key: cord-0925449-wad7uob1
authors: Chellasamy, Gayathri; Arumugasamy, Shiva Kumar; Govindaraju, Saravanan; Yun, Kyusik
title: Analytical insights of COVID-19 pandemic
date: 2020-10-17
journal: Trends Analyt Chem
DOI: 10.1016/j.trac.2020.116072
sha: 95eb8f111187c21483b80662de5171cf78f57582
doc_id: 925449
cord_uid: wad7uob1

Coronavirus disease-19 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (2019-nCoV or SARS-CoV-2). Genomic analysis has revealed that bat and pangolin coronaviruses are phylogenetically related to SARS-CoV-2. The actual origin and passage history of the virus are unknown, but human-human transmission of the virus has been confirmed. Several diagnostic techniques have been developed to detect COVID-19 in this prevailing pandemic period. In this review, we provide an overview of SARS-CoV-2 and other coronaviruses. The origin, structure, current diagnostic techniques, such as molecular assays based on oligonucleotides, immunoassay-based detection, nanomaterial-based biosensing, and distinctive sample based detection are also discussed. Furthermore, our review highlights the admissible treatment strategies for COVID-19 and future perspectives on the development of biosensing techniques and vaccines for the diagnosis and prevention of the disease, respectively.

The pandemic outbreak of COVID-19 was caused by SARS-CoV-2, which belongs to the Coronaviridae family in the order Nidovirales and the subfamily of Orthocoronavirinae.

Alpha-coronavirus (α-CoV), beta-coronavirus (β-CoV), gamma-coronavirus (γ-CoV), and delta-coronavirus (δ-CoV) are the four genera of coronaviruses in the subfamily Orthocoronavirinae [1, 2] . Among these, αand β-CoVs mainly infect mammals, while δand γ-CoVs infect birds. β-CoVs include SARS-CoV, the Middle East respiratory syndrome (MERS) virus (MERS-CoV), and the current SARS-CoV-2 . Globally, SARS-CoV and MERS-CoV have infected more than 8000 and 2428 individuals, respectively. The comparative analysis of SARS-CoV, MERS-CoV, and SARS-CoV-2 are presented in Table 1 .

Patients infected with these viruses develop pneumonia, followed by acute respiratory distress syndrome (ARDS) and renal failure [3] . Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 attacks the lower respiratory system, leading to viral pneumonia. Additionally, it affects the gastrointestinal (GI) system, liver, central nervous system, and kidney [4] . The incubation period of COVID-19 is 5-25 d, and its symptoms include fever and severe respiratory symptoms, such as cough, dyspnea, and muscle soreness. Headaches and diarrhea are reported in less than 10 % of patients, with recent confirmation of loss of smell and taste [5, 6] .

Understanding the origin and transmission of SARS-CoV-2 is an important factor that can help in determining the preventive measures for COVID-19. In the case of SARS-CoV, researchers initially focused on palm civets and raccoon dogs. Samples from civets showed a positive result for viral RNA detection, implicating them as secondary hosts for SARS-CoV.

For MERS-CoV, researchers predicted camels as the zoonotic source for viral infection and recently identified the same RNA sequence in Pipistrellus and Perimyotis bats. Before J o u r n a l P r e -p r o o f determining the genomic similarities between coronavirus and SARS-like bat viruses, some researchers suggested snakes as a possible host. However, after genomic identification, the bat virus was shown to have more similarities to the coronavirus [7] (Fig. 1) . For COVID-19, researchers still do not have a precise theory explaining the natural origin of the COVID-19 pandemic. They have suggested bats and pangolins as the possible natural hosts of SARS-CoV-2 and that the virus crossed from these animals to humans, posing a risk to human health. In addition, the virus developed the ability to spread from human to human and cause more serious and life-threatening diseases [8] . Research is ongoing to identify the exact natural host, diagnostic techniques, and treatment strategies for COVID-19.

Different testing strategies have been used by different countries based on the availability of reagents and laboratories. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay plays a main role in the diagnosis of COVID-19 from respiratory specimens. In addition, computed tomography (CT), serological testing, viral culture, immunoassay testing, laboratory specimen test, and thermal scanning have been performed to diagnose COVID-19 [9] . Various bodies, including the Centers for Disease Control and Prevention (CDC) of many countries and the World Health Organization (WHO), have presented different measures for the prevention of the spread of COVID-19. Avoiding travel, social distancing, hand washing, and wearing of face masks are some of the preventive measures that have been recommended to the public [10] . To date, no antiviral treatment or vaccine has been developed for COVID-19. WHO announced that 12 potential drug trials are underway, including those for drugs already used for the treatment of HIV and malaria. Some other experimental treatments have been employed to reduce inflammation associated with COVID-19 [11] . In this review, we briefly discuss the origin of SARS-CoV-2, the genome organization, and its comparison with other coronaviruses. Here, we present the principle, J o u r n a l P r e -p r o o f design, accuracy, sensitivity, selectivity, and response time for currently available COVID-19 diagnostic tools.

Coronaviridae viruses possess an envelope and crown-like viral particles around their genetic materials. Despite the efforts of researchers to treat infectious diseases, a new disease with high mortality has emerged and spread worldwide [12] . In the past two decades, the CoV family has caused serious illnesses, such as SARS in the years 2002 -2003 and MERS in 2012 . Now, the unexplained respiratory infection caused by a novel CoV family member was reported in December 31, 2019, and was named COVID-19 by WHO.

Since COVID-19 was identified to be highly contagious due to its spread by human-human transmission, Chinese researchers have rapidly sequenced its genome [ 13 ] . Genome sequencing of SARS-CoV-2 revealed that it has a single strand positive-sense RNA (+ssRNA) molecule of approximately 29,903 nt, containing genes in the order of 5′ to 3′ as follows:

ORF1ab, spike (S), envelope (E), membrane (M), and nucleocapsid (N). The analysis shows that SARS-CoV-2 has terminal sequences that are typical of beta-coronaviruses, containing 265 nt at the 5′ end and 229 nt at the 3′ end. The ORF1ab gene codes for 16 predicted nonstructural proteins, followed by 13 downstream ORFs. The predicted S, ORF3a, E, M, and N genes of SARS-CoV-2 are 3822, 828, 228, 669, and 1260 nt in length, respectively. In addition, SARS-CoV-2 carries an ORF8 gene of 366 nt between the M and N ORF genes [14] . The genome data of SARS-CoV-2 is publicly available to facilitate the search for the original host of this virus. Fig. 2 shows the structure of COVID-19 and its genome organization.

J o u r n a l P r e -p r o o f 2.1. Proximal host genome of SARS-CoV-2: a comparative analysis Rapid genome sequencing of SARS-CoV-2 at an early stage by Chinese researchers paved the way for the identification of the proximal host and tracing of the evolutionary relationship between SARS-CoV-2 and former coronaviruses. Phylogenetic analysis of coronaviruses shows that SARS-CoV-2 shares a highly conserved domain with SARS-CoV in nsp1 (LLRKNGNKG: amino acids 122-130). Further studies on evolutionary research suggested that SARS-CoV-2 shows 82.3 % and 77.2 % amino acid identity with the bat coronaviruses, SL-CoVZC45 and SARS-CoV, respectively [13] . A comparative study on the receptor-binding domain (RBD) of its spike protein and those of SARS-CoVs and bat SARSlike CoVs reported 73.8-74.9 % and 75.9-76.9 % amino acid similarity, respectively. In addition, the RBD of SARS-CoV-2 is only one amino acid longer than that of SARS-CoV.

These findings suggest that SARS-CoV-2 has the ability to use human angiotensin converting enzyme 2 (ACE2) receptors for cell entry, similar to SARS-CoV. Another comparative study identified SARS-CoV-2 S protein to possess a furin-like cleavage site, which is absent in SARS-like CoVs [15] .

Coronaviruses have the ability to cross species barriers from wild animals to humans, and hence, there is a need to identify the exact host of SARS-CoV-2 to facilitate the development of effective therapy for COVID-19. Bats are considered the reservoir hosts of the previous SARS-CoV and MERS-CoV outbreaks, while their intermediary hosts are Palm civets and Dromedary camels, respectively. In the case of SARS-CoV-2, researchers believe that there are two possibilities for species transmission; it may have been directly transmitted from bats to humans, or indirectly through an intermediary host. Because bat CoVs show 96 % similarity to SARS-CoV-2 at the whole-genome level, researchers believe that transmission might have occurred directly from the bat reservoir host [16] . Meanwhile, in another study, J o u r n a l P r e -p r o o f Guangzhou Chinese researchers suggested that pangolins long-snouted, ant-eating animals may also be a plausible host of SARS-CoV-2, with 99 % sequence similarity observed.

However, this research is ongoing and has not yet been published. Another study on coronaviruses isolated from Malayan pangolins (Manis javanica) showed 100 %, 98.2 %, 96.7 %, and 90.4 % amino acid similarity with SARS-CoV-2 in the E, M, N, and S genes, respectively. SimPlot analysis of the whole genome sequence of the Pangolin-CoV showed sequence similarities of 80 % and 98 % with SARS-CoV-2 and bat SARSr-CoV RaTG13, respectively, except for the S gene, suggesting that pangolin coronavirus is capable of entering the human cells [17] . The findings of these studies suggest that both pangolins and bats are nocturnal animals and share overlapping ecological niches, which make pangolins a possible intermediary host for SARS-CoV-2. In addition, the genomic comparison suggests that SARS-CoV-2 might be a chimera of pre-existing coronaviruses (one close to pangolin CoVs and another close to RaTG13 bat CoVs). It is known that for recombination to occur, the two divergent viruses must have infected the same organism simultaneously, which has been described previously for SARS-CoV infection [18] . However, the organism in which this recombination takes place and the conditions under which this recombination happens are still unknown.

The availability of the genetic information of SARS-CoV-2 and the detailed understanding of its biological properties have enabled researchers to develop detection techniques within a short period of the prevailing pandemic. Such developed detection techniques are categorized into four major classes i) Molecular assays based on oligonucleotides ii) Immunoassay-based detection iii) Nanomaterial-based biosensing and iv) Distinctive sample-based detection. Herein, the key elements emphasizing technique J o u r n a l P r e -p r o o f principle, processing, accuracy, and response time were analyzed and discussed. An outline of the diagnostic techniques for COVID-19 is presented in Fig. 3 3.1. COVID-19 diagnosis based on molecular assays

The accurate diagnosis of COVID-19 on the basis of the expressed symptoms is difficult because the symptoms are nonspecific and similar to those of the common flu.

Therefore, detecting SARS-CoV-2 at the molecular level would be more suitable for the accurate diagnosis of COVID-19 [19] . Molecular assays including rRT-PCR, loop-mediated isothermal amplification, and clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques were designed and applied for the detection of SARS-CoV-2.

The most widely used gold standard technique for SARS-CoV-2 detection is rRT-PCR, which has the ability to amplify even a small amount of genetic material [20] . More advanced rRT-qPCR-based techniques have been developed by researchers in recent times.

Ishige and team developed a multiplex rRT-PCR to detect two regions of the SARS-CoV-2 genome (E and N genes) and the human ABL1 gene simultaneously, where ABL1 serves as an internal control (IC) [21] . Compared with the general rRT-PCR, the multiplex assay provides higher sensitivity with a detection limit of ˃ 25 copies/reaction. This technique of detecting SARS-CoV-2 does not involve the use of the most abundantly expressed genes, ACTB and GAPDH, as ICs because they prevent the amplification of less abundant targets and thus lowers the sensitivity of target detection. Thus, the multiplex rRT-PCR technique facilitates highly sensitive detection of SARS-CoV-2 RNA, with reduced cost and amounts of reagents.

A study by Penarrubia et al. expresses the importance of multiple assays in RT-PCR targeting more than one region in the SARS-CoV-2 genome to mitigate the risk of sensitivity loss due to unknown genetic variability [22] . They suggested that viral mutation and recombination rates increase due to the genetic variability of the SARS-CoV-2 genome. Therefore, it is J o u r n a l P r e -p r o o f essential to perform multiple rRT-PCR assays targeting at least two regions to monitor genomic variations. Currently, several rRT-PCR-based COVID-19 kits are developed by different manufacturers, which have been commercialized. Van Kasteren research group carried out a study on COVID-19 diagnosing RT-PCR kits to assess their clinical and analytical performance [23] . The findings of this study showed that the rRT-PCR kits gave satisfactory PCR efficiency (≥ 96 %) and the estimated 95 % limit of detection (LOD95)

varied within a 6-fold range between kits (3.8-23 copies/mL). In addition, they reported that the commercially available kits can be used for routine diagnosis of symptomatic COVID-19.

Wang et al. developed a novel one-step single-tube nested quantitative real-time PCR for the sensitive detection of SARS-CoV-2 [24] . This technique targeted ORF1ab and N genes and showed detection sensitivity of 1 copy/reaction, with 100 % specificity. It was officially approved by the National Medical Products Administration and used in the detection of COVID-19 nationwide. This method has the advantage of high specificity and sensitivity, with the ability to detect asymptomatic infected persons with low viral load.

Though RT-PCR-based detection is considered a standard technique, it faces several obstacles, such as time-consuming amplification process and high-cost reagents, and requires sophisticated thermal cycling equipment [19] . To overcome these obstacles, loop-mediated isothermal nucleic acid amplification (LAMP) was used as an alternative strategy for rapid and cost-effective detection of SARS-CoV-2. Park et al., designed and evaluated a one-step reverse transcription LAMP (RT-LAMP) method to detect SARS-CoV-2 [ 25 ] , and the detection principle involved the use of leuco crystal violet method for colorimetric detection of SARS-CoV-2. They reported LOD of approximately 100 copies/reaction, with no crossreactivity with other human coronaviruses. The research group of Baek reported a single-tube RT-LAMP assay to detect novel coronavirus N gene through colorimetric visualization [26] .

Their method focused on targeting the N protein, which has the most abundant expression of J o u r n a l P r e -p r o o f subgenomic mRNA (sgmRNA) during infection, lacks the glycosylation site, and possesses distinctly unaltered immunological characteristics. It exhibited high sensitivity and specificity, with the ability to discriminate circulating human coronaviruses, including OC43, 229E, NL64, and MERS-CoV, as well as other respiratory viruses. Furthermore, this method yields rapid results within 30 min, with a detection limit of 10 2 RNA copies.

The other leading molecular technique for COVID-19 diagnosis is CRISPR-based detection, which offers precise and rapid detection of viral nucleic acid nearly at the attomolar (10 -18 ) level [27] . According to the study by Jinek et al, CRISPR-Cas technology relies on small RNAs for sequence-specific detection and silencing of foreign nucleic acids [ 28 ] . Kellner et al. described a protocol for the rapid detection of nucleic acids via fluorescence and colorimetric readouts using CRISPR-Cas13. This method provides results in < 1 h with a setup time of less than 15 min [29] . Recently, Broughton et al. developed and validated a CRISPR-Cas12-based SARS-CoV-2 detection, combined with isothermal amplification [ 30 ] . The developed technique was rapid (30-40 min) with a LOD of approximately 10 copies/µL input. This method limits the need for expensive laboratory instrumentation, but involves routine protocols and commercially available off-the-shelf reagents. The advantages of CRISPR-Cas12-based detection system are rapid turnaround time, single nucleotide target specificity, integration with accessible and easy-to-use reporting formats, such as lateral flow strips, and no requirement for complex laboratory infrastructure.

Huang and colleagues also demonstrated a diagnostic technology in their study for COVID-of ~50 min. This method of SARS-CoV-2 detection obtained comparable results with CDCapproved quantitative rRT-PCR assay; however, it failed to provide clear positive or negative results and was not able to quantify viral load.

The detection of COVID-19 is based on the presence of specific immune proteins, which could be broadly classified into enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and chemiluminescence enzyme immunoassay (CLIA). These approaches follow different strategies, but the basic principle is the antigen-antibody interaction. Although the techniques provide partial quantitative results, rRT-PCR is considered a standard analysis. However, to screen a large number of individuals for infection, alternative detection methods to the immunosorbent assay testing should be adopted. LFIA is more focused than ELISA as it requires trained personnel and sophisticated laboratories, whereas LFIA is simpler as well as more portable and rapid. For ELISA, samples are collected from nasopharyngeal (NP) sites and incubated in 96-well plates for 1 h; it is a time-consuming process and there is a high chance of producing false-positive responses upon cross-reactivity [33] . Zhang et al. demonstrated the procedure for COVID-19 detection using ELISA consisting of SARS-CoV-2 Rp3 N protein, antihuman IgG modified with horseradish peroxidase, and 3,3',5,5'-tetramethylbenzidine [ 34 ] . The detection mechanism is based on peroxidase interaction with the adsorbed nucleoprotein that produces a color change in the plate. The specificity and sensitivity of such detection range from 81 to 100 % after 5 days of infection. The performance of EUROIMMUN ELISA for semiquantitative detection with respect to the S1 domain of the spike protein for both IgA and IgG antibodies was reported by Beavis et al. [35] . The samples were diluted and incubated in wells containing IgA/IgG antibodies, followed by incubation with the detection antibodies i.e. J o u r n a l P r e -p r o o f 13 enzyme-labeled antihuman IgA or IgG, which catalyzed a color change. The results were determined as a ratio of excitation of the control or patient samples over the excitation of the calibrator. This ratio was inferred as follows < 0.8 negative, > 0.8 < 1 borderline (but considered as positive), and > 1.1 positive. The results showed a specificity of 90.5 % for IgA antibody and 100 % for IgG antibody after incubation for 4 days.

The use of immunochromatographic (ICG) strip to target IgG/IgM antibodies for the detection of viral loads in patients was investigated [36] . IgM antibody is the first line of defense produced by the immune system when the virus attacks, whereas IgG antibodies take almost 7-10 days after infection. The disease infection stages include the initial stage (from 1-7 days), intermittent stage (8-10 days) , and late stage (> 15 days). The positive response ratings were 3 %, 26 %, and 30 %, respectively. Overall, the specificity and selectivity for the detection assay range from 97.1 to 100 % depending on the duration of infection. Nicol et al.

compared three immunoassays, ELISA (Euroimmun), CLIA (Abbott), and LFIA (NG Biotech) for the detection of SARS-CoV-2 [37] . In ELISA, the target analyte was spike protein, and for CLIA, the target was N protein. In ELISA, Euroimmun anti-SARS-CoV-2

IgG and IgA assays were utilized against the recombinant S1 structural protein, whereas the CLIA IgG antibodies of SARS-CoV-2 were solely utilized against the nucleoproteins in the serum or plasma. For LFIA, an immune colloidal assay for IgG and IgM antibodies of SARS-CoV-2 against nucleoprotein was performed. A comparative study was performed for samples collected after 14 days of infection, with IgG antibodies, and the results showed a specificity of 98 % for CLIA and LFIA and 95.8 % for ELISA.

Although RT-PCR and immunoassay-based techniques are the standard procedures for COVID-19 diagnosis, the use of nanoparticle-based biosensing for SARS-CoV-2 detection is considerably on the rise due to its precision and speed. Biosensors are emerging alternative tools to conventional laboratory equipment for medical and environmental analyses. The outstanding electrical and optical properties of nanomaterials are typically combined with those of biological or synthetic molecules by biosensors to selectively detect any kind of analyte [38] . Nanoparticles, including gold nanoparticles (AuNPs), carbon-based nanoparticles and other nanomaterials, play a significant role in the development of SARS-CoV-2 sensor. In particular, properties, such as quantum size effects, high adsorption, high surface-to-volume ratio, and high reactive capacity are important for the design of biosensing techniques. Moreover, the surface, size, and shape of nanomaterials can easily be tailored to enhance the detection limit and selectivity towards the analytes [ 39 ] . Based on these physicochemical properties, researchers have been developing accurate, rapid and costeffective COVID-19 sensors. The schematic representation of nanomaterials-based biosensing techniques for COVID-19 is presented in AuNPs based colorimetric assay to detect SARS-CoV-2 by the naked eye [40] . The ASOs are specific to the N gene (nucleocapsid phosphoprotein) of SARS-CoV-2, and the sensor was developed based on the agglomeration of ASO-capped AuNPs with the target RNA sequence of SARS-CoV-2. This phenomenon changes the surface plasmon resonance (SPR) signal and the addition of RNaseH led to the agglomeration of AuNPs. During the aggregation of AuNPs, the violet color changed to dark blue, which led to an increase in the absorbance at 660 nm with a redshift of 40 nm. The sensitivity of SARS-CoV-2 to ASO-capped AuNPs was monitored based on the increase in absorbance at 660 nm. The absorbance difference observed was due to thiol-modified ASOs with a comparative ratio of ASOs to the AuNPs, which play a major role in the detection of SARS-CoV-2 by the AuNPs. This technique could be used to detect COVID-19 cases within 10 min with a detection limit of 0.18 ng/μL of RNA. Moitra's biosensing system satisfies most of the assured criteria mentioned by WHO, which includes affordability, sensitivity, and low cost. Kumar et al. reported a similar rapid colorimetric assay for the detection of SARS-CoV-2 using NP RNA samples [41] . sheet to detect SARS-CoV-2 with a specific antibody [44] . Antigen proteins, cultured virus, and NP swab specimens from patients with COVID-19 were used to determine the performance of the sensor. The LOD of SARS-CoV-2 varied depending on the samples: phosphate-buffered saline showed 1 fg/mL for SARS-CoV-2 spike protein, 100 fg/mL for clinical transport medium and SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 × 10 1 pfu/mL) and clinical samples (LOD: 2.42 × 10 2 copies/mL). The performance of SARS-CoV-2 antibodies was verified using ELISA before immobilization. The antibody bound only to SARS-CoV-2, with a LOD of 4 ng/mL. The advantage of this sensing platform is that pretreatment is not required for the samples. Moreover, the device provides high sensitivity as well as simple, rapid, and high response for COVID-19 detection. [46] . The Gr-FET can rapidly identify and easily interact with COVID-19 spike protein. The study involved a direct comparison between SARS-CoV spike S1 subunit protein antibody (CSAb) and ACE2, which have affinity for RBD. Going by the results, the CSAb had higher affinity for the LOD at a concentration down to 0.2 pM. The developed sensor was able to easily identify and screen the neutralizing antibodies.

Mavrikou et al. reported the detection of SARS-CoV-2 S1 spike protein in membrane engineered mammalian cells bearing the spike S1 antibody [47] . The sensor was developed based on the considerable and selective changes in cell bioelectric properties, which were measured by using the bioelectric recognition assay. This advanced technique showed rapid detection lasting for approximately 3 min, with a detection limit of 1 fg/mL. Furthermore, the biosensor was configured as ready-to-us platform, which can be conceivably applied for the mass screening of SARS-CoV-2 surface antigens without prior sample processing.

Li et al. developed AuNPs, a nitrocellulose membrane, and a plastic pad-based sensor kit to detect IgM and IgG antibodies against SARS-CoV-2 [48] . The detection was based on the interaction of antigens with the antibodies and the complex flow through the membrane. As the antigens proceed further, they get immobilized by capturing antibodies, making the red/blue line visible red in case of only AuNPs and blue due to plasmon coupling. using rRT-PCR techniques from fecal samples of asymptomatic or mildly infected patients [52] . GI symptoms and shedding of virus in feces could also be helpful for COVID -19 detection. The sensitivity of this procedure was estimated to be 35 % and it could persist for > 50 days. The viral loads in respiratory and fecal samples are comparable. A similar study reported a detection rate of 33.7 %, and the measurement of SARS-CoV-2 RNA was carried out in female patients having higher detection rate. Specific control strategies against fecal contamination are urgently needed [53] .

A high binding affinity aptamer specifically aiming at SARS-CoV-2 RBD was identified using an ACE2 competition-based aptamer selection and machine learning screening algorithm. An aptamer-based approach targeting the spike glycoprotein was developed for COVID-19 detection, and the Kd values were 5.8 nM and 19.9 nM for CoV2-RBD-1C (51 nt) and CoV2-RBD-4C (67 nt) aptamers, respectively, upon molecular dynamics simulation [54] . Chest computed tomography (CT) images could also be used for the preliminary identification of COVID-19 in symptomatic or asymptomatic patients. Y.

Soon et al., reported pulmonary lesions, which included patchy lesions as well as large and small nodular lesions. These lesions are not well-defined and the lower lung zones are patchy with an opacity of 80 % [55] . The overall developed detection techniques for COVID-19 are summarized in Table 2 .

The basic principle of treating patients positive for COVID-19 is to initially provide comfort to avoid worsening of their symptoms and prevent the emergence of secondary infections and potential complications from underlying diseases. In the most critical situations, patients are provided with organ function support, such as oxygen ventilation, in a timely manner [56] . Research on vaccines and drugs for coronavirus-related diseases has J o u r n a l P r e -p r o o f been ongoing since the SARS and MERS outbreak; however, no appropriate drugs or treatment regimens currently exist to specifically treat COVID-19. However, doctors have used various lines of treatment ranging from corticosteroid therapy, antibiotic therapy, antiviral therapy, antibody therapy, oxygen therapy, vaccines, and plasma transfusion [57] .

The treatment strategy for COVID-19, lines of attack of different drugs, and coronavirus replication cycle are schematically presented in Fig. 5 [11] .

Glucocorticoid therapy ease body temperature, improve oxygenation, and prevent the onset of pneumonia, and hence, it was adopted during SARS and MERS outbreak, and it is being used in the current outbreak of COVID-19 as the first line of treatment. A recent study on patients treated with corticosteroids suggests that they induced secondary complications associated with fungal and bacterial infections due to exudate accumulation in the lungs. In addition, other complications observed were hyperglycemia, GI bleeding, venous thrombosis, hypertension, and hypokalemia. Therefore, the efficacy of glucocorticoids against COVID-19 needs further elucidation, and it is strongly recommended only as an early, low-dose, and short-term application to improve clinical symptoms [ 58 ] . Recently, a breakthrough in COVID-19 treatment was achieved by a small team at Oxford University, led by Prof Peter Horby, as dexamethasone, a steroid found to be effective in severe forms of COVID-19, was discovered [59] . Dexamethasone acts on the immune system to dampen its response and reduce the cytokine storm, thereby preventing the inflammation of the lungs and heart.

Reportedly, dexamethasone reduces the mortality rate of patients who were on ventilator from 25 % to 20 %.

As the majority of patients are susceptible to secondary bacterial infections during first-line treatment with corticosteroids, doctors were urged to administer antibiotics such as moxifloxacin, levofloxacin, cephalosporins, hydroxychloroquine, azithromycin, and chloroquine to patients. Several antibiotic-based trials have been performed worldwide to determine an effective therapeutic regimen for COVID-19. China has used aminoquinolines, such as chloroquine and hydroxychloroquine, which have been able to reduce the viral load in patients with COVID-19. However, hydroxychloroquine is preferred over chloroquine because it is less toxic and more soluble than chloroquine, and it also has lower ocular toxicity [60] . 

The greatest hope for the treatment of COVID-19 is the administration of antiviral therapy, which is adopted by many countries around the world. Antiviral drugs are administered to infected patients as first-line treatment to reduce viral shedding in respiratory secretions [62] . Similar to the treatment of SARS and MERS, COVID-19 treatment also involves the use of antiviral drugs, such as remdesivir, as suggested by the FDA. Remdesivir is a promising antiviral drug that inhibits viral replication effectively and has been administered to patients since it passed the initial clinical trials [63] . Therapies without any J o u r n a l P r e -p r o o f supporting therapeutic evidence, including lopinavir, nitazoxanide, oseltamivir, baloxavir, interferon, ribavirin, or IVIG, have not been used for COVID-19 treatment [64].

SARS-CoV-2 enters the host cells through the binding of its S protein to angiotensinconverting enzyme 2 (ACE2) receptors, and hence, the development of neutralizing antibodies against these receptors has been adopted as a strategy to reduce the severity of the disease [65] . It has been demonstrated that the FDA-approved tocilizumab, a recombinant human IL-6 monoclonal antibody that effectively blocks the IL-6 signal transduction pathway, is effective in the treatment of patients with COVID-19 [66, 10] .

The last line of therapy provided to patients with COVID-19 at the severe stage of infection is oxygen therapy. Oxygen therapy includes the provision of nasal catheter or mask oxygen therapy during the early stage of respiratory distress (hypoxemia). High-flow nasal oxygen or non-invasive ventilation is provided if hypoxemia cannot be relieved by standard oxygen therapy. If respiratory distress worsens (within a short time, 1-2 h), endotracheal intubation and invasive mechanical ventilation are undertaken in the patient. If patientventilator asynchrony occurs during the time of ventilation, sedation and muscle relaxation strategies should be considered in patients [56] .

Treatment with convalescent plasma is believed to be the most consistent and responsive treatment for infectious diseases since the Spanish influenza epidemic to the more recent outbreaks of SARS and MERS. Thus, the same treatment has been used for COVID-19.

A preliminary study suggests that treating patients with COVID-19 with convalescent plasma J o u r n a l P r e -p r o o f improves clinical outcomes [67] . However, the treatment requires a convalescent plasma donor, which has remained quite difficult, and the efficacy and safety of the procedure are still unclear and require further elucidation [68] .

To (AY297028), GD01 (AY278489), and GZ50 (AY304495) have been used as inactivated or live-vectored vaccines to control the viral load in animal models [7] . Neutralizing antibodies have been developed against SARS-CoV by using recombinant protein from the Urbani (AY278741) strain of SARS-CoV [69, 70] . Recently, a microneedle array-based vaccine delivery platform was developed by Eun kim et al., who evaluated the immunogenicity of trimeric MERS-S1 subunit vaccines delivered into mice by microneedle injection. All microneedle array-delivered vaccines generated a higher level of neutralizing antibodies [71] . [72, 73] .

Initially, upon the outbreak of COVID-19 in Wuhan, an outbreak of pneumonia cases was suspected based on clinical inspection through rRT-PCR and CT scans. Based on the sudden increase in the number of similar cases being reported, bronchoalveolar lavage samples from patients were analyzed, and the results showed that the samples were positive for pan-betacoronavirus. The different types of specimens used for COVID-19 testing are presented in Table 3 . Whole genomic sequences were obtained though Illumina and Nanopore sequencing. The genomic sequences were analyzed using bioinformatics tools, and the virus was classified under the family of coronavirus (2B lineage). The genetic configuration of the virus had a 96 % similarity to that of BatCoV RaTG13, a bat SARS-like coronavirus strain, and it was documented in Global Initiative on Sharing All Influenza Data platform. As the next step in analyzing the cellular morphologies of the virus, human airway J o u r n a l P r e -p r o o f epithelial cells, Vero E6, and Huh-7 cell lines were infected with the virus. The isolated virus-infected cells were injected into human ACE2 mice and Rhesus monkeys to induce multifocal pneumonia with interstitial hyperplasia, and the virus was detected in the lung and intestinal tissues of the transgenic animals. Histopathological analysis of a human patient who died in Wuhan revealed bilateral diffuse alveolar damage and lung tissues displaying desquamation of pneumocytes, partially indicating ARDS [74, 75] . Using the testing kit manufactured by China CDC, the targets for screening COVID-19 were the nucleoprotein (N) and ORF1ab genes. The primers used for COVID-19 detection are presented in Table 4 .

The screened patients tested as positive (Ct < 37), negative (Ct ~0/Ct = 40), or suspicious . In the case of a suspicious result, the test was repeated, and the sample was classified as positive if Ct < 40 [76] . A study from Wuhan comparing PCR technique with CT method demonstrated that CT is more effective than PCR in diagnosing COVID-19, despite its low specificity (overlapping with other cases of pneumonia) [77] . Table 5 .

This review provides an overview of coronaviruses and COVID-19 disease. It includes the details on the origin, transmission, and structure of the novel coronavirus, a comparative analysis of SARS and MERS coronaviruses, and an overview of the symptoms, diagnostic techniques, and available treatments for COVID-19. Currently, nucleic acid-based testing, antibody-based testing, and nanomaterial-based biosensing are used for the rapid diagnosis of COVID-19. Controlling the spread of COVID-19 and adopting measures to avoid the regeneration of coronaviruses are crucial for the development of vaccines. There is a need for the development of more reliable, rapid, low cost, and widely available diagnostic tools. The lack of vaccines and therapeutic agents against coronaviruses might be due to the 

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Research and development on therapeutic agents and vaccines for COVID-19 and related human coronavirus diseases

Clinical, laboratory and imaging features of COVID-19: A systematic review and meta-analysis

A New Symptom of COVID-19: Loss of Taste and Smell

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

The proximal origin of SARS-CoV-2

Source: FIND Because diagnosis matters

World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19)

Race to find COVID-19 treatments accelerates

Bat Coronaviruses in China

Features, evaluation and treatment coronavirus (COVID-19)

A new coronavirus associated with human respiratory disease in China

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

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

Isolation and characterization of 2019-nCoV-like coronavirus from Malayan pangolins

Recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission

Diagnosing COVID-19: the disease and tools for detection

Assay techniques and test development J o u r n a l P r e -p r o o f for COVID-19 diagnosis

Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories

Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak

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

A novel one-step single-tube nested quantitative Real-Time PCR assay for highly sensitive detection of SARS-CoV-2

Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays targeting SARS-CoV-2

Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. science

SHERLOCK: nucleic acid detection with CRISPR nucleases

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

-sensitive and high-throughput CRISPR-Powered COVID-19 diagnosis

Clinically practiced and commercially viable nanobio engineered analytical methods for COVID-19 diagnosis

Evaluation of the EDI enzyme linked immunosorbent assays for the detection of SARS-CoV

IgM and IgG antibodies in human plasma

Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies

Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients

Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech)

Toward Nanotechnology-Enabled Approaches against the COVID-19 Pandemic

Electrochemical sensor and biosensor platforms based on advanced nanomaterials for biological and biomedical applications

Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles

Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples

eCovSens-Ultrasensitive Novel In-House Built Printed Circuit Board Based Electrochemical Device for Rapid Detection of nCovid-19 antigen, a spike protein domain 1 of SARS-CoV-2. bioRxiv

Dual-functional plasmonic photothermal biosensors for highly accurate severe acute respiratory syndrome coronavirus 2 detection

SARS-CoV-2) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor

Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19

Electrical probing of COVID-19 spike protein receptor binding domain via a graphene field-effect transistor

Development of a Portable, Ultra-Rapid and Ultra-Sensitive Cell-Based Biosensor for the Direct Detection of the SARS-CoV-2 S1 Spike Protein Antigen

Development and clinical application of a rapid IgM-IgG J o u r n a l P r e -p r o o f combined antibody test for SARS-CoV-2 infection diagnosis

Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence

Rapid and Sensitive Detection of anti-SARS-CoV-2 IgG, Using Lanthanide-Doped Nanoparticles-Based Lateral Flow Immunoassay

Detection of SARS-CoV-2 by bronchoscopy after negative nasopharyngeal testing: Stay vigilant for COVID-19

Detection of SARS-CoV-2 in Fecal Samples from Patients with Asymptomatic and Mild COVID-19 in Korea

Detection of SARS-CoV-2 RNA in fecal specimens of patients with confirmed COVID-19: a meta-analysis

Discovery of Aptamers Targeting Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein

Chest radiographic and CT findings of the 2019 novel coronavirus disease (COVID-19): analysis of nine patients treated in Korea

Chinese experts' consensus on the Internet of Things-aided diagnosis and treatment of coronavirus disease 2019

The epidemiology, diagnosis and treatment of COVID-19

Early, lowdose and short-term application of corticosteroid treatment in patients with severe COVID-19 pneumonia: single-center experience from Wuhan

Aminoquinolines Against Coronavirus Disease 2019 (COVID-19): Chloroquine or Hydroxychloroquine

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

Use of antiviral drugs to reduce COVID-19 transmission

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

COVID-19: a promising cure for the global panic

The cytokine release syndrome (CRS) of severe COVID-19 and Interleukin-6 receptor (IL-6R) antagonist Tocilizumab may be the key to reduce the mortality

Treatment with convalescent plasma for critically ill patients with SARS-CoV-2 infection

The feasibility of convalescent plasma therapy in severe COVID-19 patients: a pilot study

Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein

Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro

Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid translational development

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

The New York Times

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

Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases

Guidelines for Laboratory Diagnosis of Coronavirus Disease