1 INTRODUCTION

Coronaviruses (CoVs) are large, positive-sense, enveloped and single-stranded RNA viruses. They belong to the family; Coronaviridae, subfamily; Coronavirinae, order; Nidovirales, that infect a wide range of host.1 They include viruses causing common cold and several animal and birds’ coronaviruses, for example “Infectious Bronchitis Virus (IBV), that causes infection in poultry. CoVs characteristically produce gastrointestinal, neurological and respiratory sickness.2 The diseases caused by these viruses vary from normal cold to severe/deadly ailments. Coronavirus Study Group of International Committee on Taxonomy of Viruses (ICTV) changed the initial name of virus from “2019-nCoV” to “SARS-CoV-2” as it was discovered to be the sister virus of SARS-CoV i.e. “Severe Acute Respiratory Syndrome Coronavirus.1 SARS-CoV-2 is found to be seventh coronavirus that is identified to infect human beings.3 It is important to know that the COVID-19 virus (SARS-CoV-2) is not linked with poultry or poultry products. Coronaviruses are divided into four genera; Alpha- (α), Beta- (β), Gamma- (δ) and Delta- (γ) coronavirus groups.4 The coronavirus in avian Gamma-coronavirus group affects poultry (IBV) and causes respiratory disease in chickens and does not infect humans.5 While, COVID-19 virus is in the Beta-coronavirus group together with MERS-CoV (Middle East Respiratory Syndrome Coronavirus) and SARS-CoV that infects mammals.6

2 SOURCE OF COVID-19

Bats are extensively believed to be the reservoir for mammalian (Alpha- and Beta-) coronaviruses. There are around 1,240 different bat species harboring different coronavirus types. SARS-CoV and MERS-CoV arose from a bat reservoir, infected an intermediate host (not identified yet) and then transferred to humans. Probably, the COVID-19 virus originated from bats. Moreover, some initial data shows some viruses, isolated from bats, to be close relatives.5 Genomic sequence of COVID-19 virus is relevant and closest to coronaviruses of bat.2 However, the genetic data of SARS-CoV-2 shows that it is not manipulated and derived from the backbone of any previously used virus.7

3 HISTORY AND EPIDEMIOLOGY

Coronaviruses were first revealed in 1960s and have been around for many years, even during 1990s, causing no severe disease in human beings. Though, in 2002-03, a new coronavirus “SARS-CoV” emerged, and then MERS-CoV in 2012 in Saudi Arabia.2 In December 2019, a large group of pneumonia cases appeared in Wuhan, Hubei province, China, that were triggered by a newly recognized β-coronavirus. World Health Organization (WHO) firstly named this as the “2019-novel coronavirus” (2019-nCoV) on 12 January 2020.8 Later the name was changed to SARS-CoV-2. Now cases of coronavirus are increasing day by day all around the world except Antarctica, while about above 81,000 cases have been stated in China.9 According to WHO, a rise in this pandemic was seen at the end days of January and at the start of February, 2020.10 Whereas, a decrease in the rate of these cases was noted by the start of March, 2020. Starting from China, currently it has prevailed globally, over 213 countries or areas of the world, some with minor effects while some with severe deadly conditions like USA, Italy, Spain and France.  According to the report of South China Morning Post (SCMP) at June 2, 2020, there are more than 6 million COVID-19 cases worldwide. USA has highest mortality rate i.e. more than 100,000 deaths, followed by UK with more than 39,000 deaths, Italy with above 33,000 deaths and Brazil with more than 30,000 deaths. Coronavirus cases in Pakistan are also continuously increasing. To the writing of this manuscript, there have been 76,398 confirmed COVID-19 cases in Pakistan (Punjab; 27,850, Sindh; 29,647, KP; 10,485, Balochistan; 4514) and 1621 deaths over the country.  
The most current data for the study of global disease transmission of this developing pandemic can be found at the WHO COVID-19 Situation Board which use open sources to see the dispersion of this epidemic.11

4 SYMPTOMS OF COVID-19

Coronavirus infection showed its symptoms in patients after an incubation period of 5 days approximately.12 It is noticed and suggested that COVID-19 showed its symptoms in a person within 6 to 41 days approximately with an average of about 14 days.13 This said period totally depend on the patients’ immune system and its age.14 It was rare among patients of age less than 70 years but higher in patients having age of 70 or near it.13 The symptoms are high fever (body temperature of 39°C), dry cough, difficulty in breathing and coarse breathing sounds of both lungs,15 fatigue, severe headache, diarrhoea, lymphopenia, haemoptysis, production of sputum and dyspnea13,16-18 (Figure 1).
Chest CT scan of COVID-19 patients showed similar appearance as in the case of pneumonia patients, as well as showed some abnormal properties like “Acute Respiratory Distress Syndrome (ARDS), acute cardiac injury, RNAemia and incidence of grand-glass opacities that causes death.17 Whereas, presence of many peripheral ground-glass opacities in subpleural regions were observed in the lungs of some patients as well and ultimately, results in inflammation due to induction of systemic and localized immune response. No clinical effects were seen in patients with inhalation of interferon, instead, it appeared problematic by causing progression in pulmonary opacities.15 When patient’s chest CT scans were observed, it was also noticed that symptoms of COVID-19 and beta-coronavirus (dry cough, bilateral ground-glass opacities, fever and dyspnea) were similar to each other.17 It was also noticed that COVID-19 targets mostly the lower airway and upper respiratory tract and showed symptoms like rhinorrhoea and cause sore throat and sneezing.19 While in some cases, presence of infiltrate in upper lob of lung was seen that increases dyspnea with hypoxemia.20

5 TRANSMISSION

It was noticed that the initial cases had exposure with the animals that were infected by COVID-19. Because in Wuhan city, people worked and visited seafood market where they sold live animals. By this way, COVID-19 transmitted from animals to human and then, human to human transmission started.21 But the main and quick transmission occured through close contact between person to person. This person to person transmission mainly occured through exposure with infected person’s respiration droplets during sneezing, coughing and close contact with him/her. An infected person can transmit it into two individuals approximately11 (Figure 2).
It can also be transmitted when a person touches an infected surface and then, touches his/her nose, mouth and eyes etc. It was also noticed that the infected person transmits this virus or viral infection to his/her families as well.11 Increased transmission rate was also observed in travel related places, workplaces and through import export of infected goods. It was also noted that these droplets do not stay in air and cannot travel more than two meters. It might be transmitted through respiration, skin, feces and urine of the patient.22

6 PATHOLOGICAL CHARACTERISTICS AND EXAMINATION OF COVID-19

COVID-19 had same pathological characteristics as of MERS (Middle Eastern respiratory syndrome) and SARS.23 Both SARS-CoV and SARS-CoV-2 have ability to infect epithelial cells by fundamental interaction of viral S proteins with type 2 receptors (angio-tensin converting enzyme) present on cells of human.24 That is why, these were assumed to cause similar pathological alterations. In addition to its presence in respiratory system, it may also be present in skin sweat glands, epithelial lining of kidney convoluted tubules and digestive tract. By the examination of infected lungs of COVID-19 patient, it was noticed that there was presence of many fibromyxoid exudation, and some sticky secretions released from alveoli.25 According to Xu et al. (2020) samples of patients’ lungs, heart and liver tissues were taken for further pathological examination.26 After examination they came to know that alveolar structure was completely diffused and damaged with the presence of cellular fibromyxoid exudation. They also observed a noticeable peeling of pneumocytes and formation of hyaline membrane. Presence of some mucinous secretions in the lungs was also observed22 (Figure 3).
Pulmonary edema with formation of hyaline membrane was observed in left lung and it was suggested that this was the symptom of early ARDS. In both lungs of the patient, interstitial mono-nucleate inflammatory infiltrates, submissive by the lymphocytes were present. Viral alterations (prominent nucleoli within the alveolus space, large pneumocytes and cytoplasm with granules) were observed in multinucleated cells. They also noted mild lobular with micro vesicular steatosis in patients’ liver samples, indicating that this injury was either due to injury of liver induced by drugs or SARS-CoV-2. They also observed little inflammation in heart tissues but overall no other damage at all. They conducted flow cytometric analysis, prepared peripheral blood and noticed decrease number of T cells i.e. CD8 and CD4 (cluster of differentiation 8 and 4) as well but these cells were super activated as CD38 (CD8 39.4%) and CD4 (3.47%). In addition to it, the number of pro-inflammatory CCR6 and Th17 in CD4 type T cells was also increased. Number of cytotoxic granules was maintained by CD8 T cells, having perforin positive cells (31.6%), granulysin positive cells (64.2%) and double-positive cells (30.5%). Due to the over-activation, high toxicity of CD8 T cells and increase in Th17, patients faced severe immune injury and progressive pneumonia26 (Figure 4).
In addition to it, greater concentration of pro-inflammatory cytokines like IL1, IL2, IL6, IL7, IL8, IL10, MCP1, MIP1α, GCSF, IP10, Tnf-α and Tnf-beta that boost severity in this disease, were also seen in some severe cases.17 According to Chan et al. (2020) the most common feature and critical factor in COVID-19 patients is Lymphopenia and it may cause huge severity in infection and increased death rate.27 Tian et al. (2020) also presented the histopathological data of some patients who undergo lung lobectomies for adenocarcinoma and observed infection during their surgery.28 Xu et al. (2020) suggested that these pathological outcomes not only help to solve intense cases of COVID-19 but can also be used to determine the cause of death and explore new remedial strategies to minify death rate.26

7 ASYMPTOMATIC CARRIER TRANSMISSION OF COVID-19

It was observed that COVID-19 is transmitted during introductory period when infested persons are slightly ill, and keep on doing normal activities, contributing to the blowout of infection.29,30 As stated by a report on “Diamond Princess”, out of 1,723, 189 confirmed travelers were asymptomatic (with no visible symptoms), while they were carrier of COVID-19 virus as of February 17, 2020.31 In a previous study, a 10-year old boy was reported to be an asymptomatic, with COVID-19 infection, but his chest CT showed abnormalities.27 In another study, a 20 year old woman (assumed asymptomatic carrier) travelled from Wuhan to Anyang (China) on January 10, 2020, and met with few people, causing development of COVID-19 infection in them and severe visible symptoms. While she, herself had no symptoms including sore throat and cough. All her tests (RT-PCR, lymphocyte count and C-reactive protein level) were normal till January 28, 2020. But before the day, she had spread virus to five other people (with no travelling history) as an asymptomatic virus carrier.32 This indicates that many mild patients and asymptomatic carriers remain unexplored in the society. To study the detailed asymptomatic carrier transmission, an investigation was also carried out on 24 cases in Nanjing, China, describing their clinical characteristics. These infections were separated from nearby contacts showing the transmission capability of asymptomatic COVID-19 virus carriers. These asymptomatic carriers showed no clear symptoms, while were confirmed to be positive for Covid-19 by nucleic acid tests of pharyngeal swab samples. Not a single of these 24 cases had serious pneumonia or expired by COVID-19. All these cases had a chest CT scans. Seven of these carrier individuals were younger (average age; 14 years) as compared to others, with ordinary CT image and no symptoms even during hospitalization. Among these 24 cases, epidemiological history and medical record of one (case 13) was reviewed. Case 13 had a travelling history to Huanggang, Hubei province and was an asymptomatic carrier of virus; however, his nucleic acid test came positive for COVID-19. All his close contacts carried severe infection of COVID-19 with clear symptoms such as severe pneumonia, cough, high fever and fatigue etc. and were admitted in hospital. These results direct that mild patients and asymptomatic carriers could also be a source of COVID-19 infection as they can result in transmission from one person to another. It is critical to identify and isolate them to contain the epidemics in far ahead stages.33

8 CLINICAL CHARACTERISTICS

Among cluster of cases recorded in Wuhan, China,13 first reported infection caused by SARS-CoV-2 was a serious respiratory illness and has spread throughout the world.34 On January 2, 2020, it is examined the clinical features of 41 patients believed to be COVID-19 infected, which included 13 cases of ICU and 28 cases of non-ICU.17 On computed tomography imaging, almost all patients had bilateral lung ground glass opacity. The signs initially included fever in most of the infected patients, coughing (76%), shortness of breath (55%), myalgia (44%), production of thick mucus (28%), headache (8%), hemoptysis (5%) and diarrhea (3%).  In the early stage of the illness, fever was not tested in just one patient. ARDS was developed in 29 % of cases, 12 % had acute heart injury, approximately 7% had acute kidney injury (AKI) and shock. Chen et al. (2020) confirmed 99 cases of SARS-CoV-2–infected pneumonia on 20 January 2020.35 This case series revealed that older males with comorbidities as a result of weaker immune function were the most susceptible to COVID-19 incidence. In this study, the symptoms, complications and treatments were similar to the previous studied record, 31 % were discharged, 11 % were died and 58 % of patients were still hospitalized. Fever, cough and dyspnea are the main clinical manifestations of COVID-19. Gastrointestinal symptoms have a small but important subset.36
During the initial outbreak in the province of Hubei, Pan et al. (2020) identified clinical features from 18 January to 28 February 2020 in three seriously affected hospitals that had 97 out of 103 patients acquired respiratory symptoms together with digestive symptoms.37 Digestive symptom patients had a slightly longer duration from start of hospital admission than patients without digestive symptom. We found that patients with digestive symptoms had a variety of digestive manifestations including diarrhea, lack of appetite, abdominal pain and vomiting. Guan et al. (2020) extracted data from 552 hospitals in China regarding 1099 patients with laboratory-confirmed COVID-19.38 The patients’ median age was 47 years and 41.9 % were female. Fever (43.8 % after admission and 88.7 % during hospitalization) and cough (67.8%) were the most serious symptoms. The uncommon sign was diarrhea (3.8%) in patients, 83.2 % of patients on admission had lymphocytopenia. Patients with serious illness were by a mean of 7 years older than those with no severe illness.39 In addition, the presence of some coexisting disease was more common among patients with severe illness than among those without serious illness. On treatment, lymphocytopenia was in 83.2 % of patients, thrombocytopenia in 36.2 % and leukopenia in 33.7 %. Patients with COVID-19 infection have a lower case of mortality rate than that of MERS and SARS-CoV. The cases in Jiangsu displayed mild or moderate symptoms relative to the cases in Wuhan and no apparent weakness of the gender.40 Throughout Wuhan, there was slightly lower number of patients with liver failure and an abnormal CT imaging. Notably, infected patients may be falsely excluded based on 2 consecutively negative respiratory pathogenic nucleic acid test results.41 In pregnant women, the clinical features of COVID-19 pneumonia were similar to those identified in non-pregnant adult patients. Findings from this small group of cases suggest that there is currently no evidence for intrauterine infection caused by vertical transmission in women.42 Children who had only coronavirus detected were more likely to have underlying chronic diseases than children who had dual respiratory infections. This list may not be all-inclusive, medical warning signs include recurrent chest pain or strain, frustration or failure to excite and bluish lips or face.12

9 STRUCTURE OF CORONA VIRUS

The name CoV is a derivative of the Latin word corona that means crown. This is because of the characteristic structure of the virus whereby surface projections on the viral envelope give it a similar appearance to a crown.43

9.1 Morphology of corona virus

Enveloped, pleomorphic or spherical coronaviruses are 150 to 160 nm in size, associated with positive single stranded RNA, with 5’-Cap structure and 3’-poly-A tail,44 which acts itself as an mRNA, un-segmented, nucleoprotein, capsid, matrix, and S-protein.45 Nucleocapsid protein (N), membrane glycoprotein (M), and spike glycoprotein (S) are important viral proteins that are necessary for COVID-19 assembly and infection. COVID-19 differs from other coronaviruses by encoding an extra glycoprotein with properties of acetyl esterase and hemagglutination (HE).46 For coronaviruses in all four genera, coronavirus entry into host cells is directed by an envelope-anchored spike protein.47
            The virus is a single-stranded positive-sense RNA virus with a genome of around 30kb in length. The coronavirus RNA genome (ranging from 26 to 32 kb) is the largest of all RNA viruses and the viral particle has a diameter of about 125 nm.48 Due to this, they are the largest known RNA viruses.49 The RNA genome codes for both structural proteins (SPs) and non-structural proteins (NSPs). All known CoVs share a similar structure made of four main structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. Both structural (SP) and non-structural (NSP) proteins are coded by RNA genome. The S-protein help in binding to the host receptors, M protein helps to form proper shape of the virion particles and in binding to nucleocapsid, E-protein perform an important role in the assembly and release of particles while N-protein help in binding of the genome to a replication transcription complex which is essential for the genomic material replication. Among the structural proteins, four protein has special importance namely spike (S), envelope (E), membrane (M), and nucleocapsid (N). Within the viral membrane S, E, and M proteins are present. The proteins M and E are involved in viral assembly, while the N protein is necessary for assembly of the RNA genome.The S protein, a surface located trimeric glycoprotein of CoVs, plays a functional role in viral entry into host cells, viral infection and pathogenesis The S protein, a trimeric CoV glycoprotein found on the surface, plays an important role in viral entry into the host cells, viral infection and pathogenesis.50 Coronavirus recognizes the receptor on the target cell through the S protein on its surface and enters into the cell, then allow the infection to occur (Figure 5).

9.2 Spike protein

The S protein is the common target when designing vaccines based on neutralizing antibodies. It contains a receptor binding domain (S-RBD) in the S1 subunit, which mediates receptor binding and membrane fusion.8,51 The spike is in very two various forms: pre-fusion (the form on mature virions) and post-fusion (the form that has been completed after membrane fusion). The structure of the pre-fusion form is a homo-trimer, with three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk.52-58 The structure of the post-fusion form is a coiled-coil structure, containing S2 only.59,60 The pre-fusion form is a metastable state: due to the structural constraints imposed by S1, S2 is prevented from transitioning to the post-fusion structure. However, the spike is sequentially cleaved at two sites during cell entry by host proteases: first at the S1/S2 boundary (i.e., S1/S2 site) and second within S2.61,62 S1 dissociates from S2 after the cleavages, allowing S2 to transition to the post-fusion structure. The transition from pre-fusion to post-fusion phase is irreversible and this process is thus strictly controlled throughout the entry process.47 The spike surface glycoprotein plays a key role in its attachment to host cells and can be further cleaved by host proteases into an N-terminal S1 subunit and a membrane-bound C-terminal S2 region The spike surface glycoprotein perform an important function in its binding to host cells and can be further split into an N-terminal S1 subunit and a membrane bound C-terminal S2 region by host proteases.55

9.3 Nucleocapsid (N) protein of virus

The N-protein in coronaviruses is the most abundant, conservative protein; hence, it is also used as a diagnostic antigen. Our analysis revealed that the antigenicity of the COOH terminus of the SARS-CoV N protein is higher than that of the NH2 terminus, and that the former N protein fragment may have the same antigenicity as that seen with the full length N protein.63 Antibodies may cross react with the COVID -19 that are produced against the N protein of SARS-CoV.64 The heterophilic antibodies of SARS-CoV may not provide cross protection to COVID-19. Nevertheless, they can be used for diagnostic purposes. Another important function of SARS-CoV N protein is its ability to circumvent host immune response as an RNA viral suppressor protein (VSR).65 The VSRs suppress the RNA at the pre-dicer or post-dicer level to control the host defensive mechanism to establish infection66 (Figure 6).