Structure and Function of COVID-19 Proteins in the Replication and Transcription Mechanism with Its Preventive Measures, and Treatments; a Critical Systematic Review

IAR Journal of Medical Sciences (An Open Access, International, Indexed, Peer-Reviewed Journal) A Publication of International Academic & Research Consortium, Kenya https://iarconsortium.org/

Review Article

Structure and Function of COVID-19 Proteins in the Replication and Transcription Mechanism with Its Preventive Measures, and Treatments; A Critical Systematic Review

Oguh C.E2*, Obiwulu E.N.O3, Oniwon W.O1, Okekeaji U5, Ugwu C.V2, Umezinwa O.J4 and Osuji C.A2 1Department of Biochemistry, Kogi State University, Anyigba, Nigeria 2Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria 3Department of Integrated Science, Delta State College of Education Agbor, Delta State Nigeria 4Department of Science Laboratory Technology, University of Nigeria, Nsukka, Enugu State, Nigeria 5Department of Pharmaceutical Microbiology and Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria

*Corresponding Author Oguh C.E Email: [email protected]

Article History Received: 15.04.2020 | Accepted: 18.05.2020 | Published: 22.05.2020 Abstract: The outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) poses a serious threat to the world global public health and local economies. Such huge numbers of infected and dead people call for an urgent demand of effective, available, and affordable drugs to control and diminish the pandemic. COVID-19 (coronavirus disease 2019) is a public health emergency of international concern. As of this time, there is no known effective pharmaceutical treatment, although it is much needed for patient contracting the severe form of the disease. There is an urgent need to develop new strategies to prevent or control coronavirus infections, and understanding the biology, replication, and pathogenesis of these viruses. Therefore, this systematic review was to know the biochemical effect of the virus in human, symptoms, prevention, statistics cases and summarize the evidence regarding chloroquine and hydroxychloroquine for the treatment of COVID-19. This information may lead to a better understanding of the virus structure and function of these proteins in the replication and transcription mechanism, preventive measures, treatment, genome organization of coronaviruses and then it summarizes the structure and function of these proteins in the replication and transcription mechanism. Keywords: Coronavirus, mechanism, prevention, structure, treatment.

INTRODUCTION animals such as bats that host the largest variety of Coronavirus disease 2019 (COVID-19) is a coronaviruses appear to be immune to coronavirus- respiratory illness that can transfer from person to induced illness (Anthony et al., 2017). Researchers person. The virus that causes COVID-19 is a novel have been racing to find possible treatments to save coronavirus that was first identified during an lives and produce vaccines for future prevention. These investigation into an outbreak in Wuhan, China in viruses infect a variety of human and animal host cells, December 2019, this disease has spread to more than and also carry out their infection and replication. Also, 100 countries with over 100 000 confirmed cases and many proteins have most important role in the over 3,800 confirmed deaths worldwide as of March 9, replication mechanism, although that role is, as yet, 2020 (WHO, 2020). The name “coronavirus,” coined in poorly defined. In this case, it is necessary to know the 1968, is derived from the “corona”-like or crown-like definition of these proteins in terms of this mechanism. morphology observed for these viruses in the electron Therefore; this review explains the structure, microscope (318). The risk of infection from the virus classification, symptoms, prevention, statistics cases that causes COVID-19 is higher for people who are and summarize the evidence regarding chloroquine and close contacts of someone known to have COVID-19, hydroxychloroquine for the treatment of COVID-19. for example healthcare workers, or household members. Other people at higher risk for infection are those who STRUCTURE OF COVID-19 live in or have recently been in an area with ongoing Coronaviruses are enveloped viruses with spread of COVID-19. Risk of death is only higher in round and sometimes pleiomorphic virions of older people (above an age of 60 years) and people with approximately 80 to 120 nm in diameter (Fig. 1). pre-existing health conditions. While coronaviruses Coronaviruses contain positive-strand RNA, with the infect both humans and animals, certain types of largest RNA genome (approximately 30 kb) reported to Copyright @ 2020: This is an open-access article distributed under the terms of the Creative Commons Attribution license which permits unrestricted use, distribution, and reproduction in any medium for non commercial use (NonCommercial, or CC-BY-NC) provided the original author and source are credited. Publisher: International Academic & Research Consortium, Kenya 5

Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 date (178, 196). Corona viruses (COVID-19) are Hemagglutinin Esterase (HE) and Nucleocapsid (N) relatively large viruses containing a single-stranded protein, (Figure 1). All envelope proteins and N protein positive-sense RNA genome encapsulated within a is present in all virion but HE is only present in some membrane envelope. The viral membrane is studded beta coronaviruses (Lissenberg et al., 2005). In with glycoprotein spikes that give coronaviruses their addition to that, it is thought the virus particles are crown like appearance. Coronaviruses encode five huddled together owing to interaction between these structural proteins in their genomes. These are the Spike proteins (De Haan and Rottier, 2005; Master, 2006). (S), Membrane (M), Envelope (E) glycoproteins,

Figure 1: Coronavirus virion. (A) Electron micrograph of mouse hepatitis virus MHV particles. (B) Schematic of virion

1. S Glycoproteins: S Glycoproteins are located outside RNA and M protein triggers the formation of the virion and give the virion the typical shape. The S interacting virions in this endoplasmic reticulum-Golgi proteins form homotrimers, which allow the formation apparatus intermediate compartment (ERGIC) with this of sun-like morphologies that give the name of complex (Narayanan and Makino, 2001; Escors et al., Coronaviruses (Tan et al, 2005). S proteins bind to the 2001). virion membrane via the C-terminal transmembrane regions and they also interact with M proteins (Chinese 3. E Glycoproteins: E Glycoproteins are small proteins SMEC, 2004). Virions can be bound to specific surface that are composed of approximately 76 to 109 amino receptors in the plasma membrane of the host cell via acids. About 30 amino acids in the N-terminus of the E the N-terminus of the S proteins. This S protein proteins allow attachment to the membrane of viruses mediates host cell invasion by both SARS-CoV and (Raamsman et al., 2000). In addition, coronavirus E SARS-CoV-2 via binding to a receptor protein called proteins play a critical role in the assembly and angiotensin-converting enzyme 2 (ACE2) located on morphogenesis of virions within the cell. In one study the surface membrane of host cells (Wrapp et al., 2019; coronavirus E and M proteins were expressed together Hoffmann et al., 2020). A recent study also revealed with mammalian expression vectors to form virus-like that this invasion process requires S protein priming structures within the cell (Vennema et al., 1996). In which is facilitated by the host cell produced serine another study, there was a significant decrease in the protease TMPRSS211. In addition, the viral genome ability of the recombinant mouse hepatitis virus (MHV) also encodes several nonstructural proteins including and SARS viruses to elicit E protein expression in the RNA-dependent RNA polymerase (RdRp), coronavirus genome to support this status (DeDiego et al., 2007; main protease (3CLpro), and papain-like protease Kuuo and Masters, 2003) (PLpro) (Gorbalenya et al., 2015; Baez-santos et al., 2015). 4. N Proteins: N proteins are phosphoproteins that are capable of binding to helix and have flexible structure 2. M Glycoproteins: M Glycoproteins have three of viral genomic RNA. It plays an important role in transmembrane regions. M proteins are glycosylated in virion structure, replication and transcription of the Golgi apparatus (Niemann et al., 1984). This coronaviruses, because the N protein localizes in both modification of the M protein is crucial for the virion to the replication/ transcriptional region of the fuse into the cell and to make protein antigenic. The M coronaviruses and the ERGIC region where the virus is protein plays a key role in regenerating virions in the collected (DeDiego et al., 2007; Kuuo and Masters, cell. N protein forms a complex by binding to genomic 2003). (Figure 2).

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19

Figure 2: Cartoon illustration of the coronavirus structure and viral receptor ACE2 on the host cell surface.

CLASSIFICATION OF CORONAVIRUSES Coronavirus) and the canine CCoV. Alpha The classification of Coronaviruses has been coronaviruses also compromise human CoVs such as based on genomic organization, similarities in genomic HCoV-229E and HCoVNL63, but various bat sequence, antigenic properties of viral proteins, Coronaviruses. replication strategies, and structural characteristics of virions, pathogenic, cytopathogenic and Beta coronaviruses also infect a wide range of physicochemical properties (Lai and Cavanagh, 1997). mammalians, with species such as mice, human with The Coronaviruses (CoVs) are species of virus SARS-CoV, HCoV-OC43, HCoV-HKU1, and MERS- belonging to the Nidovirales order, which includes CoV, Murine coronavirus (MHV) and Bovine Coronaviridae, Arteriviridae, Roniviridae and Coronavirus (BCoV). Similar to SARS-CoV and Mesoniviridae families (Zirkel et al., 2011). The MERS-CoV, SARS-CoV-2 attacks the lower Arteviridae family includes swine and equine respiratory system to cause viral pneumonia, but it may pathogens, and the Roniviridae family is composed of also affect the gastrointestinal system, heart, kidney, invertebrate viruses. The Coronaviridae family is the liver, and central nervous system leading to multiple largest one of the four families, by its genomic sizes of organ failure (Su et al., 2019; Zhu et al., 2020). Current coronaviridae range from 26 to 32 kb (Gorbalenya et information indicates that SARSCoV-2 is more al., 2006). Coronaviridae virus family subdivided into transmissible/contagious than SARS-CoV (Tang et al., two subfamilies, coronavirinae and torovirinae. 2020). The beta coronavirus genome encodes several Coronavirinae is divided into four genera, Alpha structural proteins, including the glycosylated spike (S) coronavirus, Beta coronavirus, Gamma coronavirus and protein that functions as a major inducer of host Delta coronavirus (figure 3). Alpha coronaviruses type immune responses. Gamma coronaviruses are specific 1 species are classified feline FCoV, FECV (Feline of birds, with one exception of a beluga whale Enteric Coronavirus) and FIPV (Feline Infectious Coronavirus. The delta coronavirus genus was created Peritonitis Virus), the porcine TGEV (Transmissible in 2012 and regroups various (HKU11, HKU12, Gastro- Enteritis Virus), Porcine PEDV (Epidemic HKU13) Coronavirus from mammals to birds (Susanna Diarrhea Virus), PRCoV (Porcine Respiratory et al., 2012) figure 3.

Figure 3: Classification of COVID-19

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 COVID-19 MECHANISM OF ACTION After entering the cytoplasm, the virus particle releases (REPLICATION AND TRANSCRIPTION) the RNA genome. This genome is a single-stranded, Coronavirus entry starts with the S protein non-segmented RNA virus with the largest known RNA binding to a target receptor on the cell surface, where genome (gRNA), which is approximately 26-32 kb. The after fusion is mediated at the cell membrane, genome consists of seven genes. It is organized into 5’ delivering the viral nucleocapsid inside the cell for non-structural protein coding regions comprising the subsequent replication. The replication of coronaviruses replicase genes (gene 1), which are two-thirds of the occurs in host cell cytoplasm. The viruses primarily genome, and 3’ structural and nonessential accessory bind to the receptor on the host cell surface via the protein coding regions comprising the gene 2-7 (Susan, spike (S) protein. When S protein is bound to the 2005). The replicase gene 1 products are encoded two receptor, a conformational structure occurs in the very large open reading frames ORF1a and 1b, which structure and the process of entry into the virus cell are translated into two large polypeptides pp1a and begins (Bosch et al., 2003). This process with pp1b, which are synthesized directly from the 5’ two- endocytosis is dependant of pH through the receptor. thirds of the genomic RNA of CoV (figure 4).

Figure 4: MHV genome organization and replicase proteins.

After synthesis of these proteins, consisting of from sub genomic mRNA. Sub genomics RNAs encode 16 units, non-structural protein (nsp1to nsp16) is the major viral Structural proteins (S), Envelope protein converted with the contribution of viral proteases pp1a (E), Membrane protein (M), Nucleocapsid protein (N), and pp1b (Baranov et al., 2005). These 16 proteins form and the accessory proteins, which are essential for Double-Membrane Vesicles (DMV). At the same time, virus-cellreceptor binding. The newly structural this DMV is virus Replication and Transcription synthesized proteins are released into the endoplasmic Complex (RTC) (Cynthia et al., 2004; Gosert et al., reticulum. All of these proteins, along with the N 2002). These nsp proteins, especially non-structural protein, are linked to the viral genomic RNA and protein3 (nsp3), have an important role in the virion localized in the ERGIC region (Cynthia et al., 2004; structure, the replication and transcription of CoV Stertz et al.,2007) (Figure 4). (Miller, 2008; Mark, 2008). Genes 2 to 7 are translated

Figure 5: life cycle of coronavirus in a host cell

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 Although, N protein is known to be necessary addition, although the PLpro sequences of SARS-CoV- for coronavirus replication, the specific role that this 2 and SARSCoV are only 83% similar, they share protein plays in this process remains unknown. But, similar active sites (Morse et al., 2020). many studies suggest that N protein interaction with nsp3 plays a critical role in the virus replication early in TRANSMISSION OF COVID-19 infection. The interaction between viral S protein and The virus that causes COVID-19 probably ACE2 on the host cell surface is of significant interest emerged from an animal source, but is now spreading since it initiates the infection process. Cryo-EM from person to person. The virus is thought to spread structure analysis has revealed that the binding affinity mainly between people who are in close contact with of SARS-CoV-2 S protein to ACE2 is about 10−20 one another (within about 6 feet) through respiratory times higher than that of SARS-CoV S protein (Wrapp droplets produced when an infected person coughs or et al., 2019; Lu et al., 2020). It is speculated that this sneezes. It also may be possible that a person can get may contribute to the reported higher transmissibility COVID-19 by touching a surface or object that has the and contagiousness of SARS-CoV-2 as compared to virus on it and then touching their own mouth, nose, or SARS-CoV (Tang et al., 2020). The prospect also exists possibly their eyes, but this is not thought to be the for discovery of therapeutic agents targeting the highly main way the virus spreads. conserved proteins associated with both SARS-CoV and SARS-CoV-2 proteins (Lu et al., 2020; Morse et SYMPTOMS OF COVID-19 al., 2020; Chan et al., 2020; Dong et al., 2020). RdRp Patients with COVID-19 have had mild to severe and 3CLpro protease of SARS-CoV-2 share over 95% respiratory illness with symptoms of: of sequence similarity with those of SARS-CoV despite  Fever the fact that these two viruses demonstrate only 79%  Cough sequence similarity at the genome level proteins (Lu et  Running nose al., 2020; Morse et al., 2020; Chan et al., 2020; Dong  Sore throat et al., 2020). On the basis of sequence alignment and  Body Ache homology modeling, SARS-CoV and SARS-CoV-2  Shortness or difficulty breathing share a highly conserved receptor-binding domain  At severe complications from this virus (Some (RBD), a domain of S protein, and 76% of sequence patients have pneumonia in both lungs, multi-organ similarity in their S proteins (Lu et al., 2020; Morse et failure and in some cases death). al., 2020; Chan et al., 2020; Dong et al., 2020) In

Figure 6: common and uncommon symptoms of COVID-19

PREVENTIVE ACTIONS AND SPREADING OF  People with symptoms of acute respiratory COVID-19 TO OTHERS infection should practice cough etiquette (maintain  Avoid close contact with people who are sick. distance, cover coughs and sneezes with disposable  Avoid touching your eyes, nose, and mouth with tissues or clothing, and wash hands). unwashed hands.  Clean and disinfect frequently touched objects and  Wash your hands often with soap and water for at surfaces. least 20 seconds. Use an alcohol-based hand  Limit human-to-human transmission including sanitizer that contains at least 60% alcohol if soap reducing secondary infections among close and water are not available. contacts and health care workers, preventing  Stay home when you are sick. transmission amplification events, and preventing further international spread from China.

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19  Identify, isolate and care for patients early, instructions on how to get care without exposing including providing optimized care for infected other people to your illness. While sick, avoid patients. contact with people, don’t go out and delay any  Identify and reduce transmission from the animal travel to reduce the possibility of spreading illness source. to others.  Address crucial unknowns regarding clinical severity, extent of transmission and infection SITUATION IN NUMBERS BY WHO AS AT 25 treatment options, and accelerate the development MARCH 2020 of diagnostics, therapeutics and vaccines. Total (new) cases in last 24 hours  Communicate critical risk and event information to Globally: 414 179 confirmed (40 712), and 18 440 all communities and counter misinformation. deaths (2202)  Minimize social and economic impact through Western Pacific Region: 97 766 confirmed (1186) and multispectral partnerships. 3518 deaths (16)  Avoiding unprotected contact with farm or wild European Region: 220 516 confirmed (25 007) and 11 animals. 986 deaths (1797)  If you have traveled from an affected area, there South-East Asia Region: 2344 confirmed (354) and 72 may be restrictions on your movements deaths (7) for up to 2 weeks. If you develop symptoms during Eastern Mediterranean Region: 29 631 confirmed (2416) that period (fever, cough, and trouble breathing), and 2008 deaths (131) seek medical advice. Call the office of your health Region of the Americas: 60 834 confirmed (11 390) and care provider before you go, and tell them about 813 deaths (248) your travel and your symptoms. They will give you African Region: 1664 confirmed (359) and 29 deaths (3

Figure 7: Countries, territories or areas with reported confirmed cases of COVID-19, 25 March 2020 (Map was reproduced from WHO Coronavirus Disease (COVID-2019) Situation Reports. Used with permission from ref. Copyright 2020 World Health Organization)

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 Table 1. Countries, territories or areas with reported laboratory-confirmed COVID-19 cases and deaths. Data as of 25 March 2020* Days Total Total Reporting Country/ Total Total new Transmission since last confirmed confirmed † deaths deaths § reported Territory/Area ‡ new cases classification cases case Western Pacific Region China 81848 101 3287 4 Local transmission 0 Republic of Korea 9137 100 126 6 Local transmission 0 Australia 2252 543 8 1 Local transmission 0 Malaysia 1624 106 16 2 Local transmission 0 Japan 1193 65 43 1 Local transmission 0 Singapore 558 51 2 0 Local transmission 0 Philippines 552 90 35 2 Local transmission 0 New Zealand 189 87 0 0 Local transmission 0 Viet Nam 134 11 0 0 Local transmission 0 Brunei Darussalam 104 13 0 0 Local transmission 0 Cambodia 91 4 0 0 Local transmission 0 Mongolia 10 0 0 0 Imported cases only 3 Fiji 4 1 0 0 Local transmission 0 Lao People's Democratic 2 2 0 0 Under investigation 0 Republic Papua New Guinea 1 0 0 0 Imported cases only 4 Territories** Guam 32 3 1 0 Local transmission 0 French Polynesia 25 7 0 0 Local transmission 0 New Caledonia 10 2 0 0 Local transmission 0 European Region Italy 69176 5249 6820 743 Local transmission 0 Spain 39673 6584 2696 514 Local transmission 0 Germany 31554 2342 149 23 Local transmission 0 France 22025 2410 1100 240 Local transmission 0 Switzerland 8789 774 86 20 Local transmission 0 The United 8081 1427 422 87 Local transmission 0 Kingdom Netherlands 5560 811 276 63 Local transmission 0 Austria 5282 796 30 5 Local transmission 0 Belgium 4269 526 122 34 Local transmission 0 Norway 2566 195 10 2 Local transmission 0 Portugal 2362 302 33 10 Local transmission 0 Sweden 2272 256 36 11 Local transmission 0 Israel 2170 932 5 4 Local transmission 0 Turkey 1872 343 44 7 Local transmission 0 Denmark 1591 131 32 8 Local transmission 0 Czechia 1394 158 3 2 Local transmission 0 Ireland 1329 204 7 1 Local transmission 0 Luxembourg 1099 224 8 0 Local transmission 0 Poland 901 152 10 2 Local transmission 0 Finland 792 92 1 0 Local transmission 0 Romania 762 186 11 4 Local transmission 0 Greece 743 48 20 3 Local transmission 0 Russian Federation 658 220 0 0 Local transmission 0 Iceland 648 60 2 0 Local transmission 0 Slovenia 480 38 3 2 Local transmission 0 Croatia 382 76 1 1 Local transmission 0 Estonia 369 17 0 0 Local transmission 0 Serbia 303 54 3 1 Local transmission 0 Armenia 265 30 0 0 Local transmission 0 Hungary 226 39 10 2 Local transmission 0 Bulgaria 220 19 3 0 Local transmission 0 Lithuania 209 30 2 1 Local transmission 0

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 Slovakia 204 13 0 0 Local transmission 0 Latvia 197 17 0 0 Local transmission 0 Andorra 188 24 1 0 Local transmission 0 San Marino 187 0 21 1 Local transmission 1 Bosnia and 164 33 2 1 Local transmission 0 Herzegovina North Macedonia 148 12 2 0 Local transmission 0 Albania 146 23 5 1 Local transmission 0 Republic of 125 16 1 0 Local transmission 0 Moldova Cyprus 124 8 3 3 Local transmission 0 Malta 120 13 0 0 Local transmission 0 Ukraine 113 29 4 1 Local transmission 0 Azerbaijan 87 15 1 0 Local transmission 0 Belarus 81 0 0 0 Local transmission 1 Kazakhstan 79 16 0 0 Imported cases only 0 Georgia 73 6 0 0 Local transmission 0 Uzbekistan 50 4 0 0 Local transmission 0 Liechtenstein 47 1 0 0 Imported cases only 0 Kyrgyzstan 42 26 0 0 Local transmission 0 Montenegro 29 7 0 0 Imported cases only 0 Monaco 23 0 0 0 Local transmission 2 Holy See 1 0 0 0 Under investigation 18 Territories** Faroe Islands 122 4 0 0 Local transmission 0 Kosovo[1] 63 2 1 0 Local transmission 0 Guernsey 23 3 0 0 Local transmission 0 Isle of Man 23 10 0 0 Imported cases only 0 Jersey 16 0 0 0 Local transmission 1 Gibraltar 15 0 0 0 Local transmission 2 Greenland 4 0 0 0 Under investigation 1 South-East Asia Region Thailand 934 107 4 0 Local transmission 0 Indonesia 686 107 55 6 Local transmission 0 India 562 128 9 0 Local transmission 0 Sri Lanka 102 5 0 0 Local transmission 0 Bangladesh 39 6 4 1 Local transmission 0 Maldives 13 0 0 0 Local transmission 9 Myanmar 3 1 0 0 Imported cases only 0 Bhutan 2 0 0 0 Imported cases only 5 Nepal 2 0 0 0 Imported cases only 1 Timor-Leste 1 0 0 0 Imported cases only 4 Eastern Mediterranean Region Iran (Islamic 24811 1762 1934 122 Local transmission 0 Republic of) Pakistan 991 104 7 1 Local transmission 0 Saudi Arabia 767 205 1 1 Local transmission 0 Qatar 526 25 0 0 Local transmission 0 Egypt 402 36 20 1 Local transmission 0 Bahrain 392 15 3 1 Local transmission 0 Iraq 316 50 27 4 Local transmission 0 Lebanon 304 37 4 0 Local transmission 0 United Arab 248 50 2 0 Local transmission 0 Emirates Kuwait 195 4 0 0 Local transmission 0 Morocco 170 27 5 1 Local transmission 0 Jordan 153 26 0 0 Imported cases only 0

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 Tunisia 114 25 3 0 Local transmission 0 Oman 99 15 0 0 Local transmission 0 Afghanistan 74 32 1 0 Imported cases only 0 Djibouti 3 0 0 0 Imported cases only 1 Sudan 3 1 1 0 Imported cases only 0 Libya 1 1 0 0 Imported cases only 0 Somalia 1 0 0 0 Imported cases only 8 Syrian Arab 1 0 0 0 Imported cases only 2 Republic Territories** occupied 60 1 0 0 Local transmission 0 Palestinian territory Region of the Americas United States of 51914 9750 673 202 Local transmission 0 America Brazil 2201 655 46 21 Local transmission 0 Canada 1739 307 25 5 Local transmission 0 Ecuador 1049 259 27 12 Local transmission 0 Chile 922 176 2 1 Local transmission 0 Peru 416 21 5 3 Local transmission 0 Mexico 370 0 4 0 Local transmission 1 Panama 345 0 6 0 Local transmission 1 Dominican 312 67 6 3 Local transmission 0 Republic Colombia 306 29 3 0 Local transmission 0 Argentina 301 35 4 0 Local transmission 0 Costa Rica 177 19 2 0 Local transmission 0 Uruguay 162 0 0 0 Imported cases only 1 Venezuela 77 7 0 0 Local transmission 0 (Bolivarian Republic of) Trinidad and 57 6 0 0 Imported cases only 0 Tobago Cuba 48 8 1 0 Local transmission 0 Honduras 30 0 0 0 Local transmission 1 Bolivia 28 1 0 0 Local transmission 0 (Plurinational State of) Paraguay 27 5 2 1 Local transmission 0 Guatemala 21 1 1 0 Local transmission 0 Jamaica 21 2 1 0 Local transmission 0 Barbados 18 1 0 0 Local transmission 0 Haiti 7 1 0 0 Imported cases only 0 Suriname 6 4 0 0 Imported cases only 0 El Salvador 5 2 0 0 Imported cases only 0 Guyana 5 0 1 0 Local transmission 6 Bahamas 4 0 0 0 Local transmission 4 Antigua and 3 2 0 0 Imported cases only 0 Barbuda Saint Lucia 3 0 0 0 Imported cases only 1 Dominica 2 1 0 0 Imported cases only 0 Nicaragua 2 0 0 0 Imported cases only 3 Belize 1 0 0 0 Imported cases only 1 Grenada 1 0 0 0 Imported cases only 2 Saint Vincent 1 0 0 0 Imported cases only 12 and the Grenadines

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Territories** Guadeloupe 73 11 0 0 Imported cases only 0 Martinique 57 4 0 0 Imported cases only 0 Puerto Rico 39 8 2 0 Imported cases only 0 French Guiana 23 3 0 0 Local transmission 0 United States 17 0 0 0 Imported cases only 1 Virgin Islands Aruba 12 3 0 0 Local transmission 0 Saint Martin 8 0 0 0 Under investigation 1 Bermuda 6 0 0 0 Imported cases only 1 Curaçao 6 2 1 0 Imported cases only 0 Cayman Islands 5 0 1 0 Imported cases only 1 Saint 3 0 0 0 Under investigation 9 SintBarthélemy Maarten 2 0 0 0 Imported cases only 1 Montserrat 1 0 0 0 Imported cases only 7 Turks and 1 0 0 0 Imported cases only 1 Caicos Islands African Region South Africa 554 152 0 0 Local transmission 0 Algeria 264 33 17 0 Local transmission 0 Burkina Faso 114 15 3 0 Local transmission 0 Senegal 86 7 0 0 Local transmission 0 Cameroon 72 0 1 1 Local transmission 1 Côte d’Ivoire 72 47 0 0 Imported cases only 0 Ghana 53 26 2 0 Local transmission 0 Democratic 45 9 2 0 Local transmission 0 Republic of the MauritiusCongo 42 6 2 2 Imported cases only 0 Nigeria 42 20 0 0 Imported cases only 0 Rwanda 40 4 0 0 Local transmission 0 Kenya 25 9 0 0 Local transmission 0 Togo 20 2 0 0 Imported cases only 0 Madagascar 19 6 0 0 Imported cases only 0 Ethiopia 12 1 0 0 Imported cases only 0 United Republic 12 0 0 0 Imported cases only 2 of Tanzania Uganda 9 0 0 0 Imported cases only 1 Seychelles 7 0 0 0 Imported cases only 3 Equatorial 6 0 0 0 Imported cases only 3 GuineaGabon 6 0 1 0 Imported cases only 2 Benin 5 0 0 0 Imported cases only 1 Central African 4 0 0 0 Imported cases only 2 Republic Congo 4 0 0 0 Imported cases only 3 Eswatini 4 0 0 0 Imported cases only 2 Guinea 4 0 0 0 Imported cases only 1 Namibia 4 1 0 0 Imported cases only 0 Cabo Verde 3 0 0 0 Imported cases only 3 Chad 3 0 0 0 Imported cases only 1 Liberia 3 0 0 0 Local transmission 3 Mozambique 3 2 0 0 Imported cases only 0 Zambia 3 0 0 0 Imported cases only 2 Angola 2 0 0 0 Imported cases only 3 Gambia 2 1 0 0 Imported cases only 0 Mauritania 2 0 0 0 Imported cases only 6 Niger 2 0 0 0 Imported cases only 1

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 Zimbabwe 2 0 1 0 Imported cases only 3 Eritrea 1 0 0 0 Imported cases only 3 Territories** Réunion 83 12 0 0 Local transmission 0 Mayotte 30 6 0 0 Local transmission 0 Subtotal for all 412755 40712 18426 2202 regions International conveyance 712 0 7 0 Local transmission 9 (Diamond GrandPrincess) total 414179 40712 18440 2202

Figure 8: Epidemic curve of confirmed COVID-19, by date of report and WHO region through 25 March 2020

TREATMENT studied the effect of chloroquine in vitro, using Vero E6 Efficacy and safety of chloroquine, remdesivir (GS- cells infected by SARS-CoV-2 at a multiplicity of 5734) and Hydroxychloroquine for the treatment of infection (MOI) of 0.05. The study demonstrated that COVID-19 chloroquine was highly effective in reducing viral COVID-19 (Coronavirus Disease-2019) is a replication, with an effective concentration (EC)90 of public health emergency of international concern. 6.90 μM that can be easily achievable with standard dosing, due to its favourable penetration in tissues, As of this time there is no known specific, including in the lung (Wang et al., 2020). The authors effective, proven, pharmacological treatment. Invitro described that chloroquine is known to block virus studies have suggested that chloroquine, an infection by increasing endosomial pH and by immunomodulant drug traditionally used to treat interfering with the glycosylation of cellular receptor of malaria, is effective in reducing viral replication in SARSCoV. The authors also speculated on the other infections, including the SARS-associated possibility that the known immunomodulant effect of coronavirus (CoV) and MERS-CoV (Savarino et al., the drug may enhance the antiviral effect in vivo (Wang 2003; Colson et al., 2020; WHO, 2020). Chloroquine et al., 2020). has been used worldwide for more than 70 years, and it is part of the World Health Organization (WHO) model A narrative letter by Chinese authors reported list of essential medicines. It is also cheap and has an that a news briefing from the State Council of China established clinical safety profile (Colson et al., 2020). had indicated that “Chloroquine phosphate had However, the efficacy and safety of chloroquine for demonstrated marked efficacy and acceptable safety in treatment of SARS-CoV-2 (the new virus causing treating COVID-19 associated pneumonia in COVID-19) pneumonia remains Unclear. A research multicentre clinical trials conducted in China”. The letter, written by a group of Chinese researchers, authors also stated that these findings came from “more

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 than 100 patients” included in the trials (Gao et al., structures and mechanisms of acting as a weak base and 2020). We sought for evidence of such data in the trial immunomodulator, it is easy to conjure up the idea that registries we reviewed and found none. Since cases HCQ may be a potent candidate to treat infection by were reported in 85 countries so far (5th March 2020), SARS-CoV-2. Actually, as of February 23, 2020, seven the low cost of chloroquine is a major benefit for both clinical trial registries were found in Chinese Clinical the highly stressed healthcare systems of involved high- Trial Registry (http://www.chictr.org.cn) for using HCQ income countries and the underfunded heath care to treat COVID-19. Whether HCQ is as efficacious as systems of middle- and low-income counties (WHO, CQ in treating SARS-CoV-2 infection still lacks the 2020). experimental evidence.

The expert consensus was published on 20th Liu et al., 2020 evaluated the antiviral effect of February by a multicentre collaboration group of the HCQ against SARS-CoV-2 infection in comparison to Department of Science and Technology of Guangdong CQ in vitro. First, the cytotoxicity of HCQ and CQ in Province and Health Commission of Guangdong African green monkey kidney VeroE6 cells (ATCC- Province paper and related specifically to the use of 1586) was measured by standard CCK8 assay, and the chloroquine phosphate. No information was provided result showed that the 50% cytotoxic concentration on the method used to achieve consensus (Zhonghua, (CC50) values of CQ and HCQ were 273.20 and 249.50 2020). Based on in vitro evidence and still unpublished μM, respectively, which are not significantly different clinical experience, the panel recommended from each other. To better compare the antiviral activity chloroquine phosphate tablet, at a dose of 500 mg twice of CQ versus HCQ, the dose–response curves of the per day for 10 days, for patients diagnosed as mild, two compounds against SARS-CoV-2 were determined moderate and severe cases of SARS-CoV-2 pneumonia, at four different multiplicities of infection (MOIs) by provided that there were no contraindications to the quantification of viral RNA copy numbers in the cell drug. supernatant at 48 h post infection (p.i.). The data show that, at all MOIs (0.01, 0.02, 0.2, and 0.8), the 50% Researches have shown that two drugs, maximal effective concentration (EC50) for CQ (2.71, remdesivir (GS-5734) and chloroquine (CQ) phosphate, 3.81, 7.14, and 7.36 μM) was lower than that of HCQ efficiently inhibited SARS-CoV-2 infection in vitro. (4.51, 4.06, 17.31, and 12.96 μM). The differences in Remdesivir is a nucleoside analog prodrug developed EC50 values were statistically significant at an MOI of by Gilead Sciences (USA). A recent case report showed 0.01 (P < 0.05) and MOI of 0.2 (P < 0.001). that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 Consequently, the selectivity index (SI = in the United States (Holshue et al., 2020), and a phase CC50/EC50) of CQ (100.81, 71.71, 38.26, and 37.12) III clinical trial of remdesivir against SARSCoV-2 was was higher than that of HCQ (55.32, 61.45, 14.41, launched in Wuhan on February 4, 2020. However, as 19.25) at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. an experimental drug, remdesivir is not expected to be These results were corroborated by largely available for treating a very large number of immunofluorescence microscopy as evidenced by patients in a timely manner. Therefore, of the two different expression levels of virus nucleoprotein (NP) potential drugs, CQ appears to be the drug of choice for at the indicated drug concentrations at 48 h p.i. large-scale use due to its availability, proven safety (Supplementary Fig. S1). Taken together, the data record, and a relatively low cost. CQ (N4-(7-Chloro-4- suggest that the anti-SARS-CoV-2 activity of HCQ quinolinyl)-N1, N1-diethyl-1,4-pentanediamine) has seems to be less potent compared to CQ, at least at long been used to treat malaria and amebiasis. certain MOIs. Both CQ and HCQ are weak bases that However, Plasmodium falciparum developed are known to elevate the pH of acidic intracellular widespread resistance to it, and with the development of organelles, such as endosomes/lysosomes, essential for new antimalarials, it has become a choice for the membrane fusion. In addition, CQ could inhibit SARS- prophylaxis of malaria. In addition, an overdose of CQ CoV entry through changing the glycosylation of ACE2 can cause acute poisoning and death (Weniger, 1979). receptor and spike protein (Savarino et al., 2006). In the past years, due to infrequent utilization of CQ in Time-of-addition experiment confirmed that HCQ clinical practice, its production and market supply was effectively inhibited the entry step, as well as the post- greatly reduced, at least in China. entry stages of SARS-CoV-2, which was also found upon CQ treatment. Hydroxychloroquine (HCQ) sulfate, a derivative of CQ, was first synthesized in 1946 by To further explore the detailed mechanism of introducing a hydroxyl group into CQ and was action of CQ and HCQ in inhibiting virus entry, co- demonstrated to be much less (~40%) toxic than CQ in localization of virions with early endosomes (EEs) or animals (McChesney, 1983). More importantly, HCQ is endolysosomes (ELs) was analyzed by still widely available to treat autoimmune diseases, such immunofluorescence analysis (IFA) and confocal as systemic lupus erythematosus and rheumatoid microscopy. Quantification analysis showed that, at 90 arthritis. Since CQ and HCQ share similar chemical min p.i. in untreated cells, 16.2% of internalized virions

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Oguh C.E et al., IAR J Med Sci; Vol-1, Iss-1 (May-June, 2020): 5-19 (anti-NP, red) were observed in early endosome antigen out, although HCQ is less toxic than CQ, prolonged and 1 (EEA1)-positive EEs (green), while more virions overdose usage can still cause poisoning. Although the (34.3%) were transported into the late endosomal– use of chloroquine can also support expert opinion, lysosomal protein LAMP1+ ELs (green) (n > 30 cells clinical use of this drug in patients with COVID-19 for each group). By contrast, in the presence of CQ or should adhere to the MEURI framework or after ethical HCQ, significantly more virions (35.3% for CQ and approval as a trial as stated by the WHO. 29.2% for HCQ; P < 0.001) were detected in the EEs, while only very few virions (2.4% for CQ and 0.03% REFERENCES for HCQ; P < 0.001) were found to be co-localized with 1. Anthony, S. J., Johnson, C. K., Greig, D. J., LAMP1+ ELs (n > 30 cells). 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