Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic

Jila Yavarian; Ahmad Nejati; Vahid Salimi; Nazanin Zahra Shafiei Jandaghi; Kaveh Sadeghi; Adel Abedi; Ali Sharifi Zarchi; Mohammad Mehdi Gouya; Talat Mokhtari-Azad

doi:10.1371/journal.pone.0267847

. 2022 May 2;17(5):e0267847. doi: 10.1371/journal.pone.0267847

Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic

Jila Yavarian ¹, Ahmad Nejati ¹, Vahid Salimi ¹, Nazanin Zahra Shafiei Jandaghi ¹, Kaveh Sadeghi ¹, Adel Abedi ², Ali Sharifi Zarchi ³, Mohammad Mehdi Gouya ⁴, Talat Mokhtari-Azad ^1,^*

Editor: Etsuro Ito⁵

PMCID: PMC9060343 PMID: 35499994

Abstract

Purpose

Whole genome sequencing of SARS-CoV2 is important to find useful information about the viral lineages, variants of interests and variants of concern. As there are not enough data about the circulating SARS-CoV2 variants in Iran, we sequenced 54 SARS-CoV2 genomes during the 5 waves of pandemic in Iran.

Methods

After viral RNA extraction from clinical samples collected during the COVID-19 pandemic, next generation sequencing was performed using the Nextseq platform. The sequencing data were analyzed and compared with reference sequences.

Results

During the 1st wave, V and L clades were detected. The second wave was recognized by G, GH and GR clades. Circulating clades during the 3rd wave were GH and GR. In the fourth wave GRY (alpha variant), GK (delta variant) and one GH clade (beta variant) were detected. All viruses in the fifth wave were in clade GK (delta variant). There were different mutations in all parts of the genomes but Spike-D614G, NSP12-P323L, N-R203K and N-G204R were the most frequent mutants in these studied viruses.

Conclusions

These findings display the significance of SARS-CoV2 monitoring to help on time detection of possible variants for pandemic control and vaccination plans.

1 Introduction

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) was detected in Wuhan, China which has known as the cause of coronavirus disease 2019 (COVID-19) pandemic [https://covid19.who.int/].

To date, Iran has experienced five waves of the pandemic since the first detection of SARS-CoV2 on 19 February 2020 in Iran [1]. As of 16 November 2021, 253,640,693 cases of SARS-CoV2 with 5,104,899 deaths were reported worldwide and in Iran 6,045,212 laboratory confirmed cases with 128,272 deaths reported [https://covid19.who.int/].

SARS-CoV2 is a virus in coronaviridae family with a single stranded RNA. The genome is about 30 kb and consists of genes encoding multiple non-structural, structural and accessory proteins. The non-structural proteins include NSP1-16 which are necessary for virus transcription and replication. These proteins are encoded by ORF1ab with about 21300 nucleotides length [2].

NSP1 helps virus to evade innate host antiviral response and promote viral growth. NSP2 can bind to two host proteins prohibitin 1 and prohibitin 2 that are recognized to be important in cell migration, cell cycle progression, apoptosis, cellular differentiation, and mitochondrial biogenesis [3]. NSP3 is a papain-like protease and also it can suppress host protein synthesis. NSP3 and NSP4 with other cofactors are important for virus replication by prompting membrane rearrangement. NSP5 is a cysteine-like protease which is the virus main protease and cleaves NSP4-NSP16. NSP6 is involved in autophagy. NSP7 and NSP8 form complex with NSP12 for viral replicase machinery. NSP7 has primer-independent RNA polymerase activity. NSP8 has primase activity- Primase is an enzyme that synthesizes primers during replication. NSP9, an RNA-binding protein, in complex with NSP8, is involved in RNA replication and virulence. NSP10 is a cofactor for the 2′ O-methyltransferase activity of NSP16 that increases evasion of the innate immune system, and the N7-guaninemethyltransferase/exoribonuclease activities of NSP14. NSP11 and NSP15 are involved in endoribonuclease activity and essential for replication. NSP12 has RNA polymerase activity. NSP13 has helicase and NTPase activity [2].

The structural proteins include spike (S), envelope (E), membrane (M) and nucleoprotein (N). Spike with a length of 1273 amino acid (aa), mediates attachment and entry. Envelope (75 aa) is a small membrane protein and important in virus infectivity. Membrane (222 aa) is important in virion morphogenesis and nucleoprotein (419 aa) is a viral genome packaging protein [2].

There are nine accessory proteins encoded by ORF3a, 3b, 6, 7a, 7b, 8, 9a, 9b and 10, which seem to be important in virus pathogenesis [2].

During virus replication, mutations can occur which might lead to alteration in protein functions and virus transmission and pathogenesis. Nowadays next-generation sequencing (NGS) is an effective method to identify different mutations and new variants for epidemiological and surveillance studies. SARS-CoV2 has different variants known as variants of concern (VOC), variants of interests (VOI) and variants under monitoring (VUM). It has also classified to different clades that currently 9 clades have been recognized based on markers mutations of the genome: S, L, V, G, and later of G into GH, GR and GV, and more recently GR into GRY and GRA [4]. Different variants of the SARS-CoV2 have been identified during the pandemic. Some spread worldwide, while others quickly faded away. Identification of the circulation of variants in a society is important, therefore, we set up NGS for SARS-CoV2 detected during five waves of pandemic for better understanding of circulation of different variants, genetic diversity and mutations in all non-structural, structural and accessory genes.

2 Materials and methods

2.1 Sample selection

Throat swab specimens from COVID-19 suspected patients were sent for sequencing to National Influenza Center (NIC) located at Virology Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. After primary detection by Real time PCR the samples from different parts of the country with ct value lower than 25 were selected for NGS. Iran experienced the 1st wave of SARS-CoV2 pandemic in February until May 2020. The 2nd wave began in late June until September 2020. The 3rd wave occurred during October to December 2020. The 4th wave started on early April until June 2021 and the 5th wave was from August until October 2021. This study was approved by ethics committee of National Institute for Medical Research Development, Tehran, Iran (IR.NIMAD.REC.1399.119).

2.2 Next generation sequencing (NGS)

After viral RNA extraction using High Pure Viral Nucleic Acid kit (Roche,Germany) according to the manufacturer’s instruction, cDNA synthesis was performed. Library construction was done by using the Nextera DNA Flex kit (Illumina,USA), then used for hybridization using Respiratory Virus Oligo Panel kit (Illumina,USA). This was followed by bead-based capture of hybridized probes, amplification and clean-up. After quality control assessment for library concentration by Qubit (Thermo Fisher,USA), sequencing was performed using NextSeq 550 machine (Illumina,USA).

2.3 Data & phylogenetic analysis

For data analysis, all collection of reads were mapped against the reference genome assembly of SARS-CoV2. We had high quality of assembled full viral genome coverage without undetermined nucleotides. The assembled genomes were analyzed using CoVsurver mutations App in GISAID [4] and aligned by BioEdit sequence alignment software. After sequence alignment, a phylogenetic tree was drawn by neighbor joining method (bootstrap 1000) using MEGA v7 software.

Then all 54 Iranian full genome SARS-CoV2 of different waves were submitted to GISAID with accession numbers: EPI-ISL-1014676-87, EPI-ISL-959275-84, EPI-ISL-862075-81, EPI-ISL-1993547-557, EPI-ISL-2360250-57, EPI-ISL-4803556-58, EPI-ISL-4803554, EPI-ISL-4803538 and EPI-ISL-4803528.

3 Results

Fifty four COVID-19 confirmed cases were subjected to NGS selecting 10, 10, 9, 20 and 5 samples from 1st, 2nd, 3rd, 4^th and 5th waves respectively. Here we analyzed the mutations of each genes separately by comparing with hCoV-19/Wuhan/WIV04/2019 in GISAID.

3.1 Nonstructural proteins (ORF1ab)

The mutations of NSP genes are illustrated in the Fig 1. It is important to note that not all the mutations mentioned in the Fig 1 were found in all viruses during the related waves. Meanwhile in NSP10 and NSP11 no changes have been detected.

3.2 Structural proteins (S-E-M-N)

Spike

As shown in Table 1, amino acid substitutions in S glycoprotein were increased with pandemic progression. During the 1st wave there was no special amino acid substitutions on the S glycoprotein but viruses detected during the 4th and 5^th waves had the most frequent amino acid substitutions. It should be noted that D614G was detected in all viruses after the 1st wave.

Table 1. Amino acid changes detected in spike glycoprotein during the five waves of SARS-CoV2 pandemic in Iran.

Sample name		Amino acid changes in Spike			Sample name							Amino acid changes in Spike
*Wave 1*					*Wave 2*
hCoV-19/Iran/Tehran-08/2020					hCoV-19/Iran/Gilan-NIC189/2020							D614G
hCoV-19/Iran/Tehran-09/2020					hCoV-19/Iran/Ghilan-S15-319/2020							D614G
hCoV-19/Iran/Qom-907/2020					hCoV-19/Iran/Ghilan-T15-191/2020							D614G
hCoV-19/Iran/Gorgan-NIC992/2020					hCoV-19/Iran/Tehran-055M/2020							D614G			F1256L	I210del	L5F
hCoV-19/Iran/Gorgan-NIC118/2020					hCoV-19/Iran/Gilan-NIC184/2020							D614G
hCoV-19/Iran/Gorgan-NIC028/2020		G142S	I210del		hCoV-19/Iran/Gilan-NIC230/2020							D614G				I210del
hCoV-19/Iran/Gorgan-NIC140/2020					hCoV-19/Iran/Ghilan-M30-241/2020							D614G				I210del
hCoV-19/Iran/Hamedan-66H/2020					hCoV-19/Iran/Gilan-NICS1-58/2020							D614G
hCoV-19/Iran/Urmia-715U/2020					hCoV-19/Iran/Ghilan-S1-32/2020							D614G
hCoV-19/Iran/Urmia-931U/2020		G142S	I210del		hCoV-19/Iran/Qom-629/2020							D614G				I210del
Sample name					Amino acid changes in Spike
*Wave 3*
hCoV-19/Iran/Tehran-NIC45RC/2020	D614G		I210del
hCoV-19/Iran/Tehran-NIC12RC/2020	D614G			A845S	A846V	D138Y
hCoV-19/Iran/Tehran-04/2020	D614G	Q314R	I210del
hCoV-19/Iran/Tehran-06/2020	D614G					D138Y	M177I			S477N
hCoV-19/Iran/Tehran-01/2020	D614G					D138Y				S477N
hCoV-19/Iran/Tehran-02/2020	D614G
hCoV-19/Iran/Tehran-03/2020	D614G		I210del									D574N		D950N	Q677H
hCoV-19/Iran/Tehran-05V/2020	D614G					D138Y				S477N
hCoV-19/Iran/Kashmar-15K/2020	D614G		I210del
Sample name					Amino acid changes in Spike
*Wave 4*
hCoV-19/Iran/Tehran-NIC-K23/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Hormozghan-NIC-15/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Tehran-NIC-24/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del	I100T	L699I
hCoV-19/Iran/Tehran-NIC-V30/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Tehran-NIC-SH17/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Tehran-NIC-30/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del		L699I
hCoV-19/Iran/Tehran-NIC-23K/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Tehran-NIC-28/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del	I100T	L699I
hCoV-19/Iran/Tehran-NIC-31/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Tehran-NIC-17/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del	I100T	L699I
hCoV-19/Iran/Tehran-NIC-15/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del	I100T	L699I
hCoV-19/Iran/Kerman-NIC-K1/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del
hCoV-19/Iran/Kerman-NIC-K2/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del	I100T	L699I
hCoV-19/Iran/Tehran-NIC-S7/2021	D614G	A570D	D1118H	H69del	N501Y	P681H	S982A	T716I	V70del	Y144del	I100T	L699I	K1191N
hCoV-19/Iran/Hormozghan-NIC-B7/2021	D614G	A243del	A701V	D80A	D215G	E484K	K417N	L242del	L244del	N501Y
hCoV-19/Iran/Boshehr-NIC-P10/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K
hCoV-19/Iran/Boshehr-NIC-P11/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K
hCoV-19/Iran/Yazd-NIC-Y3/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K	E1202Q	T95I	C1250R	D1257F	F1256L
hCoV-19/Iran/Maku-NIC-M5/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K		T95I					S255F
hCoV-19/Iran/Yazd-NIC-Y6/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K	E1202Q	T95I		L938F
*Wave 5*
hCoV-19/Iran/Maragheh-NIC-S2/2021	D614G	D950N					P681R		T19R	T478K		T95I					I850L
hCoV-19/Iran/Ardakan-NIC-S5/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K		T95I				D574Y	T299I
hCoV-19/Iran/North Khorasan-NIC-S6/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K					T29A	T250I
hCoV-19/Iran/Ahvaz-NIC-S8/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K		T95I		A262S			I850L
hCoV-19/Iran/Ahvaz-NIC-S9/2021	D614G	D950N	E156G	F157del	G142D	L452R	P681R	R158del	T19R	T478K		T95I		A262S			I850L

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Envelope

No mutation was found in viruses studied during the 1st, 2nd, 3rd and the 5^th waves but in two samples in the 4^th wave there were mutations encoding the amino acid substitution P71L in one sample belonged to beta variant and S68F in one sample belonged to alpha variant.

Membrane

During the 1st and the 2nd waves, there were no mutations in M, but a mutation causing the amino acid substitution I73M was detected in three samples during the 3rd wave. Four samples during the 4^th wave and 4 samples of the 5^th wave had I82T which all these 8 samples belonged to delta variant.

Nucleoprotein

During the 1st wave, one sample had N192K in N and two samples had A35V. During the 2nd wave, two samples had D3Y and M234I, three samples had S194L substitutions. One sample had P13T and S194L. Another sample had A220V and P326L. hCoV-19/Iran/Gilan-NICS1-58/2020 along with four viruses during the 3rd wave, had R203K and G204R. hCoV-19/Iran/Tehran-05V/2020 had P162S beside R203K and G204R. One sample during the 3rd wave, had S201I. Three samples had S194L along with another substitution including G34W, P383L and P13T. During the 4^th wave, 13 samples of alpha variant had D3L, R203K, G204R and S235F. One sample of alpha variant beside these four substitutions had also L219F. Seven samples belonged to delta variant had D63G, D377Y, G215C and R203M. One sample of delta variant during the 5^th wave had D63G, R203M and D377Y. One sample had just R203M and D377Y and one sample had D63G, R203M and G215C. The only beta variant had T205I and T362I.

3.3 Accessory proteins

First wave

One sample had T151I in NS3, another sample had A105V in NS7a and two samples had T40I in NS7b.

Second wave

In NS3, five samples had Q57H and one sample had G18V along with Q57H. One sample had G254V and one sample had T223A and V112F, therefore during the 2nd wave eight samples had just mutation in NS3 among all accessory proteins.

Third wave

In NS3, two samples had Q57H the same as five samples in the 2nd wave. Two more samples had one more substitution along with Q57H in NS3, including W131C and T223I. One sample had just N58G in NS3. Three samples had a deletion at G66 (G66del) and S67 (S67del) and K68E in NS8. One sample had T223I and L4F in NS3.

Fourth wave

During the fourth wave, amino acid substitutions detected in NS3 were W131C, S171L, G100C, D238Y, S26L, Q57H and S171L in different samples. There was just W27L in NS6 in one sample. T39I, T120I, V82A and S83L were detected in NS7a. NS7b had T40I and a stop codon at E39 (E39stop). NS8 had the most frequent amino acid substitutions in viruses circulating during the 4^th wave including stop codons at K68 (K68stop) and Q27 (Q27stop) and R52I, V62L, Y73C, C90F. These stop codons at NS7b and NS8 may lead to producing the truncated proteins. During the 5^th wave, amino acid substitutions were detected in NS3a, NS7a and NS7b which K16T, S26L and L41F in NS3a, L49P, A50D, D51H, T120I, V82A in NS7a and T40I in NS7b were found.

3.4 Phylogenetic analysis

Fig 2 shows the phylogenetic analysis of 54 SARS-CoV2 viruses circulating during the different waves in Iran. Analyses showed that during the 1st wave clades V and L were circulating which V was dominant. During the 2nd wave, GH was dominant, but G and GR were also detected. Surprisingly one V clade was also detected in the 2^nd wave. GH and GR were the clades during the 3rd wave. During the 4^th wave, 14 viruses of alpha variant were belonged to GRY, five delta variants were in GK clade and one beta variant was in GH clade and finally five delta variants of the 5^th wave were in GK clade.

4 Discussion

In this study, we reported the circulation of distinct clades of SARS-CoV2 during the five waves in Iran. The V clade was found during the 1st wave. Clade V is characterized by NSP6-L37F plus NS3-G251V. At the end of the first wave, one sample detected in clade L (reference clade). The 2nd wave was recognized by G, GH and GR clades. G clade shows D614G in spike which is one of the most important substitutions in spike. Clade GH has S-D614G plus NS3-Q57H substitutions and clade GR characterized by S-D614G and N-G204R. One sample belonged to the V clade was also detected in the 2^nd wave. Circulating clades during the 3rd wave were GH and GR. During the fourth wave, GH (1 beta), GRY (alpha) and GK (delta) clades were detected. GRY clade (alpha) has S-H69del, S-V70del, S-Y144del, S-N501Y plus S-D614G and N-G204R. All viruses in the 5th wave were in clade GK (S-D614G plus S-T478K).

In different clades NGS of 54 samples selected during the five waves in Iran showed important mutations in the different parts of the genome which all were analyzed and compared with circulating variants worldwide. The effects of important mutations were discussed here.

ORF1ab is more than two thirds of the SARS-CoV2 genome with 21,290 nucleotides at the 5’ end which encodes 16 non-structural proteins (NSP1-NSP16). Among these NSPs, NSP3 had the highest number of mutations. NSP3 is important for virus replication and it can suppress host protein synthesis, then amino acid substitutions in NS3 deserve greater study in vitro.

Suppression of type I interferon (IFN) response is a consequence of infection of SARS-CoV2. NSP5, main protease, is an IFN antagonist. NSP5 variant K90R seen in SARS-CoV2 retained the IFN-antagonizing activity. The suppressive outcome of NSP5 on IFN-β gene transcription induced by IKKϵ, TBK1, RIG-I and MAVS suggested that NSP5 probably involved at a stage downstream of IRF3 phosphorylation in the cytoplasm [5]. In this study K90R was detected just in one sample during the 4^th wave while it has been more often found in Icelandic and Chinese strains [6].

NSP6 is important for viral assembly, viral protein folding and replication. NSP6-L37F leads to asymptomatic transmission and reduced virulence which we can see in the most of the viruses detected during the 1st wave and one in the 2nd wave [7].

In hCoV-19/Iran/Gilan-NIC230/2020, E50G in NSP7 was detected in the 2nd wave which is important in immune response as shown in a study that amino acid residues 36–50 (HNDILLAKDTTEAFE) of NSP7 and also NSP13 and N are SARS-CoV2 specific T cell epitopes recognized by CD8 T cells [8].

NSP12 is necessary for the replication/transcription of the SARS-CoV2 genome, and this protein is considered as a target for the treatment of COVID-19. The P323L substitution of NSP12, could induce structural changes and adverse effect on proofreading during the replication of the virus. Meanwhile, the P323L is located in a pocket that might be the site of drug function [9]. In our research NSP12-P323L located in the NSP8 binding cleft [10] was detected after the first wave. NSP12-P323L is the most common detected substitution with increasing in occurrence over time [11].

A study by Mohammad., et al. showed that remdesivir has higher binding affinity to NSP12 with P323L than the wild-type RdRp, therefore they suggested using remdesivir in patients infected with SARS-CoV2 carrying P323L in NSP12 [12].

In this study there was no virus seen to contain F480, V557 and S861 in NSP12, which have shown to be important in decreasing susceptibility to remdesivir [11].

As all viruses studied in this research, did not have any mutations in the genes for NSP10 and NSP11, therefore these two nonstructural proteins could be considered as good targets for diagnosis and targets for therapeutic agents. Furthermore, there were some mutations in other NSPs with that of unknown importance. The effect of these substitutions need to be studied in vitro.

After the 1st wave, all viruses studied in this research had S-D614G. Studies showed that the S-protein amino acid substitution, D614G, evolved concurrently with P323L in NSP12. NSP12- P323L might decrease the replication of viral RNA and therefore increase the probability of asymptomatic infections and/or change in incubation period. Meanwhile the infectivity-enhancing D614G substitution in S protein could compensate this reduction in virus replication and help virus transmission even by asymptomatic individuals [13]. Flores-Alanis., et al. showed that D614G substitution in S protein, P323L in NSP12, and R203K and G204R in N protein had a substantial association with the disease severity [9]. Another study showed that D614G in the S protein increased the infectivity of SARS-CoV2 and associated with lower RT-PCR cycle thresholds. It showed high viral load in the upper respiratory tract, but not enhanced disease severity. Also they showed that G614-bearing virions are not intrinsically more resistant to neutralization by convalescent sera [14]. G614 of S protein is responsible for making the virus 2.4 times more contagious with higher viral load [9, 15]. hCoV-19/Iran/Tehran-055M/2020 in the 2nd wave had D614G and L5F. One study showed that variants with D614G and L5F had increased infectivity [16].

Evaluation of the mutations which lead to amino acid substitutions, showed that Spike-D614G, NSP12-P323L, N-R203K and N-G204R were the most frequent amino acid substitutions in these studied viruses and also worldwide which we could consider that they may increase SARS-CoV2 transmissibility as the pandemic progressed.

The S protein is crucial factor for the entry of SARS-CoV2 to the host cell which interacts with the angiotensin-converting enzyme 2 (ACE-2) receptor through its receptor binding domain (RBD) [17]. The S477N is the part of an epitope recognized by human neutralizing antibodies and located in the RBD. A study showed that S477N increases the affinity for the ACE-2 receptor [9, 18]. Singh., et al. showed that S477N strengthen the binding of SARS-COV2 spike to the ACE-2 receptor [19]. In this study, two samples had S477N which both belonged to GR clade.

Among the variations in the alpha variant, S-N501Y, is in the receptor binding site which was shown to increase the binding of SARS-CoV2 to the ACE-2 host receptor, leading to increased viral fitness and transmission [20–22].

One study showed that N439K, N501Y and S477N significantly reduced the neutralization activity of some monoclonal antibodies [23]. Ostrov showed that B.1.1.7 with N501Y had increased affinity for ACE-2 and A570D, D614G and S982A substitutions might increase virus fusion by decreasing the intermolecular stability of S1 and S2 [24].

The delta variant (GK clade) has ten amino acid substitutions, T19R, G142D, 156del, 157del, R158G, L452R, T478K, D614G, P681R and D950N in the spike protein [25]. T19R removes an N-glycosylation site at position 17 that might also affect antigenic properties. A study by Li., et al. showed that variants with L452R were resistant to some neutralizing antibodies [16]. Tchesnokova., et al. showed that L452R might result in stronger binding to the ACE-2 and escape from neutralizing antibodies [26].

Overall L452R reduces sensitivity to neutralizing antibodies, increases viral infectivity, transmissibility, spike stability, ACE-2 binding affinity and viral fusogenicity, therefore it supports viral replication [27–30].

Delta variant circulation was started at the end of the 4^th wave and all viruses detected during the 5^th wave in Iran were the delta variant.

The beta variant (GH clade) includes L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, and A701V in the spike protein, which K417N, E484K, and N501Y are in the RBD and increase the binding affinity for the ACE-2 receptor and eventually the infectivity [31]. K417N and E484K have also essential role in viral escape from neutralizing antibodies [32]. Overall K417N, E484K and L452R are vaccine escape mutants [33]. The only beta variant of this study did not have L18F and R246I but it had a deletion of three amino acids 242–244 (L242del, A243del and L244del) in spike. L242del, A243del and L244del showed reduced sensitivity to some neutralizing antibodies [22]. The beta variant is reported to have an increased risk of transmission and reduced neutralization by monoclonal antibody therapy, convalescent sera, and post-vaccination sera [34].

Some studies showed that most serum samples from vaccinated people or patients recovering from COVID-19 have shown full or slightly decreased capacity to inactivate SARS-CoV2 variants, except for variants with N501Y, K417N and E484K substitutions [22, 35–37]. We had just one beta variant during the 4^th wave which had E484K in the S protein. As seen in different studies, E484K plays a crucial role in increasing virus transmission and decreasing antibody neutralizing titers [37–39], thus continuous screening for emerging variants with substitutions such as E484K is necessary for public health.

hCoV-19/Iran/Tehran-03/2020 in the 3rd wave had Q677H. Grabowski., et al. showed that E484K, F490S, S494P (in the RBD of spike) and Q677H and Q675H (in the vicinity of the polybasic cleavage site at the S1/S2 border) may limit efficiency of vaccines [40].

Liu., et al. showed that substitutions at residues T345, R346, K444, G446, N450, L452, S477, T478, E484, F486, and P499 were each related to the resistance to more than one monoclonal antibody, of which substitutions at S477 and E484 residues showed wide resistance [41].

Viruses with H69del and V70del had 2-fold higher infectivity compared to wildtype and also showed reduced neutralization sensitivity to mAb, targeting an as yet undefined epitope outside the RBD [42]. Sixteen samples of this study during the 4^th wave had H69del, V70del and also Y144del. In one study it was shown that viruses with Y144del in spike had decreased sensitivity to convalescent sera [16].

Li., et al. showed that, among spike mutations, the most characteristic ones are substitutions such as D614G, N501Y, Y453F, N439K/R, P681H, K417N/T, and E484K, and deletions of ΔH69/V70 and Δ242–244, which enhanced viral infectivity, transmissibility, and resistance to neutralization [43]. Some of these mutations were found after the 2nd wave of pandemic in Iran.

P681R in the furin cleavage site may help in increased rate of membrane fusion, internalization and so better transmissibility which was found in delta variants in this study and worldwide [44]. Voss., et al. found that P681H of spike and S235F of nucleoprotein in the alpha variant changed the specificity of the corresponding epitopes [45]. During the 4^th wave, 14 samples had P681H and S235F.

Mutations in the E gene were somewhat uncommon as in this study just two mutations in 2 different samples in the 4^th wave were detected which needs more investigation to find out their effects on virus life cycle.

All viruses of delta variant in this study had I82T in M gene. Shen., et al. showed that this M gene substitution was more naturally fit, probably connected to glucose uptake during virus replication, and it is better to be included in genomic surveillance [25].

Nucleoprotein was mainly expressed in the initial stages of infection, and is important in viral RNA transcription and replication. Nucleoprotein has been shown to affect some basic cellular processes, inflammatory responses to upregulate the expression of the proinflammatory factor COX2, and it inhibits the innate immune responses in the host cell [46]. Therefore, amino acid substitutions in the nucleoprotein might have significant effect in immune response. R203K and G204R were the most common substitutions in the nucleoprotein in viruses studied in this research and worldwide. These mutations are important in disease severity [47].

There are some studies about the accessory gene’s mutations and their impact on the virus cell cycle. The results of research by Wu et al., showed that Q57H and G251V in NS3a, S194L and R203K/G204R in N made changes in the structure of proteins and also had effect on the binding affinity of intraviral protein-protein interactions during assembly and release of coronavirus. So these changes might be associated with virus evolution and beneficial for the virus and its virulence [48]. Some viruses during the waves 2,3 and 4 had NS3a-Q57H. In primary human respiratory cells, viruses with NS3a-Q57H evade stimulation of chemokine, cytokine, and interferon-stimulated gene expression [49]. Some studies have described that NS3a can competently induce apoptosis in the cell and affect virus uptake and release [50, 51]. Besides ORF3, some mutations were detected in different accessory genes of viruses in this study which more research is needed to explore their importance in virus pathogenesis. For instance, NS8 supposed to suppress immune responses [52], then substitutions of this protein can be imperative. Of special importance, stop codon mutations which lead to absence of NS8 can extend the duration of symptoms, then increase the virus transmissibility. Spike mutations such as D614G, HV69-70 del and L5F which affect the receptor binding affinity of the S protein and increase the virus transmission when associated with NS8 stop codons which extend the period of signs and symptoms might enhance the chance of transmission [53]. The combination of these variations were found especially in some viruses detected during the 4^th wave which should raise concern.

In this study we did not have data on relationship between different SARS-CoV2 lineages and patient’s signs and symptoms and disease severity which was an important limitation of this research.

In conclusion, we detected different lineages of SARS-CoV2 contributing to all five waves and showed that all viruses circulating during the 5^th wave belonged to delta variant. We compared the mutations identified in our complete genomes study with those reported in GISAID. The findings of this study showed that with progression of the pandemic, the number of mutations were considerably increased which showed the adaptive evolution of SARS-CoV2 in human to increase transmissibility. Therefore genomic surveillance is an important tool to screen the progression of the COVID-19 pandemic. It should be noted that for variant detection, we partially sequenced S glycoprotein of more than 1000 samples with Sanger sequencer of which the results were compatible with NGS results during all 5 waves (unpublished data).

Acknowledgments

Authors would like to express their great thanks to all data contributors, i.e. the authors and their originating laboratories responsible for obtaining the specimens, and their submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID Initiative, on which this research is based. We thank GISAID for all their support.

Data Availability

All submitted files are available from the GISAID database (Accession numbers: EPI-ISL-1014676-87, EPI-ISL-959275-84, EPI-ISL-862075-81, EPI-ISL-1993547-557, EPI-ISL-2360250-57, EPI-ISL-4803556-58, EPI-ISL-4803554, EPI-ISL-4803538, and EPI-ISL-4803528).

Funding Statement

A part of this study is supported by National Institute for Medical Research Development (NIMAD) under grant number 994376. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PONE-D-21-38222Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemicPLOS ONE

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Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Regarding the manuscript entitled “Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic”:

The authors performed an extensive analysis of the SARS-CoV-2 genomes in Iran to find information about the SARS-CoV2 lineages, variants of interests and variants of concerns during five waves of pandemic. Overall it is a well-organized manuscript and a nice contribution to the field and the information presented in this manuscript can be beneficial to the other researchers.

Below are some suggestions that the authors may wish to consider to improve their manuscript:

-Please mention to the COVID-19 in the introduction section.

-Please mention to the different known clades of SARS-CoV-2 in the introduction section.

-In the figure legend please replace “constricted” by “constructed”

-The number of viruses in the phylogenetic tree is 53.

-Since the purpose of the manuscript was finding information about the viral lineages, variants of interests and variants of concern, it would be suggested to describe them briefly.

-Please add the following reference “Usage of peptidases by SARS-CoV-2 and several human coronaviruses as receptors: A mysterious story” in the line 366.

Reviewer #2: The manuscript presents valuable information about SARS-CoV2 viruses circulating in Iran during 5 waves of the pandemic. It is well written and organized. Only few revisions are suggested:

- The Introduction mostly explains the virus proteins and structure, while it might be preferred that first the Introduction part talks also about why this virus is important why this study was designed.

- It is suggested that the mutations in different proteins (non-structural, structural and accessory) are entered and organized in a table, so the reader can easily see and follow different mutations in different proteins during different waves of the pandemic in Iran.

- Line 486: … which the results were compatible with … -- needs to be revised to: … of which the results were compatible with …

Reviewer #3: This is a thorough analysis of the genome sequences of SARS-CoV-2 viruses in circulation in Iran over five waves of the pandemic. The full genome sequence of five to twenty viruses (10,10, 9, 5 and 20) from each wave were analysed by next generation sequencing and the results analysed to report here ion what variation has been seen.

The data quality has not been described fully: for example, how complete each genome sequence turned out to be and what proportion of undetermined nucleotides were in the genome sequence assembly. This would provide some kind of quality assessment of the gene sequence data. The gene sequence data have been shared in the EpiCov database of GISAID, but I have not checked the sequences fully.

The catalogue of changes seen and described in the results is comprehensive, but going through the results for each virus polypeptide is demanding for the reader and I wondered if the authors could come up with some kind of graphical display to supplement the description of what was seen and elaborated on in the text. I think for most virus polypeptides this could be relatively easily done, but, like in spike (for which there is a dedicated table), the variation in NSP3 seems quite extensive. Nevertheless, I think a graphical representation would enhance the message of the manuscript greatly.

In the discussion the authors summarise a lot of literature on the likely effects of amino acid substitutions. My feeling is that the authors should differentiate between results and conclusions made by experiment from those that have been generated by modelling. Moreover, there is frequent use of phrases like ‘viral oligomerisation interface’ (e.g. lines 136, 140, 141, 147, 150, 151 etc.). Here I was not clear in many cases what the evidence for this conclusion was. I might have missed the references to many of these. In addition, it was not what was meant precisely by a ‘viral oligomerisation interface’. I was not sure of this meant oligomerisation of the relevant polypeptide or something else.

There are also two places in which antibody recognition sites are described for the NSP7 and NSP8 (lines 175 and 179). Do the authors know if this recognition is by post-infection serum or something else? Also, references should be given for this. NSP7 was also stated to undergo antigenic drift. I wonder if this is better defined as antigenic change, or change in a T-cell epitope. This is also referred to on line 314.

The phylogenetic tree shown in the figure uses the GISAID clade nomenclature. I wonder if it is possible to correlate these clades and subclades with the PANGO lineages.

I think it might also be useful to include a graph of the five waves of COVID-19 in Iran and indicate when the samples were taken on the graph.

Minor points.

In many cases the authors refer to mutations in the virus polypeptides. I think, strictly speaking, mutations occur in genes that encode mutant proteins that have amino acid substitutions. I would recommend that this is considered by the authors.

There are a number of places where the English is not correct – the indefinite and the definite articles are missing in some number of places. This should be corrected by asking for additional help from colleagues.

Some other points include:

Generally, use family names only when referring to work done by others.

Also, the word ‘data’ has been used both as a singular term (incorrectly) and as a plural (correctly). There should be consistency.

I would suggest that when referring to a series of amino acid substitutions the order of these substitutions is always from the N-terminal to C-terminal of the polypeptide.

Line 62, define what a ‘primase’ is.

Line 69. I suggest saying that Spike ‘mediates’ attachment and entry rather than is ‘important for’ attachment and entry.

Line 88. I think ‘designated’ is not the right word here – perhaps ‘selected’.

Line 98. I suggest ‘Library construction was done by using…’

Line 99. Explain what ‘captured’ means in this context.

Line 236. Define in which wave hCoV-19/Iran/Gilan/NICS1-58/2020 was isolated.

Line 295. The word ‘third’ should be ‘thirds’.

Line 305. “Recommended’ seems to be the wrong word here.

Line 346. Add the words ‘change in’ ahead of ‘incubation period’.

Line 366. I think this should be Receptor Binding Domain (not Receptor Binding Motif).

Lines 455 to 456. I am not clear what intraviral connections refers to. Lines 453 to 457 might be better composed.

Line 481. The authors comment on the ‘rate’ of mutation. However, mutations rates have not been addressed in this work. This needs re-wording to reflect more accurately what has been observed in this study.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 May 2;17(5):e0267847. doi: 10.1371/journal.pone.0267847.r002

Author response to Decision Letter 0

13 Feb 2022

Dear Editor-in-Chief of PLOS ONE,

Thank you very much for the review of our manuscript entitled: “Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic”.

We sincerely appreciate all valuable comments and suggestions, which helped

us to improve the quality of the article. Our responses to the Reviewers’ comments

are described below in a point-to-point manner. Appropriated changes, suggested

by the Reviewers, have been introduced to the manuscript (highlighted within the

document). The language is improved.

Dear Reviewer 1:

The changes were highlighted in yellow.

- Please mention to the COVID-19 in the introduction section.

The sentence was added to the beginning of the Introduction.

- Please mention to the different known clades of SARS-CoV-2 in the introduction section.

Was added to the last paragraph of Introduction.

- In the figure legend please replace “constricted” by “constructed”

Done.

- The number of viruses in the phylogenetic tree is 53.

Phylogenetic tree was corrected.

- Since the purpose of the manuscript was finding information about the viral lineages, variants of interests and variants of concern, it would be suggested to describe them briefly.

Was described at the end of Introduction.

- Please add the following reference “Usage of peptidases by SARS-CoV-2 and several human coronaviruses as receptors: A mysterious story” in the line 366.

Was added.

Dear Reviewer 2:

The changes were underlined.

It was added to the end of Introduction.

Mutations of nonstructural genes illustrated in the figure.

- Line 486: … which the results were compatible with … -- needs to be revised to: … of which the results were compatible with …

Corrected.

Dear Reviewer 3:

The changes were highlighted in green.

This is a thorough analysis of the genome sequences of SARS-CoV-2 viruses in circulation in Iran over five waves of the pandemic. The full genome sequence of five to twenty viruses (10,10, 9, 5 and 20) from each wave were analyzed by next generation sequencing and the results analyzed to report here on what variation has been seen.

- The data quality has not been described fully: for example, how complete each genome sequence turned out to be and what proportion of undetermined nucleotides were in the genome sequence assembly. This would provide some kind of quality assessment of the gene sequence data. The gene sequence data have been shared in the EpiCov database of GISAID, but I have not checked the sequences fully.

Added to the materials and methods section.

- The catalogue of changes seen and described in the results is comprehensive, but going through the results for each virus polypeptide is demanding for the reader and I wondered if the authors could come up with some kind of graphical display to supplement the description of what was seen and elaborated on in the text. I think for most virus polypeptides this could be relatively easily done, but, like in spike (for which there is a dedicated table), the variation in NSP3 seems quite extensive. Nevertheless, I think a graphical representation would enhance the message of the manuscript greatly.

Mutations of nonstructural genes illustrated in the figure.

- In the discussion the authors summarize a lot of literature on the likely effects of amino acid substitutions. My feeling is that the authors should differentiate between results and conclusions made by experiment from those that have been generated by modelling. Moreover, there is frequent use of phrases like ‘viral oligomerisation interface’ (e.g. lines 136, 140, 141, 147, 150, 151 etc.). Here I was not clear in many cases what the evidence for this conclusion was. I might have missed the references to many of these. In addition, it was not what was meant precisely by a ‘viral oligomerisation interface’. I was not sure of this meant oligomerisation of the relevant polypeptide or something else.

That is really important point. There is not any reference about the role of some proteins as viral oligomerization interfaces, we added this, just extracted from GISAID mutation analysis, then we deleted them.

- There are also two places in which antibody recognition sites are described for the NSP7 and NSP8 (lines 175 and 179). Do the authors know if this recognition is by post-infection serum or something else? Also, references should be given for this. NSP7 was also stated to undergo antigenic drift. I wonder if this is better defined as antigenic change, or change in a T-cell epitope. This is also referred to on line 314.

It is another important point which is written just according to GISAID mutation analysis and there is not publication or in vitro studies, then we removed those from the article.

- The phylogenetic tree shown in the figure uses the GISAID clade nomenclature. I wonder if it is possible to correlate these clades and subclades with the PANGO lineages.

We could change the clades to PANGO in the tree, but because we used mostly

clades in the text, then we prefer to put clades, if it is necessary we could

change them.

- I think it might also be useful to include a graph of the five waves of COVID-19 in Iran and indicate when the samples were taken on the graph.

It is useful to include a graph, but because of the article length and number of tables and figures, we just wrote the date of each wave and the number of assessed samples in each wave.

Minor points.

- In many cases the authors refer to mutations in the virus polypeptides. I think, strictly speaking, mutations occur in genes that encode mutant proteins that have amino acid substitutions. I would recommend that this is considered by the authors.

All were corrected.

- There are a number of places where the English is not correct – the indefinite and the definite articles are missing in some number of places. This should be corrected by asking for additional help from colleagues.

Reviewed and corrected as much as possible.

Some other points include:

- Generally, use family names only when referring to work done by others.

All were corrected.

- Also, the word ‘data’ has been used both as a singular term (incorrectly) and as a plural (correctly). There should be consistency.

Corrected.

I would suggest that when referring to a series of amino acid substitutions the order of these substitutions is always from the N-terminal to C-terminal of the polypeptide.

Corrected

- Line 62, define what a ‘primase’ is.

Added.

- Line 69. I suggest saying that Spike ‘mediates’ attachment and entry rather than is ‘important for’ attachment and entry.

Corrected.

- Line 88. I think ‘designated’ is not the right word here – perhaps ‘selected’.

Corrected.

- Line 98. I suggest ‘Library construction was done by using…’

Corrected.

- Line 99. Explain what ‘captured’ means in this context.

The sentence was changed to clear explanation.

- Line 236. Define in which wave hCoV-19/Iran/Gilan/NICS1-58/2020 was isolated.

Cleared.

- Line 295. The word ‘third’ should be ‘thirds’.

Corrected.

- Line 305. “Recommended’ seems to be the wrong word here.

Corrected.

- Line 346. Add the words ‘change in’ ahead of ‘incubation period’.

Added.

- Line 366. I think this should be Receptor Binding Domain (not Receptor Binding Motif).

Corrected.

- Lines 455 to 456. I am not clear what intraviral connections refers to. Lines 453 to 457 might be better composed.

Corrected.

- Line 481. The authors comment on the ‘rate’ of mutation. However, mutations rates have not been addressed in this work. This needs re-wording to reflect more accurately what has been observed in this study.

It corrected. We mean the number of mutations.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(18.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0267847.r003

Decision Letter 1

Etsuro Ito

18 Mar 2022

PONE-D-21-38222R1Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemicPLOS ONE

Dear Dr. Mokhtari-Azad,

Please submit your revised manuscript by May 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Etsuro Ito

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: The manuscript by Yavarian et al. has addressed many of the points I raised in my report. However, I think that there are still various sections of the manuscript where improvements to the flow or meaning can be made. I will attach a scan of a handwritten marked-up document for the authors to consider. However, there are places that the authors still need to address.

The main places where the authors need to further modify the text are as follows. (Minor changes suggested are in the marked-up copy).

Lines 108 to 110. The term ‘mutations’ remain in this section to be used for amino acid substitutions. The same is also seen in lines 113 and 116. I suggest the authors re-check for the appropriate usage. I have made some suggestions in the marked-up scanned copy.

Line 140. Introduce the concept here of amino acid deletions e.g. using the phrase ‘a deletion of G66 (G66del) and S76 (S67del)…’ See also lines 162 to 164, and line 240

Lines 144 and 145. Introduce the concept of stop codons in NS7b and NS8 - and the effect of the stop codons on what can be expected to be translated.

Line 171 is not clear what the authors are trying to say here. Perhaps it is that amino acid substitutions in NS3 deserve greater study in vitro.

Lines 195 to 197. I have made suggestions for changes here in the marked copy.

Line 211. It was not clear what the authors meant by ‘missense mutations’.

Line 243. A reference is needed.

Line 271. It was unclear what therapeutic effects were envisaged here.

Line 276. Here the authors are presumably referring to amino acid substitutions in the nucleoprotein, not in the gene.

Line 277. A reference is needed.

Line 287. A reference is needed.

Lines 289 to 291. It is not clear what the authors are saying that termination of the NS8 gene somehow affects spike affinity for the receptor. The authors should elaborate on this.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Attachment

Submitted filename: Yavarian markup.pdf

Click here for additional data file.^{(1MB, pdf)}

PLoS One. 2022 May 2;17(5):e0267847. doi: 10.1371/journal.pone.0267847.r004

Author response to Decision Letter 1

13 Apr 2022

Dear Dear Editor-in-Chief of PLOS ONE,

Thank you very much for the review of our manuscript entitled: “Whole genome

sequencing of SARS-CoV2 strains circulating in Iran during five waves of

pandemic”.

We sincerely appreciate all valuable comments and suggestions, especially dear reviewer 3, which helped us to improve the quality of the article. Our responses to the Reviewers’ comments are described below in a point-to-point manner. Appropriated changes, suggested by the Reviewers, have been introduced to the manuscript. The language is improved by all corrections made by reviewer 3.

The main places where the authors need to further modify the text are as follows. (Minor changes suggested are in the marked-up copy).

They all corrected.

Line 140. Introduce the concept here of amino acid deletions e.g. using the phrase ‘a deletion of G66 (G66del) and S76 (S67del)…’ See also lines 162 to 164, and line 240.

They all corrected.

Lines 144 and 145. Introduce the concept of stop codons in NS7b and NS8 - and the effect of the stop codons on what can be expected to be translated.

We added that stop codons lead to truncated proteins.

Line 171 is not clear what the authors are trying to say here. Perhaps it is that amino acid substitutions in NS3 deserve greater study in vitro.

It was re-written.

Lines 195 to 197. I have made suggestions for changes here in the marked copy.

Correction was made.

Line 211. It was not clear what the authors meant by ‘missense mutations’.

We wrote change in amino acid.

Line 243. A reference is needed.

Added.

Line 271. It was unclear what therapeutic effects were envisaged here.

It was not appropriate sentence, we removed it.

Line 276. Here the authors are presumably referring to amino acid substitutions in the nucleoprotein, not in the gene.

Corrected.

Line 277. A reference is needed.

Added.

Line 287. A reference is needed.

Added.

Lines 289 to 291. It is not clear what the authors are saying that termination of the NS8 gene somehow affects spike affinity for the receptor. The authors should elaborate on this.

We re-wrote it.

Attachment

Submitted filename: Response to Reviewers2.docx

Click here for additional data file.^{(14.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0267847.r005

Decision Letter 2

Etsuro Ito

18 Apr 2022

Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic

PONE-D-21-38222R2

Dear Dr. Mokhtari-Azad,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Etsuro Ito

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0267847.r006

Acceptance letter

Etsuro Ito

22 Apr 2022

PONE-D-21-38222R2

Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic

Dear Dr. Mokhtari-Azad:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Etsuro Ito

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(18.7KB, docx)}

Attachment

Submitted filename: Yavarian markup.pdf

Click here for additional data file.^{(1MB, pdf)}

Attachment

Submitted filename: Response to Reviewers2.docx

Click here for additional data file.^{(14.9KB, docx)}

Data Availability Statement

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PERMALINK

Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic

Jila Yavarian

Ahmad Nejati

Vahid Salimi

Nazanin Zahra Shafiei Jandaghi

Kaveh Sadeghi

Adel Abedi

Ali Sharifi Zarchi

Mohammad Mehdi Gouya

Talat Mokhtari-Azad

Roles

Abstract

Purpose

Methods

Results

Conclusions

1 Introduction

2 Materials and methods

2.1 Sample selection

2.2 Next generation sequencing (NGS)

2.3 Data & phylogenetic analysis

3 Results

3.1 Nonstructural proteins (ORF1ab)

Fig 1. The substitutions of nonstructural proteins during the five waves of SARS-CoV2 pandemic in Iran.

3.2 Structural proteins (S-E-M-N)

Spike

Table 1. Amino acid changes detected in spike glycoprotein during the five waves of SARS-CoV2 pandemic in Iran.

Envelope

Membrane

Nucleoprotein

3.3 Accessory proteins

First wave

Second wave

Third wave

Fourth wave

3.4 Phylogenetic analysis

Fig 2. Phylogenetic tree of SARS-CoV2 full-length genomes constructed by MEGA 7.

4 Discussion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Etsuro Ito

Roles

Author response to Decision Letter 0

Decision Letter 1

Etsuro Ito

Roles

Author response to Decision Letter 1

Decision Letter 2

Etsuro Ito

Roles

Acceptance letter

Etsuro Ito

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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