Histopathologic Evaluation and Scoring of SARS-CoV-2 Infection

Muhammad Mazhar Ayaz^{1, *}, Ihtisham Ulhaq², Kashif Rahim³

¹ Department of Parasitology, Faculty of Veterinary Science, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan

² Department of Biosciences, COMSATS University Islamabad (CUI)45550, Pakistan

³ Department of Microbiology, Faculty of Veterinary Science, Cholistan University of Veterinary and Animal Sciences (CUVAS), Punjab, Bahawalpur 63100, Pakistan

Abstract

The recent emergent coronaviruses in the 21^st century, such as Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV), Middle East Respiratory Syndrome-Coronavirus (MERS-CoV), and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), has caused significant morbidity and mortality around the world. The lung is the most affected organ in the infection of human pathogenic coronaviruses. There is always a scarcity of human signs, symptoms, and modes of transmission. So to study the viral pathogenesis and evaluated interventions of therapies and vaccines, animals need to be used as models, especially at early epidemics. Lesions scoring can be identified from histopathological studies, and it can be helpful to understand the viral pathogenesis and damages to the cells to design effective therapies or vaccines. Histopathology uses the cells to determine viral host receptors and viral host tropism to relate with disease severity and lesions. Moreover, histopathology also plays a role in the qualitative description of affected organs to determine the micro-anatomic location of cells, type of cells, and cellular consequences during and post-infection. Comparatively, this approach has various limitations, but still, it is significant in comparing treatment groups. In comparing various groups, semi-quantitative and quantitative tissue scores are used for statistical analysis to increase the reproducibility of the study. This chapter refers to different features, including the importance of histopathology, principles, technique, scoring methods, and pathological characteristics of COVID-19, which can be valuable to assess the lung infection caused by SARS-CoV-2 and animal models and real situations.

Keywords: COVID-19, Infection, Lung, Pathology, RNA, Scoring.

^* Corresponding author Dr. Muhammad Mazhar Ayaz: Department of Parasitology, Faculty of Veterinary Science, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. E-mail: mmazharayaz@cuvas.edu.pk

INTRODUCTION

Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) belongs to the group of betacoronaviruses, enveloped, and genetic material is present in the form of single-stranded RNA which size is approximate ~30 kb. A single-stranded RNA genome codes a variety of structural and non-structural proteins. The non-structural proteins are comprised of various vital enzymes, including protease, helicase, and RNA-dependent RNA polymerase, which play a key role in the pathogenesis and survival of the virus inside the cell. The sequence analysis of pair-wise non-structural proteins indicated that the pathogen belongs to the Severe Acute Respiratory Syndrome-Coronavirus (SARS) coronaviruses group [1]. However, structural proteins comprised of glycoproteins of spikes and other accessory proteins role in the virus binding to the host receptor and its entry into the cell [2]. The structural proteins reported in the outbreak of SARS and the Middle East Respiratory Syndrome-Coronavirus (MERS) can help develop antiviral drugs as a potential target. Therefore, these proteins are documented as potential targets to develop antiviral drugs against SARS and MERS [3], and possibly representing in coronavirus disease-19 (COVID-19) case, according to the genetic analysis of recent SARS-COV-2 isolated from Wuhan [4]. However, new antiviral drugs or vaccines can be developed by using structural proteins as recommended. The phylogenetic analysis of the full-length genome is suggested that SARS-CoV-2 shares 79.5% sequence similarity with SARS-CoV [5^-7], which indicated that SARS-CoV-2 belongs to subgenus Sarbecovirus [5^-8]. Various novel coronaviruses have been identified from animals and humans after the emergence of SARS and MERS coronaviruses. It delivers track for the evolutionary origin of incipient coronaviruses from bats source only. Though, camels were also recognized as immediate hosts for the MERS-CoV outbreak. The emergence of SARS-CoV and MERS-CoV from zoonotic source indicate zoonosis in the case of COVID-19 as well [9, 10]. The sputum sample of a patient who suffered from acute pneumonia and kidney failure was found with MERS-CoV. It was isolated for the first time and genetically closed to PiBatCoV HKU5 (Pipistrellus bat CoV HKU5) of japan and Ty-BatCoV HKU4 (Tylonycteris bat CoV HKU4) in Hong Kong from Tylonycteris pachypus (bamboo bat). The lineage C β-CoVs associated to MERS-CoV was detected in bats, together with Hypsugo BatCoV HKU25 and BtVsβCoV/SC2013 from China bats, and coronavirus Bat CoV PREDICT/PDF-2180, BtCoVNeo5038/KZN/RSA/2015, and Neoromicia/PML-PHE1/RSA/2011 (NeoCoV), from African bats [11^-15]. Additionally, a lineage C β-CoVs, Erinaceus CoV VMC/DEU, afterward well-defined as a novel species, Hedgehog coronavirus 1 was detected in European hedgehogs that are closely related to bats phylogenetically [16]. Likewise, palm civets and raccoon dogs were found with similar coronavirus like the SARS-CoV

isolated from China during the early outbreak, which indicated that these animals have the potential of intermediate host between bats and humans [17].

Humans appeared to be susceptible to bat-sourced SARS-CoVs without before variations that indicated the re-emergence of SARS-CoVs or related to it [18, 19]. There is a similarity in the spike proteins of Ty-Bat CoV HKU4 and MERS-CoV, which indicated that the primary host of the ancestor of coronaviruses in bats [20, 21]. Two different highly human pathogenic coronaviruses, such as SARS and MERS emerged in the 21^st century in 2002 and 2012 respectively [22, 23]. At the end of December 2019, another coronavirus that was named SARS-CoV-2 has emerged in the Chinese population who had epidemiologically linked to the seafood market in Wuhan, Hubei Province [24]. The SARS-CoV-2 appeared to be transmitted to humans by nasal route, orally and through the membrane of eyes, which indicated that it has broad-scale transmission modes [25]. The recently identified SARS-CoV-2 has 88% sequence similarity with two bat-derived SARS coronaviruses such as bat-SL-CoVZXC21 and bat-SL-CoVZC45 and 79% genetic similarity with SARS-CoV and 50% with MERS-CoV [4]. After phylogenetic analysis, it was revealed that SARS-CoV-2 belongs to the genus of betacoronavirus, which comprises SARS/SARS-like-CoV and others isolated from bats and few wild animals [26]. Spikes proteins (also called S proteins) determined the host and tissue tropism of the SARS-CoV-2. S1 and S2 are the two domains of spikes proteins, which have a role in the virus attachment to host cell and membrane fusion, respectively. There is a high sequence similarity found in the S proteins of SARS-CoV-2 and SARS-CoV and both coronaviruses using the same cellular receptor for pathogenesis, which may be due to similarity in the S proteins [27]. Compared with SARS and MERS coronaviruses, the receptor domain of SARS-CoV-2 has a high mutation rate but still uses the ACE2 receptor during pathogenesis. The transmissibility and pathogenesis of SARS-CoV-2 are reported to enhance significantly because of mutation at Furin protease. The lessons learned through experiences result in tremendous advances, and valuable insight near separating the physiognomies of SARS-CoV-2 has been accomplished at an extraordinary speed from coronaviruses' epidemics, occurrence over the last two decades. It is still able to use this binding receptor. Whether mutations in ACE2 affect its binding or change receptor tropism need to explore in detail [4]. This chapter refers to different features, including the importance of histopathology, principles, technique, scoring methods, recent case studies of coronavirus and its types, virological examination, blood cell examination, and counts and pathological characteristic of COVID-19, which can be valuable to assess the lung infection caused by SARS-CoV-2 and animal models and real situation.

Importance of Histopathology Technique

Some years ago, the technique of histopathology was developed to study the abnormal activities happening inside the cell. The pioneer of the very first published book on histopathology is Johannes Miller, who published the book “On Nature and Structure Characteristics of Cancer” in 1938. In 1591, the compound microscope was constructed for the first time but writhed from various problems, including optical problems [28]. The simple microscopes were upgraded by Anton van Leeuwenhoek in 1673 with single lenses by improving the resolution and magnification [29]. Popular manufacturers such as Cambridge Rocker, Minot, and Sledge microtomes solved the issues of sectioning with the first microtome, which is ideal for animal tissues sectioning [28]. During the mid of 19^th century, embedding Paraffin wax for infiltration and maintenance throughout sectioning was introduced [30]. From time to time, various chemicals in the laboratory have been investigated to be used as preservatives [31], including formalin introduced in 1893 for the first time. In 1945, hand processing was replaced by automatic tissue processors through the industrial revolution. In 1951, the cryostat was manufactured for the first time [32]. From the past 50 years, histochemistry, electron, and polarizing microscopy have been used as diagnostic tools [28]. In 1980, a cancer diagnosis was revolutionized with the beginning of frequent use of immune-histochemistry but still under progress (Michael Titford, 2006). The human pathogenic coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2, are associated with high morbidity and mortality rates. However, WHO did not permit the postmortem of dead body organs infected with SARS-CoV-2 to observe pathologic changes among them due to transmission to healthy individuals [33].

Principles

The principles for histopathology were based on the differential coloring of the cells present in the tissue(s). The coloring ability of many dyes resides in either their acid or basic radical [34]. The acid components of a cell nucleus chromatin or chromosomes stain with basic dyes (mostly by hematoxylin). These components are referred to as basophilic or hematoxylin-ophilic dye (blue). The characteristics of cells and tissues fluctuated when they were examined by microscope due to inappropriate techniques. Vigilant fixation can kill the cells due to the reduction of adjustments in vivo morphology. The most reliable method to directly observe the living cells' interior is to separate them for the organism, make a smear, staining and observes them by dark-field or phase-contrast microscopy. Following dispensation ends with tissues implanted in a material that simplifies thin sectioning through microtome; for light microscopy, this is usually done with the help of paraffin [35].

Techniques and Types

For non-invasive tissue collection and slight harm, numerous methods have been evaluated and for the depth study of cellular activities earlier, throughout, and at the end of the pathological process, which is either created intentionally or during the study of any infection [36, 37]. Here in the pathological study, few are discussed, like histopathology and immune-histopathology, to study the COVID-19 model. The point to point procedures of histology processes are as follows:

1. Elimination or Biopsy taking of the tissue: The first work for histopathology starts from tissue removal [38]. The tissues can be removed for cellular histopathology by various methods, including (a) Living cell biopsy (b) Autopsy samples for this COVID-19 issue.

2. Fixation of sample: The sample, preferably a small piece of tissue, is employed in a chemical or reagent, most commonly formalin, a 10% aqueous solution of 40% formaldehyde that preserves substances structures in the cells and tissues. It also prevents autolytic changes. Depending on the size and nature of the tissue, it should be fixed from 4 to 24 hours [39].

3. Dehydration and Washing: Washing is done before the fixation of the specimen, and this is done to remove excess water prior [31]. The tissue is then placed in increasing strengths of alcohol or other dehydrating agents. So to remove the original water in the tissue.

4. Clearing: The process of removing the dehydrating agent with some fluid that is miscible both with the dehydrating and embedding medium [31]. Usually, it is an oil-based chemical (paraffin) that is clearing agents frequently substitute oils that are not advanced by formalin, including triglycerides in fat cells.

5. Embedding and Infiltration: In this process, the clearing agent is usually replaced with the embedding medium, paraffin [31].

6. Slicing or Cutting: Slicing is usually done with 3 µ and 10 µ (micrometers, microns) thick sections of the tissues with the help of a microtome [31].

7. Section Growing: Section growing is accomplished by transferring sections to a clean glass microscope slide [31].

8. Removal of Embedding Material or Decoration: This is the removal of the embedding agent is very much important. After these eight exhausting steps then we start the process of staining [31].

9. Staining: A process for increasing the visibility of cells by the application of dyes or by their reaction(s) with chemical or staining agent(s) to form visual substances or real happenings at the cellular level [31].

10. Mounting: This is done after clearing to cover the thin tissue section with a bit of drop of mounting medium like DPX and with the help of a thin glass cover-slip, thus making the preparation permanent [31].

Technique for Immuno-histopathology, RNA scope, or immunofluorescent staining

This technique is a colorimetric immune-alkaline phosphatase immune-histochemistry method that requires certain different that requires certain chemicals and specific antibodies (Abs) developed with a mouse anti-SARS-CoV antibody COVID-19 purpose [40^-42]. In brief, the following steps can be followed which are already established, and the first step starts with:

1. Tissues and Sections: 3 μm sections from formalin-fixed.

2. Deparaffinization and Rehydration: Paraffin-embedded tissues may be de-paraffinized, rehydrated.

3. Staining: Placed in an auto-stainer.

4. Enzyme Digestion: The sections may be digested in 0.1 mg of proteinase K per ml and then,

5. Incubation with hyper-immune Serum: Incubate for 1 hour with an ascetic fluid of hyper-immune mouse reactive with SARS-CoV antigen at a 1:1,000 dilution.

6. Dilution: Optimal dilutions of the antibody and the requirement for predigestion may be determined (already performed by various workers by a series of pilot studies performed on SARS-CoV-infected Vero cells).

7. Immunoglobulin-associated incubation: The slides are washed and evaluated for incubation with a biotinylated anti-mouse immunoglobulin antibody.

8. Visualization: Streptavidin-alkaline phosphatase complex is used to visualize the attached antigen; subsequently, for colorimetric detection, a naphthol-fast red substrate is used [43].

9. Counterstaining: Sections may be counterstained with Mayer's hematoxylin [44].

Scoring Methods

Evaluating the damage(s) caused by any etiology through histopathology is not an easy job. No such method has been developed even several hundreds of thousands of studies based on histopathology here before. It is a mere attempt to enumerate the COVID-19 pathological signs and score the damaging caused, as shown in Table 1. In the COVID-19 infection, the most important clinical feature is lung-associated symptoms that indicate the severity of the disease [45]. During the emergence of any novel disease that has no treatment option or vaccines, different animals are used as a model to test the newly developed drug or vaccines against the disease. Animal models are often required to study viral infections and therapies, especially during an initial outbreak [46]. Histopathological studies can identify better understanding of viral pathogenesis, replication, and destruction of lesions of affected cells. It can be helpful to develop any efficient therapeutics. The detection of presumed viral receptors and viral tropism for cells can be evaluated to correlate with lesions using immune-staining [47]. In the lung, lesions and immune-staining can be qualitatively described to define the cell types involved, micro-anatomic location(s), and type of pathological changes seen at the cellular level. There is also acute diffuse alveolar damage (DAD), a common clinical feature of MERS-CoV infection in the form of lung lesions characterized by septal injury in alveoli, inflammation, and edema. SARS-CoV-2 caused different lesions in the lung, such as thrombi congestion, as shown in Fig. (1).

Table 1 Lesions recording as seen in COVID-19 or MERS-CoV in lung infection(s).

S.No	Pathological Condition	Mild	Moderate	Severe
1.	Lesions in Necrosis/cell death	---	---	---
2.	Inflammation	---	---	---
3.	Hyaline membranes/fibrin	---	---	---
4.	Edema	---	---	---
5.	Thrombi	---	---	---
6.	Congestion	---	---	---
7.	Hemorrhage	---	---	---
8.	Pneumonia	---	---	---
9.	Type II hyperplasia	---	---	---
10.	Syncytia	---	---	---
11.	Blackening of lung tissues	---	---	---

Notably, the scoring principle(s) can be applied to tissue lesions at a gross or histopathological level (Table 1). Immune-stained sections can be used to evaluate the severity or pathogenicity of infection(s) for statistical analysis or comparison purposes. Such robust methods are the need for precise calculation of damage at the site of tissue or cell. This is the reason that scientists remained unable to calculate or enumerate the exact loss. The qualitative loss sometimes cannot be quantified for scoring purposes (s). The mostly used semi-quantitative method(s) produce ordinal scores [48]. The progression of disease severity is defined by assigning grades, which define every score by characteristically 4-5 grades or more being optimal such as 0, 1, 2, 3, 4,5… as presented in Table 2. The uncertainty must be minimum in the assigned samples, which required each grade to be defined well. The reproducibility score is limited with the use of simple descriptive modifiers including usual, infrequent, acute, slight, modest, and severe, thus making less impact on calculations.

Fig. (1))

Some sequences in lung tissues caused by COVID-19.

Quantification of the viral lesion(s) and immune-staining in tissues is an option, though quantification is not commonly performed in tissue sections because of potential confounding factors. Potential confounding factors include random distribution of viral inoculum and difficulty in objectively quantifying lesions. A group of statisticians evaluates the various scoring methods already used in bio-statistics to identify the most applicable tests that help assess differences and benefits in a group [49]. Examples of lung lesions observed in COVID-19 or MERS-CoV infections with quantification that can be numerically analyzed.

Table 2 Scoring of the lesion through the quantitative method.

S.No	Pathological Condition	0	1	2	3
1.	Lesions Necrosis/cell death	---	---	---	---
2.	Edema	---	---	---	---
3.	Hyaline membranes/fibrin	---	---	---	---
4.	Inflammation	---	---	---	---
5.	Thrombi	---	---	---	---
6.	Congestion	---	---	---	---
7.	Hemorrhage	---	---	---	---
8.	Pneumonia	---	---	---	---
9.	Type II hyperplasia	---	---	---	---
10.	Syncytia	---	---	---	---

Recent Case Studies of Coronavirus and its Types

The viruses of coronvirinae have spike protein that given a crown-like appearance under the microscope for the first time, so named coronavirus. All these coronaviruses are classified in Nidovirales order and Coronaviridae family [50, 51]. The alpha, beta, gamma, and delta are the subgroups of Coronvirinae that are characterized based on genomic structure. However, these subgroups have different host tropisms. Furthermore, alpha and beta coronaviruses cause respiratory distress, gastroenteritis in humans, and animals, respectively. There were 6 human pathogenic coronaviruses until December 2019. Among 6 viruses, 4 viruses, such as HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1, cause mild respiratory disease, while others, such as SARS-CoV and MERS-CoV associated with a severe type of respiratory disease [52]. The SARS-CoV emerged in 2002, and the outbreak continues till 2003, causes 10% mortality. The mortality rate was 37% in the case of the MERS-CoV outbreak that was emerged in 2012 [50]. According to the literature, SARS-CoVs and related other coronaviruses related to spikes made of proteins are protruded from the envelope having the receptor-binding domain (RBD). The RBD helps in the binding with the cellular receptor that is angiotensin-converting enzyme-2 (ACE-2), generally present in the respiratory system, kidneys, heart, and gastrointestinal tract (GIT), to which viruses bind and cause infection [53]. The SARS-CoV and SARS-CoV-2 use the same receptor during pathogenesis and having the same biochemical pathways as well. A cascade of inflammation is triggered in the lower respiratory tract when SARS-CoV reaches to lungs and bind with type-II pneumocytes through ACE-2. TMPRSS2 (type-2 trans-membrane protease) is a proteolytic enzyme that cleaves the SARS spike protein and ACE-2 receptor complex. This complex was formed by viral binding to the ACE-2 receptor, and after cleavage spikes, proteins are activated [53]. According to initial studies, in the early outbreak of COVID-19, 41 patients were enrolled in hospital, 98% were having fever, 76% have a cough, and 55% of patients had shortness of breath. During the initial two weeks of infection, the symptoms were mild, and the infection was contagious; however, the shortness of breath symptom increase and dyspnea as well [54]. According to chest CT scan results, 13 out of 41 had developed clinical pneumonia and suffered from hypoxic respiratory failure, and required ventilation. Among them, 2 patients received extracorporeal membrane oxygenation due to refractory hypoxia, 10 patients received mechanical ventilation.

In comparison, 6 patients died due to disease severity. This high CFR triggered alarming situations around the world. Although, most of the patients were having underlying diseases and aged people with an average of 49 age [50,55,56].

Virological Investigations

The flow diagram shows how the spikes get an attachment with receptors on the cell's body (Fig. 2). It is hypothesized that the mycoplasma-like organisms first paves the way for virus entry at the mucosa level [57]. The mycoplasma-like microorganism removes the villi from the mucous membrane. It denudes where the virus can get entry, and the cell's defense system starts mucous production, especially in the lungs. The whole mechanism of replication has been shown in Fig. (2).

Fig. (2))

Replication of SARS-CoV-2 inside the cell.

Blood Cell Examination and Counts

According to the complete blood count of the new hospital admitted patient, the patient has normal white blood cells (WBCs) and platelet counts. Lymphocytes level was reduced, and inadequate normal-sized red blood cells (RBCs). The WBCs and immature neutrophil levels were reported to increase after three days [58, 59], whereas the monocytes level was increased slightly and lymphocytes level reduced. The leuko-erythroblastic showed a reduced level of RBCs but normal in shape and size. Pathologically this is termed normocytic anemia. Moreover, anisocytosis conditions (a mild presence of RBCs with different sizes), nucleated immature RBCs, and dacrocytes (rare tear-drop shaped cells) were observed in the haemogram of the patient. WBCs appeared to be primarily involved in the fight with pathogens as its level was normal when it was analyzed after infection recovery.

Pathological Characteristics of COVID-19

The WHO strictly prohibits the postmortem of dead bodies with COVID-19 infection or die from it [60]. Various organs observed were lung, GIT, heart, liver, kidney, skin, spleen, and blood vessels, and they showed the characteristics of COVID-19.

Lung

Since SARS-CoV-2 is considered respiratory viruses, the lung is one of the well-studied organs in postmortem, autopsies, and biopsies. In postmortem or autopsy of the patient, the visual observation revealed an increasing abundance of gray-white viscous fluid or thick mucus, as discussed earlier. Engagement of vascular, consolidation or thickness, inflammation or edema of pleura developing to pleurisy, mildly erythematous trachea, and evidence of white mucous in lungs along with frothy pink material in airways with aggravation to dark-colored hemorrhage(s) were observed [60]. Moreover, in interstitial regions lymphocytes infiltration, diffused alveolar damage, intra-alveolar fibrin, hyperplasia of pneumocytes, thick exudate formation, and lymphocytic inflammation were also observed. Additionally, in various studies, electron microscopy reported the hyaline membrane formation, presence of fibroblasts around intra-alveolar fibrin, and loose connective tissue [61]. The other changes observed were pulmonary arteries cytoplasmic vacuolization, presence of megakaryocyte in branches of the small vessel, pulmonary microangiopathy, clotting of fibrin in small capillaries around alveoli, congestion of capillary, thrombosis of a small vessel, and alveolar hemorrhage. There was thickening of alveolar capillaries in lungs also.

Gastrointestinal (GI) tract

COVID-19 infection may also affect the GIT due to the presence of ACE2 in the enterocytes. Reports for the presence of SARS-CoV-2 in stool and diarrhea have been reported. No damage has been observed through a histopathology study. The esophagus revealed the lymphocytes intrusion in the squamous epithelium, stomach lamina propria, duodenum, and interstitial edema [62].

Liver

There were high rise in the level of hepatic enzymes, such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alanine aminotransferase (ALT) in COVID 19 patients. It had led to liver function failure. Upon autopsy, it showed a dark red liver with hepatomegaly. Upon histopathological examinations, hepatocyte degeneration, lobular focal necrosis, congested hepatic sinuses, and the microthrombus. It was also noticed that the lung had portal tract fibrosis and mononuclear leukocyte along with neutrophil infiltration [63, 64].

Kidney

Proximal renal tissues have been reported in COVID-19 patients according to histopathological findings of the biopsy. Degeneration in the vacuole, Luminal brush border sloughing, necrosis in tubules, lymphocyte infiltrations in the tubule-interstitial and/or sub-capsular area were also observed. Other kidney-related damages are glomerular epithelial cells hypertrophy, mild focal tubular atrophy, fibrosis in interstitial and cortical parenchyma, and complement membrane attack complex (MAC) deposit in tubules along with glomerular walls [65, 66]. The vascular analysis revealed the infiltration of inflammatory cells in the arcuate artery and glomerulus capillary vessels are dilated. In glomerular capillary loops, fibrin thrombus is shown, which gives a foamy appearance of endothelial cells, and hyperplasia is also observed under the microscope. The reason for kidney failure was collapsing glomerulopathy (CG) in COVID-19 patients.

Skin

Skin tissue alterations have been reported to have clinical features such as petechial rash with thrombocytopenia, widespread urticarial and vesicles on the skin, and erythematous-to-purpuric coalescing macules COVID-19 patients. Moreover, vasculopathy, including peripheral cyanosis leading to bullae and dry gangrene with red papules on fingers resembling chilblains, was also observed. Recently, a study presented skin classification based on lesions that include acral areas of erythema with vesicles or pustules and vesicular eruptions in early cases. There were common lesions such as urticarial lesions and necrosis and maculopapular eruptions [67, 68]. Other skin-associated histopathological findings are focal acantholytic suprabasal clefts, ballooning herpes-like keratinocytes, mucin deposition in the dermis and hypodermis, and nests of Langerhans cells within the epidermis have been reported frequently. Superficial and deep perivascular dermatitis, lymphocytes around blood vessels, dyskeratotic, thrombus formation, and extravasation of erythrocyte in mid-dermis blood vessels were also reported frequently [69].

Heart

The macroscopic and microscopic features of the heart are also reported to changes with the COVID-19 infection, included cardiomegaly and dilation of right ventricular were prominent. Autopsies showed firm myocardium with a red-brown appearance. Microscopic examination revealed myocyte necrosis in the dispersed area near lymphocytes [70, 71].

Blood

Blood specimens showed coagulation, leukopenia, and lymphocytopenia in infected/suspected live cases in peripheral blood examination [72].

Spleen and lymph nodes

Splenic tissues showed viral nucleocapsid protein (NP) in the red and white pulp. Histopathological examinations showed a reduction of cell composition, atrophied white pulp, neutrophilia, plasma cell infiltration, reduction or absence of lymph follicles, increased red pulp to white pulp proportion, and atrophy of corpuscles in the spleen COVID-19 cases [73]. CD20+B cells were found in abundance.

Brain

Viral particles of SARS-CoV-2/COVID-19 were observed in the frontal lobe and capillary endothelial cells of the brain. There were no recordable findings by histopathologic examination of the brain, despite mild hypoxic changes [74].

Blood vessels and placenta

On postmortem, there was the presence of inflammatory cells and endothelitis. Low-grade fetal vascular malperfusion is reported in the pregnant woman. Other histopathological changes of the pregnant woman were fetal thrombotic vasculopathy, meconium macrophages, intramural nonocclusive thrombi, and intramural fibrin deposition [75].

Animal Models and Real Situation

So far, various animals have been evaluated to study the histopathological changes, but no animal presented similar COVID-19 disease symptoms to a human. Mice were killed by euthanasia to study the effect of COVID-19 on various organs of mice. In the laboratory, access and deal with laboratory animals is easy as they are cheap, but in the case of COVID-19, it is still in infancy. The SARS-CoV-2 replicated potentially in the respiratory tract of young inbred mice and data available in sufficient number(s) for statistical evaluation. Pneumonitis and clinical illness develop in almost 1-year-old mice, but its acquisition is hard, and immune senescence complicates pathogenesis studies [76].

A rat model changed the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of the young virus adopted in BALB/c mice. Later, intranasal inoculation, 15 passages caused in a virus (MA15). The quick and high titer of replicating the virus in the lungs increases disease lethality that causes viremia, lymphopenia, and neutrophilia. Besides, the virus spread to extra-pulmonary sites leads to pathological changes in the lungs. However, focal and mild pneumonitis, airways, and alveoli with debris were also reported due to the extensive distribution of the virus. Bronchial epithelial cells and alveolar pneumocytes were also found with viral antigens. Mice have died from a devastating viral infection, and it was suggested that death occurs due to the destruction of ciliated epithelial cells and pneumocytes [76]. Primate animal models other than humans are required urgently to answer the other critical questions or the questions that have not been answered in clinical patients, such as testing antiviral drugs and vaccine evaluation. In response to this hypothesis, old-world monkeys as primates animal models were inoculated with SARS-CoV-2 experimentally. The monkeys include Macaca mulatta (M. mulatta) (12), Macaca fascicularis (M. fascicularis) (6) and 6 were from new world monkeys such as Callithrix jacchus (C. jacchus). Infection develops after inoculation in the Macaca mulatta were found with the symptoms of fever, 2 out of 6M. fascicularis develop fever, and none of the C. jacchus found with fever. The abnormality in chest regions included lungs, bronchus, and spleen, was reported in all of the M. mulatta and M. fascicularis.C. jacchus was not found with the genome of the virus. However, severe gross and histopathological changes were seen in the stomach, heart, and lungs [77]. According to another study report, the viral genome in the range of 3.0×10⁴ to 1.5×10⁷ copies/g was found in the Rhesus monkey till post-infection. In the lower lobes regions of the lungs, the titer of the viral genome identified up to 2.0×10⁷ copies/g. The pathologists estimated lung lobes damaged by lesions through necropsy for histopathological analysis. It was identified that the right and left inferior lobe is critically injured and the surroundings of the small bronchus as well. The majority of alveoli had thickened walls due to the proliferation of fibroblasts, formation of pulmonary hyaline-membrane, edema, and hemorrhage also observed in the alveoli [78].

According to the studies of cats and ferrets as animal models, the post-infection consequences were the same as humans. However, they were not observed with hyaline membranes and syncytia. The pathogenesis and consequences of SARS-CoV-2 in cats have primarily been associated with tubercle tracheobronchial lymphadenitis. Lastly, in vivo results suggested the ACE2 importance in SARS-CoV-2 infection. In cats, pneumocytes type I and II expressed the ACE2 and facilitated the SARS-CoV-2 infection. In ferrets, ACE2 is expressed only on pneumocytes II [79]. These animal models and techniques like histopathology or immune-histopathology help better understand and develop future strategies for COVID-19 control. Scientists should find the best route to overcome the COVID-19 pandemic and develop vaccines and therapeutics remedies, especially the etiology of innovative origin that happened the first time.

CONCLUSION

Conclusively, the virus induces clinical and histopathological changes that produce vascular harm that caused the failure of multiple organs. Observed the lesions of various organs, including the heart, kidney, brain, lymph nodes, skin, lungs, liver, and GIT simplified the pathophysiology. Our final suggestion is to uphold the speculation that far-reaching endothelial contamination by SARS-CoV-2 could have a function in the pathogenesis of serious type infection and susceptible genes at particular organs. More immune pathological studies are needed to reveal why kids, teenagers, adults, and aged present with restricted skin types of COVID-19, interestingly with the serious multi-organ introductions seen in more aged patients with prior infections.

CONSENT FOR PUBLICATION

Not Applicable.

CONFLICT OF INTEREST

The author confirms that this chapter contents have no conflict of interest.

ACKNOWLEDGEMENT

Declared none.

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