Overview of COVID-19 Disease: Virology, Epidemiology, Prevention Diagnosis, Treatment, and Vaccines

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Review Overview of COVID-19 Disease: Virology, Epidemiology, Prevention Diagnosis, Treatment, and Vaccines

Iman Salahshoori 1,*, Noushin Mobaraki-Asl 2,*, Ahmad Seyfaee 3,*, Nasrin Mirzaei Nasirabad 2, Zahra Dehghan 4, Mehrdad Faraji 5 , Mina Ganjkhani 4, Aziz Babapoor 4,*, Seyede Zahra Shadmehr 6 and Ali Hamrang 6

1 Department of Chemical Engineering, Science and Research Branch, Islamic Azad University, Tehran 14778-93855, Iran 2 Department of Obstetrics and Gynecology, School of Medicine and Allied Medical Sciences, Alavi Hospital, Ardabil University of Medical Sciences, Ardabil 56189-85991, Iran; [email protected] 3 School of Mechanical Engineering, University of Adelaide, Adelaide 5005, Australia 4 Department of Chemical Engineering, University of Mohaghegh Ardabili, Ardabil 56199-11367, Iran; [email protected] (Z.D.); [email protected] (M.G.) 5 Micro and Nanotechnology Graduate Program, TOBB University of Economics and Technology, Sogutozu Caddesi No 43 Sogutozu, Ankara 06560, Turkey; [email protected] 6 Department of Applied Chemistry, Faculty of Science, University of Mohaghegh Ardabili, Ardabil 56199-11367, Iran; [email protected] (S.Z.S.); [email protected] (A.H.) * Correspondence: [email protected] (I.S.); [email protected] (N.M.-A.); [email protected] (A.S.); [email protected] (A.B.)

Citation: Salahshoori, I.; Abstract: Coronaviruses belong to the “Coronaviridae family”, which causes various diseases, Mobaraki-Asl, N.; Seyfaee, A.; from the common cold to SARS and MERS. The coronavirus is naturally prevalent in mammals Mirzaei Nasirabad, N.; Dehghan, Z.; and birds. So far, six human-transmitted coronaviruses have been discovered. Severe acute res- Faraji, M.; Ganjkhani, M.; Babapoor, piratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, A.; Shadmehr, S.Z.; Hamrang, A. China. Common symptoms include fever, dry cough, and fatigue, but in acute cases, the disease Overview of COVID-19 Disease: can lead to severe shortness of breath, hypoxia, and death. According to the World Health Orga- Virology, Epidemiology, Prevention nization (WHO), the three main transmission routes, such as droplet and contact routes, airborne Diagnosis, Treatment, and Vaccines. Biologics 2021, 1, 2–40. https:// transmission and fecal and oral for COVID-19, have been identified. So far, no definitive curative doi.org/10.3390/biologics1010002 treatment has been discovered for COVID-19, and the available treatments are only to reduce the complications of the disease. According to the World Health Organization, preventive measures Academic Editors: at the public health level such as quarantine of the infected person, identification and monitoring Vasso Apostolopoulos and of contacts, disinfection of the environment, and personal protective equipment can significantly Majid Hassanzadeganroudsari prevent the outbreak COVID-19. Currently, based on the urgent needs of the community to control this pandemic, the BNT162b2 (Pfizer), mRNA-1273 (Moderna), CoronaVac (Sinovac), Sputnik V Received: 10 February 2021 (Gamaleya Research Institute, Acellena Contract Drug Research, and Development), BBIBP-CorV Accepted: 23 April 2021 (Sinofarm), and AZD1222 (The University of Oxford; AstraZeneca) vaccines have received emergency Published: 12 May 2021 vaccination licenses from health organizations in vaccine-producing countries. Vasso Apostolopoulos, Majid Hassanzadeganroudsari Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: coronavirus; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); vaccines published maps and institutional affil- iations.

1. Introduction Small parasitic particles that are unable to reproduce on their own are called viruses. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. If they enter the host cell, they can infect the host cell and replicate. Genetic material This article is an open access article (DNA or RNA strand structure), a capsid protein coat (protection of genetic material), distributed under the terms and and an outer sheath of lipids are the known components of a virus structure. The funda- conditions of the Creative Commons mental particle of an infectious virus composed of nucleic acid and an external protein Attribution (CC BY) license (https:// shell is called a virion [1]. In the modern world, a record for detecting respiratory infec- creativecommons.org/licenses/by/ tions caused by the virus in children and adults date back to the 1960s [2]. One of the 4.0/). essential viruses available that cause respiratory syndromes are coronaviruses. This family

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viruses available that cause respiratory syndromes are coronaviruses. This family is called isCoronaviridae. called Coronaviridae. Many people Many are people exposed are to exposed the coronavirus to the coronavirus once in their once lifetime, in their which lifetime,often causes which diseases often causes such diseasesas pneumonia such asor pneumoniabronchitis. Coronaviruses or bronchitis. are Coronaviruses single-stranded, are single-stranded,enveloped RNA envelopedviruses with RNA a 120–80 viruses nm with di aameter 120–80 and nm diameterare divided and into are four divided groups: into fourAlpha groups: coronaviruses, Alpha coronaviruses, Beta coronaviruses, Beta coronaviruses, Gamma coronaviruses, Gamma coronaviruses, and Delta coronaviruses and Delta coronaviruses[3,4]. The four [3 ,4types]. The fourof coronavirus, types of coronavirus, HCoV-OC43, HCoV-OC43, HCoV-229E, HCoV-229E, HCoV-NL63, HCoV-NL63, and andHCoVHKU1 HCoVHKU1 are less are lesspathogenic pathogenic and andcause cause only only mild mild respiratory respiratory illness illness [5]. [Nevertheless,5]. Neverthe- less,two twotypes types of coronavirus, of coronavirus, severe severe acute acute re respiratoryspiratory syndrome syndrome co coronavirusronavirus (SARS-CoV) and MiddleMiddle EastEast respiratoryrespiratory syndromesyndrome coronaviruscoronavirus (MERS-CoV),(MERS-CoV), causedcaused twotwo deadlydeadly epi-ep- demicsidemics [ 6[6].]. In In 2003, 2003, due due to theto the SARS-CoV SARS-CoV emergency emergency in southern in southern China China and its and widespread its wide- prevalence,spread prevalence, extensive extensive research research was conducted was cond to controlucted to and control treat and the disease.treat the According disease. Ac- to thecording World to Healththe World Organization, Health Organization, more than 8000 more people than 8000 were people infected, were and infected, 774 deaths and were 774 reporteddeaths were [7]. reported In addition, [7]. inIn 2012,addition, another in 2012 outbreak, another of theoutbreak acute respiratoryof the acute disease respiratory was reporteddisease was in Saudi reported Arabia in Saudi where Arabia MERS-COV where wasMERS-COV identiﬁed was as theidentified cause of as the the epidemic. cause of According to published reports, the mortality rate was over 35% [8]. It was stated that the epidemic. According to published reports, the mortality rate was over 35% [8]. It was market civets and dromedary camels were the primary sources of SARS-CoV transmission stated that market civets and dromedary camels were the primary sources of SARS-CoV and MERS-CoV transmission to humans [6]. The known animal origins of coronaviruses transmission and MERS-CoV transmission to humans [6]. The known animal origins of are demonstrated in Figure1. coronaviruses are demonstrated in Figure 1.

Figure 1. Animal origins of human coronaviruses (Created(Created withwith BioRender.comBioRender.com., accessedaccessed onon 11 AugustAugust 2020).2020).

On 1111 MarchMarch 2020—The2020—The WorldWorld Health Health Organization Organization (WHO) (WHO) declared declared COVID-19 COVID-19 as as a pandemica pandemic caused caused by by SARS-CoV-2 SARS-CoV-2 [9]. [9]. The The main main effect effect of SARS-CoV-2of SARS-CoV-2 is in is human in human respira- res- torypiratory system system cells. cells. However, However, new new studies studies have have revealed revealed the impact the impact of the of virus the virus on the on cells the ofcells the of gastrointestinal the gastrointestinal tract, kidneytract, kidney system, sy liver,stem, pancreas,liver, pancreas, eyes, and eyes, brain and [ 10brain]. The [10]. SARS- The CoV-2SARS-CoV-2 virus is virus about is 79%about and 79% 50%, and similar50%, similar to SARS to SARS and MERS and MERS viruses, viruses, respectively respectively [11]. Widespread[11]. Widespread prevalence prevalence due to thedue high to the transmission high transmission strength andstrength complexity and complexity of COVID-19 of treatmentCOVID-19 compared treatment to compared SARS-CoV to and SARS-CoV MERS-CoV and has MERS-CoV led to a global has crisisled to and a global preventing crisis theand disease’s preventing spread the disease’s is a necessity. spread This is studya necess providesity. This an study overview provides of all an aspects overview of the of dis- all ease,aspects such of asthe virology, disease, modessuch as of virology, transmission, modes and of transmission, diagnosis. New and therapeutic diagnosis. guidelinesNew ther- andapeutic vaccines guidelines approved and byvaccines the World approved Health Organizationby the Worldare Health also mentioned.Organization are also mentioned.

2. Virology Coronaviruses are virions that have viral envelopes. The diameter of these virion particles is 120 nm. Cloverleaf structures such as glycoproteins and proteins on the virus’s

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2. Virology Coronaviruses are virions that have viral envelopes. The diameter of these virion surface have created a crown-like structure in it. These viruses are also known as corona- particles is 120 nm. Cloverleaf structures such as glycoproteins and proteins on the virus’s viruses becausesurface of their have crown created structure. a crown-like A section structure called nucleocapsid in it. These viruses is made are of also capsid- known as coro- coated proteinsnaviruses in these viruses because and of is their placed crown inside structure. the virus’s A section genetic called material nucleocapsid (Figure 2) is made of [12]. The coronaviruscapsid-coated has a genomic proteins genus in these of RNA. viruses This and genetic is placed material inside is the seen virus’s as a spiral genetic material or circular in the(Figure nucleocapsid2)[ 12]. The of coronavirusthe virus. The has coronavirus a genomic genus genome of RNA. consists This geneticof a single material is seen strand of positiveas a RNA spiral (ribonucleic or circular in acid). the nucleocapsid This virus’s of genomic the virus. RNA The has coronavirus a methylated genome consists warhead in its of5′ aregion single and strand is rich of positive in adenine RNA nucleotide (ribonucleic at acid). the end This of virus’s its ‘3’. genomicCorona- RNA has a viruses encode methylateda protein called warhead the replicase in its 50 region enzyme and in is their rich ingenome, adenine which nucleotide functions at the by end of its ‘3’. transcribing theCoronaviruses virus genome encode and producing a protein callednew copies the replicase using the enzyme host cell’s in their capabilities genome, which func- [13]. Accordingtions to studies, by transcribing SARS-CoV-2 the virusis a beta-corona genome and virus producing genus newmember. copies The using SARS- the host cell’s CoV-2 virus is capabilitiescomposed [of13 ].four According structural to studies,proteins, SARS-CoV-2 including isnucleocapsid a beta-corona (N), virus spike genus member. (S), membrane The(M), SARS-CoV-2and envelope virus (E) (Figure is composed 2). Protein of four M structuralplays a vital proteins, role in includingintroducing nucleocapsid the virus into the(N), body spike and (S), forming membrane envelopes (M), and [14]. envelope Protein (E) E (Figureis responsible2). Protein for M the plays prolif- a vital role in introducing the virus into the body and forming envelopes [14]. Protein E is responsible for eration, germination, envelope formation, and spread of the virus [15]. Increasing tran- the proliferation, germination, envelope formation, and spread of the virus [15]. Increasing scription and assembly of viruses is the responsibility of multipurpose N protein [16]. In transcription and assembly of viruses is the responsibility of multipurpose N protein [16]. addition, the virusIn addition, binding to the host virus cells binding is the responsibility to host cells is of the the responsibility Spike (S) protein. of the There- Spike (S) protein. fore, it has a uniqueTherefore, rank itin has pharmaceutical a unique rank and in pharmaceutical vaccine research. and It vaccine should research. be noted It shouldthat, be noted due to the lackthat, of response due to the to lack neutralizing of response or to immune neutralizing antibodies, or immune proteins antibodies, N, M, proteins and E N, M, and E are not consideredare notas drug considered targets as [17]. drug targets [17]. As depicted in AsFigure depicted 2, the in S Figureglycoprotein2, the S of glycoprotein the newly ofdiscovered the newly SARS-CoV-2 discovered SARS-CoV-2is composed of twois composedsubunits, S1 of and two S2, subunits, and is commonly S1 and S2, andrepresented is commonly as a sword-like represented spike. as a sword-like The real structurespike. of this The protein, real structure however, of this can protein, be observed however, via cr canystallography. be observed The via crystallography.Pro- tein Data Bank The(PDB) Protein model Data of this Bank glycopro (PDB) modeltein reveals of this how glycoprotein the subunits reveals are comprised how the subunits are of different regionscomprised that are of different fundamental regions to that the are infection fundamental process. to the S1 infectionand S2 are process. linked S1 and S2 are together by a polybasiclinked together amino by acid a polybasic bridge, aminowhich acidmay bridge, be necessary which mayfor studying be necessary viral for studying targeting [18]. Theviral virus targeting binds [ 18to ].the The host virus cell bindswith the to thehelp host of an cell S-protein with the and help penetrates of an S-protein and the cell. After thepenetrates penetration the cell. process, After the the tr penetrationanscript process process, begins, the transcript and the processvirus multi- begins, and the virus multiplies until the host cell is wholly infected and destroyed [19]. plies until the host cell is wholly infected and destroyed [19].

. Figure 2. An in-depth look into the SARS-CoV-2 Spike Glycoprotein (Reprinted from “An In-depth Look into the Structure of the SARS-CoV-2Figure 2. SpikeAn in-depth Glycoprotein”, look into by theBioRender.com SARS-CoV-2, accessed Spike Glycoprotein on 1 August 2020)(Reprinted [20]. from “An In-depth Look into the Structure of the SARS-CoV-2 Spike Glycoprotein”, by BioRender, accessed on 1 Au- gust 2020) [20].

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Angiotensin-convertingAngiotensin-converting enzyme 2 (ACE2) enzyme receptors 2 (ACE2) in mammalian receptors in mammalianlung cells act lung as cells act as recipients of therecipients S-spike prot of theein, S-spike releasing protein, endogenous releasing endogenousviral RNA genetic viral RNA material genetic into material into host cells (Figurehost 3). cells (Figure3).

Figure 3. SARS-CoV-2Figure 3. SARS-CoV-2 targeting ACE2targeting receptor ACE2 and receptor entry inand the entr infectedy in the cell infected (Adopted cell from (Adopted “SARS-CoV-2 from “SARS- Targeting of ACE2 ReceptorCoV-2 and Targeting Entry in of Infected ACE2 Receptor Cell”, by BioRender.comand Entry in In,fected accessed Cell”, on 1 by August BioRender, 2020) [accessed21]. on 1 August 2020) [21]. One of the crucial functions of angiotensin-converting enzymes (ACE2) is to regulate One of the thecrucial renin–angiotensin functions of angiotensin- system (RAS).converting In general, enzymes the angiotensin-converting (ACE2) is to regulate enzyme 2 the renin–angiotensin(ACE2) actssystem as a cell(RAS). surface In receptorgeneral, andthe causesangiotensin-converting SARS-CoV-2 to enter enzyme host cells 2 and plasma (ACE2) acts as anda cell is expressedsurface receptor in organs and such causes as the SARS-CoV-2 lungs, kidneys, to enter and heart. host Moreover,cells and one of the plasma and is expressedreasons for in the organs development such as of the COVID-19 lungs, kidneys, is the disturbance and heart. of Moreover, the ACE/ACE2 One balance and of the reasons forthe the activation development of RAAS of byCOVID-19 SARS-CoV-2, is the especially disturbance in patients of the ACE/ACE2 with underlying bal- cardiovas- ance and the activationcular disease of RAAS problems, by SARS-CoV hypertension,-2, especially and diabetes in patients [22]. Figure with4 presents underlying the expression of ACE2 Receptor in Human Host Tissues. ACE2 expression in alveolar epithelial cells cardiovascular disease problems, hypertension, and diabetes [22]. Figure 4 presents the types 1 and 2 in the human lung is much higher than elsewhere in the body [23]. Accord- expression of ACE2 Receptor in Human Host Tissues. ACE2 expression in alveolar epi- ing to research, the level of ACE2 expressions in men is much higher than in women in thelial cells typesalveolar 1 and cells2 in [the24]. human Moreover, lung in is their much alveolar higher cells, than ACE2 elsewhere expression in the levels body in Asians are [23]. Accordingmuch to research, higher thanthe level in whites of ACE2 and African-Americansexpressions in men [25 is]. much It is stated higher that than Increasing in ACE2 women in alveolarexpression cells [24]. due Moreover, to SARS-CoV-2 in their virus alveolar binding cells, via spikeACE2 protein expression could levels lead to in alveolar cell Asians are muchdamage higher and than followed in whites by systemic and African-Americans reactions and even [25]. death It is [26 stated]. that In- creasing ACE2 expressionNevertheless, due to theSARS-CoV-2 prerequisite virus for coronavirus binding via attack spike on protein host cells could isreceptor lead binding. to alveolar cell damageAfter binding and followed to the receptor, by systemic the viral reactions spike protein and even is cleaved death by [26]. acid-dependent proteoly- Nevertheless,sis bythe Cathepsin, prerequisite TMPRRS2, for coronavirus or Furin Protease,attack on followed host cells by is fusion receptor of the binding. viral coating with After binding tocell the membranes. receptor, the In viral general, spike the protein spike protein is cleaved is structurally by acid-dependent divided into prote- two parts of the olysis by Cathepsin,nS1 terminal TMPRRS2, unit or containing Furin Protease, a binding followed site to ACEby fusion 2 receptor of the in viral human coating cells (RBD) and with cell membranes.the nS2 In terminal general, region the spike containing protein fusion is structurally protein and divided transmembrane into two anchorparts (T.A.) and of the nS1 terminalintercellular unit containing tail (I.T.). a Spike binding protein site hasto ACE a variable 2 receptor amino in acid human sequence cells that(RBD) makes it more and the nS2 terminalcompatible region with containing its host [ 27fusion]. protein and transmembrane anchor (T.A.) and intercellular tail (I.T.). Spike protein has a variable amino acid sequence that makes it more compatible with its host [27].

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Figure 4.Figure Multiple 4. Multiple human human host tissues host tissues that that express express ACE2 ACE2 receptor receptor highlighting lungs, lungs, heart, heart, and and vasculature. vasculature. Note clinical Note clinical consequencesconsequences of viral of infection viral infection in cardiovascular in cardiovascular and and respir respiratoryatory tissue. tissue. ARDS, ARDS, acute acute respiratory respiratory distressdistress syndrome syndrome (Re- printed from(Reprinted “Expression from “Expression of ACE2 ofRece ACE2ptor Receptor in Human in Human Host Tissues”, Host Tissues”, by BioR by BioRender.comender, accessed, accessed on 10 August on 10 August 2020) [28]. 2020) [28]. 3. Symptoms3. Symptoms SymptomsSymptoms of ofCOVID-19 COVID-19 disease disease varyvary from from patient patient to patient.to patient. Sometimes Sometimes it may it be may be asymptomatic.asymptomatic. Typically, Typically, in in the the early stagesstages of of COVID-19 COVID-19 infection, infection, the most the commonmost common infection symptoms can be fever, dry cough, and tiredness [29]. Less common symptoms infectionare nausea symptoms or vomiting, can be muscle fever, or dry joint coug pain,h, sore and throat, tiredness loss of [29]. sense Less of smell common or taste symptoms or are nauseaboth, nasal or vomiting, congestion, muscle conjunctivitis, or joint headache, pain, sore different throat, types loss of of skin sense rashes, of smell diarrhea, or taste or both,shivering, nasal congestion, and dizziness conjunctivitis, [30]. In the disease’s headache progression, different stages, types the of patientskin rashes, will face diarrhea, shivering,severe shortnessand dizziness of breath, [30]. decreased In the disease’s blood oxygen progression (hypoxia), stages, destruction the patient of the lungs,will face severe andshortness several organsof breath, dysfunction decreased [31]. blood More severe oxygen and (hypoxia), rare neurological destruction complications of the such lungs, and severalas stroke, organs encephalitis, dysfunction delirium, [31]. More and nerve severe damage and arerare other neurological complications complications of COVID-19 such as disease [32]. Figure5 demonstrates the common symptoms of COVID-19. stroke, encephalitis,Acute respiratory delirium, distress syndromeand nerve (ARDS) damage is the are leading other cause complications of COVID-19 inducedof COVID-19 diseasemortality [32]. Figure [33]. One 5 ofdemonstrates the most inﬂuential the common factors in symptoms developing ARDSof COVID-19. with COVID-19 is a cytokineAcute respiratory storm [34]. “Cytokine distress storm”syndrome is an aggressive(ARDS) is inﬂammatory the leading response cause of associated COVID-19 in- ducedwith mortality the secretion [33]. of largeOne amounts of the ofmost cytokines influential caused byfactors the host’s in immunedeveloping response ARDS to with COVID-19the SARS-CoV-2 is a cytokine virus andstorm is directly [34]. “Cytokine associated with storm” lung damage,is an aggressive multiple organ inflammatory failure, re- and severe COVID-19 prognosis [35]. sponse associated with the secretion of large amounts of cytokines caused by the host’s immune response to the SARS-CoV-2 virus and is directly associated with lung damage, multiple organ failure, and severe COVID-19 prognosis [35].

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Figure 5. TheFigure most minor5. The andmost major minor symptoms and major of symptoms COVID-19 of (Created COVID-19 with (Created BioRender.com with BioRender.com., accessed on 15ac- March 2021). cessed on 15 March 2021). After the virus enters the body through the mouth or nose, SARS-CoV-2 makes its way After theinto virus the enters lungs the and body uses through its distinctive the mouth spike or proteins nose, SARS-CoV-2 to infect alveolar makes cells. its In response, way into the thelungs immune and uses system its distinctive attacks the spike infected proteins area, killingto infect healthy alveolar alveolar cells. cellsIn re- in the process. sponse, the immuneReduced system surfactant attacks from the alveolar infected epithelial area, killing type IIhealthy cells, along alveolar with cells fluid in accumulation the due process. Reducedto the surfactant destruction from of cellsalveolar in the epithelial alveoli, causestype II reduced cells, along or severely with fluid hindered accu- gas exchange. mulation due However,to the destruction when cytokines of cells in arethe triggeredalveoli, causes without reduced breaks, or severely they can hindered cause damage to the gas exchange.cells’ However, responses when to thecytokines cytokines are andtriggered shut down without the organs’breaks, function.they can cause This is known as a damage to thecytokine cells’ responses storm, which to the mediatescytokines severe and shut disease, down including the organs’ COVID19 function. [36 This]. The following is known as asteps cytokine describe storm, how which a cytokine mediates storm severe occurs disease, in the lungsincluding (Figure COVID196): [36]. The following(1) stepsInfection describe of how lung a cellscytokine by COVID-19. storm occurs in the lungs (Figure 6): (1) Infection(2) of lungCytokine cells by production COVID-19. through virus detection by immune cells (macrophages). (2) Cytokine(3) productionCreating through a cycle virus of inflammation detection by inimmune lung cells cells by (macrophages). further uptake of immune cells (3) Creating a cycle(white of inflammation blood cells) through in lung the cells cytokine by further phenomenon. uptake of immune cells (white blood(4) cells)Fibrin through formation the andcytokine further phenomenon. damage. (4) Fibrin formation(5) Filling and of further the lung damage. cavities due to the infiltration of fluids into the weak blood vessels, (5) Filling of the lungfollowed cavities by due respiratory to the infilt damage.ration of fluids into the weak blood vessels, followed by respiratory damage.

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Patients with mild symptoms improved afterafter one week, while severe cases reportedreported experiencing progressive respiratory failure due to alveolar damage caused by the virus, experiencing progressive respiratory failure due to alveolar damage caused by the virus, which may lead to death [37]. Mortality rates have been assigned averagely between which may lead to death [37]. Mortality rates have been assigned averagely between mid- middle-aged and elderly patients with the disease. According to WHO, recovery time for dle-aged and elderly patients with the disease. According to WHO, recovery time for mild mild infections is about two weeks, and for severe infections three to six weeks [19]. infections is about two weeks, and for severe infections three to six weeks [19].

Figure 6. AA cytokine cytokine storm storm in in the the lungs lungs due due to toCOVID-19 COVID-19 disease: disease: (1) infection, (1) infection, (2) Cytokine (2) Cytokine pro- production,duction, (3) Creating (3) Creating a cycle a cycle of inflammation of inﬂammation in lu inng lung cells,(4) cells, Fibrin (4) Fibrin formation formation and and (5) Filling (5) Filling of the of lung cavities (Reprinted from “Cytokine Storm”, by BioRender, accessed on 3 July 2020) [38]. the lung cavities (Reprinted from “Cytokine Storm”, by BioRender.com, accessed on 3 July 2020) [38].

4. Transmission Routes Due to the lack of specific specific treatment and preventing the disease’s outbreak, knowing the transmission transmission methods methods can can reduce reduce the the diseas disease’se’s prevalence prevalence [39]. [39 Epidemiologically,]. Epidemiologically, the theSARS-CoV-2 SARS-CoV-2 outbreak outbreak could could be berelated related to toth thee wholesale wholesale market market of of Hua Hua Nan Nan seafood and wet animals in Wuhan. Communities, health carecare and the family are thethe primaryprimary settingsetting of human-to-human transmission of SARS-CoV-2SARS-CoV-2 [[40].40]. SARS-CoV-2 can bebe detecteddetected inin various organs such as the eye, nasopharynx, saliva, alveolar lavage fluid, fluid, blood, intestine, and feces after infection [[41].41]. Due to the high rate of transmission of SARS-CoV-2 andand thethe uncertainty of the main routes of transmission transmission,, infection control and prevalence are facing significantsignificant challenges. According According to research, close contact and respiratoryrespiratory dropletsdroplets areare one of thethe mainmain waysways ofof transmission.transmission. Therefore, the relevantrelevant expertsexperts stronglystrongly advisedadvised maintaining social social distance distance and and used used a mask. a mask. Touching Touching the theface’s face’s T-zone T-zone after after contact contact with withcontaminated contaminated surfaces surfaces is also is a also mode a mode of transmission of transmission that emphasizes that emphasizes the need the for need hand for handhygiene hygiene and handwashing. and handwashing. Other Other routes routes of transmission of transmission are through are through contaminated contaminated sur- surfacesfaces as well as well as airborne, as airborne, fecal-oral fecal-oral transmission transmission [42]. In [42 general,]. In general, droplet droplet and contact and contact route, route,airborne airborne transmission, transmission, and fecal and and fecal oral and have oral been have identified been identified to transmit to transmit the SARS-CoV- the SARS- CoV-22 virus virus[43] (Figure [43] (Figure 7). 7).

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FigureFigure 7. 7. SchematicSchematic representation representation of SARS-CoV-2 of SARS-CoV-2 Transmission Transmission Routes Routes (Created (Created with BioRender.com. with BioRender.com accessed, accessed on 5 Feb- on ruary5 February 2021). 2021).

DropletDroplet and and contact contact routs: AsAs mentioned, mentioned, the the main main routes routes of of transmission transmission of of the the COVID-19 virus are respiratory droplets and close contact [44]. In general, the transmission COVID-19 virus are respiratory droplets and close contact [44]. In general, the transmis- of the COVID-19 virus through droplets and contact can be done in two ways: (1) direct sion of the COVID-19 virus through droplets and contact can be done in two ways: (1) contact with infected people and (2) indirect contact with surfaces used by an infected direct contact with infected people and (2) indirect contact with surfaces used by an in- person. If the infected person does not observe the social distance (1 m) can make a healthy fected person. If the infected person does not observe the social distance (1 m) can make person sick through coughing or sneezing, the virus entering the mucous membranes of the a healthy person sick through coughing or sneezing, the virus entering the mucous mem- mouth or nose, or Conjunctiva (eyes). Fomite can also be transmitted through household branes of the mouth or nose, or Conjunctiva (eyes). Fomite can also be transmitted items such as clothing, utensils, and furniture around the infected person [45,46]. According through household items such as clothing, utensils, and furniture around the infected per- to the Centers for Disease Control and Prevention (CDC) instructions in this section, it is son [45,46]. According to the Centers for Disease Control and Prevention (CDC) instruc- necessary to observe the following points [47]: tions in this section, it is necessary to observe the following points [47]: (1) Make sure the patient is in a convenient place. (2) The necessity of proper use of personal protective equipment (PPE). (3) Restrict the transportation and movement of patients as much as possible. (4) Utilization of disposable or patient care equipment as much as possible.

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(1) Make sure the patient is in a convenient place. (2) The necessity of proper use of personal protective equipment (PPE). (3) Restrict the transportation and movement of patients as much as possible. (4) Utilization of disposable or patient care equipment as much as possible. (5) Prioritization disinfection and scouring of rooms. Airborne transmission: Transmission of the virus through airborne particles is one of the main transmission routes of COVID-19 disease [48]. According to published research, if the suspended particles remain in the air for a long time, they will spread the virus [49]. In addition, World Health Organization stated that the virus could also be spread through aerosols in poorly ventilated indoors [50]. In closed environments (ICU rooms), airborne droplets or suspended particles in the air may infect the lungs when large doses of aerosols are inhaled [51]. The CDC’s essential recommendations in this section are as follows [52]: (1) The necessity of keeping the patient in the air infection isolated room (AIIR). (2) limitation on the movement of health care personnel to patients’ rooms. (3) The need for proper use of personal protective equipment (PPE). (4) Conﬁne movement and transport of patients. (5) Immunization of people suspected of COVID-19 from unguarded contact. Fecal and oral: As mentioned, gastrointestinal symptoms are a common symptom of COVID-19 disease [53]. Some patients with SARS-CoV-2 have the RNA virus in their stools [54]. Recent research has shown that SARS-CoV-2 in fecal samples from patients with COVID-19 can be another way of transmitting the virus. This transmission can occur through contact with food and contaminated water with fecal secretions [55]. Zhang et al. reported that the molecular diagnostic value of COVID-19 in a stool sample was equivalent to that of an oropharyngeal swab [56].

5. Prevention Preventive measures due to lack of deﬁnitive treatment are of particular importance to prevent the spread of COVID-19 [57]. Several preventative measures have been taken at the public health level that can prevent or delay the transmission of COVID-19. These include quarantine of the infected person, identiﬁcation and monitoring of contacts, disinfection of the environment, and utilization of personal protective equipment [58]. Table1 reports the CDC recommendations for avoiding the outbreak of COVID-19 [59]:

Table 1. The CDC recommendations for preventing the spread of COVID-19.

Everyone Should Description • Washing hands for at least 20 s with soap and water • Prior to preparing or consuming the food • In advance of touching your face • Posterior to utilization the toilet • When you leave a public place Wash your hands • After coughing, blowing your nose, and sneezing • Posterior to touching your mask • After changing a diaper • When caring for a sick person • Posterior to handling pets and animals • Use hand sanitizer (at least 60% alcohol) if the soap is not available • Indoors: prevent close contact with sick people and maintain a distance of 6 feet between the patient and other members of the home. Stay away from close contact • Outdoors: Maintain a 6-foot distance between yourself and people. • Warning: Asymptomatic disease vectors spread the virus. Biologics 2021, 1 11

Table 1. Cont.

Everyone Should Description • Prevent the high prevalence and infection of the virus by masks. • Due to the transmission of COVID-19 disease to others even if there are no symptoms. • The necessity of wearing masks in public areas and around people in society due to The necessity of using masks to the difficulty of observing social distancing. cover the mouth and nose in the • Do NOT use masks for employees of health centers and hospitals due to the face of others importance of surgical masks and N95 respirators to prevent disease in staff. • Everyone should maintain social distancing in society because masks are not a suitable alternative to social distancing. • Everyone should cover their mouth and nose with a handkerchief or hand when coughing or sneezing, and refrain from spitting in public places. Cover coughs and sneezes • Never leave used napkins in public places and be sure to put them in the trash bin • Wash your hands regularly for at least 20 s and in the absence of soap and water, be sure to disinfect your hands with disinfectant solutions containing at least 60% alcohol. • Do NOT touch contaminated surfaces AND regularly clean, and disinfect surfaces that are frequently touched, such as telephones, keyboards, light switches, handles, faucets, Clean and disinfect sinks, toilets, tables, door buttons, and countertops. • Clean dirty surfaces with detergent or soap and water before disinfecting. • Use a household disinfectant as much as possible. • Check your symptoms regularly, such as fever, cough, shortness of breath, or other symptoms of COVID-19, especially when travelling to high-risk areas. • If your symptoms develop, check your body temperature regularly. Be careful not to Monitor Your Health Daily check your body temperature within 30 min of exercising or after taking fever medications such as acetaminophen. • If your symptoms develop, try to follow THE CDC guideline. • The importance of influenza vaccination in winter and autumn months in 2020–2021 Protect Your Health This for the following reasons: Flu Season • Reduction of the risk of influenza, hospitalization, and mortality. • Saving health resources to care for patients with COVID-19.

Studies have also fulfilled on the prevention of nosocomial infections and the psychological health issues associated with COVID-19. Nosocomial infections can be controlled by increasing awareness or prevention via personal protective equipment such as face and eye protection masks by medical staff at hospitals, disinfection of the tools, and plac- ing classified protocols based on the infectious environment. Regarding mental health, some suggested psychological intervention in suspected and confirmed cases and medical staff [60] Figure8 demonstrates different safety information about the COVID-19 infectious disease caused by the SARS-CoV-2 virus. Biologics 2021, 1 12 Biologics 2021, 1, FOR PEER REVIEW 11

FigureFigure 8. 8.Infographic Infographic of variousof various safety safety information information about about the COVID-19 the COVID-19 infectious infectious disease disease caused caused by the by SARS-CoV-2 the SARS-CoV-2 virus (Adoptedvirus (Adopted from “COVID-19 from “COVID-19 Safety Information”, Safety Information”, by BioRender.com by BioRender,, accessed accessed on 18on March 18 March 2020) 2020) [61]. [61].

6.6. Epidemiology Epidemiology Epidemiologists’Epidemiologists’ duties duties are are essential essential when when discovering discovering a a new new infectious infectious disease disease in in collaborationcollaboration with with other other scientistsscientists to determine determine the the reasons reasons for for the the spread spread of ofdisease. disease. Ep- Epidemiologistsidemiologists were were initially initially present present at the virusvirus observationobservation sitesite forfor the the ﬁrst first time time (Wuhan, (Wuhan, China)China) [62 [62].]. Then, Then, theythey investigatedinvestigated thethe origin of the outbreak, control, control, and and follow-up follow-up of ofthe the disease disease (such (such as asnew new cases, cases, hospitalizat hospitalizations,ions, deaths, deaths, demograp demographichic information information (such (suchas symptoms, as symptoms, age, age,and andgender, gender, race/ethnicity race/ethnicity,, and andtreatment treatment methods). methods). In addition, In addition, they theyconducted conducted clinical clinical studies studies (including (including information information from from antibody antibody testing, testing, risk risk factors factors for forsevere severe illness, illness, effective effective medical medical treatments) treatments) [62]. [62 ].Finally, Finally, it it takes takes necessary necessary measures measures to toslow slow the the disease’s disease’s spread spread (Supporting (Supporting and and assi assistingsting people people in in high-risk high-risk groups groups such such as ashealth health care care workers workers or or the the elderly to staystay safesafe inin environmentsenvironmentssuch such as as grocery grocery stores, stores, home,home, or or school) school) [62 [62].]. An An overview overview of of the the various various epidemiological epidemiological statistics statistics associated associated withwith seasonal seasonal inﬂuenza, influenza, SARS, SARS, MERS, MERS, and and COVID-19 COVID-19 outbreaks outbreaks are are reported reported in in Table Table2. 2. AccordingAccording to to clinical clinical research, research, and and all all ages ages are ar sensitivee sensitive to to COVID-19. COVID-19. It It should should be be noted noted

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that both symptomatic and asymptomatic patients could transmit the disease [63]. Studies thatthatthat bothboth both symptomaticsymptomatic symptomatic andand and asymptomaticasymptomatic asymptomatic patients patients patients could could could transmit transmit transmit the the the disease disease disease [ 63[63]. [63].]. StudiesStudies Studies havethat bothshown symptomatic that the viral and loadasymptomatic is not sign patientsificantly could different transmit between the disease symptomatic [63]. Studies and havehavehave shownshown shown thatthat that thethe the viralviral viral loadload load isis is notnot not significantlysign significantlyificantly differentdifferent different betweenbetween between symptomaticsymptomatic symptomatic andand and asymptomatichave shown that individuals the viral [64]. load is not significantly different between symptomatic and asymptomaticasymptomaticasymptomatic individualsindividuals individuals [[64].64 [64].]. asymptomaticDrops infected individuals with the [64]. SARS-CoV-2 virus can be spread up to one to two meters DropsDropsDrops infectedinfected infected with with with the the the SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 virus virus virus can can can be spreadbe be spread spread up toup up one to to one toone two to to meterstwo two meters meters and and canDrops be placed infected on with surfaces the SARS-CoV-2in favorable weathervirus can conditions be spread for up many to one days. to two Neverthe- meters canandand be can can placed be be placed placed on surfaces on on surfaces surfaces in favorable in in favorable favorable weather weather weather conditions conditions conditions for many for for many many days. days. days. Nevertheless, Neverthe- Neverthe- less,and incan the be face placed of common on surfaces disinfectants in favorable such weather as sodium conditions hypochlorite, for many hydrogen days. Neverthe-peroxide, inless, the in face the offace common of common disinfectants disinfectants such such as sodium as sodium hypochlorite, hypochlorite, hydrogen hydrogen peroxide, peroxide, etc., less, in the face of common disinfectants such as sodium hypochlorite, hydrogen peroxide, itetc.,etc., is quickly itit isis quicklyquickly eliminated eliminatedeliminated [65]. According[65].[65]. AccordingAccording to a study toto aa studystudy on other onon other COVID-19other COVID-19COVID-19 traits, traits,traits, the disease’s thethe dis-dis- etc., it is quickly eliminated [65]. According to a study on other COVID-19 traits, the dis- sensitivityease’sease’s sensitivitysensitivity on people onon peoplepeople depends dependsdepends upon age,uponupon physical age,age, physicalphysical health, health,health, and biological andand biologicalbiological characteristics. character-character- ease’s sensitivity on people depends upon age, physical health, and biological character- Statistically,istics.istics. Statistically,Statistically, most adult mostmost patients adultadult patientspatients are between araree betweenbetween 35 and 3535 55 andand years 5555 yearsyears old and oldold are andand less areare common lessless com-com- istics. Statistically, most adult patients are between 35 and 55 years old and are less com- inmonmon infants inin infantsinfants and children. andand children.children. Among AmongAmong them, them,them, people peoplepeople with withwith weaker weakerweaker immune immuneimmune function, function,function, the elders thethe el-el- mon in infants and children. Among them, people with weaker immune function, the el- withdersders anwithwith average anan averageaverage age of ageage 60 of yearsof 6060 yearsyears and older, andand older,older, people peoplepeople with kidneywithwith kidneykidney and liver andand liver dysfunctionliver dysfunctiondysfunction [60] ders with an average age of 60 years and older, people with kidney and liver dysfunction and[60][60] hypertension, andand hypertension,hypertension, diabetes, diabetes,diabetes, asthma, asthma,asthma, chronic chronicchronic pulmonary pulmonarypulmonary obstruction, obstruction,obstruction, heart patients, heartheart patients,patients, smok- [60] and hypertension, diabetes, asthma, chronic pulmonary obstruction, heart patients, erssmokerssmokers [63], pregnant [63],[63], pregnantpregnant women, women,women, and people andand peoplepeople with disabilities withwith disabilitiesdisabilities are at higherareare atat higherhigher risk and riskrisk more andand likely moremore smokers [63], pregnant women, and people with disabilities are at higher risk and more tolikelylikely be exposed toto bebe exposedexposed to the virus toto thethe [66 virusvirus]. Currently, [66].[66]. Currently,Currently, there is there athere high isis potential aa highhigh potentialpotential for epidemics forfor epidemicsepidemics among likely to be exposed to the virus [66]. Currently, there is a high potential for epidemics humans;amongamong humans;humans; in other in words,in otherother people words,words, of peoplepeople any age ofof become ananyy ageage infected becomebecome with infectedinfected the coronavirus withwith thethe coronaviruscoronavirus infection. among humans; in other words, people of any age become infected with the coronavirus Ininfection.infection. older people, InIn olderolder the people,people, average thethe mortality averageaverage rate mortalitmortalit was higheryy raterate waswas with higherhigher the diseases withwith thethe than diseasesdiseases in the than youngthan inin infection. In older people, the average mortality rate was higher with the diseases than in peoplethethe youngyoung [11]. peoplepeople There have[11].[11]. There beenThere no havehave reports beenbeen of nono pregnant reportsreports ofof women pregnantpregnant receiving womenwomen prenatal receivingreceiving transplants prenatalprenatal the young people [11]. There have been no reports of pregnant women receiving prenatal ortransplantstransplants intrauterine oror [ intrauterine63intrauterine]. According [63].[63]. to According theAccording WHO, the toto the consequencesthe WHO,WHO, thethe of consequencesconsequences not breastfeeding ofof notnot and breast-breast- sep- transplants or intrauterine [63]. According to the WHO, the consequences of not breast- arationfeedingfeeding between andand separationseparation mother and betweenbetween child aremothermother more andand significant childchild areare than moremore the significant risksignificant of COVID-19 thanthan thethe infection riskrisk ofof feeding and separation between mother and child are more significant than the risk of inCOVID-19COVID-19 infants because infectioninfection the inin infectioninfantsinfants becausebecause in infants thethe infectioninfection is usually inin mild infantsinfants or isis asymptomatic. usuallyusually mildmild oror WHO asympto-asympto- rec- COVID-19 infection in infants because the infection in infants is usually mild or asympto- ommendsmatic.matic. WHOWHO that recommendsrecommends mothers with thatthat suspected mothersmothers or withwith confirmed suspectedsuspected COVID-19 oror confirmedconfirmed should COVID-19COVID-19 be encouraged shouldshould matic. WHO recommends that mothers with suspected or confirmed COVID-19 should tobebe initiate encouragedencouraged or continue toto initiateinitiate to breast oror continuecontinue feed. Motherstoto breastbreast should feed.feed. MothersMothers be counselled shouldshould that bebe counselledcounselled breastfeeding thatthat be encouraged to initiate or continue to breast feed. Mothers should be counselled that benefitsbreastfeedingbreastfeeding substantially benefitsbenefits outweigh substantiallysubstantially the outweighoutweigh potential the risksthe potentialpotential for transmission risksrisks forfor transmissiontransmission [67–69]. Important [67–69].[67–69]. breastfeeding benefits substantially outweigh the potential risks for transmission [67–69]. epidemiologicalImportantImportant epidemiologicalepidemiological indicators associated indicatorsindicators with associatedassociated COVID-19 withwith areCOVID-19COVID-19 reported areare in reported Tablereported3. inin TableTable 3.3. Important epidemiological indicators associated with COVID-19 are reported in Table 3. TableTableTable 2. 2.2.Epidemiological EpidemiologicalEpidemiological comparison comparisoncomparison of ofof respiratory respiratoryrespiratory viral viralviral infection infectioninfection [ 70[70–73].[70–73].–73]. Table 2. Epidemiological comparison of respiratory viral infection [70–73]. RO CFR RROO CFRCFR Disease-Causing RO CFR IncubationIncubation HospitalizationHospitalization CommunityCommunity AnnualAnnual Infected Disease Disease-Causing BasicBasic Reproductive ReproductiveRO CaseCase Fatality CFRFatality Incubation Hospitalization Community Annual Disease Disease-CausingPathogen Basic Reproductive Case Fatality IncubationTime HospitalizationRate AttackCommunity Rate GlobalAnnual Disease Disease-CausingPathogen BasicNumber Reproductive CaseRate Fatality IncubationTime HospitalizationRate AttackCommunity Rate InfectedAnnual Global Disease Pathogen BasicNumber Reproductive CaseRate Fatality Time Rate Attack Rate Infected Global Pathogen Number Rate Time Rate Attack Rate Infected Global Number Rate

SARS 33 9.6–11%9.6–11% 2–72–7 days days MostMost cases cases 10–60%10–60% 80988098 (in (in 2003) 2003) SARS 3 9.6–11% 2–7 days Most cases 10–60% 8098 (in 2003) SARS 3 9.6–11% 2–7 days Most cases 10–60% 8098 (in 2003)

SARS-CoV SARS-CoV SARS-CoV

MERS 0.3–0.8 34.4% 6 days Most cases 4–13% 420 MERS 0.3–0.80.3–0.8 34.4%34.4% 6 days6 days MostMost cases cases 4–13%4–13% 420420 MERS 0.3–0.8 34.4% 6 days Most cases 4–13% 420

MERS-CoV MERS-CoV MERS-CoV

Flu 1.3 0.05–0.1% 1–4 days 2% 10–20% ~1 billion Flu 1.31.3 0.05–0.1%0.05–0.1% 1–41–4 days days 2%2% 10–20%10–20% ~1~1 billion billion Flu 1.3 0.05–0.1% 1–4 days 2% 10–20% ~1 billion

Influenza virus Influenza virus InﬂuenzaInfluenza virus virus

COVID- COVID- 2.0–2.5 ~3.4% 4–14 days ~19% 30–40% N/A ongoing COVID-19 2.0–2.5 ~3.4% 4–14 days ~19% 30–40% N/A ongoing COVID-1919 2.0–2.52.0–2.5 ~3.4%~3.4% 4–144–14 days days ~19%~19% 30–40%30–40% N/AN/A ongoing ongoing 19

SARS-CoV-2 SARS-CoV-2 SARS-CoV-2SARS-CoV-2

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Table 3.3. LeadingLeading epidemiologicalepidemiological indicatorsindicators ofof COVID-19COVID-19 researchresearch articlesarticles inin JanuaryJanuary 2020.2020.

IndicatorsIndicators Description Description • Elderly (over 60 years old) [60]. • Elderly (over 60 years old) [60]. • Mean• ageMean of 59 age years of 59 (research years (research conducted conducted between between patients patients 50 to 89 50 years) to 89 years)[74]. [74]. • Risk• ofRisk a severe of a severeillness illnesswith increasing with increasing age of about age of 40 about years 40 [75]. years [75]. • The• lowThe number low number of patients of patients with mean with age mean 0–49 age years 0–49 [76]. years [76]. • Average• Average age of patients age of patients was 55 was.5 years; 55.5 age years; distribution: age distribution: ≤39: 10%;≤39: 40–49: 10%; 22%, 40–49: 50–59: 22%, 30%; 50–59: 60–69: 30%; 22%, ≥70: 15%60–69: [77]. 22%, ≥70: 15% [77].  AgeAge of patients of patients • In• termsIn of terms age distribution of age distribution,, 1% under 1% 10, under 1% 10,10–19, 1% 10–19,8% 20–29, 8% 20–29,87% 30–79, 87% 30–79,and 3% and >80 3% [78]. >80 [78]. • 50–60• (More50–60 than (More half than a million half a million COVID-19 COVID-19 patients patients from different from different countries countries participated participated in this inmeta- this analysis)meta-analysis) [79]. [79]. • Cases range from 2 to 72 years [80]. • Cases range from 2 to 72 years [80]. • Increased incidence with increasing age over 60 years [81]. • Increased incidence with increasing age over 60 years [81]. • The median age of was 74 years [82]. • The• medianCases age range of was aged 74 between years [82]. 35 and 55 years [83]. • Cases range aged between 35 and 55 years [83]. Sex of patients • More cases were males [84,85].  Sex of patients • More cases were males [84,85]. • • Increased mortality between 60 and 80 years [86–88] Mortality rate Increased mortality between 60 and 80 years [86–88]  Mortality rate • The mortality rate was significantly high in the age range of >60 years [75,89]. • The mortality rate was significantly high in the age range of >60 years [75,89]. • • AverageAverage of 7 days of 7(2–14 days days) (2–14 [60,90]. days) [ 60,90]. • Average of 10 days [91]. • Average of 10 days [91].  IncubationIncubation time • 6.4 days (95% CI 5.6–7.7 days), with a range of 2.1–11.1 days [92]. • 6.4• days5.0 (95% days CI (95% 5.6–7.7 CI 4.4–5.6days), with days), a withrange a of range 2.1–11.1 of 2–14 days days [92]. [92 ]. time • 5.0• days4.0 (95% days, CI with 4.4–5.6 a range days), of with 1–7 daysa range [93 ].of 2–14 days [92]. • 4.0 days, with a range of 1–7 days [93]. 7. Diagnosis 7. Diagnosis Because of the lack of definitive curative treatment for this disease, the most effective solutionBecause after preventingof the lack of and definitive controlling curative is the tr timelyeatment diagnosis for this of disease, the disease the most and isolatingeffective thesolution illnesses. after Therepreventing are several and controlling ways to diagnose is the timely the diseasediagnosis early, of the such disease as the and RT-PCR isolat- method,ing the illnesses. CT-Scan, There Serological are several antibody ways blood to diagnose test, and the Artificial disease early, intelligence. such as the RT-PCR method, CT-Scan, Serological antibody blood test, and Artificial intelligence. 7.1. RT-PCR Method 7.1. RT-PCROne of theMethod most important ways to detect the SARS-CoV-2 virus in upper and lower respiratoryOne of specimensthe most important is the Real-Time ways to Reverse detect the Transcriptase SARS-CoV-2 (RT)–PCR virus in Diagnosticupper and Panel.lower Therespiratory basis of specimens the PCR is copyingis the Real-Time the RNA Reverse and DNA Transcriptase structure of (RT)–PCR the sample, Diagnostic which can Panel. diag- noseThe basis infectious of the origin PCR andis copying various the genetic RNA and and blood DNA diseases structure [94 of]. the Figure sample,9 demonstrates which can COVID-19diagnose infectious diagnostic origin testing and through various real-time genetic RT-PCR.and blood As diseases depicted [94]. in Figure Figure9, there9 demon- are fivestrates necessary COVID-19 steps diagnostic to perform testing the test: through sample real-time collection, RT-PCR. RNA extraction,As depicted RT-qPCR in Figure set 9, up,there and are test five results, necessary all of steps which to can perform be customized the test: sample to explain collection, both this RNA and otherextraction, RT-qPCR RT- diagnosticqPCR set up, protocols. and test Theresults, steps all for of performingwhich can be the customized RT-qPCR testto explain are as followsboth this [ 95and]: other RT-qPCRNasopharyngeal diagnostic protocols. swab <15The steps min: forCotton performing swab the is RT-qPCR inserted test into are theas follows nostril [95]: to absorbNasopharyngeal sections. swab <15 min: Cotton swab is inserted into the nostril to absorb sections.Collected specimen 0–72 h specimen is stored at 2–8 ◦C for up to 72 h or proceed to RNACollected extraction. specimen 0–72 h specimen is stored at 2–8 ℃ for up to 72 h or proceed to RNARNA extraction. extraction ~45 min purified RNA is extracted from the deactivated virus. RT-qPCR,RNA extraction ~1 h per~45 primer,min purified set purified RNA is RNA extracted is reverse from the transcribed deactivated to cDNAvirus. and amplifiedRT-qPCR, by qPCR. ~1 h per primer, set purified RNA is reverse transcribed to cDNA and amplifiedTest results by qPCR. real-time positive SARS-CoV-2 patients cross the threshold line within 40.00Test cycles results (<40.00 real-time Ct). positive SARS-CoV-2 patients cross the threshold line within 40.00This cycles method (<40.00 aims Ct). to detect the nucleic acid present in the nasal swab sampling or the respiratoryThis method tract aims using to detect the PCRthe nucleic process acid in present real-time. in the It isnasal confirmed swab sampling based on or thethe reproductionrespiratory tract function using and the sequencePCR process of the in virusreal-time. in the It sample is confirmed [96]. based on the repro- ductionBecause function the infectiousand sequence virus of infects the virus the in host’s the sample respiratory [96]. system, the necessary samples areBecause taken the from infectious the lower virus and infects upper the respiratory host’s respiratory tract. The system, swab test the is necessary sampling withsam- aples special are taken swab from from the the lower throat and and upper nose ofrespir a person.atory tract. The samplingThe swab of test the is nasalsampling aspirates with anda special lungs swab is implementing from the throat by and injection nose of a person. saline solution The sampling into the of nose, the nasal and aspirates then the

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and lungs is implementing by injection of a saline solution into the nose, and then the samplesample isis collecting collecting by by suction. suction. Finally, Finally, if it if is it necessary is necessary to continue to continue the sampling, the sampling, the lower the respiratory tract, i.e., Bronchoalveolar lavage or chip aspiration, which is sputum sampling, lower respiratory tract, i.e., Bronchoalveolar lavage or chip aspiration, which is sputum is performing [19,63]. The RT-PCR is widely used in the diagnostic ﬁeld, and the error sampling, is performing [19,63]. The RT-PCR is widely used in the diagnostic field, and percentage of the method is meagre [97]. the error percentage of the method is meagre [97].

FigureFigure 9.9. TheThe protocol protocol template template COVID-19 COVID-19 diagnostic diagnostic testing testing th throughrough real-time real-time RT-PCR: RT-PCR: (1) (1) Nasopharyngeal Nasopharyngeal swab, swab, (2) (2)Collected Collected specimen, specimen, (3) (3)RNA RNA extraction, extraction, (4) purified (4) puriﬁed RNA RNA and and(5) Test (5) Testresults results real-time real-time (Adopted (Adopted from from“COVID-19 “COVID-19 Diag- Diagnosticnostic Test Testthrough through RT-PCR”, RT-PCR”, by BioR by BioRender.comender, accessed, accessed on 9 April on 2020) 9 April [98] 2020). [98].

7.2.7.2. CT-ScanCT-Scan ComputedComputed tomographytomography (CT) (CT) is is a a suitable suitable diagnostic diagnostic method method that that sheds sheds light light on severalon sev- stageseral stages of disease of disease diagnosis diagnosis and and development development [99]. [99]. One One way way to look to look at the at the morphological morpholog- patternsical patterns of lung of lung lesions lesions associated associated with with COVID-19 COVID-19 is through is through chest chest scans scans such such as X-rays as X- and computed tomography (CT) scans. It should be noted that the accuracy of diagnosis depends heavily on specialists [100]. In the early stages of the epidemic in any country,

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rays and computed tomography (CT) scans. It should be noted that the accuracy of diagnosis depends heavily on specialists [100]. In the early stages of the epidemic in any coun- thetry, use the of use CT of imaging CT imaging methods methods was morewas more critical critical than RT-PCRthan RT-PCR due to due the to lack the of lack RT-PCR of RT- technologyPCR technology or the lackor the of kitslack and of kits diagnostic and diag equipmentnostic equipment suitable for suitable accurate for sampling accurate [ 101sam-]. Inpling addition, [101]. CTIn addition, images are CT a images valuable are tool a valuab to helple physicianstool to help identify physicians internal identify structures internal andstructures examine and their examine shape, their size, shape, density, size, and density, texture and [102 ].texture [102]. Moreover,Moreover, CTCT imagingimaging cancan help to reveal reveal the the abnormalities abnormalities caused caused by by COVID-19. COVID-19. In Inabout about 85% 85% of of patients patients with with superimposed superimposed ir irregularregular lines and interfaces,interfaces, ChestChest CTCT inin COVID-19COVID-19 pneumonia pneumonia cases cases shows shows bilateral, bilateral,peripheral, peripheral, andand basalbasalpredominant predominant ground-ground- glassglass opacities opacities (GGOs) (GGOs) and/or and/or consolidationconsolidation [ 103[103].]. InIn addition,addition, in in patience patience without without severe severe respiratoryrespiratory distress distress who who recovered recovered from from coronavirus coronavirus disease disease 2019, 2019, chest chest CT scans CT revealedscans re- thatvealed the that greatest the greatest severity severity in lung in abnormalities lung abnormalities occurred occurred after aboutafter about ten days ten days from from the initialthe initial symptoms symptoms [104 ].[104]. It is essentialIt is essential to pay to pay close close attention attention to asymptomatic to asymptomatic patience patience as theyas they can can transmit transmit the the infection infection quite quite easily easily if theyif they are are undetected. undetected. CT CT imaging imaging of of these these patientspatients hashas uniqueunique features th thatat can can be be detected detected even even in inasymptomatic asymptomatic patience patience with with neg- negativeative nucleic nucleic testing testing [105]. [105 Figure]. Figure 10 shows10 shows the thepositive positive and and negative negative COVID-19 COVID-19 CT scan CT scanresults results of the of patient. the patient.

FigureFigure 10. 10.Examples Examples of of CT CT images images that that are are positive positive for for COVID-19 COVID-19 (top (top) and) and non-COVID-19 non-COVID-19 (bottom (bot- ) tom) from the COVID-CT dataset, reprinted from [106], Copyright 2020, Elsevier. from the COVID-CT dataset, reprinted from [106], Copyright 2020, Elsevier.

7.3.7.3. The The Serological Serological Antibody Antibody Blood Blood Test Test SerologySerology testingtesting isis a diagnostic method method for for detecting detecting antibody-mediated antibody-mediated immune immune re- responsessponses against against infectious infectious agents agents [107]. [107 The]. The European European Center Center for forDisease Disease Control Control and and Pre- Preventionvention (ECDC) (ECDC) has has approved approved the the COVID-19 COVID-19 serological test forfor epidemiologicalepidemiological andand monitoringmonitoring purposespurposes onlyonly becausebecause itit doesdoes notnot detectdetect thethe earlyearly stagesstages ofof infectioninfection [[108].108]. RapidRapid serological serological testing testing can can be be considered considered an an alternative alternative to to molecular molecular testing testing to to identify identify COVID-19COVID-19 patientspatients when access access to to PCR PCR testing testing is islimited limited or ornon-existent. non-existent. The Theuse of use sero- of serologicallogical tests tests with with low low prevalence prevalence is not is notappropriate appropriate because because this thismethod method is likely is likely to have to havefalse-positive false-positive results results compared compared to the to actual the actual positive positive [109]. [This109]. protocol This protocol template template (Figure (Figure11) demonstrates 11) demonstrates COVID-19 COVID-19 serologic serologic diagnostic diagnostic testing testing through through antibody antibody detection. detection. It Itdepicts depicts sample sample loading, loading, SARS-CoV-2 SARS-CoV-2 antibody-antigen antibody-antigen detection, detection, and and qualitative qualitative test test re- results.sults. This This can can be be customized customized as as a whole to explainexplain otherother serologicserologicdiagnostic diagnostic protocols protocols forfor differentdifferent viral,viral, bacterial, bacterial, or or parasitic parasitic pathogens pathogens [110]. [110 The]. The steps steps for performing for performing a serol- a serology test shown in Figure 11 are as follows [111]: ogy test shown in Figure 11 are as follows [111]: (1) Sample loading: add a drop of blood or serum in the sample well (S). (1) Sample loading: add a drop of blood or serum in the sample well (S). (2) Buffer loading: add dilution phosphate saline buffer to sample well. (2) Buffer loading: add dilution phosphate saline buffer to sample well. (3) Sample incubation: capillary action moves sample across lateral ﬂow test. (3) Sample incubation: capillary action moves sample across lateral flow test. (4) Antibody-antigen recognition: antibodies with speciﬁcity for COVID-19 bind to (4) Antibody-antigen recognition: antibodies with specificity for COVID-19 bind to gold COVID-19-antigen conjugates in the conjugate pad. gold COVID-19-antigen conjugates in the conjugate pad. (5) COVID-19 antibody detection: sample enters testing well (T), and COVID-19 antibody– antigen complex binds to immobilized anti-human lgG/IgM antibodies.

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(5) COVID-19 antibody detection: sample enters testing well (T), and COVID-19 antibody–antigen complex binds to immobilized anti-human lgG/IgM antibodies. (6) ControlControl antibody detection: rabbit antibody-goldantibody-gold conjugateconjugate binds toto immobilizedimmobilized anti-rabbitanti-rabbit lgG lgG antibodies. antibodies. (7) InterpretingInterpreting results: results: Positive Positive:: one one strip strip each each in in C well and TT well,well, NegativeNegative == oneone stripstrip in in C C well. well.

FigureFigure 11.11. SchematicSchematic ofof serological serological testing testing steps: steps: (1) (1) Sample Sample loading, loadin (2)g, (2) Buffer Buffer loading, loading, (3) Sample(3) Sample incubation, incubation, (4) Antibody- (4) Anti- antigenbody-antigen recognition, recognition, (5) COVID-19 (5) COVID-19 antibody antibody detection, detection, (6) Control (6) Control antibody antibody detection detection and (7) Interpretingand (7) Interpreting results: Positive results: (ReprintedPositive (Reprinted from “COVID-19 from “COVID-19 Serologic Serologic Diagnostic Diagnostic Test through Test through Antibody Antibody Detection”, Detection”, by BioRender.com by BioRender,, accessedaccessed onon 22 JanuaryJanuary 2020)2020) [[112].112].

7.4. Artificial Artificial Intelligence (AI) In the COVID-19COVID-19 pandemic,pandemic, the developmentdevelopment of newnew technologiestechnologies toto monitormonitor andand control the outbreak of COVID-19 (coronavirus)(coronavirus) is a significant significant step taken by medical researchers. ArtificialArtificial intelligenceintelligence (AI) (AI) in in medicine medicine has has recently recently led led to to significant significant advances advances in diagnosis,in diagnosis, biotechnology, biotechnology, and and drug drug production production [113 [113].]. AI technologyAI technology has shownhas shown promising prom- prospectsising prospects for various for various epidemics epidemics (SARS, (SARS, EBOLA, EBOLA, and and HIV) HIV) [114 [114].]. The The COVID-19 COVID-19 crisis cri- andsis and the the rapid rapid increase increase in thein the number number of newof new and and suspected suspected cases cases have have led led to theto the rise rise in AIin AI technology technology used used to to control control and and diagnose diagnose the the disease disease [ 99[99,115].,115]. ArtificialArtificial intelligenceintelligence can quickly detect the symptoms of the diseasedisease and alert patients and health authorities. In this way, it can reduce the decision time in traditional traditional disease disease diagnosis diagnosis processes processes [116]. [116]. In these studies,studies, artificialartificial intelligenceintelligence mainlymainly usesuses medicalmedical imagingimaging technologiestechnologies suchsuch asas computed tomography (CT), X-ray imagingimaging (X-ray),(X-ray), and magneticmagnetic resonanceresonance imagingimaging (MRI) toto diagnosediagnose infected infected cases cases [117 [117].]. Moreover, Moreover, AI AI can can take take practical practical steps steps in tracking in tracking the coronavirus’sthe coronavirus’s spread, spread, identifying identifying high-risk high-ris patients,k patients, adequately adequately analyzing analyzing patients’ patients’ previ- ousprevious data, anddata, predicting and predicting the risk the of risk death of [ 118death]. Table [118].4 demonstratesTable 4 demonstrates the main the applications main ap- of AI in Pandemic COVID-19. plications of AI in Pandemic COVID-19.

Biologics 2021, 1, FOR PEER REVIEW 13

Table 3. Leading epidemiological indicators of COVID-19 research articles in January 2020.

BiologicsIndicators2021, 1 Description 18 • Elderly (over 60 years old) [60]. • Mean age of 59 years (research conducted between patients 50 to 89 years) [74]. • Risk of a severe illness with increasing age of about 40 years [75]. Table 4. Main applications of AI in COVID-19 pandemic. • The low number of patients with mean age 0–49 years [76]. • Properties Average age of patients Description was 55.5 years; age distribution: ≤39: 10%; 40–49: 22%, 50–59: 30%; 60–69: 22%, ≥70: 15% [77]. • Rapid analysis of patients’ irregular symptoms  AgePrimary of patients diagnosis • In and terms detection of age distribution, 1% under 10, 1% 10–19, 8% 20–29, 87% 30–79, and 3% >80 [78]. • Facilitating to identify suspected infection cases through medical imaging such as of the infection• [119,120] 50–60 (More than half acomputed million COVID-19 tomography patients (CT), from magnetic different resonance countries imaging participated (MRI). in this meta- analysis) [79]. • Assist in automated outbreak monitoring and prediction • Monitoring the treatmentCases range from 2• to 72Establish years [80]. a neural network to extract visual features and assist in appropriate • [121,122] Increased incidence withmonitoring increasing and age treatment over 60 years of patients [81]. • The median age of• was Follow-up74 years [82]. patient updates and follow-up solutions for the COVID-19 pandemic. • Contact tracing of Cases the range aged between• Analyze 35 and infected 55 years surfaces [83]. and hotspots  Sexindividuals of patients [117•, 123More] cases were males• Provide[84,85]. to predict disease progression • Increased mortality between 60 and 80 years [86–88]  Mortality rate • Predict the nature of the virus through existing data, social media, and media platforms Projection of cases• The and mortality mortality rate •was significantlyTracking the high risks in of the infection age range and theof >60 possibility years [75,89]. of spread [124,125] • Average of 7 days •(2–14Predict days) [60,90]. the number of positive cases and deaths in different areas • Average of 10 days• [91].Identify vulnerable areas and help the people of that area  Incubation • 6.4 days (95% CI 5.6–7.7• Analysis days), with of existing a range data of 2.1–11.1 on COVID-19 days [92]. for pharmaceutical research time Development of• drugs5.0 days and (95% CI 4.4–5.6• Provide days), with real-time a range acceleration of 2–14 days of drug [92]. testing vaccines [126–129• ]4.0 days, with a range• ofHelp 1–7 identifydays [93]. drugs useful for treating COVID-19 patients • Design diagnostic tests and assist in the production of vaccines • Reducing the workload of healthcare workers Reducing the workload of7. Diagnosis • Training students and physicians in early diagnosis and providing early-stage healthcare workers [130–133] Because oftreatment the lack usingof definitive digital methods curative treatment for this disease, the most effective solution after• preventingUpdate information and controlling is the timely diagnosis of the disease and isolat- Prevention of the diseaseing [117 the] illnesses.• Predict There possible are several sites of ways infection to diagnose the disease early, such as the RT-PCR method, CT-Scan,• Help Serological prevent viruses antibody and diseases blood intest, the and future, Artificial with the intelligence. help of previous data

7.1. RT-PCR Method 8. The Role of Nanotechnology in Diagnostics and Treatment of COVID-19 One of the most important ways to detect the SARS-CoV-2 virus in upper and lower Nanotechnology is currently used to diagnose, treat, control, and prevent medical respiratory specimens is the Real-Time Reverse Transcriptase (RT)–PCR Diagnostic Panel. science diseases [134]. Figure 12 represents the applications of nanotechnology in the face The basis of the PCR is copying the RNA and DNA structure of the sample, which can of COVID-19 disease. Properties of nanoparticles such as high solubility, small size, surface

adaptability,diagnose infectious and versatility origin and are used various to produce genetic safeand and blood high-quality diseases [94]. drugs, Figure targeted 9 demon- tissue therapies,strates COVID-19 personalized diagnostic nanomedicines, testing through and early real-time diagnosis RT-PCR. and debarment As depicted diseases in Figure [135 9,]. Nanotechnologythere are five necessary can play steps a unique to perform role in the identifying, test: sample treating, collection, and preventing RNA extraction, COVID-19. RT- EssentialqPCR set propertiesup, and test of results, nanoparticles all of which that cancan bebe mentionedcustomized in to the explain ﬁght againstboth this COVID-19 and other areRT-qPCR [135]: diagnostic protocols. The steps for performing the RT-qPCR test are as follows [95]: Nasopharyngeal swab <15 min: Cotton swab is inserted into the nostril to absorb (1) Design of safe personal protective equipment (PPE) to prevent infection and increase sections. healthcare workers’ safety. Collected specimen 0–72 h specimen is stored at 2–8 ℃ for up to 72 h or proceed to (2) Production of antiviral disinfectants and surface coatings that inactivate the virus and RNA extraction. prevent its spread. RNA extraction ~45 min purified RNA is extracted from the deactivated virus. (3) Design of precise and sensitive nano-based sensors for rapid detection of infection or RT-qPCR, ~1 h per primer, set purified RNA is reverse transcribed to cDNA and immunological response. amplified by qPCR. (4) Production of new drugs to increase activity, reduce toxicity, and continuous release. (5) TestTargeting results drug real-time delivery. positive SARS-CoV-2 patients cross the threshold line within (6)40.00Vaccination cycles (<40.00 production Ct). (enhancement of humoral and cellular immune responses). This method aims to detect the nucleic acid present in the nasal swab sampling or the Table5 reported the advantages and disadvantages of nanotechnology in the face respiratory tract using the PCR process in real-time. It is confirmed based on the repro- of COVID-19. duction function and sequence of the virus in the sample [96]. Because the infectious virus infects the host’s respiratory system, the necessary samples are taken from the lower and upper respiratory tract. The swab test is sampling with a special swab from the throat and nose of a person. The sampling of the nasal aspirates

Biologics 2021, 1, FOR PEER REVIEW 13

Biologics 2021, 1 19 Table 3. Leading epidemiological indicators of COVID-19 research articles in January 2020.

Indicators Description • ElderlyTable 5.(overThe 60 advantages years old) and[60]. disadvantages of nanotechnology in COVID-19. • Mean age of 59 years (research conducted between patients 50 to 89 years) [74]. Properties• Risk of a severe Advantages illness with increasing age of about 40 years [75]. Disadvantages • The low number• Progression of patients of with effective mean and age promising0–49 years [76]. • Average age ofdisinfection patients was process 55.5 years; (Antimicrobial age distribution: activity), ≤39: 10%; 40–49:• Expandability 22%, 50–59: 30%; and 60–69: 22%, ≥70: 15% [77]. Development of new materials, expansion of manufacture costs  AgeNanomaterials of patients for• In terms of ageself-cleaning distribution, properties1% under 10, of surfaces 1% 10–19, (Release 8% 20–29, of 87% •30–79,Issues and 3% related >80 [78]. to intellectual and surface decontamination• 50–60 (More thanchemical half a disinfectantsmillion COVID-19 to increase patients their from duration different of countriesregulatory participated characteristics in this meta- analysis) [79]. action) [136]. • Toxicity and environmental • • Cases range fromAntiseptic, 2 to 72 years Antimycotic [80]. Agent, and antiviral impacts of these systems [138] activity of metal nanoparticles [137] • Increased incidence with increasing age over 60 years [81]. • The median• ageParticipate of was 74 inyears the production[82]. of appropriate masks and gloves [139] • Skin irritation • Cases range aged between 35 and 55 years [83]. Development of • Use equipment more efﬁciently, more resilient, • Allergies  Sex of patients • More cases were males [84,85]. nanomaterials for and safely against biological and chemical • Toxic effects in humans • Increased mortality between 60 and 80 years [86–88]  Mortalitypersonal rate protective hazards [140] • Environmental pollution equipment (PPE)• The mortality• rateLack was of effectsignificantly on texture high and in breathabilitythe age range of of >60 years [75,89].(release of nanoparticles during • Average of 7 daysmaterials (2–14 days) due to [60,90]. antimicrobial activity and washing from clothes) [135] • Average of 10 dayshydrophobicity [91]. [141]  Incubation • 6.4 days (95%• CIImproving 5.6–7.7 days), the quality with a ofrange SARS-CoV-2 of 2.1–11.1 diagnosis days [92]. time • 5.0 days (95% CI(Reduce 4.4–5.6 the days), duration with ofa range diagnosis of 2–14 and days increase [92]. the nanotechnology• for scope of diagnosis) [142] 4.0 days, with a range of 1–7 days [93]. • high operating expenses and virus detection and • A useful tool for the production and development capital expenditures disease diagnosis of various diagnostic kits in COVID-19 [143] 7. Diagnosis • develop of the sensors for the detection of BecauseSARS-CoV-2 of the [ 135lack,144 of] definitive curative treatment for this disease, the most effective solution• Transfer after preventing of the drug toand the controlling target organ is the timely diagnosis of the disease and isolat- ing• theEnhance illnesses. the There antiviral are effectsseveral of ways drugs to diagnose the disease early, such as the RT-PCR Carriers and drug method,• Very CT-Scan, effective Serological antiviral formulation antibody blood test, and Artificial intelligence. delivery systems • Reduction of side effects and toxicity of the drug • 7.1. RT-PCRControl Method the cytokine storm [145] • Increase the half-life of the drug • OneImprove of the action most andimportant reaction ways with viralto detect particles the SARS-CoV-2 virus in upper and lower respiratory• Interference specimens with is their the entry Real-Time into cells Reverse Transcriptase (RT)–PCR Diagnostic Panel. Nanoparticles design The• basisEnhance of the bioactivity PCR is copying and consistency the RNA and DNA structure of the sample, which can for virus inhibition diagnoseof compoundsinfectious origin and various genetic and blood diseases [94]. Figure 9 demonstrates• ReleaseCOVID-19 of antiviral diagnostic agents testing in a controlled through manner real-time RT-PCR. As depicted in Figure 9, • Inhibition of the virus in the reproductive stage there are five necessary steps to perform the test: sample collection, RNA extraction, RT- • qPCR setProtects up, and antigens test results, versus precociousall of which demolition can be customized to explain both this and other • Continuous release RT-qPCR• Increase diagnostic antigen protocols. consistency The steps for performing the RT-qPCR test are as follows [95]: • NasopharyngealTargeted immunogenic swab delivery<15 min: Cotton swab is inserted into the nostril to absorb sections.• Increased uptake by antigen-presenting cells (APCs) [146] ℃ Development of vaccines Collected specimen 0–72 h specimen is stored at 2–8 for up to 72 h or proceed to RNA• extraction.Use of various materials to produce Nanocarriers RNAsuch extraction as polysaccharides, ~45 min polymers, purified and RNA lipids is [extracted147] from the deactivated virus. • Protect DNA or RNA versus enzymatic RT-qPCR,demolition ~1 and h enhancementper primer, cellset absorption purified [RNA148] is reverse transcribed to cDNA and amplified• Release by qPCR. of genetic material in target cells • TestIncreasing results the real-time compatibility positive of vaccines SARS-CoV-2 by patients cross the threshold line within 40.00 cycleschanging (<40.00 the properties Ct). of nanoparticles This method aims to detect the nucleic acid present in the nasal swab sampling or the respiratory tract using the PCR process in real-time. It is confirmed based on the reproduction function and sequence of the virus in the sample [96]. Because the infectious virus infects the host’s respiratory system, the necessary samples are taken from the lower and upper respiratory tract. The swab test is sampling with a special swab from the throat and nose of a person. The sampling of the nasal aspirates

Biologics 2021, 1 20 Biologics 2021, 1, FOR PEER REVIEW 19

Figure 12.FigureSchematic 12. Schematic representation representation of the of nanotechnologies the nanotechnologies tools tool tos preventto prevent and and control control COVID-19COVID-19 (Created (Created with with Bio- BioRender. Render.com. accessed on 13 February 2021). com, accessed on 13 February 2021). It should be noted that nanoparticles (NP) have been widely used in many medical applicationsIt should be such noted as biosynthetic that nanoparticles sensors, drug (NP) delivery, have imaging, been widely and antimicrobial used in many ther- medical applicationsapy [149]. suchTable as6 reports biosynthetic the types sensors, of NPs and drug their delivery, applications imaging, in the detection, and antimicrobial control, therapy [and149 vaccination]. Table6 reports against thecoronaviruses. types of NPs and their applications in the detection, control, and vaccination against coronaviruses. Table 6. The types of NPs and their applications in the detection, control, and vaccination against coronaviruses.

TableProperties 6. The types Type of NPs of NPs and their Description applications in the detection, control, and vaccination against coronaviruses. • Development of a colorimetric assay based on gold nanoparticles (AuNPs) [150]. • Metal NPs Properties Type of NPs Description• Development of the nanoplasmonic pillar arrays (NPA) comprise gold Nano islands [151]. • • DevelopmentFabrication of nanosensor of a colorimetric based on assaynon-mediated based onSWCNTs gold nanoparticleswith ACE2. These (AuNPs) ACE2- [150]. • Metal NPs • DevelopmentSWCNT nanosensors of the nanoplasmoniccan be turned into pillar an op arraystical tool (NPA) for prompt comprise diagnosis gold Nano of SARS- islands [151]. • Carbon nano- CoV-2 [152]. • tubes • FabricationUse in electrochemical of nanosensor sensors basedbased on on fu non-mediatednctional cobalt TiO2 SWCNTs nanotubes with (Co-TNTs) ACE2. Thesefor prompt ACE2-SWCNT diagnosis of SARS-CoV-2 nanosensors via can sensing be turned the spike into (receptor-binding an optical tool domain for prompt • Carbon diagnosis(RBD)) present of SARS-CoV-2 at the virus surface [152]. [153]. Nanoparticle nanotubes • • UseAntiviral in electrochemical effects against SARS sensors-CoV-2 based on face on masks functional [154]. cobalt TiO2 nanotubes (Co-TNTs) • Silica NPs for diagnostic • forUse prompt of silica-coated diagnosis superparamagnetic of SARS-CoV-2 nanoparticles via sensing to detect the spike the virus (receptor-binding [155]. domain • (RBD))Use of QD present nanoparticle at the probes virus surfaceto identify [153 and]. confirm SARS-CoV-2 Spike and ACE2 • Quantum • Antiviralreceptor binding effects inhibito againstrs in SARS-CoV-2 human cells [156]. on face masks [154]. • • Silicadots NPs (QDs) • UseDesignation of silica-coated of human IgG superparamagnetic and to investigate severe nanoparticles acute respiratory to detect syndrome the virus corona- [155]. virus 2 (SARS-CoV-2)-specific IgM and IgG [157]. • Use of QD nanoparticle probes to identify and conﬁrm SARS-CoV-2 Spike and ACE2 • Fabrication of the surface-functionalized magnetic nanoparticles (MNP’s) and viral • Nanoparticle Quantum receptorRNA-extraction binding protocol inhibitors for pote inntial human detection cells of [ 156COVID-19]. [158]. • Magnetic NPs for diagnostic dots (QDs) • • DesignationDevelopment of pcMNPs-based human IgG and viral to RNA investigate elicitation severe method acute for the respiratory sensitive diagno- syndrome coronavirussis of COVID-19 2 (SARS-CoV-2)-speciﬁc causing virus, the SARS-CoV-2 IgM and[159]. IgG [157]. • Fabrication of the surface-functionalized magnetic nanoparticles (MNP’s) and viral RNA-extraction protocol for potential detection of COVID-19 [158]. • Magnetic NPs • Development of pcMNPs-based viral RNA elicitation method for the sensitive

diagnosis of COVID-19 causing virus, the SARS-CoV-2 [159]. • The role of graphene-based materials and strategies in ﬂuid biopsy and diagnosis of viral diseases in the diagnosis of COVID-19 [160]. • Use of graphene effect transistors (GFET) to detect SARS-CoV-2 in human • Graphene nasopharyngeal swab samples. • Use of laser engraved graphene for rapid and remote evaluation of COVID-19 biomarkers [161]. Biologics 2021, 1 21

9. Treatment The treatments available for people with COVID-19 are based on their symptoms, and there is no exact treatment available for complete recovery in patients with COVID-19. Researchers and physicians are making efforts to provide proper treatment for COVID- 19 patients [162]. Researchers are testing a wide range of possible therapies, including antiviral medicines, immunosuppressants, monoclonal antibodies, and vaccines [163]. In the early stages of the disease, the patient’s immune system is challenged to prevent replication of the SARS-CoV-2 virus; however, in the acute stages, maybe experience tissue damage due to severe immune/inflammatory reactions [164]. According to clinical research, antiviral therapies are most effective in the early stages of the disease. In contrast, immunosuppressive/anti-inflammatory treatments are likely to be most effective in the severe stages of COVID-19 [165]. It should be noted that anti-SARS-CoV-2 antibody-based therapies are more effective in the early stages of infection before the patient enters the acute phase. Therefore, physicians have recommended receiving monoclonal antibodies against SARS-CoV-2 [166]. The drugs approved by the US Food and Drug Administration (FDA) are Dexamethasone and Remdesivir. It is recommended for hospitalized patients who need supplemental oxygen [167]. Remdesivir is an intravenous nucleotide drug from the adenosine analogue [168]. The mechanism of action of Remdesivir against the SARS-CoV-2 virus is presented in Figure 13. As depicted in Figure 13, Remdesivir binds to RNA- dependent RNA polymerase and prevents virus replication by premature termination of RNA transcription [168]. In the acute phase of the disease, when patients need a Biologics 2021, 1, FOR PEER REVIEWventilator, dexamethasone, a corticosteroid, significantly affects patients’21 recovery [169].

All recommended treatments for COVID-19 are reported in Table7.

FigureFigure 13. TheThe potential potential mechanism mechanism of action of of Remdesivir action of against Remdesivir coronavirus against replication coronavirus (Re- replication printed from “Remdesivir: Potential Repurposed Drug Candidate for COVID-19 (Portrait)”, by Bi- (ReprintedoRender, accessed from on “Remdesivir: 1 April 2020) [170]. Potential Repurposed Drug Candidate for COVID-19 (Portrait)”, by BioRender.com, accessed on 1 April 2020) [170]. Table 7. All recommended treatments for COVID-19.

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Table 7. All recommended treatments for COVID-19.

Properties Type Description • Due to its ability to inhibit SARS-CoV-2, one of the promising drugs for COVID-19 [171,172]. • Adults who are hospitalized and pediatric patients (aged ≥12 years and weighing ≥40 kg). • Pediatric patients who are hospitalized (aged <12 years and weighing 3.5 kg to <40 kg) [173]. • Remdesivir • Side effects (gastrointestinal symptoms (e.g., nausea), increased transaminase levels, increased prothrombin time and hypersensitivity reactions, control of renal function, drug interaction with chloroquine or hydroxychloroquine) [168,174]. • Low side effects in pregnant women [175]. • Favipiravir is a promising drug for COVID-19 that decreases hospital stay and the need for mechanical ventilation [176]. • Favipiravir may emerge as a valuable drug in the treatment of mild to moderate symptomatic SARSCoV-2 infected • Favipiravir cases [177]. • The recommended dosage of favipiravir for adults is 1800 mg orally twice daily on 1st day, followed by 800 mg orally twice daily, up to a maximum of 14 days. The 14-day course in India costs Rs 10,200 [177]. • Chloroquine is an antimalarial drug (manufactured in 1934). Hydroxychloroquine, an analogue of chloroquine (manufactured in 1934). • Less toxicity of hydroxychloroquine compared to chloroquine [178]. • Prevents acute coronavirus 2 (SARS-CoV-2) syndrome by increasing endosomal pH [179]. • Chloroquine is a suitable inhibitor of Angiotensin-converting enzyme 2 (ACE2) [180]. Drug treatment • Inhibits the transfer of SARS-CoV-2 from primary endosomes to end lysosomes and prevents the spread of viral genomes [181]. • The COVID-19 Treatment Guidelines Panel recommends against the use of chloroquine or hydroxychloroquine with • Chloroquine or or without azithromycin for the treatment of COVID-19 in Hydroxychloroquine hospitalized patients (AI) [182]. • Non-recommendation for use in nonhospitalized patients [178]. • high-dose chloroquine (600 mg twice daily for ten days). • Side effects: QTc prolongation, Torsade de Pointes, ventricular arrhythmia, and cardiac deaths [183], ﬂuoroquinolone antibiotics [184], Prolongation of QTc interval with concomitant use of hydroxychloroquine and azithromycin [179,185], Increased risk of cardiac arrest if concomitant use of hydroxychloroquine with azithromycin [186]. • Drug-Drug Interactions: Caution in concomitant use with drugs metabolized by CYP2D6 (e.g., certain antipsychotics, beta-blockers, selective serotonin reuptake inhibitors, methadone) [187]. • Prevent severe acute respiratory syndrome-associated coronavirus (SARS-CoV-2) IN VITRO condition [173,188,189]. • No recommendation for use by the COVID-19 Treatment Guidelines for the treatment of COVID-19, except in a • Lopinavir/ritonavir clinical trial [190]. • Side effects (Nausea, vomiting, diarrhea (common), QTc prolongation, Hepatotoxicity) [190]. • Drug and drug interactions (Increased drug concentration with concomitant use of drugs metabolized by cytochrome P450 3A enzyme and increased toxicity) [190]. Biologics 2021, 1 23

Table 7. Cont.

Properties Type Description • Ivermectin, an FDA-approved anti-parasitic (broad-spectrum anti-viral activity in vitro, an inhibitor of the causative virus (SARS-CoV-2)) [174,191,192]. • Ivermectin • No recommendation for use by the COVID-19 Treatment Guidelines for the treatment of COVID-19, except in a clinical trial [192]. • Use of blood-derived products of people recovered from SARS-CoV-2 infection(convalescent plasma, immunoglobulin products) [179] (The Food and Drug • Blood-derived Administration (FDA) has issued an Emergency Use products Authorization (EUA) for the use of convalescent plasma for hospitalized patients with COVID-19). Immune-Based • Neutralizing monoclonal antibodies (clinical trials) [193,194]. Therapy [165] • Use of drugs to treat immune and/or inflammatory syndromes such as corticosteroids (e.g., glucocorticoids) [195]. • Immunomodulatory • Targeted anti-inflammatory treatments such as interleukin therapies inhibitors [184], interferons [196], kinase inhibitors [197]. • Tocilizumab is a suggested anti-inflammatory drug to treatment COVID-19 [198]. • Baricitinib plus remdesivir was superior to remdesivir alone in reducing recovery time and accelerating clinical status improvement among patients with COVID-19, • Baricitinib notably among those receiving high-flow oxygen or noninvasive ventilation. The combination was associated with fewer serious adverse events [199]. • Bamlanivimab and etesevimab are neutralizing monoclonal antibodies that bind to different but overlapping epitopes in the receptor-binding domain of the spike protein of Anti-SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 Antibody Products (SARS-CoV-2). The bamlanivimab plus etesevimab combination blocks SARS-CoV-2 entry into host cells and is being evaluated for the treatment of COVID-19 [200]. • Bamlanivimab • On 9 February 2021, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) to make bamlanivimab 700 mg plus etesevimab 1400 mg available for the treatment of outpatients with mild to moderate COVID-19 who are at high risk for progressing to severe disease and/or hospitalization (see the EUA criteria for use of the products below). The issuance of an EUA does not constitute FDA approval of a product [200]. • In hospitalized patients with severe COVID-19 who required oxygen support, using dexamethasone 6 mg daily for up to 10 days reduced mortality at 28 days, with the • Dexamethasone [201] greatest benefit seen in those who were mechanically ventilated at baseline. • There was no observed survival benefit of dexamethasone in patients who did not require oxygen support at baseline. • If dexamethasone is not available, alternative Corticosteroids glucocorticoids such as prednisone, methylprednisolone, or hydrocortisone can be used [202]. • Methylprednisolone: [A total of 102 severe and critically ill • Methylprednisolone COVID-19 patients were included in this study. The results • Prednisone showed that methylprednisolone treatment did not • Hydrocortisone improve the prognosis] [203]. • Hydrocortisone is commonly used to manage septic shock in patients with COVID-19 (The hydrocortisone dose was adjusted according to clinical response). Biologics 2021, 1 24

Table 7. Cont.

Properties Type Description • Chronic Anticoagulant and Antiplatelet Therapy. • Not used for non-hospitalized patients with COVID-19. • Antithrombotic • Undecomposed heparin, low molecular weight heparin, Therapy [204] and warfarin are not contraindicated in children and pregnant women. • Prohibition of oral anticoagulants with direct action. • The potential designation of high doses of vitamin C in • Vitamin C [187] ameliorating inﬂammation and vascular injury in patients with COVID-19. • Increased risk of pneumonia in patients with low levels of Adjunctive Therapy vitamin D [190,206]. • Vitamin D [205] • Use of vitamin D supplementation to protect against acute respiratory tract infection [207]. • There are insufﬁcient data to recommend either for or against the use of zinc to treat COVID-19 [208]. • The COVID-19 Treatment Guidelines Panel recommends against using zinc supplementation above the • Zinc [208] recommended dietary allowance to prevent COVID-19, except in a clinical trial (BIII) [208]. • Evaluation in clinical trials of zinc supplement alone or in combination with hydroxychloroquine for the prevention and treatment of COVID-19 [209].

10. Vaccines A vaccine is a biological product that produces an acquired active immunity against a specific microbial disease [192]. Vaccines are very vital to save the lives of millions of people every year. The primary function of vaccines is to train and prepare the immune system to identify and fight the target viruses and bacteria. Common components of vaccines are as follows (Figure 14): (1) Active ingredients Viral or bacterial antigens that directly stimulate the immune system but cannot cause disease. (2) Adjuvants Aluminum salts in small quantities that help to boost the immune response to the vaccine. (3) Antibiotics prevent contamination by bacteria during the vaccine manufacturing process. (4) Stabilizers Sugar/gelatin keeps the valuable vaccine until it is administered to a patient. (5) Preservatives Thimerosal prevents dangerous bacterial or fungal contamination (only used for influenza vaccines). (6) Trace components Residual inactivating ingredients such as formaldehyde, and residual cell culture materials (present in small quantities that do not pose a safety concern). Biologics 2021, 1, FOR PEER REVIEW 24

Biologics 2021, 1 25 (6) Trace components Residual inactivating ingredients such as formaldehyde, and residual cell culture materials (present in small quantities that do not pose a safety concern).

FigureFigure 14. CommonCommon components components of of Vaccines Vaccines (Adopted (Adopted from from “Common “Common Components Components of Vaccines”, of Vaccines”, by BioRender, by BioRender.com accessed, accessedon 7 April on 2020) 7 April 2020). According to the WHO, there are currently more than 50 COVID-19 vaccine candidates According to the WHO, there are currently more than 50 COVID-19 vaccine candi- in trials. To accelerate and distribute the vaccine equitably, WHO is working with scientists, dates in trials. To accelerate and distribute the vaccine equitably, WHO is working with businesses, and global health organizations through the COVAX (Working for global scientists, businesses, and global health organizations through the COVAX (Working for equitable access to COVID-19 vaccines) project (led by WHO, GAVI CEPI). BNT162b2 global equitable access to COVID-19 vaccines) project (led by WHO, GAVI CEPI). (Pfizer-BioNTech) developed by BioNTech and manufactured and distributed by Pfizer, BNT162b2 (Pfizer-BioNTech) developed by BioNTech and manufactured and distributed and Fosun Pharmaceutical is the first EUA-approved COVID-19 vaccine for emergency by Pfizer, and Fosun Pharmaceutical is the first EUA-approved COVID-19 vaccine for use. Clinical trials were performed on 44,000 people, which is reported to be more than emergency use. Clinical trials were performed on 44,000 people, which is reported to be 95% successful [210,211]. It should be noted that the Moderna vaccine was authorized for more than 95% successful [210,211]. It should be noted that the Moderna vaccine was au- emergency use on 18 December 2020, by the US Food and Drug Administration (EUA) to thorized for emergency use on 18 December 2020, by the US Food and Drug Administra- prevent COVID-19 disease. The AZD1222 vaccine developed by Oxford [212] is another tion (EUA) to prevent COVID-19 disease. The AZD1222 vaccine developed by Oxford effective vaccine that has just received an emergency license. The vaccine has been clinically tested[212] is on another 65,000 effective people andvaccine is reported that has tojust be received more than an emergency 70% effective license. against The COVID- vaccine 19has Acute been clinically Respiratory tested Syndrome on 65,000 [213 people]. Authorized/approved and is reported to be COVID-19more than 70% vaccines effective are tabulatedagainst COVID-19 in Table8 Acute. The Respiratory mechanism Syndrome and type of [213]. process Authorized/approved of the approved BNT162b2, COVID-19 Sinopharmvaccines are and tabulated Oxford/AstraZeneca in Table 8. The vaccines mechan areism presented and type in of Figure process 15 .of the approved BNT162b2, Sinopharm and Oxford/AstraZeneca vaccines are presented in Figure 15.

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Table 8. Authorized/approved COVID-19 vaccines.

Name Vaccine Type Primary Developers Country of Origin Authorization/Approval Storage Description BNT162b2 is a modified nucleoside mRNA-based vaccine developed by BioNTech and Pfizer. Fosun Pharma has licensed BNT162b2 in China. The vaccine is given as an intramuscular injection in Pfizer, BioNTech; UK, Bahrain, Canada, −70 ◦C BNT162b2 mRNA-based vaccine Fosun Pharma Multinational Mexico, US two doses 21 days apart. BNT162b2 generates an immune response against SARS-CoV-2, the virus that causes COVID-19, by encoding a mutated form of the virus’s full spike protein. A safe and efficacious vaccine with more than 70% The University of Oxford; impact against severe acute respiratory syndrome AstraZeneca/Oxford Adenovirus AstraZeneca; IQVIA; The UK 2–8 ◦C coronavirus 2 (SARS-CoV-2). AZD1222 Serum Institute of India Mechanism: Replication-deficient viral vector vaccine (adenovirus from chimpanzees) CoronaVac (formerly PiCoVacc) is a Inactivated vaccine formalin-inactivated and alum-adjuvanted vaccine Sinovac CoronaVac (formalin with Sinovac China China 2–8 ◦C developed by the China-based biotechnology alum adjuvant) company Sinovac Biotech. The vaccine is administered in two doses 14 days apart. The Gamaleya Research Institute in Russia and Gamaleya Research Health Ministry of the Russian Federation evaluates Institute, Acellena Contract Russia Russia −18 ◦C Sputnik V Non-replicating viral vector Drug Research and their non-replicating viral vector vaccine, Sputnik V Development (formerly Gam-COVID-Vac), in a Phase 3 trial in Russia and internationally. Moderna developed mRNA-1273 based on prior the U.S. Food and Drug studies of related coronaviruses such as those that Administration’s (FDA) has cause severe acute respiratory syndrome (SARS) and Moderna mRNA-1273 mRNA-based vaccine Moderna USA authorized the emergency 2–8 ◦C the Middle East respiratory syndrome (MERS). use of mRNA-1273, Moderna predicts it will be able to distribute Moderna’s vaccine 20 million doses to the United States in December 2020, and 100 m doses globally Beijing Institute of Sinopharm is developing a second inactivated Sinopharm Biological Products; China China, 2–8 ◦C BBIBP-CorV Inactivated vaccine China United Arab Emirates COVID-19 vaccine candidate, BBIBP-CorV, with the National Pharmaceutical Beijing Institute of Biological Products. Group (Sinopharm) Biologics 2021, 1 27

Table 8. Cont.

Name Vaccine Type Primary Developers Country of Origin Authorization/Approval Storage Description COVAXIN is an inactivated vaccine obtained from the SARS-CoV-2 strain isolated at the NIV, Pune, an Indian virology research institute. The vaccine is used along with immune stimulants, commonly known as vaccine adjuvants (Alhydroxiquim-II), to improve immune response Bharat Biotech, the Indian and longer-lasting immunity. The vaccine candidate is produced through the formulation of the β Council of Medical Bharat Biotech’s Bharat Biotech BBV152 whole-virion - Research (ICMR) and India ‘COVAXIN™’ by 2–8 ◦C inactivated virus with Kansas-based ViroVax’s propiolactone-inactivated National Institute of DCGI-CDSCO, MoH&FW Alhydroxiquim-II adjuvant. Virology (NIV) COVAXIN mainly contains 6 µg of whole-virion inactivated SARS-CoV-2 antigen (Strain: NIV-2020-770), and the other inactive components such as 250 µg aluminium hydroxide gel, 15 µg TLR 7/8 agonist (imidazoquinolinone), 2.5 mg TM 2-phenoxyethanol, and phosphate buffer saline up to 0.5 mL. The J&J/Janssen vaccine was 66.3% effective in clinical trials (efficacy) at preventing laboratory-confirmed COVID-19 illness in people who had no evidence of prior infection 2 weeks after Johnson & Johnson receiving the vaccine. People had the most protection Janssen Pharmaceuticals COVID-19 Vaccine Johnson and Johnson Companies of USA 2–8 ◦C 2 weeks after getting vaccinated. vaccine (JNJ-78436735) Viral vector Authorized by U.S. FDA Johnson & Johnson for Emergency Use The vaccine had high efficacy at preventing hospitalization and death in people who did get sick. No one who got COVID-19 at least 4 weeks after receiving the J&J/Janssen vaccine had to be hospitalized. Biologics 2021, 1 28

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Figure 15. The mechanism and type of process of the approved (a) BNT162b2, (b) Sinopharm and (c) Oxford/AstraZeneca Figure 15. The mechanism and type of process of the approved (a) BNT162b2, (b) Sinopharm and (c) Oxford/AstraZeneca vaccine (Reprinted from “Clinical Phase Vaccine Candidates for COVID-19”, by BioRender, accessed on 15 April 2020) vaccine[214]. (Reprinted from “Clinical Phase Vaccine Candidates for COVID-19”, by BioRender.com, accessed on 15 April 2020) [214]. 11. The Effect of COVID-19 on Pregnant Women 11. The Effect of COVID-19 on Pregnant Women Due to the complications of COVID-19, pregnant women are expected to be at risk of developingDue to severe the complications COVID-19 compared of COVID-19, to non-pregnant pregnant women women. are It should expected be noted to be atthat risk ofthe developing easy spread severe of viral COVID-19 respiratory compared diseases, to such non-pregnant as influenza, women. duringIt pregnancy should be indi- noted thatcates the that easy pregnant spread women of viral are respiratory more vulnerable diseases, to COVID-19 such as influenza, and require during more pregnancy medical indicatescare [215,216]. that pregnant Generally, women mechanical are more and vulnerable physiological to COVID-19 changes in and pregnancy require moregained medical sus- careceptibility [215,216 to]. COVID-19, Generally, significantly mechanical andwhen physiological it affects cardiorespiratory changes in pregnancy and gravida, gained and sus- ceptibilityit increases to the COVID-19, rate of progression significantly in respiratory when it affects failure. cardiorespiratory Due to physiological and gravida, changes and in it increasesthe immune the rateand ofcardiopulmonary progression in respiratorysystem in pregnant failure. Due women to physiological (e.g., improved changes dia- in thephragm, immune increased and cardiopulmonary oxygen consumption system and in respiratory pregnant women mucosal (e.g., oedema), improved they diaphragm, are very increasedsusceptible oxygen to respiratory consumption pathogens and respiratory severe pneumonia mucosal [217]. oedema), Additionally, they are verypregnant susceptible peo- tople respiratory with COVID-19 pathogens might severebe at increased pneumonia risk [ 217of adverse]. Additionally, pregnancy pregnant outcomes, people such withas COVID-19preterm births. might Figure be at 16 increased demonstrates risk ofan adverseoverview pregnancy of pregnant outcomes, women with such COVID-19 as preterm births.disease Figure in diagnosis, 16 demonstrates prevention, an and overview potential of pregnanttreatments. women with COVID-19 disease in diagnosis,A wide prevention, range of vaccines and potential is routinely treatments. and safely administered during pregnancy. As mentionedA wide in range the vaccine of vaccines section, is routinely Pfizer-BioNTech and safely vaccines administered are an effective during pregnancy. vaccine Asagainst mentioned COVID-19. in the However, vaccine section,as clinical Pfizer-BioNTech trials of the COVID-19 vaccines are vaccine an effective in pregnant vaccine againstwomen COVID-19.have not yet However, been performed, as clinical there trials is insufficient of the COVID-19 evidence vaccine to recommend in pregnant routine women haveuse of not the yet COVID-19 been performed, vaccine to there pregnant is insufficient women. evidenceHowever, to given recommend the mechanism routine useand of thefunction COVID-19 of these vaccine vaccines to pregnant (mRNA vaccine), women. However,experts believe given that the it mechanism is unlikely andto pose function a risk of theseto pregnant vaccines women. (mRNA Moreover, vaccine), expertsmRNA believevaccines that are it not is unlikely expected to poseto pose a risk a risk to pregnant to the women.breastfed Moreover, infant. It should mRNA be vaccines noted that are notmRNA expected vaccines to pose do not a risk contain to the a breastfedlive virus infant.that Itcauses should COVID-19 be noted and that therefore, mRNA vaccines cannot give do nota person contain COVID-19. a live virus In addition, that causes mRNA COVID-19 vac- andcines therefore, do not interact cannot givewith agenetic person DNA COVID-19. because In mRNA addition, does mRNA not enter vaccines the docell not nucleus. interact withHowever, genetic the DNA potential because risks mRNA of mRNA does vacci not enternes for the pregnant cell nucleus. women However, and their the fetus potential are risksunknown of mRNA because vaccines they have for pregnantnot been studied women in and pregnant their fetuswomen. are According unknown becauseto the CDC they haverecommendation, not been studied Acetaminophen in pregnant may women. be offere Accordingd as an option to the for CDC pregnant recommendation, women ex- Acetaminophenperiencing other post-vaccinati may be offeredon assymptoms an option as well for pregnant[218]. women experiencing other post-vaccination symptoms as well [218].

Biologics 2021, 1 29 Biologics 2021, 1, FOR PEER REVIEW 28

FigureFigure 16. An 16. overviewAn overview of pregnant of pregnant women women with COVID-19 with COVID-19 disease in disease terms of in diagnosis, terms of diagnosis,prevention, prevention, and potential and treatments. potential treatments. ThereThere isis nono evidence of of neonates neonates infectio infectionn in in women women who who have have given given vaginal vaginal birth. birth. ToTo understand understand thethe riskrisk of infection, further further studies studies are are required, required, and and guidelines guidelines should should be be taken for the method and timing of delivery in pregnant women with COVID-19 infection. taken for the method and timing of delivery in pregnant women with COVID-19 infection. The previous investigations on the severe acute respiratory syndrome (SARS) virus have The previous investigations on the severe acute respiratory syndrome (SARS) virus have Biologics 2021, 1, FOR PEER REVIEW represented that the virus can cause intrauterine fetal demise, premature birth, and intra- 13 represented that the virus can cause intrauterine fetal demise, premature birth, and in- uterine growth limitation; hence, testing suspected cases and continuous control of the trauterine growth limitation; hence, testing suspected cases and continuous control of the patients their infants is importantly necessary [219,220]. Additionally, there is no evidence patients their infants is importantly necessary [219,220]. Additionally, there is no evidence of mother-to-child transmission risk through cesarean and delivery section [221]. Table 9 Table 3. Leadingof mother-to-child epidemiological transmission indicators of COVI riskD-19 through research cesarean articles and in January delivery 2020. section [221]. Table9 summarizes the published articles on the relationship between pregnancy and COVID-19. Indicators Descriptionsummarizes the published articles on the relationship between pregnancy and COVID-19. • ElderlyTable 9.(over A summary 60 years of old) published [60]. research on pregnant women with COVID-19. Table 9. A summary of published research on pregnant women with COVID-19. • Mean age of 59 years (research conducted between patients 50 to 89 years) [74]. Title Objective Description • Title Risk of Determination a Objectivesevere illness of the with clinical increasing • ageA case of about study in40 obstetrics Descriptionyears [75]. and gynecology ward of Tongji Hospital • The lowcharacteristics number of of patients COVID-19 with in mean age(Huazhong 0–49 years University •[76]. Aof caseScience study and Technology, in obstetrics Wuhan, and gynecology China).  Coronavirus Disease • Averagepregnancy age of andpatients their newbornwas 55.5 years;• ageEvaluation distribution: of demographic, ≤39:ward 10%; clinical, of 40–49: Tongji laboratory 22%, Hospital 50–59: and (Huazhong radiological 30%; 60–69: 22%, 2019 (COVID-19) Dur- infant. characteristics of SARS-CoV-2University infection of Science series. and Technology, ing Pregnancy: A Case ≥70: 15% [77]. Determination of the clinical  Evaluation of transmission of • Sampling: oropharyngealWuhan, swab, placenta China). tissue, vaginal mucus, and  AgeSeriesCoronavirus of [222]patients Disease• In terms of agecharacteristics distribution, 1% of COVID-19under 10, 1% in 10–19, 8% 20–29, 87% 30–79, and 3% >80 [78]. SARS-CoV-2 virus vertically breast milk of mothers• Evaluationand oropharyngeal of demographic, swab, umbilical clinical, cord 2019 (COVID-19)• During50–60 (More thanpregnancy half a million and their COVID-19 newborn patients infant. from different countries participated in this meta- Pregnancy: A Case through theEvaluation intrauterine. of transmissionblood, of and serum of newborns.laboratory and radiological characteristics of analysis) [79]. SARS-CoV-2 infection series. Series [222] SARS-CoV-2 virus vertically• Sampling: through amniotic fluid, cord blood, and neonatal pharyngeal swab. • • Sampling: oropharyngeal swab, placenta Cases range fromthe 2 intrauterine. to 72 years [80]. • Clinical evaluation of 116 pregnant women with coronavirus tissue, vaginal mucus, and breast milk of  Coronavirus disease• Increased Clinical incidence characteristics with increasingof SARS- age pneumonia.over 60 years [81]. mothers and oropharyngeal swab, umbilical 2019 in pregnant • CoV-2 infection. • The most common symptoms: fever (50.9%), cough (28.4%); patients The median age of was 74 years [82]. cord blood, and serum of newborns. women: a report based• Cases Verticalrange aged transmission between potential 35 and of 55 yearspresented [83]. without• symptoms (23.3%). • Sampling: amniotic ﬂuid, cord blood, and on 116 cases [223] • SARS-CoV-2infection. Severe pneumonia (6.9%).  Sex of patients More cases were males [84,85]. • neonatal pharyngeal swab. Preterm delivery• (21.2%).Clinical evaluation of 116 pregnant women • Increased mortality between 60 and 80• years [86–88]  MortalityCoronavirus rate disease Clinical characteristics of Negative newborns (86%).with coronavirus pneumonia. • 2019 in pregnant The mortality rateSARS-CoV-2 was significantly infection. high in the age range• ofThe >60 mostyearscommon [75,89]. symptoms: fever (50.9%), women: a report• basedAverage of 7 daysVertical (2–14 transmission days) [60,90]. potential of cough (28.4%); patients presented without on 116 cases [223•] Average of 10 SARS-CoV-2infection.days [91]. symptoms (23.3%).  Incubation • 6.4 days (95% CI 5.6–7.7 days), with a range of 2.1–11.1• daysSevere [92]. pneumonia (6.9%). time • Preterm delivery (21.2%). • 5.0 days (95% CI 4.4–5.6 days), with a range of 2–14 days [92]. • Negative newborns (86%). • 4.0 days, with a range of 1–7 days [93].

7. Diagnosis Because of the lack of definitive curative treatment for this disease, the most effective solution after preventing and controlling is the timely diagnosis of the disease and isolat- ing the illnesses. There are several ways to diagnose the disease early, such as the RT-PCR method, CT-Scan, Serological antibody blood test, and Artificial intelligence.

7.1. RT-PCR Method One of the most important ways to detect the SARS-CoV-2 virus in upper and lower respiratory specimens is the Real-Time Reverse Transcriptase (RT)–PCR Diagnostic Panel. The basis of the PCR is copying the RNA and DNA structure of the sample, which can diagnose infectious origin and various genetic and blood diseases [94]. Figure 9 demonstrates COVID-19 diagnostic testing through real-time RT-PCR. As depicted in Figure 9, there are five necessary steps to perform the test: sample collection, RNA extraction, RT- qPCR set up, and test results, all of which can be customized to explain both this and other RT-qPCR diagnostic protocols. The steps for performing the RT-qPCR test are as follows [95]: Nasopharyngeal swab <15 min: Cotton swab is inserted into the nostril to absorb sections. Collected specimen 0–72 h specimen is stored at 2–8 ℃ for up to 72 h or proceed to RNA extraction. RNA extraction ~45 min purified RNA is extracted from the deactivated virus. RT-qPCR, ~1 h per primer, set purified RNA is reverse transcribed to cDNA and amplified by qPCR. Test results real-time positive SARS-CoV-2 patients cross the threshold line within 40.00 cycles (<40.00 Ct). This method aims to detect the nucleic acid present in the nasal swab sampling or the respiratory tract using the PCR process in real-time. It is confirmed based on the reproduction function and sequence of the virus in the sample [96]. Because the infectious virus infects the host’s respiratory system, the necessary samples are taken from the lower and upper respiratory tract. The swab test is sampling with a special swab from the throat and nose of a person. The sampling of the nasal aspirates

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Biologics 2021, 1 Table 3. Leading epidemiological indicators of COVID-19 research articles in January 2020. 30 Indicators Description • Elderly (over 60 years old) [60]. • Mean age of 59 years (research conductedTable 9. Cont.between patients 50 to 89 years) [74]. • Risk of a severe illness with increasing age of about 40 years [75]. Title• The low number Objective of patients with mean age 0–49 years Description[76]. • Average age of patients was 55.5 years; age distribution:• ≤39:comprehension 10%; 40–49: 22%, of pathophysiology50–59: 30%; 60–69: 22%, ≥70: 15% [77]. and susceptibility. • Diagnostic challenges with real-time  AgeCoronavirus of patients disease• In terms of age distribution, 1% under 10, 1% 10–19, 8% 20–29, 87% 30–79, and 3% >80 [78]. A comprehensive study of the effects reverse transcription-polymerase chain 2019 (COVID-19)• 50–60 (More than half a million COVID-19 patients from different countries participated in this meta- pandemic and of COVID-19 on pregnant women reaction (RT-PCR). • Therapeutic controversies. pregnancy [217] analysis) [79]. and their infants. • Cases range from 2 to 72 years [80]. • Intrauterine transmission. • Maternal-fetal complications. • Increased incidence with increasing age over 60 years [81]. • Antiviral therapy and vaccine development. • The median age of was 74 years [82]. • The priority of caring for pregnant women • Cases range aged between 35 and 55 years [83]. with physiological conditions of  Sex of patients • More cases were males [84,85]. immunodeficiency in a higher-level • health center. Increased mortality between 60 and 80 years [86–88] • Treatment and maintenance of pregnant  Mortality rate • The mortality rate was significantly high in the age range ofwomen >60 years in the[75,89]. prenatal, postpartum or A Review on the• EffectAverage of 7 daysEvaluation (2–14 days) of COVID-19 [60,90]. and its postpartum period, in an isolated of COVID-19 in • Average of 10 severitydays [91]. in pregnant women and its environment or negative pressure.  IncubationPregnant Women [224] • Attempts to prevent mother-to-child • 6.4 days (95% CIcomparison 5.6–7.7 days), with with SARS a range and MERS. of 2.1–11.1 days [92]. transmission of SARS-CoV-2 or iatrogenic time • 5.0 days (95% CI 4.4–5.6 days), with a range of 2–14 days [92].infection during and after delivery. • 4.0 days, with a range of 1–7 days [93]. • Separation of infants from the mother of COVID-19 after postnatal stage. • 7. Diagnosis Need to read more about breastfeeding health of mothers with COVID-19. Because of the lack of definitive curative• trTheeatment study for conducted this disease, on 16 the pregnant most womeneffective solution after preventing and controlling is the withtimely COVID-19 diagnosis and of 18 the pregnant disease women and isolat- suspected of COVID-19. ing the illnesses. There are several ways to •diagnoseVaginal the delivery disease (2), early, Cesarean such deliveryas the RT-PCR (32). Maternal and Neonatal method, CT-Scan, Serological antibody blood• test,Having and symptomsArtificial ofintelligence. fever and cough at the Outcomes of Pregnant A case-control study to compare time of admission, the presence of symptoms Women With 7.1. RT-PCRclinical Method characteristics and maternal of COVID-19 pneumonia in their CT scan. Coronavirus Disease and neonatal outcomes of pregnant • Lower counts of white blood cells (WBCs), 2019 (COVID-19) One of the most important ways to detect the SARS-CoV-2 virus in upper and lower Pneumonia: women with and without neutrophils, C-reactive protein (CRP), A Case-Control respiratoryCOVID-19 specimens pneumonia. is the Real-Time Reverse andTranscriptase alanine aminotransferase (RT)–PCR Diagnostic on admission Panel. Study [225] (Patients with COVID-19 pneumonia). The basis of the PCR is copying the RNA• and IncreaseDNA structure the levels of WBCs,the sample, neutrophils, which can diagnose infectious origin and various genetic eosinophils,and blood anddiseases CRP in[94]. postpartum Figure 9 blood demonstrates COVID-19 diagnostic testing through real-timetests of pneumonia RT-PCR. patients.As depicted in Figure 9, there are five necessary steps to perform the• test:No sample COVID-19 collection, infection RNA in infants. extraction, RT- • Evaluation of clinical records, laboratory qPCR set up, and test results, all of which can beresults customized and chest to CTexplain scan ofboth 9 pregnant this and other RT-qPCR diagnostic protocols. The steps for performingwomen withthe RT-qPCR COVID-19 test pneumonia. are as follows [95]: Clinical characteristics Nasopharyngeal swab <15 min: Cotton• swabCesarean is inserted delivery. into the nostril to absorb • Observed symptoms: fever, cough, myalgia, and intrauterine vertical sections. Evaluation of clinical features of transmission potential of sore throat and lethargy. COVID-19 in pregnancy and the COVID-19 infection in Collected specimen 0–72 h specimen •is storedFetal distressat 2–8 ℃ control for up in twoto 72 cases. h or proceed to nine pregnant women: RNA extraction.potential for vertical intrauterine • Observation of lymphopenia in five patients transmission of COVID-19 infection. a retrospective review of RNA extraction ~45 min purified RNA is extracted(<1.0 × 10 9fromcells perthe L).deactivated virus. medical records [226] RT-qPCR, ~1 h per primer, set purified• RNAIncreased is reverse aminotransferase transcribed concentration to cDNA inand three patients. amplified by qPCR. • No observation of neonatal asphyxia Test results real-time positive SARS-CoV-2in infants.patients cross the threshold line within 40.00 cycles (<40.00 Ct). • Such a model has classified lesions as This method aims to detect the nucleic acidpermissive present in at the term, nasal but swab catastrophic sampling early or the first-trimester pregnancy or near An Experimental Model respiratory tract using the PCR process in real-time.embryo It implantation. is confirmed based on the repro- for Peri-conceptual duction function and sequence of the virus• in theTo resolvesample this [96]. challenge, recurrent pregnancy COVID-19 Pregnancy BecauseReport anthe experimental infectious virus model infects for the host’sloss respiratory suggests workable system, interventions the necessary utilizing sam- Loss and Proposed COVID-19 infection loss and suggest clinical experience, but success strongly Interventions ples areinterventions taken from to the optimize lower outcomes.and upper respiratorydepends tract. upon The accurate swab test and promptis sampling with to Optimize a special swab from the throat and nose of a person.SARS-CoV-2 The sampling recognition. of However,the nasal aspirates Outcomes [227] this experimental model can warrant a high-risk determination for such cases, the patients should be received priority access to treatment and screening resources. Biologics 2021, 1, FOR PEER REVIEW 13

Table 3. Leading epidemiological indicators of COVID-19 research articles in January 2020.

Indicators Description Biologics 2021, 1 31 • Elderly (over 60 years old) [60]. • Mean age of 59 years (research conducted between patients 50 to 89 years) [74]. • Risk of a severe illness with increasing age of about 40 years [75]. • The low number of patients with Tablemean 9.ageCont. 0–49 years [76]. • Average age of patients was 55.5 years; age distribution: ≤39: 10%; 40–49: 22%, 50–59: 30%; 60–69: 22%, Title≥70: 15% Objective[77]. Description  AgeImpact of patients of COVID-19 • In terms of age distribution, 1% under 10, 1% 10–19, 8% 20–29, 87% 30–79, and 3% >80 [78]. infection on pregnancy• 50–60 (More than half a million COVID-19 patients from• differentSwab samplingcountries fromparticipated all three in patients this meta- using outcomes and the risk of A complete study of three pregnant maternal-to-neonatalanalysis) [79]. qRT-PCR. intrapartum • Cases range fromwomen 2 to 72 with years COVID-19 [80]. in Renmin • Vaginal delivery. Hospital. • transmission of • Increased incidence with increasing age over 60 years [81]. One case of premature birth. COVID-19 during• The median age of was 74 years [82]. natural birth [228] • Cases range aged between 35 and 55 years [83].  Sex of patients • More cases were males [84,85]. • Increased12. mortality Conclusions between 60 and 80 years [86–88]  Mortality rate • The mortalityThe rate prevalence was significantly of COVID-19 high in the has age recently range of been >60 identifiedyears [75,89]. as a global health emergency. • AverageAccording of 7 days (2–14 to the days) WHO, [60,90]. ~131M people have been infected, of which ~74.4M have recovered, • Averageand of 10 unfortunately, days [91]. more than ~3M people have died so far. Quarantine alone is not enough  Incubation • 6.4 daysto (95% prevent CI 5.6–7.7 the outbreak days), with of a COVID-19, range of 2.1–11.1 and thedays global [92]. impact of this viral infection on the time • 5.0 dayseconomy (95% CI 4.4–5.6 is a significant days), with concern. a range Althoughof 2–14 days researchers [92]. are trying to find an utterly effective • 4.0 days,drug with for a range the definitive of 1–7 days treatment [93]. of COVID-19, no 100% effective drug has been discovered for complete recovery. Fortunately, due to researchers and pharmaceutical companies’ efforts,7. Diagnosis many effective vaccines to prevent the prevalence of this deadly disease have been approvedBecause by of the the World lack of Health definitive Organization. curative treatment However, for itthis takes disease, a long the time most for effective these effectivesolution vaccinesafter preventing to reach and all the controlling world’s people is the andtimely all diagnosis people to beof vaccinated.the disease Untiland isolat- then, alling the the points illnesses. recommended There are several by the ways World to Organizationdiagnose the disease to prevent early, the such prevalence as the RT-PCR of this diseasemethod, must CT-Scan, be fully Serological observed. antibody blood test, and Artificial intelligence.

Author7.1. RT-PCR Contributions: Method I.S. Conceptualization, designed the figures, writing original draft, data validation, conceived the original idea, and supervised the project; N.M.-A. Data validation, conceived the originalOne of idea, the most and investigated important ways and supervised to detect the the SARS-CoV-2 findings of this virus work; in A.S.upper Took and the lower lead inrespiratory writing the specimens manuscript, is developmentthe Real-Time of Reverse methodology, Transcriptase conceived (RT)–PCR the original Diagnostic idea, and investi- Panel. gatedThe basis and supervisedof the PCR the is findingscopying of the this RNA work; an N.M.N.d DNA Contributed structure toof samplethe sample, preparation, which con-can tributeddiagnose to theinfectious interpretation origin of and the resultsvarious and genetic development and blood of methodology; diseases [94]. Z.D., Figure M.F., M.G., 9 demon- S.Z.S., A.H.strates (contributed COVID-19 to diagnostic sample preparation, testing through aided inreal-time interpreting RT-PCR. the results, As depicted and worked in Figure on the 9, manuscript);there are five A.B. necessary Formal data steps analysis, to perform writing the original test: draft,sample writing collection, review RNA and editing, extraction, conceived RT- theqPCR original set up, idea, and and test supervised results, all the of project.which can All authorsbe customized have read to andexplain agreed both to this the publishedand other versionRT-qPCR of thediagnostic manuscript. protocols. The steps for performing the RT-qPCR test are as follows [95]: Funding:NasopharyngealThis research received swab <15 no externalmin: Cotton funding. swab is inserted into the nostril to absorb sections. Institutional Review Board Statement: Not applicable. Collected specimen 0–72 h specimen is stored at 2–8 ℃ for up to 72 h or proceed to InformedRNA extraction. Consent Statement: Not applicable. Data AvailabilityRNA extraction Statement: ~45 minNot purified applicable. RNA is extracted from the deactivated virus. RT-qPCR, ~1 h per primer, set purified RNA is reverse transcribed to cDNA and Conflicts of Interest: All authors state that there is no conflict of interest. amplified by qPCR. References Test results real-time positive SARS-CoV-2 patients cross the threshold line within 40.00 cycles (<40.00 Ct). 1. Khaykelson, D.; Raviv, U. Studying viruses using solution x-ray scattering. Biophys. Rev. 2020, 12, 41–48. [CrossRef] 2. Gardner, P. Virus infections and respiratoryThis method disease aims of to childhood. detect theArch. nucleic Dis. Child.acid present1968, 43 ,in 629. the [ CrossRefnasal swab][PubMed sampling] or the 3. Payne, S. Family Coronaviridae.respiratoryViruses 2017 tract, 149 using, 149–158. the PCR process in real-time. It is confirmed based on the repro- 4. Pal, M.; Berhanu, G.; Desalegn,duction C.; Kandi, function V. Severe and sequence Acute Respiratory of the virus Syndrome in the sample Coronavirus-2 [96]. (SARS-CoV-2): An Update. Cureus 2020, 12, e7423. [CrossRef][BecausePubMed the] infectious virus infects the host’s respiratory system, the necessary sam- 5. Su, S.; Wong, G.; Shi, W.; Liu,ples J.; Lai, are A.C.; taken Zhou, from J.; Liu, the W.;lower Bi, Y.; and Gao, upper G.F. Epidemiology, respiratory tract. genetic The recombination, swab test is and sampling pathogenesis with of coronaviruses. Trends Microbiol.a special2016 swab, 24, 490–502. from the [CrossRef throat ]and nose of a person. The sampling of the nasal aspirates 6. Cui, J.; Li, F.; Shi, Z.-L. Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 2019, 17, 181–192. [CrossRef] [PubMed] 7. Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu, R. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 2020, 382, 727–733. [CrossRef] Biologics 2021, 1 32

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