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Go Back       IAR Journal of Medical Sciences | Iar J Med Sci, 2020; 1(1):5-19. | Volume:1 Volume:1 ( May 22, 2020 ) : NA
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            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: collinsoguh@gmail.com

 

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

Coronavirus disease 2019 (COVID-19) is a respiratory illness that can transfer from person to person. The virus that causes COVID-19 is a novel coronavirus that was first identified during an investigation into an outbreak in Wuhan, China in December 2019, this disease has spread to more than 100 countries with over 100 000 confirmed cases and over 3,800 confirmed deaths worldwide as of March 9, 2020 (WHO, 2020). The name “coronavirus,” coined in 1968, is derived from the “corona”-like or crown-like morphology observed for these viruses in the electron microscope (318). The risk of infection from the virus that causes COVID-19 is higher for people who are close contacts of someone known to have COVID-19, for example healthcare workers, or household members. Other people at higher risk for infection are those who live in or have recently been in an area with ongoing spread of COVID-19. Risk of death is only higher in older people (above an age of 60 years) and people with pre-existing health conditions. While coronaviruses infect both humans and animals, certain types of animals such as bats that host the largest variety of coronaviruses appear to be immune to coronavirus-induced illness (Anthony et al.,  2017).  Researchers have been racing to find possible treatments to save lives and produce vaccines for future prevention. These viruses infect a variety of human and animal host cells, and also carry out their infection and replication. Also, many proteins have most important role in the replication mechanism, although that role is, as yet, poorly defined. In this case, it is necessary to know the definition of these proteins in terms of this mechanism. Therefore; this review explains the structure, classification, symptoms, prevention, statistics cases and summarize the evidence regarding chloroquine and hydroxychloroquine for the treatment of COVID-19.

 

STRUCTURE OF COVID-19

Coronaviruses are enveloped viruses with round and sometimes pleiomorphic virions of approximately 80 to 120 nm in diameter (Fig. 1). Coronaviruses contain positive-strand RNA, with the largest RNA genome (approximately 30 kb) reported to date (178, 196). Corona viruses (COVID-19) are relatively large viruses containing a single-stranded positive-sense RNA genome encapsulated within a membrane envelope. The viral membrane is studded with glycoprotein spikes that give coronaviruses their crown like appearance.  Coronaviruses encode five structural proteins in their genomes. These are the Spike (S), Membrane (M), Envelope (E) glycoproteins, Hemagglutinin Esterase (HE) and Nucleocapsid (N) protein, (Figure 1). All envelope proteins and N protein is present in all virion but HE is only present in some beta coronaviruses (Lissenberg et al.,  2005). In addition to that, it is thought the virus particles are huddled together owing to interaction between these proteins (De Haan and Rottier, 2005; Master, 2006).

 

 

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 the virion and give the virion the typical shape. The S proteins form homotrimers, which allow the formation of sun-like morphologies that give the name of Coronaviruses (Tan et al, 2005). S proteins bind to the virion membrane via the C-terminal transmembrane regions and they also interact with M proteins (Chinese SMEC, 2004). Virions can be bound to specific surface receptors in the plasma membrane of the host cell via the N-terminus of the S proteins. This S protein mediates host cell invasion by both SARS-CoV and SARS-CoV-2 via binding to a receptor protein called angiotensin-converting enzyme 2 (ACE2) located on the surface membrane of host cells (Wrapp et al., 2019; Hoffmann et al.,  2020). A recent study also revealed that this invasion process requires S protein priming which is facilitated by the host cell produced serine protease TMPRSS211. In addition, the viral genome also encodes several nonstructural proteins including RNA-dependent RNA polymerase (RdRp), coronavirus main protease (3CLpro), and papain-like protease (PLpro) (Gorbalenya et al., 2015; Baez-santos et al., 2015).              
  2. M Glycoproteins: M Glycoproteins have three transmembrane regions. M proteins are glycosylated in the Golgi apparatus (Niemann et al., 1984). This modification of the M protein is crucial for the virion to fuse into the cell and to make protein antigenic. The M protein plays a key role in regenerating virions in the cell. N protein forms a complex by binding to genomic RNA and M protein triggers the formation of interacting virions in this endoplasmic reticulum-Golgi apparatus intermediate compartment (ERGIC) with this complex (Narayanan and Makino, 2001; Escors et al., 2001).

 

  1. E Glycoproteins: E Glycoproteins are small proteins that are composed of approximately 76 to 109 amino acids. About 30 amino acids in the N-terminus of the E proteins allow attachment to the membrane of viruses (Raamsman et al., 2000). In addition, coronavirus E proteins play a critical role in the assembly and morphogenesis of virions within the cell. In one study coronavirus E and M proteins were expressed together with mammalian expression vectors to form virus-like structures within the cell (Vennema et al., 1996). In another study, there was a significant decrease in the ability of the recombinant mouse hepatitis virus (MHV) and SARS viruses to elicit E protein expression in the genome to support this status (DeDiego et al., 2007; Kuuo and Masters, 2003)

 

  1. N Proteins: N proteins are phosphoproteins that are capable of binding to helix and have flexible structure of viral genomic RNA. It plays an important role in virion structure, replication and transcription of coronaviruses, because the N protein localizes in both the replication/ transcriptional region of the coronaviruses and the ERGIC region where the virus is collected (DeDiego et al., 2007; Kuuo and Masters, 2003). (Figure 2).

 

 

 

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

 


CLASSIFICATION OF CORONAVIRUSES

The classification of Coronaviruses has been based on genomic organization, similarities in genomic sequence, antigenic properties of viral proteins, replication strategies, and structural characteristics of virions, pathogenic, cytopathogenic and physicochemical properties (Lai and Cavanagh, 1997). The Coronaviruses (CoVs) are species of virus belonging to the Nidovirales order, which includes Coronaviridae, Arteriviridae, Roniviridae and Mesoniviridae families (Zirkel et al., 2011). The Arteviridae family includes swine and equine pathogens, and the Roniviridae family is composed of invertebrate viruses. The Coronaviridae family is the largest one of the four families, by its genomic sizes of coronaviridae range from 26 to 32 kb (Gorbalenya et al.,  2006). Coronaviridae virus family subdivided into two subfamilies, coronavirinae and torovirinae. Coronavirinae is divided into four genera, Alpha coronavirus, Beta coronavirus, Gamma coronavirus and Delta coronavirus (figure 3). Alpha coronaviruses type 1 species are classified feline FCoV, FECV (Feline Enteric Coronavirus) and FIPV (Feline Infectious Peritonitis Virus), the porcine TGEV (Transmissible Gastro- Enteritis Virus), Porcine PEDV (Epidemic Diarrhea Virus), PRCoV (Porcine Respiratory Coronavirus) and the canine CCoV. Alpha coronaviruses also compromise human CoVs such as HCoV-229E and HCoVNL63, but various bat Coronaviruses.

 

Beta coronaviruses also infect a wide range of mammalians, with species such as mice, human with SARS-CoV, HCoV-OC43, HCoV-HKU1, and MERS-CoV, Murine coronavirus (MHV) and Bovine Coronavirus (BCoV). Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 attacks the lower respiratory system to cause viral pneumonia, but it may also affect the gastrointestinal system, heart, kidney, liver, and central nervous system leading to multiple organ failure (Su et al., 2019; Zhu et al., 2020). Current information indicates that SARSCoV-2 is more transmissible/contagious than SARS-CoV (Tang et al., 2020). The beta coronavirus genome encodes several structural proteins, including the glycosylated spike (S) protein that functions as a major inducer of host immune responses. Gamma coronaviruses are specific of birds, with one exception of a beluga whale Coronavirus. The delta coronavirus genus was created in 2012 and regroups various (HKU11, HKU12, HKU13) Coronavirus from mammals to birds (Susanna et al., 2012) figure 3.

 

 

Figure 3: Classification of COVID-19

 


 

 

COVID-19 MECHANISM OF ACTION (REPLICATION AND TRANSCRIPTION)

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

 

 

Figure 4:  MHV genome organization and replicase proteins.

 

 

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

 

 

Figure 5: life cycle of coronavirus in a host cell


 

 

Although, N protein is known to be necessary for coronavirus replication, the specific role that this protein plays in this process remains unknown. But, many studies suggest that N protein interaction with nsp3 plays a critical role in the virus replication early in infection. The interaction between viral S protein and ACE2 on the host cell surface is of significant interest since it initiates the infection process. Cryo-EM structure analysis has revealed that the binding affinity of SARS-CoV-2 S protein to ACE2 is about 10−20 times higher than that of SARS-CoV S protein (Wrapp et al., 2019; Lu et al., 2020). It is speculated that this may contribute to the reported higher transmissibility and contagiousness of SARS-CoV-2 as compared to SARS-CoV (Tang et al., 2020). The prospect also exists for discovery of therapeutic agents targeting the highly conserved proteins associated with both SARS-CoV and SARS-CoV-2 proteins (Lu et al., 2020; Morse et al., 2020; Chan et al., 2020; Dong et al., 2020). RdRp and 3CLpro protease of SARS-CoV-2 share over 95% of sequence similarity with those of SARS-CoV despite the fact that these two viruses demonstrate only 79% sequence similarity at the genome level proteins (Lu et al., 2020; Morse et al.,  2020; Chan et al.,  2020; Dong et al.,  2020). On the basis of sequence alignment and homology modeling, SARS-CoV and SARS-CoV-2 share a highly conserved receptor-binding domain (RBD), a domain of S protein, and 76% of sequence similarity in their S proteins (Lu et al.,  2020; Morse et al.,  2020; Chan et al.,  2020; Dong et al.,  2020) In addition, although the PLpro sequences of SARS-CoV-2 and SARSCoV are only 83% similar, they share similar active sites (Morse et al.,  2020).

 

TRANSMISSION OF COVID-19 

The virus that causes COVID-19 probably emerged from an animal source, but is now spreading from person to person. The virus is thought to spread mainly between people who are in close contact with one another (within about 6 feet) through respiratory droplets produced when an infected person coughs or sneezes. It also may be possible that a person can get COVID-19 by touching a surface or object that has the virus on it and then touching their own mouth, nose, or possibly their eyes, but this is not thought to be the main way the virus spreads.

 

SYMPTOMS OF COVID-19

Patients with COVID-19 have had mild to severe respiratory illness with symptoms of:

  • Fever
  • Cough
  • Running nose
  • Sore throat
  • Body Ache
  • Shortness or difficulty breathing
  • At severe complications from this virus (Some patients have pneumonia in both lungs, multi-organ failure and in some cases death).

 

 

Figure 6: common and uncommon symptoms of COVID-19

 


PREVENTIVE ACTIONS AND SPREADING OF COVID-19 TO OTHERS

  • Avoid close contact with people who are sick.
  • Avoid touching your eyes, nose, and mouth with unwashed hands.
  • Wash your hands often with soap and water for at least 20 seconds. Use an alcohol-based hand sanitizer that contains at least 60% alcohol if soap and water are not available.
  • Stay home when you are sick.
  • People with symptoms of acute respiratory infection should practice cough etiquette (maintain distance, cover coughs and sneezes with disposable tissues or clothing, and wash hands).
  • Clean and disinfect frequently touched objects and surfaces.
  • Limit human-to-human transmission including reducing secondary infections among close contacts and health care workers, preventing transmission amplification events, and preventing further international spread from China.
  • Identify, isolate and care for patients early, including providing optimized care for infected patients.
  • Identify and reduce transmission from the animal source.
  • Address crucial unknowns regarding clinical severity, extent of transmission and infection treatment options, and accelerate the development of diagnostics, therapeutics and vaccines.
  • Communicate critical risk and event information to all communities and counter misinformation.
  • Minimize social and economic impact through multispectral partnerships.
  • Avoiding unprotected contact with farm or wild animals.
  • If you have traveled from an affected area, there may be restrictions on your movements
    for up to 2 weeks. If you develop symptoms during that period (fever, cough, and trouble breathing), seek medical advice. Call the office of your health care provider before you go, and tell them about your travel and your symptoms. They will give you instructions on how to get care without exposing other people to your illness. While sick, avoid contact with people, don’t go out and delay any travel to reduce the possibility of spreading illness to others.

 

SITUATION IN NUMBERS BY WHO AS AT 25 MARCH 2020

Total (new) cases in last 24 hours

Globally: 414 179 confirmed (40 712), and 18 440 deaths (2202)

Western Pacific Region: 97 766 confirmed (1186) and 3518 deaths (16)

European Region: 220 516 confirmed (25 007) and 11 986 deaths (1797)

South-East Asia Region: 2344 confirmed (354) and 72 deaths (7)

Eastern Mediterranean Region: 29 631 confirmed (2416) and 2008 deaths (131)

Region of the Americas: 60 834 confirmed (11 390) and 813 deaths (248)

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)

 

 

Table 1. Countries, territories or areas with reported laboratory-confirmed COVID-19 cases and deaths. Data as of 25 March 2020*

Reporting Country/ Territory/Area†

Total confirmed

cases

Total confirmed new cases

Total deaths

Total new deaths

Transmission classification§

Days since last reported 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 Republic

2

2

0

0

Under investigation

0

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 Kingdom

8081

1427

422

87

Local transmission

0

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

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 Herzegovina

164

33

2

1

Local transmission

0

North Macedonia

148

12

2

0

Local transmission

0

Albania

146

23

5

1

Local transmission

0

Republic of Moldova

125

16

1

0

Local transmission

0

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 Republic of)

24811

1762

1934

122

Local transmission

0

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 Emirates

248

50

2

0

Local transmission

0

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

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 Republic

1

0

0

0

Imported cases only

2

Territories**

occupied Palestinian territory

60

1

0

0

Local transmission

0

Region of the Americas

United States of America

51914

9750

673

202

Local transmission

0

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 Republic

312

67

6

3

Local transmission

0

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 (Bolivarian Republic of)

77

7

0

0

Local transmission

0

Trinidad and Tobago

57

6

0

0

Imported cases only

0

Cuba

48

8

1

0

Local transmission

0

Honduras

30

0

0

0

Local transmission

1

Bolivia (Plurinational State of)

28

1

0

0

Local transmission

0

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 Barbuda

3

2

0

0

Imported cases only

0

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 and the Grenadines

1

0

0

0

Imported cases only

12

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 Virgin Islands

17

0

0

0

Imported cases only

1

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 Barthélemy

3

0

0

0

Under investigation

9

Sint Maarten

2

0

0

0

Imported cases only

1

Montserrat

1

0

0

0

Imported cases only

7

Turks and Caicos Islands

1

0

0

0

Imported cases only

1

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 Republic of the Congo

45

9

2

0

Local transmission

0

Mauritius

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 of Tanzania

12

0

0

0

Imported cases only

2

Uganda

9

0

0

0

Imported cases only

1

Seychelles

7

0

0

0

Imported cases only

3

Equatorial Guinea

6

0

0

0

Imported cases only

3

Gabon

6

0

1

0

Imported cases only

2

Benin

5

0

0

0

Imported cases only

1

Central African Republic

4

0

0

0

Imported cases only

2

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

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 regions

412755

40712

18426

2202

 

 

International conveyance (Diamond Princess)

 

712

 

0

 

7

 

0

 

Local transmission

 

9

Grand 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

Efficacy and safety of chloroquine, remdesivir (GS-5734) and Hydroxychloroquine for the treatment of COVID-19

COVID-19 (Coronavirus Disease-2019) is a public health emergency of international concern.

 

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

 

A narrative letter by Chinese authors reported that a news briefing from the State Council of China had indicated that “Chloroquine phosphate had demonstrated marked efficacy and acceptable safety in treating COVID-19 associated pneumonia in multicentre clinical trials conducted in China”. The authors also stated that these findings came from “more than 100 patients” included in the trials (Gao et al.,  2020). We sought for evidence of such data in the trial registries we reviewed and found none. Since cases were reported in 85 countries so far (5th March 2020), the low cost of chloroquine is a major benefit for both the highly stressed healthcare systems of involved high-income countries and the underfunded heath care systems of middle- and low-income counties (WHO, 2020).

 

The expert consensus was published on 20th February by a multicentre collaboration group of the Department of Science and Technology of Guangdong Province and Health Commission of Guangdong Province paper and related specifically to the use of chloroquine phosphate. No information was provided on the method used to achieve consensus (Zhonghua, 2020). Based on in vitro evidence and still unpublished clinical experience, the panel recommended chloroquine phosphate tablet, at a dose of 500 mg twice per day for 10 days, for patients diagnosed as mild, moderate and severe cases of SARS-CoV-2 pneumonia, provided that there were no contraindications to the drug.

 

Researches have shown that two drugs, remdesivir (GS-5734) and chloroquine (CQ) phosphate, efficiently inhibited SARS-CoV-2 infection in vitro. Remdesivir is a nucleoside analog prodrug developed by Gilead Sciences (USA). A recent case report showed that treatment with remdesivir improved the clinical condition of the first patient infected by SARS-CoV-2 in the United States (Holshue et al.,  2020), and a phase III clinical trial of remdesivir against SARSCoV-2 was launched in Wuhan on February 4, 2020. However, as an experimental drug, remdesivir is not expected to be largely available for treating a very large number of patients in a timely manner. Therefore, of the two potential drugs, CQ appears to be the drug of choice for large-scale use due to its availability, proven safety record, and a relatively low cost. CQ (N4-(7-Chloro-4-quinolinyl)-N1, N1-diethyl-1,4-pentanediamine) has long been used to treat malaria and amebiasis. However, Plasmodium falciparum developed widespread resistance to it, and with the development of new antimalarials, it has become a choice for the prophylaxis of malaria. In addition, an overdose of CQ can cause acute poisoning and death (Weniger, 1979). In the past years, due to infrequent utilization of CQ in clinical practice, its production and market supply was greatly reduced, at least in China.

 

Hydroxychloroquine (HCQ) sulfate, a derivative of CQ, was first synthesized in 1946 by introducing a hydroxyl group into CQ and was demonstrated to be much less (~40%) toxic than CQ in animals (McChesney, 1983). More importantly, HCQ is still widely available to treat autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ and HCQ share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, it is easy to conjure up the idea that HCQ may be a potent candidate to treat infection by SARS-CoV-2. Actually, as of February 23, 2020, seven clinical trial registries were found in Chinese Clinical Trial Registry (http://www.chictr.org.cn) for using HCQ to treat COVID-19. Whether HCQ is as efficacious as CQ in treating SARS-CoV-2 infection still lacks the experimental evidence.

 

Liu et al., 2020 evaluated the antiviral effect of HCQ against SARS-CoV-2 infection in comparison to CQ in vitro. First, the cytotoxicity of HCQ and CQ in African green monkey kidney VeroE6 cells (ATCC-1586) was measured by standard CCK8 assay, and the result showed that the 50% cytotoxic concentration (CC50) values of CQ and HCQ were 273.20 and 249.50 μM, respectively, which are not significantly different from each other. To better compare the antiviral activity of CQ versus HCQ, the dose–response curves of the two compounds against SARS-CoV-2 were determined at four different multiplicities of infection (MOIs) by quantification of viral RNA copy numbers in the cell 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% maximal effective concentration (EC50) for CQ (2.71, 3.81, 7.14, and 7.36 μM) was lower than that of HCQ (4.51, 4.06, 17.31, and 12.96 μM). The differences in EC50 values were statistically significant at an MOI of 0.01 (P < 0.05) and MOI of 0.2 (P < 0.001).

 

Consequently, the selectivity index (SI = CC50/EC50) of CQ (100.81, 71.71, 38.26, and 37.12) was higher than that of HCQ (55.32, 61.45, 14.41, 19.25) at MOIs of 0.01, 0.02, 0.2, and 0.8, respectively. These results were corroborated by immunofluorescence microscopy as evidenced by different expression levels of virus nucleoprotein (NP) at the indicated drug concentrations at 48 h p.i. (Supplementary Fig. S1). Taken together, the data suggest that the anti-SARS-CoV-2 activity of HCQ seems to be less potent compared to CQ, at least at certain MOIs. Both CQ and HCQ are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion. In addition, CQ could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein (Savarino et al., 2006). Time-of-addition experiment confirmed that HCQ effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ treatment.

 

To further explore the detailed mechanism of action of CQ and HCQ in inhibiting virus entry, co-localization of virions with early endosomes (EEs) or endolysosomes (ELs) was analyzed by immunofluorescence analysis (IFA) and confocal microscopy. Quantification analysis showed that, at 90 min p.i. in untreated cells, 16.2% of internalized virions (anti-NP, red) were observed in early endosome antigen 1 (EEA1)-positive EEs (green), while more virions (34.3%) were transported into the late endosomal–lysosomal protein LAMP1+ ELs (green) (n > 30 cells for each group). By contrast, in the presence of CQ or HCQ, significantly more virions (35.3% for CQ and 29.2% for HCQ; P < 0.001) were detected in the EEs, while only very few virions (2.4% for CQ and 0.03% for HCQ; P < 0.001) were found to be co-localized with LAMP1+ ELs (n > 30 cells). This suggested that both CQ and HCQ blocked the transport of SARS-CoV-2 from EEs to ELs, which appears to be a requirement to release the viral genome as in the case of SARS-CoV (Mingo et al., 2015). Interestingly, CQ and HCQ treatment caused noticeable changes in the number and size/morphology of EEs and ELs. In the untreated cells, most EEs were much smaller than ELs. In CQ and HCQ-treated cells, abnormally enlarged EE vesicles were observed, arrows in the upper panels), many of which are even larger than ELs in the untreated cells. This is in agreement with previous report that treatment with CQ induced the formation of expanded cytoplasmic vesicles (Zheng et al.,  2011). Within the EE vesicles, virions (red) were localized around the membrane (green) of the vesicle. CQ treatment did not cause obvious changes in the number and size of ELs; however, the regular vesicle structure seemed to be disrupted, at least partially. By contrast, in HCQ-treated cells, the size and number of ELs increased significantly, arrows in the lower panels). Since acidification is crucial for endosome maturation and function, we surmise that endosome maturation might be blocked at intermediate stages of endocytosis, resulting in failure of further transport of virions to the ultimate releasing site. CQ was reported to elevate the pH of lysosome from about 4.5 to 6.5 at 100 μM (Ohkuma and Poole, 1978).

 

It has been reported that oral absorption of CQ and HCQ in humans is very efficient. In animals, both drugs share similar tissue distribution patterns, with high concentrations in the liver, spleen, kidney, and lung reaching levels of 200–700 times higher than those in the plasma (Popert, 1976). It was reported that safe dosage (6–6.5 mg/kg per day) of HCQ sulfate could generate serum levels of 1.4–1.5 μM in Humans. Therefore, with a safe dosage, HCQ concentration in the above tissues is likely to be achieved to inhibit SARS-CoV-2 infection.

 

CONCLUSION

Coronaviruses (CoVs) are a diverse family of viruses that interact at multiple levels with components of host cells taking this advantage of some of the cellular machineries for replication and proliferation. In conclusion, HCQ can efficiently inhibit SARS-CoV-2 infection in vitro. In combination with its anti-inflammatory function, we predict that the drug has a good potential to combat the disease. This possibility awaits confirmation by clinical trials. We need to point out, although HCQ is less toxic than CQ, prolonged and overdose usage can still cause poisoning. Although the use of chloroquine can also support expert opinion, clinical use of this drug in patients with COVID-19 should adhere to the MEURI framework or after ethical approval as a trial as stated by the WHO.

 

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