vaccine that can produce cross-reactive antibodies.

However, the success of such a vaccine relies greatly

on its ability to provide protection not only against

present versions of the virus but also the ones that

are likely to emerge in the future. This can be

achieved by identifying antibodies that can recognize

relatively conserved epitopes that are maintained as

such even after the occurrence of considerable

variations (362). Even though several vaccine

clinical trials are being conducted around the world,

pregnant women have been completely excluded

from these studies. Pregnant women are highly

vulnerable to emerging diseases such as COVID-19

due to alterations in the immune system and other

physiological systems that are associated with

pregnancy. Therefore, in the event of successful

vaccine development, pregnant women will not get

access to the vaccines (361). Hence, it is

recommended that pregnant women be included in

the ongoing vaccine trials, since successful

vaccination in pregnancy will protect the mother,

fetus, and newborn.

The heterologous immune effects induced by Bacillus Calmette Guérin (BCG) vaccination is a promising
strategy for controlling the COVID-19 pandemic and requires further investigations. BCG is a widely used
vaccine against tuberculosis in high-wrought havoc in China and caused a pandemic

situation in the worldwide population, leading to

disease outbreaks that have not been controlled to

date, although extensive efforts are being put in

place to counter this virus (25). This virus has been

proposed to be designated/named severe acute

respiratory syndrome coronavirus 2 (SARS-CoV-2)

by the International Committee on Taxonomy of

Viruses (ICTV), which determined the virus belongs

to the Severe acute respiratory syndrome-related

coronavirus category and found this virus is related

to SARS-CoVs (26). SARS-CoV-2 is a member of

the order Nidovirales, family Coronaviridae,

subfamily Orthocoronavirinae, which is subdivided

into four genera, viz., Alphacoronavirus,

Betacoronavirus, Gammacoronavirus, and

Deltacoronavirus

(3, 27). The genera

Alphacoronavirus and Betacoronavirus originate

from bats, while Gammacoronavirus and

Deltacoronavirus have evolved from bird and swine

gene pools (24, 28, 29, 275).

Coronaviruses possess an unsegmented, single- stranded, positive-sense RNA genome of around 30 kb,
enclosed by a 5'-cap and 3'-poly(A) tail (30). The genome of SARS-CoV-2 is 29,891 bp long, with a G+C
content of 38% (31). These viruses are encircled with an envelope containing viral exponentially in other
countries including South Korea, Italy and Iran. Of those infected, 20% are in critical condition, 25% have
recovered, and 3310 (3013 in China and 297 in other countries) have died [2]. India, which had reported
only 3 cases till 2/3/2020, has also seen a sudden spurt in cases. By 5/3/2020, 29 cases had been
reported; mostly in Delhi, Jaipur and Agra in Italian tourists and their contacts. One case was reported in
an Indian who traveled back from Vienna and exposed a large number of school children in a birthday
party at a city hotel. Many of the contacts of these cases have been quarantined.
These numbers are possibly an underestimate of the infected and dead due to limitations of surveillance
and testing. Though the SARS-CoV-2 originated from bats, the intermediary DIAGNOSIS OF SARS-CoV-2
(COVID- 19)

RNA tests can confirm the diagnosis of SARS-

CoV-2 (COVID-19) cases with real-time RT-PCR or

next-generation sequencing (148, 149, 245, 246). At

present, nucleic acid detection techniques, like RT-

PCR, are considered an effective method for

confirming the diagnosis in clinical cases of COVID-

19 (148). Several companies across the world are

currently focusing on developing and marketing

SARS-CoV-2-specific nucleic acid detection kits.

Multiple laboratories are also developing their own

in-house RT-PCR. One of them is the SARS-CoV-2

nucleic acid detection kit produced by Shuoshi

Biotechnology (double fluorescence PCR method)

(150). Up to 30 March 2020, the U.S. Food and Drug

Administration (FDA) had granted 22 in vitro

diagnostics Emergency Use Authorizations (EUAs),

including for the RT-PCR diagnostic panel for the

universal detection of SARS-like betacoronaviruses

and specific detection of SARS-CoV-2, developed

by the U.S. CDC (Table 1) (258, 259). developed for rapid and colorimetric detection of this virus (354).
RT-LAMP serves as a simple, rapid, and sensitive diagnostic method that does not require sophisticated
equipment or skilled personnel (349). An interactive web-based dashboard for tracking SARS-CoV-2 in a
real-time mode has been designed (238). A smartphone-integrated home-based point- of-care testing
(POCT) tool, a paper-based POCT combined with LAMP, is a useful point-of-care diagnostic (353). An
Abbott ID Now COVID-19 molecular POCT-based test, using isothermal nucleic acid amplification
technology, has been designed as a point-of-care test for very rapid detection of SARS-CoV-2 in just 5
min (344). A CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) diagnostic
for rapid detection of SARS-CoV-2 without the requirement of specialized instrumentation has been
reported to be very useful in the clinical diagnosis of COVID-19 (360). A CRISPR-Cas12-based lateral flow
assay also has been developed for rapid detection of SARS-CoV-2 (346). Artificial intelligence, by means
of a three- dimensional deep-learning model, has been developed for sensitive and specific diagnosis of
COVID-19 via CT images (332). Tracking and mapping of the rising incidence

rates, disease outbreaks, community spread, The exploration of fully human antibodies

(human single-chain antibodies; HuscFvs) or humanized nanobodies (single-domain antibodies; sdAb,

VH/VHH) could aid in blocking virus replication, as these agents can traverse the virus- infected cell
membranes (transbodies) and can interfere with the biological characteristics of the replicating virus
proteins. Such examples include transbodies to the influenza virus, hepatitis C virus, Ebola virus, and
dengue virus (206). Producing similar transbodies against intracellular proteins of coronaviruses, such as
papain-like proteases (PLpro), cysteine-like protease (3CLpro), or other nsps, which are essential for
replication and transcription of the virus, might formulate a practical move forward for a safer and
potent passive immunization approach for virus-exposed persons and rendering therapy to infected
patients.

In a case study on five grimly sick patients having symptoms of severe pneumonia due to COVID-19,
convalescent plasma administration was found to be helpful in patients recovering successfully. The
convalescent plasma containing a SARS-CoV-2-specific ELISA (serum) antibody titer higher than 1:1,000
and neutralizing antibody titer more significant than 40 was collected from the recovered patients and
used for plasma transfusion encircled with an envelope containing viral

nucleocapsid. The nucleocapsids in CoVs are

arranged in helical symmetry, which reflects an

atypical attribute in positive-sense RNA viruses (30).

The electron micrographs of SARS-CoV-2 revealed

a diverging spherical outline with some degree of

pleomorphism, virion diameters varying from 60 to

140 nm, and distinct spikes of 9 to 12 nm, giving the

virus the appearance of a solar corona (3). The CoV

genome is arranged linearly as 5'-leader-UTR-

replicase-structural genes (S-E-M-N)-3' UTR-

poly(A) (32). Accessory genes, such as 3a/b, 4a/b,

and the hemagglutinin-esterase gene (HE), are also

seen intermingled with the structural genes (30).

SARS-CoV-2 has also been found to be arranged

similarly and encodes several accessory proteins,

although it lacks the HE, which is characteristic of

some betacoronaviruses (31). The positive-sense

genome of CoVs serves as the mRNA and is

translated to polyprotein la/lab (ppla/lab) (33). A

replication-transcription complex (RTC) is formed in

double-membrane vesicles (DMVs) by nonstructural

proteins (nsps), encoded by the polyprotein gene

(34). Subsequently, the RTC synthesizes a nested set

of subgenomic RNAs (sgRNAs) via discontinuous

transcription (35). and chest discomfort, and in severe cases dyspnea and bilateral lung infiltration".
Among the first 27 docu- mented hospitalized patients, most cases were epidemi- ologically linked to
Huanan Seafood Wholesale Market a wet market located in downtown Wuhan, which sells not only
seafood but also live animals, including poultry and wildlife. According to a retrospective study, the
onset of the first known case dates back to 8 December 2019 (REF.). On 31 December, Wuhan Municipal
Health Commission notified the public of a pneumonia out- break of unidentified cause and informed
the World Health Organization (WHO) (FIG. 1).

By metagenomic RNA sequencing and virus isola- tion from bronchoalveolar lavage fluid samples fromm
patients with severe pneumonia, independent teams of Chinese scientists identified that the causative
agent of this emerging disease is a betacoronavirus that had never been seen before6,10,11. On 9
January 2020, the result of this etiological identification was publicly announced (FIG. 1). The first
genome sequence of the novel coro- navirus was published on the Virological website om 10 January,
and more nearly complete genome sequences determined by different research institutes were then
released via the GISAID database on 12 January Later, more patients with no history of exposure to
Huanan Seafood Wholesale Market were identified. Several familial clusters of infection were reported.
and nosocomial infection also occurred in health-care facilities. All these cases provided clear evidence
for human-to-human transmission of the new virus 4.12-14 As the outbreak coincided with the
approach of the lunar New Year, travel between cities before the festival facilitated virus transmission in
China. This novel coro- navirus pneumonia soon spread to other cities in Hube province and to other
parts of China. Within 1 month route warrants the introduction of negative fecal viral nucleic acid test
results as one of the additional discharge criteria in laboratory-confirmed cases of COVID-19 (326).

The COVID-19 pandemic does not have any novel factors, other than the genetically unique pathogen
and a further possible reservoir. The cause and the likely future outcome are just repetitions of our
previous interactions with fatal coronaviruses. The only difference is the time of occurrence and the
genetic distinctness of the pathogen involved. Mutations on the RBD of CoVs facilitated their capability
of infecting newer hosts, thereby expanding their reach to all corners of the world (85). This is a
potential threat to the health of both animals and humans. Advanced studies using Bayesian
phylogeographic reconstruction identified the most probable origin of SARS-CoV-2 as the bat SARS-like
coronavirus, circulating in the Rhinolophus bat family (86).

Phylogenetic analysis of 10 whole-genome sequences of SARS-CoV-2 showed that they are related to
two CoVs of bat origin, namely, bat-SL- CoVZC45 and bat-SL-CoVZXC21, which were reported during
2018 in China (17). It was reported that SARS-CoV-2 had been confirmed to use ACE2 as an entry
receptor while exhibiting an RBD similar only a matter of time before another zoonotic coronavirus
results in an epidemic by jumping the so-called species barrier (287).

The host spectrum of coronavirus increased when

a novel coronavirus, namely, SW1, was recognized

in the liver tissue of a captive beluga whale

(Delphinapterus leucas) (138). In recent decades,

several novel coronaviruses were identified from

different animal species. Bats can harbor these

viruses without manifesting any clinical disease but

are persistently infected (30). They are the only

mammals with the capacity for self-powered flight,

which enables them to migrate long distances, unlike

land mammals. Bats are distributed worldwide and

also account for about a fifth of all mammalian

species (6). This makes them the ideal reservoir host

for many viral agents and also the source of novel

coronaviruses that have yet to be identified. It has

become a necessity to study the diversity of

coronavirus in the bat population to prevent future

outbreaks that could jeopardize livestock and public

health. The repeated outbreaks caused by bat-origin

coronaviruses calls for the development of efficient

molecular surveillance strategies for studying

Betacoronavirus among animals (12), especially in

the Rhinolophus bat family (86). Chinese bats have

high commercial value, since they are used in another study, the average reproductive number of
COVID-19 was found to be 3.28, which is significantly higher than the initial WHO estimate of 1.4 to 2.5
(77). It is too early to obtain the exact Ro value, since there is a possibility of bias due to insufficient
data. The higher Ro value is indicative of the more significant potential of SARS-CoV-2 transmission in a
susceptible population. This is not the first time where the culinary practices of China have been blamed
for the origin of novel coronavirus infection in humans. Previously, the animals present in the live-animal
market were identified to be the intermediate hosts of the SARS outbreak in China (78). Several wildlife
species were found to harbor potentially evolving coronavirus strains that can overcome the species
barrier (79). One of the main principles of Chinese food culture is that live- slaughtered animals are
considered more nutritious (5).

After 4 months of struggle that lasted from December 2019 to March 2020, the COVID-19 situation now
seems under control in China. The wet animal markets have reopened, and people have started buying
bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins (scaly anteaters), minks, soup from palm
civet, ostriches, hamsters, snapping turtles, ducks, fish, Siamese crocodiles, and other major problem
associated with this diagnostic kit is that it works only when the test subject has an active infection,
limiting its use to the earlier stages of infection. Several laboratories around the world are currently
developing antibody-based diagnostic tests against SARS-CoV-2 (157).

Chest CT is an ideal diagnostic tool for

identifying viral pneumonia. The sensitivity of chest

CT is far superior to that of X-ray screening. The

chest CT findings associated with COVID-19-

infected patients include characteristic patchy

infiltration that later progresses to ground-glass

opacities (158). Early manifestations of COVID-19

pneumonia might not be evident in X-ray chest

radiography. In such situations, a chest CT

examination can be performed, as it is considered

highly specific for COVID-19 pneumonia (118).

Those patients having COVID-19 pneumonia will

exhibit the typical ground-glass opacity in their chest

CT images (154). The patients infected with

COVID-19 had elevated plasma angiotensin 2 levels.

The level of angiotensin 2 was found to be linearly

associated with viral load and lung injury, indicating

its potential as a diagnostic biomarker (121). The

chest CT imaging abnormalities associated with

COVID-19 pneumonia have also been observed even

in asymptomatic patients. These abnormalities fever, cough, and sputum (83). Hence, the clinicians must
be on the look-out for the possible occurrence of atypical clinical manifestations to avoid the possibility
of missed diagnosis. The early transmission ability of SARS-CoV-2 was found to be similar to or slightly
higher than that of SARS-CoV, reflecting that it could be controlled despite moderate to high
transmissibility (84).

Increasing reports of SARS-CoV-2 in sewage and

wastewater warrants the need for further

investigation due to the possibility of fecal-oral

transmission. SARS-CoV-2 present in environmental

compartments such as soil and water will finally end

up in the wastewater and sewage sludge of treatment

plants (328). Therefore, we have to reevaluate the

current wastewater and sewage sludge treatment

procedures and introduce advanced techniques that

are specific and effective against SARS-CoV-2.

Since there is active shedding of SARS-CoV-2 in the

stool, the prevalence of infections in a large

population can be studied using wastewater-based

epidemiology. Recently,

reverse transcription-

quantitative PCR (RT-qPCR) was used to enumerate

the copies of SARS-CoV-2 RNA concentrated from

wastewater collected from a wastewater treatment

plant (327). The calculated viral RNA copy numbers

determine the number of infected individuals. The viruses in nasal washes, saliva, urine and faeces for
up to 8 days after infection, and a few naive ferrets with only indirect contact were positive for viral
RNA, suggest- ing airborne transmission. In addition, transmission of the virus through the ocular surface
and prolonged presence of SARS-CoV-2 viral RNA in faecal samples were also documented 101,102.
Coronaviruses can persist on inanimate surfaces for days, which could also be the case for SARS-CoV-2
and could pose a prolonged risk of infection 103. These findings explain the rapid geographic spread of
COVID-19, and public health interventions to reduce transmission will provide benefit to mitigate the
epidemic, as has proved successful in China and several other countries, such as South Korea
89,104,105,

Diagnosis

Early diagnosis is crucial for controlling the spread of

COVID-19. Molecular detection of SARS-CoV-2 nucleic

acid is the gold standard. Many viral nucleic acid detec-

tion kits targeting ORF1b (including RdRp), N, E or

S genes are commercially available ¹1,106-109. The detection

time ranges from several minutes to hours depending

on the technology 106,107,109-111. The molecular detection

can be affected by many factors. Although SARS-CoV-2

has been detected from a variety of respiratory sources,

including throat swabs, posterior oropharyngeal saliva,

nasopharyngeal swabs, sputum and bronchial fluid,

the viral load is higher in lower respiratory tract sam-

ples 11,96,112-115. In addition, viral nucleic acid was also

found in samples from the intestinal tract or blood even

when respiratory samples were negative¹16. Lastly, viral

load may already drop from its peak level on disease

onset 96.97. Accordingly, false negatives can be common

when oral swabs and used, and so multiple detection

methods should be adopted to confirm a COVID-19

diagnosis 117,118. Other detection methods were there-

fore used to overcome this problem. Chest CT was

used to quickly identify a patient when the capacity of

molecular detection was overloaded in Wuhan. Patients Recently, 95 full-length genomic sequences of
SARAS-CoV-2 strains available in the National Center for Biotechnology Information and GISAID
databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying
variations in the viral genome (260). All the viral strains revealed high homology of 99.99% (99.91% to
100%) at the nucleotide level and 99.99% (99.79% to 100%) at the amino acid level. Overall variation
was found to be low in ORF regions, with 13 variation sites recognized in 1a, 1b, S, 3a, M, 8, and N
regions. Mutation rates of 30.53% (29/95) and 29.47% (28/95) were observed at nt 28144 (ORF8) and nt
8782 (ORF1a) positions, respectively. Owing to such selective mutations, a few specific regions of SARS-
CoV-2 should not be considered for designing primers and probes. The SARS-CoV-2 reference sequence
could pave the way to study molecular biology and pathobiology, along with developing diagnostics and
appropriate prevention and control strategies for countering SARS-CoV-2 (260).

Nucleic acids of SARS-CoV-2 can be detected from samples (64) such as bronchoalveolar lavage fluid,
sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal swabs, feces, blood, and
urine, with different levels of diagnostic performance (Table 2) (80, 245, 246). The viral loads range of
hosts, producing symptoms and diseases ranging from the common cold to severe and ultimately fatal
illnesses, such as SARS, MERS, and, presently, COVID-19. SARS-CoV-2 is considered one of the seven
members of the CoV family that infect humans (3), and it belongs to the same lineage of CoVs that
causes SARS; however, this novel virus is genetically distinct. Until 2020, six CoVs were known to infect
humans, including human CoV 229E (HCOV-229E), HCOV-NL63, HCOV-OC43, HCOV- HKUI, SARS-CoV,
and MERS-CoV. Although SARS-CoV and MERS-CoV have resulted in outbreaks with high mortality,
others remain associated with mild upper-respiratory-tract illnesses (4).

Newly evolved CoVs pose a high threat to global

public health. The current emergence of COVID-19

is the third CoV outbreak in humans over the past 2

decades (5). It is no coincidence that Fan et al.

predicted potential SARS- or MERS-like CoV

outbreaks in China following pathogen transmission

from bats (6). COVID-19 emerged in China and

spread rapidly throughout the country and,

subsequently, to other countries. Due to the severity

of this outbreak and the potential of spreading on an

international scale, the WHO declared a global

health emergency on 31 January 2020: subsequently samples obtained from lower respiratory tracts.
Hence, based on the viral load, we can quickly evaluate the progression of infection (291). In addition to
all of the above findings, sequencing and phylogenetics are critical in the correct identification and
confirmation of the causative viral agent and useful to establish relationships with previous isolates and
sequences, as well as to know, especially during an epidemic, the nucleotide and amino acid mutations
and the molecular divergence. The rapid development and implementation of diagnostic tests against
emerging novel diseases like COVID-19 pose significant challenges due to the lack of resources and
logistical limitations associated with an outbreak (155).

SARS-CoV-2 infection can also be confirmed by isolation and culturing. The human airway epithelial cell
culture was found to be useful in isolating SARS-CoV-2 (3). The efficient control of an outbreak depends
on the rapid diagnosis of the disease. Recently, in response to the COVID-19 outbreak, 1-step
quantitative real-time reverse transcription-PCR assays were developed that detect the ORF1b and N
regions of the SARS-CoV-2 genome (156). That assay was found to achieve the rapid detection of SARS-
CoV-2. Nucleic acid-based assays offer high accuracy in the diagnosis of SARS-The results of the studies
related to SARS-CoV-2

viral loads reflect active replication of this virus in

the upper respiratory tract and prolonged viral

shedding after symptoms disappear, including via

stool. Thus, the current case definition needs to be

updated along with a reassessment of the strategies

to be adopted for restraining the SARS-CoV-2

outbreak spread (248). In some cases, the viral load

studies of SARS-CoV-2 have also been useful to

recommend precautionary measures when handling

specific samples, e.g., feces. In a recent survey from

17 confirmed cases of SARS-CoV-2 infection with

available data (representing days 0 to 13 after onset),

stool samples from nine cases (53%; days 0 to 11

after onset) were positive on RT-PCR analysis.

Although the viral loads were lower than those of

respiratory samples (range, 550 copies per ml to

1.21 × 105 copies per ml), this has essential biosafety

implications (151). The samples from 18 SARS-CoV-2-positive patients in Singapore who had traveled
from Wuhan to Singapore showed the presence of viral RNA in stool and whole blood but not in urine by
real-time RT-PCR (288). Further, novel SARS-CoV-2 infections have been detected in a variety of clinical
specimens, like bronchoalveolar lavage fluid, adaptive evolution, close monitoring of the viral mutations
that occur during subsequent human-to- human transmission is warranted.

M Protein

The M protein is the most abundant viral protein present in the virion particle, giving a definite shape to
the viral envelope (48). It binds to the nucleocapsid and acts as a central organizer of coronavirus
assembly (49). Coronavirus M proteins are highly diverse in amino acid contents but maintain overall
structural similarity within different genera (50). The M protein has three transmembrane domains,
flanked by a short amino terminus outside the virion and a long carboxy terminus inside the virion (50).
Overall, the viral scaffold is maintained by M-M interaction. Of note, the M protein of SARS-CoV-2 does
not have an amino acid substitution compared to that of SARS-CoV (16).

E Protein
The coronavirus E protein is the most enigmatic and smallest of the major structural proteins (51). It
plays a multifunctional role in the pathogenesis, assembly, and release of the virus (52). It is a small
integral membrane polypeptide that acts as a viroporin (ion channel) (53). The inactivation or
Coronaviruses are a diverse group of viruses infecting

many different animals, and they can cause mild to

severe respiratory infections in humans. In 2002 and

2012, respectively, two highly pathogenic coronaviruses

with zoonotic origin, severe acute respiratory syndrome

coronavirus (SARS-CoV) and Middle East respiratory

syndrome coronavirus (MERS-CoV), emerged in

humans and caused fatal respiratory illness, making

emerging coronaviruses a new public health concern

in the twenty-first century¹. At the end of 2019, a novel

coronavirus designated as SARS-CoV-2 emerged in the

city of Wuhan, China, and caused an outbreak of unusual

viral pneumonia. Being highly transmissible, this novel

coronavirus disease, also known as coronavirus disease

2019 (COVID-19), has spread fast all over the world23.

It has overwhelmingly surpassed SARS and MERS in

terms of both the number of infected people and the

spatial range of epidemic areas. The ongoing outbreak of

COVID-19 has posed an extraordinary threat to global

public health. In this Review, we summarize the cur-

rent understanding of the nature of SARS-CoV-2 and

COVID-19. On the basis of recently published findings,

this comprehensive Review covers the basic biology

of SARS-CoV-2, including the genetic characteristics,

the potential zoonotic origin and its receptor binding.

Furthermore, we will discuss the clinical and epide-

miological features, diagnosis of and countermeasures

against COVID-19.

Emergence and spread

In late December 2019, several health facilities in Wuhan, in Hubei province in China, reported clusters
of patients with pneumonia of unknown cause. Similarly to patients with SARS and MERS, these patients
showed symptoms of viral pneumonia, including fever, cough or even die, whereas most young people
and children have only mild diseases (non-pneumonia or mild pneumonia) or are asymptomatic9,81,82.
Notably, the risk of disease was not higher for pregnant women. However, evidence of transplacental
transmission of SARS-CoV-2 from an infected mother to a neonate was reported, although it was an
isolated case83,84. On infection, the most common symptoms are fever, fatigue and dry cough
13.60,80,81, Less common symptoms include sputum production, headache, haemoptysis, diarrhoea,
anorexia, sore throat, chest pain, chills and nausea and vomiting in studies of patients in China
13,60,80,81. Self-reported olfac- tory and taste disorders were also reported by patients in Italy5. Most
people showed signs of diseases after an incubation period of 1-14 days (most commonly around 5
days), and dyspnoea and pneumonia developed within a median time of 8 days from illness onset.

In a report of 72,314 cases in China, 81% of the cases were classified as mild, 14% were severe cases that
required ventilation in an intensive care unit (ICU) and a 5% were critical (that is, the patients had
respiratory failure, septic shock and/or multiple organ dysfunction or failure)9,86. On admission,
ground-glass opacity was the most common radiologic finding on chest computed tomography (CT)
13,60,80,81. Most patients also developed marked lymphopenia, similar to what was observed in
patients with SARS and MERS, and non-survivors devel- oped severer lymphopenia over time
13,60,80,81. Compared with non-ICU patients, ICU patients had higher levels 216 countries and regions
from all six continents had reported more than 20 million cases of COVID-19, and more than 733,000
patients had died. High mortality occurred especially when health-care resources were overwhelmed.
The USA is the country with the largest number of cases so far.

Although genetic evidence suggests that SARS-CoV-2 is a natural virus that likely originated in animals,
there is no conclusion yet about when and where the virus first entered humans. As some of the first
reported cases in Wuhan had no epidemiological link to the seafood market, it has been suggested that
the market may not be the initial source of human infection with SARS-CoV-2. One study from France
detected SARS-CoV-2 by PCR in a stored sample from a patient who had pneumonia at the end of 2019,
suggesting SARS-CoV-2 might have spread there much earlier than the generally known starting time of
the outbreak in France. However, this individual early report cannot give a solid answer to the origin of
SARS-CoV-2 and contamination, and thus a false positive result cannot be excluded. To address this
highly controversial issue, further retrospective inves- tigations involving a larger number of banked
samples from patients, animals and environments need to be conducted worldwide with well-validated
assays.

Genomics, phylogeny and taxonomy

As a novel betacoronavirus, SARS-CoV-2 shares 79% genome sequence identity with SARS-CoV and 50%
with MERS-CoV24. Its genome organization is shared with other betacoronaviruses. The six functional
open reading frames (ORFs) are arranged in order from 5' to 3': replicase (ORF1a/ORF1b), spike (S),
envelope (E), membrane (M) and nucleocapsid (N). In addition, seven putative ORFs encoding accessory
proteins are interspersed between the structural genes. Most of the proteins encoded by SARS-CoV-2
have a similar helicase activity.

Among the evaluated compounds, 4-(cyclopent- 1-en-3-ylamino)-5-[2-(4-

iodophenyl)hydrazinyl]-4H-1,2,4-triazole-3-thiol and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-

chlorophenyl)hydrazinyl]-4H-1,2,4-triazole-3-thiol were found to be the most potent. These compounds

were used for in silico studies, and molecular docking was accomplished into the active binding site of
MERS-CoV helicase nsp13 (21). Further studies are required for evaluating the therapeutic potential of
these newly identified compounds in the management of COVID-19 infection.

Passive Immunization/Antibody Therapy/MAb Monoclonal antibodies (MAbs) may be helpful in the

intervention of disease in CoV-exposed individuals. Patients recovering from SARS showed robust
neutralizing antibodies against this CoV infection (164). A set of MAbs aimed at the MERS- CoV S
protein-specific domains, comprising six specific epitope groups interacting with receptor- binding,
membrane fusion, and sialic acid-binding sites, make up crucial entry tasks of S protein (198, 199).
Passive immunization employing weaker and strongly neutralizing antibodies provided considerable
protection in mice against a MERS- mask and practice cough hygiene. Caregivers should be asked to
wear a surgical mask when in the same room as patient and use hand hygiene every 15-20 min.

The greatest risk in COVID-19 is transmission to healthcare workers. In the SARS outbreak of 2002, 21%
of those affected were healthcare workers [31]. Till date, almost 1500 healthcare workers in China have
been infected with 6 deaths. The doctor who first warned about the virus has died too. It is important to
protect healthcare workers to ensure continuity of care and to prevent transmission of infection to
other patients. While COVID-19 transmits as a droplet pathogen and is placed in Category B of infectious
agents (highly pathogenic H5N1 and SARS), by the China National Health Commission, infection control
measures recommended are those for might be lower. Further genetic analysis is required between
SARS-CoV-2 and different strains of SARS-CoV and SARS-like (SL) CoVs to evaluate the possibility of
repurposed vaccines against COVID-19. This strategy will be helpful in the scenario of an outbreak,
since much time can be saved, because preliminary evaluation, including in vitro studies, already
would be completed for such vaccine candidates.

Multiepitope subunit vaccines can be considered

a promising preventive strategy against the ongoing

COVID-19 pandemic. In silico and advanced

immunoinformatic tools can be used to develop

multiepitope subunit vaccines. The vaccines that are

engineered by this technique can be further evaluated

using docking studies and, if found effective, then

can be further evaluated in animal models (365).

Identifying epitopes that have the potential to

become a vaccine candidate is critical to developing

an

effective vaccine against COVID-19. The

immunoinformatics approach has been used for

recognizing essential epitopes of cytotoxic T

lymphocytes and B cells from the surface

glycoprotein of SARS-CoV-2. Recently, a few

epitopes have been recognized from the SARS-CoV-

2 surface glycoprotein. The selected epitopes

explored targeting molecular dynamic simulations, Based on molecular characterization, SARS- CoV-2
is considered a new Betacoronavirus belonging to the subgenus Sarbecovirus (3). A few other critical
zoonotic viruses (MERS-related CoV and SARS-related CoV) belong to the same genus. However, SARS-
CoV-2 was identified as a distinct virus based on the percent identity with other Betacoronavirus;
conserved open reading frame la/b (ORFla/b) is below 90% identity (3). An overall 80% nucleotide
identity was observed between SARS-CoV-2 and the original SARS-CoV, along with 89% identity with
ZC45 and ZXC21 SARS- related CoVs of bats (2, 31, 36). In addition, 82% identity has been observed
between SARS-CoV-2 and human SARS-CoV Tor2 and human SARS-CoV BJ01 2003 (31). A sequence
identity of only 51.8% was observed between MERS-related CoV and the recently emerged SARS-CoV-
2 (37). Phylogenetic analysis of the structural genes also revealed that SARS-CoV-2 is closer to bat
SARS-related CoV. Therefore, SARS-CoV-2 might have originated from bats, while other amplifier
hosts might have played a role in disease transmission to humans (31). Of note, the other two
zoonotic CoVs (MERS-related CoV and SARS-related CoV) also originated from bats (38, 39).
Nevertheless, for SARS and MERS, civet understanding of the lung inflammation associated with this
infection (24).
SARS is a viral respiratory disease caused by a formerly unrecognized animal CoV that originated from
the wet markets in southern China after adapting to the human host, thereby enabling transmission
between humans (90). The SARS outbreak reported in 2002 to 2003 had 8,098 confirmed cases with
774 total deaths (9.6%) (93). The outbreak severely affected the Asia Pacific region, especially
mainland China (94). Even though the case fatality rate (CFR) of SARS-CoV-2 (COVID-19) is lower than
that of SARS-CoV, there exists a severe concern linked to this outbreak due to its epidemiological
similarity to influenza viruses (95, 279). This can fail the public health system, resulting in a pandemic
(96).

MERS is another respiratory disease that was first reported in Saudi Arabia during the year 2012. The
disease was found to have a CFR of around 35% (97). The analysis of available data sets suggests that
the incubation period of SARS-CoV-2, SARS-CoV, and MERS-CoV is in almost the same range. The
longest predicted incubation time of SARS-CoV-2 is 14 days. Hence, suspected individuals are isolated
for 14 days to avoid the risk of further spread (98). Even though a high similarity has been reported
lower respiratory tracts. Acute viral interstitial pneu-

monia and humoral and cellular immune responses were observed 48.75. Moreover, prolonged virus
shedding peaked early in the course of infection in asymptomatic macaques, and old monkeys
showed severer intersti- tial pneumonia than young monkeys, which is similar to what is seen in
patients with COVID-19. In human ACE2-transgenic mice infected with SARS-CoV-2, typ- ical interstitial
pneumonia was present, and viral anti- gens were observed mainly in the bronchial epithelial cells,
macrophages and alveolar epithelia. Some human ACE2-transgenic mice even died after infection
70.71. In wide-type mice, a SARS-CoV-2 mouse-adapted strain with the N501Y alteration in the RBD of
the S protein was generated at passage 6. Interstitial pneumonia and inflammatory responses were
found in both young and aged mice after infection with the mouse-adapted strain. Golden hamsters
also showed typical symptoms after being infected with SARS-CoV-2 (REF.7"). In other animal models,
including cats and ferrets, SARS-CoV-2 could efficiently replicate in the upper respiratory tract but did
not induce severe clinical symptoms 13.78. As trans- mission by direct contact and air was observed in
infected ferrets and hamsters, these animals could be used to model different transmission modes of
COVID-19 (REFS77-79). Animal models offer important information for understanding the
pathogenesis of SARS-CoV-2 infection and the transmission dynamics of SARS- CoV-2, and are
important to evaluate the efficacy of antiviral therapeutics and vaccines.

Clinical and epidemiological features

It appears that all ages of the population are susceptible to SARS-CoV-2 infection, and the median age
of infection is around 50 years9.13,60,80,81, However, clinical manifesta- tions differ with age. In
general, older men (>60 years old) with co-morbidities are more likely to develop severe respiratory
disease that requires hospitalization was linked to a family member and 26 children had history of
travel/residence to Hubei province in China. All the patients were either asymptomatic (9%) or had
mild disease. No severe or critical cases were seen. The most common symptoms were fever (50%)
and cough (38%). All patients recovered with symptomatic therapy and there were no deaths. One
case of severe pneumonia and multiorgan dysfunction in a child has also been reported [19]. Similarly
the neonatal cases that have been reported have been mild [20].

Diagnosis [21]

A suspect case is defined as one with fever, sore throat and cough who has history of travel to China
or other areas of persistent local transmission or contact with patients with similar travel history or
those with confirmed 6.5 Specimen collection and storage

A Nasopharyngeal and oropharyngeal swab should be collected using Dacron or polyester flocked
swabs. It should be transported to the laboratory at a temperature of 4°C and stored in the laboratory
between 4 and -70°C on the basis of the number of days and, in order to increase the viral load, both
nasopharyngeal and oropharyngeal swabs should be placed in the same tube. Bronchoalveolar lavage
and nasopharyngeal aspirate should be collected in a sterile container and transported similarly to the
laboratory by maintain a temperature of 4°C.

Sputum samples, especially from the lower respiratory tract, should be collected with the help of a
sterile container and stored, whereas tissue from a biopsy or autopsy should be collected using a
sterile container along with saline. However, both should be stored in the laboratory at a temperature
that ranges between 4 and -70°C. Whole blood for detecting the antigen, particularly in the first week
of illness, should be collected in a collecting tube and stored in the laboratory between 4 and -70°C.
Urine samples must also be collected using a sterile container and stored with SARS and MERS (117).

SARS-CoV-2 invades the lung parenchyma, resulting in severe interstitial inflammation of the lungs.
This is evident on computed tomography (CT) images as ground-glass opacity in the lungs. This lesion
initially involves a single lobe but later expands to multiple lung lobes (118). The histological
assessment of lung biopsy samples obtained from COVID-19-infected patients revealed diffuse
alveolar damage, cellular fibromyxoid exudates, hyaline membrane formation, and desquamation of
pneumocytes, indicative of acute respiratory distress syndrome (119). It was also found that the SARS-
CoV-2-infected patients often have lymphocytopenia with or without leukocyte abnormalities. The
degree of lymphocytopenia gives an idea about disease prognosis, as it is found to be positively
correlated with disease severity (118). Pregnant women are considered to have a higher risk of getting
infected by COVID-19. The coronaviruses can cause adverse outcomes for the fetus, such as
intrauterine growth restriction, spontaneous abortion, preterm delivery, and perinatal death.

Nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical transmission) of

CoVs is low and was not seen during either the SARS- or MERS-CoV outbreak (120). However, All of
these therapeutic approaches have revealed both in vitro and in vivo anti-CoV potential. Although in
vitro research carried out with these therapeutics showed efficacy, most need appropriate support
from randomized animal or human trials. Therefore, they might be of limited applicability and require
trials against SARS-CoV-2 to gain practical usefulness. The binding of SARS-CoV-2 with ACE2 leads to
the exacerbation of pneumonia as a consequence of the imbalance in the renin- angiotensin system
(RAS). The virus-induced pulmonary inflammatory responses may be reduced by the administration of
ACE inhibitors (ACEI) and angiotensin type-1 receptor (AT1R) (207).
Several investigations have suggested the use of small-molecule inhibitors for the potential control of
SARS-CoV infections. Drugs of the FDA-approved compound library were screened to identify four
small-molecule inhibitors of MERS-CoV (chlorpromazine, chloroquine, loperamide, and lopinavir) that
inhibited viral replication. These compounds also hinder SARS-CoV and human CoVs (208).
Therapeutic strategies involving the use of specific antibodies or compounds that neutralize cytokines
and their receptors will help to restrain the host inflammatory responses. Such drugs acting
specifically in the respiratory tract will help to Interestingly, disease in patients outside Hubei province
has been reported to be milder than those from Wuhan [17]. Similarly, the severity and case fatality
rate in patients outside China has been reported to be milder [6]. This may either be due to selection
bias wherein the cases reporting from Wuhan included only the severe cases or due to predisposition
of the Asian population to the virus due to higher expression of ACE2 receptors on the respiratory
mucosa [11].

Disease in neonates, infants and children has been also reported to be significantly milder than their
adult counterparts. In a series of 34 children admitted to a hospital in Shenzhen, China between
January 19th and February 7th, there were 14 males and 20 females. The median age was 8 y 11 mo
and in 28 children the infection was linked to a family member and 26 there, there is an increase in
the outbreak of this virus through human-to-human transmission, with the fact that it has become
widespread around the globe. This confirms the fact similar to the previous epidemics, including SARS
and MERS, that this coronavirus exhibited potential human-to-human transmission, as it was recently
declared a pandemic by WHO.26

Respiratory droplets are the major carrier for coronavirus transmission. Such droplets can either stay
in the nose or mouth or enter the lungs via the inhaled air. Currently, it is known that COVID-19's
transmission from one person to another also occurs through touching either an infected surface or
even an object. With the current scant awareness of the transmission systems however, airborne
safety measures with a high-risk procedure have been proposed in many countries. Transmission
levels, or the rates from one person to another, reported differ by both location and interaction with
involvement in infection control. It is stated that even asymptomatic individuals or those individuals
in their incubation period can act as carrier of SARS-CoV2.27, 28 With the data and evidence provided
by the CDC, the usual incubation period is probably 3 to 7 days, sometimes being prolonged up to
even 2 weeks, and the typical symptom occurrence assessed intrauterine vertical transmission of
COVID-19 infection in nine infants born to infected mothers, found that none of the infants tested
positive for the virus.45 Likewise, there was no evidence of intrauterine infection caused by vertical
transmission in the SARS and MERS epidemics.43

The CDC asserts that infants born to mothers with confirmed COVID-19 are considered persons under
investigation (PUI) and should be temporarily separated from the mother and isolated.46

7.1 Breastfeeding and infant care

The data available to date is limited and cannot confirm whether or not COVID-19 can be transmitted
through breast milk.40 Assessing the presence of COVID-19 in breast milk samples from six patients
showed negative result.45 The CDC points out that in case of a confirmed or suspected COVID-19
infection, the decision of whether or how to start or continue breastfeeding should be made by the
mother in collaboration with the family and healthcare practitioners.47 Careful precautions need to
be taken by the mother to prevent transmitting the disease to her infant through respiratory droplets
during breastfeeding. This includes wearing a facemask and practising hand vaccine, and li-Key
peptide COVID-19 vaccine are under preclinical trials (297). Similarly, the WHO, on its official website,
has mentioned a detailed list of COVID-19 vaccine agents that are under consideration. Different
phases of trials are ongoing for live attenuated virus vaccines, formaldehyde alum inactivated vaccine,
adenovirus type 5 vector vaccine, LNP-encapsulated mRNA vaccine, DNA plasmid vaccine, and S
protein, S-trimer, and li-Key peptide as a subunit protein vaccine, among others (298). The process of
vaccine development usually takes approximately ten years, in the case of inactivated or live
attenuated vaccines, since it involves the generation of long-term efficacy data. However, this was
brought down to 5 years during the Ebola emergency for viral vector vaccines. In the urgency
associated with the COVID-19 outbreaks, we expect a vaccine by the end of this year (343). The
development of an effective vaccine against COVID-19 with high speed and precision is the combined
result of advancements in computational biology, gene synthesis, protein engineering, and the
invention of advanced manufacturing platforms (342).

The recurring nature of the coronavirus outbreaks calls for the development of a pan-coronavirus
vaccine that can produce cross-reactive antibodies. nsps and Accessory Proteins

Besides the important structural proteins, the SARS-CoV-2 genome contains 15 nsps, nspl to nsp10
and nsp12 to nsp16, and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and ORF14) (16). All these
proteins play a specific role in viral replication (27). Unlike the accessory proteins of SARS-CoV, SARS-
CoV-2 does not contain 8a protein and has a longer 8b and shorter 3b protein (16). The nsp7, nsp13,
envelope, matrix, and p6 and 8b accessory proteins have not been detected with any amino acid
substitutions compared to the sequences of other coronaviruses (16).

The virus structure of SARS-CoV-2 is depicted in

Fig. 2.

polymorphism at nucleotide position 28,144, which results in amino acid substitution of Ser for Lys at
residue 84 of the ORF8 protein. Those variants with this muta- tion make up a single subclade labelled
as 'clade S'33,34. Currently, however, the available sequence data are not sufficient to interpret the
early global transmission his- tory of the virus, and travel patterns, founder effects and public health
measures also strongly influence the spread of particular lineages, irrespective of potential biological
differences between different virus variants.

Animal host and spillover

Bats are important natural hosts of alphacoronavi- ruses and betacoronaviruses. The closest relative
to SARS-CoV-2 known to date is a bat coronavirus detected in Rhinolophus affinis from Yunnan
province, China, named 'RaTG13', whose full-length genome sequence is 96.2% identical to that of
SARS-CoV-2 (REF."). This bat virus shares more than 90% sequence identity with SARS-CoV-2 in all
ORFs throughout the genome, including the highly variable S and ORF8 (REF."). Phylogenetic analysis
confirms that SARS-CoV-2 closely clusters with RaTG13 (FIG. 2). The high genetic similarity between
SARS-CoV-2 and RaTG13 supports the hypothesis that SARS-CoV-2 likely originated from bats35.
Another related coronavirus has been reported more recently in a Rhinolophus malayanus bat
sampled in Yunnan This novel bat virus denoted 'RmYN02' involved in the COVID-19 outbreak is of
great importance, because the strain on their mental well- being will affect their attention,
concentration, and decision-making capacity. Hence, for control of the COVID-19 outbreak, rapid steps
should be taken to protect the mental health of medical workers (229).

Since the living mammals sold in the wet market

are suspected to be the intermediate host of SARS-

CoV-2, there is a need for strengthening the

regulatory mechanism for wild animal trade (13).

The total number of COVID-19 confirmed cases is

on a continuous rise and the cure rate is relatively

low, making disease control very difficult to achieve.

The Chinese government is making continuous

efforts to contain the disease by taking emergency

control and prevention measures. They have already

built a hospital for patients affected by this virus and

are currently building

several more for

accommodating the continuously increasing infected

population (230). The effective control of SARS-

CoV-2/COVID-19 requires high-level interventions

like intensive contact tracing, as well as the

quarantine of people with suspected infection and the

isolation of infected individuals. The implementation

of rigorous control and preventive measures together

might control the Ro number and reduce the

transmission risk (228). Considering the zoonotic involved in the COVID-19 outbreak is of great
importance, because the strain on their mental well- being will affect their attention, concentration,
and decision-making capacity. Hence, for control of the COVID-19 outbreak, rapid steps should be
taken to protect the mental health of medical workers (229).

Since the living mammals sold in the wet market

are suspected to be the intermediate host of SARS-

CoV-2, there is a need for strengthening the

regulatory mechanism for wild animal trade (13).

The total number of COVID-19 confirmed cases is

on a continuous rise and the cure rate is relatively

low, making disease control very difficult to achieve.

The Chinese government is making continuous

efforts to contain the disease by taking emergency

control and prevention measures. They have already

built a hospital for patients affected by this virus and

are currently building

several more for

accommodating the continuously increasing infected

population (230). The effective control of SARS-

CoV-2/COVID-19 requires high-level interventions

like intensive contact tracing, as well as the

quarantine of people with suspected infection and the

isolation of infected individuals. The implementation

of rigorous control and preventive measures together

might control the Ro number and reduce the

transmission risk (228). Considering the zoonotic There is a new public health crises

threatening the world with the

emergence and spread of 2019 novel

coronavirus (2019-nCoV) or the severe

acute respiratory syndrome

coronavirus 2 (SARS-CoV-2). The virus

originated in bats and was transmitted

to humans through yet unknown

intermediary animals in Wuhan, Hubei

province, China in December 2019.

There have been around 96,000

reported cases of coronavirus disease

2019 (COVID-2019) and 3300 reported

deaths to date (05/03/2020). The disease

is transmitted by inhalation or contact

with infected droplets and the

incubation period ranges from 2 to 14

d. The symptoms are usually fever,

cough, sore throat, breathlessness,

fatigue, malaise among others. The

disease is mild in most people; in some

(usually the elderly and those with

comorbiditias) it may progress to the United States, tilorone dihydrochloride (tilorone),

was previously found to possess potent antiviral activity against MERS, Marburg, Ebola, and
Chikungunya viruses (306). Even though it had broad-spectrum activity, it was neglected for an
extended period. Tilorone is another antiviral drug that might have activity against SARS-CoV-2.

Remdesivir, a novel nucleotide analog prodrug,

was developed for treating Ebola virus disease

(EVD), and it was also found to inhibit the

replication of SARS-CoV and MERS-CoV in

primary human airway epithelial cell culture systems

(195). Recently, in vitro study has proven that

remdesivir has better antiviral activity than lopinavir

and ritonavir. Further, in vivo studies conducted in

mice also identified that treatment with remdesivir

improved pulmonary function and reduced viral

loads and lung pathology both in prophylactic and

therapeutic regimens

compared

to

lopinavir/ritonavir-IFN-y treatment in MERS-CoV

infection (8). Remdesivir also inhibits a diverse

range of coronaviruses, including circulating human

CoV, zoonotic bat CoV, and prepandemic zoonotic

CoV (195). Remdesivir is also considered the only

therapeutic drug that significantly reduces

pulmonary pathology (8). All these findings indicate

that remdesivir has to be further evaluated for its (173, 174). Hence, knowledge and understanding of
S protein-based vaccine development in SARS-CoV will help to identify potential S protein vaccine
candidates in SARS-CoV-2. Therefore, vaccine strategies based on the whole S protein, S protein
subunits, or specific potential epitopes of S protein appear to be the most promising vaccine
candidates against coronaviruses. The RBD of the S1 subunit of S protein has a superior capacity to
induce neutralizing antibodies. This property of the RBD can be utilized for designing potential SARS-
CoV vaccines either by using RBD-containing recombinant proteins or recombinant vectors that
encode RBD (175). Hence, the superior genetic similarity existing between SARS-CoV-2 and SARS- CoV
can be utilized to repurpose vaccines that have proven in vitro efficacy against SARS-CoV to be utilized
for SARS-CoV-2. The possibility of cross- protection in COVID-19 was evaluated by comparing the S
protein sequences of SARS-CoV-2 with that of SARS-CoV. The comparative analysis confirmed that the
variable residues were found concentrated on the S1 subunit of S protein, an important vaccine target
of the virus (150). Hence, the possibility of SARS-CoV-specific neutralizing antibodies providing cross-
protection to COVID-19 might be lower. Further genetic analysis is required INTRODUCTION

Over the past 2 decades, coronaviruses (CoVs) have been associated with significant disease
outbreaks in East Asia and the Middle East. The severe acute respiratory syndrome (SARS) and the
Middle East respiratory syndrome (MERS) began to emerge in 2002 and 2012, respectively. Recently, a
novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing
coronavirus disease 2019 (COVID-19), emerged in late 2019, and it has posed a global health threat,
causing an ongoing pandemic in many countries and territories (1).

Health workers worldwide are currently making efforts to control further disease outbreaks caused by
the novel CoV (originally named 2019-nCoV), which was first identified in Wuhan City, Hubei Province,
China, on 12 December 2019. On 11 February 2020, the World Health Organization (WHO) announced
the official designation for the current CoV-associated disease to be COVID-19, caused by SARS-CoV-2.
The primary cluster of patients was found to be connected with the Huanan South China Seafood
Market in Wuhan (2). CoVs belong to the family Coronaviridae (subfamily Coronavirinae), the
members of which infect a broad