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v1

Clinical, molecular and epidemiological characterization of the SARS-

CoV2 virus and the Coronavirus disease 2019 (COVID-19), a
comprehensive literature review

Esteban Ortiz-Prado*1, Katherine Simbaña-Rivera1, Lenin Gómez- Barreno1, Mario Rubio-

Neira2, Linda P. Guaman3, Nikolaos kyriakidis4, Claire Muslin1, Ana María Gómez
Jaramillo5, Carlos Barba1, Doménica Cevallos6, Hugo Sanches-SanMiguel1, Luis Unigarro7,
Rasa Zalakeviciute8,9, Naomi Gadian10, Andrés López-Cortés11,12

1
One Health Research Group, Faculty of Medicine, Universidad de Las Americas (UDLA),
Quito, Ecuador
2
Hospital Baca Ortiz, Pediatric and Cardiology Department, Quito, Ecuador
3
Centro de Investigación Biomédica, Facultad de Ciencias de la Salud Eugenio Espejo,
Universidad UTE, Quito, Ecuador
4
Grupo de Investigación en Biotecnología Aplicada a Biomedicina (BIOMED),
Universidad de la Americas, Quito, Ecuador
5
Faculty of Medicine, Pontificia Universidad Católica del Ecuador, Quito, Ecuador
6
Intensive Care Unit, Hospital Quito Sur IESS, Quito, Ecuador
7
Intensive Care Unit, Hospital SOLCA Quito., Quito, Ecuador
8
Grupo de Biodiversidad Medio Ambiente y Salud (BIOMAS), Universidad de Las
Americas, Quito, Ecuador.
9
Intelligent and Interactive Systems Lab (SI2 Lab) Universidad de Las Americas (UDLA),
Quito, Ecuador
10
University of Southampton, Department of Public Health, Southampton, United
Kingdome
11
Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio
Espejo, Universidad UTE, Quito, Ecuador
12
Red Latinoamericana de Implementación y Validación de Guías Clínicas
Farmacogenómicas (RELIVAF-CYTED), Quito, Ecuador

© 2020 by the author(s). Distributed under a Creative Commons CC BY license.

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Esteban Ortiz-Prado: [email protected]

Katherine Simbaña-Rivera: [email protected]
Lenin Gómez- Barreno: [email protected]
Mario Rubio: [email protected]
Linda P. Guaman: [email protected]
Nikolaos kyriakidis: [email protected]
Claire Muslin: [email protected]
Carlos Barba: [email protected]
Ana María Gómez Jaramillo: [email protected]
Carlos Barba: [email protected]
Doménica Cevallos: [email protected]
Hugo Sanches-SanMiguel: [email protected]
Luis Unigarro: [email protected]
Rasa Zalakeviciute: [email protected]
Naomi Gadian: [email protected]
Andrés López-Cortés: [email protected]

Corresponding author: Esteban Ortiz-Prado One Health Research Group, Universidad de

las Américas, Quito, Ecuador Calle de los Colimes y Avenida De los Granados, Quito 170137,
Ecuador. Email: [email protected] Phone: +593995760693
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Abstract

Coronaviruses are an extensive family of viruses that can cause disease in both animals and
humans. The current classification of coronaviruses recognizes 39 species in 27 subgenera
that belong to the family Coronaviridae. From those, at least seven coronaviruses are known
to cause respiratory infections in humans. Four of these viruses can cause common cold-like
symptoms, while others that infect animals can evolve and become infectious to humans.
Three recent examples of this viral jumps include SARS CoV, MERS-CoV and SARS CoV-
2 virus. They are responsible for causing severe acute respiratory syndrome (SARS), Middle
East respiratory syndrome (MERS) and the most recently discovered coronavirus disease
during 2019 (COVID-19).
COVID-19, a respiratory disease caused by the SARS-CoV-2 virus, was declared a pandemic
by the World Health Organization (WHO) on 11 March 2020. The rapid spread of the disease
has taken the scientific and medical community by surprise. Latest figures from 14 April
2020 show more than 2 million people had been infected with the virus, causing more than
120,000 deaths in over 210 countries worldwide.

The large amount of information we receive every day concerning this new disease is so
abundant and dynamic that medical staff, health authorities, academics and the media are
not able to keep up with this new pandemic.
In order to offer a clear insight of the extensive literature available, we have conducted a
comprehensive literature review of the SARS CoV-2 Virus and the Coronavirus Diseases
2019 (COVID-19).

Key words: Covid-19; Coronavirus; SARS-CoV-2; Review; Pandemic;

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Background

The new COVID-19 disease is caused by a novel coronavirus (SARS-CoV2) probably

originated in Wuhan, China. In mid- December 2019, the Wuhan health authorities detected
few cases of an atypical pneumonia that eventually was discovered to be caused by a novel
coronavirus that probably jumped from an animal reservoir to a human during the first week
of November 2019 [1].
Subsequent investigations discovered that the etiological agent was a RNA virus related to
same family of coronavirus that caused the Severe Acute Respiratory Syndrome (SARS) and
to Respiratory Syndrome of Middle East (MERS) pandemic during 2003 and 2012
respectively[2].
The specific origin of this new pandemic is not totally understood. At the beginning of the
outbrake it was believed that a viral jump occurred between a wild animal and a human being
in one of the most populated wet market in Wuhan, China during the November 2019. Further
investigations were focused in finding which animals were responsible for this new zoonotic
diseases, although still unclear which animal is the intermediary host, is well-known that bats
are the main reservoirs for these type of virus and they probably emerged in one of the local
wild-animal farms[3, 4].

Chronology of the pandemic

The Center for Disease Control in China (CDCC) reported that during the last week of
December 2019, the first cases of an atypical pneumonia were seen in Wuhan, the capital of
Central China’s Hubei province. Days later, after the first cases were reported, the Chinese
health authorities decided to close the Huanan's “wet market” after some research suggested
this place as the probable initial source of contagion[5].
During the first week of January, China’s authorities announced that the new atypical
pneumonia was not caused by either the SARS or the MERS coronavirus, but a new variant
of the Coronaviriade family, a newly discovered virus called SARS-CoV2[5].
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In January 11th the first SARS CoV-2 related death was reported and one day later, a group
of Chinese researchers reveal the genome of the virus implicated in the Wuhan pneumonia
outbreak.
From the initial case reported in China, the SARS-CoV-2 virus spread worldwide. At the
beginning of the outbrake it started to move through Asia but only days later the first
suspicious cases were reported in Europe and North America. In March 11th the World
Health Organization (WHO) declare this disease a worldwide distributed pandemic. Since
the first case and using the latest figures from April the 14th , 2020 more than 2 million
people had been infected with the virus, causing more than 120,000 deaths in over 210
countries worldwide[6].

Structure and genome of the SARS-CoV-2 virion

The family Coronaviridae is a large group of viruses infecting animals and humans. There
are seven types of human coronaviruses that are primarily respiratory pathogens: 229E, NL63,
OC43, KHU1, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute
respiratory syndrome coronavirus (SARS-CoV), and severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2). MERS-CoV, SARS-CoV and SARS-CoV-2 belong to genus
Betacoronavirus and all have high mutation rates that result in viral genetic diversity,
plasticity, and adaptability to invade a wide range of hosts[7].

Like other coronaviruses, SARS-CoV-2 is an enveloped virus with roughly spherical or

moderately pleomorphic virions of approximately 60 to 140 nm in diameter (Figure 1a) [8].
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Figure 1 Overall structure and mechanism of infection of SARS-CoV-2. A) Structure and mechanism of infection of the novel
coronavirus into human cells through the spike glycoprotein, the ACE2 receptor protein, and the CD147 receptor. The
structure of the spike glycoprotein was taken from RCSB PDB 6VXX according to Walls et al. [9]; the structure of the ACE2-
BoAT1 complex was taken from RCSB PDB 6M17 according to Yan et al. [10]; lastly, the structure of the main protease
(Mpro) was taken from RCSB PDB 6Y84 according to Zhang et al. [11]. B) Genomic structure and proteins encoded by SARS-
CoV-2. C) Genomic structure and proteins encoded by SARS-CoV-2. B) Most frequent amino acid replacements in genomes
analyzed worldwide.

The viral membrane contains the spike (S) glycoprotein that forms the peplomers on the
virion surface, giving the virus its ‘corona’- or crown-like morphology in the electron
microscope. The membrane (M) glycoprotein and the envelope (E) protein provide the ring
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structure. Within the virion interior lies a helical nucleocapsid comprised of the nucleocapsid
(N) protein complexed with a single positive-strand RNA genome of about 30 kb in length
[12].

The first genome of SARS-CoV-2 named Wuhan-Hu-1 (NCBI reference sequence

NC_045512) was isolated and sequenced in China in January 2020 [8, 12]. The SARS-CoV-
2 genome has similarities to other viruses: approximately 96% similarity to the bat
coronavirus BatCoV RaTH13; an estimated 80% similarity with SARS-CoV [12], and an
estimated 50% identity with MERS-CoV [13, 14]. SARS-CoV-2 has a positive-sense single-
stranded RNA genome. It is approximately 30,000 bases in length and comprises of a 5′
terminal cap structure and a 3′ poly A tail. According to Wu et al. [15], this novel coronavirus
(IVDC-HB-01/2019 strain) has 14 open reading frames (ORFs) encoding 27 proteins. The 5’
terminus of the genome contains the ORF1ab and ORF1a genes. ORF1ab is the largest gene
and encodes the pp1ab protein that contains 15 non-structural proteins named nsps (nsp1-
nsp10 and nsp12-nsp16). ORF1a encodes the pp1a protein and also has 10 nsps (nsp1-nsp10)
[15]. The 3’ terminus of the genome contains four structural proteins: spike (S) glycoprotein;
envelope (E) protein; membrane (M) glycoprotein and nucleocapsid (N) phosphoprotein. It
also contains 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and ORF14) [16] (Figure 1b).

The global scientific community from 52 countries have united to study this novel
coronavirus by sequencing and submitting 2,500 SARS-CoV-2 genomes to the Global
Initiative on Sharing All Influenza Data (GISAID) (https://www.gisaid.org/) between
December 2019 and March 2020 [17, 18]. SARS-CoV-2 has accumulated mutations in its
RNA genome as the outbreak progresses.

From the 2,500 viral genomes of SARS-CoV02 sequences analyzed to date in the outbreak,
the CoV-GLUE project (http://cov-glue.cvr.gla.ac.uk/#/home) has identified 1,539 amino
acid replacements, 3 insertions, and 18 coding region deletions.. Regarding amino acid
replacements, 206 mutations were found in nsp3 (ORF1a) corresponding to the papain-like
protease (PLpro) / transmembrane domain 1; 146 were found in the S glycoprotein; 89 were
found in nsp2 (ORF1a); 61 were found in the N phosphoprotein; 59 were found in nsp12
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(ORF1ab) corresponding to the RNA-dependent RNA polymerase (RdRp); 56 were found in

nsp14 (ORF1ab) corresponding to the 3’-5’ exonuclease; 50 were found in nsp4 (ORF1a)
corresponding to the transmembrane domain 2; 48 were found in nsp13 (ORF1ab)
corresponding to the Zinc-binding domain / helicase domain; 43 were found in nsp15
(ORF1ab) corresponding to the endoRNAse; 39 were found in ORF3a, 41 were found in
nsp16 (ORF1ab) corresponding to the 2’-O-ribose methyltransferase; 41 were found in nsp6
(ORF1a) corresponding to the putative transmembrane domain; 38 were found in ORF7a; 27
were found in nsp5 (ORF1a) corresponding to the 3C-like proteinase; 29 were found in nsp1
(ORF1a); 26 were found in the M glycoprotein; 22 were found in ORF8; 17 were found in
nsp10 (ORF1a); 15 were found in ORF10 and ORF6; 14 were found in nsp8 corresponding
to the putative primase; 13 were found in the E protein; 9 were found in nsp9 corresponding
to the ssRNA-binding domain; 8 were found in nsp7; 7 were found in ORF7b; and 2 were
found in nsp11. The most prevalent amino acid replacements were D614G (S glycoprotein)
in 1,188 genomes, P323L (RdRp) in 1,181 genomes, L84S (ORF8) in 576 genomes, L37F
(putative transmembrane domain) in 415 genomes, and Y541C (Zinc-binding domain) in 402
genomes (Figure 1c and Supplementary Table 1).

SARS-CoV-2 replication cycle

As an intracellular obligate microorganism, the coronavirus exploits the host cell machinery
for its own replication and spread. Since virus–host interactions form the basis of diseases,
knowledge about their interplay is of great importance, particularly when identifying key
targets for antivirals.

SARS-CoV-2 entry into host cells is mediated by the transmembrane S glycoprotein that
forms homotrimers protrunding from the viral surface (Figure 1a) [9]. Coronavirus S protein
consists of two functional subunits: S1 subunit, where the receptor-binding domain (RBD) is
found and is responsible for binding host cell surface receptors and S2 subunit, which
mediates subsequent fusion between the viral and host cellular membranes [19, 20].
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SARS-CoV-2 RBD directly binds to the peptide domain of angiotensin-covering enzyme 2

(ACE2), which is also the cellular receptor for SARS-CoV [9, 10, 21, 22]. RBD is the most
variable part of SARS-CoV-2 genome [12, 23]. Six RBD amino acids (L455, F486, Q493,
S494, N501 and Y505) are involved in the binding to ACE2 receptors [24], and five of these
six residues differ between SARS-CoV and SARS-CoV-2 [25] (Figures 1a and 1b).

Figure 2 . SARS-CoV-2 replication cycle and its inhibitors. SARS-CoV-2 infection begins with the attachment of the spike (S)
protein with the host cell receptor. Two cellular receptors have been identified for SARS-CoV-2 so far: angiotensin-
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

converting enzyme 2 (ACE2) and CD147. After receptor interaction, the cleavage of S protein by the cell surface-associated
transmembrane protease serine 2 TMPRSS2 promotes the fusion of viral and cell membranes. Following the release of the
nucleocapsid to the cytoplasm, the viral genomic RNA is translated through ribosomal frameshifting to produce
polyproteins pp1a and pp1ab, which undergo cotranslational proteolytic processing into the 15 non-structural proteins
(nsp1-nsp10 and nsp12-nsp16) that form the replication-transcription complex (RTC). The RTC is involved in the genomic
RNA replication and in the transcription of a set of nested subgenomics mRNAs required to express the structural and
accessory protein genes. New virions are assembled by budding into the intracellular membranes of the ER - Golgi
intermediate compartment membranes and released through exocytosis. Additionally, there are detailed host-based
treatment options in blue and viral-based treatment options in pink.

ACE2 is a type I membrane protein that participates in the maturation of angiotensin, a

peptide hormone that controls vasoconstriction and blood pressure [26]. In the respiratory
tract, ACE2 is widely expressed on the epithelial cells of alveoli, trachea, bronchi, bronchial
serous glands [27], and alveolar monocytes and macrophages [28]. Xu et al. reported the [29]
RNA-seq profiling data of 13 organs with para-carcinoma normal tissues from The Cancer
Genome Atlas (TCGA; https://www.cancer.gov/tcga) and 14 organs with normal tissue from
FANTOM5 CAGE (https://fantom.gsc.riken.jp/). These were used to validate the expression
of the human cell receptor ACE2 in the virus and may indicate the potential infection routes
of SARS-CoV-2 [30]. Interestingly, the ACE2 receptor is expressed more in oral cavity than
lung. This potentially could indicate that susceptibility and infectivity of SARS-CoV-2 is
greater from oral mucosa surfaces. [29].

Following the binding of the RBD in the S1 subunit to the receptor ACE2, SARS-CoV-2 S
protein is cleaved by the cell surface-associated transmembrane protease serine 2 TMPRSS2,
which activates S2 domain for membrane fusion between the viral and cell membrane [31].
A functional polybasic (furin) cleavage site was found at the S1-S2 boundary through the
insertion of 12 nucleotides [9, 25, 32]. The S673, T678 and S686 residues of O-linked glycans
flank the cleavage site and are unique in SARS-CoV-2 [25].

In addition to the S glycoprotein - ACE2 receptor complex, Wang et al. reported an

alternative route where SARS-CoV-2 invades host cell through the S glycoprotein – CD147
complex. These findings were validated using co-immunoprecipitation, ELISA, and in vitro
antiviral tests with meplazumab. This anti-CD147 humanized antibody significantly
inhibited the viruses from invading host cells (https://doi.org/10.1101/2020.03.14.988345)
Paper: SARS-CoV-2 invades host cells via a novel route: CD147-spike protein.
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Like SARS-CoV and other coronaviruses, SARS-CoV-2 likely enter target cells through
receptor-mediated endocytosis, where fusion of the virus envelops the endosome membranes
and leads to the release of the viral nucleocapsid into the cytosol of the infected cell [33].

Following the release and uncoating of viral RNA to the cytoplasm, coronavirus replication
starts with the translation of ORF1a and ORF1b into polyproteins pp1a and pp1ab via a
frameshifting mechanism (Figure 2) [34]. Subsequently, polyproteins pp1a and pp1ab are
processed by internal viral proteases, including the main protease Mpro, a potential drug target
whose crystal structure was recently determined for SARS-CoV-2 [11]. Polyprotein cleavage
yields 15 mature replicase proteins, which assemble into a replication-transcription complex
that engages in negative-strand RNA synthesis. Both full-length and multiple subgenomic
negative-strand RNAs are produced. The former serves as template for new full-length
genomic RNAs and the latter template the synthesis of the subgenomic mRNAs required to
express the structural and accessory protein genes residing in the 3′-proximal quarter of the
genome [33]. Coronavirus RNA replication occurs on a virus-induced reticulovesicular
network of modified endoplasmic reticulum (ER) membranes [35].

The assembly of virions is quickly ensued with the accumulation of new genomic RNA and
structural components. The N protein complexes with genome RNA, forming helical
structures. Then, the transmembrane M protein, localized to the intracellular membranes of
the ER - Golgi intermediate compartment (ERGIC), interacts with the other viral structural
proteins (S, E and N proteins) to allow the budding of virions [36, 37]. Following assembly
and budding, virions are transported in vesicles and eventually released by exocytosis.

SARS CoV-2 and human immune responses

Normal immune responses against the majority of viruses involves a rapid containment
response mediated by innate immunity components. These include antiviral Type I IFNs,
pro-inflammatory cytokine production and NK cells, and a delayed virus-tailored adaptive
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immune response aiming to eradicate the pathogen and produce long-lasting memory. The
latter involves antigen specific CD8+ cytotoxic T cells (CTLs), the Th1 subset of CD4+ T
helper cells that orchestrates the immune response against viruses and other intracellular
pathogens, specific antibody producing plasma cells, and finally the production of memory
T and B cell subsets.

Immune system responses following SARS-CoV-2 infection can be a double-edged sword.

The response can lead to virus clearance and immune memory or, for others, cause severe
pathology that can lead to pneumonia, ARDS, septic shock, multi-organ failure and,
eventually, death.

Accordingly, patients who have immune system is weakened or otherwise dysregulated, such
as older men with comorbidities severe COVID-19 is clearly more likely to occur [38–40].

Innate immunity:

Type I IFNs are mainly produced by plasmacytoid dendritic cells (pDCs) and have a plethora
of antiviral effects such as blocking cell entry and trafficking of viral particles, inducing
RNase and DNase expression to degrade virus genetic material, enhancing presentation of
viral antigens by MHC-I, inhibiting protein synthesis and inducing apoptosis of infected cells
[41].

Pathogen recognition receptors like cytosolic RIG-I and MDA-5 [42, 43] or endosomal Toll
like receptors (TLRs) 7 and 8 that recognize viral RNA [44] are responsible for the activation
of signaling cascades that activate the transcription factors NF-kB, interferon regulatory
factor (IRF) 3 and IRF7 that translocate to the nucleus and induce proinflammatory cytokines
and Type I interferon (IFN) production. In turn, Type I IFNs activate the downstream JAK-
STAT signal pathway resulting in expression of IFN-stimulated genes (ISGs) [45, 46].

Our experience from SARS-CoV and MERS-CoV infection has shown that delayed type I
IFN production and excessive recruitment and activation of infiltrating proinflammatory cells
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

(neutrophils and monocytes-macrophages) are possible mediators of lung dysfunction and

bad prognostic factors for the outcome of the infection. Delayed type I IFN production allows
for highly efficient viral replication that, in turn, results in recruitment of hyperinflammatory
neutrophils and monocytes. Therefore, the pathogen recognition receptors (PRRs) of these
proinflammatory cells recognize high numbers of their ligands and subsequently secrete
excessive amounts of proinflammatory cytokines that lead to septic shock, lung pathology,
pneumonia or acute respiratory distress syndrome [47–49].

It has been shown that in severe cases both SARS-CoV and MERS-CoV fruitfully employ
an immune evasion mechanism whereby early type I IFN responses to viral infection are
dampened [48]. This can be achieved by blocking signaling both upstream, as well as
downstream of type I IFN expression. SARS-CoV can inhibit IRF3 nuclear translocation,
whereas MERS-CoV can impede histone modification [50]. Additionally, both viruses can
inhibit IFN signaling by decreasing STAT1 phosphorylation [51]. Due to the many sequence
similarities of SARS-CoV-2 with SARS-CoV and MERS-CoV it would be enticing to
speculate that similar mechanisms are also present, however further studies are needed to
shed light to this hypothesis.
Hyperactivated neutrophils and monocytes-macrophages are the usual source of the cytokine
storm. In this aspect, absolute neutrophil counts and neutrophil to lymphocyte ratio (NLR)
were strongly associated with disease severity in a large cohort of COVID-19 patients and
were proposed as markers of adverse disease prognosis [52].

Interestingly, the increased amounts of proinflammatory cytokines in serum associated with

pulmonary inflammation and extensive lung damage described both in SARS [53] and MERS
diseases [54] were also reported in the early study of 41 patients with COVID-19 in Wuhan
[39]. Evidence shows that the leading cause of COVID-19 mortality is respiratory failure
caused by acute respiratory distress syndrome (ARDS). There is an association with a
cytokine storm mediated by high-levels of proinflammatory cytokines including IL-2, IL-7,
IL-10, G-CSF, IP-10, MCP-1, MIP-1A and TNF-α. ARDS was associated with increased
fatality and subsequent studies confirmed IL-6 and C-reactive protein are significantly
upregulated in patients that died compared to convalescent patients [50]. Moreover, a recent
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

study of 452 patients in Wuhan identified that severe cases showed significantly higher
cytokines and chemokines such as tumor necrosis factor-α (TNF-α), IL-2, IL-6, IL-8 and IL-
10 expressed [52].

In accordance with these findings, therapeutic strategies are being tested. A phase 3
randomized controlled trial of IL-1 blockade (anakinra) in sepsis has shown significant
survival benefit in patients with hyperinflammation, without apparent increased adverse
events [55]. Currently, a multicenter, randomized controlled trial of tocilizumab (IL-6
receptor blockade, licensed for cytokine release syndrome), is being trialled in patients with
COVID-19 pneumonia presenting with high levels of IL-6 in China (ChiCTR2000029765)
[56]. Moreover, several clinical trials are exploring if the well-established antiviral [57] and
anti-inflammatory effects of hydroxychloroquine will be effective in treating patients with
COVID-19 as has previously been suggested for SARS-CoV infection [58]. This has also
been demonstrated in vitro for SARS-CoV-2 [59]. In contrast, Janus kinase (JAK) inhibition
has been proposed as a potential treatment in order to reduce both inflammation and cellular
viral entry in COVID-19 [60]. Thus, it comes as no surprise that in a recent correspondence,
Lancet authors have identified the following potential therapeutic options for cytokine storm
syndrome including ARDS the use of corticosteroids, selective cytokine blockade (eg,
anakinra or tocilizumab) and JAK inhibition [61].

Adaptive immunity:

Virus presentation to the different T cell subsets stands on the crossroads between innate and
adaptive immune responses. Studies on SARS-CoV [62–65] and MERS-CoV [66]
presentation have identified several susceptibility and protection conferring HLA alleles. The
dearth of similar data regarding SARS-CoV-2 antigen presentation to T cells and possible
virus evasion mechanisms of this process suggests it is a virgin investigation field to be
explored.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Apart from the sustained inflammation and cytokine storm, lymphopenia has been implicated
as a major risk factor for ARDS and mortality in the context of COVID-19 [67]. Similar
findings were described for SARS-CoV infected patients who had considerable decreases of
CD4+ T and CD8+ T cells [63]. However, in convalescent patients specific T-cell memory
responses to SARS-CoV were still found six years post infection [68]. Though it is still very
early to trace memory responses against SARS-CoV-2, the observations linking lymphopenia
with severe pathology are similar to patients diagnosed with severe acute respiratory
syndrome (SARS) during the 2003 epidemic.

In a study of 452 Chinese patients in Wuhan, severe cases tended to have lower lymphocyte
counts. This dearth of lymphocytes was mainly attributed to significantly lower T cell counts
in severe cases. Numbers of CD8+ T lymphocytic cells responsible for recognizing and
killing infected cells were found to be significantly lower in patients with severe
manifestations of COVID-19. Additionally, severely affected patients presented with a
higher naïve CD4+ to memory T cells ratio, suggesting that the adaptive immune system in
the severe infection subgroup was less activated. Furthermore, these patients had less
numbers of regulatory T cells (Tregs), especially induced Tregs. Tregs form the T cell subset
responsible for controlling excessive inflammatory responses and their absence can lead to
production of cytokine storm and enhancement of tissue pathology. Overall, this data suggest
that dysregulation of T cell mediated immune responses may play a pivotal role in COVID-
19 pathogenesis and severity [52].

Production of protective antiviral antibodies and long-lived memory B cells are fundamental
for avoiding reinfection with the virus and form the basic principles behind vaccination. Less
research has been completed relating to humoral immunity compared to than cellular against
coronaviruses. However, in view of COVID-19 patient sera portraying some cross-reactivity
with SARS-CoV, but not with other coronavirus, it might imply that similar mounting of
humoral responses could be expected [8]. Studies conducted during the SARS epidemic have
revealed that seroconversion is induced as early as day 4 after disease onset and that IgG
protective antibodies lasted for as long as 2 years after infection [69] Anti-SARS-CoV IgM
in turn disappeared after 12 weeks [70].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Preliminary data suggests that humoral responses are robust and follow a similar pattern. A
study including 173 COVID-19 positive Chinese patients showed that 93.1% of the patients
demonstrated anti-SARS-CoV-2 seroconversion. There was no late stage data available for
the remainder of patients. Anti-SARS-CoV-2 antibodies were detected as early as 4 days’
post disease onset, with a median time of positivity for IgM and IgG seroconversion being
11- and 14-days after disease onset, respectively. Interestingly, high antibody levels were not
always found to be enough to clear the virus, as critically ill patients were found to have
significantly higher virus specific antibody titers. However, the authors argue that combining
viral nucleic acid and seroconversion detection significantly raised the detection sensitivity
for patients [71]. Another recent study where a new ELISA assay for anti-SARS-CoV-2
specific antibody detection was developed reported the existence of IgA specific antibody in
patients’ serum apart from the expected IgM and IgG isotypes. Notably, among IgG subtypes
tested IgG3 exhibited the highest reactivity followed by IgG1, while IgG4 showed no
reactivity with viral antigens. However, the small number of sera used (n=4) implies that
further investigation is needed to corroborate these results [72]. Nonetheless, since we are
currently in early stages of SARS-CoV-2 pandemic more studies need to be carried out to
shed light on antibody persistence (both IgM and IgG) and protective effects.

Recently, macaques re-challenged with SARS-CoV-2 after a primary infection did not show
signs of re-infection, suggesting that protective immunity and memory responses were
fruitfully mounted. This finding can also impact vaccine production strategies [73].

Importantly, COVID-19 convalescent sera was shown to hold promise as a passive immune
therapy alternative to facilitate disease containment [74]. To the best of our knowledge, at
least one pharmaceutical company, Takeda, is preparing to purify antibody preparations from
COVID-19 convalescent sera against SARS-CoV-2 [75].
A recently published case report of a patient with mild-to-moderate COVID-19 revealed the
presence of an increased activated CD4+ T cells and CD8+ T cells, antibody-secreting cells
(ASCs), follicular helper T cells (TFH cells), and anti-SARS-CoV-2 IgM and IgG antibodies,
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

suggesting that both cellular and humoral responses are important in containing the virus and
inhibiting severe pathology [76].

Antibody dependent enhancement (ADE) is a mechanism whereby non-protective antibodies

produced during an infection with an agent cross-recognize a different pathogen and facilitate
its entrance to target cells [77]. Evidence emerging over the past two decades suggests that
antibodies against different coronavirus can cross-react to some extent and mediate ADE [78].
ADE in the context of SARS-CoV was thought to be mediated by antibodies produced
against 229E-CoV [79] and was contemplated as contributing to high mortality rates in China
[80]. The described mechanism suggests that anti-Spike protein antibodies mediate the
infection of immune cells, further aggravating the dysregulation of anti-SARS-CoV immune
responses [81]. Indeed, in vitro as well as in vivo experimental models have shown that ADE
hinders the ability to manage inflammation in the lung and elsewhere. This may lead to
ARDS and other hyperinflammation-induced clinical manifestations also observed in several
of the documented cases of severe COVID-19 [82, 83]. While the molecular and
immunological host response to SARS-CoV-2 infection has not yet been fully elucidated to
confirm ADE is occurring, anti-SARS-CoV-2 have been shown to partially cross-react with
SARS-CoV, suggesting enhancement is a possibility. With this in mind, ADE in populations
previously exposed to other coronavirus can partially explain the geographic discrepancies
observed in COVID-19 pathogenesis and severity.

Molecular diagnosis methods to detect COVID-19

RT-qPCR:
Detection methods based on nucleic amplification tests (NAT) are usually preferred in the
case of MERS-CoV and other viruses, because they have demonstrated the highest sensitivity
at the earliest time point in the acute phase of infection [84]. Detection and surveillance of
COVID-19 spread is currently carried out by one-step quantitative RT-PCR (RT-qPCR)
targeting SARS-CoV-2 sequences. Recently, the WHO compiled a list of various protocols
for detection of SARS-CoV-2, developed by researchers in China, Germany, Hong Kong,
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Japan, Thailand, France, and USA (WHO 2020). Relative positions of RT-qPCR primer-
probe sets on the SARS-CoV-2 genome are shown in Figure 3 and detailed in Table 4.

Figure 3 Relative positions of qRT-PCR primer-probe set on the SARS-CoV-2 listed by WHO. Institut Pasteur, Paris, France
(nCoV_IP2, IP4 and E), China CDC (Orf1ab and N), Charité universitätsmedizin Berlin institute of virology in Germany
(RdRp_SARSr and E), the University of Hong Kong (HKU-ORF1b_nsp14 and HKU-N), USA CDC (2019-nCoV_N1, N2,
and N3), National Institute of Health in Thailand (WH-NIC N), National Institute of Infectious Disease in Japan
(NIID_2019-nCoV_N). Orf1: open reading frame 1; RdRp: RNA-dependent RNA polymerase gene; Nsp14:
non-structural protein 14 gene; S: spike protein gene; E: envelope protein gene, N: nucleocapsid protein gene.
The number below amplicons are genome positions according to SARS-CoV-2, GenBank MN908947.3

Although RT-qPCR assay is considered the gold-standard method to detect viruses such as
SARS-CoV and MERS-CoV [85, 86], currently available RT-qPCR assays targeting SARS-
CoV-2 have important considerations. Firstly, due to the genome similarity of SARS-CoV-2
to SARS-CoV (82% of nucleotide identity [87]), some of the primer-probe sets described by
different groups and listed in the WHO Coronavirus disease (COVID-19) technical guidance
[88], have cross-reaction with SARS-CoV and other bat-associated SARS-related viruses,
therefore, it is important to run confirmatory tests.

Table 1 Information of primers and probes recommended by WHO.

Target Country Institute Name Position Reference

RdRp/Orf 1 China China CDC ORF1ab - F 13342 - 13362 [89]
ORF1ab - R 13442 - 13460
ORF1ab - P 13377 - 13404
Germany Charité RdRp_SARSr - F 15431 - 15452 [90]
RdRp_SARSr - R 15505 - 15530
RdRp_SARSr - P2 15470 - 15494
Hong Kong HKU HKU - ORF1b - nsp14F 18778 - 18797 [91]
HKU - ORF1b - nsp14R 18889 - 18909
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HKU - ORF1b - nsp14P 18849 - 18872

France Institut nCoV_IP2-12669F 12621- 12641 [92]
Pasteur, Paris nCoV_IP2-12759R 12727 - 12747
nCoV_IP2-12696bP 12696 - 12716
nCoV_IP4-14059F 14010 - 14030
nCoV_IP4-14146R 14116 - 14136
nCoV_IP4-14084P 14084 - 14104
N Japan National NIID_2019 - nCOV_N_F2 29125 - 29144 [93]
Institute of NIID_2019 - nCOV_N_R2 29263 - 29282
Infectious NIID_2019 - nCOV_N_P2 29222 - 29241
Diseases
Thailand National WH - NIC N - F 28320 - 28339 [94]
Institute of WH - NIC N - R 28358 - 28376
Health WH - NIC N - P 28341 - 28356
USA CDC 2019 - nCoV_N1 - F 28287 - 28306 [95]
2019 - nCoV_N1 - R 28335 - 28358
2019 - nCoV_N1 - P 28309 - 28332
2019 - nCoV_N2 - F 29164 - 29183
2019 - nCoV_N2 - R 29213 - 29230
2019 - nCoV_N2 - P 29188 – 29210
2019 - nCoV_N3 - F 28681 - 28702
2019 - nCoV_N3 - R 28732 - 28752
2019 - nCoV_N3 - P 28704 - 28727

Most of the tests enlisted in this review are currently available for use under an EUA by the
FDA, a policy that aims to quicken the approval process for US labs developing tests for
COVID-19. The approval is part of a concerted effort to make up for a lost time after delays
and then a global shortage of the essential chemicals needed to make new tests (Table 5).

Table 2 Commercially Available COVID-19 Diagnostic Tests with EUA status

Company/ Test Name Instrument Test type Time Ref.

Organization
Carbon Health COVID-19 Home Test Kits NA PCR 3 hours [96]
IDbyDNA Explify Platform for respiratory diseases NA NGS 24 hours [97]
Cepheid Xpert® SARS-CoV-2 GeneXpert® System PCR 45 minutes [98]
Roche cobas SARS-CoV-2 Test Cobas 6800 and 8800 PCR 4 hours [99]
Abbott Abbott RealTime SARS-CoV-2 EUA test m2000 RealTime PCR 1200 in 24 [100]
system hours
CDC USA CDC 2019-Novel Coronavirus (2019- NA PCR 4 hours [101]
nCoV) Real-Time RT-PCR Diagnostic
Panel (CDC)
DiaSorin Molecular Simplexa COVID-19 Direct LIAISON® MDX PCR 6 hours [102]

Thermo Fisher TaqPath COVID-19 Combo Kit Applied Biosystems PCR 3.5 hours [103]
7500
Hologic Panther Fusion® SARS-CoV-2 test, Panther Fusion ® PCR 1150 in 24 [104]
System, hours
Quidel Lyra SARS-CoV-2 Applied Biosystems PCR 75 minutes [105]
7500 Fast DX
GenMark Diagnostics. ePlex SARS-CoV-2 Test ePlex system PCR 2 hours [106]
Integrated DNA IDT 2019-novel coronavirus kit NA PCR 5 hours [107]
Technologies
LGC, Biosearch 2019-nCoV CDC-qualified Probe and NA PCR - [108]
Technologies Primer Kits for SARS-CoV-2
Wadsworth Center New York SARS-CoV-2 Real-time RT- NA PCR - [109]
PCR Diagnostic Panel
Quest Diagnostics Coronavirus Disease 2019 (COVID-19) NA PCR 4 days [107]
Test
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BioMérieux/BioFire BioFire COVID-19 test Filmarray® 2.0 and PCR 45min [107]
Defense Torch
Laboratory Corporation LabCorp 2019 Novel Coronavirus test NA PCR 4 hours [110]
of America
Novacyt/Primerdesign COVID-19 Genesig Real-Time PCR NA PCR - [111]
assay
PerkinElmer PerkinElmer New Coronavirus Nucleic NA PCR - [112]
Acid Detection Kit
Abbot ID NOW™ COVID-19 test ID NOW platform Isothermal 5 min. [100]
amplification
BGI Real-Time Fluorescent RT-PCR kit for NA PCR 3 hours [111]
detecting SARS-2019-nCoV
Cellex qSARS-CoV-2 IgG/IgM Rapid Test NA Serological 10 min. [113]
Ipsum Diagnostics COV-19 IDx assay NA PCR 4 hours [114]
Luminex Molecular NxTAGCoV Extended Panel Assa ARIES® M1 Systems PCR 4 hours [115]
Diagnostics
Mesa Biotech Accula SARS-CoV-2 test Accula System PCR 30 min. [116]
NeuMoDx Molecular NeuMoDx SARS-CoV-2 Assay NeuMoDx™ Molecular PCR 80 min. [117]
Systems
Qiagen QiaStat-Dx Respiratory SARS-CoV-2 QIAstat-Dx Analyzer, PCR 1 hour [118]
Panel

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

Loop-mediated isothermal amplification (LAMP) is a one-step isothermal amplification

reaction that couples amplification of a target sequence with four to six primers, to ensure
high sensitivity and specificity, under isothermal conditions (63-65°C), using a polymerase
with high strand displacement activity[119]. In the case of an RNA sample, LAMP, is
preceded by the reverse transcription of the sample RNA. RT-LAMP has been used before
for the detection of various pathogens[120]. including SARS-CoV-2 [53,54] and other
respiratory viruses[121, 122]. Recently, it received emergency use authorization (EUA) from
the U.S. Food and Drug Administration (FDA) for a point-of-care test for the detection of
novel coronavirus (COVID-19), delivering positive results in as little as five minutes and
negative results in 13 minutes[100].

Serological tests:

Serological tests also, called immunoassays, are rapid and simple alternatives for screening
of SARS-CoV-2 infected individuals based on the qualitative or quantitative detection of
SARS-CoV-2 antigens and/or anti-SARS-CoV-2 antibodies. There are several types of
serological tests available, including ELISA (enzyme-linked immunosorbent assay), IIFT
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(indirect immunofluorescence test) and neutralization tests. Immunoassays assays are very
useful because they allow us to study the immune response(s) to SARS-CoV-2 in a
qualitative and quantitative manner. In addition, help us to determine the precise rate of
infection [72, 123], and to determine the fatality rate of the infection [72]. Several SARS-
CoV-2 targeted serological tests are commercially available or in development [124]. A
recently developed kit, reported a sensitivity of 88.66% and specificity of 90.63% [125] using
SARS-CoV-2 IgG-IgM combined antibody rapid (within 15 minutes) test [125]. Despite their
simple and fast readout and their potential for being used outside laboratory environments
(bedside, small clinics, airports, train stations, etc.), serological tests have a critical
disadvantage; given the fact that antibodies specifically targeting the virus would normally
appear after 6 days or longer [126] after the illness onset [127], tests based on this principle
have a lag period of approximately 4 to 7 days post-infection. During this lag period, infected
and non-infected individuals will both result in a negative output. In addition, it is important
to highlight that because serological tests depend on the ability to produce antibodies,
intrinsic immunological differences and/or responses between individuals, can significantly
affect the outcome of these tests. Recently, some commercially available immunoassays
received CE Mark for professional use [128, 129], and therefore are registered as in vitro
diagnostic devices.

Alternative methods:

Even though COVID-19 can be diagnosed using qPCR as the gold standard, inadequate
access to reagents and equipment has slowed disease detection even in developed countries
such as the US. Several low cost and rapid tests using different approaches have been
described.
The CRISPR-based SHERLOCK (Specific High Sensitivity Enzymatic Reporter
UnLOCKing) technique for the detection of COVID-19 and the DETECTR (developed by
Mammoth Biosciences) prototype rapid detection diagnosis kit using CRISPR to detect the
SARS-COV-2 in human samples have been described[130].
The use of RNA aptamers, have recently emerged as a powerful background-free technology
for live-cell RNA imaging due to their fluorogenic properties upon ligand binding, a
technology that could be use to diagnose SARS-CoV-2 infection [131].
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Finally the use of next generation sequence (Explify®) might be used to detect and identify
bacterial, viral, fungal, and parasitic pathogens by their unique genome sequences[97].

Clinical features of COVID-19

In COVID-19 symptomatic infection, the clinical presentation can range from mild to critical
scenarios. The symptoms of a lower respiratory infection, pneumonia, is the most serious
manifestation of COVID-19 infection.

Studies derived from the Wuhan population have established the most common clinical
characteristics at the beginning of the disease: fever, fatigue and cough [132]. Other
descriptive studies of Wuhan patients with confirmed COVID-19 have reported a similar
range of clinical findings. In cohorts of patients outside of Wuhan, this clinical behavior is
similar. At Zhejian province cohort of 62 people, only 1 case required mechanical ventilation
assistance [133].

Evolution of the disease: spectrum of clinical manifestations

The spectrum of symptoms of COVID19 infection are characteristic of a mild disease in most
of the cases, however, there is important to point that the progression could lead to a severe
respiratory distress.

Asymptomatic infection:

Asymptomatic infection (while incubation occurs) was described both in the first cases in
Wuhan and in other cohorts. A group of isolated patients were screened for SARS-CoV-2,
where 17% (629 cases) were positive for the test, and half of these cases had no symptoms.
On the other hand, there are reports of cases without overt symptoms in which there were
ground glass images in the chest tomography in up to 50% of patients [134].
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Of the asymptomatic cases studied in Wuhan city, the 2.5% of people exposed developed
specific symptoms in 2.2 days, and the remaining 97.5% were symptomatic in the following
11.5 days (CI, 8.2 to 15.6 days). The median estimated incubation period was 5.1 days (95%
CI, 4.5 to 5.8 days) [135].

Acute infection: mild and moderate

Some patients with initially mild symptoms had symptom progression over the course of one
week [136]. The descriptive studies available so far have concluded that the majority of cases
are mild infections (more than 80% of cases); with up to 15% of patients being sever in most
cohorts, and less than 5% have been considered as critical cases with high vital risk [137].

In a study describing 138 patients with COVID-19 pneumonia in Wuhan, the most common
clinical characteristics at the onset of the disease were described. This is consistent with
other international cohorts (Table 1) [132].

Table 3 Clinical Manifestations of COVID 19 infection.

Clinical manifestations ICU*

Presentation n=138 Non-ICU n=102
n=36
n (%) n (%)
n (%)
Fever 136 (98.6) 36 (100) 100 (98)
Fatigue 96 (70) 29 (80.6) 67 (65.7)
Dry Cough 82 (59.4) 21 (58.3) 61 (59.8)
Anorexia 55 (40) 24 (66.7) 31 (30.4)
Myalgia 48 (34.8) 12 (35.3) 36 (35.3)
Dyspnea 43 (31.2) 23 (63.9) 20 (19.6)
Sputum production 37 (27) 8 (22.2) 29 (28.4)
Pharyngalgia 24 (17.4) 12 (33.3) 12 (11.8)
Diarrhea 14 (10.1) 6 (16.7) 8 (7.8)
Nausea 14 (10.1) 4 (11.1) 10 (9.8)
Dizziness 13 (9.4) 8 (22.2) 5 (4.9)
Headache 9 (6.5) 3 (8.3) 6 (5.9)
Abdominal pain 5 (3.6) 3 (8.3) 0 (0)
Vomiting 5 (3.6) 3 (8.3) 2 (2.0)

*ICU: intensive care unit

Source: Wang D et al., 2020 [132].
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It is important to note that fever is not always present and up to 20% of patients could had a
low grade temperature between 37.5 to 38 degrees Celsius or normal temperature. [138] If
these patients required hospitalization, 89% developed a fever during the course of the illness.
Rarer accompanying symptoms included headache without warning signs, odynophagia and
rhinorrhea. Gastrointestinal symptoms such as nausea and watery diarrhea were relatively
rare [133].

Dyspnea develops after a median of 5 to 8 days from the onset of symptoms. It is important
to notice that, if dyspnea is an important clinical finding, not all the patients with this
symptom will develop respiratory distress ore require oxygen supplementation [132].

According to World Health Organization (WHO) guidelines, COVID-19 infection can

present as pneumonia without signs of severity, and could be managed in the outpatient
setting; this applies to those patients who do not need supplemental oxygen [139].

Severe infection and critical state:

As previously mentioned, the most serious manifestation of COVID 19 infection is

pneumonia, characterized by cough, dyspnea, and infiltrates on chest images; the latter is
indistinguishable from other viral lung infections.

Acute respiratory distress syndrome (ARDS) is a major complication of COVID pneumonia

in patients with severe disease. This develops in 20% after a median of eight days.Mechanical
ventilation is implemented in 12.3% of cases [140].

In different case reports, the need for supplemental oxygen via the nasal cannula was required
in approximately 50% of hospitalized patients. 30% required non-invasive mechanical
ventilation, and less than 3% required invasive mechanical ventilation with or without
Extracorporeal Membrane Oxygenation (ECMO) [141].
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It is important to mention that the proportion of severe cases is highly dependent on the study
population and may be related to the epidemiological behavior of the infection in each
country. Additionally, the number of people tested will influence the denominator. In Italy,
the average age of people infected with COVID-19 is between 60 and 65 years, and 16% of
those hospitalized require admission to the intensive care unit (ICU) [142].

The WHO recommendations had stablished that severe COVID-19 disease could be defined
by the following parameters in table 2 [139].

Table 4 Severe COVID-19 disease definitions in adults

Clinical scenario Criteria

Adolescent or adult: fever Respiratory rate > 30 breaths/min

or suspected respiratory Severe respiratory distress; or
infection, plus one of: SpO2 ≤ 93% on room air.
Acute respiratory distress Chest imaging (radiograph, CT scan, or lung ultrasound): bilateral opacities, not fully explained
syndrome (ARDS): by volume overload, lobar or lung collapse, or nodules.
Onset: within 1 week of a Origin of pulmonary infiltrates: respiratory failure not fully explained by cardiac failure or fluid
known clinical insult or overload.
new or worsening Need objective assessment (e.g. echocardiography) to exclude hydrostatic cause of
respiratory symptoms. infiltrates/edema if no risk factor is present.
Oxygenation Mild ARDS: 200 mmHg < PaO2/FiO2 a ≤ 300 mmHg (with PEEP
impairment in adults or CPAP ≥ 5 cmH2O, or non-ventilated)
Moderate ARDS: 100 mmHg < PaO2/FiO2 ≤ 200 mmHg (with
PEEP ≥ 5 cmH2O, or non-ventilated)
Severe ARDS: PaO2/FiO2 ≤ 100 mmHg (with PEEP ≥ 5 cmH2O,
or non-ventilated).
When PaO2 is not available, SpO2/FiO2 ≤ 315 suggests ARDS
(including in non-ventilated patients).
Adapted from: WHO, 2020. Clinical management of severe acute respiratory infection (SARI) when
COVID-19 disease is suspected [143].

The Surviving Sepsis Campaign (SSC) has directed some recommendations to the population
with COVID 19. This guideline focuses on the critical management of severe cases and
makes recommendations through an exhaustive review of the literature. For more details, the
clinical algorithm includes those recommendations in the critical scenario [144].

Risk factors for severe disease:

Among the established risk factors for the development of ARDS is age greater than 65 years,
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diabetes mellitus and hypertension, in at least 40% of patients [133].

It should be clarified that, although advanced age is identified as a risk factor for a severe
infection, those of any age may suffer from severe illness from COVID-19. The descriptions
made so far of the patients from China have determined that almost 90% of the patients were
between the ages of 30 and 79 years (cohort of 44,500 cases) [137].

In other population settings, such as in the United States, more than 60% of confirmed
patients were older than 45 years. (CDC, et al. 2020) In most of the described cohorts,
mortality was associated with age, with 80% of the deceased in China being over 65 years
old, and in the USA the case fatality rate was up to 15% in adults over 70 years.

The Massachusetts General Hospital has suggested additional factors that can be considered
risk for severe COVID 19 infection, detailed in Table 3 [145].

Table 5 Risk factors for severe COVID-19 infection Adapted from: Ginsberg, L. E. (2010). “If clinically indicated:” Is it?
Radiology, 254(2), 324–325. https://doi.org/10.1148/radiol.09091736

Epidemiological - Category 1 Vital signs – Category 2 Laboratory – Category 3

Age > 55 years Respiratory rate > 24 breaths/min D-Dimer > 1000ng/mL
Diabetes Mellitus Heart rate > 125 beats/min CPK > 2 folds over upper limit
Hypertension and high cardiovascular risk Spo2 < 90% at room air LDH > 245 U/L
Immunosuppression and use of biological Elevated troponin
drugs
HIV patients regardless CD4 count High Troponin
Lymphocyte count < 0.8
Ferritin > 300ug/L

The document was developed by the Infectious Diseases division in conjunction with the
front-line support departments. Their recommendations are continually updated as more data
comes out.
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Figure 4 Clinical features of patients with Covid-19

Clinical diagnosis and screening:

The clinical characteristics of symptomatic cases and their severity has been described. In
addition to the symptoms reported by the patients, the findings on physical examination may
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be absent during mild COIVD-19 infection. Those with moderate to severe COVID-19
infection have various signs on the pulmonary auscultation, however the most common
findings include: wet rales; global decrease in respiratory sounds and increased thrill. [146].

Early recognition is essential to classify cases as potential cases and initiate one of the most
important measures to contain the pandemic, isolation.

The Center of Disease Control (CDC) and the WHO have established clinical scenarios that
should be considered as a high suspicion of COVID 19 infection:

1. Close contact with a confirmed or suspected COVID-19 case, including through work
in healthcare settings. Close contact includes being within approximately two meters
of a patient for an extended period of time without wearing personal protective
equipment or having direct contact with infectious secretions without wearing
personal protective equipment.
2. Anyone who has resided or been traveling in areas where widespread community
transmission has been reported.
3. Any patient who has had potential exposure through attending events or has spent
time in specific settings where cases of COVID-19 have been reported.

The scenarios described respond to the context of a high suspicion of COVID-19 infection.
The world health authorities (CDC, WHO) continually update these contexts, that is why
they have made several clarifications regarding who to perform the test:

• They have pointed out the importance of fever, cough and dyspnea as sentinel
symptoms, since these should form part of the clinical judgment that guides doctors.
This allows to expand the group of suspicious patients.
• In cases of severe respiratory distress of undetermined etiology and that do not meet
the previously indicated criteria, a screening for COVID-19 would be indicated.
• In areas of limited resources, the suggestion is to prioritize cases that require hospital
care, and in this way guide the epidemiological fence to order isolation and protect
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the most vulnerable people (chronically ill and over 65 years of age), as well as test
those with the greatest possibility of exposure (travelers and health personnel).

Laboratory findings:

At the moment, there is no laboratory data profile that is framed in COVID 19 infection.
From a cohort of 43 patients confirmed with COVID 19, these findings were classified as
mild, moderate and severe disease [147].

IL-6, D-Dimer, glucose, TTP, fibrinogen and PCR values were associated with the greatest
difference in the deviation of their values. Thus, the optimal threshold and area under the
ROC curve for IL-6 were 24.3 pg / mL and 0.795 respectively, while for D-Dimer they were
0.28 µg / L and 0.750, respectively. The area under the ROC IL-6 curve (AUC) combined
with D-Dimer was 0.840. The specificity of IL-6 and D-Dimer was up to 93.3%, while the
sensitivity of IL-6 and D-Dimer in severe COVID was 96.4%, especially in early stages of
severe infection.

High levels of D-dimer and more severe lymphopenia have been associated with mortality
due to a prothrombotic state that determines multi-organ failure.

In general, leukopenia and / or leukocytosis can be found in the interpretation of blood

biometry, however, the most widely described finding is lymphopenia [148]. It should be
considered that in the context of viral pneumonia biomarkers such as Procalcitonin and PCR
are not useful, in most patients since these biomarkers are in ranges of the normal.

Among other findings, descriptive studies have reported considerable elevations of lactate
dehydrogenase and ferritin as well as alteration in aminotransferases; although elevation
ranges for these parameters have not been established [149].
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Imaging findings:

About the imaging findings, COVID 19 viral pneumonia shows images similar to other viral
infections.

Although computed tomography (CT) is the test of choice, it is not useful for a definitive
diagnosis due to the wide variety of images that can be found in COVID 19 infection. This
statement is derived from a large cohort of more than 1000 Wuhan patients, where RT-PCR
confirmation of COVID 19 and chest CT images of these patients were correspondingly
analyzed. CT images were determined to have a sensitivity of 98%; however, the specificity
was only 25% [150].

In general, the majority of descriptive studies concur that the finding of ground glass
opacifications is most common. It is typically basal and bilateral, and rarely associated with
underlying consolidation. A multicenter Chinese study that retrospectively reviewed the CT
scans of 101 patients found that 87% had typical ground-glass images and up to 53% had this
finding along with consolidations. These findings were more frequent in the most severe and
older age groups of patients [151].

These findings were compared between 205 viral pneumonia patients with a respiratory panel
positive for other viruses versus 219 SARS-CoV-2 positive patients. The most uncommon
findings on CT images of patients with COVID 19 were: central distribution of opacifications
(14%), air bronchogram (14 %), pleural thickening (15%), pleural effusion (4%), and
lymphadenopathy (2.7%) [152].

Diagnosis methods to detect COVID-19

The emergence and outbreak of SARS-CoV-2, the causative agent of COVID-19, has rapidly
become a global concern that highlights the need for fast, sensitive, and specific tools to
surveil the spread of this infectious agent.
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Diagnostic protocols to detect SARS-CoV-2 using real-time quantitative polymerase chain

reaction (RT-qPCR) were listed on the World Health Organization (WHO) website as
guidance, however, various institutions and governments have chosen to establish their own
protocols that might not be publicly available or listed by WHO.

There are important challenges associated with close surveillance of the current SARS-CoV-
2 outbreak. Firstly, the rapid increase of cases has overwhelmed diagnostic testing capacity
in many countries, underscoring the need for a high-throughput, scalable pipeline for sample
processing [153, 154]. Secondly, given that SARS-CoV-2 is closely related to other
coronaviruses [87], some of the currently available nucleic acid detection assays can result
in false positives [155]. Thirdly, critical concern for molecular detection is the low sensitivity
reported for RT-qPCR assays [150] and serological tests [125], particularly in the early stages
of infection. Additionally, most of the available RT-qPCR assays require sample processing
and equipment only available in diagnostic and/or research laboratories.

The most common tests for COVID-19 involve taking a swab from a patient’s nose and throat
and checking these swabs for the genetic footprint of the virus. They are called “PCR tests”.
The first PCR test for COVID-19 was developed within two weeks of the disease being
identified[125].

Even though most of the available diagnostics have focused on RT-PCR, additional methods
include using microarray or microfluidic technologies, CRISPR to isolate gene segments for
diagnostics, serological and full genetic sequencing are available. It is important to note that
the FDA has so far granted Emergency Use Authorization (EUA) status only to some PCR-
based tests.

Differential diagnosis

COVID-19 pneumonia presents a clinical picture that, as previously stated, may be

indistinguishable from other viral pneumonias. Any viruses that causes pneumonia must be
in the differential diagnosis of COVID-19 and include influenza, parainfluenza, adenovirus,
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respiratory syncytial virus, rhinovirus, human metapneumovirus, SARS-CoV, etc.

It is important to mention that coinfection is a possibility, as some reports from Italy and
China had described, the most common pathogen in coinfection was Influenza virus (H1N1,
H3N2), Rhinovirus and Respiratory syncytial virus (A/B). In contrast, bacterial coinfection
was infrequent [156, 157].

The bacterial etiology that may have clinical and radiographic similarities to COVID-19 is
that caused by bacteria such as mycoplasma and chlamydia. Among the pictures that cause
non-infectious lung lesions are those autoimmune diseases with lung involvement such as
vasculitis, dermatomyositis and other pneumonitis.

COVID-19 in pregnant women

Regarding SARS-nCoV infection in pregnant women, there is currently limited evidences

about the effect of the virus on the mother or fetus. However, due to the physiological changes
typical of pregnancy, especially on the immune system (immunosuppression) and the
cardiopulmonary system, pregnant women are thought to be more susceptible to developing
severe symptoms when they acquire viral respiratory disease. In 2009, when Influenza A
H1N1 infection occurred, pregnant women were 1% of the infected population, yet accounted
for 5% of infection-related deaths [158].

Some of the guidance related to the effects of the coronavirus in pregnant women and the
fetus is due to previous studies of various viruses. During the SARS-CoV pandemic in 2002
and 2003, in a very small study of 12 patients, women infected during their first trimester
had high a miscarriage rate (57%). During their second and third trimesters they developed
intrauterine growth restriction (40%), and preterm delivery (80% [one spontaneous and three
induced by maternal condition]), and three women died during pregnancy (25%) [159]. In
another study of 11 pregnant patients infected with MERS-CoV, 9 presented adverse results
(91%), 6 neonates were admitted to the neonatal intensive care unit (55%) and three of them
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died (27%) [160].

With information obtained so far from the Wuhan SARS-CoV-2 outbreak, the infection
appears to be less severe for pregnant women, compared to previous SARS-CoV and MERS-
CoV outbreaks [158]. However, it is important to take into account that the data obtained
are from reviews consist of a small number of patients. Additionally, the majority of
pregnancies with confirmed SARS-Cov-2 pneumonia were in the third trimester and there
were very few within the first and second trimesters . Therefore, more information should be
collected with larger numbers of pregnant women with the infection. Follow-up of positively
diagnosed pregnant women during in the first and second trimesters should be encouraged,
to understand the impact of the new coronavirus infection on the pregnant mother, the fetus
and the course of pregnancy [161, 162]

Mullins et al, carried out a bibliographic review of all the evidence collected until March 10,
2020, relating to any pregnant women with coronavirus diagnosed during her pregnancy or
puerperium. 23 studies were included but there is a high probability that reported cases
overlap. In total, they found 32 women affected by COVID-19, including one with a twin
pregnancy. Delivery of 30 newborns was reported, 27 by Caesarean section 3 by vaginal
delivery[163].
The management of pregnant patients with COVID-19, in general, follows the same
principles as for the wider population. It is vital to consider that the mother, fetus and,
subsequently, the newborn are always considered a high-risk population. Management
should include early isolation, oxygen therapy if necessary, avoid fluid overload, empirical
antibiotic therapy (due to the risk of bacterial infection), maternal fetal monitoring, Doppler
ultrasound is recommended within obstetric surveillance. In patients who are asymptomatic,
home management can be done, indicating that they should seek further medical advice if
their symptoms develop into more severe disease. All mothers recovering from COVID-19
infection should be monitored with a Doppler ultrasound every two weeks, due to the risk of
developing intrauterine growth restriction [164, 165].

The time of termination of the pregnancy, as well as the method, also depend on several
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factors, including gestational age, maternal condition in relation to SARS-CoV-2 infection,

presence of maternal comorbidities, and fetal condition. It must be taken within a
multidisciplinary team, with individualized management for each patient [166].

Prematurity conditions neonatal morbidity and mortality, so the diagnosis of COVID-19 is

not an indication on its own of termination of pregnancy, and the use of corticosteroids is
recommended for antenatal fetal lung maturation, with betamethasone or dexamethasone
[167]; taking special care in critically nursing patients, as this may worsen their condition,
and may delay delivery, which is necessary for the management of these patients [164, 168].

COVID-19 in children

The symptoms presented by children are in themselves similar to adults with an incubation
period ranging from 1 to 14 days (mean of 5.2). Cough is the most frequent symptom (65%)
followed by fever (60%) with the difference of gastrointestinal symptoms diarrhea (15%),
nausea, vomiting (10 %) and abdominal pain, which are usually more flowery than in the
adult stage and, sometimes only manifestations along with fever [169, 170].

The clinical manifestations in pediatric patients vary markedly from adults, particularly
relating to general progression and severity of the disease. Over 90% of affected children
are asymptomatic or have mild to moderate disease [169]. The majority of serious cases in
children are related to those with significant comorbidities such as heart disease,
immunosuppression, etc. To date of this review, only a few cases of a child without
comorbidities died as a result of COVID 19 are reported. This difference of severity of illness
between adults and children has not been clarified, however, several theories have been
postulated. These include that children express more ACE2 receptors in their lungs which
confer some protection to severe injuries such as those caused by RSV and which would
decrease dramatically with age [171, 172].

Immunological factors could may also influence outcomes, as in childhood we are most
exposed to frequent challenges with recent seasonal viruses such as RSV in the winter months.
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Most likely, it is multifactorial and depends on factors from both the host and the virus itself
[172].
Abnormal radiological (CT) findings are found in asymptomatic children and consist of
bilateral lung lesions (50%). Elevated CRP (C – reactive protein), Procalcitonin PCT (80%),
and liver enzymes are present in most affected children, unlike adults in whom PCT is not a
reliable marker.
Virus elimination via the stool even after the negativity in the nasopharyngeal mucosa and
the disappearance of symptoms makes them a source of contagion through the fecal-oral
route [173].

SARS-COV-2 infection and cancer

Patients with cancer are generally more susceptible to infections than healthy people, because
they have a state of systemic immunosuppression that is exacerbated during chemotherapy
or radiotherapy [174].

In China, according to national surveillance data, coronavirus infection occurs in 1.3% of

patients with malignant tumors, a proportion higher than the general incidence of 0.3% of
malignant tumors in the country [175]. When comparing non-malignant tumors patients with
malignant tumors patients have a higher risk of developing a more serious infection (OR 5.34;
95% CI: 1.80–16.18; p = 0.0026) and health deterioration is accelerated (HR 3.56; 95% CI:
1.65-7.69; p<0.0001) even after adjusting for age [174]. To back up this findings, in a tertiary
hospital in Wuhuan - China, it was found that 25% of patients with cancer and SARS-COV-
2 infection died, most of them over 60 years of age [176].

Due to these findings, it has been proposed by many international entities that during the
pandemic, for prevention it should be developed an individualized plan based on the specific
conditions of each patient and treat to minimize the number of visits to health institutions.
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• For early-stage patients with need of post-surgical adjuvant chemotherapy, especially

those whose clinical, pathologic, and molecular biologic staging suggest a better
prognosis, the start time of adjuvant chemotherapy may be delayed up to 90 days after
surgery without affecting the overall effect of treatment [177].

• For patients with advanced cancer, the main approach should be to minimize
hospitalization in COVID-19 positive installations. Replacing the existing
intravenous treatment regimen with oral chemotherapy during this special period may
be considered, to ensure that treatment is not interrupted for a long time during the
pandemic [178].

However, if there is a suspicion of COVID-19 infection in this population group, it should

be followed the same updated diagnostic guidelines and the corresponding management
according to their state of severity. Moreover, it should be considered an individualized
follow-up due to more likely of complications in this group of population [179].
It should be noted that cancer out-patients have different levels of anxiety, depression and
other mental health problems than general population. Studies have shown that
approximately 50% of malignant tumor survivors have a moderate to severe fear of tumor
recurrence [180]. For this reason, psychologist surveillance of out-patients in quarantine or
during hospitalization should be considered.

Complications SARS-CoV-2 infection

Reported complications derived from COVID-19 describe a severe disease that requires
management in an intensive care unit (ICU) in approximately 5% of proven infections. Main
ones were respiratory failure, cardiovascular dysfunction, cardiomyopathy and acute kidney
injury; the average duration between symptom onset and dyspnea and ICU admission has
been 7 and 10 days, respectively. Suggesting gradual deterioration in most cases, with older
patients (mean> 60 years) the most susceptible. The risk of patient-to-patient transmission in
the ICU is currently unknown, therefore adherence to infection control precautions is
paramount [181, 182].
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Progressive deterioration of respiratory function is undoubtedly the main and worst

complication of the infection. The prevalence of hypoxic respiratory failure in COVID-19
patients is 19%, and it can progress to acute respiratory distress syndrome (ARDS), with the
need of mechanical ventilation support at 10.5 days on average; between 10 and 32% of
hospitalized patients require admission to the ICU due to respiratory deterioration [182]. As
the respiratory complication is the main and most severe, its early diagnosis will undoubtedly
help in timely support, taking into account risk factors such as advanced age, neutrophilia
and organic dysfunction for the development of ARDS. The diagnostic support of pulmonary
tomography is undoubtedly a valid tool; images in patients with different clinical types of
COVID-19 have characteristic manifestations, but it can become an operational problem due
to the difficulty in performing it in critically ill patients; On the contrary, the performance of
lung ultrasound at the foot of the bed may replace the performance of radiographs and
tomography for its diagnosis [183, 184].

Since more than 70% of hospitalized patients will require supplemental oxygen, it is
recommended that it should be started with pulse oximetry values less than 90% with a target
of no more than 96%, since higher values have been shown to be harmful [185, 186].
Regarding the use of high-flow nasal cannula (HFNC) oxygen therapy, great variability of
results were recorded, because it was not possible to determine whether the progression to
orointubation, mortality, or the risk of contamination to health personnel had decreased, but
it still should be used instead of non-invasive mechanical ventilation (NIMV). HFNC use
should be closely monitored and cared for in an environment where intubation can be
facilitated in case of decompensation, due to the failure rate can be high and emergency
intubation in an uncontrolled environment increase the risk of nosocomial infection of health
providers [187–189].

The recommendation for starting with NIMV is of very low quality, and it is of high risk for
both patients and health personnel. In adults with COVID-19 hypoxic respiratory failure,
there is no direct evidence to support the use of NIMV; Furthermore, some previous studies
suggested that it may be associated with an increased risk of transmission of infections to
healthcare workers and may worsen severe forms of lung injury as a result of harmful
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transpulmonary pressures and large tidal volumes (TV), in addition to delaying initiation of
invasive mechanical ventilation, leading to emerging intubations that may increase the risk
of transmission to the healthcare team with increased risk to the patient [190–192].

For the initiation of invasive mechanical ventilation, the recommendation for highly
protective ventilation is maintained, with the use of low TV (6 ml / kg of ideal weight),
plateau pressure less than 30 cm H2O, conduction pressure between 13-15 cmH2O,
respiratory rate can be carried up to 35 per minute, as needed. If hypoxemia progresses to
values less than 100–150 mmHg of PaFiO2, there are several therapeutic options, initially
increasing positive expiratory pressure (PEEP) by 2–3 cmH2O every 15 to 30 minutes to
improve oxygen saturation to 88–90%, maintaining a plateau of less than 30 cm H2O.
Recruitment maneuvers are probably of little value, but could be used in selected cases in the
presence of a physician to control hemodynamics. If there is considerable asynchrony with
positive pressure ventilation, accompanied by an increase in plateau pressure and refractory
hypoxemia, deep sedation should be used followed by prompt institution of neuromuscular
block. If hypoxemia has been reached refractory to the aforementioned measures, it is
recommended to move quickly to ventilation in the prone position and as a final measure
venous venous ECMO (VV) should be considered if available or to refer the patient to an
ECMO center [193–198]. Routine use of corticosteroids has been discouraged, and
restricting it exceptionally for patients who develop ARDS, although without reports of
improvement in survival, with discrepancy in results of shorter mechanical ventilation time
and ICU stay [199].

Hemodynamic deterioration has a variability of presentation, this depends on the study

population and the definition [200], the presence of shock in the intensive care unit may be
present between 25 to 35% [181, 201]. Cardiomyopathy related to viral infection is one of
the main causes of hemodynamic detriment, occurring in up to 23% of patients with COVID-
19 [202]. Hemodynamic failure is one of the main causes of death in these patients, with
percentages of up to 40%, inconclusive risk factors are associated to date such as diabetes,
hypertension, lymphopenia, and elevation of D-dimer [203]. Acute kidney injury (AKI) is
present in up to 12% of critically ill patients, podocytes and proximal tubule cells are
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potential host cells for SARS-CoV-2, caused by the virus induced cytopathic effect. The
diagnosis is based on markers of early kidney injury and urinary output [187].

Initial management of shock is based on fluid resuscitation, based on the application of

dynamic parameters to predict response to fluids, such as variation in stroke volume (SVV),
variation in pulse pressure volume (PPV) and change in stroke volume with passive leg
elevation or fluid challenge above static parameters [203]. Variables such as skin temperature,
capillary refill time and/or serum lactate measurement are currently valid tools. The amount
of liquids used in resuscitation should be restricted and administered in relation to dynamic
assessment, a liberal water resuscitation strategy is not recommended, preferring balanced
crystalloids over colloids as resuscitation liquids, avoiding the use of hydroxyethyl starches,
albumin, dextrans or gelatins [204, 205]. Indirect evidence suggests that the target mean
arterial pressure (TAM) for patients with septic shock is 65 mmHg using vasoactive support
[206]. The recommendation of norepinephrine use as the first agent is maintained. If
norepinephrine is unavailable, vasopressin or epinephrine could be used, avoiding the use of
dopamine as the initial vasopressor due to the potential development of arrhythmias [207,
208]. In patients with COVID-19 and shock with evidence of cardiac dysfunction and
persistent hypoperfusion despite fluid resuscitation and norepinephrine use, dobutamine as
inotropic is recommended. Given the development of refractory septic shock, the suggestion
of the use of hydrocortisone in continuous infusion is maintained, as indirect evidence, this
in favor of reducing the length of stay in the ICU and the resolution time of the shock [207].

Clinical prognosis

According to the investigative mission of the WHO in China, the case-fatality rate ranged
from 5.8 percent in Wuhan to 0.7 percent in the rest of China. Of these cases, the deaths were
mostly in patients with chronic diseases (cardiovascular disease, diabetes mellitus, chronic
lung disease, hypertension and cancer) and the elderly. (WHO, et al. 2020).

Other reports from China have coincided with this clinical risk profile, for example, a study
that included 41 confirmed cases, 12 patients who had ARDS had as main underlying
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diseases: diabetes and high blood pressure. Of these cases, 6 patients died [138].

Recovery from COVID-19 infection

According to WHO, the recovery time is estimated to be two weeks for mild infections and
three to six weeks for serious illnesses. On the other hand, CDC established that people who
had symptoms in the mild to moderate spectrum and maintained home isolation have a
resolution of 3 days after the fever decrease, and there was a substantial improvement in
respiratory symptoms, even without use of medications.

Isolation may be limited to 7 days from resolution of symptoms, however, it must be adapted
to the population circumstances of the epidemic [140].

Current treatment strategies

Non - pharmacological measures:

The evolution of epidemiological curve in COVID-19 outbreak makes consider containment

strategies in China primarily, and other countries based on non-pharmaceutical interventions
(NPIs). According WHO, the most effective measure is hands washing. In general, the
recommendations are: “If hands are not visibly dirty, the preferred method is to perform hand
hygiene with an alcohol-based hand rub for 20−30 seconds using the appropriate technique.
When hands are visibly dirty, they should be washed with soap and water for 40−60 seconds
using the appropriate technique” [209].
Five different non-pharmaceutical interventions (NPI) implemented individually and in
combination as public health measures reduced contact rates in the population and therefore
reduce virus transmission (Table 6) [210].

Table 6 Non - pharmacological measures

Measure Description
Home isolation Symptomatic cases stay at home for 7 days, reducing non-household contacts by 75% for this
period. Household contacts remain unchanged. Assume 70% of household comply with the
policy.
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Voluntary home quarantine Following identification of a symptomatic case in the household, all household members
remain at home for 14 days. Household contact rates double during this quarantine period,
contacts in the community reduce by 75%. Assume 50% of household comply with the policy.

Social distancing of those over 70 Reduce contacts by 50% in workplaces, increase household contacts by 25% and reduce other
years of age contacts by 75%.

Social distancing of entire All households reduce contact outside household, school or workplace by 75%. School contact
population rates unchanged, workplace contact rates reduced by 25%. Household contact rates assumed to
increase by 25%.
Closure of schools and universities Closure of all schools, 25% of universities remain open. Household contact rates for student
families increase by 50% during closure. Contacts in the community increase by 25% during
closure.

Increasing the level of hand cleanliness to 60% in places with a high concentration of people,
like all airports in the world would have a reduction of 69% in the impact of a potential
disease spreading [211].

The specific recommendations from WHO are social distancing and hand washing. About
rational use of masks, WHO recommends: “If the person is healthy, only need to wear a
mask if he/she is taking care of a person with suspected SARS-CoV-2 infection”. In Japan,
the statement in this topic was that effectiveness of wearing a face mask to protect from
contracting viruses is thought to be limited. If the use of a face mask in confined, badly
ventilated spaces, it might help avoid catching droplets emitted from others but if you are in
an open-air environment, the use of face mask is not very efficient. CDC does not
recommend that people who are well wear a face mask (including respirators) to protect
themselves from respiratory diseases, including COVID-19. Thus, the recommendation is to
optimize face mask distribution and priories the needs of frontline health-care workers and
the most vulnerable populations in communities who are more susceptible to infection and
mortality if infected, including older adults (particularly those older than 65 years) and
people with underlying health conditions [212].

Pharmacological treatment:

Therapeutic strategies are urgently needed to be applied in the context of COVID-19, as a

pandemic. In terms of this public health urgency is important to consider two important
definitions: Drug repurposing and compassionate use of drugs. The first one, drug
repurposing is an emerging strategy where pre- existing medicines, having already been
tested safe in humans, in similar virus or targets in the infection process, are redirected
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against unique objective: SARS-Cov 2 (viral structure, infection process). The second one,
compassionate use of drugs is the use of a new, unapproved drug to treat a seriously ill
patient when no other treatments are available. These concepts have been applied with
COVID-19 treatment.

Identifying targets for pharmacological use has been important to develop therapeutically
drugs with roles in virus structure and infection process (Figure 2). Some representative
existing drugs act on targets in similar RNA viruses like Ebola, hepatitis C, influenza, and
others as MERS and SARS viruses. The most important studied targets are 3CLpro and
PLpro, the two viral proteases responsible for cleavage viral peptides into functional units
for virus replication and packaging within the host cells. Thus as drug repurposing appears
Lopinavir and Ritonavir [213]. RdRp is other important target as the RNA polymerase
responsible for viral RNA synthesis, blocked by Remdesivir and Favipiravir. About
endocytosis process into host cells, viral spike protein and its interaction with ACE2 receptor
constitute other important target blocked by arbidol, used also in Influenza. ACE2 is a negative
regulator, receptor of renin-angiotensin system, involved in pressure control and
inflammatory lung disease. By the knowledge of physiopathology of covid-19 infection, we
know that activities of ACE2, AT1 and AT2 receptors are altered, thus some drugs are being
studied around these targets, but also in vitro and experimental way. Some homologue target-
drug models have been purposed between SARS-CoV and SARS-CoV-2 due to the receptor-
binding domain (RBD) in S protein with 76% of sequence similarity. In the same way with
PLpro sequences with 83% similar active sites [213].

Other drugs like Chloroquine and analogues (Hydroxicloroquine) acts directly on endosomal
pH and interfere with ACE2 glycosylation. In general, the most studied pharmaceutical
interventions found for COVID-19 treatment include arbidol, remdesivir, oseltamivir,
favipiravir, human immunoglobulin, interferons, chloroquine, hydroxychloroquine,
methylprednisolone, ritonavir, darunavir, lopinavir, tocilizumab and convalescent plasma.
Drugs listed with their mechanisms of action on COVID 19, and adverse effects can be found
on Table 7.
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Table 7 Drugs and targets in SARS-CoV-2

TYPE OF DRUG TARGET OTHER DISEASES MECHANISM OF ACTION IN COVID 19 ACTIVITY AGAINST SIDE EFFECTS
INDICATION (Drugs Repurposing) SARS-COV-2
ANTIVIRAL DRUGS

Favipiravir RdRp, RNA dependent RNA Influenza. Ebola, yellow fever, Inhibitor of viral RNA-dependent RNA IN VITRO ND
polimerase chikungunya, norovirus. polymerase. Pyrazinecarboxamide derivative
viral RNA polymerase inhibitor.
Arbidol S protein, ACE2 Influenza Entrance. S protein- AC2 receptor IN VITRO Gastrointestinal effects
ANTIRETROVIRAL DRUGS

Lopinavir + Viral proteases: 3CLpro or Combination for HIV infection HIV reverse transcriptase inhibitors. Rito IN VITRO. IN VIVO Rash, GI upset,
Ritonavir PLpro enhance the action of other drugs by inhibition of abnormal liver tests
CYP3A4
May inhibit the viral proteases: 3CLpro or PLpro
Remdesivir RdRp, RNA dependent RNA Ebola and Marburg viruses, Inhibe viral replication IN VITRO, IN VIVO Abnormal liver tests, GI
polimerase SARS-CoV-1 and MERS
Darunavir Protease inhibitor HIV protease inhibitor In combination with cobicistat, a CYP3A ND Rash, GI upset,
inhibitor, abnormal liver tests
ANTIMALARIAL DRUGS

Chloroquine endosome/ ACE2 Antimalarial actions, chloroquine Glycosilation Inhibition and elevate endosomal IN VITRO Retinopathy, QT
has some efficacy in HIV-AIDS pH and interfere with ACE2 glycosylation prologation,
Hidroxichloroquin endosome/ ACE2 Antimalarial actions, chloroquine inhibiting virus entry into host cells IN VITRO QT
e has some efficacy in HIV-AIDS prolongation
ANTIBIOTICS

Azitromicin Bacterial protein sybthesis, Bacterial infections For suspected bacterial superinfection ND GI effects
blocking 50S ribosomal
ANTIVIRAL DRUGS. NON-SPECIFIC

Interferon PKR, Mx protein Hepatitis B virus and HCV Inhibite viral replication by inhibition of PKR ND Depression,
injection site
reaction, flu
like
syndrome
NEUROAMINID
ASA INHIBITOR
Oseltamivir Neuroaminidasa Inhibitor Influenza Not well studied In vitro
TYPE OF DRUG TARGET OTHER DISEASES MECHANISM OF ACTION IN COVID 19 ACTIVITY SIDE EFFECTS
INDICATION (Drugs Repurposing)
MONOCLONAL ANTIBODY
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Tocilizumab IL-6 receptors ( soluble and rheumatoid arthritis, systemic Inhibit IL-6. Taper immune system in critical ND Abnormal liver tests, GI
membrane-bound) juvenile idiopathic arthritis, patients perforation
juvenile
idiopathic polyarthritis, giant -
cell arteritis
ANTIINFLAMATORY DRUGS

Corticosteroids Inflammation cascade Inflammatory responses For patients with refractory shock or acute ND Cushing Sd., diabetes,
respiratory distress syndrome weigh gain.
OTHERS

Acetylcysteine Mucolytic Symptomatic relief; Syntomatic relief ND Nausea, fver, vomiting,

Angiotensin ACE2 Receptors ND Diziness, nausea,
receptor blockers diarrea, headache
Thalidomide Immunosuppressant Myeloma Inhibite production of TNF-α, antiangiogenic ND Fever, low cell counts,
activity. anxiety, weigh gain or
loss
Pirfenidone idiopathic pulmonary fibrosis reduces fibroblast proliferation, production of ND
fibrosis-associated proteins and cytokines
Vitamin C Antioxidant Sepsis, chronic process, Module redoz signaling ND

*There are several drugs in study to be considering in treatment for Covid-19. This table summarizes the most important in terms of principal outcomes in clinical trials or activity
in vitro. ND= Non Data
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Actually, there is a great effort to build strong evidence. There are 382 clinical trials in
progress. Some antiviral, antimalarial and antibiotic drugs have also been shown to have in
vitro activity against SARS CoV 2, but it does not guarantee clinical efficacy. For these there
are several completed and in progress clinical studies. Some of them like Darunavir are in
phase II, Remdesivir, cloroquine and hydroxycloroquine are in phase III of clinical trials,
Lopinavir and Ritonavir (Kaletra) and Umifenovir or Arbidol in phase IV. In order to collect
data quickly and get information from many countries on March 20, 2020, the WHO
announced a large global trial, called SOLIDARITY. The treatments included in this big trial
are: remdesivir, chloroquine and hydroxychloroquine, ritonavir / lopinavir-ritonavir /
lopinavir and interferon beta. The completed and clinical trials with evidence that favors them
are listed in (Supplementary Table 7) and its relation with clinical features in Table 8 [143].
The clinical trials evaluate some important outcomes. A systematic review of Lopinavir
/Ritonavir assess treatment in terms of mortality, mechanical ventilation or extracorporeal
membrane oxygenation (ECMO)[214] development of acute respiratory distress syndrome
and serious adverse effects [215]. None of the outcomes favors the intervention; nevertheless,
in terms of development of the respiratory syndrome, the pharmacological intervention is
effective, although the certainty of the evidence was very low. Another systematic review,
that included six articles and 23 ongoing clinical trials in China about the use of chloroquine
in COVID-19 [216]. Studies indicates chloroquine reduced progression of disease and
decreased duration of symptoms, but none of the studies favors the use of cloroquine because
of the lack of strong evidence in ramdomized trials. In a recent trial, 100% of patients treated
with hydroxychloroquine in combination with azithromycin were “virologically cured”
comparing with 57.1% in patients treated with hydroxychloroquine alone, and 12.5% in the
control group, but these cannot be completely extrapolated because it requires more quantity
and quality studies. The use of chloroquine or hydroxychloroquine in primary health care is
not recommended for the management of COVID 19. These drugs are associated with an
increased risk of heart damage, especially when administered concurrently with macrolides
(QT interval prolongation). Drugs like Tocilizumab has been included in severe or critical
patients. Remdesivir is effective against the 2019-nCoV in vitro in Vero E6 cells through
mechanism of involving the host cells' post-entry stage. Several randomized trials are
underway to evaluate the efficacy of remdesivir for moderate or severe COVID-19.
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Oseltamivir inhibits the viral neuraminidase, drug approved for influenza A and B treatment.
Its use was reported during the COVID-19 epidemic in China, but it has no effective
outcomes. Tocilizumab, an inhibitor of IL-6 is considered in a group of critical patients, in
which 75% cursed with improved respiratory function after treatment. The last treatment
reported in a 5 patient case series is convalescent plasma. Following plasma transfusion
normalized temperature within 3 days in 4 of 5 patients, decreased SOFA score, increase
PAO2/FIO2 within 12 days and viral loads also decreased and became negative within 12
days after the transfusion. A promising drug, although the evidence level is low [217].
Bevacizumab is a monoclonal antibody that targets vascular endothelial growth factor
(VEGF) that might suppress the edema in patients with COVID-19.

The other drugs studied in pharmaceutical interventions for COVID-19 treatment include
arbidol, human immunoglobulin, interferons, chloroquine, methylprednisolone, tocilizumab,
vitamin C, pirfenidone, bromhexine, danoprevir, darunavir, cobicistat, convalescent plasma,
biological therapies and traditional Chinese medicines (TCM), which are studied in clinical
trials in progress and do not appear as strong evidence to recommend them in practice.

Regarding corticosteroids, are beneficial in treating SARS-CoV patients; it prolongs the

survival time of clinical cases. Due to the cytokine storm some authors described the use of
corticosteroids, but others describe its use in the early stages of SARS infection with
increasing values of viral load. Other drugs like vitamin C has been used to prevent but there
is no good evidence to support this. All of the evidence including clinical trials, randomized
clinical trials, favors the use or not about the therapeutically drugs are detailed in Table 8.

This review summarized some drug repurposing agents currently known to be effective
against other RNA viruses including SARS-CoV, MERS-CoV, influenza. Actually, exist
some new drugs with high potential impact of biologics targets for Covid-19 treatment. It is
important to notice that there is no specific treatment for the coronavirus approach. In context
of the scientific evidence and the particular clinical features of each patient, the reader will
be able to make clinical and therapeutically decisions.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Vaccines development

When it comes to vaccine design and manufacturing, the main objectives are its safety, its
efficacy in activating specific adaptive immune responses and the production of -ideally-
long term memory. Thus, eliciting protective immune responses including neutralization
antibodies and/or CTL generation is of paramount importance.

Huge challenges need to be tackled in order to minimize the long and cumbersome process
of vaccine generation. Among them, candidate antigen targets need to be identified,
immunization routes and delivery systems investigated, animal models set, adjuvants
optimized, scalability and production facility considered, target population selected, and
vaccine safety and long-term efficiency evaluated.

Currently there are no approved vaccines against any human coronavirus, suggesting that
their generation is quite trivial. Several candidate vaccines against SARS-CoV had shown
promise reaching Phase I or Phase II clinical trials [77, 78], but the rapid containment of
SARS-CoV expansion rendered them redundant, did not allow for a test population for Phase
III trials and, therefore, put their further assessment to a halt.

However, the accumulated experience from previous coronavirus vaccine designs and the
sequence and structural similarity of SARS-CoV and SARS-CoV-2 are significant
advantages in the current endeavor. Thorough studies conducted in SARS-CoV-specific T
cells of SARS convalescent patients have shown that all memory T cell responses are directed
at SARS‐CoV structural proteins. T cell epitope mapping showed that CD8+ responses were
targeting SARS-CoV membrane (M) and Nucleocapsid (N) proteins and CTL memory could
last up to 11 years after infection [218]. These data suggest that vaccine strategies employing
viral structural proteins that can elicit effective, long‐term memory T cell responses could
yield fruitful results.

On the other hand, the S1 spike protein region containing the ACE receptor binding domain
(RDB) is the obvious option when neutralizing antibody responses are considered [219–221].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Indeed, a candidate SARS vaccine antigen consisting of the RBD of SARS-CoV Spike
protein was created and found it could elicit robust neutralizing antibody responses and long-
term protection in vaccinated animals [222].

The fact that COVID-19 convalescent sera shows potential as a therapeutic approach [67]
argues that efficient B cell responses are mounted and lead to production of protective
antibodies. Two different groups, using an immunoinformatic approach mapped several CTL
and B cell epitopes on different proteins of the virus [223, 224]. Moreover, various CTL
epitopes were found to be binding MHC class I peptide‐binding grooves via multiple contacts,
illustrating their probable capacity to elicit immune responses [82]. Consequently, these
identified B and T cell epitopes could be potential targets for therapeutic vaccines.

However, important safety considerations should be taken into account before releasing a
new vaccine in the market. Previous studies on macaque models have shown that a vaccine-
induced anti-Spike protein antibody at the acute stage of SARS-CoV infection can provoke
severe acute lung injury [225]. Similar observations of SARS-CoV vaccine-induced
pulmonary injury have also been described in multiple several murine and monkey animal
models [226].

Classic vaccine strategies like use of attenuated virus or recombinant protein subunit
administration begin to lose support in the scientific community. COVID-19 mainly affects
older patients with underlying pathologies that debilitate their immune system. Use of
attenuated virus vaccines is contraindicated in these populations as weakened immune
systems can permit the reversion of the attenuated pathogen to its wild type state, therefore
causing the pathology it was designed to prevent. On the other hand, subunit vaccine design
can be challenging when the protein used contains extended glycosylation. Interestingly,
nucleic acid-based vaccines showed great promise in response to emerging pathogens like
the DNA vaccine designed for Zika virus, entering in Phase I clinical trials [227]. Another
nucleic acid-based platform for vaccine development, mRNA vaccines, seems a quite
revolutionary strategy. Being designed to possess improved stability and protein translation
efficiency these vaccine platforms can act both as adjuvants and antigen sources alike,
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

inducing potent immune responses [228, 229]. The optimization of the delivery system, such
as lipid nanoparticles makes them excellent design candidates [230]. Finally, delivery
systems such as recombinant vesicular stomatitis virus particles or the administration of
mRNA molecules that codify for virus-like particles have been proven extremely efficient as
testified by the recent FDA approved vaccine against Ebola [231].

In an unprecedentedly swift response to develop and manufacture an anti-SARS-CoV-2

vaccine, more than 40 companies and academic institutions are exploring the aforementioned
strategies. An example illustrating the rapid reaction of the scientific community to the
SARS-CoV-2 outbreak is that of the the biopharmaceutical company Moderna, the first
vaccine manufacturer that entered in Phase I clinical trials for one candidate vaccine for
COVID-19. On the night of Saturday, January 11, 2020, in the headquarters of the National
Institute of Allergies and Infectious Diseases (NIAID) of USA Barney Graham, Deputy
Director of the Vaccine Research Center, received the SARS-CoV-2 sequence. During the
weekend his group analyzed the data and on Monday, 13 of January he discussed his
observations with a group of investigators of the biopharmaceutical company Moderna. On
the same day Moderna’s infectious disease research team finalized the sequence for mRNA-
1273, the company’s first vaccine candidate against SARS-CoV-2. On February 7, 2020, the
first clinical batch of Moderna was completed. On February 24, 2020, the clinical batch was
shipped from Moderna to the NIH to be used in their own Phase I clinical study. On March
4, 2020, the U.S. FDA gave the green light for mRNA-1273 to begin clinical trials. Twelve
days later, on March 16, 2020, the NIH announced that the first participant in its Phase I
clinical study received the first dose of mRNA-1273. The time between virus sequencing to
beginning of Phase I trials was a record total of 63 days.

The pharmaceutical companies that are currently on a race to produce a vaccine for COVID-
19 along with the vaccine developing strategies they are using are summarized in Table 9.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Table 8 COVID-19 vaccine development update by manufacturer. Vaccination strategies employed, delivery platforms
used, and current development status are presented if official data are provided.

Manufacturer Vaccine Vaccination Delivery Current stage of Status ¥ Viruses targeted

candidate Strategy Platform development/Trial Phase ¥ by candidate
vaccines using
same strategy
Moderna/ NIAID mRNA- mRNA LNP* Phase 1 Recruiting completed SARS-CoV,
1273 codifying encapsulated 19 March 2020 MERS-CoV
for full- mRNA NCT04283461 §

length S
protein
CanSino Ad5-nCoV Recombinan Adenovirus Type Phase 1 Currently recruiting Ebola, MERS-
Biological Inc./ t virus/Non- 5 Vector CoV
Beijing Institute replicating ChiCTR2000030906§
of Biotechnology

Inovio INO-4800 DNA Plasmid- Pre-clinical development Phase 1 clinical trials Lassa, Nipah,
Pharmaceuticals vaccine Electroporation are expected to begin HIV, Filovirus,
facilitated entry in April 2020 HPV, Zika,
Hepatitis B
Takis Biotech & Not DNA DNA Pre-clinical development Phase 1 clinical trials Lassa, Nipah,
Applied DNA announced/4 vaccine are expected to begin HIV, Filovirus,
Sciences/ candidates in fall 2020 HPV, Zika,
Evvivax for COVID- Hepatitis B
19
Zydus Cadila Not 1. DNA 1. Plasmid Pre-clinical development Not announced Lassa, Nipah,
announced/2 vaccine HIV, Filovirus,
strategies 2. Recombinant HPV, Zika,
employed 2. Live replicating Hepatitis B
attenuated measles virus
recombinant
vaccine
Sinovac Not Formalin Inactivated virus Pre-clinical development Not announced SARS-CoV
announced inactivated
& alum
adjuvant
Serum Institute Not Live Live Attenuated Pre-clinical development In vivo testing pending HAV, InfA,
of India & announced Attenuated Virus ZIKV, FMD,
Codagenix Virus SIV, RSV,
DENV
Geovax/ Not Recombinan Modified Pre-clinical development Narrowing the vaccine LASV, EBOV,
BravoVax announced t viral vaccinia ankara candidates down from MARV, HIV
vector/Non- virus like three to one
replicating particles encoded
(MVA-CLP)
Janssen Not Recombinan Ad26 (alone or Pre-clinical development Vaccine candidate is Ebola, HIV, RSV
Pharmaceutical announced t viral with MVA expected end of March
Companies of vector/Non- boost) - AdVac 2020/ Clinical testing
Johnson & replicating and PER.C6 starting in November
Johnson/Barda systems 2020
University of ChAdOx1 Recombinan Chimpanzee Pre-clinical development Not announced Influenza strains,
Oxford t viral adenovirus Mycobacterium
vector/Non- vaccine vector tuberculosis,
replicating Chikungunya,
Zika, MenB,
plague
Altimmune Intranasal Recombinan Adenovirus - Pre-clinical development Animal testing Influenza strains
COVID-19 t viral based NasoVAX imminent /Clinical (NasoVAX
vaccine vector/Non- expressing testing is initially vaccine)
replicating SARS2-CoV S scheduled for August
protein 2020
Greffex Adenovirus- Recombinan Adenovirus- Pre-clinical development Animal testing has MERS-CoV
based vector t viral based vector begun
vaccine for vector/Non- vaccine
COVID-19 replicating
Vaxart Not Recombinan Oral Vaccine Pre-clinical development Not announced InfA, CHIKV,
announced t viral platform LASV, NORV;
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

vector/Non- EBOV, RVF,

replicating HBV, VEE
ExpreS2ion Not Protein Drosophila Pre-clinical development Phase 1/2a clinical -
announced Subunit Schneider 2 testing to begin within
insect cell 12 months
expression
system VLPs#
Walter Reed Not Protein Antigen + Pre-clinical development Several vaccine MERS-CoV
Army Institute of announced Subunit/S adjuvant candidates developed/
Research/United protein Animal testing has
States Army begun
Medical
Research
Institute of
Infectious
Diseases
Clover COVID-19 Protein Antigen + Pre-clinical development Pre-clinical trials HIV, REV
Biopharmaceutic S-Trimer Subunits/S- adjuvant pending Influenza
als Inc./Glaxo Trimer
Smith Kline
Vaxil Bio Protein Protein Antigen + Pre-clinical development Candidate
subunit Subunit/ adjuvant identified/Beginning
COVID-19 signal of trials not announced
vaccine peptide
candidate technology
(Patented)
AJ Vaccines Not Protein Antigen + Pre-clinical development Not announced
announced Subunit adjuvant
Generex Ii-Key Protein li-key/antigenic Pre-clinical development Intention is to begin Influenza strains,
Biotechnology/E peptide Subunit epitope hybrid human testing within 3 HIV, SARSCoV
piVax COVID-19 peptide vaccine months
vaccine
EpiVax/Universit Ii-Key Protein li-key/antigenic Pre-clinical development Not announced H7N9
y of Georgia peptide Subunit/S epitope hybrid
COVID-19 protein peptide vaccine
vaccine
Sanofi Not Protein Antigen + Pre-clinical development Not announced Influenza strains,
Pasteur/BARDA announced Subunit/S adjuvant SARS-CoV
protein
produced in
baculovirus
Novavax Not Protein Recombinant Pre-clinical development Several candidates RSV; CCHF,
announced Subunit nanoparticles currently tested in HPV, VZV,
animals/Clinical EBOV
testing to begin in late
spring 2020
Heat gp96-based Protein Antigen + Pre-clinical development Not announced NSCLC, HIV,
Biologics/Univers vaccine Subunit/gp- adjuvant malaria, Zika
ity of Miami 96 heat-
shock
protein
backbone
University of Molecular Protein Antigen + Pre-clinical development Further development Nipah, influenza,
Queensland/CSL clamp Subunit/ adjuvant prior to pre-clinical Ebola, Lassa
vaccine for Molecular testing required
COVID-19 clamp
stabilized
Spike
protein
Baylor College of Re-purposed Protein Antigen + Pre-clinical development Not announced SARS-CoV
Medicine SARS Subunit/S1 adjuvant
vaccine for or RBD
COVID-19 protein
iBio/CC- Plant-based Subunit Antigen + Pre-clinical development Not announced
Pharming COVID-19 protein/Plant adjuvant
vaccine produced
VIDO-InterVac/ Not Protein Adjuvanted Pre-clinical development Not announced
University of announced Subunit microsphere
Saskatchewan peptide
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Institute Not Recombinan Measles Vector Pre-clinical development Not announced West nile, Ebola,
Pasteur/Themis/ announced t replicating Lassa, Zika
Univ. of Viral Vector
Pittsburg Center
for Vaccine
Research
Tonix Horsepox Recombinan Horsepox vector Pre-clinical development Not announced Smallpox,
Pharma/Souther vaccine with t replicating expressing S- monkeypox
n Research percutaneou Viral Vector protein
s (used also in
administrati TNX-1800
on vaccine)
Fudan mRNA mRNA 1. LNP* Pre-clinical development Not announced
University/ vaccine vaccine/2 encapsulated
Shanghai Jiao candidate candidates mRNA cocktail
Tong for COVID- encoding SARS-
University/RNA 19 CoV-2 VLP#
Cure Biopharma
2. LNP*
encapsulated
mRNA encoding
RBD of S-
protein
China Not mRNA Not announced Pre-clinical development Not announced
CDC/Tongji announced vaccine
University/Sterm
ina
Arcturus/Duke- Not mRNA Self-replicating Pre-clinical development Not announced Various
NUS Medical announced vaccine RNA and candidates
School nanoparticle
non-viral
delivery system
BioNTech/Fosun BNT162 mRNA Not announced Pre-clinical development Clinical testing is Influenza strains
Pharma/Pfizer vaccine expected to begin in
April 2020
Curevac Not mRNA Not announced Pre-clinical development Clinical trials expected RABV, LASV,
announced vaccine to begin in summer YFV; MERS,
2020 InfA, ZIKV,
DengV, NIPV
Imperial College Self- saRNA Not announced Pre-clinical development Animal testing is EBOV; LASV,
London amplifying vaccine underway/ Clinical MARV, Inf
(sa) RNA trials expected to begin (H7N9), RABV
vaccine in summer 2020
Medicago Inc. Plant-based Plant- VLP# Pre-clinical development Human testing Influenza,
COVID-19 derived expected to begin in Rotavirus,
vaccine VLP# July or August 2020 Norovirus, West
Nile virus,
Cancer
* LNP: Lipid nanoparticle system, § Clinical Trial Registry Identifier, ¥ According to manufacturer, # VLP: Virus like particle, Table
updated until 22/03/2020; Several more companies have announced their intention to manufacture COVID-19 vaccines without
disclosing further information.

As can be easily deduced from Table 9, optimistic predictions dictate that a vaccine for
COVID-19 will not be ready in the next 12-18 months. An indirect course of action that could
help to mitigate the impact of COVID-19 pandemic would be a plan of vaccination against
influenza strains and Strepococcus pneumoniae. Influenza is a major universal health
problem accounting for 3 to 5 million cases of severe illness and about 350 000 to 650 000
respiratory deaths yearly. For the time period from 17 February 2020 to 01 March 2020 alone
the WHO laboratories tested positive for influenza viruses 62423 samples [232]. On the other
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

hand, Streptococcus pneumoniae is the most common cause of community acquired

pneumonia. In the present context of COVID-19 global outbreak vaccination against the most
prevalent strains of influenza and Streptococcus pneumoniae would have a multifaceted
effect. Firstly, it would lower the risk of severe disease, reduce hospitalization and admission
to already heavily charged ICUs due to these pathologies that could prove critical for weaker
health systems that would struggle to carry the burden of combined outbreaks. Moreover,
vaccinating health care workers is crucial for reducing the risk of absence due to disease,
thereby strengthening the healthcare workforce and minimizing the risk to infect COVID-19
hospitalized patients with additional pneumonia-causing pathogens. Lastly, COVID-19
patients vaccinated for influenza and Streptococcus pneumoniae allow their immune system
to focus on one pathogen and, therefore, give it a better fighting chance against SARS-CoV-
2 infection [233]. High risk groups prioritized for vaccination for these two pathogens include
pregnant women, persons with immunocompromised immune systems (either due to
congenital or acquired immunodeficiencies), children, adults ≥65 years and health care
professionals.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Figure 4. Strategies used or proposed for COVID-19 vaccine development and delivery. A) and B) Adenoviral and measles
recombinant viral vectors can be manipulated to express and therefore elicit robust immune responses against the Spike
(S) protein of SARS-CoV-2. C. Recombinant subunit vaccine strategies use the Sf9-baculovirus insect cell expression system
resulting in the production of high-quality antigen that can be used to elicit immune responses. D) Purified antigen vaccine
strategies implicate the replication of large numbers of virus in cell cultures and the subsequent purification of viral
antigens to be used for vaccination. E) Attenuated vaccines contain whole pathogen that has been submitted to heat or
chemical treatment inactivation. F) Attenuated live pathogen vaccine strategies consist in administering a live pathogen
that due to cell culture passaging has lost its virulence. They usually elicit robust and long-term memory immune responses
without the need to administer an adjuvant. G) In DNA vaccines the DNA codifying a highly immunogenic antigen is
administered and captured by professional antigen presenting cells (APCs) leading to antigen production and presentation
by these cells. H. Moderna’s vaccine candidate already in Phase I clinical trials uses an mRNA vaccine approach whereby
the genetic information codifying for the S protein of SARS-CoV-2 is delivered in LNPs to enhance absorption by APCs. Once
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

uptaken by APCs the mRNA induces the expression of S antigen that is subsequently mounted on and presented by MHC
molecules to elicit adaptive immune response.

Climate and SARS-CoV-2

Numerous studies confirm that climate has an impact on virus (i.e., influenza, coronavirus,
etc.) spread through manipulating the conditions of i) its diffusion, ii) the virus survival
outside the host, and iii) the immunity of host population [234]. Meteorological conditions,
such as temperature, humidity, wind speed and direction, atmospheric pressure, solar
radiation (including ultraviolet (UV) spectrum) and precipitation amount and intensity
depend on the latitude and the elevation of the location, thus creating distinct climatic zones
in the planet. While in some regions, such as temperate climate zones, human influenza peaks
have clear seasonal cycles, in others it is not as predictable [234–238].

An array of studies, investigating the relationship between climatic factors and the activity
of influenza all over the world, concluded that at the high latitudes of the world the peaks of
influenza correlate with cold and dry weather conditions (i.e., winter season), while around
the equatorial zone, it is more common during the months of high humidity and precipitation
[239–245]. Essentially, it depends on explicit threshold conditions based on monthly
averages of specific humidity and temperature. When specific humidity drops below 11-12
g/kg and temperature drops below 18-21°C, the peak of influenza is stimulated during the
cold-dry season, however, for tropical and subtropical (always humid and warm) regions, it
is likely to prevail during the high precipitation (≥150 mm) months [239]. The “cold-dry” set
of climatic conditions endorses a greater survival of the virus outside human body, and, thus,
results in better transmission [237, 246]. Similar temperature dependency was concluded for
SARS (strain CoV-P9) coronavirus. Laboratory experiments testing virus stability,
demonstrated a decreasing infectivity with increasing ambient temperatures, where at 4°C,
56°C and 75°C the survival rates outside host decreased from at least 96 to 1.5 and to 0.5
hours, respectively [247]. In addition, cold air cools nasal epithelium which, in turn,
decreases mechanical defenses of the respiratory and immune systems [248].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Duan et. al., (2003) concluded that, even a relatively short exposure (1 hour) to UV radiation
destroys viral infectivity of SARS (strain CoV-P9) coronavirus. Other studies also correlate
vitamin D secretion and influenza immunity, due to the UV role in vitamin D production
[249, 250]. The latter, and the reduced immune system due to melatonin oscillations during
the dark (lack of sunlight hours) winter seasons could further explain winter outbreaks of
influenza at high latitude regions [251].

Finally, wind speed may contribute to the spread of influenza nanoparticles. While low winds
might improve its transmission from one host to another, strong winds contribute to its
dispersion and ventilation [252], which could be a positive effect depending on wind
direction.

Conclusions

The authors of this study examined the most important literature available in terms of the
genetic, virologic, clinical and therapeutic evidence on the SARS-CoV-2 virus and the novel
coronavirus diseases 2019 (COVID-19).
This extensive and comprehensive literature review tries to offer a good insight of the most
recent information available. This review was designed to offer a good insight of the virus
and the diseases to the entire medical community. This document although summarized,
tries to bring well-supported information on this new disease. A disease that has been keeping
us on a partial or total lockdown all over the planet.

List of Abbreviations:
ACE2: Angiotensin Converting Enzyme-2
ARDS: acute respiratory distress syndrome
CDCC: The Center for Disease Control in China
COVID-19: Coronavirus disease 2019
ELISA: The enzyme-linked immunosorbent assay
GISAID: Global Initiative on Sharing All Influenza Data
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

INF: Interferon
ISGs: IFN-stimulated genes
MERS: Middle East Respiratory Syndrome
MHC-I: Major Histocompatibility complex I
ORF: Open reading frames
PDB: Protein Data Bank archive
pDCs: plasmacytoid dendritic cells
RCSB: Research Collaboratory for Structural Bioinformatics
RNA: Ribonucleic acid
RdRp: RNA-dependent RNA polymerase
RBD: receptor-binding domain
SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2
SARS: Severe acute respiratory syndrome
TCGA: The Cancer Genome Atlas
TLRs: endosomal Toll like receptors
WHO: World Health Organization

Declarations
Ethics approval and consent to participate

According to the local and international regulation, this project did no required ethical
approval.

Consent to publish
Not Applicable.

Availability of data and materials

All the information used for this analysis can be found

Competing interests
The authors declare that they have no competing interests.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0283.v1

Funding
This work did not receive financial support of any kind.

Authors' Contributions

EOP was responsible for the full conceptualization and he was in charge of drafting the
document in all of the stages. KSR and LGB contributed with the Cancer and Covid-19
section. MRN completed the section about covid-19 in children. LG and CBO completed the
diagnosis section of the review. NK completed the immunological response to SARS CoV2.
CM completed the virologic aspects of the manuscript. AMG, DC, HSS and LU completed
the clinical section of the manuscript, the therapeutic strategies and the gynecological and
complication section of the manuscript. RZ completed the environmental effects and Covid-
19. NG critically review the manuscript and ALC was partially responsible for the
conceptualization of the study, the genetic aspects of the virus and he completed all the
figures for this work.

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