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 Table of Contents  
REVIEW ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 5  |  Page : 13-18

The importance of genomic changes of SARS-CoV-2 and its comparison with Iranian-reported COVID-19 sequencing; Whether each country has to design its treatment and vaccine production


1 Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Biotechnology, School of Advanced Technology in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission10-Jun-2020
Date of Acceptance15-Jul-2020
Date of Web Publication13-Aug-2020

Correspondence Address:
Prof. Parissa Farnia
Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_122_20

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  Abstract 


Coronavirus (COVID-19), a type of RNA virus, has a positive, single, sense stranded RNA. Studies of coronaviruses have shown high mutations in the virus's RNA. The more the virus infects people, the more the genome will mutate. In addition, evolutionary and genetic studies have the potential to recombine and easily jump from one host to another host. This means flexibility to adapt to new conditions and proliferation in new host cells. Examination of the hosts of this virus shows their extent. Studies show that the virus closely resembles hosts such as bats and pangolins. Genomic studies on severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and bat coronavirus (RaTG13) showed 96.2% genetic similarity although there are differences in the nucleotides of different hosts. The importance of examining mutated areas in coronaviruses for diagnosis and treatment is necessary because molecular methods such as RT-PCR, although having a specific probe, may not cause a connection between the primer and the specific probe and cause a false-negative response be reported. This issue has led various countries to sequence SARS-CoV-2 for their own countries, eventually leading to the identification of specific drugs and vaccines for their country.

Keywords: COVID-19, mutations, severe acute respiratory syndrome coronavirus-2


How to cite this article:
Velayati AA, Farnia P, Besharati S, Farnia P, Ghanavi J. The importance of genomic changes of SARS-CoV-2 and its comparison with Iranian-reported COVID-19 sequencing; Whether each country has to design its treatment and vaccine production. Biomed Biotechnol Res J 2020;4, Suppl S1:13-8

How to cite this URL:
Velayati AA, Farnia P, Besharati S, Farnia P, Ghanavi J. The importance of genomic changes of SARS-CoV-2 and its comparison with Iranian-reported COVID-19 sequencing; Whether each country has to design its treatment and vaccine production. Biomed Biotechnol Res J [serial online] 2020 [cited 2022 Jan 25];4, Suppl S1:13-8. Available from: https://www.bmbtrj.org/text.asp?2020/4/5/13/292074




  Introduction Top


COVID-19 is a viral disease caused by the infections with severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and was introduced as a pandemic on March 11, 2020, by the World Health Organization.[1] In addition to causing respiratory infections, coronaviruses can cause infections in the gastrointestinal tract. This virus classified into four major genera: Alpha coronavirus and Beta coronavirus can cause infection in mammals, and Gamma coronavirus and Delta coronavirus can cause infection in birds.[2]

The genome size of coronaviruses ranges between 26,000 and 32,000 bases and includes 6–11 open reading frames (ORFs). ORF1 is the largest amount in the genome and encodes 16 nonstructural proteins (NSPs). Other ORFs encode other proteins, including accessory and structural proteins. Proteins such as surface glycoprotein (S), envelope protein (E), matrix protein (M), and nucleocapsid protein (N) are among the most important structural proteins.[3] Genomic analysis performed on SARS-CoV-2 showed that it is a SARS-CoV-like β-lineage coronavirus. Further, this virus most likely originates from bats. Genomic studies on SARS-CoV-2 and bat coronavirus (Bat-CoV) (RaTG13) showed 96.2% genetic similarity although up to 1200 nucleotides may be different.[4]

Due to the change in the number and position of nucleotides in different hosts of COVID-19 and the possibility of mutation in this virus, either naturally after each amplification or acquired after transmission, it seems necessary to study the genome sequence of COVID-19 in each country.


  Classification and Characteristics of Covid-19 Type A, B, and C Top


Coronaviruses typically cause respiratory and enteric infections. This virus affects humans and animals. Six coronaviruses are known to have two species, SARS-CoV and Middle East respiratory syndrome coronavirus, which can cause severe respiratory illness.[5] In the case of classification between COVID-19, it is surprising that in addition to examining the sequence of the virus and genome organization, which represents the three types A, B, and C, some researchers have suggested that it can be based on cases such as course of treatment and prognosis of COVID-19 and also should be done in the absence or presence of comorbidities [Table 1].[6],[7],[8],[9]
Table 1: Investigating various aspects of COVID-19 classification

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Studies on three different types of coronavirus have shown that type A has two subclusters: one linked to Wuhan and one common in America and Australia. These two subclusters are distinguished by the synonymous mutation T29095C. Study by Forster et al. Showed that type A COVID 19 was the closest type to a bat, the “main genome of the human virus” in Wuhan. Surprisingly, Type A was not the dominant type in Wuhan. In the United States, mutant versions of “A” have lived in Wuhan, and a large number of type A viruses have been found in patients in the United States and Australia. Type “A” is closely related to the virus in bats and penguins and can be described as the “the root of the outbreak.”

There are two subclusters of A which are distinguished by the synonymous mutation T29095C (in N protein, codons TTT to TTC, silent mutation). Wuhan's major virus was type “B.” To examine type B, the genomes of patients in parts of Eastern China were sampled from neighboring Asian countries and in the non-East Asian region from the United States, Canada, Mexico, France, Germany, Italy, and Australia.[6] The N protein gene of the SARS-CoV-2 strain that found in the USA has three mutations (28881G > A, 28882G > A, and 28883G > C). Studies showed that the N protein from SARS-CoV is responsible for the formation of the spiral structure during virion assembly. In addition, it is important to make a vaccine because it may trigger an immune response.[10],[11]

It was shown that type B is obtained by two mutations from type A: the synonymous mutation T8782C (in ORF1a, codons AGT to AGC, serine to serine, silent mutation) and the nonsynonymous mutation C28144T (in ORF8b, codons TCA to TTA, serine to leucine, nonsilent mutation). Type B genome outside of Asia has evolved mutations. There is little chance that this phenomenon is due to a 1-month delay and a simultaneous mutation in the viral genome before it spreads outside China. However, it cannot be ignored that the type B virus is compatible with a large part of the East.[6],[12] Type C is the daughter of type B that obtained by the nonsynonymous mutation G26144T (in ORF3a protein) which changes a glycine to a valine. The nonsynonymous mutation G26144T distinguishes type C from type B.

Studies on the data set have shown that the type C is the main type in Europe and has been observed in countries such as the USA, France, Italy, Sweden, England, and Brazil. This type has not been observed in China, but there is evidence of its presence in some countries in Asians such as Hong Kong, Singapore, Taiwan, and South Korea.[6]

The important point in common treatments for patients with COVID-19 is that the drugs used to treat COVID-19 can vary depending on the treatment of different types of COVID-19 (type A, B, and C) in different countries and the patient's clinical conditions and clinical symptoms. Sixty patients were examined at Wuhan Tongji Hospital in China with three types A, B, and C. Patients with pneumonia were classified as type A. Antivirals, antibiotics, oxygen therapy, and glucocorticoids were used to treat them.

For the treatment of patients with COVID-19 type B, pneumonia monitoring, individual evaluations and development of special treatment programs such as treatment of hypoglycemia, treatment of hypertension are performed. COVID-19 type C seems to be due to exacerbation of type B and A disease and also Dissatisfied treatment of patients with COVID-19 type A occurs. When the initial therapeutic effects for the treatment of type A disease are not satisfactory, it leads to organ injuries.[13]

In a study conducted in Singapore, among 18 patients admitted with SARS-CoV-2 infection with PCR approval, 28% of patients had no clinical symptoms of fever, which is much higher than in China, which is about 1.6%–17%. Because most cases reported in Singapore are type C, it can be hypothesized that patients with type C could require less supplemental oxygen for treatment than patients with type A (the most common type in Wuhan in China). Further, fever in patients with type C in comparisons with type A and B was less observed.[14],[15],[16],[17]

Treatment results in 54 patients in Hunan, China, showed that treatment of patients with COVID-19 with interferon-α2b was effective, but novafron was not effective. There was no need for antibiotic treatment, and corticosteroids should be used with caution. All patients received oxygen therapy.[18] Due to the fact that patients in China improved with interferon-α2b, favipiravir, ribavirin, remdesivir, and galidesivir, it is possible that these drugs can be effective in the treatment of type B and A.

Different types of this virus have been identified in some countries, but some countries, such as Iran, do not have enough information about COVID-19 types. For better studies on this virus, it is necessary to analyze the data of specific patterns of different types. These patterns (type A, B, and C) can help identify, select the right medication, and treat COVID-19.[6]


  Investigation of the Presence of Severe Acute Respiratory Syndrome Coronavirus-2-Like Cov in Bat Coronavirus and Pangolin Species Top


The analysis SARS-CoV-2 showed that it is a SARS-CoV-like β-lineage coronavirus. Further, this virus most likely originates from bats (horseshoe bat). The horseshoe bat (Rhinolophus ferrumequinum) has a vast distribution from Western Palearctic to Eastern Asia, Southern Europe, and Northern Africa and through the Mediterranean Sea including all Central European large islands.[19]

Genomic studies on SARS-CoV-2 and Bat-CoV (RaTG13) showed 96.2% genetic similarity although up to 1200 nucleotides may be different. Due to the prevalence of horse breed bats in different cities of Iran, including West Azerbaijan, East Azerbaijan, Urmia, Zanjan, Ardabil and Khuzestan, there is a concern that in addition to human-to-human transmission, COVID-19 may also be transmitted through bats to human.

A study by Zhang et al. showed that, like bats, penguin species are a natural reservoir of CoVs such as SARS-CoV-2, which can host the SARS-CoV-2 interface to transmit the disease. Genomic and evolutionary findings of SARS-CoV-2-like CoV and pangolins show that penguins are similar to SARS-CoV-2 and Bat-CoV (RaTG13) 91.02% and 90.55% respectively. Worryingly, instead of bats, SARS-CoV-2 may once again spread around the world through pangolin.[12] Especially, because the first patient of coronavirus 2019 (COVID-19) did not report any exposure to the seafood market, SARS-CoV-2 was probably transmitted to humans by other animals. Finding the intermediate SARS-CoV-2 hosts is critical to preventing transmission between species.[20]

To confirm our assumption, we downloaded raw RNA sequencing data (SRA: PRJNA573298) for those two lung samples from the SRA and conducted consistent quality control and contaminant removal.[21] Examination of the samples mapped to the SARS-CoV-2 reference genome (GenBank: MN908947) showed that they covered 76.02% of the SARS-CoV-2 genome. Finally, the results showed that Malay pangolin may carry the CoV novel (here called Pangolin-CoV), which is similar to SARS-CoV-2.[22]


  Hcov-19 Genome Data Processing Top


The genome of COVID-19 reported from different laboratories and institute across the world has been sequenced and deposited to Global Initiative on Sharing All Influenza Data (GISAID) database and NCBI database. COVID-19 is a positive, single, sense stranded RNA virus. Several mutations that have been identified are shown in [Table 2].[6],[11],[23]
Table 2: Different characteristics of mutation of severe acute respiratory syndrome coronavirus-2

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Until July 5, 2020, 60,028 sequences of SARS-CoV-2 genome have been identified in patients with COVID-19 in different countries around the world and intermediate host saved in the GISAID database. Download the published SARS-CoV-2 genome Iranian (29,761–29,828 bp) from GISAID, with sequence numbers (virus names): EPI_ISL_424349 (hCoV-19/ Iran/HGRC-01-IPI-8206/2020), EPI_ISL_437512 (hCoV-19/ Iran/HGRC-2-2162/2020), EPI_ISL_442044 (hCoV-19/Iran/ KHGRC-2-2162/2020), EPI_ISL_442523 (hCoV-19/Iran/ KHGRC-1.1-IPI-8206/2020), EPI_ISL_445088 (hCoV-19/ Iran/KHGRC-3-2178/2020) For investigation of nucleotide. The patterns of codon used CLC Genomics Workbench (QIAGEN, Aarhus, Denmark) (https://www.qiagenbioinformatics.com/), and the Graph Pad prism software (GraphPad Inc., San Diego, CA, USA) used for correlation analysis. Investigation mutation in five nucleotide hCoV-19 in Iran with hCoV-19/Wuhan/WIV04/2019 shows several codons mutation such as: AGG to CGG (in ORF1ab, silent mutation), CGG to UGG (in ORF1ab, nonsilent mutation), GUA to UUA (in ORF1ab, nonsilent mutation), AGA to GGA (in ORF1ab, silent mutation), AAC to AAU (in S, silent mutation), UUU to CUU (in S, nonsilent mutation), UCU to CCU (in S, silent mutation), UUC to UUU (in N, silent mutation) [Table 3]. Analysis of five nucleotide sequences in the hCoV-19 population of Iran showed that variable genomics in genes such as nonstructure protein (Nsp) 2, Nsp16, spike, Nsp6, and Nsp14 S had more mutations than other genes. A study conducted by Wen et al. about nucleotide sequences showed results similar to us, but genes such as nsp1, nsp3, and nsp15 of ORF1ab and gene S had more mutations than other genes.[7] Due to the increase in mutations in the viral genome, more may occur in the near future.[23]
Table 3: Investigation mutation 5 whole genome sequence in Iran with first patient with hCov-19 in Wuhan

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  Genome Sequences Covid-19 Top


The virus genome has six main ORFs that are common to coronavirus and some other accessories gene. ORF1a/b (The first ORFs) about 70% (the two-third) of the whole genome length that encode 16 non-structure proteins (nsp1-16). Other ORFs About 30% (the one-third of the genome) encodes the main structural proteins: S, M, E, and N proteins. Special structural and accessory proteins encode by these four proteins.[24]

The genome structure of the SARS-CoV-2 is arranged in the order of 5'-replicas (ORF1ab)-structural proteins [S-E-M-N]-3' as shown in [Figure 1].[25] Analysis of the 2019-nCoV genes indicates that some of this gene shared <80% nucleotide sequence identity to SARS-CoV. However, the amino acid sequences of the seven conserved replicase domains in ORF1ab that were used for CoV species between 2019-nCoV and SARS-CoV had 94.4% identical sequences. Maybe, these two viruses belong to the same species, SARSr-CoV. An integrated study showed that a short region of RNA-dependent RNA polymerase from Bat-CoV (RaTG13) (previously detected in Rhinolophus affinis from the city of Greece) identified high sequence identities as 2019-nCoV has it. The close relationship between phylogeny and RaTG13 is evident that 2019-nCoV may have originated in bats.[12]
Figure 1: Genome organization of the SARS-CoV-2 genome compared to other betacoronaviruses

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  Nucleotide Sequence Statistics Top


Investigation of nucleotide sequence showed that EPI_ISL_445088, EPI_ISL_437512, EPI_ISL_442523 at the middle of sequence have less conservative between nucleotide 19,240 and 20,174 bp. Frequencies of C + G and A + T in three genomes were same, respectively, 10,972 bp and 17,853 bp. Nucleotides A, T, C, and G in hCoV19_iran_HGRC-2-2162_2020-1 were more than another [Table 4]. The nucleotide sequences between hCoV-19/Iran/KHGRC-3-2178/2020, hCoV-19/Iran/KHGRC-1.1-IPI-8206/2020, and hCoV-19/Iran/KHGRC-2-2162/2020 had 100% similarities. These three viruses had different length (bp). The homology among five viral strains from Iran was generally high. Information about location, host, patient statues, and specimen source is shown in [Table 5].
Table 4: Counts of nucleotides and frequency in 5 whole genome COVID-19 in Iran

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Table 5: Sequence information of 5 genome COVID-19 in Iran

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The reference sequence (hCoV-19/Wuhan/IVDC-HB-01/2019) from the GISAID database compared with several genome sequence of COVID-19 in Iran such as hCoV-19/Iran/ KHGRC-3-2178/2020 | EPI_ISL_445088|, hCoV-19/Iran/ KHGRC-1.1-IPI-8206/2020 | EPI_ISL_442523|, hCoV-19/ Iran/KHGRC-2-2162/2020 | EPI_ISL_442044|, hCoV-19/ Iran/HGRC-2-2162/2020 | EPI_ISL_437512|, and hCoV-19/ Iran/HGRC-01-IPI-8206/2020 | EPI_ISL_424349 | that showed several mutations. Mutations such as silent and nonsilent were observed in Iranian genomes. The hCoV-19/Iran/HGRC-01-IPI-8206/2020 EPI_ISL_424349 | 2020-03-09 virus has fissures at the beginning and end of the genome, while the hCoV-19/Iran/KHGRC-3-2178/2020 EPI_ISL_445088 | 2020-03-26, hCoV-19/Iran/KHGRC-2-2162/2020 EPI_ISL_442044 | 2020-03-26, and hCoV-19/Iran/HGRC-01-IPI-8206/2020 EPI_ISL_424349 | 2020-03-09 have the most gaps in the middle of the genome.


  The Reliability of the Rt-Pcr Molecule Method With the Mutation Occurring Top


Mutations occur in COVID-19 both naturally and in human-to-human transmission. These mutations may cause greater severity of the disease, different clinical symptoms, and problems in diagnosing with molecular methods such as RT-PCR. Due to mutations in areas such as ORF, S proteins, and N proteins in the coronavirus [Table 2], it is possible that the specificity of the primers, the specific probe, and the mutation in these areas may lead to the nonconnection of the primer or probe and the lack of PCR response, which leads to a false-negative result of the experiment.


  Phylogenetic Analysis Top


For genome analysis, we selected five published full-length human coronavirus genomes from Iran, several hCovid-19 in Asia, and bat, pangolin, tiger, cat, and one published full-length human coronavirus genomes from China. We identified the sequences of genome by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). CLC Main Workbench software (Qiagen Bioinformatics, CA, USA) was used for alignments of the sequences of genome, and phylogenetic trees were then inferred using likelihood method.

Arrows cluster A [Figure 2] shows that sequence Iran (hCoV-19_Iran_HGRC-01-IPI-8206) has homology with bat (hCoV-19_bat_Yunnan_RaTG13), bat (hCoV-19_bat_Yunnan_RmYN02_2019), pangolin (hCoV-19_pangolin_Guangxi_P4 L), and cat (hCoV-19_cat_France_53_2020). Cluster B [Figure 2] shows homology sequence between Taiwan (hCoV-19_Taiwan_2_2020), India (hCoV-19_India_1-27_2020), and Malaysia (hCoV-19_Malaysia_MKAK-CL-2020-5045_2020).
Figure 2: Phylogenetic analysis. CLC Main Workbench software was used for alignments of the sequences of genome and Phylogenetic trees was done by likelihood method

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The similarity of nucleotide sequences due to the proximity of these three countries to each other makes it possible to transfer as a result of travel to each of these countries. A Pakistani sequence study reported that the patient had traveled to Iran 5 days before sampling. The survey also showed similar sequences between Pakistan and Iran.


  Discussion Top


Because ORF1 and protein S are important structures of the virus, mutations in these areas may exacerbate the disease, and due to the use of these areas for primer design, the diagnosis may be false negative. Mutations in these and other important areas of the virus may increase the severity of the disease.


  Conclusion Top


This study showed the relationship of hCoV-19/Wuhan with hCoV-19/Iran on the genome. The mutations in five Iranian genomes were studied. The silent and nonsilent mutations were observed in the sequence of the genome. Most of mutations in hCoV-19/Iran with hCoV-19/Wuhan (the first hCoV-19 in Wuhan) were nsp2 and nsp16. Mutations can lead to more severe illness, different clinical symptoms, and problems with molecular diagnosis. Studies have shown that despite the similarities in some nucleotide sequences, they have mutations that can vary from country to country. As a result, it is likely that the vaccine produced in each country will be based on the characteristics of the coronavirus in that country.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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