Book Volume 1
Page: i-i (1)
Author: Muhammad Sajjad Khan
Page: ii-iii (2)
Author: Kamal Niaz* and Muhammad Farrukh Nisar*
List of Contributors
Page: iv-iv (1)
Author: Kamal Niaz and Muhammad Farrukh Nisar
Coronavirus Disease-2019 (COVID- 19) Epidemiology
Page: 1-41 (41)
Author: Ihtisham Ulhaq, Abdul Basit, Ijaz Ali, Firasat Hussain, Zahid Ali, Faisal Siddique, Haroon Ahmed, Amjad Islam Aqib and Kashif Rahim*
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At the end of December 2019, patients were diagnosed with a pneumonialike infection in the Wuhan wholesale market of seafood, Hubei Province, China. Laboratory diagnosis revealed a novel coronavirus named severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), that causes coronavirus disease-19 (COVID- 19). Initially, t he novel virus was reported in bats. Due to the highly contagious nature of pathogens and the susceptibility of every human the virus spread rapidly across China then Globally. Respiratory droplets of infected patients played a significant role in the transmission of COVID-19 from human to human. Wuhan being a transport hub, and the crowd of people during New Chinese Year played a considerable role in the virus spread across the country. In link with earlier coronaviruses, the SARS-CoV-2 was noticed with a more contagious nature, and it quickly spread throughout the world. It was declared a pandemic by the World Health Organization (WHO) on March 12, 2020. In March 2021, the spread of infection decreased in China but increased globally, mainly in Europe. In April 2020, the disease burden increased in the USA. Till April 17 2020, China reported 84,149 cases with 4642 deaths, while worldwide cases reached 2,074,5279 with 139378 deaths. Europe reported confirmed cases 1,050,871 with 93,480 deaths, 743,607 patients in USA regions, Western Pacific regions with 127,595 patients, Eastern Mediterranean Regions reached 115,824 cases, and South-East Asia Regions reported 23,560 cases while African regions had 12,360 cases. The below figure illustrates the analysis of the epidemiological studies of COVID-19 (Fig. 1).
Studying Evolutionary Adaptation of SARS-CoV-2
Page: 42-71 (30)
Author: Samina Ejaz*, Yasir Hameed, Waqas Nazir Malik, Muhammad Usman and Uzma Karamat
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Although the acute respiratory syndromes causing SARS-Coronaviruses are not new to humanity, the recent SARS-CoV-2 based epidemic has spread to almost every part of the world and claimed a large number of human lives without any discrimination of race, gender, and color. However, multiple issues related to its origin, its transfer time in humans, evolutionary patterns, and underlying forces that derived the SARS-CoV-2 outbreak and pandemic remain unclear. Knowing the pathogen is an essential step to devise appropriate strategies for controlling and treating associated infection. This chapter attempts to enhance knowledge regarding the history of SARSCoV- 2 origin, zoonotic transfer events, and related evolutionary adaptions. This manuscript also provides an overview of various factors that contributed to making this virus more compatible with infecting the human cell and evaluated the possibility of its engineered / laboratory-based emergence. Our in-depth literature analysis demonstrated that SARS-CoV-2 was possibly pre-adopted in different animal species. Molecular fingerprints and phylogenetic analysis have confirmed high similarity (96% and 84%, respectively) of SARS-CoV-2 with bats (RaTG13) and pangolins SARS-CoV-like coronavirus. The genomic similarities of SARS-CoV-2 are due to the spike glycoprotein and RBD domain and poly cleavage site with bats and pangolin coronaviruses. It conclusively suggests that it is not a man-made bioweapon but rather emerged naturally through the recombination process. Thus generated information may help develop effective treatment strategies for SARS-CoV-2 and avoid the high risk of its re-emergence in the future.
Evaluating SARS-CoV-2 Entry Pathways SARSCoV- 2 Entry Pathways
Page: 72-97 (26)
Author: Muhammad Kalim*, Firasat Hussain*, Hayat Khan, Kashif Rahim, Muhammad Shuaib, Amjad Islam Aqib and Jinbiao Zhan
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Coronaviruses (CoVs) are members of the Coronaviridae family that possess positive-sense RNA. These are enveloped viruses causing severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS), and currently coronavirus disease-19 (COVID-19) in humans. Still, there is less information available about the biology of Severe Acute Respiratory Syndrome- Coronavirus-2 (SARS-CoV-2). Recently, it was suggested that endocytosis mechanism studies and autophagy implicate importance in the viral entrance and an infection. These suggestions ascertain that endocytosis and intracellular trafficking studies have become essential target sites for developing therapeutic approaches. Initially, it was thought that coronaviruses possibly enter the host cell through direct diffusion, evading the membrane barriers. Laterally, it was found that the virus may enter the cell through the mechanism of endocytosis. Entry pathways and endocytosis of other viruses and especially SARS-CoV discussed here may expand the cellular range of viral endocytosis studies, pathogenesis, and infection. It may provide new information for pharmacokinetic studies and vaccine development. This chapter discussed some current advances in our understanding of cellular pathways of SARS-CoV-2 attachment, molecular signaling during virus entry, and trafficking mechanism studies.
Genomic Characterization of SARS-CoV-2
Page: 98-132 (35)
Author: Faisal Siddique*, Muhammad Farrukh Nisar, Maida Manzoor, Firasat Hussain, Muhammad Saeed, Kashif Rahim and Asif Javaid
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Coronaviruses is associated with three big public health nightmares in the 21st century globally, such as acute respiratory syndrome coronavirus (SARS-CoV, in 2002), Middle East respiratory syndrome coronavirus (MERS-CoV, in 2012), and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, in 2020). They have caused respiratory diseases in humans, particularly in the elderly, children, and pre-existing comorbidities and immunocompromised patients. SARS-CoV-2 was first recorded in Wuhan city, Hubei Province, Chinathat severely affecting the world economy by more than $1 trillion. It consists of an enveloped lipid bilayer and a positive-sense RNA genome. The genome size is approximately 30 kb. The SARSCoV- 2 structure consists of many essential proteins such as spike glycoprotein (S), membrane (M), envelope (E), and nucleocapsid (NC). This chapter highlights the updated knowledge of SARS-CoV-2 infections such as current and background history, phylogenetic tree analysis of SARS-CoV-2, expression of ACE2 genes in human tissues, phylogenic of S surface glycoprotein gene, M protein, E protein, NC protein, evolutionary resemblance/comparison with SARS-CoV-2 and MERS gene, the role of replication of novel strain lead to COVID-19, factors involve in COVID-19 pathogenesis and conserved and non-conserved gene of SARS-CoV-2. This study may be a little supportive of the battle against COVID-19 infection worldwide.
Mutation of SARS-CoV-2
Page: 133-161 (29)
Author: Umair Younas* and Kashif Prince
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Coronavirus undergoes more frequent mutations over time. It is well understood with previous outbreaks of the diseases, like the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome-coronavirus (MERSCoV). The behavior and pattern of diseases change with the mutation, and the virus becomes a new virus of its out kind regarding morbidity and mortality. Virus from different origins has been observed to have mutations at various places of their genome resulting in changed disease pattern resulting in the worldwide outbreak of the coronavirus disease-19 (COVID-19). Mutation in coronavirus is a slow and emerging process that may be responsible for the modulation of viral transmission, virulence, and replication efficiency in different regions of the globe. SARS-CoV-2 is going to spread around the world, and novel mutation hotspots are emerging in the genome. There has been increasing in the genetic diversity of coronavirus in human hosts. Random mutations cause the diversification of the virus leading to drug resistance, the changed pathogenicity and infectivity. This variation could remain in the virus, but it could be transferred to the next generation. The study was conducted to focus on viral evolution through mutation in various geographic regions of the world.
Biochemical Characterization of SARS-CoV-2 Spike Protein Proteolytic Processing
Page: 162-194 (33)
Author: Hayat Khan*, Firasat Hussain*, Muhammad Kalim, Shafi Ullah, Kashif Rahim and Faisal Siddique
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Coronavirus spike (S) glycoproteins belong to class I viral fusion protein. Spike protein has S1 and S2 domain that respectively are involved in receptor-binding and fusion of virus-host membrane. Spike protein of SARS-CoV-2 interacts with human angiotensin-converting enzyme-2 (hACE-2), expressing predominantly on the lungs and intestinal cells as a receptor. Although the receptor-binding motif of SARSCoV- 2 shares only 50% homology with that of SARS-CoV, its affinity towards hACE- 2 are many folds higher. The host proteases mediate the membrane fusion reaction through the cleavage of S protein between S1 and S2. The S of SARS-CoV-2 harbors cleavage site for furin or furin-like protease, which sets the SARS-CoV-2 apart from SARS and SARS-like other coronaviruses. The S protein is cleaved either by one or several host proteases depending upon the infecting cells and virus strains. Based on the presence of a different type of host protease, the CoVs decides whether to enter cells via the cell membrane route or endocytosis. Unlike SARS-CoV, S of SARS-Co- -2 causes typical syncytium formation (cell-cell fusion) among the infected cells. Besides receptor binding, S protein is a major target of neutralizing antibodies following infection. However, antibodies raised against SARS-CoV either weakly or fail to neutralize the SARS-CoV-2, suggesting no cross-protection between them. Being is a driver of viral entry, the spike protein of SARS-CoV-2 is a good candidate for vaccine development. Furthermore, the role of host proteases in the membrane is also very significant for the designing of entry inhibitors. The purpose of this chapter is to summarize the detailed structure, fusogenic potential, protease-driven activation, and cross-neutralization of spike protein with antibodies from SARS-CoV and sera of recovered patients.
Crystal Structure Determination and Receptor Recognition Basis of SARS-CoV-2 Spike Glycoprotein
Page: 195-225 (31)
Author: Maher Darwish*
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A new coronavirus outbreak has emerged in Wuhan, China, in 2019 and turned into a global pandemic posing a massive public health concern. Its genome has been sequenced and showed pairwise percent identities of approximately 79.5% and 50% compared to severe acute respiratory syndrome-coronavirus (SARS-CoV) and the Middle East respiratory syndrome-coronavirus (MERS-CoV), respectively. The coronavirus spike glycoprotein is the primary determinant of viral tropism and is responsible for receptor binding and membrane fusion. The receptor-binding domain of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has been characterized and structurally identified through X-ray crystallography and cryoelectron microscopy. The variations in residual sequences, hidden receptor binding domain, and furin-like cleavage nature of the spike clarified the higher receptor affinity and efficient spread among humans compared to SARS-CoV. The spike glycoprotein is a vital target for vaccines and therapeutic antibodies. Correspondingly, the potential implication of its structure elucidation in developing new and cross-neutralizing antibodies targeting the spike epitopes has been briefly discussed. Here, it is expected that the elaborated details of the molecular structure may facilitate the opportunity of developing therapeutics against SARS-CoV-2.
Structural Elucidation of SARS-CoV-2 Accessory Proteins
Page: 226-249 (24)
Author: Tahir Shah* and Kamal Niaz
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The coronaviruses (CoVs) are a huge family of positive-sense RNA viruses with a single-strand. These CoVs consist of 38% G+C content and around 29,900 nucleotides, which encodes about 9,860 amino acids. It includes open reading frames yielding two polyproteins which are cleaved into 16 nonstructural proteins (nsps) proteolytically. These nsps play pivotal roles inside the lifestyles cycle of CoVs. To deal with the present-day outbreak, using wide-spectrum inhibiting drugs against CoVs is an appealing tactic. Still, for this reason, the knowledge of specific target places of nsps within the CoVs genus is necessary. The position of each nsp is well defined. The main protease (Mpro) forms replicase polyprotein complex. The papain-like protein (PLpro) also suppresses type 1 interferon signaling, thus risking the host's immunity. The NSP-3 takes part in the processing of polyprotein as a protease. Also, the NSP-8 and NSP-9 attach to the cis-acting parts of RNA of the virus. NSP-10 is a vital cofactor forming compounds with NSP-14 and 16. The NSP-12 encodes RNA-dependent RNA polymerase (RdRp). At the same time, the catalyzing and unwinding of duplex oligonucleotides into single strands is done by NSP-13 helicase. The NSP-14 of CoVs is important for viral replication and transcription, and NSP-16 is activated when the bond with NSP-10 preventing host recognition and decreasing the immune response. The purpose of this chapter is to elucidate the accessory proteins of SARS-CoV-2 structurally. Also explains the inflammatory cytokines produced due to SARS-CoV-2, residues involved in the interaction between viral spike and ACE2, the structure of receptor-binding domain (RBD) of ACE2 in SARS-CoV-2, and possible potential compounds that have been studied and found effective against SARS-CoV-2.
Antigen Capture Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of the Novel SARS-CoV-2 in Humans
Page: 250-272 (23)
Author: Muhammad Farooq* and Zia Ullah
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Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is the reason behind the transmission of the recently prevailing disease worldwide and is known as coronavirus disease 19 (COVID-19). Coronavirus was first diagnosed in Wuhan, China 2019, which followed a worldwide spread. Whereas assays associated with the examination of virus molecules only detect the hereditary viral substance in the patient's body. On the other hand, the novel coronavirus requires the diagnosis of high serological parameters to examine specific antibodies of SARS-CoV-2. In this case, the only solution is the -linked immunosorbent assay (ELISA) test based on hunting the antigens. In this technique, the combination of antibodies is derived from the S proteins present in the cells of infected coronavirus patients. By applying these test assays for the COVID-19 patients, it is obvious that the nature of the testing is specific and sensitive. This test allows the detection of serum through seroconverts since the visible emergence of symptoms of SARS-CoV-2 in patients. The major benefit of this method is that it takes less time during the examination. Instead, it deals with the identifications of different antigens found in the human body. These tests play a pivotal role in characterizing introduction and recognize profoundly receptive human donors. Herein, we developed an ELISA assay for the easy detection of SARS-CoV-2 and nucleocapsid protein expression levels as an early tool for this disease.
Quantification of the SARS-CoV-2 RNA in Tissues by Quantitative Real Time-PCR
Page: 273-300 (28)
Author: Abhinav Anand, Neha Sharma, Sonali Bajaj, Naman Wahal, Devesh Tewari and Navneet Khurana*
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Severe Acute Respiratory Syndrome-coronavirus-2 (SARS-CoV-2) has become infamous recently due to its capability to cause a severe disease known as coronavirus disease-19 (COVID-19). Believed to have originated from Wuhan, Hubei province, China, SARS-CoV-2 has impacted a global scale, thereby making the World Health Organization (WHO) declare COVID-19 a pandemic. The range of R0 values is between 2.43 and 3.10, showing the severity of the disease, highlighting this concern for health and public administration authorities. The major preventive steps from the governments and public health authorities are to conduct aggressive diagnostic tests and isolate the patients until the complete recovery of COVID-19. Out of various diagnostic methods, reverse transcription-polymerase chain reaction (RT-PCR) is an appropriate technique. The amplified RNA/DNA molecules can be quantified through quantitative real-time PCR (qRT-PCR). In this chapter, the basics of PCR shall be discussed along with the quantification of RNA from SARS-CoV-2 infected tissue samples.
Evaluation of SARS-CoV-2 Neutralizing Antibodies in Sera Using Live Virus Microneutralization Assay
Page: 301-314 (14)
Author: Shaukat Hussain Munawar*, Zahid Manzoor, Muhammad Farrukh Nisar, Muhammad Yasir Waqas and Imran Ahmad Khan
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Neutralizing antibodies (nAbs) make a defense line against viral attacks by binding to viruses to shield infections. A neutralizing antibody interferes with the virus in different ways as it may block the cell receptor or bind to viral capsid by inhibiting genome un-coating. The micro-neutralization (MN) assay is a basic technique for detecting viruses in epidemiological, immunological, virological studies, and vaccine assessment tests. The underlying mechanism of this assay is based on the detection of specific antigen-antibody reactions. It only detects the antibodies involved in the blockage of the virus replication. This technique is specifically helpful in evaluating specific micro-organism serotype neutralizing antibodies in the sera of humans and animals. It describes the neutralization or inhibition of replicating virus strains by antibodies in the sera of humans and animals. But highly constraint provisions may be required while working with live virus-based micro-neutralization techniques, particularly when handling precarious micro-organisms like severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Herein, all the possible data regarding isolation, amplification, and titration of SARS-CoV-2 and MN assay to measure nAbs level in the sera of mammalian species have been summarized.
Pseudovirus Neutralization Assay for SARS-CoV-2
Page: 315-336 (22)
Author: Ankush Sharma, Sneha Joshi, Pooja Patni, Navneet Khurana and Devesh Tewari*
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The World Health Organization (WHO) has indicated coronavirus disease- 19 (COVID-19) as a global pandemic. A virus that is formed by the recombination process and whose genetic material as well as protein-rich envelope are resulting out of various viruses is known as pseudovirus. There are certain advantages of using pseudovirus over native viruses. Due to several novel viruses e.g., Marburg virus, Ebola virus, Lassa fever virus, immunodeficiency virus, and coronavirus, there is a development of numerous packing arrangements to produce pseudotype virus having envelope in their structure. This chapter deals with various virus packaging systems with a focus on production and neutralization assay of pseudovirus.
Quantitative and Qualitative Determination of COVID-19 (SARS-CoV-2) Specific Antibodies Using ELISA
Page: 337-371 (35)
Author: Mohammad Ejaz, Shahzad Ali and Muhammad Ali Syed*
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After the emergence of coronavirus disease-19 (COVID-19) in late 2019 spread to different countries, becoming a global health concern. It belongs to the betacoronavirus family notorious for causing two outbreaks of severe acute respiratory syndrome (SARS-CoV) and the Middle East respiratory syndrome (MERS-CoV) in the last decade, causing huge morbidities and mortalities in a large number of population. For the current pandemic of COVID-19, accurate and precise diagnosis and surveillance of the infected individuals must be tackled by providing healthcare facilities and preventive measures in hotspot regions. COVID-19 patients were observed with non-specific symptoms like dry cough, sore throat, and fever in the early phase of infection that can worsen to serve acute respiratory syndrome and shock in the later phase of infection. Besides clinical symptoms, the radiographical finding of lungs and other definitive tests like real-time-polymerase chain reaction (RT-PCR) confirms the presence of a causative agent of COVID-19. After the infection of COVID-19, the human immune system activates and tries to neutralize the infection by forming virusspecific neutralizing antibodies. These antibodies can be detected in the patient's serum and can serve as the diagnostic biomarker. Several different types of serological assays, including enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, chemiluminescence enzyme immunoassays, immunofluorescence assay, and lateral flow immune assay, used to detect the presence of antibodies in the serum of infected individuals. This chapter will focus on the quantitative and qualitative determination of SARS-CoV-2 specific antibodies by different serological assays like ELISA in COVID-19 patients.
Chest CT Scan: An Ideal Diagnostic Tool for COVID-19
Page: 372-404 (33)
Author: Noor Muhammad Khan*, Muhammad Adil, Amar Nasir and Arbab Sikandar
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Diagnosis of any disease requires careful assessment. Diagnostic procedures might play a pivotal role in making the treatment protocols more powerful/potent efficient, and effective by highlighting the clinical findings of the disease. Along with other diagnostic techniques, a computed tomography (CT) scan has been employed to diagnose coronavirus disease-19 (COVID-19) patients since the outbreak of this disease and therefore promises it as a crucial diagnostic tool. A CT-scan is a specialized medical imaging technique that produces cross-sectional images of specific areas of a targeted object utilizing a combination of multiple X-ray measurements taken from multiple angles. CT scan help diagnose COVID-19 individuals display severe clinical features and advanced forms of the disease. Pulmonary CT images of COVID-19 patients had common diagnostic manifestations such as ground-glass opacities (GGO), consolidation, reticular pattern, and fibrosis. It also includes nodular lesions reversed halo sign. and thickening of the pleura as the less common findings. The receiver operative characteristic (ROC) curve has been successfully applied for determining the accuracy of the CT-scan-based diagnosis of COVID-19. Artificial intelligence (AI) techniques, particularly deep learning, are extensively used for processing and evaluating imaging data, thereby improving the diagnostic performance of radiologists and clinicians. Despite its emergence as an effective method for screening COVID-19 patients, a CT scan is not recommended as a primary tool for diagnosing COVID-19 and must be used with utmost caution as it may cause the transmission of COVID-19 pathogen in the current epidemic. Overall, the current chapter focuses on CT-scan implications in diagnosing the COVID-19 infection and its comparison with the other diagnostic tools.
Nucleic Acid-Based Detection of COVID-19
Page: 405-419 (15)
Author: Abdul Basit, Ihtisham Ulhaq, Firasat Hussain, Zia-ud-Din and Kashif Rahim*
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Current coronavirus disease-19 (COVID-19) pandemic is caused by a novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), posing a major health problem worldwide. Therefore, rapid and accurate identification of COVID-19 is playing an important role in preventing subsequent secondary spread from controlling this epidemic. Different diagnostic tools and nucleic acid (NA)-based technologies for COVID-19 have been adopted. Polymerase chain reaction (PCR) is widely used for molecular diagnosis at prime level. However, it exhibits low sensitivity and falsepositive results in clinical applications. Therefore, loop-mediated isothermal amplification (LAMP) assay is considered a prominent NA amplification method for diagnosing COVID-19. Similarly, microarray and RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) with advancements in instrumentation are recently adapted for rapid, high throughput, and portable sensing of COVID-19 NA detection. Besides, nanopore technology, rolling circle amplification-based method and silicon-based integrated point-of-need (PoN) transducer are also promising diagnostic tools for detecting COVID-19. This chapter will discuss the potential of all these latest technologies for COVID-19 diagnosis in detail.
Page: 420-428 (9)
Author: Kamal Niaz and Muhammad Farrukh Nisar
COVID-19: Epidemiology, Biochemistry, and Diagnostics explains COVID-19 from multidisciplinary angles such as the evolution of SARS-COV, genetic techniques to study the virus, and diagnostic methodologies widely used in the global COVID-19 pandemic. The chapters in this book provide the reader with up-to-date literature about research on SARS-CoV-2 through three parts: I) Evolution and Entry of SARS-CoV-2 into the host II) Genetic Alteration and Structural Determination of SARS-CoV-2 Proteins III) Quantitative Analysis of SARS-CoV-2 for research and medical diagnosis Key Features: - 15 chapters on SAR-CoV-2 in a multidisciplinary context - Provides a comprehensive overview of SARS-CoV-2 evolution and genetics - Provides biochemical information about SARS-CoV-2 proteins and receptor targets (both structural and non-structural proteins) - Includes an overview of several methods of detecting SARS-CoV-2 virus particles (ELISA, PCR, Neutralizing Antibodies - Covers some critical diagnostic modalities for COVID-19 diagnosis - Provides bibliographic references for further reading Readers will understand the significance of phylogenetic analysis of coronaviruses, along with the pathogenesis of COVID-19 and related diseases such as SARS and MERS. Applications of biochemical technologies such as RT-PCR and CRISPR are also demonstrated in the text. This book is a comprehensive introduction to COVID-19 research for medical researchers, microbiologists and virologists. Students in academic programs in life sciences and medicine will also benefit from the information provided in the book.