key: cord-0812717-3c8of563
authors: Rahimi, Azadeh; Mirzazadeh, Azin; Tavakolpour, Soheil
title: Genetics and genomics of SARS-CoV-2: A review of the literature with the special focus on genetic diversity and SARS-CoV-2 genome detection
date: 2020-09-30
journal: Genomics
DOI: 10.1016/j.ygeno.2020.09.059
sha: f4676daf08df66dd9f6cbeb06e28a4cba620face
doc_id: 812717
cord_uid: 3c8of563

The outbreak of 2019-novel coronavirus disease (COVID-19), caused by SARS-CoV-2, started in late 2019; in a short time, it has spread rapidly all over the world. Although some possible antiviral and anti-inflammatory medications are available, thousands of people are dying daily. Well-understanding of the SARS-CoV-2 genome is not only essential for the development of new treatments/vaccines, but it also can be used for improving the sensitivity and specificity of current approaches for virus detection. Accordingly, we reviewed the most critical findings related to the genetics of the SARS-CoV-2, with a specific focus on genetic diversity and reported mutations, molecular-based diagnosis assays, using interfering RNA technology for the treatment of patients, and genetic-related vaccination strategies. Additionally, considering the unanswered questions or uncertainties in these regards, different topics were discussed.

In the late December 2019, a novel virus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), was reported with an unidentified source. [1] The genomic sequence of this newly emerged virus is highly similar to that of severe acute respiratory syndrome coronavirus (SARS-CoV) with a 79.6% sequence identity. [2] The SARS-CoV-2 causes coronavirus disease 2019 (COVID-19), a respiratory tract infection, with a clinical spectrum ranging from asymptomatic to acute respiratory distress syndrome. On March 11, 2020, COVID-19 was declared as a global pandemic and the sixth public health emergency worldwide by the World Health Organization (WHO). During the recent months, some antivirals (e.g., remdesivir) and anti-inflammatory medications (e.g., tocilizumab) showed acceptable outcomes to viral clearance and amelioration of patients' symptoms. [3, 4] Additionally, following the initial release of some researches for coronavirus vaccine, the spectrum of hope moves from cure to hope for prevention. As of September 19, 2020, 27 vaccines are in Phase 1, 15 in Phase 2, nine in Phase 3, and five have been approved for limited use. [5, 6] However, no approval has been granted by the United States Food and Drug Administration (FDA) so far.

Understanding the genetics of SARS-CoV-2 may open some new avenues for the development of more effective and safer targeted therapies and even new generations of vaccines for this highly mutable coronavirus. In this review, we tried to cover the most critical and up-to-date findings related to the genetics of SARS-CoV-2 to be used for developing the current strategies of diagnosis, and treatment platforms. each mutation in the identical sequences causes a sharp increase in Shannon entropy, and also to omit biases in collection time and location in Shannon entropy analysis. [38] In a recent study, ten hotspot mutations including D614G (23403A>G) on S, L84S (28144T>C) on ORF8, S5932F on nsp14, M5865V on nsp13, L37F (10818G>C) on nsp6, T85I (1059C>T) of nsp2, Q57H (25563G>T) on ORF3a, G251V (26144G>T) on ORF3a, R203K (28881G>A) on N, as well as G204R (28883G>C) on N with a frequency of over 0.10 were reported in SARS-CoV-2 genomes. [35] Due to the role of more mutable regions in viral replication, transmission, and the induced immune responses accordingly, further surveys required to determine the effects of these mutations in the outbreak of SARS-CoV-2. [30, 38, 43] Compared to the genome sequence of SARS-CoV-2 isolated from Asia, coinciding mutation 14408C >T (P323L) on nsp12 with more spot mutations was shown in European viral genomes. [26] Reportedly, co-mutations 241C>T (in 5′ UTR) with 3037C>T (F105F), 23403A>G (D614G), and 28144T>C (L84S), as well as 8782C>T (S75S) with 28144T>C (L84S) and 18060C>T>C (L6L), were found. Also, the leader sequence mutation 241C >T tends to coincide with three mutations including 3037C > T (F105F), 14408C >T(P323L), and 23403A>G (D614G) in the virus isolates extracted from European population with a high COVID-19 infection rate reports, thus, these four comutations may have a pivotal role in raising virus transmission. [31] In a recent study, the co-occurrence of 8782C > T, 29095C > T, and 28144T > C variants were observed in various samples. Moreover, both single-base substitutions (8782C > T and 29095C > T) are synonymous variants; in contrast, 28144T > C caused a nonsynonymous substitution, where lysine was replaced with serine (L84S) on ORF8. [14] Since publishing complete SARS-CoV-2 sequences, various studies have been conducted to categorize globally circulating SARS-CoV-2 population, based on mutation profiles. [41, [50] [51] [52] For instance, a recent study on the genomic distribution of SARS-CoV-2 mutations has grouped detected mutations into five distinct clades, G (241C>T, 3037C>T, 14408C>T, and 23403A>G), GH(241C>T, 3037C>T, 14408C>T, 23403A>G, and 25563G>T), GR (241C>T, 3037C>T, 14408C>T, 23403A>G, and J o u r n a l P r e -p r o o f 28881GGG>AAC), S (8782C>T and 28144T>C), and V (11083G>T and 26144G>T), each clade being identified by a specific set of mutations. Considerably, clades allow tracking of the SARS-CoV-2 genetic diversity and prevalence over time. [33] There is presently little evidence on the clinical and molecular effects of mutations detected in the SARS-CoV-2 genome. The results of several preliminary studies suggested a strong link between raising the case fatality rate with the proportion of viruses bearing mutation 23403A > G (D614G), a mutation outside the receptor-binding domain (RBD), over a time period in different regions, as well as the association of this variant with viral infectivity and greater transmissibility. [53] [54] [55] [56] [57] [58] Using in silico methods, the effect of mapped mutations R408I, L455Y, F486L, Q493N, Q498Y, N501T on RBD, and A930V, D936Y on heptad repeat 1 (HR1) domain identified deleterious and cause instability of spike glycoprotein. [59] Furthermore, the substitution of proline to leucine (P323L) caused by the 14408C>T mutation on RdRp appears to increase mutation rates. The results of studies have suggested that this mutation leads to rigidify the RdRp protein structure and subsequently change interactions of RdRp with other components of the replication or transcription machinery. [26, [60] [61] [62] In addition to P323L, A97V and A185V mutations on RdRp have also been reported to alter the secondary structure of the protein. [62] According to a cohort study, COVID-19 patients with a 382-nucleotide deletion on ORF8 have reportedly illustrated a milder infection compared with patients infected with wild-type virus only. [63] palindromic repeats/CRISPR associated (CRISPR/Cas)-based approach have been employed for genetically detection of SARS-CoV-2. [64] 

Real-time RT-PCR, which has been recommended by WHO could be applied as the effective, straightforward, and gold standard method for the detection of SARS-CoV-2 genome in respiratory secretions, serum, stool, sputum, or ocular secretions, [65] [66] [67] although a study indicated that sampling by nasopharyngeal swab is associated with more accurate results than other types of samples. [68] Generally, RT-PCR has been performed as a one-step or two-step format. In the one-step procedure, which is also preferred to identify SARS-CoV-2, a single tube is used for the whole RT-PCR reaction. This method has been considered in SARS-CoV-2 detection due to the possibility of quick start-up as well as reducing the possibility of contamination and pipetting technique errors. [69, 70] However, the possibility of false-negative results is considered to be one of the major challenges in real-time RT-PCR test, [71] which has been reported to range between 21% and 67%. [72] [73] [74] [75] [76] The most important suggested underlying causes include thermal inactivation, storage time and temperature of specimen preservation, inadequate viral material, laboratory error, and test sample transfer limitation. [77, 78] It was also suggested that inappropriate timing of sample collection and deficiency in sampling technique, especially of nasopharyngeal swabs are other reasons for false-negative results of real-time RT-PCR. [79] Furthermore, mutations in the primer-and probe-target regions caused by the rapid evolution of the SARS-CoV-2 genome, infection routes, issues related to sampling (timing and method), and co-infection with other viruses would probably have specific effects on RT-PCR test accuracy. [71, 72, 80] According to the need to test asymptomatic patients or test before quarantine release as well as the high false-negative rate of RT-PCR for detection of SARS-CoV-2, digital, and digital-droplet PCR methods have received attention due to their high sensitivity. In a study, digital PCR has demonstrated 96.3% accuracy for SARS-CoV-2 detection J o u r n a l P r e -p r o o f from pharyngeal swab samples and identified four patients' samples, which were considered negative based on the RT-PCR test. [81] It was shown that digital-droplet PCR capability for SARS-CoV-2 detection with lower minimum detection range is 500 times more sensitive than RT-PCR. [82] The use of the latest laboratory standards, quality sampling at different times from multiple sample types, and selection of RT-PCR kits with the lowest rate of reported falsenegative results can effectively prevent the occurrence of false-negative results. Moreover, considering real-time RT-PCR results along with clinical features, especially computed tomography imaging, can be impactful in the timely diagnosis of SARS-CoV-2 infection and COVID-19 management. [83, 84] According to the association between the quality of specimens and satisfactory RT-PCR results, evaluating cellular content and considering an internal reference such as RPP30 has been suggested. Indeed, the reliability of the SARS-CoV-2 RNA test and detection of false-negative results could be assessed using the cycle threshold cutoff values of RPP30 RT-PCR. [85] Until now, myriads of real-time RT-PCR kits with different qualities and methods of optimization have been introduced for SARS-CoV-2 detection by clinical laboratories and companies around the world, [84, 86, 87] Also, many SARS-CoV-2 primers and probes were designed and published by WHO as well as many laboratories worldwide. Employed primers and probes address different SARS-CoV-2 genomic regions target, including ORF1ab or ORF8 regions as well as E, N, S, and RdRP genes. [88] [89] [90] [91] [92] Additionally, a two-target system with one universally primer for detection of diverse coronaviruses and a second primer set for the only identification of SARS-CoV-2 can be employed in RT-PCR assay. [1, 91, 93] 

J o u r n a l P r e -p r o o f

As another diagnostic technique, RT-LAMP is considered as a rapid, effective, and one-step nucleic acid amplification method used to identify different viruses including influenza virus, MERS-CoV, Ebola virus, Zika virus, yellow fever virus, and West Nile virus. [94] [95] [96] [97] This method is performed at a constant temperature using DNA polymerase and four to six specific primers to detect six to eight sequences of the target gene in the only 60 minutes. [98] Recently, RT-LAMP has been applied as an alternative test for SARS-CoV-2 detection. [99] [100] [101] [102] Compared with the RT-PCR method, the RT-LAMP assay is faster, easier, more cost-effective as well as less time-consuming for diagnosing COVID-19. [103] Besides, the sensitivity of this assay has been reported to be similar to the RT-PCR assay. [93] Importantly, the RT-LAMP method can be used as a point-of-care diagnostic test. load. Additionally, they developed direct swab-to-RT-LAMP assay, a simplified version of RT-LAMP without the need for prior RNA isolation. [104] In different studies, in order to use RT-LAMP assay to diagnose SARS-CoV-2 in patients, several primer sets have been designed that target the ORF1ab, nsp3, S, and N genes/regions for optimization of the assay, which are summarized in supplementary Table 1 .

As another alternative approach, metagenomic next-generation sequencing (mNGS) allows unbiased detection of pathogenic genomes for epidemiological aims, as well as identification of co-infections that may lead to increase morbidity and mortality in emerging infectious diseases. [106] Additionally, this offers an opportunity to identify unknown pathogens with sudden onset in a particular area, similar to the Ebola virus in West Africa. [107] In recent studies, mNGS has been reported as a feasible and efficient approach for detecting a wide range of pathogens, especially the SARS-CoV-2 in clinical samples. [108, 109] Moreover, the amplicon-based metagenomic sequencing approach which is a combination of amplicon-based sequencing and metagenomic sequencing has been utilized to recognize the genome sequence of SARS-CoV-2 and the other microbiome from nasopharyngeal swabs of patients with COVID-19. [108] J o u r n a l P r e -p r o o f 4.5. CRISPR/Cas systems CRISPR/Cas systems are recently characterized as rapid and versatile platforms for nucleic acid detection. [109] Further, the analysis of the results of CRISPR-based methods can be done using fluorescent, lateral flow strips, or agarose gels in less than an hour. [110, 111] Compared to the PCR strategies, CRISPR-Cas diagnostic tools are significantly more sensitive and less costly than PCR-based approaches. [112] Additionally, in a study on the performance of the three technology platforms including mNGS, RT-PCR, and CRISPR in identifying the SARS-CoV-2, results indicated a great sensitivity and specificity of CRISPR-based assay for SARS-CoV-2 detection in clinical specimens with a shorter turn-around time than other methods. [113] Since the beginning of the COVID-19 outbreak, several CRISPR-Cas-based platforms have been developed to detect the SARS-CoV-2, which are compared in Table 2 . For instance, using a combination of CRISPR-Cas12-based assay and LAMP, Broughton et al. were able to reduce the SARS-CoV-2 detection time to around 30 minutes, while maintaining a relatively low sensitivity (10 copies of RNA per ??L). [114] In contrast, All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR ) platform was able to detect 4.6 copies of SARS-CoV-2 RNA per ??L of input in about one hour. [115] 

In contrast to the singleplex reaction, in the multiplex diagnostic methods, more than one viral gene can be detected in a short time, which makes these methods acceptable for routine diagnostic tests. Furthermore, the multiplex approach has some advantages including increased sample throughput, as well as a reduced amount of sample required and turnaround time than a singleplex diagnostic approach. [116] Until now, several multiplex assays including FTD21 kit, [117] GeXP assay, [118] Qiagen ResPlex II V2.0 kit, [119] and FilmArray multiplex PCR system [120] have been developed for the detection of respiratory viruses as well as some common human coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) in J o u r n a l P r e -p r o o f particular. According to the operation assessment results of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, this assay has shown high specificity (98.4%) and considerable concordance with the reference laboratory for the identification of SARS-CoV-2 by the ORF1ab gene. [121] The QIAstat-Dx Respiratory SARS-CoV-2 panel (QIAstat-SARS) has also been indicated the ability to detect 1000 copies per milliliter with 100% sensitivity and 93% specification by targeting the E and ORF1ab genes from SARS-CoV- which has been shown to be useful for COVID-19 screening. [124] In addition, HCR technology features including the lack of thermal cycler or enzymes need for amplification, as well as the possibility of testing at room temperature, have made HCR technology as a suitable method for quick detection of some viruses such as the hepatitis B and influenza A. [125] J o u r n a l P r e -p r o o f

RNA interference (RNAi) is a molecular process in which small noncoding RNAs (of endogenous or exogenous origin) match with the target mRNA in a sequence-specific manner and silence its expression. [126] During the past decade, based on the therapeutic potential of RNAi and their abilities to limit gene expression, many of them have been investigated and employed for medical application. NucB1000 against four separate targets (Pre-C, Pre-S1, Pre-S2, and X) of chronic hepatitis B virus (phase I), [142] SPC3649 for blocking miR-122 and its interaction with hepatitis C virus RNA (phase II), [143] pHIV7-shI-TAR-CCR5RZ for targeting several human immunodeficiency virus genes (Tat, Tar, and CCR5) (phase I), [144] and TKM-Ebola against some transcripts (L, VP24, and VP35) of Ebola virus (phase I). [145] J o u r n a l P r e -p r o o f [152] In another study, two siRNAs targeted the SARS-CoV genome at S and nsp12 regions, which led to the depletion of fever caused by the virus as well as SARS-CoV viral loads. Likewise, it reduced acute diffuse alveoli damage in Rhesus macaques. [140] Furthermore, the previous study reported the N protein of SARS-CoV as a VSR in mammalian cells by a cellular reversal-of-silencing assay regarding viral infection. [153] 

In order to target the RNA genome of SARS-CoV-2 and as the first step in the production of antiviral siRNAs, Chen et al. [154] suggested nine potential siRNA targets with 21-25 nucleotides in length within ORF1ab, S, ORF3a, M, and N regions of the SARS-CoV-2 genome and detected those regions as conserved areas. In another study, considering siRNA as a potential therapy for COVID-19, nsp5, and nsp12 were introduced to be targeted using siRNA based therapeutics. [147] Besides, in a study related to the interaction between antiviral RNAi immunity and SARS-CoV-2 has been reported structural protein N of the SARS-CoV-2 as an indicator of viral suppressor of RNAi (VSR) activity, which is a challenge in developing siRNA therapeutic and a factor which opposes the antiviral activity of RNAi in different steps. [155] J o u r n a l P r e -p r o o f A recent study found that host cell entry of SARS-CoV-2 is contingent on ACE2, as well as the transmembrane protease, serine 2 (TMPRSS2) produced by the host cell is needed to cleave and activate spike protein in the invasive process of the SARS-CoV-2. [156] Therefore, siRNA targeting TMPRSS2 and ACE2 can be considered as therapeutic options for blocking SARS-CoV-2 infections, as shown in Figure 2 . Regarding the strategy of inhibiting viral entry for antiviral treatment, Lu et al. [157] reported a reduction in the replication of SARS-CoV in the ACE2-silenced cells using siRNA technology. However, there is a concern that the selective elimination of ACE2 in vulnerable organs to SARS-CoV-2 may have unanticipated outcomes because ACE2 has critical roles in a variety of pathological and physiological processes. [158] Moreover, siRNA targeting of TMPRSS2 has formerly indicated a significant decrease in SARS-CoV entry into Calu-3 cells. [159] Figure 2 briefly shows the use of RNAi as a preventive or therapeutic agent against SARS-CoV-2.

In viral infections, many known human miRNAs appear to play an important role in regulating immune responses and interfering with functions of viral genes including reproduction, translation, and expression by targeting viral genes. Consequently, miRNA targeting can be considered as an antiviral therapy approach. [160] In a recent computational study, a list of predicted possible host genes targeted by viral miRNAs and viral genes targeted by cellular miRNAs was presented by computational analysis to understand the basic mechanisms of SARS-CoV-2 infection. [161] Additionally, in a recent study, six antiviral host-miRNAs comprising hsa-let-7a, hsa-miR101, hsa-miR126, hsa-miR23b, hsa-miR378, and hsa-miR98 were predicted to act on nsps, S, and N genes of SARS-CoV2 by cleaving their target sites or translation inhibition. The association between these miRNAs and several viruses, including Hepatitis C virus, Herpes simplex virus 1, and Enterovirus 71 has already been identified. [44] Moreover, in a current study on the prediction of miRNAs in SARS-CoV-2 genomes, seven miRNAs, including miR-8066, miR-5197-3p, miR-3611, miR-3934-3p, miR-1307-3p, miR-3691-3p, and miR-1468-5p were introduced that J o u r n a l P r e -p r o o f were related to viral pathogenesis and host response using KEGG pathway analysis. [162] In another in silico prediction-based study, the SARS-CoV-2 genome was searched for finding virus-encoded miRNA seed sponges that could bind to human miRNA seed sites and subsequently disrupt the interaction of host miRNA with their native targets. Nearly 80 human miRNAs that can interact with the SARS-CoV-2 genes have been identified. According to the literature, these miRNAs are related to pulmonary and cardiac disorders. [163] 

The past few years have witnessed a surge in the development of highly efficacious delivery materials for nucleic acids with some remarkable results. Following viral sequencing technology, nucleic acid therapeutics have emerged as promising alternatives to conventional vaccine approaches. Regarding previous strains of coronavirus, some studies had been performed to develop DNA-vaccines. [166, 167] One of the most successful DNA-based vaccines for MERS is INO-4700, which led to durable neutralizing antibodies and T cell immune responses through targeting MERS-CoV S protein. 

RNA-based therapies and vaccines have remarkable potential for the treatment and prevention of a wide range of diseases. [172] RNA vaccines offer a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development, safe administration, and low-cost production. However, their application has been restricted until recently by the instability and inefficient in vivo delivery of mRNA. [173] Gradually, some techniques served to reduce toxicity and improve the translation of the mRNA. Various modifications including the incorporation of modified nucleosides (particularly modified uridine), optimization of coding sequences, and stringent purification of in vitro transcribed mRNA by high-performance liquid chromatography have been applied to remove doublestranded RNA contaminants. [174] Given that the 5' and 3' UTRs of mRNA can significantly influence the rate of translation and half-life of the transcript, optimization of the UTRs is of paramount value in the design of mRNA vaccines. [173] As of September 2020, the phase III, (NCT04470427) clinical trial of a novel lipid nanoparticleencapsulated mRNA-based vaccine, mRNA-1273, encoding the S protein of SARS-CoV-2, began in the United States by the Moderna Inc. [175] The German company BioNTech has recently entered into collaborations with Pfizer, based in New York, and the Chinese drug maker Fosun Pharma to develop their mRNA vaccine. They found that one version, called BNT162b2, produced significantly fewer side effects, such as fevers and fatigue, and so they chose it to move into Phase 2/3 trials. On September 12, the companies announced the launch of a Phase 2/3 trial with 43,000 volunteers in the United States and other countries including Argentina, Brazil, and Germany. [171] J o u r n a l P r e -p r o o f In addition to these two RNA-based vaccines, which are entered into the clinical trials, there are at least sixteen other ongoing studies in pre-clinical evaluation (based on WHO's report on September, 2020). In addition to the aforementioned active clinical trials, there is a competition between those in pre-clinical for entering the clinic. Imperial College London, which has developed a "self-amplifying" RNA vaccine with the aim of boosting the production of a viral protein to stimulate the immune system. They begun Phase I/II trials on June 15, and have partnered with Morningside Ventures to manufacture and distribute the vaccine through a new company called VacEquity Global Health. Also, CureVac is working on a similar vaccine and launched a Phase II trial of its mRNA vaccine in August. [171] 

In addition to the DNA and RNA-based vaccines, epitope-based vaccines are other approaches, which somehow rely on the genetic aspect of SARS-CoV-2. In the new era of medicine, the immunoinformatics approaches have been desperately used to provide putative epitopes using a genome database. Recently, a group of computational scientists obtained immunogenic epitopes for all critical proteins of the virus.

This list is composed of top-ranked cytotoxic T cell helper and epitopes common across MHC alleles, covering all predominant human leukocyte antigen (HLA) supertypes in population. [176] By immunogenicity predictive models, researchers provide the immunogenicity of detected peptides and their binding potential to HLA alleles. [177] Currently, a total of 63 peptides with a high immunogenicity potential have been identified for SARS-CoV-2. Identification of a ranked list of immunogenic peptides shows that they can be used as potential targets for SARS-CoV-2 vaccine development, and it accelerates the development pipeline. A detailed screen of candidate peptides based on comparison with immunogenic peptides was used using deposited data in the immune epitope database, which resulted in a de novo prediction from SARS-CoV-2associated 9-mer peptides. [177] It has been shown that these peptides bind various HLA alleles (both class I and class II), although with a higher tendency towards HLA-A:02:01, and can cause activation of effector T cells. In another study to provide a fast-immunogenic profile of these epitopes, linear B-cell J o u r n a l P r e -p r o o f Journal Pre-proof epitopes, along with their sequence, position, and length, were introduced for SARS-CoV-2. In a recent study, 13 Major Histocompatibility Complex-(MHC) I and 3 MHC-II epitopes were recognized to have antigenic properties. [178] These epitopes molecularly dock on toll-like receptor-5 to get binding affinity and are usually linked to specific linkers to build vaccine components. Reportedly, a list of 22 SARS-CoV-2 peptides has been revealed, which have a higher predicted immunogenicity score than their target peptides. Further predictive models and algorithms for the characterization of immunogenic peptides, as well as several in vitro and in vivo validations, are required for the most efficient vaccine development. [177] 

Currently, the genome of SARS-CoV-2 has been comprehensively studied. Without a doubt, our understanding of this regard caused molecular-based diagnosis, some targeted therapies, and vaccine developments. Focusing on targeting the SARS-CoV-2 genome via RNAi technology could lead to emerging effective and safe treatments, which probably could prevent disease severity and decrease COVID-19 associated mortality. Although many efforts have been made to reveal the behavior of SARS-CoV-2, based on its genetics, there are still some concerns regarding emerging variation/mutations. These could significantly affect targeted therapies and, more importantly, vaccine development. Moving forward, we must recognize that we have had a new major coronavirus epidemic every decade in the twenty-first century SARS in the 2000s, MERS in the 2010s, and now COVID-19. It is, therefore, a global security priority to advance coronavirus vaccines and to identify international funding support resources, as well as other data regarding their development, manufacture, and storage. This recent outbreak should be considered as a sine qua non to alert the international research community to both react and prepare for the next coronavirus transmission to mammals. We urgently need to manufacture a pan-coronavirus vaccine, and it is something that appears feasible if sufficient resources are provided in Tables   Table 1 the SARS-CoV-2 can either be directed against the SARS-CoV-2 itself or against the ACE2 receptor or TMPRSS2, whose silencing will inhibit

A Novel Coronavirus from Patients with Pneumonia in China

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

Effective treatment of severe COVID-19 patients with tocilizumab

COVID-19 Vaccine: A comprehensive status report

How close are we to a vaccine for COVID-19?

A new coronavirus associated with human respiratory disease in China

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Coronaviruses: an overview of their replication and pathogenesis

Origin and evolution of pathogenic coronaviruses

Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection

The molecular biology of coronaviruses

Coronavirus genomics and bioinformatics analysis

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan, Emerging microbes & infections

Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China

Complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in Wuhan, China, bioRxiv

The structure and functions of coronavirus genomic 3' and 5' ends

Mechanisms of viral emergence

Evolutionary Trajectory for the Emergence of Novel Coronavirus SARS-CoV-2, Pathogens

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

The receptor binding domain of SARS-CoV-2 spike protein is the result of an ancestral recombination between the bat-CoV RaTG13 and the pangolin-CoV MP789

GISAID: Global initiative on sharing all influenza data -from vision to reality

Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and emergence of RBD mutant with lower ACE2 binding affinity, bioRxiv

The evolution of Ebola virus: Insights from the 2013-2016 epidemic

An unusually high substitution rate in transplant-associated BK polyomavirus in vivo is further concentrated in HLA-C-bound viral peptides

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

Global genetic diversity patterns and transmissions of SARS-CoV-2, medRxiv

Characterizing SARS-CoV-2 mutations in the United States

Moderate mutation rate in the SARS coronavirus genome and its implications

The establishment of reference sequence for SARS-CoV-2 and variation analysis

Genotyping coronavirus SARS-CoV-2: methods and implications

Variant analysis of COVID-19 genomes

Geographic and Genomic Distribution of SARS-CoV-2 Mutations

Four SARS-CoV-2 Genome Sequences from Late

Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations, bioRxiv

Missense mutations in SARS-CoV2 genomes from Indian patients

Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission

Identification of the hyper-variable genomic hotspot for the novel coronavirus SARS-CoV-2

Emergence of genomic diversity and recurrent mutations in SARS-CoV-2, Infection

Evidence for strong mutation bias towards, and selection against, U content in SARS-CoV-2: implications for vaccine design

Variant analysis of SARS-CoV-2 genomes

Mutation landscape of SARS-CoV-2 reveals three mutually exclusive clusters of leading and trailing single nucleotide substitutions, bioRxiv

Genetic diversity and evolution of SARS-CoV-2, Infection

Comparative analyses of SAR-CoV2 genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis, bioRxiv

Genome Wide Analysis of Severe Acute Respiratory Syndrome Coronavirus-2 Implicates World-Wide Circulatory Virus Strains Heterogeneity, in, Preprints.org

An 81 base-pair deletion in SARS-CoV-2 ORF7a identified from sentinel surveillance in Arizona

Direct RNA sequencing and early evolution of SARS-CoV-2, bioRxiv

Identification of multiple large deletions in ORF7a resulting in in-frame gene fusions in clinical SARS-CoV-2 isolates

Genomic variance of the 2019-nCoV coronavirus

s. laboratories, G.E. group*, Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region

Phylogenetic network analysis of SARS-CoV-2 genomes

A genetic barcode of SARS-CoV-2 for monitoring global distribution of different clades during the COVID-19 pandemic

SARS-CoV-2 viral spike G614 mutation exhibits higher case fatality rate

Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2, bioRxiv

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

The D614G mutation in SARS-CoV-2 Spike increases transduction of multiple human cell types, bioRxiv

SARS-CoV-2 genomic variations associated with mortality rate of COVID-19

Multiple Virus Lineages and Spike Protein Mutation Associated with Higher Transmission and Pathogenicity

Insights into the structural and dynamical changes of spike glycoprotein mutations associated with SARS-CoV-2 host receptor binding

Specific mutations in SARS-CoV2 RNA dependent RNA polymerase and helicase alter protein structure, dynamics and thus function: Effect on viral RNA replication, bioRxiv

RdRp mutations are associated with SARS-CoV-2 genome evolution

Identification of novel mutations in RNA-dependent RNA polymerases of SARS-CoV-2 and their implications on its protein structure

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study

Clinical display, diagnostics and genetic implication of novel Coronavirus (COVID-19) epidemic, European review for medical and pharmacological sciences

Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the United States

SARS-CoV-2-Positive Sputum and Feces After Conversion of Pharyngeal Samples in Patients With COVID-19

Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

Real-time PCR for mRNA quantitation

Assay Techniques and Test Development for COVID-19 Diagnosis

Real-time RT-PCR in COVID-19 detection: issues affecting the results, Expert review of molecular diagnostics

False-Negative Results of Real-Time Reverse-Transcriptase Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2: Role of Deep-Learning-Based CT Diagnosis and Insights from Two Cases

Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure

Chest CT for Typical 2019-nCoV Pneumonia: Relationship to Negative RT-PCR Testing

A case of SARS-CoV-2 carrier for 32 days with several times false negative nucleic acid tests, medRxiv

Diagnosis of the Coronavirus disease (COVID-19): rRT-PCR or CT?

Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19

Potential False-Negative Nucleic Acid Testing Results for Severe Acute Respiratory Syndrome Coronavirus 2 from Thermal Inactivation of Samples with Low Viral Loads

Interpreting Diagnostic Tests for SARS-CoV-2

Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome

SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge, medRxiv

ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens, medRxiv

Application and optimization of RT-PCR in diagnosis of SARS-CoV-2 infection, medRxiv

Diagnostic efficacy of three test kits for SARS-CoV-2 nucleic acid detection], Zhejiang da xue xue bao. Yi xue ban

Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV

An assessment of real-time RT-PCR kits for SARS-CoV-2 detection

Comparison of seven commercial RT-PCR diagnostic kits for COVID-19

Diagnosing COVID-19: The Disease and Tools for Detection

Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa™ COVID-19 direct assay

Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Test Tracker: Commercially Available COVID-19 Diagnostic Tests. 360Dx, 2020. www.360dx.com/coronavirus-test-tracker-launched-covid-19-tests

Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia

A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus

Survey and Visual Detection of Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone

Rapid and specific detection of Asian-and Africanlineage Zika viruses

Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device

Loop-mediated isothermal amplification of DNA

A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry

Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP, medRxiv

Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP, medRxiv : the preprint server for health sciences

Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

Recombinase polymerase amplification: Basics, applications and recent advances

Metagenomic analysis identified co-infection with human rhinovirus C and bocavirus 1 in an adult suffering from severe pneumonia

Evaluating Novel Diagnostics in an Outbreak Setting: Lessons Learned from Ebola

Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs

CRISPR/Cas Systems towards Next-Generation Biosensing

Rapid, field-deployable nucleobase detection and identification using FnCas9, bioRxiv

SHERLOCK: nucleic acid detection with CRISPR nucleases

Nucleic acid detection with CRISPR-Cas13a/C2c2

Development and Evaluation of A CRISPR-based Diagnostic For 2019-novel Coronavirus, medRxiv

CRISPR-Cas12-based detection of SARS-CoV-2

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus, bioRxiv

Clinical Evaluation of the New High-Throughput Luminex NxTAG Respiratory Pathogen Panel Assay for Multiplex Respiratory Pathogen Detection

Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season

The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes

Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections

Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens

Clinical evaluation of AusDiagnostics SARS-CoV-2 multiplex tandem PCR assay

Massively multiplexed nucleic acid detection with Cas13

Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract

Hybridization Chain Reactions Targeting the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Cycling of Rational Hybridization Chain Reaction To Enable Enzyme-Free DNA-Based Clinical Diagnosis

Biogenesis of small RNAs in animals

A review on current status of antiviral siRNA

Universal and mutation-resistant anti-enteroviral activity: potency of small interfering RNA complementary to the conserved cis-acting replication element within the enterovirus coding region

Selection of hyperfunctional siRNAs with improved potency and specificity

siRNA efficiency: structure or sequence-that is the question

Slicing and dicing viruses: antiviral RNA interference in mammals

RNA interference as an antiviral approach: targeting HIV-1

Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference

Silencing of hepatitis A virus infection by small interfering RNAs

A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference

Construction of influenza virus siRNA expression vectors and their inhibitory effects on multiplication of influenza virus

Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections

Inhibition of adenovirus infections by siRNAmediated silencing of early and late adenoviral gene functions

Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque

A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus

RNA interference and its potential applications to chronic HBV treatment: results of a Phase I safety and tolerability study

Miravirsen (SPC3649) can inhibit the biogenesis of miR-122

Current prospects for RNA interference-based therapies

Emerging targets and novel approaches to Ebola virus prophylaxis and treatment

Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases

siRNA could be a potential therapy for COVID-19

Inhibition of SARS-associated coronavirus infection and replication by RNA interference

Attenuation of SARS coronavirus by a short hairpin RNA expression plasmid targeting RNAdependent RNA polymerase

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

Chang, siRNA targeting the leader sequence of SARS-CoV inhibits virus replication

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells

Computational Identification of Small Interfering RNA Targets in SARS-CoV-2

SARS-CoV-2-encoded nucleocapsid protein acts as a viral suppressor of RNA interference in cells

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

siRNA silencing of angiotensin-converting enzyme 2 reduced severe acute respiratory syndrome-associated coronavirus replications in Vero E6 cells

The emerging role of ACE2 in physiology and disease

Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry

On the Importance of Host MicroRNAs During Viral Infection

Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection, bioRxiv

The Prediction of miRNAs in SARS-CoV-2 Genomes: hsa-miR Databases Identify 7 Key miRs Linked to Host Responses and Virus Pathogenicity-Related KEGG Pathways Significant for Comorbidities

What is the potential function of microRNAs as biomarkers and therapeutic targets in COVID-19?

Competing endogenous RNA network profiling reveals novel host dependency factors required for MERS-CoV propagation, Emerging microbes & infections

Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection

A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice

Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial

Immunogenicity of a DNA vaccine candidate for COVID-19

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

World Health Organization. Draft landscape of COVID-19 candidate vaccines

Coronavirus Vaccine Tracker

Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery

mRNA vaccines -a new era in vaccinology

Recent advances in mRNA vaccine technology

An Evidence Based Perspective on mRNA-SARS-CoV-2 Vaccine Development, Medical science monitor : international medical journal of experimental and clinical research

T Cell Epitope-Based Vaccine Design for Pandemic Novel Coronavirus 2019-nCoV, in, ChemRxiv

In silico identification of vaccine targets for 2019-nCoV

Development of epitope-based peptide vaccine against novel coronavirus 2019 (SARS-COV-2): Immunoinformatics approach

Isolation and phylogenetic analysis of SARS-CoV-2 variants collected in Russia during the COVID-19 outbreak

Genome-wide analysis of Indian SARS-CoV-2 genomes for the identification of genetic mutation and SNP

Global cataloguing of variations in untranslated regions of viral genome and prediction of key host RNA binding protein-microRNA interactions modulating genome stability in SARS-CoV-2

Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material, bioRxiv

SARS-CoV-2 detection with CRISPR diagnostics, bioRxiv

Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification, medRxiv

Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform