Benedettietal. J Transl Med (2020) 18:329
Emerging ofaSARS-CoV-2 viral strain
Francesca Benedetti1,2†, Greg A. Snyder1,3†, Marta Giovanetti4, Silvia Angeletti5, Robert C. Gallo1,6,7,
Massimo Ciccozzi5* and Davide Zella1,2,7*
Background: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was ﬁrst detected in
Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed
that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV.
Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode
for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important
pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and
hampering the innate immune system response. Detailed experiments of site-speciﬁc mutagenesis and in vitro
reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of speciﬁc amino
acid residues of nsp1 and (b) the interaction between the protein and the host’s small ribosomal unit. In fact, substitu-
tion of certain amino acids resulted in reduction of its negative eﬀects.
Methods: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July
2020) from patients infected by SARS-CoV-2 from diﬀerent areas around the world. Genomes alignment was per-
formed using MAFFT (REFF) and the nsp1 genomic regions were identiﬁed using BioEdit and veriﬁed using BLAST.
Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER.
Results: We identiﬁed SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown dele-
tion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in
diﬀerent geographical areas. Structural prediction modelling suggests an eﬀect on the C-terminal tail structure.
Conclusions: Modelling analysis of a newly identiﬁed deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests
that this deletion could aﬀect the structure of the C-terminal region of the protein, important for regulation of viral
replication and negative eﬀect on host’s gene expression. In addition, substitution of the two amino acids (KS) from
nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe
indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) conﬁrm the spreading of
this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of
asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral
© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
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*Correspondence: M.firstname.lastname@example.org; Dzella@ihv.umaryland.edu
†Francesca Benedetti and Greg Snyder equally contributed to this work
1 Institute of Human Virology, School of Medicine, University of Maryland,
5 Medical Statistic and Molecular Epidemiology Unit, University
of Biomedical Campus, Rome, Italy
Full list of author information is available at the end of the article
Page 2 of 6
Benedettietal. J Transl Med (2020) 18:329
Severe Acute Respiratory Syndrome Coronavirus-2
(SARS CoV-2) belongs to the realm Riboviria, order
Nidovirales, suborder Cornidovirineae, family Coro-
naviridae, subfamily Orthocoronavirinae, genus Beta-
coronavirus (lineage B), subgenus Sarbecovirus, and
the species Severe acute respiratory syndrome-related
coronavirus, and is the virus responsible for the current
global pandemic [1–3]. e genome of SARS-CoV-2 
is highly homologous to the coronavirus that caused
the SARS epidemic in 2003, SARS-CoV [5, 6] and to the
coronavirus responsible for the Middle-Eastern Respir-
atory Syndrome, MERS-CoV .
Coronavirus Diseases (COVID-19) comprises symp-
toms reported by patients infected by SARS-CoV-2,
ranging from mild to severe, and some cases result in
death. Severe acute respiratory illness with fever and
respiratory symptoms, such as cough and shortness
of breath, are the primary case deﬁnition, but recently
patients without respiratory symptoms are becoming
more recognized, with manifestations such as gastro-
intestinal, olfactory, cardiovascular, and neurological.
Cases resulting in death are primarily middle-aged and
elderly patients with obesity and/or pre-existing dis-
eases (tumor surgery, cirrhosis, hypertension, coronary
heart disease, diabetes, and Parkinson’s disease) [8–11].
Given the similarity among the viruses, the data
about biological functions, characteristics and eﬀects
on the host of the proteins expressed by SARS-CoV-2
are mostly inferred by the previous studies on SARS-
CoV and other related human (e.g. MERS-CoV) [12–14]
and animal coronaviruses (e.g. mouse hepatitis virus)
. In SARS-CoV two large polyproteins, ORF1a and
ORF1ab, are cleaved by a speciﬁc protease to form 16
nonstructural proteins (nsp), four structural proteins,
namely spike (S), envelope (E), membrane (M), and
nucleocapsid (N), and eight accessory proteins: ORF3a,
ORF3b (absent in SARS CoV-2), ORF6, ORF7a, ORF7b,
ORF8a, ORF8b, and ORF9b (absent in SARS-CoV-2).
Experimental data indicate that some accessory proteins
are considered not essential for viral replication, while
others have been demonstrated to be important for
virus-host interactions both invitro and invivo [16, 17].
Among these proteins, SARS-CoV, nonstructural
protein 1, nsp1 also known as the leader protein, plays a
central role in hampering the anti-viral innate immune
response, in particular Interferon-alpha expression
, and it has been considered as a possible target
for therapeutic interventions aimed at reducing viral
pathogenicity . Further indicative of its preserved
biological function, nsp1 from alpha- and beta-CoVs
have diﬀerent size, but show comparable biological
activities in their ability to reduce host gene expres-
sion, even though the mechanism seems diﬀerent [15,
SARS-CoV nsp1 almost completely blocks host pro-
tein translation by binding the 40S ribosome of the host
cell, which stops canonical mRNA translation at diﬀer-
ent steps during the initiation process [23–25]. is in
turn results in template-dependent endonucleolytic
cleavage, followed by degradation of mRNAs of infected
cells, while viral mRNA shutdown is avoided through a
still not clear mechanism involving interaction between
nsp1 with a conserved 5′ untranslated region of the
SARS-CoV mRNA . By blocking expression of sev-
eral components of the innate immune system, including
the interferon response, SARS-CoV is thus able to main-
tain viral expression and escape immune system detec-
Critical for this mechanism are certain amino acid
residues of nsp1. For example, in the case of SARS-
CoV several residues have been identiﬁed that diﬀeren-
tially inhibit host gene expression, like interferon alpha,
responsible for antiviral activity . More recently, a
region in the C-terminal domain of nsp1 of SARS-CoV-2
has been demonstrated to interfere with host expression
Here we describe a deletion identiﬁed in the C-ter-
minal region of nsp1 observed in certain genomes from
SARS-CoV-2 patients, from diﬀerent areas of the word.
e deletion did result in removal of three amino acid
residues (KSF). Two of them (KS) have been shown to
be responsible for nsp1 of SARS-CoV partial attenua-
tion of both inhibition of signal transduction and inhibi-
tion of gene expression, including Interferon-alpha .
Our data indicate that a small percentage of SARS-CoV-2
viruses is actually harboring a deletion in an important
protein responsible for pathogenesis, possibly adapting
toward a decrease pathogenicity.
We analyzed 17,928 genomic sequences obtained from
the GISAID database (updated on 07/24/2020) derived
from patients infected by SARS-CoV-2 from diﬀerent
areas around the world. e genomes were collected
from December 2019 to July 2020. SARS-CoV-2 refer-
ence genome (RefSeq: NC_045512.2) was obtained from
Keywords: SARS-CoV-2, COVID-19, nsp1, Deletion, Pathogenic, Viral adaptation
Page 3 of 6
Benedettietal. J Transl Med (2020) 18:329
the GenBank database. Genomes alignment was per-
formed using MAFFT .
Nsp1 sequence belonging to SARS-CoV-2 were iden-
tiﬁed using BioEdit and veriﬁed by using BLAST .
Nsp1 protein of SARS-CoV-2 with and without dele-
tion have been subsequently modelled using I-TASSER
We identiﬁed genomic sequences, from speciﬁc Coun-
tries, carrying a deletion of 9 nucleotides in position
686-694, corresponding to AA position 241-243 (KSF)
(Fig. 1). e list of Countries with the related num-
ber of sequences available analyzed and the number of
sequences carrying the deletion is listed in Table1. e
Fig. 1 Nsp1 alignment between sequences from SARS-CoV-2 wild type and strains carrying the KSF deletion. The amino acid sequences of
SARS-CoV-2 wild type (WT) and SARS-CoV-2 with the 3 amino acids deletion (DEL) were aligned using Clustal Omega. The deletion is shown
Table 1 List ofCountries analyzed andnumber ofsequences examined which carry theamino acid deletion
Country Number ofsequences examined Number ofsequences carrying
Austria 387 0 0.00
Belgium 754 2 0.27
Brazil 630 4 0.63
Denmark 601 1 0.17
England 8300 37 0.45
France 378 1 0.26
Germany 230 0 0.00
Ireland 16 0 0.00
Israel 222 2 0.90
Italy 146 0 0.00
Netherland 1363 3 2.21
Portugal 501 1 0.22
Spain 1195 2 0.17
Sweden 527 10 1.90
Switzerland 401 0 0.00
Total 15,651 63 0.40
Utah 275 2 0.73
New York 1345 10 0.74
New Jersey 219 2 0.91
Connecticut 155 1 0.65
Texas 234 0 0.00
Nebraska 49 0 0.00
Total 2277 15 0.66
Page 4 of 6
Benedettietal. J Transl Med (2020) 18:329
overall presence of genomes carrying the deletion in the
cases analyzed was 0.44%, though it was not homogelouly
distributed. In fact, we did not found it in certain Coun-
tries, such as Italy, Germany and Austria., while in oth-
ers it was clearly present, for example in Sweden with 10
out of 527 genomes (1.90%), Israel (0.90), Brazil (0.63%)
and England (0.45%). Among the States analyzed in the
United States, we could detect it in New Jersey (0.91%),
New York (0.74), Utah (0.73), and Connecticut (0.65),
while we could not detect it in Texas and Nebraska. We
note that some of the areas where the deletion could not
be detected had a very low number of genomic sequences
available for analysis, making the negative results diﬃ-
cult to interpret. Furthermore, the dataset available did
not allow us to determine whether this deletion hap-
pened as a series of independent events in diﬀerent tem-
poral moments and geographical areas, as if the virus
has an intrisecally fragile site, or it emerged from a sin-
gle transforming event originating from a unique clus-
ter. More data are needed to diﬀerentiate between these
We next used I-TASSER to model nsp1 protein of
SARS-CoV-2 carrying the deletion. A structure compari-
son of nsp1 from SARS-CoV-2 models with and without
the deletion is represent in Fig.2. Cartoon depiction of
the nsp1 from SARS-CoV-2 with and without the dele-
tion show the superimposed core (AA1-127) and the
C-terminal tails (AA128-148) . e structure of the
C-terminal tail is unresolved in the NMR structure of
SARS-CoV (PDB code 2GDT) and this region is pre-
dicted to be highly ﬂexible and disordered, with a few
secondary helical elements predicted . Prediction
models for both nsp1 SARS-CoV and nsp1 SARS-CoV-2
indicate a possibility of a short helical secondary struc-
ture for KSY and KSF amino acids, respectively, and this
terminal tail was found to be very important for expres-
sion of nsp1 itself . e ﬂexibility, lack of struc-
ture and disorder in this region is speculated to allow
for availability of the protease recognition seuquence
between nsp1 and nsp2 . Indeed, the C-terminal tail
was found to be dispensable for MHV (murine hepatitis
virus) viral replication but necessary for proteolysis of
nsp1 and nsp2 . e newly described deletion of KSF
amino acids may inﬂuence potential secondary structure
in this region of SARS-CoV-2, thereby altering activity of
nsp1 interactions and consequent activity on viral pro-
tein and host’s gene expression regulation.
Our analysis shows the emergence of a deletion in nsp1,
one of the most important determinants of pathogenic-
ity of SARS-CoV-2. is is quite surprising, since corona
viruses typically experience a moderate rate of muta-
tions, due to the presence of a protein with proofreader
activity (ExonN, also called nsp14), calculated in about
26 mutations per year (https ://nexts train .org/ncov/globa
l?l=clock ). ough the number of sequences detected
was a small fraction of the total analyzed, our data clearly
identify a new SARS-CoV-2 viral strain present in sub-
jects from diﬀerent areas (Europe, North and South
America). However, our analysis also indicates that this
deletion is not homogeneously present in all the Coun-
tries analyzed. For this reason, it would be important to
monitor its presence over time, and to determine its pen-
etrance and probability to spread and compete with the
current viral strains. Nonetheless, our results suggest the
possibility of the evolution of a new viral quasi-specie,
but further data are necessary to conﬁrm this hypothe-
sis and explore the possibility of a developing intra-host
e new viral strain that we describe carries a deﬁning
characteristic deletion of 9 nucleotides in the C-terminal
region of the nsp1 gene, translating into a protein lack-
ing three amino acids (KSF). Substitution of two of these
amino acids (KS) reduced the inhibitory eﬀect of innate
immune response to SARS-CoV, and by predicted struc-
ture analysis we show that these amino acids compromise
proper folding of nsp1. Consequently, we hypothesize
that viruses harboring this deletion are likely to be less
pathogenic than commonly observed viral strains. To this
regard, we note that the two common endemic human
coronaviruses, HCoV-OC43  and HCoV-299E ,
have extensive deletions in the C-terminal region of nsp1.
ought crystallization and biological data are needed
to conﬁrm our hypothesis, our observations, together
Fig. 2 Comparison of NSP1 SARS-CoV and SARS-CoV-2. Comparison
of core structure with prediction models of full length nsp1 SARS-CoV
(cyan) and SARS-CoV-2 are superimposed in diﬀerent colors
(magenta and light pink). The prediction models for both C-terminal
tails of nsp1 SARS-CoV with KSY (blue) and nsp1-SARS-CoV-2 with
KSF present (blue) and KSF deleted (green) are predicted to be highly
disordered compared with nsp1 Core elements (yellow). R.M.S.D
is 0.78Å for core elements. Note that the core structure has been
previously resolved for SARS -CoV (PDB code 2GDT), while the C-tail
structure has not
Page 5 of 6
Benedettietal. J Transl Med (2020) 18:329
with the recent ﬁndings of two viral strains carrying in
one case an extensive deletion in the orf7a gene , a
deletion in the nsp2 gene  and deletions in nsp1
gene also identiﬁed by other groups [37, 38], indicate
that SARS-CoV-2 genome may be undergoing a signiﬁ-
cant evolutionary process, which may result in virus-
host adaptation . Since the overwhelming majority of
genomic sequences collected so far are from symptomatic
subjects, it seems logical to characterize in detail SARS-
CoV-2 genomes from the asymptomatic population. If
our hypothesis is correct, this is the proper population
where we should be able to identify more in detail fur-
ther viral evolutionary steps, which may indicate reduc-
tion of pathogenicity. Understanding the diﬀerent steps
that characterize the pathogenicity of this virus, as well
as the spreading and changes of these pathogenic deter-
minants among the population, may help determining
proper strategies of containment of SARS-CoV-2 spread
and identify better drugs for treatment of COVID-19.
We identiﬁed the emergence in infected subjects of a new
viral strain of SARS-CoV-2 with a deletion of 3 amino
acids (KSF) in the C-terminal region of nsp1. I-TASSER
structure analysis indicates that this deletion may aﬀects
the structure of the C-terminal region, important for
regulation of nsp1 activity. Substitution of two of these
amino acids (KS) was also previously reported to revert
the loss of interferon-alpha expression in cells trans-
fected with mutated nsp1 from SARS-CoV. is deletion
in nsp1, together with deletions previously described in
other parts of SARS-CoV-2 genome by diﬀerent groups,
indicates that the virus is undergoing profound genomic
changes. It should be noted that mutations of the virus
are not very common, due to its proofreading mecha-
nism, and that collection of the sequencing data is cur-
rently biased toward symptomatic subjects. It would be
of interest to monitor over time and conﬁrm the spread-
ing of this particular viral strain, and potentially of strains
with other deletions in the nsp1 protein, in the popula-
tion of asymptomatic and pauci-symptomatic subjects
and to correlate these changes in nsp1 with a possible
decreased viral pathogenicity.
SARS-CoV: Severe acute respiratory syndrome coronavirus; SARS-CoV-2: Severe
acute respiratory syndrome coronavirus 2 (COVID-19); MERS-CoV: Middle east
respiratory syndrome coronavirus; COVID-19: Coronavirus disease-19.
FB and MG conducted the sequence analysis. GS performed the predicted
structural analysis. FB, MC and DZ wrote the paper. GS, MG, SA, RCG revised
the paper. MC and DZ supervised the project. All authors read and approved
the ﬁnal manuscript.
MG is supported by Fundação de Amparo à Pesquisa do Estado do Rio de
Availability of data and materials
All data utilized, generated or analyzed during these studies are included in
this published article.
Ethics approval and consent to participate
Consent for publication
We consent to publish our data.
The authors have declared that no competing interests exist.
1 Institute of Human Virology, School of Medicine, University of Maryland, Bal-
timore, USA. 2 Department of Biochemistry and Molecular Biology, University
of Maryland, Baltimore, USA. 3 Department of Microbiology and Immunology,
University of Maryland, Baltimore, USA. 4 Flavivirus Laboratory, Oswaldo Cruz
Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. 5 Medical Statistic
and Molecular Epidemiology Unit, University of Biomedical Campus, Rome,
Italy. 6 Department of Medicine, University of Biomedical Campus, Rome, Italy.
7 Global Virus Network, Baltimore, USA.
Received: 2 August 2020 Accepted: 26 August 2020
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