key: cord-0773319-yf81o44d
authors: Al-Qaaneh, Ayman M.; Alshammari, Thamer; Aldahhan, Razan; Aldossary, Hanan; Alkhalifah, Zahra Abduljaleel; Borgio, J. Francis
title: Genome composition and genetic characterization of SARS-CoV-2
date: 2021-01-06
journal: Saudi J Biol Sci
DOI: 10.1016/j.sjbs.2020.12.053
sha: b96ea6d2d8cf55a27632155e28e05ffcf495fe01
doc_id: 773319
cord_uid: yf81o44d

SARS-CoV-2 is a type of Betacoronaviruses responsible for COVID-19 pandemic disease, with more than 1.745 million fatalities globally as of December-2020. Genetically, it is considered the second largest genome of all RNA viruses with a 5′ cap and 3′ poly-A tail. Phylogenetic analyses of coronaviruses reveal that SARS-CoV-2 is genetically closely related to the Bat-SARS Like-Corona virus (Bat-SL-Cov) with 96% whole-genome identity. SARS-CoV-2 genome consists of 15 ORFs coded into 29 proteins. At the 5′ terminal of the genome, we have ORF1ab and ORF1a, which encode the 1ab and 1a polypeptides that are proteolytically cleaved into 16 different nonstructural proteins (NSPs). The 3′ terminal of the genome represents four structural (spike, envelope, matrix, and nucleocapsid) and nine accessory (3a, 3b, 6, 7a, 7b, 8b, 9a, 9b, and orf10) proteins. As the number of COVID-19 patients increases dramatically worldwide, there is an urgent need to find a quick and sensitive diagnostic tool for controlling the outbreak of SARS-CoV-2 in the community. Today, molecular testing methods utilizing viral genetic material (e.g., PCR) represent the crucial diagnostic tool for the SARS-CoV-2 virus despite its low sensitivity in the early stage of viral infection. This review summarizes the genome composition and genetic characterization of the SARS-CoV-2.

As a human pathogen, coronaviruses (CoVs) have been drawing a great consideration as a global health threat. They are types of viruses that can attack many systems in humans and vertebrates, such as the respiratory system, digestive system, nervous system, and liver . It belongs to the Coronavirinae subfamily, a subset of the Coronavirdiae family and Nidovirales order . Genetically they are categorized into four distinct genera; Alpha, Beta, Gamma, and Deltacoronavirus . While bats and rodents consider the primary sources of alpha and Betacoronaviruses, the avian species are the primary sources of Gamma and Deltacoronaviruses (Z. W. Ye et al., 2020) .

Among four betacoronaviruses that can infect humans, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are considered the most serious pathogens able to cause severe lower respiratory tract infection. They are responsible for both the 2003 Severe acute respiratory syndrome (SARS) endemic and 2012 Middle East respiratory syndrome (MERS) endemic, respectively (Ahmed et al., 2020; Cui et al., 2019; Shereen et al., 2020) . Recently we had a new addition to the betacoronavirus family, a novel coronavirus-2019 (2019-nCoV) that has been named on February 12, 2019 by World Health Organization (WHO) as SARS-CoV-2, which is responsible for the pneumonia-like disease since December 2019 Gurwitz, 2020) . It is well documented and approved that these three betacoronaviruses have shown the possibility of animal to human and human to human transmission (J. Y. . As of today, December 25, 2020, around 79 million patients have been diagnosed with SARS-CoV-2 and 1.745 million deaths all over the world " n.d.) . In addition, the reported cases geographically vary and are related to local responses divergence of hosts. Yet, if the biological consequences of hosts are different due to alterations in the SARS-CoV-2 genome, this could lead to serious complications for combating the pandemic (Garvin et al., 2020) . Like any other Betacoronaviruses, Bats represent the primary host of SARS-CoV-2, which then transfers to a secondary amplification host (e.g., palm civets and Racoon dogs) before being able to infect humans (C. Shereen et al., 2020) . This review will discuss the genome composition and genetic characteristics of SARS-CoV-2, the mechanism of SARS-CoV-2 genome replication and translation, the function of different proteins expressed by these genes, and using molecular genetic techniques in diagnosis SARS-CoV-2.

Since1960s (the date of the first recognition of human coronaviruses), seven types of human coronaviruses have been identified (Seah and Agrawal, 2020) . They are classified based on their protein sequence into; Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Delta coronavirus. The alpha and betacoronaviruses are the two genera that infect mammals, whereas Gamma and Delta coronaviruses infect birds .

Generally, RNA viruses encode for proteins that are required to control the host cells. For instance, they encode for the protein types needed to assemble the new viral particles. Further, RNA viruses are well-known for their high mutation rate, which supports them to rapidly adapt in host cells (Manfredonia et al., 2020) .

Human coronaviruses, in general, share core genomic features, e.g., I. They are vast and highly conserved RNA genomes viruses. II. Genome structure is organized as a 5 0 -untranslated region (UTR)-replicase-S-M-N-UTR-3 0 with the genome function as a replicase gene. III. All family members have two enveloped (E) protein species, helical Nucleocapsid (N), spike (S) and membrane (M) proteins. They all share the expression of various nonstructural genes (Snijder et al., 2016) . In addition, they all are required to achieve an infectious cycle of replication, starting from entering, assembling, packaging, and releasing the new virus particle inside of the host (Hassan et al., 2020) . The two overlapping open reading frames (ORFs) ORF1a&1ab represent the two-third of the genome's upstream region (Fig. 1) . The general genetic characteristic that distinguishes Alphacoronavirus from other coronaviruses is that they have a unique type of nonstructural protein NSP1 that differ in sequence and size from other Betacoronavirus NSP1 (~9 kDa for Alphacornavirus and~20 kDa for Betacoronavisrus). In contrast, Gamma and Deltacoronaviruses lack NSP1 moiety (Jaimes et al., 2020) (''Virus Taxonomy À 1st Edition," n.d.). NSP1 protein plays a crucial role in inhibits host gene expression, and its location is essential for virus virulence (Shen et al., 2019) . [ Table 1 ] shows different genomic structures for human coronavirus genera (Fehr and Perlman, 2015; Jansson, 2013; Payne, 2017; Snijder et al., 2016) .

Scientists explored seven Coronavirus strains able to infect humans (HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2) (Arabi et al., 2020; Gaunt et al., 2010; Z.-W. Ye et al., 2020) . While HCoV-229E and HCoV-NL63 belong to alphacoronaviruses family and use different host proteins as a target receptor (HCoV-229E binds with host aminopeptidase N (hAPN) as a receptor, and HCoV-NL63 binds angiotensin-converting enzyme II (ACE 2) in addition to host hAPN as a receptor), the other five beta coronaviruses bind to different host receptors (HCoV-OC43 and HCoV-HKU1 use 9-O-acetylsialic acids as a receptor, SARS-CoV and SARS-CoV-2 bind ACE2 receptors, and MERS-CoV uses Dipeptidyl peptidase-4 (DPP4) receptor Z. W. Ye et al., 2020) .

Clinically, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 cause common cold-like symptoms (e.g., sneezing, fever, dry cough, sore throat, Dyspnea, Myalgia, and diarrhea), which may be progressed to pneumonia in cardiopulmonary and immunocompromised patients. However, SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic viruses and can cause respiratory system failure in immunocompromised or cardiopulmonary patients (Seah and Agrawal, 2020) . [ Table 2 ] represents the classification of different human coronaviruses with their animal reservoir, intermediate host, cellular receptors, and their main sign and symptoms.

Coronaviruses (CoVs) genome represents the second largest genome of all RNA viruses. It is a single-stranded, positive-sense RNA, with genome size ranging from 26 Kb to 32 Kb in length and has a cap and poly-A at its 5 0 and 3 0 tail, respectively Wu et al., 2020) . In general, the number of ORFs in the CoVs genome ranging from 6 to 15 ORFs (Song et al., 2019) . The 1a & 1ab ORFs represent the biggest gene in the coronavirus's genome and cover almost two-thirds of its entire genome. These two genes encode different NSPs like replication-transcription complex (RTC), which responsible for synthesizing and transcrib-ing the subgenomic RNA (sgRNA) (Gorbalenya et al., 2000; Hussain et al., 2005; Snijder et al., 2006) .

Transcription regulatory sequence that mediates the transcription process is located between ORFs in the sgRNA, and serves as a template for the mRNA synthesis (Sawicki et al., 2007) . The Frameshift mutation between ORF1a and ORF1b results in two polypeptide synthesis (pp1a and pp1ab) that will then be processed into 16 NSPs by the aid of either chymotrypsin-like proteases (3CLpro) or papain-like proteases with the main protease (Mpro) (Masters, 2006) . The remaining one-third of the coronaviruses genome is responsible for encoding at least four structural proteins like spike protein (S), nucleocapsid protein (N), envelope protein (E), and the membrane (M) proteins, besides some accessory proteins such as 3a/b, 4a/b, and Hemagglutinin-Esterase (HE) proteins (Hussain et al., 2005) . Sequence alignment of all CoVs genome illustrates an identity of 43% for structural proteins coding regions and 58% for nonstructural proteins coding regions. In comparison, the identity in the entire genome among all CoVs is about 54%. These results suggest that the structural proteins have more diversity than the other nonstructural proteins . Nonstructural protein 14 (NSP-14) is a 3 0 to 5 0 exoribonuclease enzyme distinctive for all CoVs. Its function is related to maintaining the whole RNA genome of CoVs and proofreading the replicationtranscription complex (Eckerle et al., 2010; Smith et al., 2013) . and National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Amino acid sequences of both N and E proteins in SARS-CoV-2 were found to have a~92% sequence identity (Azeez et al., 2020) .

SARS-CoV-2 genome consists of 15 ORFs coded into 29 proteins (Srinivasan et al., 2020) ; at the 5 0 terminal of the genome, we have ORF1ab and ORF1a that encode 1ab and 1a Polypeptide, respectively [ Fig. 1 ]. The 3 0 terminal of the genome represents four structural (spike, envelope, matrix, and nucleocapsid) and nine accessory (3a, 3b, 6, 7a, 7b, 8b, 9a, 9b, and orf10) proteins. [Table 3 ] represents the coding region, Base length, amino acid length, and functions of different ORFs in SARS-CoV-2, and [ Table 4 ] represents the coding region, amino acid length, and functions of different NSPs in the SARS-CoV-2 virus genome.

Phylogenetic analyses of CoVs [ Fig. 2 ] reveal that the SARS-CoV-2 is related to SARS-CoV and very closely related to both bat-SL-CoV ZC45 and bat-SL-CoV ZXC21. As previously reported, SARS-CoV and MERS-CoV are bat-originated (Cui et al., 2019) ; bats continue to be the prime suspected origin of SARS-CoV-2 since SARS-CoV-2 has a 96% resemblance of the whole genome identity with the bat coronavirus RaTG13 isolated from Rhinolophus affinis . However, there are some important differences in genomic factors between SARS-CoV-2 and RaTG13, including the burin cleavage near the junction of the spike (S) protein subunits; S1 and S2, induced by the insertion of amino acids residues (PRRA) (Coutard et al., 2020) . This insertion, which is absent in other related betaoronaviruses, may the reason for the increased infectivity of the virus (Zhang and Holmes, 2020) . Also, there is a decreased similarity between SARS-CoV-2 and RaTG13 in the receptor-binding domain (RBD) protein encoded by the (S) gene (Wrapp et al., 2020) .

This divergence region within the RBD corresponds to the receptor-binding motif (RBM), which determines the receptor binding specificity. All these variabilities, along with the fact that animal species like civets and camels generally host SARS-CoV and MERS-CoV before infecting humans, suggest the existence of an intermediate host between bats and humans for SARS-CoV-2 (Cui et al., 2019; Wong et al., 2020) . This suggestion opposes the early indication that SARS-CoV-2 can leaps directly from bats to humans . Full genome sequencing of coronaviruses isolated from Malayan pangolins (Liu et al., 2019) revealed 90% identity to RaTG13 and~91% to SARS-CoV-2 (Zhang and Holmes, 2020) . Although the percentage identity between SARS-CoV-2 and Pangolin-CoV is lower than that between SARS-CoV-2 and RaTG13, the S1 protein in Pangolin-CoV showed high similarity to the one in SARS-CoV-2 compared to that of RaTG13. Furthermore, the RBM segment is highly conserved in both SARS-CoV-2 and Pangolin-CoV, sharing all five key residues. Unlike RaTG13, where only one of the five residues is shared (Zhang and Holmes, 2020; Zhou et al., 2020) . Accordingly, the RBM introduced in SARS-CoV-2 might be resulting from a recombination event between Pangolin-CoV and RaTG13, and it is therefore unexpected that RaTG13 has obtained the same mutations found in the genome of Pangolin-CoV by random chance. The low nucleic acid identity between Pangolin-CoV and SARS-CoV-2 compared to the high conservation of amino-acid sequences suggests that the recombination events have developed a long time in the past, which enabled genetic drift to take place as the time passes . Also, it was discovered that the SARS-CoV-2 helicase amino acid sequence is similar to bat and SARS-like coronaviruses. . [Table 5 ] represent the percent similarity between different betacoronaviruses (Jaimes et al., 2020) . Once a virus is inside the body, it encounters strong immunologic responses that may trigger the virus to develop mutations to beat and bypass the immune system. Consequently, these mutations alter the virus's virulence, infectiousness, and transmission (Berngruber et al., 2013; Lucas et al., 2001) . A recent study on COVID-19 patients found a considerable level of viral diversity, with a median number of 4, suggesting rapid evolution of SARS-CoV-2 and a high frequency of mutations . It has been proposed that nucleotide substitution is a critical mechanism for evolving viruses in nature (Lauring and Andino, 2010) . Phan reported three deletions and nitty-three missense mutations within the whole genome of SARS-CoV-2 isolated from different numbers of infected patients. ORF1ab polyprotein and the 3 0 end of the genome were subjected to deletion. Apart from the envelope protein, the missense mutations were observed in the nonstructural and structural proteins. Three of these missense mutations were also in the RBD of the spike surface glycoprotein (Phan, 2020) . Moreover, Tang et al. analysis of 103 sequences of the SARS-CoV-2 genome revealed that the virus has evolved into two types; type S, which accounts for~30% of the strains, and type L accounts for 70%. Type L is derived from type S, and it is suggested that it is more aggressive and contagious than type S. However, the frequency of L type was high at the beginning of the outbreak in Wuhan, China but has decreased later (Tang et al., 2020) .

Mutations drive viral evolution and genome variability, facilitating virus scape from different immune systems and promoting antivirus drug resistance. In general, RNA viruses are characterized by a high mutation rate, which can sometimes reach up to a million times than their hosts (Pachetti et al., 2020) . For the SARS-CoV-2 virus, more than 10 thousand single nucleotide polymorphism (SNP) have been recognized and reported globally, with expected mutation rates ranging between (0.0001-0.01) substitutions per nucleotide site per cell infection (Cotten et al., 2014; Peck and Lauring, 2018) . These mutations are distributed between different genomic structure of the virus. e.g., mutations in Spike protein, nucleocapsid, ORF (7b, 8, 9b, and 14) , and RdRP (Badua et al., 2020) . Among discovered mutations, two of them located in spike protein have been reported to have a higher rate of transmissibility; D614G and N501Y (Discovered in UK) (Daniloski et al., 2020; Leung et al., 2020) . On the other hand, the newly discovered South African variant '501.V2' is characterized by N501Y, E484K, and K417N mutations in the S protein -so it shares the N501Y mutation with the UK variant, but the other two mutations are not found in the UK variant-but the impact of this variant is still under investigation (Tegally et al., 2020) . OMT, negatively regulates the innate immunity (Chen et al., 2011; Decroly et al., 2011; Shi et al., 2019) ADRP: adenosine diphosphate-ribose 1 00 -phosphatase; 3CLpro: 3C-like cysteine proteinase; RdRp: RNA-dependent RNA polymerase; NendoU: nidoviral endoribonuclease specific for U; OMT: S-adenosylmethionine-dependent ribose 2 0 -O-methyltransferase. 

Since the SARS-CoV-2 genome has the 5 0 methylated cap (where the nonstructural proteins are placed) and 3 0 polyadenylated side (where structural proteins reside), this allows direct translation after infection without needing an intermediate transcription stage. Also, the SARS-CoV-2 genome includes multiple ORFs that can be transcribed by several transcription regulating sequences (TRS). The viral life cycle begins when the virion enters the human respiratory tract and depend on the viral and epithelial cell membrane interaction. The infection initiates at the cell surface when the S1 subunit recognizes angiotensin-converting enzyme 2 (ACE2) as a targeted receptor or recognize exopeptidases as a key receptor for entry to the host cell. The entry mechanism depends on cellular proteases such as cathepsins and transmembrane protease serine 2 (TMPRSS2), and human airway trypsin protease (HAT) (Hoffmann et al., 2020) . The S1 subunit consists of two subdomains that can operate as receptor binding domains proteins (RBD); N-terminal domain (NTD) as a mediated sugar-binding and a C-terminal domain (CTD) as receptors recognition. . Mutated RBD of the S1 subunit is needed for crossspecies of SARS-CoV-2 transmitting. Once the binding occurs, conformational changes in the S protein, like pH reducing and/or S protein proteolysis, promote splitting of S1 from S2. Consequently, membrane fusion is placed by the S2 region (Han et al., 2020) . S2 subunit has two heptad repeat (HR) domains, (HR1) and (HR2), that interact together to form a six-helix bundle (6-HB) fusion core to approximate the viral and cellular interaction . The replicase/transcriptase genes encoded by ORF1a/1ab translate two polyproteins (pp1a and pp1ab) that are cleaved into individual 16 nonstructural proteins (nsp1 to 16) by the action of chymotrypsin-like cysteine proteases enzyme (3CL pro , nsp3); that control viral replication and it is crucial for SARS-CoV-2 life cycle and papine-like proteases enzyme (PL pro , nsp5 proteases) which has a crucial role in suppressing immune system by deubiquitylating the host cell proteins (Morse et al., 2020; Tahir ul Qamar et al., 2020) . RNA-dependent RNA polymerase (RdRp) that encoded by NSP12 plays a central role in the synthesis of a new genome molecule of viruses (replication), Also synthesis of subgenomic templates for messenger RNA production (transcription) . The genome replication and transcription take place at cytoplasmic membranes. The transcript proteins are inserted into the endoplasmic reticulum, and the Golgi apparatus then translated RNA packed inside the formed capsid. N protein forms the capsid while the envelope, membrane, and spike formed by E, M, and S proteins, respectively. Finally, the packed vesicles virus is released from the cell membrane by the exocytosis process (Shereen et al., 2020) .

There is no doubt that the presented outcome of the pandemic regarding morbidity is scary (Simmonds, 2020) . As the number of COVID-19 patients increased worldwide, there was an urgent need to find a quick and sensitive diagnostic tool as it is crucial for con-trolling the outbreak of SARS-CoV-2 in the community. World Health Organization (WHO) has recommended molecular testing methods like polymerase chain reaction (PCR) of respiratory tract samples for the identification and laboratory confirmation of COVID-19 cases (World Health Organization, 2020). By comparison with syndromic testing and the computed tomography (CT), molecular techniques are more suitable because of their ability to target and identify particular infectious agents. Developing the molecular techniques requires recognizing the pathogen genomics and proteomics composition and understanding gene expression changes within the host during and post-infection (Udugama et al., 2020) . Real-time reverse transcription-polymerase chain reaction (rRT-PCR), which is the most used diagnostic method for COVID-19 (''Real-time RT-PCR Primers and Probes for COVID-19 | CDC," n.d.), allows the genetic detection of SARS-CoV-2. Thus, various kits have been designed to reverse-transcribe the viral RNA genome to complementary DNA (cDNA) and amplify specific regions of the cDNA (Freeman et al., 1999; Kageyama et al., 2003) . Corman et al. have found three regions of the SARA-CoV-2 genome with conserved sequences, the RNA-dependent RNA polymerase gene in the ORF1ab region, the E and the N gene. Analytically, both RdRP and E genes provided high detection sensitiveness, while the N gene showed poorer sensitivity (Corman et al., 2020) . [ Table 6 ] represents some probes and primers used for SARS-CoV-2 detection via real-time PCR tests. Besides designing the reaction primers and probes, other factors must be optimized to achieve a proper and accurate RT-PCR assay, including reagent conditions, incubation time, and temperature (Wong and Medrano, 2005) . Also, selecting the appropriate controls is essential to ensure the results' reliability . In general, these controls can help identify the detrimental factors such as contamination and ensure pathogen recognition in the addressed samples. Thus, allowing for the detection of any false-negative or false-positive amplification. There are two main methods to perform RT-PCR, the one-step assay format where reverse transcription and amplification are combined into one reaction. This method can yield fast and reproducible results; however, it is challenging because it requires the optimization of both the reverse transcription and amplification, so they do not compete with each other. The other method is the two-steps assay, where the reaction occurs in two different tubes. This assay method is more precise than the one-step assay, yet it also consumes a longer time and needs the optimization of extra parameters (Bustin, 2002; Wong and Medrano, 2005) . Detection of SARS-CoV-2 through rRT-PCR assays requires collecting respiratory samples. The upper respiratory tract specimens, which involve nasopharyngeal swabs, oropharyngeal swabs, and nasal aspirates, are recommended for the initial diagnostic testing. However, the lower respiratory tract specimens, which involve sputum, Bronchoalveolar Lavage Fluid (BALF), and tracheal aspirates, are recommended when the patient is having a productive cough (''Interim Guidelines for Clinical Specimens for COVID-19 | CDC," n.d.). Yang et al. have reported that sputum, along with nasal swabs, are more accurate for SARS-CoV-2 diagnosis.

Further, in severe cases, BALF is necessary for detecting the viral RNAs to diagnose and monitor the viruses . Respiratory samples negative results do not eliminate the possibility of having the disease and may arise as results of poorly-handled sampling techniques or small viral amount in the sampled area (Ai et al., 2020; Winichakoon et al., 2020) . Like any other diagnostic tool, RT-PCR for SARS-CoV-2 diagnosis has some drawbacks, such as the shortage of the kits with the increased demand, its inability to detect prior infection in recovered patients who showed no symptoms of the disease, and requiring advanced equipment that are often not available in resource-limited regions. However, other alternative nucleic acid tests may overcome these limitations (X. Li Table 5 Genome sequence similarity among beta-coronaviruses. There are different ways to detect amplifying DNA either by turbidity (induce by-products of the reaction), fluorescence (a fluorescent dye binds to doublestranded DNA), or color (pH-sensitive dye), and observed either on colorimetric detection or agarose gel electrophoresis. However, when comparing the two genes, it showed that the synthetic DNA substrates are slower in amplification and recognition than the RNA pattern, confirming that RNA is proficiently transformed to cDNA through the reverse transcriptase and amplified by the DNA-dependent DNA polymerase. The result was completely comparable with the real-time detection. Also, cell lysate has been tested, and the detection sensitivity was as the synthetic RNA alone (Zhang and Holmes, 2020) . Loop-mediated isothermal amplification (LAMP) assay is rapid, conducted at a single PCR, and does not require high-priced reagents or devices (Udugama et al., 2020) . There are disadvantages to LAMP, such as the challenge reaction condition, designing primers, and the difficulty with designing the readout device. The RT-LAMP assay has been improved using a quenching probe (QProbe) to reveal signs, which has a good performance as an RT-PCR assay (Ozma et al., 2020) . On the other hand, the microarray test has been used broadly in detecting SARS-COV-2, in which the Viral RNA will produce categorized cDNA that has precise probes over reverse transcription. Those labeled cDNAs are loaded into a solid-phase oligonucleotide, which has hybridized and fixed on the microarray accompanied to remove unfastened DNAs through washing steps. The viral RNA can be distinguished through the recognition of specific probes. Luna et al. had designed a nonfluorescent low-value, low-density oligonucleotide array that can detect the whole genome, with sensitivity equal to the RT-PCR (De Souza Luna et al., 2007; Hardick et al., 2018) . Furthermore, thinking about the fast mutation of SARS-CoV 2, Guo et al. advanced the microarray test to discover mutations in 24 single nucleotide polymorphism (SNP), as well as a few mutations in the spike (S) gene (Guo et al., 2014; Shi et al., 2003) . Also, the next-generation sequencing (NGS) and electron microscope technology has a role in the initial diagnosis, which has been developed in current years and made excessive progress in the fast recognition of SARS-COV-2 via RNA-Seq. NGS can sequence millions of DNA fragments by using random primers RNA-Sequences (RNA-Seq). Frequently, most RNA-Seq are from cellular RNA, but some may be from the viral genome. Consequently, RNA-Seq is used to identify RNA viruses. NGS is currently an ideal method for virus detection to verify unbiased sequencing of bat CoVs, bearing in mind its high genetic diversity. This method is highly effective in reducing sequence cost by increasing detection sensitivity (Shi et al., 2003) . Moreover, SHERLOCK method used Cas13a ribonuclease for RNA sensing. Cas13a can be activated when the RNA guide sequence binds to an amplified RNA product, and then probes are cleaved to generate a fluorescent sign (Guo et al., 2014) .

SARS-CoV-2 via utilizing molecular techniques and based on its genetic material, represents a new addition to the betacoronavirus family. It is genetically related, with more than 96% identity, to bat-SARS-Like-coronavirus. Although it is highly conserved, new strains and mutations appeared in different parts of the world, revealing the virus's capability to adopt different environmental conditions to survive. As of today, no curable treatment or prophylactic vaccine available for this virus, which makes the success in resolving this pandemic depends on the people awareness of adopting proper safety measures to stop viral dissemination and the availability of new diagnostic tools able to detect the virus on its early stage in more efficient-fast way.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements

The Molecular Biology of Coronaviruses

Characterization of accessory genes in coronavirus genomes

Discovery of an RNA virus 3 0 ?5 0 exoribonuclease that is critically involved in coronavirus RNA synthesis

Sars-cov-2 orf8 and sars-cov orf8ab: Genomic divergence and functional convergence

Learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-nCoV

Host-membrane interacting interface of the SARS coronavirus envelope protein: Immense functional potential of C-terminal domain

Advice on the use of point-of-care immunodiagnostic tests for COVID-19: scientific brief

Clinical manifestation, diagnosis, prevention and control of SARS-CoV-2 (COVID-19) during the outbreak period

Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant

Family Coronaviridae, in: Viruses

Complexities of viral mutation rates

Coronavirus 229E-related pneumonia in immunocompromised patients

Genetic diversity and evolution of SARS-CoV-2

Gene of the month: The 2019-nCoV/SARS-CoV-2 novel coronavirus spike protein

Real-time RT-PCR Primers and Probes for COVID-19 | CDC

A contemporary view of coronavirus transcription

SARS coronavirus accessory gene expression and function

Can the Coronavirus Disease 2019 (COVID-19) Affect the Eyes? A Review of Coronaviruses and Ocular Implications in Humans and Animals

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence

Genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease 2019

COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses

PEDV nsp16 negatively regulates innate immunity to promote viral proliferation

Design and application of 60mer oligonucleotide microarray in SARS coronavirus detection

Pervasive RNA secondary structure in the genomes of SARS-CoV-2 and other coronaviruses

SARS-CoV-2 E protein is a potential ion channel that can be inhibited by Gliclazide and Memantine

Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics

The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing

Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex

From SARS to MERS, thrusting coronaviruses into the spotlight

Structural genomics of SARS-CoV-2 indicates evolutionary conserved functional regions of viral proteins

Chimeric exchange of coronavirus nsp5 Proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity

Structural basis of SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal plants

Characterization of viral proteins encoded by the SARS-coronavirus genome

Severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mRNA

The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

lineage with multiple spike mutations in South Africa

Diagnosing COVID-19: the disease and tools for detection

Development of one-step, real-time, quantitative reverse transcriptase PCR assays for absolute quantitation of human coronaviruses OC43 and 229E

Coronavirus Pathogenesis

Negative nasopharyngeal and oropharyngeal swabs do not rule out COVID-19

Evidence of recombination in coronaviruses implicating pangolin origins of nCoV-2019

Real-time PCR for mRNA quantitation

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Laboratory testing of SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV): Current status, challenges, and countermeasures

Evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-nCoV infections

Zoonotic origins of human coronaviruses

Zoonotic origins of human coronaviruses

The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19

Dimerization of coronavirus nsp9 with diverse modes enhances its nucleic acid binding affinity

Insights into SARS-CoV transcription and replication from the structure of the nsp7-nsp8 hexadecamer

Structural and biochemical characterization of endoribonuclease Nsp15 encoded by middle east respiratory syndrome coronavirus

Biological, clinical and epidemiological features of COVID-19, SARS and MERS and AutoDock simulation of ACE2

A genomic perspective on the origin and emergence of SARS-CoV-2

A pneumonia outbreak associated with a new coronavirus of probable bat origin