth-

er respiratory pathogens29,30. The mutation

L452R present in sub-lineages BA.4 and BA.5,

may confer to these Omicron VOC sub-lineag-

es an increased immune evasion31,32, and may

also contribute to the dissemination of these

lineages. The sub-lineages more frequent in

June 2022, BA.2.12.1, BA.4 and BA.5, exhibit

an increased potential of breakthrough in-

fection after vaccination and infection, even

with a previous infection with another Omi-

cron VOC33-35.

In contrast, the sub-lineage BA.3 of the

Omicron VOC did not exhibit wide dissemi-

nation, compared to the other ones. This

may be related to the absence of many key

mutations exhibited by the other sub-lineag-

es, related to increased transmissibility and

immune evasion (Figs. 1 and 2). However,

the number of mutations found in this sub-

lineage is also variable among different iso-

lates, and some of those key mutations are

also frequently found.

Several lines of evidence suggest that

the Omicron VOC is highly more transmis-

sible than all previous variants, in addition

to the reduced sensitivity to the immune

protection conferred by vaccines and previ-

ous infections. The effective and basic re-

production numbers of the Omicron variant

have been estimated to be 3.8 and 2.5 times

higher than that of the Delta variant, respec-

tively, which was the previous VOC display-

ing the highest reproduction numbers36.

From this significant increase in trans-

missibility and resistance to previous immu-

nity, it could be inferred at a first glance,

that the clinical course of the Omicron VOC

might be more severe. However, the changed

tropism of this variant, to a virus infecting

more the upper respiratory airway, com-

pared to the lungs, suggests that is the con-

trary. Indeed, several studies point to a less

severe clinical course of infection with the

Omicron VOC, compared to the infection

caused by the Delta VOC.

The first evidence comes from in vitro

and animal model infections, where the Omi-

cron VOC exhibited an attenuated pheno-

type37. The early studies of the Omicron VOC

in South Africa also suggested a reduced odd

of severity for the patients infected with this

VOC38. This trend was confirmed in a retro-

spective observational study39. The same ob-

servation was found among US Veterans40.

Children in Israel were less prone to develop

multisystem inflammatory syndrome (MISC),

a common sequel of COVID-19 in children,

during the Omicron VOC wave, than during

the Delta and Alpha VOC waves41.

The emergence of Omicron VOC and

its sub-lineages led to an abrupt epidemic

peak worldwide42. However, differences in

the intensity of the epidemic peak caused

by the Omicron VOC were however observed

between countries. For example, countries

such as South Korea, Australia, and New Zea-

land were characterized by a successful con-

trol of COVID-19 during the pandemic43-45,

until the Omicron VOC wave. The increased

transmission ability of the Omicron VOC al-

270 Zambrano et al.

Investigación Clínica 63(3): 2022

lowed it to spread widely even in countries

with previous highly successful control mea-

sures. Finally, the great disparities in vac-

cination coverage between countries46, also

had a profound influence on the intensity of

the Delta and Omicron VOC waves.

In conclusion, the Omicron VOC displays

a great number of mutations, some of them

already present in previous lineages. Although

associated with a high attack rate and im-

mune evasion, this variant seems to be associ-

ated with lower severity, compared to previous

ones. A probable explanation for this somehow

attenuated phenotype could be related to its

resistance to the cleavage of TMPRSS2 and

probably furin, and its reduced ability to form

syncytia, leading to increased tropism for epi-

thelia over the lungs. The reduced effectivity

observed for various vaccines to protect against

infection with this variant has not hampered

its effectiveness in reducing the morbidity

and mortality of COVID-19, particularly with a

booster dose, which still induces neutralizing

antibodies47. The well-known non-pharmaco-

logical preventive interventions (mask, social

distancing, ventilation and frequent hand hy-

giene), together with vaccination, are still the

most effective measures to reduce the burden

of this highly contagious variant.

Funding

This study was supported by IVIC, Min-

isterio del Poder Popular de Ciencia, Tec-

nología e Innovación de Venezuela.

Conflict of interest

The authors declared that they have no

competing interests.

Authors contribution

Design of the study and writing of the

manuscript: JLZ, RCJ, FL, FHP. Data analy-

sis: JLZ, RCJ, MH, YS, CLL, ZCM, DJG, EV,

HRR, FHP. All the authors read and approved

the final version of the manuscript.

Author’s ORCID numbers

• José Luis Zambrano:

0000-0001-9884-2940

• Rossana C Jaspe:

0000-0002-4816-1378

• Mariana Hidalgo:

0000-0002-8307-8254

• Yoneira Sulbarán:

0000-0002-3170-353X

• Carmen L Loureiro:

0000-0003-3665-1107

• Zoila C Moros:

0000-0001-6322-9230

• Domingo J Garzaro:

0000-0002-9956-5786

• Esmeralda Vizzi:

0000-0001-6865-1617

• Héctor R Rangel:

0000-0001-5937-9690

• Ferdinando Liprandi:

0000-0001-8084-8252

• Flor H Pujol:

0000-0001-6086-6883

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