Analysis of COVID-19 and SARS-CoV-2

On 31 December 2019, the WHO China Country Office was informed of cases of pneumonia unknown etiology(unknown cause) detected in Wuhan City, Hubei Province of China. From 31 December 2019 through 3 January 2020, a total of 44 case-patients with pneumonia of unknown etiology were reported to WHO by the national authorities in China. During this reported period, the causal agent was not identified.

On 11 and 12 January 2020, WHO received further detailed information from the National Health Commission China that the outbreak is associated with exposures in one seafood market in Wuhan City.

The Chinese authorities identified a new type of coronavirus, which was isolated on 7 January 2020.[1] This was the start of the recent ongoing pandemic. To date, over 30.6 million COVID-19 cases and 950 000 deaths have been reported to WHO. From 14 through 20 September, there were almost 2 million new cases of COVID-19, which represents a 6% increase compared to the previous week, and the highest number of reported cases in a single week since the beginning of the epidemic. [2]

Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2 Analysis of COVID-19 and SARS-CoV-2

This post is an upload of my Class 12th Biology Investigatory project. It provides a comprehensive analysis of COVID-19 and SARS-CoV-2; ranging from its classification, structure and origin to is symptoms, diagnosis, and high risk populations. 

CLASSIFICATION AND NOMENCLATURE

A virus is a an ultramicroscopic, metabolically inert, obligate parasites that replicates only within the cells of living hosts, composed of an RNA or DNA core, a protein coat, and, in more complex types, a surrounding envelope.

The International Committee on Taxonomy of Viruses (ICTV) is concerned with the designation and naming of virus taxa (i.e. species, genus, family, etc.) rather than the designation of virus common names or disease names. The virus' are classified according to the following features :

1. Virion morphology, including size, shape, type of symmetry, presence or absence of peplomers, and presence or absence of membranes.
2. Virus genome properties, including type of nucleic acid (DNA or RNA), size of genome, strandedness (single or double), whether linear or circular, sense (positive, negative, ambisense), nucleotide sequence, and presence of special features (repetitive elements, isomerization, 5′-terminal cap).
3. Genome organization and replication, including gene order, number and position of open reading frames, a strategy of replication (patterns of transcription, translation), and cellular sites (accumulation of proteins, virion assembly, virion release).
4. Virus protein properties, including number, size, and functional activities of structural and nonstructural proteins, amino acid sequence, modifications (glycosylation, phosphorylation, myristylation), and special functional activities (transcriptase, reverse transcriptase, neuraminidase, fusion activities).
5. Antigenic properties.
6. Physicochemical properties of the virion, including molecular mass, buoyant density, pH stability, thermal stability, and susceptibility to physical and chemical agents, especially ether and detergents.

But, the most commonly used system of virus classification was developed by Nobel Prize-winning biologist David Baltimore in the early 1970s.

BALTIMORE CLASSIFICATION :

Virologist & Nobel laureate David Baltimore developed a classification which mainly focusses on the central line below - [3] (Fig : 4.1-1, 4.1-2)

'All viruses must synthesize positive-strand mRNAs from their genomes, in order to produce proteins and replicate themselves'

The classification divides viruses into seven groups, depending upon type of nucleic acid (ss or ds, RNA or DNA), sense and method of replication. They are: [4][5] 

CLASSIFICATION AND NOMENCLATURE OF CORONAVIRUS :

During the initial outbreak in Wuhan, China, various names were used for the virus; some names used by different sources included the "coronavirus" or "Wuhan coronavirus".

In January 2020, the World Health Organisation recommended "2019 novel coronavirus" (2019-nCov) as the provisional name for the virus. This was in accordance with WHO's 2015 guideline against using geographical locations, animal species, or groups of people in disease and virus names.

On 11 February 2020, the International Committee on Taxonomy of Viruses adopted the official name "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2). To avoid confusion with the disease SARS, the WHO sometimes refers to SARS-CoV-2 as "the COVID-19 virus" in public health communications and the name HCoV-19 was included in some research articles.

So, of the realm Riboviria and order Nidovirales, the family Coronaviridae is classified as Group IV – helical enveloped ssRNA type virus. This family is further divided into two sub-families Letovirinae and Orthocoronavirinae. Orthocoronavirinae is classified into four genera – Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (based on host).

The corona virus, of genus Betacoronavirus, is named as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2). And the species to which the virus SARS-CoV-2 belongs is Severe acute respiratory syndrome-related coronavirus. The disease name has been designated as COVID-19 by the WHO. The '19' in COVID-19 stands for the year, 2019, that the virus was first seen. The number '19' has nothing whatsoever to do with virus strains, genotypes, or anything else related to the virus' genetics.[6]

STRUCTURAL BIOLOGY

Since January, structural biologists have been busy modeling the virus' vital proteins, which could lead to therapeutic breakthroughs. As soon as the genomic sequence of SARS-CoV-2, the virus that causes the COVID-19 disease, was mapped, researchers were off to the races in synthesizing its proteins and determining their structures.

Unlike the highly complex genome sequence encoded in human DNA, the new coronavirus has a much shorter sequence and stores its genetic information in a single strand of RNA.

The first structural models were uploaded to the Protein Data Bank, an international database for 3D structural data of large biomolecules, within five weeks of the earliest reported cases of COVID-19.

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are both similar to the novel coronavirus, which scientists used as a basis for identifying protein sequences and shapes.

X-ray crystallography had been used rapid and accurate data gathering., and was a key tool in structural biology. However, X-ray crystallography doesn't work for every protein, and thus a new technology was used for the study. The relatively new cryo-electron microscopy emerged as a standout method for capturing the proteins in SARS-CoV-2, reconstructing an array of 2D images into a clear 3D model which facilitated the study.

SARS-CoV-2 is an enveloped, non-segmented, positive sense RNA virus with a diameter of about 65–125 nm, containing single strands of RNA and provided with crown-like spikes on the outer surface. The proteins of SARS-CoV-2 consist of two large polyproteins: ORF1a and ORF1ab (that proteolytically cleave to form 16 nonstructural proteins), four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and six accessory proteins: ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10. The non structural proteins are NSP1. NSP2, NSP3, NSP4, NSP5, NSP6, NSP7, NSP8, NSP9, NSP10, NSP11, NSP12, NSP13, NSP14, NSP15, and NSP16.

STRUCTURAL PROTEINS : 

Spike or S Glycoprotein 

The spike or S glycoprotein is a transmembrane protein, with a molecular weight of about 150 kDa, is found in the outer portion of the virus. S protein forms homotrimers protruding on the viral surface and facilitates binding of envelope viruses to host cells via angiotensin-converting enzyme 2 (ACE2). This glycoprotein is cleaved by the host cell furin-like protease into 2 sub units namely S1 and S2. [7]

Nucleocapsid or N Protein

The nucleocapsid, known as N protein, is the structural component of CoV localizing in the endoplasmic reticulum-Golgi region that structurally is bound to the nucleic acid material of the virus. Because the protein is bound to RNA, the protein is involved in processes related to the viral genome, the viral replication cycle, and the cellular response of host cells to viral infections [8]. N protein is also heavily phosphorylated and suggested to lead to structural changes enhancing the affinity for viral RNA [9]

Envelope or E Protein

E protein which is the smallest protein in the SARS-CoV structure that plays a role in several aspects of the virus’ life cycle, such as assembly, budding, envelope formation, and pathogenesis.[8]

Membrane or M Glycoprotein

The membrane or M protein, which is the most structurally structured protein and plays a role in determining the shape of the virus envelope. This protein can bind to all other structural proteins. Binding with M protein helps to stabilize nucleocapsids or N proteins and promotes completion of viral assembly by stabilizing N protein-RNA complex, inside the internal virion.

NON STRUCTURAL PROTEINS OR NSPs :

ACCESSORY PROTEINS :

GENETIC MATERIAL

The SARS-CoV-2 virus belongs to the B lineage of the β-coronaviruses (β-CoVs); this family comprises an enveloped, non-segmented, positive-sense single-stranded RNA virus genome, with a 5′ cap structure and 3′ poly-A tail, allowing to perform as an mRNA for translation of the replicase poly proteins.

Among the known RNA viruses, coronaviruses, which are single-stranded and positive-sense RNA viruses have the largest genome between other RNA viruses, with the GC content ranging from 32% to 43%. [29] [30] The genomic organization includes 5′- leader sequence - ORF1/ab - S - ORF3a - E - M - ORF6a - ORF7a - ORF7b - ORF8 - N - ORF10 -3′ from left to right and lacks the hemagglutinin-esterase gene which is detected in some β-CoVs. [31]

About two-thirds of SARS-CoV-2's RNA comprises ORF1a/b region, which with 16 non-structural proteins (nsp1-16) is considered as the largest ORF. The remaining one-third of the genome near the 3′-terminus contains ORFs encode structural and accessory proteins. [32]

ZOONOTIC ORIGIN 

As defined by the WHO, a zoonoses is any disease or infection that is naturally transmissible from vertebrate animals to humans. Some examples are MERS, and SARS-CoV-1.

The word 'zoonoses' is derived from Greek words 'Zoon' meaning animl and 'Noses' meaning disease. The term was first used and coined by Rudolf Virchow, who used it for communicable diseases. 

CLASSIFICATION BY RESERVOIR HOST : 

Anthropozoonoses

• Infections transmitted to humans from lower vertebrates. 
• Eg - Rabies, Plague, Leptospirosis, etc. 

Zooanthroponoses

• Infections transmitted from humans to lower vertebrates. 
• Eg - Streptococci, Staphylococci, Diphtheria, Human Tuberculosis in parrots and cattles, etc. 

Amphixenoses

• Infections maintained in both humans and lower vertebrates and transmitted in either direction. 
• Eg - Salmonellosis, etc.

CLASSIFICATION BY MODE OF TRANSMISSION :

Direct Zoonoses

• From an infected vertebrate host to a susceptible host [human] by direct contact, or fomite transmission.
• Eg - Rabies, Anthrax, Leptospirosis, etc.

Cyclozoonoses

• Requires more than one vertebrate host species, but no invertebrate needed for completion of life cycle.
• Eg - Taeniasis

Metazoonoses

• Transmitted biologically by invertebrate vectors, in which the agent multiplies and(or) develops.
• Always an extrinsic incubation period before transmission to another vertebrate host.
• Eg - Plague, Leishmaniasis, etc.

Saprozoonoses

• Requires a vertebrate host and a non-animal developmental site [like soil, plant material, pigeon dropping etc] for the development of the infectious agent.
• Eg - Histoplasmosis, Zygomycosis, etc.

TRANSMISSION OF ZOONOSES :

People tend to get infected with zoonotic diseases by five major ways - (Fig : 7.3-17)

Direct Contact

• First is by coming in direct contact with saliva, blood, urine, mucous, feces, or other body fluids of an infected animal. 
• Examples include petting or touching animals, and bites or scratches. 

Indirect/Fomite

• Encountering areas where animals live and roam, or objects or surfaces that have been contaminated with germs. 
• Examples include aquarium tank water, pet habitats, chicken coops, barns, plants, and soil, as well as pet food and water dishes. 

Vector or Water or Food Borne

• Other way could be the transmission by vectors like a tick, mosquito or a flea. 
• The most common way is via faeco-oral route, i.e. the disease could be waterborne or foodborne. [33]
• Eg - 
• Being bitten by a tick, or an insect like a mosquito or a flea. 
• Eating or drinking something unsafe, such as unpasteurized (raw) milk, undercooked meat or eggs, or raw fruits and vegetables that are contaminated with feces from an infected animal.
•  Contaminated food can cause illness in people and animals, including pets. 
• Drinking or coming in contact with water that has been contaminated with feces from an infected animal.

ZOONOTIC ORIGIN OF SARS-CoV-2 : 

Jumping the Species Barrier

The first patients of SARS-CoV-2 worked in the wet animal markets in Wuhan, China. It is believed that SARS-CoV-2 was introduced from the animal kingdom to human populations during November or December 2019. The spillover of SARS-CoV-2 from animals to humans took place at the beginning of December 2019 and clinical cases appeared at the end of December 2019.

Now, several evidences based on genome sequences, the homology of the ACE2 receptor, and the presence of single intact ORF on gene 8 indicate bats as a natural reservoir of these viruses.

However, an unknown animal is yet to be unravelled as an intermediate host. [36][37]Initial investigations on animal source origin of SARS-CoV-2 have inconclusively revealed snakes, pangolins, and turtles. [34] [35]

Recently, a Pomeranian dog as a probable intermediate host was identified; however, such reports are yet to be validated, and research is underway to explore the emergence of this infectious disease at the animal-human interface. [38][39]

A study reported that the SARS-CoV-2 might infect the cats and further transmitted by the infected cat to other cats. [40] Furthermore, along with dogs and cats, the zoo animals like tigers and lions were also reported to get the SARS-CoV-2 infection and exhibit clinical signs such as vomiting, diarrhoea, dry cough, breathing difficulty and wheezing. [41][42]

Spillover of SARS-CoV-2 was also reported in mink farms of Netherlands, further increasing the concern of transmission to humans.

Zoonotic Spillover

Since the beginning of 2002 till the end of 2019, three coronaviruses viz. SARS-CoV, MERS-CoV, and SARS-CoV-2 have caused havoc in the human population globally and will continue to do so.

Earlier identified betacoronaviruses (SARS-CoV and MERS-CoV) were reported in Guangdong province of China in November 2002 and Saudi Arabia in 2012, respectively SARS-CoV-2 is the third zoonotic betacoronaviruses recognized in this century. Both SARS-CoV and SARS-CoV-2 showed prominent similarities in their pathogenesis and epidemics. In both cases, bats were considered as the natural host, and the cold temperature and low humidity in cold, dry winter provided conducive environmental conditions that promoted the survival of the virus in the environment. [43]

Zoonotic spillover is the transmission of pathogens to humans from vertebrate animals. At present, these spillovers are of significant concern as in the past, many spillovers in the form of Nipah, Hendra, Ebola, SARS, MERS, and ongoing COVID-19 involving many animal species like pigs, horses, monkeys, camels, civets, among others, were documented.

Spillover is governed by the interaction of viral-specific proteins like S protein and host ACE2 receptor. [44] Mutations in amino acid sequences of RBDs [Receptor Binding Domains] results in a change in specificity of a receptor, interaction and binding, hence alteration in transmissibility, pathogenicity and cross-species jumping with a predisposition to novel and more severe diseases. [45]

In the case of SARS-CoV-2, RBD of S protein has 10–15 times affinity ACE2 receptor. [45] The enhancement of pathogenicity has been recorded previously too, which was bestowed upon by intermediate hosts, and thus helped in a spillover event.

Transmission to Humans

(Fig : 7.4.3-18) The involvement of intermediate hosts in maintaining and transmitting the virus to susceptible host predisposes humans to novel CoVs leading to the emergence of new diseases in humans.Usually, one or more types of animal hosts are involved in the transmission cycle of CoVs to humans. [44]

Bats have been the natural hosts for most human CoVs of Alphacoronavirus and Betacoronavirus genera. Genome sequence analysis has revealed bats as a natural host for SARS-CoV-2 [44]

In natural or reservoir hosts, CoVs adapts well, however, being unstable RNA viruses, they keep multiplying continuously without producing disease thereby enabling persistence or survivability and accumulation of mutations over the time resulting in the emergence of newer and novel strains of viruses. These unique strains or viruses occasionally spill over to other species including animals or humans, adapting to their body systems and hence broaden the biological host range for evolutionary sustainability; however, results in epidemiological widening of disease sphere as well.

In the context of SARS-CoV-2, snakes, pangolins and bats have been suspected as intermediate hosts since the first cases of COVID-19 had links to Huanan Sea Food Market where different animals, birds, and wild animals were being sold along with seafood items. [46] [47]

Bats are the natural reservoir host of many CoVs.The bat coronavirus, BatCoV RaTG13, has shown higher relatedness to SARS-CoV-2 at the whole genome level and spike gene in particular. [48] Based on Resampling Similarity Codon Usage (RSCU), snakes (Bungarus multicinctus and Naja atra) were suggested as wildlife reservoirs of SARS-CoV-2 and reported to be associated with the cross-species transmission and later it was disapproved by other researchers.

Unfortunately, to date, the intermediate host of the SARS-CoV-2 is abstruse what results in its escalation in the human population around the globe.

INFECTION AND TRANSMISSION 

ROUTES OF TRANSMISSION : 

Respiratory Droplets and Aerosols 

(Fig : 8.1.1-19)The SARS-CoV-2 mainly spread from person to another through small respiratory droplets from the nose or mouth when a person confirmed COVID-19 coughs or exhales. These droplets land on objects and surfaces around the person. Other people may catch the virus by breathing in droplets or touching these objects or surfaces, then touching their eyes, nose, or mouth. [49]

These respiratory emissions can range in size, with large droplets typically referring to those above 5-10 µm in diameter, and small droplets or aerosols referring to those below 5-10 µm diameter. Forceful respiratory actions such as coughing and sneezing can produce a burst of droplets that range in size and could include both large droplets and aerosols that present an exposure risk in close proximity to an infected person.

Under experimental conditions, SARS-CoV-2 has been found to remain viable when airborne over short distances for several hours and in field studies, viable virus has been isolated from air samples at distances greater than two metres from a COVID-19 patient. [50]

Fomite Transmission

(Fig : 8.1.2-20) Contact with contaminated surfaces (fomites) followed by touching of the eyes, mouth or nose is another possible mode of SARS-CoV-2 transmission, although it is not considered to be the main route. Fomites can become contaminated by deposition of droplets, aerosols, sputum or feces, either directly or by cross-contamination by touching an object with contaminated hands. Surfaces that are frequently touched by many people, such as door handles, or faucets may be more important in fomite transmission compared to objects or surfaces that are only touched incidentally and less frequently.

The virus appears to remain viable for longer periods (one to seven days or more) on smooth hard surfaces such as stainless steel, hard plastic, glass, and ceramics and for shorter periods (several hours to two days) on porous materials such as paper, cardboard, and textiles, although viability may be dependent on other factors such as temperature. [51] [52]

Faeco-Oral Route

Generally, many respiratory pathogens, such as influenza, SARS-CoV and MERS-CoV, cause enteric symptoms, so is SARS-CoV-2. In early reports from Wuhan, 2–10% of patients with COVID-19 had gastrointestinal symptoms such as diarrhea or vomiting. [53]

The study found that the detection of SARS-CoV-2 nucleic acid positive in a few feces of patients with confirmed COVID-19 cases indicated the presence of a live virus. Considering the evidence of fecal excretion for SARS-CoV-2, the virus can also be transmitted via the fecal-oral transmission route or re-transmitted through the formation of aerosols in virus-containing feces. 

Blood-Borne Spread

Cytokines (CK) are key factors regulating the immune response caused by many infectious pathogens that can significantly damage host organs and tissues. In the early stages of SARS-CoV infection, cytokine levels in the blood are rapidly elevated, the elevation of which is associated with the progression of lung invasion and injury [54]

The concentrations of various cytokines in the blood of severe patients with SARS-CoV-2 infection were significantly higher than those of non-serious patients. The concentration of cytokines can indicate the severity of the disease.

In severe cases of infection, immune system was overstimulated, resulting in a cytokine storm that leads to severe immune damage to the lungs and other organs. More and more cases reported that the brain, kidney, and heart impairment induced by the virus infection, may contribute to multi-organ failure and death eventually. [55]

Massive accumulation of SARS-CoV-2 leads to a surge of immune cells and more and more pro-inflammatory cytokines, resulting in a rapid increase in CK levels in the blood, or releasing more virus particles into the blood circulation. The virus and cytokines positively induce high expression of ACE2 in the intestinal epithelium and other organs which accelerated over-expression of ACE2 and viral binding, causing systemic infections with the virus.

PROCESS OF INFECTION : 

Introduction

(FIG : 8.2.1-22) Viruses enter cells and initiate infection by binding to their cognate cell surface receptors. The expression and distribution of viral entry receptors therefore regulates their tropism, determining the tissues that are infected and thus disease pathogenesis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus known to co-opt the peptidase angiotensin-converting enzyme 2 (ACE2) for cell entry. The interaction between SARS-CoV-2 and ACE2 is critical to determining both tissue tropism and progression from early SARS-CoV-2 infection to severe coronavirus disease 2019 (COVID-19).

Angiotensin-converting enzyme 2 is a zinc membrane-bound metalloproteinase that acts as a carboxypeptidase able to hydrolyze Ang I to Ang-(1-9) and Ang II to Ang-(1-7). ACE converts angiotensin I to angiotensin II, a highly active octapeptide that exhibits both vasopressor (vascular contraction to increase blood pressure) and proinflammatory activities. The carboxypeptidase activity of ACE2 converts angiotensin II to the heptapeptide angiotensin-(1-7), a functional antagonist of angiotensin II.

The Renin Angiotensin System (RAS) seems to be involved in COVID-19 natural course, since studies suggest the membrane-bound Angiotensin-converting enzyme 2 (ACE2) works as SARS-CoV-2 cellular receptor. The renin–angiotensin–aldosterone system (RAAS) is a critical regulator of blood volume and systemic vascular resistance. While the baroreceptor reflex responds in a short-term manner to decreased arterial pressure, the RAAS is responsible for more chronic alterations. It is composed of three major compounds: renin, angiotensin II, and aldosterone. These three act to elevate arterial pressure in response to decreased renal blood pressure, decreased salt delivery to the distal convoluted tubule, and/or beta-agonism. Through these mechanisms, the body can elevate the blood pressure in a prolonged manner.

The ACE2 receptor is present in most organs: it is attached to the cell membrane of mainly lung type II alveolar cells, enterocytes of the small intestine, arterial and venous endothelial cells, and arterial smooth muscle cells in most organs. Since the introduction of virus is mostly through the respiratory pathway, most cases of infection are isolated in those areas.

Entry into the Cell via ACE2

(Fig : 8.2.2-23) Like all coronaviruses, SARS-CoV-2 utilizes the S glycoprotein to promote entry into the host cell. This protein contains two functional domains: an S1 receptor-binding domain (RBD), and a second S2 domain that mediates the fusion of the viral and host cell membranes.

SARS-CoV-2 S protein binds to the ACE2 receptor on the host cell, initially through the S1 receptor binding domain. The S1 domain is then shed from the viral surface, allowing the S2 domain to fuse to the host cell membrane. This process is dependent upon activation of the S protein, by cleavage at two sites (S1/S2 and S2’) via the a cellular serine protease, TMPRSS2, and, in a smaller rate, an endosomal cysteine protease, cathepsin B, and L (CatB/L) (Hoffmann et al., 2020)[70].

Furin cleavage at the S1/S2 site may lead to conformational changes in the viral S protein that exposes the RBD and/or the S2 domain. TMPRSS2 cleavage of the SARS-CoV-2 S protein is believed to enable the fusion of the viral capsid with the host cell to permit viral entry[71][72]

Exposure of the RBD in the S1 protein subunit creates an unstable subunit conformation. Consequently, during binding, this subunit undergoes conformational rearrangement between two states, known as the up and down conformations. The down state transiently hides the RBD, while the up state exposes the RBD, but temporarily destabilizes the protein subunit[73][74][75]. Within the trimeric S protein, only one of the three RBD is present in the accessible conformation to bind the human Angiotensin 2 (hACE2) host cell receptor[76].

The critical functions of the SARS-CoV-2 S protein, ACE2, and the Furin and TMPRSS2 enzymes in binding and mediating viral entry to the host cell make these proteins key targets for drug development and viral inhibition in the efforts against COVID-19.

Analysis of COVID-19 and SARS-CoV-2

Viral Gene Expression

(Fig : 8.2.3-24) The release of the coronavirus genome into the host cell cytoplasm upon entry marks the onset of a complex programme of viral gene expression, which is highly regulated in space and time.

The translation of ORF1a and ORF1b from the genomic RNA produces two polyproteins, pp1a and pp1ab, respectively. Sixteen non-structural proteins are co-translationally and post-translationally released from pp1a (nsp1–11) and pp1ab (nsp1–10, nsp12–16) upon proteolytic cleavage by two cysteine proteases that are located within nsp3 (papain-like protease; PLpro) and nsp5 (chymotrypsin-like protease).

Proteolytic release of nsp1 is known to occur rapidly[77], which enables nsp1 to target the host cell translation machinery[78],[79],[80]. Nsp2–16 compose the viral RTC and are targeted to defined sub cellular locations where interactions with host cell factors determine the course of the replication cycle[81],[82],[83]. Nsp2–11 are believed to provide the necessary supporting functions to accommodate the viral RTC (Replication/Transcription Complex), such as modulating intracellular membranes, host immune evasion and providing cofactors for replication, whereas nsp12–16 contain the core enzymatic functions involved in RNA synthesis, RNA proofreading and RNA modification [84],[82].

RNA synthesis is performed by the nsp12 RNA-dependent RNA polymerase (RdRP) and its two cofactors nsp7 and nsp8, the latter with proposed primase or 3′-terminal adenylyltransferase activity[84],[82],[85],[86]. Notably, nsp14 provides a 3′–5′exonuclease activity that assists RNA synthesis with a unique RNA proofreading function[87] .

The establishment of the viral RTC is crucial for virus replication and thus a promising target for antivirals against SARS-CoV-2. One such target is Mpro, which resides in nsp5. Mpro releases the majority of nsps from the polyproteins and is essential for the viral life cycle. Furthermore, as Mpro is very sequence specific, compounds that structurally mimic those cleavage sites can specifically target the viral protease with little or no impact on host cellular proteases[88],[89],[90].

RNA Synthesis

(Fig : 8.2.4-25) Viral genomic replication is initiated by the synthesis of full-length negative-sense genomic copies, which function as templates for the generation of new positive-sense genomic RNA. These newly synthesized genomes are used for translation to generate more nsps and RTCs or are packaged into new virions.

A hallmark of coronaviruses and most members of the order Nidovirales is the discontinuous viral transcription process, first proposed by Sawicki and Sawicki[91], that produces a set of nested 3′ and 5′ co-terminal subgenomic RNAs (sgRNAs)[91],[92].

During negative-strand RNA synthesis, the RTC interrupts transcription following the encounter of transcription regulatory sequences (TRSs) that are located upstream to most ORFs in the 3′ one-third of the viral genome. At these TRS elements, also called TRS ‘body’, the synthesis of the negative-strand RNA stops and is re-initiated at the TRS adjacent to a leader sequence (TRS-L) located at about 70 nucleotides from the 5′ end of the genome [91],[92],[93],[94],[95].

This discontinuous step of coronavirus RNA synthesis involves the interaction between complementary TRSs of the nascent negative strand RNA (negative-sense TRS body) and the positive strand genomic RNA (positive-sense TRS-L). Upon re-initiation of RNA synthesis at the TRS-L region, a negative strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs.

The discontinuous step of negative strand RNA synthesis results in the production of a set of negative-strand sgRNAs that are then used as templates to synthesize a characteristic nested set of positive-sense sg mRNAs that are translated into structural and accessory proteins. Although the coronavirus sg mRNAs are structurally polycistronic, it is assumed that they are functionally monocistronic.

Expression of Structural and Accessory Proteins 

The ORFs encoding the structural proteins (that is, S protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein) are located in the 3′ one-third of coronavirus genomes. Interspersed between these ORFs are the ORFs encoding for so-called accessory proteins.

In general, coronavirus structural proteins assemble and assist in the budding of new virions at the endoplasmic reticulum (ER)-to-Golgi compartment that are suggested to exit the infected cell by exocytosis [96],[97],[98]. These proteins are translated from positive sense sg mRNA.

At least five ORFs encoding accessory genes have been reported for SARS-CoV-2: ORF3a, ORF6, ORF7a, ORF7b and ORF8 (GenBank entry NC_045512.2). In addition, ORF10 has been postulated to be located downstream of the N gene. However, not all of these ORFs have been experimentally verified yet and the exact number of accessory genes of SARS-CoV-2 is still debated [93],[99].

Replication Compartment 

After translation, the structural proteins, along with accessory proteins and its genome, form new viruses (virion) capable of infecting other cells. The mechanism of the formation of virions is still being researched and is out of scope of this project.

SYMPTOMS of COVID-19

Infection of SARS-CoV-2 can lead to symptoms, both common and fatal. The most common symptoms are : [56]

Apart from the above symptoms, a part of the population affected shows the following symptoms :

DIAGNOSIS

There are many different types of tests available for the fast diagnosis of COVID-19, but bellow are the four main tests available in India :

RT-PCR is the test widely used in India and around the world. The test detects the virus’ RNA, which will be present in the body before antibodies form or symptoms of the disease are present, and tells whether or not someone has the infection very early on. It proceeds by converting the RNA to DNA through a process called ‘reverse transcription’, before detecting the virus. (Fig 10-27)

Rapid Antibody tests are fast, inexpensive, and can be used to gauge the extent of infection within a community. Unlike RT-PCR, antibody tests require a blood sample to determine whether the human body has antibodies for coronavirus. Since the results are not 100 per cent reliable (they can give false positives), the ICMR has advised that if a person tests positive through a rapid test, they have to undergo a confirmatory RT-PCR test before treatment. (Fig : 10-28)

Like RT-PCR, the Rapid Antigen test too, seeks to detect the virus rather than the antibodies produced by the body. It has been approved by ICMR for use in containment zones and healthcare settings. The test is reliant on the amount of virus, or the viral load, in a nasal swab. An antigen refers to any toxin in the body that triggers an immune response. (Fig : 10-29)

TruNat test, commonly used for detecting tuberculosis and HIV, works on the same principle as RT-PCR, but with a smaller kit, and produces faster results. ICMR recently approved TrueNat, manufactured by a Goa-based company, for screening and confirmation for COVID-19. (Fig : 10-30)

Other than the above tests, there are some other confirmatory tests which could be used to ensure an accurate diagnosis like:

VULNERABLE POPULATION

Severe illness means that a person with COVID-19 may require :

• hospitalization, 
• intensive care, or a 
• ventilator to help them breathe, or 
• they may even die. 

Risk for severe illness with COVID-19 increases with age, with older adults at highest risk. Certain medical conditions can also increase risk for severe illness. People at increased risk, and those who live or visit with them, need to take precautions to protect themselves from getting COVID-19.

Analysis of COVID-19 and SARS-CoV-2

AGE : 

Older adults are at greater risk of requiring hospitalization or dying if they are diagnosed with COVID-19. As you get older, your risk of being hospitalized for COVID-19 increases. (Fig 11-32)

PRE-EXISTING CONDITIONS : 

Adults of any age with the following conditions are at increased risk of severe illness from the virus that causes COVID-19 like,

• Cancer
• Chronic kidney disease
• COPD (chronic obstructive pulmonary disease) 
• Down Syndrome 
• Heart conditions (heart failure, coronary artery disease, or cardiomyopathies) 
• Immunocompromised state from solid organ transplant 
• Obesity (body mass index [BMI] of 30 kg/m2 or higher but < 40 kg/m2) 
• Severe Obesity (BMI ≥ 40 kg/m2) 
• Pregnancy 
• Sickle cell disease 
• Smoking 
• Type 2 diabetes mellitus

Moreover, adults of any age with the following conditions might be at an increased risk for severe illness from the virus that causes COVID-19 :

• Asthma (moderate-to-severe) 
• Cerebrovascular disease (affects blood vessels and blood supply to the brain) 
• Cystic fibrosis 
• Hypertension or high blood pressure 
• Immunocompromised state from blood or bone marrow transplant; immune deficiencies; HIV; use of corticosteroids, etc 
• Neurologic conditions, such as dementia 
• Liver disease 
• Overweight (BMI > 25 kg/m2, but < 30 kg/m2) 
• Pulmonary fibrosis (having damaged or scarred lung tissues) 
• Thalassemia (a type of blood disorder) 
• Type 1 diabetes mellitus 

People who also need extra precaution fall under these categories :

• People Living in Rural Communities 
• People with Disabilities 
• People with Developmental & Behavioral Disorders 
• People Experiencing Homelessness 
• Pregnant People & Breastfeeding 
• Racial & Ethnic Minority Groups (Fig 11-33)

PREVENTATIVE MEASURES

Actions to Protect Ourselves in Public : 

(Fig 12.1-34)

• Avoid touching your face , especially your eyes, nose and mouth. 
• Wash your hand frequently with soap and water or use alcohol-based rub. 
• Seek immediate medical advice if you have fever, cough or are experiencing breathing difficulties. 
• When coughing or sneezing, discard the used tissue immediately into a closed bin. 
• Maintain distance of at least 1 metre from someone who is coughing or sneezing 
• When coughing or sneezing, cover your mouth and nose with flexed elbow or tissue. 
• If you have cough, fever, and difficulty in breathing, wear a mask correctly and seek medical advice. 

Actions to Protect Ourselves at Home : 

• Clean your home regularly, particularly frequently touched surfaces like door knobs, refrigerator handles, etc. 
• Stay home stay safe. Stay physically fit. Exercise regulary. Eat a nutritious diet. Don’t smoke. 
• Follow the Golden Rule. Wash your hands frequently with soap and water or use alcohol based hand-rub. 
• Stay positive. Avoid alarmist news. Be connected to friends and family. Have a hobby. 
• If any member of the household shows symptoms of Covid-19, seek medical advice and follow your local health authority's guidance.

Message for the Elderly and those having Other Diseases : 

(Fig 12.3-35)

• Take care if you are elderly 
• Avoid Exposure: Stay at home. Wash hands or use alchohol-based rub. 
• Avoid touching your face. 
• Eat nutritious food, exercise regularly. Stay connected with friends and family. 
• Be prepared: Keep a supply of essential medicines. Keep all helpline numbers handy. 
• Keep at least 1 meter apart from others. 

Precautions for Pregnant and New Mothers : 

(Fig 12.4-36)

• Stay at home and avoid meeting outsiders. Wash your hands with soap and water or use alcohol-based hand rub. Prepare for delivery and make arrangements to reach hospital. Its safer to deliver in a hospital, even during the COVID-19 pandemic.
• Always be alert on pregnancy related warning signs. Continue visiting your doctor or midwife for regular check ups. Immediately contact the doctor in case of symptoms like pain in abdomen, bleeding, watery discharge and severe headache. In case of an emergency visit nearest hospital. 
• Wash hands before and after touching and feeding your baby. Wash clothes and sterilize all utensils & articles that come in contact with you or your baby. 
• Breastfeed within 1 hour of birth and continue. It protects your baby from infections. Keep your baby close to you. Practice skin-to-skin contact (Kangaroo care) for small or preterm babies. 
• If the mother is COVID positive, wear a medical mask while breastfeeding the baby. Wash your hands with soap and water or use alcohol-based hand rub before feeding. Routine clean and disinfect surfaces around you. Keep at least 1 meter distance from others.

Precautions at Work Places : 

(Fig 12.5-37)

• Keep physical distance of at least one metre.
• Wear a mask when close to others.
• Minimise the need for physical meetings by teleconferencing.
• Defer or suspend work place events and social gatherings.
• Take special care if you have pre-existing medical conditions, are pregnant or above 60 yrs in age.
• Wash your hands frequently with soap and water or use alcohol-based hand rub.
• Ensure respiratory hygiene by covering mouth and nose with flexed elbow/tissue during cough or sneeze.
• Avoid physical contact like hugging, touching, shaking hands, etc.
• If you show symptoms of COVID-19; such as sore throat, fever, difficulty in breathing, then isolate yourself, take medical advice and do not come to work.

Taking these extensive measures to prevent the spread of a pandemic is of utmost importance. Social distancing, wearing masks, and using disinfectants are a must nowadays. Although many are lathering themselves with sanitizers, people are not aware of the risks and hazards of the long term exposure to these chemical disinfectants. You can read about this in the post 'Hazards of Disinfectants' where we discuss about the disinfectants and their by-products, their toxicity and effects. While there is no need to panic, we should start moving towards a more sustainable and holistic alternative. You can read about one such alternative on 'Aloe Vera : A Sustainable Alternative'.

Analysis of COVID-19 and SARS-CoV-2

REFERENCES

Analysis of COVID-19 and SARS-CoV-2