key: cord-0059199-ud2rpcwm
authors: Singh, Vishwendra; Luthra, Ankur; Chauhan, Rajeev; Meena, Shyam C.
title: Basic Virology and Pathophysiology of COVID-19
date: 2020-10-06
journal: Clinical Synopsis of COVID-19
DOI: 10.1007/978-981-15-8681-1_2
sha: a3d5126bb7f03a7ce6355c892f506647218b7d18
doc_id: 59199
cord_uid: ud2rpcwm

The world welcomed the year 2020 with the spread of the novel coronavirus. The pandemic of COVID-19 has claimed numerous lives with the counts constantly rising day by day. SARS-CoV-2 is a single-stranded RNA virus containing a genome of approximately 30,000 base pairs. It belongs to the family Coronaviridae. Genomic characterization has revealed that it is a zoonotic virus that has somehow jumped to humans. SARS-CoV-2 complies with the general morphological characteristics of coronaviruses and has a diameter of about 70–90 nm. Spike, membrane, envelope, and nucleocapsid proteins comprise the main structural proteins of the virus and help in its transmission and pathogenesis. Its genome contains at least six open reading frames (ORFs) at the 5′ end which encode pp1a and pp1b proteins. These produce nonstructural proteins which are required for optimum function of the virus particle. The structural proteins are coded for by the ORFs near the 3′-end of the genome. SARS-CoV-2 displays typical viral internalization and amplification cycles inside the host cell to which it binds by the spike protein. This spike protein displays specific amino acid sequences in its receptor binding domain which enable it to form a strong bond to host receptor. Host cells produce different immune products as a reaction to SARS-CoV-2 infection. These are responsible for controlling the progress of infection. However, they can lead to acute respiratory distress and other tissue injuries if produced in uncontrolled quantities. Histopathological examinations of affected organs show cytopathic effects due to viral infection. A lot has been written on COVID-19—its typical and atypical symptoms, mode of spread, organ systems affected, and ways of prevention. However, an understanding of what the virus contains and actually looks like (structure), its classification and evolution (position on the phylogenetic tree), and the ways in which it can infect and produce disease in the host (pathophysiology) is important to develop strategies for prevention and mitigation of the ailment caused by the novel coronavirus. This chapter presents a detailed description of the basic virology and pathophysiology of SARS-CoV-2.

intermediate hosts. Genetic analysis of the genome of SARS-CoV-2 showed that it is related to the bat CoV RaTG13. It is, thus, a separate lineage than SARS and bat SARS-like CoVs. It is now suggested that the 2019 novel coronavirus is a new human-infecting coronavirus which most probably originated from bats and, thereafter, jumped to human via intermediate hosts [7] . Liu et al. [8] predicted the interaction between receptor binding domain of coronavirus spike protein and the host receptor after analyzing coronavirus genome sequences and spike protein residues. They suggest that bats are the natural host, while pangolins, snakes, and even turtles can serve as intermediate hosts for the novel coronavirus. A definite conclusion about the natural and intermediate hosts is, however, still missing.

As mentioned above, a virus has to fulfill certain criteria to be called a coronavirus. Members of Coronaviridae family are relatively large, enveloped, single-stranded RNA viruses. In fact, they are the largest known RNA viruses with the virion sizes ranging from 118 to 136 nm in diameter and genomes ranging from 25 to 32 kilobase pair (kbp) in length. The virions are spherical in shape and are characterized by relatively large spikes that emerge from the virus envelope [10] . Park et al. [11] isolated and reported the morphology of the novel coronavirus in a 35-year-old female patient. For identification, monolayers of vero cells were inoculated with the oropharyngeal samples of the patient. Five days after this first inoculation, blind passage of the culture supernatant was done. Thereafter, vero cell monolayers showing cytopathic effects were fixed using 2% formaldehyde and 2.5% glutaraldehyde. These fixed cells were sectioned and observed under an electron microscope. Observation revealed spherical particles with crown-like spikes. These spherical particles were 66-81 nm in diameter and were observed within the cytoplasmic vesicles and in the extracellular space next to the cell membrane. Four main proteins, called the structural proteins, and other accessory proteins make up and define the visible structure of the novel coronavirus. The structural proteins starting from the inside out include nucleocapsid (N) protein, membrane (M) glycoprotein, envelope (E) glycoprotein, and the spike (S) glycoprotein [12] .

The nucleocapsid also known as the N proteins functions primarily to bind to the CoV RNA genome, making up the nucleocapsid [13] . It is located in the endoplasmic reticulum-Golgi region which is bound to the nucleic acid of the virus. It is involved in functions of the viral genome, replication of the virus, and the host response to viral infections [12] . It is heavily phosphorylated and is thought to lead to structural modifications that enhance affinity for viral RNA. Transient expression of the nucleocapsid proteins has, however, been shown to increase the production of virus-like particles (VLPs) markedly in some coronaviruses, thus suggesting that this protein might be required for complete virion formation and not envelope formation [14] .

The next is the membrane or M protein. It is the most well-structured and the most abundant of all structural proteins in SARS-CoV-2. It determines the shape of the viral envelope. It is considered to be the central organizer of coronavirus assembly. It can bind and interact with all other major structural proteins [14] . Homotypic interactions among M proteins are responsible for the formation of virion envelope, but the M protein is not sufficient for virion formation by itself [15] . Interactions between M and N proteins stabilize the nucleocapsid proteins (N) and help in completion of viral assembly [12] . The interaction of spike (S) and membrane (M) proteins is essential for retention of S in the endoplasmic reticulum-Golgi intermediate compartment and also for the incorporation of S into new virions. However, it is not required for the assembly process [14, 16] . The M and E proteins together make up the viral envelope, and their interaction leads to the production and release of VLPs [14] .

The envelope (E) protein is the smallest among the major structural proteins. It is a short, integral membrane protein of 76-109 amino acid sequences and ranges from 8.4 to 12 kDa in weight [14, 17, 18] . E has a short, hydrophilic amino terminus containing 7-12 amino acids and a large hydrophobic transmembrane domain of 25 amino acids. It ends with a long, hydrophilic carboxyl terminus which makes up a majority of the protein [14, 19, 20] . The envelope protein is needed for production and maturation of the virus. It is copiously expressed inside an infected cell, but only a small portion of this is incorporated into the virion envelope [14] . It is mainly localized at sites of intracellular trafficking-the endoplasmic reticulum, Golgi apparatus, and the endoplasmic reticulum-Golgi complex. Here it partakes in the assembly and budding of the virus [21] . Its importance in virus production and maturation can be gauged from the fact that recombinant coronaviruses which lack the envelope protein display markedly reduced viral titers, affected viral maturation, or produce incompetent progeny [14, 22] .

The outermost protein responsible for giving a characteristic appearance and name to the coronavirus family is the spike (S) protein. It is a transmembrane protein and has a molecular weight of around 150 kDa. It mediates the entry of the virus into the host cell and hence also receives particular attention from scientists. The S glycoprotein forms homotrimers that project up from the viral structure and promote binding to the host cells by attraction and attachment to the angiotensinconverting-enzyme 2 (ACE2) receptors [12] . This protein is made up of two functional subunits-S 1 and S 2 . These can be recognized upon cleavage of the S protein by host cell furin-like protease [3, 16] . Subunit S 1 binds to the host cell receptor and thus determines the host range and the types of cells that a virus can affect. Subunit S 2 mediates fusion of virus with the host cell membrane. Both these subunits remain non-covalently bound in the prefusion state [3] . The distal S 1 subunit makes up the receptor-binding domains (RBDs) and stabilizes the S 2 subunit that has the machinery needed for fusion in the prefusion state [23] . The S homotrimers contain numerous N-linked glycans which are needed for achieving the correct structure and access to host proteases and neutralizing antibodies [3] . Compared to the S protein of SARS-CoV (and other beta coronaviruses), the spike protein of SARS-CoV-2 has 12 extra nucleotides at its cleavage site which is similar to a canonical furinlike cleavage site. The presence of this furin-like cleavage site might increase the efficiency of spread of SARS-CoV-2 as compared to other beta coronaviruses [24] .

Some beta coronaviruses exhibit an additional structural protein called the hemagglutinin-esterase (HE) protein. This protein binds to sialic acid present on the virion surface, and this binding and the esterase activity together help the virus enter into the host cell. This entry is further facilitated by the spike protein. The HE protein also helps in mucosal spread of the virus [24] . A difference between SARS-CoV and SARS-CoV-2 is that HE proteins are present in SARS-CoV although they lack membrane fusion activity and are accessory to the spike protein. It is also debatable whether they help in virion attachment or not [25] . However, the genome of SARS-CoV-2 lacks the hemagglutinin esterase gene [26] . A graphical representation of the structural proteins and genomic material of the novel coronavirus is given in Fig. 2 

All these proteins along with other nonstructural proteins are coded for by the genome of the virus which is discussed in the next section.

Coronaviruses possess a single-stranded positive sense RNA (+ssRNA). There is a 5′-cap and 3′-poly-A-tail. The novel coronavirus genome is about 30 kb long and has at least six open reading frames (ORFs). At the 5′ end, ORF1a and ORF1b, the first open reading frame genes, constitute around two thirds of the complete genome length and codes for pp1a and pp1b proteins, respectively. These together make up 16 nonstructural proteins (nsp1-nsp16) [27] . These nonstructural proteins are required for the maintenance, replication, and optimum function of the virion.

A summary of their purpose in the virion is presented in It is also known that these mutations in the novel coronavirus exist at the nucleotide level in S gene, nsp1, nsp3, and nsp15, and not at the amino acid level [1] .

Lu et al. [29] have described the genomic characteristics of the novel coronavirus in detail. Their analysis revealed that the novel coronavirus is most closely related to bat-SL-CoVZC45 and SARS-like betacoronavirus of bat origin, bat-SL-CoVZXC21. The sequence resemblances were more than 90% in five gene regions-E, M, 7, N, and 14. Out of these, the E gene displayed a sequence similarity of 98.7% (the highest). The lowest similarity, between the novel coronavirus Process mediated nsp1

Inhibition of interferon signaling, degradation of cellular mRNA nsp2

Not known nsp3

Cleaving of papain-like protease domains, blocking host innate immune response, promoting cytokine expression, tether RNA genome to the replicase/ transcriptase complex nsp4

Formation of double membrane vesicle (membrane remodeling), transmembrane scaffold nsp5

Inhibition of interferon signaling, splitting chymotrypsin like proteins and main protease nsp6

Formation of double membrane vesicle, restrict expansion of autophagosome nsp7

Acts as a co-factor of nsp8 and nsp12 nsp8

Acts as a co-factor of nsp7 and nsp12, primase, can function as a processivity clamp for RNA-dependent-RNA-polymerase (RdRp) nsp9

Dimerization and binding of single-stranded RNA nsp10

Acts as building protein for nsp14 and nsp16 nsp11

Not known nsp12

RNA-dependent-RNA-polymerase primer nsp13 RNA 5′ triphosphatase (cap synthesis), RNA helicase nsp14 N7-methyltransferase, exo N 3′-5′ exonuclease (provides proofreading function for coronavirus RdRp) nsp15 N endo U endonuclease (cleaves single-and double-stranded RNA downstream of uridylate residues, producing 2-3 cyclic phosphates), evasion of doublestranded DNA sensors nsp16

Methyltransferase, helps avoid MDA5 (melanoma-differentiation-associated protein 5) recognition, downregulates innate immunity and the other bat-SLCoVs, was seen in the S gene region being only around 75%. An interesting finding was the low genetic resemblance between SARS-CoV and SARS-CoV-2 (about 79%) and between SARS-CoV-2 and MERS-CoV (about 50%). Compared to previous coronaviruses-the SARS-CoV, MERS-CoV, and the bat SARS-like coronaviruses-SARS-CoV-2 codes for a longer spike protein.

As mentioned earlier, the spike (S) protein facilitates binding to host receptor and fusion with cell membrane and is functionally composed of the S1 domain and S2 domain-the functions of them both have been discussed in the previous section. The S2 protein of the novel coronavirus exhibits a 93% similarity to bat-SL-CoVZC45 and bat-SL-CoVZXC21, while the S1 protein displays a similarity of only 68% with these viruses. It is seen that the receptor binding domain of the mentioned bat coronaviruses is located in the C-terminal domain of S1; however, despite similarities in the S1 and S2 domains, the receptor binding domain of SARS-CoV-2 falls within lineage B and is hence closer to that of SARS-CoV. Thus, in terms of the whole genome sequence, the novel coronavirus is closer to bat SARS-like coronaviruses than SARS-CoV. Based on the observations above, certain differences in the genome sequences of the novel coronavirus (SARS-CoV-2) and SARS-CoV are given in Table 2 .3. However, it remains to be seen how these differences affect the pathogenesis and functionality of the novel coronavirus. A basic representation of the genomes of the novel coronavirus and that of SARS-CoV is given in Fig. 2. 3. Upon analysis and comparison of the genome of SARS-CoV-2, it is evident that it resembles the genome of bat coronaviruses. Hence, the host and intermediate hosts of this virus can also be identified. As seen earlier, bats are purported to be the natural host of this virus. However, there are several factors suggesting that the conjecture of a direct transmission from bats to humans is fraught with inadequacies as mentioned below:

1. An outbreak was reported in late December 2019. This is a period of winter when bats are hibernating. Hence, based on the above points, the hypothesis that bats are the natural hosts while some other animal acted as an intermediate host of the virus is further strengthened [29] . However, making conclusive remarks about definitive and intermediate hosts is not possible at the moment.

Viruses are like parasites which need another living body to survive. Outside this body, they either lie dormant or cannot survive for long. Similarly, the novel coronavirus too needs the human body to survive. The virus can enter the body either through direct contact, indirect transmission through aerosols, or through droplets [30] . There are different steps involved in entry and propagation of the virus inside the human body which are given below:

1. Attachment: Angiotensin-converting enzyme 2 (ACE2) receptor is a zinc-binding carboxypeptidase receptor expressed on the cell surfaces of different organ systems, viz., lungs, heart, ileum, small intestine, kidney, bladder, and others [10, 31] . This receptor has been identified as the target receptor for SARS-CoV-2.

Receptor recognition is the first step in viral infection. It is also a key determinant of host cell and tissue tropism. As discussed earlier, the spike (S) protein of SARS-CoV-2 is responsible for recognition and attachment to human ACE2 (hACE2) receptors. It contains a receptor binding domain (RBD) that specifically recognizes ACE2 as its receptor. The RBD contains a core and a receptorbinding motif (RBM) that mediates contact with the receptor. The surface of ACE2 contains two virus-binding hotspots that are essential for binding with the virus. While binding to the receptor, the S protein RBM forms a concave surface with a ridge on one side which binds to the exposed outer surface of the "clawlike" ACE2 receptor. As compared to SARS-CoV, the RBM of SARS-CoV-2 forms a larger binding interface and more contacts with its receptor. SARS-CoV-2 RBM contains a four-amino-acid residue motif: glycine-valine/glutamineglutamate/threonine-glycine. Due to these bulky residues and flexible glycine, the binding loop takes a different conformation as compared to SARS-CoV. This structural difference leads to the formation of an additional main chain hydrogen bond between Asn487 and Ala475 in the RBM of the novel coronavirus. Due to this the ridge takes an even more compact arrangement, and the loop containing Ala475 gets even closer to ACE2. As a result the ridge forms more contacts with the N-terminal helix of ACE2 [32] . Two virus-binding hotspots have been identified in ACE2-SARS-CoV-2 interface: hotspot Lys31 (also known as hotspot 31) and hotspot Lys353 (also known as hotspot 353). These hotspots are relatively weak in SARS-CoV; however, in SARS-CoV-2, they are stronger and wellstabilized due to different conformations. These hotspots are important for coronavirus binding [32] . 2. Penetration: As mentioned earlier, the S protein is made of two subunits-S 1 and S 2 -which are non-covalently bound. The presence of the four amino acid residue at the boundary between S 1 and S 2 subunits results in the introduction of a furin cleavage site. The cleavage of the subunits occurs by furin present in the Golgi compartment. One reason, why the novel coronavirus affects different organs and is highly transmissible, could be the presence of this polybasic cleavage site in the fusion glycoprotein (S) which is cleaved by furin-a protease which is found ubiquitously in the body [3] . Once attachment occurs, type II transmembrane serine protease (TMPRSS2), which is present on the host cell surface, clears the ACE2 and activates the receptor-attached S protein. S protein is also activated by furin via cleavage. Activation of the S protein causes conformational changes and allows the virus to enter host cell either by fusion of the envelope (E) protein with the host cell surface or by the endosomal pathway [12, 33] . If virions are taken up into endosomes, cathepsin L activates the spike protein. However, this cysteine protease can be blocked by lysosomotropic agents (like bafilomycin A1 or ammonium chloride inhibitors). Thus, it is not an efficient mode of viral replication. Alternatively, if the S protein is activated by TMPRSS2, the viral membrane fuses with the plasma membrane [34] . This fusion is less likely to activate host cell immunity and is thus a more efficient method of viral replication. 3. Biosynthesis: Once the virus enters a host cell, it releases its genetic material in the cytoplasm. The first synthetic event in the life cycle of coronavirus is the translation of viral genome by host cell ribosomes. As the released viral genome is positive stranded, 5′ capped, and 3′ polyadenylated, it can be directly translated to proteins by the host ribosomes. The virus has 14 open reading frames (ORFs) which code for a variety of proteins-structural as well as nonstructural.

The gene segments which code the nonstructural proteins (nsp) are translated first into ORF1a and ORF1b. This translation produces two large overlapping polyproteins-pp1a and pp1ab through a ribosomal frame-shifting mechanism. These polyproteins are supplemented by protease enzymes-papain-like proteases (PL pro ) and a serine-type M pro [chymotrypsin-like protease (3CL pro )] protease which are coded in nsp3 and nsp5. Thereafter, cleavage occurs between pp1a and pp1ab into nsp1-nsp11 and nsp1-nsp16, respectively [12] . These nsps play important roles in various processes in the virus and host cells which are mentioned in Table 2 .2. Several of these nsps form replicase-transcriptase complex (RTC) with RNA-dependent RNA polymerase (RdRp) in double membrane vesicles (DMVs). This complex leads to transcription of positive sense mRNAs, and this process is mediated by RdRp [12] .

During biosynthesis, the subgenomic proteins get translated to structural (and nonstructural and accessory) proteins-N, M, S, and E. These proteins are bound/ formed on smooth-walled vesicles in the endoplasmic reticulum and then moved to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) [12] . Genomic RNA is bound by N protein. This associates with M protein and buds into endoplasmic reticulum/Golgi membranes. M packs into membranes and is thought to produce membrane curvatures which leads to budding. S and E are acquired during budding [10] . 5. Release of virions: The synthesized virions are released from the host cell via exocytosis. The ion channel activity of E protein is that of a viroporin. It alters the cell secretory pathways to expedite the release of virions from the cell. It increases the pH of transport vesicles [10] . Once these vesicles fuse with the plasma membrane, virions are released to continue infecting other cells. 

Host Immune Response [35, 36] . TLR-4 recognizes the spike protein. Through mediation of MyD88, it triggers activation of NF-κB transcription factors and the pathogen-activated protein kinase pathway to produce pro-inflammatory proteins. Activation of endosomal receptors like the TLR-3 and TLR which can recognize the RNA or dsRNA genome of coronavirus leads to recruitment of TRIF adapter protein directly. TRIF in turn directs interferon response factor 3 (IRF 3) and nuclear factor NF-κB transcription factors to induce pro-inflammatory cytokines like interferon-α (IFN-α) and tumor necrosis factor-beta (TNF-β). Type I IFN complexes with its receptors and activates the JAK-STAT pathways. JAK1 and TYK2 kinases phosphorylate STAT1 and STAT2 followed by its complexing with IFN-9. Both these migrate to the nucleus to start transcription of IFNstimulated genes and lead to suppression of viral replication and prevent the severity of the disease [12] . This process can cause exhaustion, weakness, and [37] . It is seen that SARS-CoV-2 infection produces an aberrant immune response wherein the release of pro-inflammatory cytokines like IFN-α, IFN-γ, IL-1β, IL-6, IL-12, IL-18,  IL-33, TNF-α, and TGFβ, and chemokines CCL2, CCL3, CCL5, CXCL8 , CXCL9, and CXCL10 in excessive quantities from immune effector cells leads to hyperinflammation leading to acute respiratory distress syndrome (ARDS) [12] . (b) Cell-mediated immunity: The APCs present the CoV antigens to CD4+ T-helper (Th) cells via major histocompatibility complex (MHC) class 1. This causes release of IL-12-a stimulatory molecule which stimulates Th1 cell activation.

In addition to the stimulation of Th1, other processes like the release of IL-12 and IFN-α, increase in MHC class I expression, and activation of natural killer (NK) cells are also required for thwarting viral replication and eradication of virus-infected cells. Antigen presentation also causes production of proinflammatory cytokines via the NF-κB signaling pathway. These cytokines recruit neutrophils and monocytes to the infection site and activate other proinflammatory cytokines and chemokines including IL-1, IL-6, IL-8, IL-21, TNF-β, and MCP-1 [12] . Activation of Th1 cells stimulates CD8+ T cells which target and kill cells infected with the coronavirus. Simultaneously, CD4 T cells stimulate humoral immune response by activating T-dependent B cells [12, 38] . It is seen that in COVID-infected patients, the number and function of CD8+ T cells is greater than CD4+ T cells. Also, virus-specific T cells from severely infected patients tend to possess a central memory phenotype with a significantly higher polyfunctional CD4+ and CD8+ T cells. Strong T cell responses have been shown to have higher neutralizing antibody, while more serum Th2 cytokine secretion (e.g., IL-4, IL-5, IL-10-which increase production of antibodies) have been observed in deceased patients [37] . (c) Humoral immunity: The production of neutralizing antibodies plays a protective role in limiting infection. It also prevents re-infections in the future. The antibodies produced against SARS-CoV-2 infection are IgM and IgG which display a unique presence pattern. Usually IgM produced against SARS-CoV-2 lasts for only 12 weeks, but IgG will last longer [12] . SARS-CoV-2 infection induces production of IgG against the N protein, and this can be detected as early as day 4 after the onset of disease with most patients seroconverting by day 14 [37] . In addition to antibody formation, exposure to the novel coronavirus also leads to formation of CD4 T cells and CD8 memory cells as seen above, and these can last for up to 4 years [12] .

Over the course of evolution, viruses have developed mechanisms to protect them from immune system cells. This ability enables them to survive and infect host cells efficiently. Such an avoidance strategy can be applied to different processes both before and after entering a host cell. As seen earlier, host immune cells recognize a virus by various PRRs. The virus can avoid recognition by forming double membrane vesicles which lack these PRRs. This way they can replicate inside such vesicles without their dsRNA being detected [39] . Additionally, the virus has proteins that block IFN and thus avoid the immune system onslaught. nsp1 can suppress IFN 1 by inactivating the host translation mechanism, degrading host RNA and inhibition of STAT1 phosphorylation. This mechanism can cause IFN 1 failure and can lead to dissemination of viruses at an early stage and hence an increased severity of the disease [12, 40] . nsp14 and nsp16 can help the virus mimic the host capping mechanism. nsp14 initiates cap formation (5′ end similar to host cell RNA), and nsp16 later modifies it to viral RNA. This way the virus seems similar to host cell RNA and thus can escape being recognized by immune cells [12, 41, 42] .

nsp3 is another nonstructural protein which encodes two functional proteins, macro-domains and PL pro (which cleaves nsps). This nsp also helps the virus evade host cell immune response. Besides nonstructural proteins, the virus may also use accessory proteins to escape immune response. As an example, a protein coded for by ORF3b can antagonize the IFN signaling pathway and thus lead to inhibition of effector cell activation cascade responsible for eradication and inhibition of viral replication. Similarly, proteins coded in ORF6 can inactivate JAK-STAT signaling pathway by complexing with karyopherin-α2 and tethering karyopherin-β1 on internal membranes thus blocking nuclear translocation of transcription factor STAT-1 [12] .

The novel coronavirus is a new virus, and limited literature is available on its different attributes. Hence, conclusive remarks about its life cycle or immune responses cannot be made at the present moment. Much understanding of how it behaves inside the human body comes from research on SARS-CoV which has been studied in detail in the past years. The immune evasion methods described above are also based on SARS-CoV research. As such, readers are encouraged to update themselves about immune mechanisms and responses of SARS-CoV-2 as and when additional information is added in the literature about it.

Knowledge about the life cycle and immune response to the novel coronavirus can help us better understand the progression of the disease that it causes. Various hypotheses have been proposed to explain the course of COVID-19 in the human body. From a cellular biology perspective, COVID-19 can be divided into three stages [43] :

1. First stage: asymptomatic period (the first 1-2 days of infection).

The virus which has gained entry inside the body most likely binds to epithelial cells in the nasal cavity and begins multiplying. Through research conducted on SARS-CoV, it is surmised that ciliated cells are the first cells to be infected in conducting airways. However, this understanding remains to be validated as conducting airways show low levels of ACE2 expression. At this stage, the virus progresses locally, and there is a limited innate immune response. The virus can be detected via nasal swabs during this period.

The clinical significance of this period is that in spite of having low viral load, the infected individuals can transmit the virus to others. 2. Second stage: upper airway and conducting airway period (the next few days).

The virus multiplies and migrates downward along the conducting airways. A stronger immune response is elicited at this stage. For testing purposes, both nasal swabs and sputum samples should theoretically yield the virus. The disease COVID-19 manifests itself clinically at this stage. The host immune responses given in the previous section are activated and amplified during this stage. Thus biochemical markers (cytokines, interferons, antibodies, etc.) can be tested for during this period. A majority of the infected patients (about 80%) experience the disease till this stage only, and they can be monitored at home via symptomatic therapy. 3. Third stage: hypoxia, pulmonary infiltrates, and progression to respiratory distress. Around 20% infected patients progress to this stage of COVID-19. In this stage, the virus reaches the alveoli (gas exchange units) and infects type II pneumocytes. The virus proliferates in these cells releasing large number of viral particles which can affect other organs. The infected cells, however, undergo apoptosis and die. This way, large areas of the lungs may lose their type II cells, and thus epithelial regeneration is triggered. COVID-19 causes diffuse alveolar damage with fibrin-rich hyaline membranes and a few multinucleated giant cells. An aberrant wound healing leads to even more scarring and fibrosis.

Cytokine storm in COVID- 19 : The viral infection, if not controlled at stage 2 or 3, can lead to the death of the infected individual. A major cause of death from COVID-19 is the acute respiratory distress syndrome (ARDS). A severely lethal, uncontrolled systemic inflammatory response due to the release of large amounts of pro-inflammatory cytokines and chemokines (refer to Innate immunity under host immune response section) is called a "cytokine storm." This cytokine storm triggers a violent attack to the body by its own immune system which can lead to ARDS, multi-organ failure, and eventually death [44] .

Some other investigation parameters which have been reported from COVID-19 patients are given in Table 2 .4.

The clinical presentation of COVID-19 varies with age-most severely affecting the elderly [43] while the pediatric age group remains relatively unaffected [31] . It is interesting because infants and young children are a high-risk group for other respiratory infections like the respiratory syncytial and influenza virus infection. The reason for this finding remains unknown; however, some possible explanations have been proposed [30, 31] :

1. Immature and quantitatively less ACE2 receptors in children. 2. Growing immune system in children reacts differently to SARS-CoV-2 as compared to adults. 3. Presence of other viruses in airways and lungs of children which can limit growth of SARS-CoV-2.

The target receptor for the novel coronavirus (ACE2) is found abundantly in the lungs and small intestine and is highly expressed in endothelial cells and smooth muscle cells of virtually all organs. Thus, SARS-CoV-2 is a threat not only to the respiratory system but also to the gastrointestinal, central nervous, and circulatory systems [45] . These systems along with their specific pathophysiology and symptoms are discussed below: 1. Respiratory system: The mechanism of infection of type II pneumocytes in lungs has been discussed previously. Three classical symptoms of COVID-19 related to the respiratory system are fever, cough, and shortness of breath. Other symptoms may include sore throat, nasal congestions, and dyspnea. Radiographically, bilateral lung involvement in the form of sub-pleural and peripheral areas of ground glass opacity and consolidation is seen [46] .

Histopathological examination reveals pulmonary edema, cellular fibromyxoid exudates with diffuse alveolar damage, pneumocyte desquamation, and formation of hyaline membrane. Lungs also exhibit interstitial mononuclear patchy inflammatory infiltrates dominated by lymphocytes. Intra-alveolar spaces show multinucleate syncytial cells with atypical enlarged pneumocytes showing virusinduced cytopathic effect [33] . 2. Gastrointestinal system: The clinical features representing involvement of the digestive system are not very specific. Diarrhea, vomiting, and abdominal pain have been reported by affected patients [47] . Though these symptoms are not 100 High levels of pro-inflammatory cytokines (IL-2, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP-1A, TNF-α) during cytokine storm very severe and are also not reported by all patients, they do point to the fact that the gastrointestinal system might get affected and also contribute in transmission of the virus. The intestinal epithelium comes in direct contact with exogenous pathogens, and, hence, it possibly gets affected first after consumption of a SARS-CoV-2-infected animal. The nucleic acids of SARS-CoV-2 have also been detected in stool samples of the affected patients strengthening the assumption that gastrointestinal system is a potential route of transmission [45] .

Other than this, patients can also suffer from liver injury with raised enzymes found in blood tests. It is assumed that liver injury can occur in three waysdirect viral infection of hepatocytes, immune-related injury, or due to drug hepatotoxicity. The possible mechanism of liver involvement could be the binding of the virus to ACE2 receptors on cholangiocytes. Histopathology of liver samples from deceased COVID-19 patient has revealed microvesicular steatosis and mild lobular activity. However, no viral inclusions were seen [47] . 3. Urogenital system: COVID-19 patients exhibit increased serum creatinine, urea nitrogen, and urine protein indicating renal damage. Different studies have shown that 3-10% of COVID patients have renal insufficiency and 7% have acute kidney injury. Viral nucleic acids have also been isolated from urine samples of these patients. The CT scan of kidneys in COVID patients suggests inflammation and edema in parenchymal region [45] . Apart from renal tubular and mesenchymal cells, ACE2 is expressed in testicular and vas deferens cells as well. It is thought that SARS-CoV-2 binds to these receptors and leads to dysfunction of kidneys and testis [48] . It is, therefore, important for clinicians to assess the risk of testicular lesions in young patients to lessen the impact of COVID-related reproductive injury. 4. Central nervous system (CNS): It is known that viruses can travel along infected nerve endings. As the nasal mucosa is usually the first affected part of the body, it is possible that the olfactory tract can act as a channel for viral transmission to brain. The potential invasion of the brainstem by the novel coronavirus can lead to acute respiratory failure. Headache, epilepsy, and confusion have been reported by some COVID patients all of which point to intracranial infection. Pulmonary injury can lead to hypoxia in the CNS leading to interstitial edema, obstruction of cerebral blood flow, congestion, and even coma. CNS can also get damaged from the cytokines released by glial cells as a result of SARS-CoV-2 infection [45] . 5. Cardiovascular system: COVID patients have been known to present with circulatory system symptoms like palpitations, chest tightness, and shortness of breath. Elevated creatinine kinase, creatinine kinase-MB, and hs-cTnl have been also reported in COVID patients. It is presently believed that SARS-CoV-2 causes myocardial injury through three possible mechanisms: (a) The virus infects the heart and causes direct myocardial injury. (b) The virus binds to ACE2 receptors in the cardiovascular system and causes myocardial injury via signaling pathways. (c) The cytokine storm that occurs in COVID patients causes myocardial injury.

Additionally, hypoxemia and respiratory dysfunction caused by the virus can also damage myocardial cells [45] . Biopsies performed on COVID patients have revealed infiltration of myocardium by interstitial mononuclear inflammatory cells [46] . 6. Oral cavity: ACE2 receptors are ubiquitously present in the respiratory tract as well as salivary gland duct epithelium in the mouth [30] . As such, the oral cavity and its functions can also be affected in COVID-19. Symptoms like irregular ulcers on dorsal surface of the tongue [49] , hyposmia/anosmia and dysjeusia [50] , glossodynia, unilateral ulcers (resembling recurrent herpetic stomatitis) on keratinized as well as non-keratinized mucosa, pain in the palate, and sore throat [51] have been reported. Some authors have also speculated that these might be the first manifestations of COVID in the human body [50] . However, this claim remains to be validated. 7. Ocular manifestations: It has been previously suggested that respiratory disease could be transmitted through the nasolacrimal system. It is known that the ocular mucosal system clears and drains fluid from the eye to the inferior meatus of the nose. Hence, it is possible that if a respiratory droplet is deposited in the eye, the infected ocular fluid can enter the respiratory system. Moreover, ACE2 receptors have been demonstrated on corneal and conjunctival cells. Conjunctival hyperemia, chemosis, epiphora, unilateral or bilateral conjunctivitis, foreign body sensation, tearing without blurred vision, inferior palpebral conjunctival follicles, and tender palpable preauricular lymph nodes have all been reported as the ocular manifestations of COVID-19. These findings are consistent with acute viral conjunctivitis. However, they have not been reported in all patients, and patients who presented with these complaints have not consistently tested positive for COVID. This paradox has been attributed to low viral load in ocular secretions and improper collection techniques [52, 53] . 8. Dermatological manifestations: The commonly reported skin anomalies include chilblain-like lesions, maculopapular lesions, vesicular lesions, urticarial lesions, livedoid/necrotic lesions, pain, and burning. Skin lesions have been reported especially on acral sites like the digits of feet. These lesions begin as erythematous violaceous patches which turn to purpuric lesions, blisters, and necrotic lesions and finally return to normal [54] . Improvement in skin lesions has also been reported to be concomitant with improvement in laboratory markers (bilirubin transaminases and coagulation parameters) [55] . Once again the exact mechanisms of dermatological manifestations are not fully understood; however, a few plausible explanations have been suggested [56] : (a) Viral particles in cutaneous blood vessels lead to a lymphocytic vasculitis induced by immune complexes which activate cytokines. Immune reaction to COVID infection activates Langerhans cells leading to vasodilation and spongiosis. (b) Accumulation of microthrombosis from other organs can reduce blood flow to cutaneous microvascular system.

(c) Accumulation of deoxygenated blood in veins due to hypoxia and low-grade generalized intravascular coagulation can also cause such lesions. (d) Pauci-inflammatory thrombogenic vasculopathy with deposition of C5b- 9 and C4d can also cause such manifestations. None of the above given theories can explain the cutaneous manifestations on its own, and such features are most likely a result of these mechanisms acting together. Cutaneous manifestations could also be late manifestations of the inflammatory phase of a primary respiratory infection [55] .

Thus, it can be appreciated that once the novel coronavirus reaches the blood stream, it can affect any organ in the body; however, its target remains the samethe ACE2 receptor. This is of concern because patients might present with symptoms other than the recognized classical features of COVID, and misclassification or incorrect diagnosis of patients can negatively impact community transmission control efforts. Therefore, a sound knowledge and understanding of the pathophysiological mechanisms and manifestations of SARS-CoV-2 can help in its early diagnosis and management.

The novel coronavirus has ravaged the world since it first came to light in December 2019. It is a beta-coronavirus approximately 66-81 nm in diameter. Having originated in bats, it has jumped from its natural host into humans via an intermediate host which is still not known. Four main structural proteins-N, M, E, and S-provide the necessary framework for establishing its characteristic morphology and also define its pathologic ability. These as well as other nonstructural and accessory proteins are coded for by the genome of the virus which displays significant differences from its predecessor-the SARS-CoV. The virus can enter the body by direct contact, aerosols, and droplet transmission and attaches to its target receptor ACE2 via the spike protein. This spike protein exhibits considerable differences from other coronaviruses which might explain its increased transmissibility. Post attachment, it multiplies and also escapes detection by host immune cells via specific strategies. SARS-CoV-2 can affect pulmonary as well as extrapulmonary sites due to the ubiquitous distribution of ACE2 receptors. An understanding of its structure, immune response, and pathophysiology can help in better management of affected patients and possibly arrest the spread of this pandemic.

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