The interferon response is one of the major innate immunity defences against virus invasion.

Interferons induce the expression of diverse interferon-stimulated genes, which can interfere with
every step of virus replication. Previous studies identified type I interferons as a promising
therapeutic candidate for SARS149. In vitro data showed SARS-CoV-2 is even more sensitive to type I
interferons than SARS-CoV , suggesting the potential effectiveness of type I interferons in the early
treatment of COVID-19 (REF.150). In China, vapor inhalation of interferon-a is included in the
COVID-19 treatment guideline151. Clinical trials are ongoing across the world to evaluate the efficacy
of different therapies involving interferons, either alone or in combination with other agents152.

Immunoglobulin therapy

Convalescent plasma treatment is another potential adjunctive therapy for COVID-19. Preliminary
findings have suggested improved clinical status after the treatment153 154. The FDA has provided
guidance for the use of COVID-19 convalescent plasma under an emergency investigational new drug
application. However, this treatment may have adverse effects by causing antibody-mediated
enhancement of infection, transfusion-associated acute lung injury and allergic transfusion reactions.

Monoclonal antibody therapy is an effective immunotherapy for the treatment of some viral
infections in select patients. Recent studies reported specific monoclonal antibodies neutralizing
SARS-CoV-2 infection.

Inhibition of virus replication

Replication inhibitors include remdesivir (GS-5734), favilavir (T-705 ), ribavirin, lopinavir and ritonavir.
Except for lopinavir and ritonavir, which inhibit 3CLpro, the other three all target RdRp128135(FIG.
5). Remdesivir has shown activity against SARS-CoV-2 in vitro and in vivo128136 A clinical study
revealed a lower need for oxygen support in patients with COVID-19 (REF137). Preliminary results of
the Adaptive COVID-19 Treatment Trial (ACTT) clinical trial by the National Institute of Allergy and
Infectious Diseases (NIAID) reported that remdesivir can shorten the recovery time in hospitalized
adults with COVID-19 by a couple days compared with placebo, but the difference in mortality was
not statistically significant138 . The FDA has issued an emergency use authorization for remdesivir for
the treatment of hospitalized patients with severe COVID-19. It is also the first approved option by
the European Union for treatment of adults and adolescents with pneumonia requiring supplemental
oxygen. Several international phase III clinical trials are continuing to evaluate the safety and efficacy
of remdesivir for the treatment of COVID-19.

Favilavir (T-705), which is an antiviral drug developed in Japan to treat influenza, has been approved
in China, Russia and India for the treatment of COVID-19. A clinical study in China showed that
favilavir significantly reduced the signs of improved disease signs on chest imaging and shortened the
time to viral clearance139. A preliminary report in Japan showed rates of clinical improvement of
73.8% and 87.8% from the start of favilavir therapy in patients with mild COVID-19 at 7 and 14 days,
respectively, and 40.1% and 60.3% in patients with severe COVID-19 at 7 and 14 days, had >95%
homology with the bat coronavirus and > 70% similarity with the SARS- CoV. Environmental samples
from the Huanan Sea food market also tested positive, signifying that the virus originated from there
[7]. The number of cases started increasing exponentially, some of which did not have exposure to
the live animal market, suggestive of the fact that human-to-human transmission was occurring [8].
The first fatal case was reported on 11th Jan 2020. The massive migration of Chinese during the
Chinese New Year fuelled the epidemic. Cases in other provinces of China, other countries (Thailand,
Japan and South Korea in quick succession) were reported in people who were returning from
Wuhan. Transmission to healthcare workers caring for patients was described on 20th Jan, 2020. By
23rd January, the 11 million population of Wuhan was placed under lock down other clinical trials in
different phases are still ongoing elsewhere.

Immunomodulatory agents

SARS-CoV-2 triggers a strong immune response which may cause cytokine storm syndrome6061.
Thus, immunomodulatory agents that inhibit the excessive inflammatory response may be a
potential adjunctive therapy for COVID-19. Dexamethasone is a corticosteroid often used in a wide
range of conditions to relieve inflammation through its anti-inflammatory and immunosuppressant
effects. Recently, the RECOVERY trial found dexamethasone reduced mortality by about one third in
hospitalized patients with COVID-19 who received invasive mechanical ventilation and by one fifth in
patients receiving oxygen. By contrast, no benefit was found in patients without respiratory
support146.

Tocilizumab and sarilumab, two types ofinterleukin-6 (IL-6) receptor-specific antibodies previously
used to treat various types of arthritis, including rheumatoid arthritis, and cytokine release
syndrome, showed effectiveness in the treatment of severe COVID-19 by attenuating the cytokine
storm in a small uncontrolled trial147. Bevacizumab is an anti-vascular endothelial growth factor
(VEGF) medication that could potentially reduce pulmonary oedema in patients with severe
COVID-19. Eculizumab is a specific monoclonal antibody that inhibits the proinflammatory
complement protein CS. Preliminary results showed that it induced a drop of inflammatory markers
and C-reactive protein levels, suggesting its potential to be an option for the treatment of severe
COVID-19 [REF148. Article gives a bird’s eye view about this new virus. Since knowledge about this
virus is rapidly evolving, readers are urged to update themselves regularly.

History

Coronaviruses are enveloped positive sense RNA viruses ranging from 60 nm to 140 nm in diameter
with spike like projections on its surface giving it a crown like appearance under the electron
microscope; hence the name coronavirus [3]. Four corona viruses namely HKU1, NL63, 229E and
OC43 have been in circulation in humans, and generally cause mild respiratory disease.

There have been two events in the past two decades wherein crossover of animal betacorona viruses
to humans has resulted in severe disease. The first such instance was in 2002-2003 when a

Inhibition of virus entry

SARS-CoV-2 uses ACE2 as the receptor and human proteases as entry activators; subsequently it
fuses the viral membrane with the cell membrane and achieves invasion. Thus, drugs that interfere
with entry may be a potential treatment for COVID-19. Umifenovir (Arbidol) is a drug approved in
Russia and China for the treatment of influenza and other respiratory viral infections. It can target
the interaction between the S protein and ACE2 and inhibit membrane fusion (FIG. 5). In vitro
experiments showed that it has activity against SARS-CoV-2, and current clinical data revealed it may
be more effective than lopinavir and ritonavir in treating COVID-19 (REFS122'123. However, other
clinical studies showed umifenovir might not improve the prognosis of or accelerate SARS-CoV-2
clearance in patients with mild to moderate COVID-19 (REFS12 125J. Yet some ongoing clinical trials
are evaluating its efficacy for COVID-19 treatment. Camostat mesylate is approved in Japan for the
treatment of pancreatitis and postoperative reflux oesophagitis. Previous studies showed that it can
prevent SARS-CoV from entering cells by blocking TMPRSS2 activity and protect mice from lethal
infection with SARS-CoV in a pathogenic mouse model (wildtype mice infected with a
mouse-adapted SARS-CoV strain)126 127. Recently, a study revealed that camostat mesylate blocks
the entry of SARS-CoV-2 into human lung cells47. Thus, it can be a potential antiviral drug against
SARS-CoV-2 infection, although so far there are not sufficient clinical data to support its efficacy.

Abstract

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 days. 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 comorbidities) it may progress to and Middle
East respiratory syndrome coronavirus (MERS-CoV), but has lower fatality. The global impact of this
new epidemic is yet uncertain.

Keywords: 2019-nCOV, SARS-CoV-2, COVID-19, Pneumonia, Review

Introduction

The 2019 novel coronavirus (2019- nCoV) or the severe acute respiratory syndrome corona virus 2
(SARS-CoV-2) as it is now called, is rapidly spreading from its origin in Wuhan City of Hubei Province
of China to the rest of the world [1]. Till 05/03/20 20 around 96,000 cases of coronavirus disease
2019 (COVID-1 9) and 3300 deaths have been reported [2]. India has reported 29 cases till date.
Fortunately, so far, children have been infrequently affected with no deaths. But the future course of
this virus is unknown. This article gives a bird's eye view about with COVID-19 showed typical
features on initial CT, including bilateral multilobar ground-glass opacities with a peripheral or
posterior distribution118 119. Thus, it has been suggested that CT scanning combined with repeated
swab tests should be used for individuals with high clinical suspicion of COVID-19 but who test
negative in initial nucleic acid screening118 Finally, SARS-CoV-2 serological tests detecting antibodies
to N or S protein could complement molecular diagnosis, particularly in late phases after disease
onset or for retrospective studies116,120,121 However, the extent and duration of immune
responses are still unclear, and available serological tests differ in their sensitivity and specificity, all
of which need to be taken into account when one is deciding on serological tests and interpreting
their results or potentially in the future test for T cell responses.

Therapeutics

To date, there are no generally proven effective therapies for COVID-19 or antivirals against
SARS-CoV-2, although some treatments have shown some benefits in certain subpopulations of
patients or for certain end points (see later). Researchers and manufacturers are conducting large-
scale clinical trials to evaluate various therapies for COVID-19. As of 2 October 2020, there were
about 405 therapeutic drugs in development for COVID-19, and nearly 318 in human clinical trials
(COVID-19 vaccine and therapeutics tracker). In the following sections, we summarize potential
therapeutics against SARS-CoV-2 on the basis of published clinical data and experience.

Chloroquine and hydroxychloroquine are other potential but controversial drugs that interfere with
the entry of SARS-CoV-2. They have been used in the prevention and treatment of malaria and
autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. They can
inhibit the glycosylation of cellular receptors and interfere with virus-host receptor binding, as well
as increase the endosomal pH and inhibit membrane fusion. Currently, no scientific consensus has
been reached for their efficacy in the treatment of COVID-19. Sorne studies showed they can inhibit
SARS-CoV-2 infection in vitro, but the clinical data are insufficient128,129. Two clinical studies
indicated no association with death rates in patients receiving chloroquine or hydroxychloroquine
compared with those not receiving the drug and even suggest it may increase the risk of dying as a
higher risk of cardiac arrest was found in the treated patients130,131. On 15 June 2020, owing to the
side effects observed in clinical trials, the US Food and Drug Administration (FDA) revoked the
emergency use authorization for chloroquine and hydroxychloroquine for the treatment of
COVID-19. Another potential therapeutic strategy is to block binding of the S protein to ACE2 through
soluble recombinant hACE2, specific monoclonal antibodies or fusion inhibitors that target the
SARS-CoV-2 S protein132-134(FIG. 5). The safety and efficacy of these strategies need to be assessed
in future clinical trials. in vitro and in vivo155-158. Compared with convalescent plasma, which has
limited availability and cannot be amplified, monoclonal antibodies can be developed in larger
quantities to meet clinical requirements. Hence, they provide the possibility for the treatment and
prevention of COVID-19. The neutralizing epitopes of these monoclonal antibodies also offer
important information for vaccine design. However, the high cost and limited capacity of
manufacturing, as well as the problem of bioavailability, may restrict the wide application of
monoclonal antibody therapy.

Vaccines

Vaccination is the most effective method for a long-term strategy for prevention and control of
COVID-19 in the future. Many different vaccine platforms against SARS-CoV-2 are in development,
the strategies of which include recombinant vectors, DNA, mRNA in lipid nanoparticles, inactivated
viruses, live attenuated viruses and protein subunits159-161. As of 2 October 2020, ~174 vaccine
candidates for COVID-19 had been reported and 51 were in human clinical trials (COVID-19 vaccine
and therapeutics tracker). Many of these vaccine candidates are in phase II testing, and some have
already advanced to phase III trials. A randomized double-blinded phase II trial of an adenovirus type
vectored vaccine expressing the SARS-CoV-2 S protein, developed by CanSino Biologicals and the
Academy of Military Medical Sciences of China, was conducted in 603 adult volunteers in Wuhan.
The vaccine has proved to be safe and induced considerable humoral and cellular immune response
in most recipients after a single immunization.162 Another vectored vaccine, ChAdOxl,

comorbidities), it may progress to pneumonia, acute respiratory distress syndrome (ARDS) and multi
organ dysfunction. Many people are asymptomatic. The case fatality rate is estimated to range from
2 to 3%. Diagnosis is by demonstration of the virus in respiratory secretions by special molecular
tests. Common laboratory findings include normal/ low white cell counts with elevated C reactive
protein (CRP). The computerized tomographic chest scan is usually abnormal even in those with no
symptoms or mild disease. Treatment is essentially supportive; role of antiviral agents is yet to be
established. Prevention entails home isolation of suspected cases and those with mild illnesses and
strict infection control measures at hospitals that include contact and droplet precautions. The virus
spreads faster than its two ancestors the SARS-CoV

by the University of Oxford. In a randomized controlled phase I/II trial, it induced neutralizing
antibodies against SARS-CoV-2 in all 1,077 participants after a second vaccine dose, while its safety
profile was acceptable as well163. The NIAID and Moderna co-manufactured mRNA-1273, a lipid
nanoparticle-formulated mRNA vaccine candidate that encodes the stabilized prefusion SARS-CoV-2 S
protein. Its immunogenicity has been confirmed by a phase I trial in which robust neutralizing
antibody responses were induced in a dose-dependent manner and increased after a second
dose164. Regarding inactivated vaccines, a successful phase I/II trial involving 320 participants has
been reported in China. The whole-virus COVID-19 vaccine had a low rate of adverse reactions and
effectively induced neutralizing antibody production165. The verified safety and immunogenicity
support advancement of these vaccine candidates to phase III clinical trials, which will evaluate their
efficacy in protecting healthy populations from SARS-CoV-2 infection.

Future perspectives

COVID-19 is the third highly pathogenic human coronavirus disease to date. Although less deadly
than SARS and MERS, the rapid spreading of this highly contagious disease has posed the severest
threat to global health in this century. The SARS-CoV-2 outbreak has lasted for more than half a year
now, and it is likely that

this emerging virus will establish a niche in humans and coexist with us for a long time166.Before
clinically approved vaccines are widely available, there is no better way to protect us from
SARS-CoV-2 than personal preventive behaviours such as social distancing and wearing masks, and
public health measures, including active testing, case tracing and restrictions on social gatherings.
Despite a flood of SARS-CoV-2 research published every week, current knowledge of this novel
coronavirus is just the tip of the iceberg. The animal origin and cross-species infection route of
SARS-CoV-2 are yet to be uncovered. The molecular mechanisms of SARS-CoV-2 infection
pathogenesis and virus-host the SARS- CoV. Environmental samples from the Huanan Sea food
market also tested positive, signifying that the virus originated from there [7]. The number of cases
started increasing exponentially, some of which did not have exposure to the live animal market,
suggestive of the fact that human-to-human transmission was occurring [8]. The first fatal case was
reported on 11th Jan 2020. The massive migration of Chinese during the Chinese New Year fuelled
the epidemic. Cases in other provinces of China, other countries (Thailand, Japan and South Korea in
quick succession) were reported in people who were returning from Wuhan. Transmission to
healthcare workers caring for patients was described on 20th Jan, 2020. By 23rd January, the 11
million population of Wuhan was placed under Iock down with restrictions of entry and exit from the
region. Soon this Iock down was 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 HSN1 and SARS), by the China National Health
Commission, infection control measures recommended are those for

Origin and Spread of COVID-19

[1, 2, 6]

In December 2019, adults in Wuhan, capital city of Hubei province and a major transportation hub of
China started presenting to local hospitals with severe pneumonia of unknown cause. Many of the
initial cases had a common exposure to the Huanan wholesale seafood market that also traded live
animals. The surveillance system (put into place after the SARS outbreak) was activated and
respiratory samples of patients were sent to reference labs for etiologic investigations. On December
31st 2019, China notified the outbreak to the World Health Organization and on 1st January the
Huanan Sea food market was closed. On 7th January the virus was identified as a coronavirus that
had >95% homology with the bat prongs, face mask, high flow nasal cannula (HFNC) or non-invasive
ventilation is indicated. Mechanical ventilation and even extra corporeal membrane oxygen support
may be needed. Renal replacement therapy may be needed in some. Antibiotics and antifungals are
required if co-infections are suspected or proven. The role of corticosteroids is unproven; while
current international consensus and WHO advocate against their use, Chinese guidelines do
recommend short term therapy with low-tomoderate dose corticosteroids in COVID-19 ARDS [24,
25]. Detailed guidelines for critical care management for COVID-19 have been published by the WHO
[26]. There is, as of now, no approved treatment for COVID-19. Antiviral drugs such as ribavirin,
lopinavir-ritonavir have been used based on the experience with SARS and MERS. In a historical

pandemic flu where patients were asked to resume work/school once afebrile for 24 h or by day 7 of
illness. Negative molecular tests were not a prerequisite for discharge.

At the community level, people should be asked to avoid crowded areas and postpone non-essential
travel to places with ongoing transmission. They should be asked to practice cough hygiene by
coughing in sleeve/ tissue rather than hands and practice hand hygiene frequently every 15- 20 min.
Patients with respiratory symptoms should be asked to use surgical masks. The use of mask by
healthy people in public places has not shown to protect against respiratory viral infections and is
currently not recommended by WHO. However, in China, the public has been asked to wear masks in
public and especially in crowded places and large-scale gatherings are prohibited (entertainment
parks etc). China also

Prevention [21, 30]

Since at this time there are no approved treatments for this infection, prevention is crucial. Several
properties of this virus make prevention difficult namely, nonspecific features of the disease, the
infectivity even before onset of symptoms in the incubation period, transmission from asymptomatic
people, long incubation period, tropism for mucosal surfaces such as the conjunctiva, prolonged
duration of the illness and transmission even after clinical recovery.

Isolation of confirmed or suspected cases with mild illness at home is recommended. The ventilation
at home should be good with sunlight to allow for destruction of virus. Patients should be asked to
wear a simple surgical mask and practice cough hygiene. themselves while examining such patients
and practice hand hygiene frequently.

• Suspected cases should be referred to government designated centers for isolation and
testing (in Mumbai, at this time, it is Kasturba hospital). Commercial kits for testing are not yet
available in India.

• Patients admitted with severe pneumonia and acute respiratory distress syndrome should be
evaluated for travel history and placed under contact and droplet isolation. Regular decontamination
of surfaces should be done. They should be tested for etiology using multiplex PCR panels if logistics
permit and if no pathogen is identified, refer the samples for testing for SARSCoV-2.

been used based on the experience with SARS and MERS. In a historical control study in patients with
SARS, patients treated with lopinavir ritonavir with ribavirin had better outcomes as compared to
those given ribavirin alone [15].
 In the case series of 99 hospitalized patients with COVID-19 infection from Wuhan, oxygen was given
to 76%, non invasive ventilation in 13%, mechanical ventilation in 4%, extracorporeal membrane
oxygenation (ECMO) in 3%, continuous renal replacement therapy (CRRT) in 9%, antibiotics in 71%,
antifungals in 15%, glucocorticoids in 19% and intravenous immunoglobulin therapy in 27% [15].
Antiviral therapy consisting of oseltamivir, ganciclovir and lopinavir ritonavir was given to 75% of the
patients. The duration of non-invasive ventilation was 4-22 d [median 9 d] interactions remain largely
unclear. Intensive studies on these virological profiles of SARS-CoV-2 will provide the basis for the
development of preventive and therapeutic strategies against COVID-19. Moreover, continued
genomic monitoring of SARS-CoV-2 in new cases is needed worldwide, as it is important to promptly
identify any mutation that may result in phenotypic changes of the virus. Finally, COVID-19 is
challenging all human beings. Tackling this epidemic is a long-term job which requires efforts of every
individual, and international collaborations by scientists, authorities and the public. such instance
was in 2002-2003 when a new coronavirus of the β genera and with origin in bats crossed over to
humans via the intermediary host of palm civet cats in the Guangdong province of China. This virus,
designated as severe acute respiratory syndrome coronavirus affected 8422 people mostly in China
and Hong Kong and caused 916 deaths (mortality rate 11%) before being contained [4]. Almost a
decade later in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV), also of bat
origin, emerged in Saudi Arabia with dromedary camels as the intermediate host and affected 2494
people and caused 858 deaths (fatality rate 34%)[5].

Origin and Spread of COVID-19

[1, 2, 6]

In December 2019, adults in Wuhan, capital city of Hubei province and a respectively140. However,
this study did not include a control arm, and most of the trials of favilavir were based on a small
sample size. For more reliable assessment of the effectiveness of favilavir for treating COVID-19,
large-scale randomized controlled trials should be conducted.

Lopinavir and ritonavir were reported to have in vitro inhibitoryactivity against SARS-CoV and

MERS-CoV141 ,142. Alone, the combination of lopinavir and ritonavir had little therapeutic benefit
in patients with COVID-19, but appeared more effective when used in combination with other drugs,
including ribavirin and interferon beta-1b143,144. The Randomized Evaluation of COVID-19 Therapy
(RECOVERY) trial, a national clinical trial program in the UK, has stopped treatment with lopinavir and
ritonavir as no significant beneficial effect was observed in a randomized trial established in March
2020 with a total of 1,596 patients145. Nevertheless,

Practice Points from an Indian Perspective

At the time of writing this article, the risk of coronavirus in India is extremely low. But that may
change in the next few weeks. Hence the following is recommended:

• Healthcare providers should take travel history of all patients with respiratory symptoms, and any
international travel in the past 2 weeks as well as contact with sick people who have travelled
internationally.

• They should set up a system of triage of patients with respiratory illness in the outpatient
department and give them a simple surgical mask to wear. They should use surgical masks
themselves while examining such (entertainment parks etc). China is also considering introducing
legislation to prohibit selling and trading of wild animals [32].
The international response has been dramatic. Initially, there were massive travel restrictions to
China and people returning from China/ evacuated from China are being evaluated for clinical
symptoms, isolated and tested for COVID-19 for 2 weeks even if asymptomatic. However, now with
rapid world wide spread of the virus these travel restrictions have extended to other countries.
Whether these efforts will lead to slowing of viral spread is not known.

A candidate vaccine is under development.

Practice Points from an Indian Perspective

[ median 17 d]. In the case series of children discussed earlier, ail children recovered with basic
treatment and did not need intensive care [17].

There is anecdotal experience with use of remdeswir, a broad spectrum anti RNA drug developed for
Ebola in management of COVID-19 [27]. More evidence is needed before these drugs are
recommended. Other drugs proposed for therapy are arbidol (an antiviral drug available in Russia
and China), intravenous immunoglobulin, interferons, chloroquine and plasma of patients recovered
from COVID-19 [21, 28, 29]. Additionally, recommendations about using traditional Chinese herbs
find place in the Chin es e guidelines [21].

Prevention [21, 30]

category A agents (cholera, plague). Patients should be placed in separate rooms or cohorted
together. Negative pressure rooms are not generally needed. The rooms and surfaces and equipment
should undergo regular decontamination preferably with sodium hypochlorite. Healthcare workers
should be provided with fit tested N95 respirators and protective suits and goggles. Airborne
transmission precautions should be taken during aerosol generating procedures such as intubation,
suction and tracheostomies. Ali contacts including healthcare workers should be monitored for
development of symptoms of COVID-19. Patients can be discharged from isolation once they are
afebrile for at least 3 d and have two consecutive negative molecular tests at 1 d sampling interval.
This recommendation is different from pandemic flu where patients were 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 mutation 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 history 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 alphacoronaviruses 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.11). 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(REF11). 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 ‘RmYNO2 '
To assess the genetic variation of different SARS Co V-2 strains, the 2019 Novel Coronavirus Resource
of China National Center for Bioinformation aligned 77,801 genome sequences of SARS-CoV-2
detected globally and identified a total of 15,018 mutations, including 14,824 single-nucleotide
polymorphisms (BIGD )31 In the S protein, four amino acid alterations, V483A, 14551, F456V and
G476S, are located near the binding interface in the RED, but their effects on binding to the host
receptor are unknown. The alteration D614G in the S1 subunit was found far more frequently than
other S variant sites, and it is the marker of major subclade of SARS-CoV-2 (clade G). Since March
2020, SARS-CoV-2 variants with G614 in the S protein have replaced the original D614 variants and
become the dominant form circulating globally. Compared with the D614 variant, higher viral loads
were found in patients infected with the G614 variant, but clinical data suggested no significant link
between the D614G alteration and disease severity32. Pseudotyped viruses carrying the S protein
with G614 generated higher infectious titres than viruses carrying the S protein with D614,
suggesting the alteration may have increased the infectivity of SARS-CoV-2 (REF.32). However, the
results of in vitro experiments based on pseudovirus models may not exactly reflect natural infection.
This preliminary finding should be validated by more studies using wild-type SARS-CoV-2 variants to
infect different target cells and animal models. Whether this amino acid change enhanced virus
transmissibility is also to be determined. Another marker mutation for SARS-CoV-2 evolution is the
single-nucleotide a polybasic cleavage site (RRAR), which enables effective cleavage by furin and
other proteases27.Such an S1-S2 cleavage site is not observed in ail related viruses belonging to the
subgenus Sarbecovirus, except for a similar three amino acid insertion (PAA) in RmYN02, a bat-
derived coronavirus newly reported from Rhinolophus malayanus in China28 (FIG. 3a). Although the
insertion in RrnYN02 does not functionally represent a polybasic cleavage site, it provides support for
the notion that this characteristic, initially considered unique to SARS-CoV-2, has been acquired
naturally28. A structural study suggested that the furin-cleavage site can reduce the stability of
SARS-CoV-2 S protein and facilitate the conformational adaption that is required for the binding

of the RBD to its receptor29 Whether the higher transmissibility of SARS-CoV-2 compared with
SARS-CoV is a gain of function associated with acquisition of the furin -like cleavage site is yet to be
demonstrated26.

An additional distinction is the accessory gene orf8 of SARS-CoV-2, which encodes a novel protein
showing only 40% amino acid identity to ORF8 of SARS-CoV. Unlike in SARS-CoV, this new ORF8
protein does not contain a motif that triggers intracellular stresspathways25. Notably, a SARS-CoV-2
variant with a 382-nucleotide deletion covering the whole of ORF8 has been discovered in a number
of patients in Singapore, which resembles the 29- or 415-nucleotide deletions in tl1e ORF8 region
observed in human SARS-CoV variants from the late phase of the 2002-2003 outbreak30. Such

ORF8 deletion may be indicative of human adaptation after cross-species transmission from an
animal host.

and other SARSr-CoVs (FIG. 2). Using sequences of five conserved replicative domains in pp lab (3C-
like protease (3CLpro), nidovirus RNA-dependent RNA polymerase (RdRp)-associated
nucleotidyltransferase (NiRAN), RdRp, zinc-binding domain (ZBD) and HELI ), the Coronaviridae Study
Group of the International Committee on Taxonomy of Viruses estimated the pairwise patristic
distances between SARS-CoV-2 and known coronaviruses, and assigned SARS-CoV-2 to the existing
species SARSr-CoV17.Although phylogenetically related, SARS-CoV-2 is distinct from ail other
coronaviruses from bats and pangolins in this species.

The SARS-CoV-2 S protein has a full size of 1,273 amino acids, longer than that of SARS-CoV (1,255
amino acids) and known bat SARSr-CoVs (1,245- 1,269 amino acids). It is distinct from the S proteins
of most members in the subgenus Sarbecovirus, sharing amino acid sequence similarities of 76.7-
77.0% with SARS-CoVs from civets and humans, length to the corresponding proteins in SARS-CoV.
Of the four structural genes, SARS-CoV-2 shares more than 90% amino acid identity with SARS-CoV
except for the S gene, which diverges11, 24. The replicase gene covers two thirds of the 5' genome,
and encodes a large polyprotein (pp lab), which is proteolytically cleaved into 16 non-structural
proteins that are involved in transcription and virus replication. Most of these SARS-CoV-2 non-
structural proteins have greater than 85% amino acid sequence identity with SARS-CoV25.

The phylogenetic analysis for the whole genome shows that SARS-CoV-2 is clustered with SARS-CoV
and SARS-related coronaviruses (SARSr-CoVs) found in bats, placing it in the subgenus Sarbecovirus
of the genus Betacoronavirus. Within this clade, SARS-CoV-2 is grouped in a distinct lineage together
with four horseshoe bat coronavirus isolates (RaTG13, RmYN02, ZC45 and ZXC21) as well as novel
coronaviruses recently identified in pangolins, which group parallel to SARS-CoV

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 died21. 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 market22, 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 France23. However, this individual early report
cannot give a solid answer to the origin of SARS-Co V-2 and contamination, and thus a false positive
result cannot be excluded. To address this highly controversial issue, further retrospective
investigations 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 navel 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 (ORFla/ORFlb),
spike (S), envelope (E), membrane (M) and nucleocapsid (N). In addition, seven putative ORFs
encoding accessory proteins are interspersed between the structural genes25. Most of the proteins
encoded by SARS-CoV-2 have a similar it hadspread massively to all 34 provinces of China. The
number of confirmed cases suddenly increased, with thousands of new cases diagnosed daily during
late January15 . On 30 January, the WHO declared the novel coronavirus outbreak public health
emergency of international concern16. On 11 February, the International Committee on Taxonomy of
Viruses named the novel coronavirus 'SARS-CoV-2', and the WHO named the disease 'COVID-19'
(REF.17 ) .The outbreak of COVID-19 in China reached an epidemic peak in February. According to the
National Health Commission of China, the total number of cases continued to rise sharply in early
February at an average rate of more than 3,000 newly confirmed cases per day. To control COVID-19,
China implemented unprecedentedly strict public health measures. The city of Wuhan was shut
down on 23 January, and all travel and transportation connecting the city was blocked. In the
following couple of weeks, all outdoor activities and gatherings were restricted, and public facilities
were closed in most cities as well as in countryside18. Owing to these measures, the daily number of
new cases in China started to decrease steadily19.

However, despite the declining trend in China, the international spread of COVID-19 accelerated from
late February. Large clusters of infection have been reported from an increasing number of
countries18. The high transmission efficiency of SARS-CoV-2 and the abundance of international
travel enabled rapid worldwide spread of COVID-19. On 11 March 2020, the WHO officially
characterized the global COVID-19 out break as a pandemic20. Since March, while COVID-19 in China
has become effectively controlled, the case numbers in Europe, the USA and other regions have
jumped sharply. According to the COVID-19 dashboard of the Center for System Science and
Engineering at Johns Hopkins University, as of 11 August 2020, (17J, 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 SARSCoV 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 crossprotection 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 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, suggesting
airborne transmission78. In addition, transmission of the virus through the ocular surface and
prolonged presence of SARS-CoV-2 viral RNA in faecal samples were also documented101, 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 infection103. 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 Korea89,104,105.

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 detection kits targeting ORF1b (including
RdRp), N, E or S genes are con1mercially available11,106-109. The detection time ranges from
several minutes to hours depending on the technology106, 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 samples11, 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 onset96,97 Accordingly, false negatives can be common when oral swabs and used, and so
multiple detection methods should be adopted to confirm a COVID-19 diagnosis117 ,118. Other
detection methods were therefore 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
areas. For example, a cohort study in London reveals 44% of the frontline health-care workers from a
hospital were infected with SARS-CoV-2 (REF.94 ) .The high transmissibility of SARS-CoV-2 may be
attributed to the unique virological features of SARS-CoV-2. Transmission of SARS-CoV occurred
mainly after illness onset and peaked following dis ease severity95. However, the SARS-CoV-2 viral
load in upper respiratory tract samples was already highest during the first week of symptoms, and
thus the risk of pharyngeal virus shedding was very high at the beginning of infection 96,97. It was
postulated that undocumented infections might account for 79% of documented cases owing to the
high transmissibility of the virus during mild disease or the asymptomatic period89. A patient with
COVID-19 spreads viruses in liquid droplets during speech. However, smaller and much more
numerous particles known as aerosol particles can also be visualized, which could linger in the air for
a long time and then penetrate deep into the lungs when inhaled by someone else98,100. Airborne
transmission was also observed in the ferret experiments mentioned above. SARS-CoV-2-infected
ferrets shed 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 cough13,60,80,81production, headache,
haemoptysis, diarrhoea, anorexia, sore throat, chest pain, chills and nausea and vomiting in studies
of patients in China13,60 ,80 ,81. Self-reported olfactory and taste disorders were also reported by
patients in Italy85. 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 onset9.

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 developed severer lymphopenia over time13, 60
80,81. Compared with non-ICU patients, ICU patients had higher levels of plasma cytokines, which
suggests an immunopathological process caused by a cytokine storm60,86,87. In this cohort of
patient, around 2.3% people died within a median time of 16 days from disease onset9,86. Men
older than 68 years had a higher risk of respiratory failure, acute cardiac injury and heart failure that
led to death, regardless of a history of cardiovascular disease86 (FIG. 4). Most patients recovered
enough to be released from hospital in 2 weeks9,80 (FIG. 4).

Early transmission of SARS- Co V-2 in Wuhan in December 2019 was initially linked to the Huanan
Seafood Wholesale Market, and it was suggested as the source of the outbreak9,22,60. However,
community transmission might have happened before that88. Later, ongoing human-to-human
transmission propagated the outbreak9. It is generally accepted that SARS-CoV-2 is more
transmissible than SARS-CoV and MERS-CoV; however, determination of an accurate reproduction
number (RO) for COVID-19 is not possible yet, as many asymptomatic infections cannot be accurately
accounted for at this stage89. An estimated RO of 2.5 (ranging from 1.8 to 3.6) has been proposed
for SARS-CoV-2 recently, compared with 2.0- 3.0 for SARS-CoV90. Notably, most of the SARS-CoV-2
human-to-human transmission early in China occurred in family clusters, and in other countries large
outbreaks also happened in other settings, such as migrant worker communities, slaughterhouses
and meat packing plants, indicating the necessity of isolating infected people9,12,91-93. Nosocomial
transmission was not the main source of transmission in China because of the implementation of
infection control measures in clinical settings. By contrast, a high risk of nosocomial transmission was
reported in some other lower respiratory tracts. Acute viral interstitial pneumonia and humoral and
cellular immune responses were observed48,75. Moreover, prolonged virus shedding peaked early in
the course of infect ion in asymptomatic macaques69, and old monkeys showed severer interstitial
pneumonia than young monkeys76, which is similar to what is seen in patients with COVID-19. In
human ACE2-transgenic mice infected with SARS-CoV-2, typical interstitial pneumonia was present,
and viral antigens were observed mainly in the bronchial epithelial cells, macrophages and alveolar
epithelia. Some human ACE2-transgenic mice even died after infection70,71. 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 strain74. Golden hamsters also showed
typical symptoms after being infected with SARS-CoV-2 (REF77). In other animal models, including
cats and ferrets, SARS-CoV-2 could efficiently replicate in the upper respiratory tract but did not
induce severe clinicalsymptoms43,78. As transmission 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 manifestations differ with age. In
general, older men (>60 years old) with co-morbidities are more likely to develop severe respiratory
disease that requires hospitalization. The pathogenesis of SARS-CoV-2 infection in humans manifests
itself as mild symptoms to severe respiratory failure. On binding to epithelial cells in the respiratory
tract, SARS-CoV-2 starts replicating and migrating down to the airways and enters alveolar epithelial
cells in the lungs. The rapid replication of SARS-CoV-2 in the lungs may trigger a strong immune
response. Cytokine storm syndrome causes acute respiratory distress syndrome and respiratory
failure, which is considered the main cause of death in patients with COVID-19 (REFS60, 61). Patients
of older age (>60 years) and with serious preexisting diseases have a greater risk of developing acute
respiratory distress syndrome and death 62-64 (FIG. 4). Multiple organ failure has also been reported
in some COVID-19 cases9, 13, 6, 5.

Histopathological changes in patients with COVID-19 occur mainly in the lungs. Histopathology
analyses showed bilateral diffused alveolar damage, hyaline membrane formation, desquamation of
pneumocytes and fibrin deposits in lungs of patients with severe COVID-19. Exudative inflammation
was also shown in some cases. Immunohistochemistry assays detected SARS-CoV-2 antigen in the
upper airway, bronchiolar epithelium and submucosal gland epithelium, as well as in type I and type
II pneumocytes, alveolar macrophages and hyaline membranes in the lungs13,60,66,67.

Animal models used for studying SARS-CoV-2 infection pathogenesis include non-human primates
(rhesus macaques, cynomolgus monkeys, marmosets and African green monkeys), mice (wild-type
mice (with mouse-adapted virus) and human ACE2- transgenic or human ACE2-knock-in mice),
ferrets and golden hamsters43, 48, 68-74. In non-human primate animal models, most species
display clinical features similar to those of patients with COVID-19, including virus shedding, virus
replication and host responses to SARS-CoV-2 infection69, 72, 73. For example, in the rhesus
macaque model, high viral loads were detected in the upper and appeared asymptomatic45. Another
serological study detected SARS-CoV-2 neutralizing antibodies in cat serum samples collected in
Wuhan after the COVID-19 outbreak, providing evidence for SARS-CoV-2 infection in cat populations
in Wuhan, although the potential of SARS-CoV-2 transmission from cats to humans is currently
uncertain46

Receptor use and pathogenesis

SARS-CoV-2 uses the same receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2) 11 ,47.
Besides human ACE2 (hACE2), SARS-CoV-2 also recognizes ACE2 from pig, ferret, rhesus monkey,
civet, cat, pangolin, rabbit and dog11, 43, 48, 49. The broad receptor usage of SARS-CoV-2 implies
that it may have a wide host range, and the varied efficiency of ACE2 usage in different animals may
indicate their different susceptibilities to SARS-CoV-2 infection. The S1 subunit of a coronavirus is
further divided into two functional domains, an N-terminal domain and a C-terminal domain.
Structural and biochemical analyses identified a 211 amino acid region (amino acids 319-529) at
the S1 C-terminal domain of SARS-CoV-2 as the RBD, which has a key role in virus entry and is the
target of neutralizing antibodies50 ,51 (FIG. 3a). The RBM mediates contact with the ACE2 receptor
(amino acids 437- 507 of SARS-CoV-2 S protein), and this region in SARS-CoV-2 differs from that in
SARS-CoV in the five residues crit- Currently, our knowledge on the animal origin of SARS-CoV-2
remains incomplete to a large part. The reservoir hosts of the virus have not been clearly proven. It is
unknown whether SARS-CoV-2 was transmitted to humans through an intermediate host and which
animals may act as its intermediate host. Detection of RaTG13, RmYN02 and pangolin coronaviruses
implies that diverse coronaviruses similar to SARS-CoV-2 are circulating in wildlife. In addition, as
previous studies showed recombination as the potential origin of some sarbecoviruses such as SARS-
CoV, it cannot be excluded that viral RNA recombination among different related coronaviruses was
involved in the evolution of SARS-CoV-2. Extensive surveillance of SARS-CoV-2 related viruses in
China, Southeast Asia and other regions targeting bats, wild and captured pangolins and other
wildlife species will help us to better understand the zoonotic origin of SARS-CoV-2.

Besides wild life, researchers investigated the susceptibility of domesticated and laboratory animals
to SARS-CoV-2 infection. The study demonstrated experimentally that SARS-CoV-2 replicates
efficiently in cats and in the upper respiratory tract of ferrets, whereas dogs, pigs, chickens and ducks
were not susceptible to SARS-CoV-2 (REF.43). The susceptibility of minks was documented by a
report from the Netherlands on an outbreak of SARS-CoV-2 infection in farmed minks. Although the
symptoms in most infected minks were mild, some developed severe respiratory distress and died of
interstitial pneumonia44. Both virological and serological testing found evidence for natural SARS-
CoV-2 infection in two dogs from households with human cases of COVID-19 in Hong Kong, but the
dogs residues for receptor binding40 (FIG. 3b). In comparison with the Guangdong strains, pangolin
coronaviruses reported from Guangxi are less similar to SARS-CoV-2, with 85.5% genome sequence
identity39. The repeated occurrence of SARS-CoV-2-related coronavirus infections in pangolins from
different smuggling events suggests that these animals are possible hosts of the viruses. However,
unlike bats, which carry coronaviruses healthily, the infected pangolins showed clinical signs and
histopathological changes, including interstitial pneumonia and inflammatory cell infiltration in
diverse organs40.These abnormalities suggest that pangolins are unlikely to be the reservoir of these
coronaviruses but more likely acquired the viruses after spillover from the natural hosts.

An intermediate host usually plays an important role in the outbreak of bat-derived emerging
coronaviruses; for example, palm civets for SARS-CoV and dromedary camels for MERS-CoV. The virus
strains carried by these two intermediate hosts were almost genetically identical to the
corresponding viruses in humans (more than 99% genome sequence identity)1. Despise an RBD that
is virtually identical to that of SARS-CoV-2, the pangolin coronaviruses known to date have no more
than 92% genome identity with SARS-CoV- 2 (REF.42 ). The available data are insufficient to interpret
pangolins as the intermediate host of SARS-CoV-2. So far, no evidence has shown that pangolins were
directly involved in the emergence of SARS-CoV-2. in Yunnan. This novel bat virus, denoted 'RmYN02’

is 93.3% identical to SARS-CoV-2 across the genome. In the long lab gene, it exhibits 97.2% identity to
SARS-CoV-2, which is even higher than for RaTG13 (REF28). In addition to RaTG13 and RmYN02,
phylogenetic analysis shows that bat coronaviruses ZC45 and ZXC21 previously detected in
Rhinolophus pusillus bats from eastern China also fall into the SARS-CoV-2 lineage of the subgenus
Sarbecovirus36 (FIG 2). The discovery of diverse bat coronaviruses closely related to SARS-CoV-2
suggests that bats are possible reservoirs of SARS-C oV-2 (REF.37). Nevertheless, on the basis of
current findings, the divergence between SARS-CoV-2 and related bat coronaviruses likely represents
more than 20 years of sequence evolution, suggesting that these bat coronaviruses can be regarded
only as the likely evolutionary precursor of SARS-CoV-2 but not as the direct progenitor of SARS-CoV-
2 (REF.38).

Beyond bats, pangolins are another wildlife host probably linked withSARS-CoV-2. Multiple SARS-
CoV-2- related viruses have been identified in tissues of Malayan pangolins smuggled from Southeast
Asia into southern China from 2017 to 2019. These viruses from pangolins independently seized by
Guangxi and Guangdong provincial customs belong to two distinct sublineages39-41.

The Guangdong strains, which were isolated or sequenced by different research groups from
smuggled pangolins, have 99.8% sequence identity with each other41. They are very closely related
to SARS-CoV-2, exhibiting 92.4% sequence similarity. Notably, the RBD of Guangdong pangolin
coronaviruses is highly similar to that of SARS-CoV-2. The receptor-binding motif (RBM; which is part
of the RBD) of these viruses has only one amino acid variation from SARS-CoV-2, and it is identical to
that of SARS-CoV-2 in all five critical differs from that in SARS-CoV in the five residues critical for ACE2
binding, namely Y4551, L486F, N493Q, D494S and T501N52 (FIG. 3b.c). Owing to these residue
changes, interaction of SARS-CoV-2 with its receptor stabilizes the two virus-binding hotspots on the
surface of hACE2 (REF.50) (FIG. 3d). Moreover, a four-residue motif in the RBM of SARS-CoV-2 (amino
acids 482-485: G-V-E-G) results in a more compact conformation of its hACE2-binding ridge than in
SARS-Co V and enables better contact witl1 the N-terminal helix of hACE2 (REF.50). Biochemical
data confirmed that the structural features of the SARS-CoV-2 RBD has strengthened its hACE2
binding affinity compared with that of SARS-CoV50,52,53.

Similarly to other coronaviruses, SARS-CoV-2 needs proteolytic processing of the S protein to activate
the endocytic route. It has been shown that host proteases participate in the cleavage of the S
protein and activate the entry of SARS-CoV-2, including transmembrane protease serine protease 2
(TMPRSS2), cathepsin Land furin47,54,55. Single-cell RNA sequencing data showed that TMPRSS2 is
highly expressed in several tissues and body sites and is co-expressed with ACE2 in nasal epithelial
cells, lungs and bronchial branches, which explains some of the tissue tropism of SARS-CoV-2

(REFS56 ,57). SARS-CoV-2 pseudovirus entry assays revealed that TMPRSS2 and cathepsin L have
cumulative effects with furin on activating virus entry55. Analysis of the cryo-electron microscopy
structure of SARS-Co V-2 S protein revealed that its RBD is mostly in the lying-down state, whereas
the SARS-CoV S protein assumes equally standing-up and lying-down conformational states50,51
58,59. A lying-down conformation of the SARS-CoV-2 S protein may not be in favour of receptor
binding but is helpful for immune evasion55.
infections clinically or through routine lab tests. Therefore, travel history becomes important.
However, as the epidemic spreads, the travel history will become irrelevant.

Treatment [21, 23]

Treatment is essentially supportive and symptomatic.

The first step is to ensure adequate isolation (discussed later) to prevent transmission to other
contacts, patients and healthcare workers. Mild illness should be managed at home with counseling
about danger signs. The usual principles are maintaining hydration and nutrition and controlling
fever and cough. Routine use of antibiotics and antivirals such as oseltamivir should be avoided in
confirmed cases. In hypoxic patients, provision of oxygen through nasal prongs, face mask, high flow
nasal glass opacities and sub segmental consolidation. It is also abnormal in asymptomatic patients/
patients with no clinical evidence of lower respiratory tract involvement. In fact, abnormal CT scans
have been used to diagnose COVID-19 in suspect cases with negative molecular diagnosis; many of
these patients had positive molecular tests on repeat testing [22].

Differential Diagnosis [21]

The differential diagnosis includes all types of respiratory viral infections [influenza, parainfluenza,
respiratory syncytial virus (RSV), adenovirus, human metapneumovirus, non COVID-19 coronavirus],
atypical organisms (mycoplasma, chlamydia) and bacterial infections. It is not possible to
differentiate COVID-19 from these infections clinically or through routine lab tests. Therefore, travel
history becomes important. However, as the epidemic spreads, the travel history epidemic
progresses, commercial tests will become available.

Other laboratory investigations are usually nonspecific. The white cell count is usually normal or low.
There may be lymphopenia; a lymphocyte count <1000 has been associated with severe disease. The
platelet count is usually normal or mildly low. The CRP and ESR are generally elevated but
procalcitonin levels are usually normal. A high procalcitonin level may indicate a bacterial
co-infection. The ALT/AST, prothrombin time, creatinine, D-dimer, CPK and LDH may be elevated and
high levels are associated with severe disease.

The chest X-ray (CXR) usually shows bilateral infiltrates but may be normal in early disease. The CT is
more sensitive and specific. CT imaging generally shows infiltrates, ground glass opacities and sub
segmental of persistent local transmission or contact with patients with similar travel history or those
with confirmed COVID-19 infection. However, cases may be asymptomatic or even without fever. A
confirmed case is a suspect case with a positive molecular test.

Specific diagnosis is by specific molecular tests on respiratory samples (throat swab/ nasopharyngeal
swab/ sputum/ endotracheal aspirates and bronchoalveolar lavage). Virus may also be detected in
the stool and in severe cases, the blood. It must be remembered that the multiplex PCR panels
currently available do not include the COVID-19. Commercial tests are also not available at present.
In a suspect case in India, the appropriate sample has to be sent to designated reference labs in India
or the National Institute of Virology in Pune. As the epidemic progresses, commercial tests 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

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

identified angiotensin receptor 2 (ACE2) as the receptor through which the virus enters the
respiratory mucosa [11].

The basic case reproduction rate (BCR) is estimated to range from 2 to 6.47 in various modelling
studies [11]. In comparison, the BCR of SARS was 2 and 1.3 for pandemic flu H1N1 2009 [2].

Clinical Features [8, 15- 18]

The clinical features of COVID-19 are varied, ranging from asymptomatic state to acute respiratory
distress syndrome and multi organ dysfunction. The common clinical features include fever (not in
all), cough, sore throat, headache, fatigue, headache, myalgia and breathlessness. Conjunctivitis has
also been described. Thus, they are indistinguishable from other respiratory infections. In a subset

on surfaces. The virus can remain viable on surfaces for days in favourable atmospheric conditions
but are destroyed in less than a minute by common disinfectants like sodium hypochlorite, hydrogen
peroxide etc. [13]. Infection is acquired either by inhalation of these droplets or touching surfaces
contaminated by them and then touching the nose, mouth and eyes. The virus is also present in the
stool and contamination of the water supply and subsequent transmission via aerosolization/feco
oral route is also hypothesized [6]. Asper current information, transplacental transmission from
pregnant women to their fetus has not been described [14]. However, neonatal disease due to post-
natal transmission is described [14].

The incubation period varies from 2 to 14 d [median 5 d]. Studies have identified angiotensin
receptor 2

Epidemiology and Pathogenesis [10, 11]

All ages are susceptible. Infection is transmitted through large droplets generated during coughing
and sneezing by symptomatic patients but can also occur from asymptomatic people and before
onset of symptoms [9]. Studies have shown higher viral loads in the nasal cavity as compared to the
throat with no difference in viral burden between symptomatic and asymptomatic people [12].
Patients can be infectious for as long as the symptoms last and even on clinical recovery. Sorne
people may act as super spreaders; a UK citizen who attended a conference in Singapore infected 11
other people while staying in a resort in the French Alps and upon return to the UK [6]. These
infected droplets can spread 1-2 m and deposit

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

Cases continued to increase exponentially and modelling studies reported an epidemic doubling time
of 1.8 d [10]. In fact, on the 12th of February, China changed its definition of confirmed cases to
include patients with negative/ pending molecular tests but with clinical, radiologic and
epidemiologic features of COVID-19 leading to an increase in cases by 15,000 in a single day [6]. As of
05/03/2020 96,000 cases worldwide (80,000 in China) and 87 other countries and 1 international
conveyance (696, in the cruise ship Diamond Princess parked off the coast of Japan) have been
reported [2]. It is important to note that while the number of new cases has reduced in China lately,
they have increased exponentially in other countries including South Korea, Italy and Iran.

Of those infected, 20% are in critical conditions

Extended to other cities of Hubei province. Cases of COVID-19 in countries outside China were
reported in those with no history of travel to China suggesting that local human-to human
transmission was occurring in these countries [9]. Airports in different countries including India put in
screening mechanisms to detect symptomatic people returning from China and placed them in
isolation and testing them for COVID-19. Soon it was apparent that the infection could be
transmitted from asymptomatic people and also before onset of symptoms. Therefore, countries
including India who evacuated their citizens from Wuhan through special flights or had travellers
returning from China, placed ail people symptomatic or otherwise in isolation for 14 d and tested
them for the virus.

Cases continued to increase exponentially and modelling studies

14-ANTIVIRAL THERAPY

COVID-19 is an infectious disease caused by SARS-CoV-2, which is also termed the navel coronavirus
and is diligently associated with the SARS virus. The Ministry of Science and Technology from the
People's Republic of China declared three potential antiviral medicines suitable for treating COVID-
19. Those three medicines are, namely, Favilavir, chloroquine phosphate and remdesivir. A clinical
trial was conducted to test the efficacy of those three drugs, and the results proved that out of the
three medicines above only Favilavir is effective in treating the patients with navel coronavirus. The
remaining two drugs were effective in treating malaria.62

Likewise, a study carried out in the United States by the National Institute of Health proved that
remdesivir is effective in treating the Middle East respiratory syndrome coronavirus (MERSCoV),
which is also a type of coronavirus that was transmitted from monkeys. The drug remdesivir was also
used in the United States for treating the patients with COVID-19. There has been a proposal to use
the combination of protease inhibitors lopinavir-ritonavir for treating the patients affected by COVID-
1 It is also evident that remdesivir was effective in treating the patients who were infected with
Ebola virus. Per this evidence, China has already started testing the efficacy of remdesivir in treating
the patients with COVID-19, especially in Wuhan, where the outbreak occurred. Chloroquine, which
is an existing drug which is currently used in treating malaria cases, was given to more than 100
patients who were affected with novel coronavirus to test its efficacy. 62

A multicentric study was conducted in China to test the effectiveness of remdesivir in treating the
patients with COVID-19. Thus, the results of the clinical trial proved that remdesivir has a
considerably acceptable level of efficacy for treating the patients with COVID-19. Therefore, the
National Health Commission of the People's Republic of China decided to include remdesivir in the
Guidelines for the Prevention, Diagnosis and Treatment of Pneumonia Caused by COVID-19.62

Chloroquine and hydroxychloroquine are existing anti-malaria drugs also given to more than 30
patients infected with COVID-19 in Guangdong province and Hunan province to test their
effectiveness and efficacy. Thus, the results of the clinical trial showed that the patients who were
given chloroquine had a significant reduction in their body temperature. The clinical trial also
showed better recovery among the patients who were given chloroquine and hydroxy
chloroquine.63-65 Hydroxychloroquine treatment is significantly associated with viral load reduction
as well as disappearance in COVID-19 patients. Further, the outcome is reinforced by azithromycin.
The role of lopinavir and ritonavir in the treatment of COVID-19 is uncertain. A potential benefit was
suggested by preclinical data, but additional data has failed to confirm it. Tocilizumab is an
immunomodulating agent used as adjunct therapy in some protocols based on a theoretical
mechanism and limited preliminary data.66

15-HOME CARE

Home management may be appropriate for patients with mild infection who can be adequately
isolated in the outpatient setting. Management of such patients should focus on prevention of
transmission to others, and monitoring for clinical deterioration, which should prompt
hospitalization. interim recommendations on home management of patients with COVID-19 can be
found on the WHO and CDC websites.67

16-CONCLUSION

The corona virus (COVID-19) spreads at an alarming rate all over the world. The outbreak of the virus
has confronted the world's economic, medical and public health infrastructure. Elderly and
immunocompromised patients also are susceptible to the virus's mortal impacts. Currently, there is
no documented cure for the virus and no vaccine has been created, although some treatment
protocols have been promising. Therefore, the virus can be controlled with the appropriate
prevention strategies. Also, attempts have to be made to formulate systematic strategies to prevent
such future zoonotic outbreaks.
including 1L2, 1L7, 1L10, GCSF, 1P10, MCP1, MIP1A, and TNFα [15]. The median time from onset of
symptoms to dyspnea was 5 d, hospitalization 7 d and acute respiratory distress syndrome (ARDS) 8
d. The need for intensive care admission was in 25-30% of affected patients in published series.
Complications witnessed included acute lung injury, ARDS, shock and acute kidney injury. Recovery
started in the 2nd or 3rd week. The median duration of hospital stays in those who recovered was 10
d. Adverse outcomes and death are more common in the elderly and those with underlying
co-morbidities (50- 75% of fatal cases). Fatality rate in hospitalized adult patients ranged from 4 to
11%. The overall case fatality rate is estimated to range between 2 and 3% [2].

Interestingly, disease in patients outside Hubei province has been

• All clinicians should keep themselves updated about recent developments including global spread
of the disease.

• Non-essential international travel should be avoided at this time.

• People should stop spreading myths and false information about the disease and try to allay panic
and anxiety of the public.

Conclusions

This new virus outbreak has challenged the economic, medical and public health infrastructure of
China and to some extent, of other countries especially, its neighbours. Time alone will tell how the
virus will impact our lives here in India. More so, future outbreaks of viruses and pathogens of
zoonotic origin are likely to continue. Therefore, apart from curbing this outbreak, efforts should be
made to recovered patients and used for plasma transfusion twice in a volume of 200 to 2501 m on
the day of collection (310). At present, treatment for sepsis and ARDS mainly involves antimicrobial
therapy, source control, and supportive care. Hence, the use of therapeutic plasma exchange can be
considered an option in managing such severe conditions. Further randomized trials can be designed
to investigate its efficacy (311).

Potential Therapeutic Agents

Potent therapeutics to combat SARS-CoV-2 infection include virus binding molecules , molecules or
inhibitors targeting particular enzymes implicated in replication and transcription process of the
virus, helicase inhibitors, vital viral proteases and proteins, protease inhibitors of host cells,
endocytosis inhibitors, short interfering RNA (siRNA), neutralizing antibodies, MAbs against the host
receptor, MAbs interfering with the S1 RBD, antiviral, peptide aimed at S2, and natural
drugs/medicines (7, 166, 186). The S protein acts as the critical target for developing CoV antivirals,
like inhibitors of S protein and S cleavage, neutralizing antibodies, RBD-ACE2 blockers, siRNAs,
blockers of the fusion core, and proteases (168).

All of the 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 (AT1 R) (207).
Severa] 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 hast inflammatory responses. Such drugs acting
specifically in the respiratory tract will help to reduce virus-triggered immune pathologies in COVID-
19 (209). The later stages of coronavirus induced inflammatory cascades are characterized by the
release of proinflammatory interleukin-1 (IL-1) family members, such as IL-1 and IL-33. Hence,
there exists a possibility that the inflammation associated with coronavirus can be inhibited by
utilizing anti-inflammatory cytokines that belong to the IL-1 family (92). It has also been suggested
that the actin protein is the host factor that is involved in cell entry and pathogenesis of SARS-CoV-2.
Hence, those drugs that modulate the biological activity of this protein, like ibuprofen, might have
some therapeutic application in managing the disease (174). The plasma angiotensin 2 level was
found to be markedly elevated in COVID-19 infection and was correlated with viral load and lung
injury. Hence, drugs that block angiotensin receptors may have potential for treating COVID-19
infection (121). A scientist from Germany, named Rolf Hilgenfeld, has been working on the
identification of drugs for the treatment of coronaviral infection since the time of the first SARS
outbreak (19).

The SARS-CoV S2 subunit bas a significant function in mediating virus fusion that provides entry into
the host cell. Heptad repeat 1 (HR1) and heptad.

Animal Models and Cell Cultures

For evaluating the potential of vaccines and therapeutics against CoVs, including SARS-CoV, MERS-
CoVs, and the presently emerging SARSCoV-2, suitable animal models that can mimic the clinical
disease are needed (211, 212). Various animal models were assessed for SARS- and MERSCoVs, such
as mice, guinea pigs, golden Syrian hamsters, ferrets, rabbits, nonhuman primates like rhesus
macaques and marmosets, and cats (185, 213- 218). The specificity of the virus to hACE2 (receptor of
SARS-CoV) was found to be a significant barrier in developing animal models. Consequently, a SARS-
CoV transgenic mouse model l1as been developed by inserting the hACE2 gene into the mouse
genome (219). The inability of MERS-CoV to replicate in the respiratory tracts of animals (mice,
hamsters, and ferrets) is another limiting factor. However, with genetic engineering, a 288-330+/+
MERS-CoV genetically modified mouse model was developed and now is in use for the assessment of
novel drugs and vaccines against MERS-CoV (220). In the past, small animals (mice or hamsters) have
been targeted for being closer to a humanized structure, such as 1nouse DPP4 altered with human
DPP4 (hDPP4), hDPP4-transduced mice. and hDPP4-Tg mice (transgenic for expressing

Virological, radiological and pathological observations indicated that the monkeys with reexposure
had no recurrence of COVID-19, like the SARS-CoV-2-infected monkeys without rechallenge. These
findings suggest that primary infection with SARS-CoV-2 could protect from later exposures to the
virus, which could help in defining disease prognosis and crucial inferences for designing and
developing potent vaccines against COVID-19 (274).

PREVENTION, CONTROL, AND MANAGEMENT

In contrast to their response to the 2002 SARS outbreak, China has shown immense political
openness in reporting the COVID-19 outbreak promptly. They have also performed rapid sequencing
of COVID-19 at multiple levels and shared the findings globally within days of identifying the novel
virus (225). The move made by China opened a new chapter in global health security and diplomacy.
Even though complete lockdown was declared following the COVID-19 outbreak in Wuhan, the large-
scale movement of people has resulted in a radiating spread of infections in the surrounding
provinces as well as to several other countries. Large-scale screening programs might help us to
control the spread of this virus. However, this is both challenging as well as time-consuming due to
the present extent of infection (226). The current scenario demands effective implementation of
vigorous prevention and control strategies owing to the prospect of COVID-19 for nosocomial
infections (68). Follow-ups of infected patients by telephone on day 7 and day 14 are advised to
avoid any further unintentional spread or nosocomial transmission (312). The availability of public
data sets provided by independent analytical teams will act as robust evidence that would guide us in
designing interventions against the COVID-19 outbreak. Newspaper reports and social media can be
used to analyze and reconstruct the progression of an outbreak. They can help us to obtain detailed
patient-level data in the early stages of an outbreak (227). Immediate travel restrictions imposed by
several countries might have contributed significantly to preventing the spread of SARS-CoV-2
globally (89, 228). Following the outbreak, a temporary ban was imposed on the wildlife trade,
keeping in mind the possible role played by wild ani1nal species in the origin of SARS-CoV-2/COVID-
19 (147). Making a permanent and bold decision on the trade of wild animal species is necessary to
prevent the possibility susceptible individuals. Hence, hand hygiene is equally as important as the
use of appropriate PPE, like face masks, to break the transmission cycle of the virus; both hand
hygiene and face masks help to lessen the risk of COVID-19 transmission (315).

Medical staff are in the group of individuals most at risk of getting COVID-19 infection. This is
because they are exposed directly to infected patients. Bence, proper training must be given to all
hospital staff on methods of prevention and protection so that they become competent enough to
protect themselves and others from this deadly disease (316). As a preventive measure, health care
workers caring for infected patients should take extreme precautions against both contact and
airborne transmission. They should use PPE such as face masks (N95 or FFP3), eye protection
(goggles), gowns, and gloves to nullify the risk of infection (299).

The human-to-human transmission reported in SARS-CoV-2 infection occurs mainly through droplet
or direct contact. Due to this finding, frontline health care workers should follow stringent infection
control and preventive measures, such as the use of PPE, to prevent infection (110). The mental
health of the medical/health workers who are involved in the COVID-19 outbreak is of great
importance, because the strain on their mental wellbeing will affect their attention, concentration,
and decision-making capacity. Bence, for control of the COVID-19 outbreak, rapid steps should be
taken to protect the mental health of medical workers (229).

Since the living 1nammals 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 R0 number
and reduce the trans1nission risk (228). Considering the zoonotic
Princess, Celebrity Apex, and Ruby Princess. The number of confirmed around the world is on the
rise. The success of preventive measures put forward by every country is mainly dependent upon
their ability to anticipate the approaching waves of patients. This will help to properly prepare the
health care workers and increase the intensive care unit (ICU) capacity (321). Instead of entirely
relying on lockdown protocols, countries should focus mainly on alternative intervention strategies,
such as large-scale testing, contract tracing, and localized quarantine of suspected cases for limiting
the spread of this pandemic virus. Such intervention strategies will be useful either at the beginning
of the pandemic or after lockdown relaxation (322). Lockdown should be imposed only to slow down
disease progression among the population so that the health care system is not overloaded.

The reproduction number (Ro) of COVID-19 infection was earlier estimated to be in the range of

1.4 to 2.5 (70); recently, it was estimated to be 2.24 to 3.58 (76). Compared to its coronavirus
predecessors, COVID-19 has an Ro value that is greater than that of MERS (Ro < 1) (108) but less
than that of SARS (Ro value of 2 to 5) (93). Still, to prevent further spread of disease at mass
gatherings, functions remain canceled in the affected cities, and persons are asked to work from
home (232). Hence, it is a relief that the current outbreak of COVID-19 infection can be brought
under control with the adoption of strategic preventive and control measures along with the early
isolation of subsequent cases in the coming days. Studies also report that since air traffic between
China and African countries increased many times over in the decade after the SARS outbreak,
African countries need to be vigilant to prevent the spread of navel coronavirus in Africa (225). Due
to fear of virus spread, Wuhan City was completely shut down (233). The immediate control of the
ongoing COVID-19 outbreaks appears a mammoth task, especially for developing countries, due to
their inability to allocate quarantine stations that could screen infected individuals’ movements
(234). Such underdeveloped countries should divert their resources and energy to enforcing the
primary level of preventive measures, like controlling the entry of individuals from China or countries
where the disease has flared up, isolating the infected individuals, and quarantining individuals with
suspected infection. Most of the sub-Saharan African countries have a fragile health system that can
be crippled in the event of an outbreak. Effective management of COVID-19 would be difficult for
low-income countries due to their inability to respond rapidly due to the lack of an efficient health
care system (65). Controlling the imported cases is critical in preventing the spread of COVID-19 to
other countries that have not reported the disease until now. The possibility of an imported case of
COVID-19 leading to sustained human-to-human transmission was estimated to be 0.41. This can be
reduced to a value of 0.012 by decreasing the meantime from the onset of symptoms to
hospitalization and can only be made possible by using intense disease surveillance systems (235).
The silent importations of infected individuals (before the manifestation of clinical signs) also
contributed significantly to the spread of disease across the 1najor cities of the world. Even though
the travel ban was implemented in Wuhan (89), infected persons who traveled out of the city just
before the imposition of the ban might have remained undetected and resulted in local outbreaks
(236). Emerging novel diseases like COVID-19 are difficult to contain within the country of origin,
since globalization has led to a world without borders. Hence, international collaboration plays a vital
role fatigue. Individuals with asymptomatic and typical clinical manifestations were also identified
recently, further adding to the complexity of disease transmission dynamics. Atypical clinical
manifestations may only express symptoms such as fatigue instead of respiratory signs such as fever,
cough, and sputum. In such cases, the clinician must be vigilant for the possible occurrence of
asymptomatic and atypical clinical manifestations to avoid the possibility of missed diagnoses.

The present outbreak caused by SARS-CoV-2 was, indeed, expected. Similar to previous outbreaks,
the current pandemic also will be contained shortly. However, the real question is, how are we
planning to counter the next zoonotic CoV epidemic that is likely to occur within the next 5 to 10
years or perhaps sooner? Our knowledge of most of the bat CoVs is scarce, as these viruses have not
been isolated and studied, and extensive studies on such viruses are typically only conducted when
they are associated with specific disease outbreaks. The next step following the control of the COVID-
19 outbreak in China should be focused on screening, identification, isolation, and characterization of
CoVs present in wildlife species of China, particularly in bats. Both in vitro and in vivo studies (using
suitable animal models) should be conducted to evaluate the risk of future epidemics. Presently,
licensed antiviral drugs or vaccines against SARS CoV, MERS-CoV, and SARS-CoV-2 are lacking.
However, advances in designing antiviral drugs and vaccines against several other emerging diseases
will help develop suitable therapeutic agents against COVID-19 in a short time. Until then, we must
rely exclusively on various control and prevention measures to prevent this new disease from
becoming a pandemic.

4-VIROLOGY

Coronaviruses, a family of viruses within the nidoviruses superfamily, are further classified according
to their genera, alpha-, beta-, gamma and deltacoronaviruses (α-, β-, γ- and δ-). Among those, alpha
and beta species are capable of contaminating only mammals, whereas the other two genera can
infect birds and could also infect mammals.13 ,14 Two of these genera belong to human
coronaviruses (HCoVs): α-coronaviruses, which comprise human coronavirus 229E (hcov229E) and
human coronavirus NL63 (hcovNL63), and β-coronaviruses, which are human coronavirus HKU1,
human coronavirus OC43, MERS-COV (known as Middle East respiratory syndrome coronavirus) and
SARS-CoV (referred to as severe acute respiratory syndrome coronavirus).15

The severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is now named novel COVID-19
(coronavirus disease 2019).16 Genome sequencing and phylogenetic research revealed that the
COVID-19-causing coronavirus is a beta-coronavirus that belongs to the same subtypes as SARS virus,
but still exists in a variant group. The receptor-binding gene region

primary anti-genic epitopes mainly those recognised by neutralising antibodies. The spike S-protein
being in a spike form is subjected to a structural rearrangement process so that fusing the outer
membrane of the virus with the host cell membrane becomes easier.19,20 Recent SARS-CoV work
has also shown that the membrane exopeptidase ACE enzyme (angiotensin-converting enzyme)
functions as a COVID-19 receptor to enter the human cell.21
Figure 1

4.2 Viral replication

Usually, replication of coronavirus occurs within the cytoplasm and is closely associated with
endoplasmic reticulum and other cellular membrane organelles. Human coronaviruses are thought
to invade cells, primarily through different receptors. For 229E and OC43, amino peptidase-N (AP-N)
and a sialic acid containing receptor, respectively, were known to function in this role. After the virus
enters the hast cell and uncoating process occurs, the genome is transcribed, and then, translated. A
characteristic feature of replication is that all mRNAs form an enclosed group of typical 3' ends; only
the special portions of the 5' ends are translated. ln total, about 7 mRNAs are produced. The shortest
mRNA codes and the others can express the synthesis of another genome segment for
nucleoprotein. At the cell membrane, these proteins are collected and genomic RNA is initiated as a
mature particle type by burgeoning from internal cell membranes.22,23

5-PATHOGENESIS

Coronaviruses are tremendously precise and mature in most of the airway epithelial cells as
observed through bath in vivo and in vitro

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 orthose 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

6.1 Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases

The assessment of the patients with COVID-19 should be based on the clinical features and also
epidemiological factors. The screening protocols must be prepared and followed per the native
context.31 Collecting and testing of specimen samples from the suspected individual is considered to
be one of the main principles for controlling and managing the outbreak of the disease in a country.
The suspected cases must be screened thoroughly in order to detect the virus with the help of
nucleic acid amplification tests such as reverse transcription polymerase chain reaction (RTPCR). If a
country or a particular region does not have the facility to test the specimens, the specimens of the
suspected individual should be sent to the nearest reference laboratories per the list provided by
WHO .32

It is also recommended that the suspected patients be tested for the other respiratory pathogens by
performing the routine laboratory investigation per the local guidelines, mainly to differentiate from
other viruses that include influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus,
rhinovirus, human

weeks, and the typical symptom occurrence from incubation period to infection takes an average of
12.5 days.29

6-CLINICAL DIAGNOSIS

The symptoms of COVID-19 remain very similar to those of the other respiratory epidemics in the
past, which include SARS and MERS, but here the range of symptoms includes mild rhinitis to septic
shock. Sorne intestinal disturbances were reported with the other epidemics, but COVID-19 was
devoid of such symptoms. When examined, unilateral or bilateral involvement compatible with viral
pneumonia is observed in the patients, and bilateral multiple lobular and sub-segmental
consolidation areas were observed in patients hospitalized in the intensive care unit.

Comorbid patients showed a more severe clinical course than predicted from previous epidemics.
Diagnosis of COVID-19 includes the complete history of travel and touch, with laboratory testing. It is
more preferable to choose serological screening, which can help to analyze even the asymptomatic
infections; several serological tests are in progress for SARS-CoV-2.14,30

observed through both in vivo and in vitro experiments. There is an enhanced nasal secretion
observed along with local oedema because of the damage of the host cell, which further stimulates
the synthesis of inflammatory mediators. ln addition, these reactions can induce sneezing, difficulty
breathing by causing airway inhibition and elevate mucosal temperature. These viruses, when
released, chiefly affect the lower respiratory tract, with the signs and symptoms existing clinically.
Also, the virus further affects the intestinal lymphocytes, renal cells, liver cells and T-lymphocytes.
Furthermore, the virus induces T-cell apoptosis, causing the reaction of the T-cell to be erratic,
resulting in the immune system's complete collapse.24,25

5.1 Mode of transmission

ln fact it was accepted that the original transmission originated from a seafood market, which had a
tradition of selling live animals, where the majority of the patients had either worked or visited,
although up to now the understanding of the COVID-19 transmission risk remains incomplete.16 ln
addition, while the newer patients had no exposure to the market and still got the virus from the
humans present there, there is an increase in the outbreak of variant group. The receptor-binding
gene region appears to be very similar to that of the SARSCoV and it is believed that the same
receptor would be used for cell entry.17

4.1 Virion structure and its genome

Coronaviruses are structurally enveloped, belonging to the positive-strand RNA viruses’ category that
has the largest known genomes of RNA. The structures of the coronavirus are more spherical in
shape, but their structure has the potential to modify their morphology in response to
environmental conditions, being pleomorphic. The capsular membrane which represents the outer
envelope usually has glycoprotein projection and covers the nucleus, comprising a matrix protein
containing a positive-strand RNA. Since the structure possesses 5'-capped and 3'-polyadenylated
ends, it remains identical to the cellular mRNAs.18 The structure is comprised of hemagglutinin
esterase (HE) (present only in some beta-coronaviruses), spike (S), small membrane (E), membrane
(M) and nucleocapsid (N), as shown (Figure 1). The envelope containing glycoprotein is responsible
for attachment to the hast cell, which possesses the primary anti-genic epitopes mainly those

CONCLUDING REMARKS

Several years after the global SARS epidemic, the current SARS-CoV-2/COVID-19 pandemic bas served
as a reminder of how novel pathogens can rapidly emerge and spread through the human population
and eventually cause severe public health crises. Further research should be conducted to establish
animal models for SARS-CoV-2 to investigate replication, transmission dynamics, and pathogenesis in
humans. This may help develop and evaluate potential therapeutic strategies against zoonotic CoV
epidemics. Present trends suggest the occurrence of future outbreaks of CoVs due to changes in the
climate, and ecological conditions may be associated with human-animal contact. Liveanimal
markets, such as the Huanan South China Seafood Market, represent ideal conditions for interspecies
contact of wildlife with domestic birds, pigs, and mammals, which substantially increases the
probability of interspecies transmission of CoV infections and could result in high risks to humans
due to adaptive genetic recombination in these viruses (323- 325).

The COVID-19-associated symptoms are fever, cough, expectoration, headache, and myalgia or
fatigue. individuals with asymptomatic and atypical

We also predict the possibility of another outbreak, as predicted by Fan et al.(6). Indeed, the present
outbreak caused by SARS-CoV-2 (COVID- 19) was expected. Similar to previous outbreaks, the current
outbreak also will be contained shortly. However, the real issue is how we are planning to counter
the next zoonotic CoV epidemic that is likely to occur within the next 5 to 10 years or even sooner
(Fig. 7).
Transmission risk (228). Considering the zoonotic links associated with SARS-CoV-2, the one Health
approach may play a vital role in the prevention and control measures being followed to restrain this
pandemic virus (317- 319). The substantial importation of COVID-19 presymptomatic cases from
Wuhan has resulted in independent, self-sustaining outbreaks across major cities both within the
country and across the globe. The majority of Chinese cities are now facing localized outbreaks of
COVID-19 (231). Hence, deploying efficient public health interventions might help to cut the spread
of this virus globally.

The occurrence of COVID-19 infection on several cruise ships gave us a preliminary idea regarding the
transmission pattern of the disease. Cruise ships act as a closed environment and provide an ideal
setting for the occurrence of respiratory disease outbreaks. Such a situation poses a significant threat
to travelers, since people from different countries are on board, which favors the introduction of the
pathogen (320). Although nearly 30 cruise ships from different countries have been found harboring
COVID-19 infection, the major cruise ships that were involved in the COVID-19 outbreaks are the
Diamond Princess, Grand Princess, Celebrity Apex, and Ruby Princess. The animal species is
necessary to prevent the possibility of virus spread and initiation of an outbreak due to zoonotic
spillover (1).

Personal protective equipment (PPE), like face masks, will help to prevent the spread of respiratory
infections like COVID-19. Face masks not only protect from infectious aerosols but also prevent the
transmission of disease to other susceptible individuals while traveling through public transport
systems (313). Another critical practice that can reduce the transmission of respiratory diseases is
the maintenance of hand hygiene. However, the efficacy of this practice in reducing the transmission
of respiratory viruses like SARS-CoV-2 is much dependent upon the size of droplets produced. Rand
hygiene will reduce disease transmission only if the virus is transmitted through the formation of
large droplets (314). Hence, it is better not to overemphasize that hand hygiene will prevent the
transmission of SARS-CoV-2, since it may produce a false sense of safety among the general public
that further contributes to the spread of COVID-19. Even though airborne spread has not been
reported in SARS-CoV-2 infection, transmission can occur through droplets and fomites, especially
when there is close, unprotected contact between infected and susceptible individuals. Hence, band
hygiene is mice, and Hdpp4-Tg mice (transgenic for expressing Hdpp4) for MERS-CoV infection (221).
The CRISPR-Cas9 gene-editing tool has been used for inserting genomic alterations in mice, making
them susceptible to MERS-CoV infection (222). Efforts are under way to recognize suitable animal
models for SARS-CoV2/COVID-l 9, identify the receptor affinity of this virus, study pathology in
experimental animal models, and explore virus-specific immune responses and protection studies,
which together would increase the pace of efforts being made for developing potent vaccines and
drugs to counter this emerging virus. Cell lines, such as monkey epithelial cell lines (LLC-MK2 and
Vero-B4), goat lung cells, alpaca kidney cells, dromedary umbilical cord cells, and advanced

ex VlVO three-dimensional tracheobronchial tissue, have been explored to study human CoVs (MERS-
CoV) (223, 224). Vero and Huh-7 cells (human liver cancer cells) have been used for isolating SARS-Co
V-2 (194). Recently, an experimental study with rhesus monkeys as animal models revealed the
absence of any viral loads in nasopharyngeal and anal swabs, and no viral replication was recorded in
the primary tissues at a time interval of 5 days post-reinfection in reexposed monkeys (274). The
subsequent virological, radiological, and pathological into the host cell. Heptad repeat 1 (HR1) and
heptad repeat 2 (HR2) can interact and cellular membranes in close proximity, facilitating its fusion.
The sequence alignment study conducted between COVID-19 and SARS-CoV identified that the S2
subunits are highly conserved in these CoVs. The HR1 and HR2 domains showed 92.6% and 100%
overall identity, respectively (210). From these findings, we can confirm the significance of COVID-19
HR1 and HR2 and their vital role in host cell entry. Hence, fusion inhibitors target the HR1 domain of
S protein, thereby preventing viral fusion and entry into the host cell. This is another potential
therapeutic strategy that can be used in the management of COVID-19. Other than the specific
therapy directed against COVID-19, general treatments play a vital role in the enhancement of host
immune responses against the viral agent. Inadequate nutrition is linked to the weakening of the
host immune response, making the individual more susceptible. The role played by nutrition in
disease susceptibility should be measured by evaluating the nutritional status of patients with
COVID-19 (205). 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 biologic 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 RBD, indicating its potential as a therapeutic agent in the
management of COVID-19. It can be used alone or in combination with other effective neutralizing
antibodies for the treatment and prevention of COVID-19 (202). Furthermore, SARSCoV-specific
neutralizing antibodies, like m396 and CR3014, failed to bind the S protein of SARS-CoV-2, indicating
that a particular level of similarity is mandatory between the RBDs of SARS-CoV and SARS-CoV-2 for
the cross-reactivity to occur.

Further assessment is necessary before confirming the effectiveness of such combination therapy. In
addition, to prevent further community and nosocomial spread of COVID-19, the postprocedure risk
management program should not be neglected (309). Development of broad-spectrum inhibitors
against the human coronaviral pathogens will help to facilitate clinical trials on the effectiveness of
such inhibitors against endemic and emerging coronaviruses (203). A promising animal study
revealed the protective effect of passive immunotherapy with immune serum from MERS immune
camels on mice infected with MERS-CoV (204). Passive immunotherapy using convalescent plasma is
another strategy that can be used for treating COVID-19-infected, critically ill patients (205).

Considerable protection in mice against a MERS-Cov lethal challenge. Such antibodies may play a
crucial role in enhancing protective humoral responses against the emerging CoVs by aiming
appropriate epitopes and functions of the S protein. The cross-neutralization ability of SARS-CoV
RBDspecific neutralizing MAbs considerably relies on the resemblance between their RBDs;
therefore, SARS-CoV RBD-specific antibodies could cross neutralized SL CoVs, i.e., bat-SL-CoV strain
WIV1 (RBD with eight amino acid differences from SARSCoV) but not bat-SL-CoV strain SHC014 (24
amino acid differences) (200).

Appropriate RBD-specific MAbs can be recognized by a relative analysis of RBD of SARS CoV-2 to that
of SARS-CoV, and cross-neutralizing SARS-CoV RBD-specific MAbs could be explored for their
effectiveness against COVID-19 and further need to be assessed clinically. The U.S. biotechnology
company Regeneron is attempting to recognize potent and specific MAbs to combat COVID-19. An
ideal therapeutic option suggested for SARS-CoV-2 (COVID-19) is the combination therapy comprised
of MAbs and the drug remdesivir (COVID-19) (201). The SARS-CoV-specific human MAb CR3022 is
found to bind with SARS-CoV-2 RBD, indicating its potential as therapeutic agent assays offer high
accuracy in the diagnosis of SARCoV-2, but the current rate of spread limits its use due to the lack of
diagnostic assay kits. This will further result in the extensive transmission of COVID-19, since only a
portion of suspected cases can be diagnosed. In such situations, conventional serological assays, like
enzyme-linked immunosorbent assay (ELISA), that are specific to COVID-19 IgM and IgG antibodies
can be used as a high-throughput alternative (149). At present, there is no diagnostic kit available for
detecting the SARS CoV-2 antibody (150). The specific antibody profiles of COVID-19 patients were
analyzed, and it was found that the IgM level lasted more than 1 month, indicating a prolonged stage
of virus replication in SARS-CoV-2-infected patients. The lgG levels were found to increase only in the
later stages of the disease. These findings indicate that the specific antibody profiles of SARS-CoV-2
and SARS-CoV were similar (325). These findings can be utilized for the development of specific
diagnostic tests against COVID-19 and can be used for rapid screening. Even though diagnostic test
kits are already available that can detect the genetic sequences of SARS-CoV- 2 (95), their availability
is a concern, as the number of COVID-19 cases is skyrocketing (155, 157). A 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). Barly
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 associate 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 progress from the initial focal unilateral to diffuse bilateral
ground-glass opacities and will further progress to or coexist with lung consolidation changes within
1 to 3 weeks (159). The role played by radiologists in the current scenario is very important.
Radiologists can help in the early diagnosis of lung abnormalities associated with COVID-19
pneumonia. They can also help in the evaluation of disease severity, identifying its progression to
acute respiratory distress syndrome and the presence of secondary bacterial infections (160). Even
though chest CT is considered an essential diagnostic tool for COVID-19, the extensive use of CT for
screening purposes in the suspected individuals might be associated with a disproportionate
risk-benefit ratio due to increased radiation exposure as well as increased risk of cross infection.
Hence, the use of CT for early diagnosis of SARS-CoV-2 infection in high-risk groups should be done
with great caution (292).

More recently, other advanced diagnostics have been designed and developed for the detection of
SARS-CoV-2 (345, 347, 350-352). A reverse transcriptional loop-mediated isothermal amplification
(RT-LAMP), namely, iLACO, has been developed for rapid and colorimetric detection of this rates,
disease outbreaks, community spread, clustered transmission events, hot spots, and superspreader
potential of SARS-CoV-2/COVID warrant full exploitation of real-time disease mapping by employing
geographical information systems (GIS), such as the GIS software Kosmo 3.1, web-based real-time
tools and dashboards, apps, and advances in information technology (356-359). Researchers have
also developed a few prediction tools/models, such as the prediction model risk of bias assessment
tool (PROBAST) and critical appraisal and data extraction for systematic reviews of prediction
modeling studies (CHARMS), which could aid in assessing the possibility of getting infection and
estimating the prognosis in patients; however, such models may suffer from bias issues and, hence,
cannot be considered completely trustworthy, which necessitates the development of new and
reliable predictors (360).

VACCINES, THERAPEUTICS, AND DRUGS

Recently emerged viruses, such as Zika, Ebola, and Nipah viruses, and their grave threats to humans
have begun a race in exploring the designing and developing of advanced vaccines, prophylactics,
therapeutics, and drug regimens to counter emerging viruses (161-163, 280). Several attempts are
being made to design and develop vaccines for CoV infection, mostly by targeting the spike
glycoprotein. Nevertheless, owing to extensive diversity in antigenic variants, cross-protection
rendered by the vaccines is significantly limited, even within the strains of a phylogenetic subcluster
(104). Due to the lack of effective antiviral therapy and vaccines in the present scenario, we need to
depend solely on implementing effective infection control measures to lessen the risk of possible
nosocomial transmission (68). Recently, the receptor for SARS-CoV-2 was established as the human
angiotensin-converting enzyme 2 (hACE2), and the virus was found to enter the host cell mainly
through endocytosis. It was also found that the major components that have a critical role in viral
entry include PIKfyve, TPC2, and cathepsin L. These findings are critical, since the components
described above might act as candidates for vaccines or therapeutic drugs against SARS CoV-2 (293).

The majority of the treatment options and strategies that are being evaluated for SARS-CoV-2
(COVID-19) have been taken from our previous experiences in treating SARS-CoV, MERS-CoV, and
other emerging viral diseases. Several therapeutic been controlled by adopting appropriate and strict
prevention and control measures, and patients for clinical trials will not be available. The newly
developed drugs cannot be marketed due to the lack of end users.

Vaccines

The S protein plays a significant role in the induction of protective immunity against SARS-CoV by
mediating T-cell responses and neutralizing antibody production (168). In the past few decades, we
have seen several attempts to develop a vaccine against human coronaviruses by using S protein as
the target (168, 169). However, the developed vaccines have minimal application, even among
closely related strains of the virus, due to a lack of cross-protection. That is mainly because of the
extensive diversity existing among the different antigenic variants of the virus (104). The
contributions of the structural proteins, like spike (S), matrix (M), small envelope (E), and
nucleocapsid (N) proteins, of SARS-CoV to induce protective immunity has been evaluated by
expressing them in a recombinant parainfluenza virus type 3 vector (BHPIV3). Of note, the result was
conclusive that the expression of M, E, or N proteins without the presence of S protein would not

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. ln 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,
evaluating their interaction with corresponding major histocompatibility complex class I molecules.
They potentially induce immune responses (176). The recombinant vaccine can be designed by using
rabies virus (RV) as a viral vector. RV can be made to express MERS-CoV S1 protein on its surface so
that an immune response is induced against MERS-CoV. The RV vector-based vaccines against
MERS-CoV can induce faster antibody response as well as higher degrees of cellular immunity than
the Gram-positive enhance matrix (GEM) particle vector-based vaccine. However, the latter can
induce a very high antibody response at lower doses (167). Hence, the degree of humoral and
cellular immune responses produced by such vaccines depends upon the vector used.

Dual vaccines have been getting more popular recently. Among them, the rabies virus-based
vectored vaccine platform is used to develop vaccines against emerging infectious diseases. The dual
vaccine developed from inactivated rabies virus particles that express the MERS-CoV S1 domain of S
protein was found to induce immune responses for both MERS-CoV and rabies virus. The vaccinated
mice were found to be completely protected from challenge with MERS-CoV (169). The intranasal
administration of the recombinant adenovirus-based vaccine in BALB/c mice was found to induce
long lasting neutralizing immunity against MERS spike pseudo typed virus, characterized by the
induction of systemic IgG, secretory IgA, and lung-resident memory T-cell responses (177).
Immunoinformatics methods have been employed for the genome-wide screening of potential
vaccine targets among the different immunogens of MERS-CoV (178). The N protein and the
potential B-cell epitopes of MERSCoV E protein have been suggested as immunoprotective targets
inducing both T-cell and neutralizing antibody responses (178, 179).

The collaborative effort of the researchers of Rocky Mountain Laboratories and Oxford University is
designing a chimpanzee adenovirus-vectored vaccine to counter COVID-19 (180). The Coalition for
Epidemic Preparedness Innovations (CEPI) has initiated three programs to design SARS-CoV-2
vaccines (181). CEPI has a collaborative project with Inovio for designing a MERS-CoV DNA vaccine
that could potentiate effective immunity. CEPI and the University of Queensland are designing a
molecular clamp vaccine platform for MERS-CoV and other pathogens, which could assist in the
easier identification of antigens by the immune system (181). CEPI has also funded Moderna to
develop a vaccine for COVID-19 in partnership with the Vaccine Research Center (VRC) of the
National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health
(NIH) (182). By employing mRNA vaccine platform technology, a vaccine candidate expressing SARS-
CoV-2 spike protein is likely to go through clinical testing in the coming months (180). On 16 March
2020, Jennifer Haller became the first person outside China to receive an experimental vaccine,
developed by Moderna, against this pandemic virus. Moderna, along with China's CanSino Biologics,
became the first research group to launch small clinical trials of vaccines against COVID-19. Their
study is evaluating the vaccine's safety and ability to trigger immune responses (296).

Scientists from all over the world are trying hard to develop working vaccines with robust protective
immunity against COVID-19. Vaccine candidates, like mRNA-1273 SARS-CoV-2 vaccine, INO-4800 DNA
coronavirus vaccine, and adenovirus type 5 vector vaccine candidate (Ad5-nCoV), are a few examples
under phase I clinical trials, while self-amplifying RNA vaccine, oral recombinant COVID-19 vaccine,
BNT162, plant-based COVID-19 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. 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).
Bence, 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-risk regions. It is derived from a live attenuated
strain of Mycobacterium bovis. At present, three new clinical trials have been registered to evaluate
the protective role of BCG vaccination against SARS CoV-2 (363). Recently, a cohort study was
conducted to evaluate the impact of childhood BCG vaccination in COVID-19 PCR positivity rates.
However, childhood BCG vaccination was found to be associated with a rate of COVID-19-positive
test results similar to that of the nonvaccinated group (364). Further studies are required to analyze
whether BCG vaccination in childhood can induce protective effects against COVID-19 in adulthood.
Population genetic studies conducted on 103 genomes identified that the SARS-CoV-2 virus has
evolved into two major types, L and S. Among the two types, L type is expected to be the most
prevalent (~70%), followed by the S type (-30%) (366). This finding has a significant impact on our
race to develop an ideal vaccine, since the vaccine candidate has to target both strains to be
considered effective. At present, the genetic differences between the L and S types are very small
and may not affect the immune response. However, we can expect further genetic variations in the
coming days that could lead to the emergence of new strains (367).

Therapeutics and Drugs

There is no currently licensed specific antiviral treatment for MERS- and SARS-CoV infections, and the
main focus in clinical settings remains on lessening clinical signs and providing supportive care (183-
186). Effective drugs to 1nanage COVID-19 patients include remdesivir, lopinavir/ritonavir alone or in
a blend with interferon beta, convalescent plasma, and monoclonal antibodies (MAbs); however,
efficacy and safety issues of these drugs require additional clinical trials (187, 281). A controlled trial
of ritonavir-boosted lopinavir and interferon alpha 2b treatment was performed on COVID-19
hospitalized patients (ChiCTR2000029308) (188). In addition, the use of hydroxychloroquine and
tocilizumab for their potential role in modulating inflammatory responses in the lungs and antiviral
effect has been proposed and discussed in many research articles. Still, no fool-proof clinical trials
have been published (194,196,197, 261-272). Recently, a clinical trial conducted on adult patients
suffering from severe COVID-19 revealed no benefit of lopinavir-ritonavir treatment over standard
care (273).

The efforts to control SARS-CoV-2 infection utilize defined strategies as followed against MERS and
SARS, along with adopting and strengthening a few precautionary measures owing to the unknown
nature of this novel virus (36, 189). Presently, the main course of treatment for severely affected
SARS-CoV-2 patients admitted to hospitals includes mechanical ventilation, intensive care unit (ICU)
admittance, and symptomatic and supportive therapies. Additionally, RNA synthesis inhibitors
(lamivudine and tenofovir disoproxil fumarate), remdesivir, neuraminidase inhibitors, peptide (EK1),
anti-inflammatory drugs, abidol, and Chinese traditional medicine (Lianhuaqingwen and
ShuFengJieDu capsules) could aid in COVID-19 treatment. However, further clinical trials are being
carried out concerning their safety and efficacy(7). It might require months to a year(s) to design and
develop effective drugs, therapeutics, and vaccines against COVID-19, with adequate evaluation and
approval from regulatory bodies and moving to the bulk production of many 1nil lions of doses at
commercial levels to meet the timely demand of mass populations across the globe (9). Continuous
efforts are also warranted to identify and assess viable drugs and immunotherapeutic regimens that
revealed proven potency in combating other viral agents similar to SARS-CoV-2.

COVID-19 patients showing severe signs are treated symptomatically along with oxygen therapy. In
such cases where the patients progress toward respiratory failure and become refractory to oxygen
therapy, mechanical ventilation is necessitated. The COVID-19-induced septic shock can be managed
by providing adequate hemodynamic support (299). Several classes of drugs are currently being
evaluated for their potential therapeutic action against SARS-CoV-2. Therapeutic agents that have
anti-SARS-CoV-2 activity can be broadly classified into three categories: drugs that block virus entry
into the host cell, drugs that block viral replication as well as its survival within the host cell, and
drugs that attenuate the exaggerated host immune response (300). An inflammatory cytokine storm
is commonly seen in critically ill COVID-19 patients. Hence, they 1n ay benefit fro1n the use of timely
anti-inflammation treat1ne nt. Anti-inflammatory therapy using drugs like glucocorticoids, cytokine
inhibitors, JAK inhibitors, and chloroquine/hydroxychloroquine should be done only after analyzing
the risk/benefit ratio in COVID-19 patients (301). There have not been any studies concerning the
application of nonsteroidal anti-inflammatory drugs (NSAID) to COVID-19-infected patients. However,
reasonable pieces of evidence are available that link NSAID uses with the occurrence of respiratory
and cardiovascular adverse effects. Bence, as a cautionary approach, it is better to recommend the
use of NSAIDs as the first-line option for managing COVID-19 symptoms (302). The use of
corticosteroids in COVID-19 patients is still a matter of controversy and requires further systematic
clinical studies. The guidelines that were put forward to manage critically ill adults suggest the use of
systemic corticosteroids in mechanically ventilated adults with ARDS (303). The generalized use of
corticosteroids is not indicated in COVID-19, since there are some concerns associated with the use
of corticosteroids in viral pneumonia. Stem cell therapy using mesenchymal stem cells (MSCs) is
another hopeful strategy that can be used in clinical cases of COVID-19 owing to its potential
immunomodulatory capacity. It may have a beneficial role in attenuating the cytokine storm that is
observed in severe cases of SARS-CoV-2 infection, thereby reducing mortality. Among the different
types of MSCs, expanded umbilical cord MSCs can be considered a potential therapeutic agent
that requires further validation for managing critically ill COVID-19 patients (304).

Repurposed broad-spectrum antiviral drugs having proven uses against other viral pathogens can be
employed for SARS-CoV-2-infected patients. These possess benefits of easy accessibility and
recognized pharmacokinetic and pharmacodynamic activities, stability, doses, and side effects (9).
Repurposed drugs have been studied for treating CoV infections, like lopinavir/ritonavir, and
interferon-1β revealed in vitro anti-MERS-CoV action. The in vivo experiment carried out in the
nonhuman primate model of common marmosets treated with lopinavir/ritonavir and interferon
beta showed superior protective results in treated animals than in the untreated ones (190). A
combination of these drugs is being evaluated to treat MERS in humans (MIRACLE trial) (191). These
two protease inhibitors (lopinavir and ritonavir), in combination with ribavirin, gave encouraging
clinical outcomes in SARS patients, suggesting their therapeutic values (165). However, in the current
scenario, due to the lack of specific therapeutic agents against SARSCoV-2, hospitalized patients
confirmed for the disease are given supportive care, like oxygen and fluid therapy, along with
antibiotic therapy for managing secondary bacterial infections (192). Patients with novel coronavirus
or COVID-19 pneumonia. who are mechanically ventilated often require sedatives, analgesics, and
even muscle relaxation drugs to prevent ventilator-related lung injury associated with
human-machine incoordination (122). The result obtained from a clinical study of four patients
infected with COVID-19 claimed that combination therapy using lopinavir/ritonavir, arbidol, and
Shufeng Jiedu capsules (traditional Chinese medicine) was found to be effective in managing COVID-
19 pneumonia (193). It is difficult to evaluate the therapeutic potential of a drug or a combination of
drugs for managing a disease based on such a limited sample size. Before choosing the ideal
therapeutic agent for the management of COVID-19, randomized clinical control studies should be
performed with a sufficient study population.

Antiviral Drugs

Several classes of routinely used antiviral drugs, like oseltamivir (neuraminidase inhibitor), acyclovir,
ganciclovir, and ribavirin, do not have any effect on COVID-19 and, hence, are not recommended
(187). Oseltamivir, a neuraminidase inhibitor, bas been explored in Chinese hospitals for treating
suspected COVID-19 cases, although proven efficacy against SARS-CoV-2 is still lacking for this drug
(7). The in vitro antiviral potential of FAD-approved drugs, viz., ribavirin, penciclovir, nitazoxanide,
nafamostat, and chloroquine, tested in comparison to remdesivir and favipiravir (broad-spectrum
antiviral drugs) revealed remdesivir and chloroquine to be highly effective against SARS-CoV-2
infection in vitro (194). Ribavirin, penciclovir, and favipiravir might not possess noteworthy in vivo
antiviral actions for SARS-CoV-2, since higher concentrations of these nucleoside analogs are needed
in vitro to lessen the viral infection. Both remdesivir and chloroquine are being used in humans to
treat other diseases, and such safer drugs can be explored for assessing their effectiveness in COVID-
19 patients.

Several therapeutic agents, such as lopinavir/ritonavir, chloroquine, and hydroxychloroquine, have

been proposed for the clinical management of COVID-19 (299). A molecular docking study,
conducted in the RNAdependent RNA polymerase (RdRp) of SARS-CoV-2 using different commercially
available antipolymerase drugs, identified that drugs such as ribavirin, remdesivir, galidesivir,
tenofovir, and sofosbuvir bind RdRp tightly, indicating their vast potential to be used against COVID-
19 (305). A broad-spectrum antiviral drug that was developed in 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-γ
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 efficacy in the treatment of
COVID-19 infection in humans. The broad-spectrum activity exhibited by remdesivir will help control
the spread of disease in the event of a new coronavirus outbreak.

Chloroquine is an antimalarial drug known to possess antiviral activity due to its ability to block virus-
cell fusion by raising the endosomal pH necessary for fusion. It also interferes with virus-receptor
binding by interfering with the terminal glycosylation of SARS-CoV cellular receptors, such as ACE2
(196). In a recent multicenter clinical trial that was conducted in China, chloroquine phosphate was
found to exhibit both efficacy and safety in the therapeutic management of SARS-CoV-2-associated
pneumonia (197). This drug is already included in the treatment guidelines issued by the National
Health Commission of the People's Republic of China. The preliminary clinical trials using
hydroxychloroquine, another aminoquinoline drug, gave promising results. The COVID-19 patients
received 600 mg of hydroxychloroquine daily along with azithromycin as a single-arm protocol. This
protocol was found to be associated with a noteworthy reduction in viral load. Finally, it resulted in a
complete cure (271); however, the study comprised a small population and, hence, the possibility of
misinterpretation could arise. However, in another case study, the authors raised concerns over the
efficacy of hydroxychloroquine azithromycin in the treatment of COVID-19 patients, since no
observable effect was seen when they were used. In some cases, the treatment was discontinued
due to the prolongation of the QT interval (307). Hence, further randomized clinical trials are
required before concluding this matter.

Recently, another FDA-approved drug, ivermectin, was reported to inhibit the in vitro replication of
SARS-CoV-2. The findings from this study indicate that a single treatment of this drug was able to
induce an ~5,000-fold reduction in the viral RNA at 48h in cell culture. (308). One of the main
disadvantages that limit the clinical utility of ivermectin is its potential to cause cytotoxicity. However,
altering the vehicles used in the formulations, the pharmacokinetic properties can be modified,
thereby having significant control over the systemic concentration of ivermectin (338). Based on the
phannacokinetic simulation, it was also found that ivermectin may have limited therapeutic utility in
managing COVID-19, since the inhibitory concentration that has to be achieved for effective anti-
SARS-CoV-2 activity is far higher than the maximum plasma concentration achieved by administering
the approved dose (340). However, ivermectin, being a host-directed agent, exhibits antiviral activity
by targeting a critical cellular process of the mammalian cell. Therefore, the administration of
ivermectin, even at lower doses, will reduce the viral load at a minor level. This slight decrease will
provide a great advantage to the immune system for mounting a large-scale antiviral response
against SARS-CoV-2 (341). Further, a combination of ivermectin and hydroxychloroquine might have a
synergistic effect, since ivermectin reduces viral replication, while hydroxychloroquine inhibits the
entry of the virus in the host cell (339). Further, in vivo studies and randomized clinical control trials
are required to understand the mechanism as well as the clinical utility of this promising drug.

Nafamostat is a potent inhibitor of MERS-CoV that acts by preventing membrane fusion.

Nevertheless, it does not have any sort of inhibitory action against SARS-CoV-2 infection (194).
Recently, several newly synthesized halogenated triazole compounds were evaluated, using
fluorescence resonance energy transfer (FRET)based helicase assays, for their ability to inhibit
helicase activity.

Among the evaluated compounds, 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-iodophenyl) hydraziny1]-4H-

l ,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 MERSCoV 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-proteins without the presence of S protein would not confer any
noticeable protection, with the absence of detectable serum SARS-CoV-neutralizing antibodies (170).
Antigenic determinant sites present over S and N structural proteins of SARS-CoV-2 can be explored
as suitable vaccine candidates (294). In the Asian population, S, E, M, and N proteins of SARS CoV-2
are being targeted for developing subunit vaccines against COVID-19 (295).

The identification of the immunodominant region among the subunits and domains of S protein is
critical for developing an effective vaccine against the coronavirus. The C-terminal domain of the S1
subunit is considered the immunodominant region of the porcine deltacoronavirus S protein (171).
Similarly, further investigations are needed to determine the immunodominant regions of SARSCoV-2
for facilitating vaccine development.

However, our previous attempts to develop a universal vaccine that is effective for both SARSCoV and
MERS-CoV based on T-cell epitope similarity pointed out the possibility of cross reactivity among
coronaviruses (172). That can be made possible by selected potential vaccine targets that are
common to both viruses. SARS-CoV-2 has been reported to be closely related to SARS-CoV(173,
174). Hence, knowledge and understanding of

other emerging viral diseases. Several therapeutic and preventive strategies, including vaccines,
immunotherapeutics, and antiviral drugs, have been exploited against the previous CoV outbreaks
(SARS-CoV and MERS-CoV) (8,104,164-167).

These valuable options have already been evaluated for their potency, efficacy, and safety, along with
several other types of current research that will fuel our search for ideal therapeutic agents against
COVID-19 (7, 9, 19, 21, 36). The primary cause of the unavailability of approved and commercial
vaccines, drugs, and therapeutics to counter the earlier SARS-CoV and MERS-CoV seems to owe to
the lesser attention of the biomedicine and pharmaceutical companies, as these two CoVs did not
cause much havoc, global threat, and panic like those posed by the SARS-CoV-2 pandemic (19).
Moreover, for such outbreak situations, the requirement for vaccines and therapeutics/drugs exists
only for a limited period, until the outbreak is controlled. The proportion of the human population
infected with SARS-CoV and MERS-CoV was also much lower across the globe, failing to attract drug
and vaccine manufacturers and producers. Therefore, by the time an effective drug or vaccine is
designed against such disease outbreaks, the virus would have been controlled by adopting
appropriate and strict 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 bas 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-Casl2-based lateral flow assay also has been developed for rapid detection of SARS-CoV-2
(346). Artificial intelligence, by means of a threedimensional 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, 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 ORFlb
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-

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 epide1niological 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

Between the genome sequence of the new coronavirus (SARS-CoV-2) and SARS-like CoVs, the
comparative analysis recognized a furin-like cleavage site in the SARS-CoV-2 S protein that is missing
from other SARS-like CoVs (99). The furin like cleavage site is expected to play a role in the life cycle
of the virus and disease pathogenicity and might even act as a therapeutic target for furin inhibitors.
The highly contagious nature of SARS CoV-2 compared to that of its predecessors might be the result
of a stabilizing mutation that occurred in the endosome-associated-protein-like domain of nsp2
protein.

Similarly, the destabilizing mutation near the phosphatase domain of nsp3 proteins in SARS-CoV-2
could indicate a potential mechanism that differentiates it from other CoVs (100). Even though the
CFR reported for COVID-19 is meager compared to those of the previous SARS and MERS outbreaks,
it has caused more deaths than SARS and MERS combined (101). Possibly related to the viral
pathogenesis is the recent finding of an 832- nucleotide (nt) deletion in ORF8, which appears to
reduce the replicative fitness of the virus and leads to attenuated phenotypes of SARS-CoV-2 (256).

Coronavirus is the most prominent example of a virus that has crossed the species barrier twice from
wild animals to humans during SARS and MERS outbreaks (79, 102). The possibility of crossing the
species barrier for the third time has also been suspected in the case of SARS-CoV-2 (COVID-19). Bats
are recognized as a possible natural reservoir host of both SARS-CoV and MERS-CoV infection. In
contrast, the possible intermediary host is the pal1n civet for SARS-CoV and the dromedary camel for
MERS-CoV infection (102). Bats are considered the ancestral hosts for both SARS and MERS (103).
Bats are also considered the reservoir host of human coronaviruses like HCoV-229E and HCoV-NL63
(104). In the case of COVID-19, there are two possibilities for primary trans1nission: it can be
transmitted either through intermediate hosts, similar to that of SARS and MERS, or directly from
bats (103). The emergence paradigm put forward in the SARS outbreak suggests that SARS-CoV
originated from bats (reservoir host) and later jumped to civets (intermediate host) and incorporated
changes within the receptor-binding domain (RBD) to improve binding to civet ACE2. This
civet-apted virus, during their subsequent exposure to humans at live markets, promoted further
adaptations that resulted in the epidemic strain (104). Transmission can also visible signs of infection,
making it challenging to identify animals actively excreting MERS-CoV that has the potential to infect
humans. However, they may shed MERS-CoV through milk, urine, feces, and nasal and eye discharge
and can also be found in the raw organs (108). In a study conducted to evaluate the susceptibility of
animal species to MERS-CoV infection, llamas and pigs were found to be susceptible, indicating the
possibility of MERS CoV circulation in animal species other than dromedary camels (109).

Following the outbreak of SARS in China, SARS-CoV-like viruses were isolated from Himalayan palm
civets (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) found in a live-animal market
in Guangdong, China. The animal isolates obtained from the live-animal market retained a
29-nucleotide sequence that was not present in most of the human isolates (78). These findings
were critical in identifying the possibility of interspecies transmission in SARS-CoV. The higher
diversity and prevalence of bat coronaviruses in this region compared to those in previous reports
indicate a host/pathogen coevolution. SARS-like coronaviruses also have been found circulating in
the Chinese horseshoe bat (Rhinolophus sinicus) populations. The in vitro and in vivo studies carried
out on the isolated virus confirmed that there is a potential risk for the reemergence of SARS-CoV
infection from the viruses that are currently circulating in the bat population (105).
CLINICAL PATHOLOGY OF SARS-CoV-2 (COVID-19)

The disease caused by SARS-CoV-2 is also named severe specific contagious pneu1nonia (SSCP),
Wuhan pneumonia, and, recently, COVID-19(110).Compared to SARS-CoV, SARS-CoV-2 has less
severe pathogenesis but has superior transmission capability, as evidenced by the rapidly increasing
number of COVID-19 cases (111). The incubation period of SARS-CoV-2 in familial clusters was found
to be 3 to 6 days (112). The mean incubation pe1iod of COVID-19 was found to be 6.4 days, ranging
from 2.1 to 11.1 days (113). Among an early affected group of 425 patients, 59 years was the median
age, of which more males were affected (114). Similar to SARS and MERS, the severity of this nCoV is
high in age groups above 50 years (2, 115). Symptoms of COVID-19 include fever, cough, myalgia or
fatigue, and, less commonly, headache, hemoptysis, and diarrhea (116, 282). Compared to the SARS-
CoV-2-infected patients in Wuhan during the initial stages of the outbreak, only mild symptoms were
noticed in those patients that are infected by human-to-human transmission (14).

The initial trends suggested that the mortality associated with COVID-19 was less than that of
previous outbreaks of SARS (101). The updates obtained from countries like China, Japan, Thailand,
and South Korea indicated that the COVID-19 patients had relatively mild manifestations compared
to those with SARS and MERS (4). Regardless of the coronavirus type, immune cells, like mast cells,
that are present in the submucosa of the respiratory tract and nasal cavity are considered the
primary barrier against this virus (92). Advanced in-depth analysis of the genome bas identified 380
amino acid substitutions between the amino acid sequences of SARS-CoV-2 and the SARS/SARS-like
coronaviruses. These differences in the amino acid sequences might have contributed to the
difference in the pathogenic divergence of SARS-CoV-2 (16). Further research is required to evaluate
the possible differences in tropism, pathogenesis, and transmission of this novel agent associated
with this change in the amino acid sequence. With the current outbreak of COVID-19, there is an
expectancy of a significant increase in the number of published studies about this emerging
coronavirus, as occurred 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, preter1n 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, SARS-CoV-2/COVID- l 9 on pregnancy. Researchers

have mentioned the probability of in utero transmission of novel SARS-CoV-2 from COVID- 19-
infected mothers to their neonates in China based upon the rise in IgM and IgG antibody levels
and cytokine values in the blood obtained from newborn infants inmediately postbirth; however,
RT-PCR failed to conftrm the presence of SARS-CoV-2 genetic material in the infants (283). Recent
studies show that at least in some cases, preterm delivery and its consequences are associated
with the virus. Nonetheless, some cases have raised doubts for the likelihood of vertical
transmission (240- 243).

COVID-19 infection was associated with pneumonia, and some developed acute respiratory
distress syndrome (ARDS). The blood biochemistry indexes, such as albumin, lactate
dehydrogenase, C reactive protein, lymphocytes (percent), and neutro phils (percent) give
an idea about the disease severity in COVID-19 infection (121). During COVID-19, patients
may present leukocytosis, leukopenia with lymphopenia (244), hypoalbuminenia, and an
increase of lactate dehydro genase, aspartate transaminase, alanine aminotransferase,
bilirubin, and, especially, D-dimer snakes, and various other wild animals ( 20 , 30, 79, 93, 124, 125,
287 ). Coronavirus infection is linked to different kinds of clinical manifestations, varying from
enteritis in cows and pigs, upper respiratory disease in chickens, and fatal respiratory infections in
humans (30).

Among the CoV genera, Alphacoronavirus and Betacoronavirus infect mammals, while
Gammacoronavirus and Deltacoronavirus mainly infect birds, fishes, and, sometimes, mammals
(27, 29, 106). Severa! novel coronaviruses that corne under the genus Deltacoronavirus have
been discovered in the past fro1n birds, like Wigeon coronavirus HKU20, Bulbul coronavirus
HKU11, Munia coronavirus HKU13, white-eye coronavirus HKU16, night-heron coronavirus
HKU19, and common moorhen coronavirus HKU21, as well as from pigs (porcine coronavirus
HKU15) (6, 29).

Transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine
hemagglutinating encephalomyelitis virus (PHEV) are some of the coronaviruses of swine.
Among them, TGEV and PEDV are responsible for causing severe gastroenteritis in young piglets
with noteworthy morbidity and mortality. Infection with PHEV also causes enteric infection but
can cause encephalitis due to its ability to infect the nervous system (30).

Bovine coronaviruses (BoCoVs) are known to infect several domestic and wild ruminants (126).
BoCoV inflicts neonatal calf diarr hea in adult cattle, leading to bloody diarrhea (winter
dysentery) and respiratory disease complex (shipping fever) in cattle of all age groups (126).
BoCoV-like viruses have been noted in humans, suggesting its zoonotic potential as well (127).
Feline enteric and feline infectious peritonitis (FIP) viruses are the two major feline CoVs (128),
where feline CoVs can affect the gastrointestinal tract, abdominal cavity (peritonitis),
respiratory tract, and central nervous system (128). Canines are also affected by CoVs that fall
under different genera, namely, canine enteric coronavirus in Alphacoronavirus and canine
respiratory coronavirus in Betacoronavirus, affecting the enteric and respiratory tract, respectively
(129, 130). IBV, under Gammacoronavirus, causes diseases of respiratory, urinary, and reproductive

systems, with substantial economic losses in chickens (131, 132). In small laboratory animals,
mouse hepatitis virus, rat sialodacryoadenitis coronavirus, and guinea pig and rabbit
coronaviruses are the major CoVs associated with disease manifestations like enteritis, hepati tis ,
and respiratory infections (10, 133).

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was first identified in suckling piglets

having severe enteritis and belongs to the genus Alphacoronavirus (106). The outbreak was
 associated with considerable scale mortality of piglets (24,693 deaths) across four farms in China
(134 ). The virus isolated from the piglets was ahnost identical to and had 95% genomic similarity
with horseshoe bat (Rhinolophus species) coronavirus HKU2, suggesting a bat origin of the pig
virus (106, 134, 135). It is also imperative to note that the SADS-CoV outbreak started in
Guangdong province, near the location of the SARS pandemic origin (134). Before this outbreak,
pigs were not known to be infected with bat-origin coronaviruses. This indicates that the bat-origin
coronavirus jumped to pig by breaking the species barrier. The next step of this jump might not
end well, since pigs are considered the mixing vessel for influenza A viruses due to their ability to
be infected by both human and avian influenza A viruses (136).

Similarly, they may act as the mixing vessel for coronaviruses, since they are in frequent contact
with both humans and multiple wildlife species. Additionally, pigs are also found to be
susceptible to infection with human SARS-CoV and MERS-CoV, making this scenario a nightmare
(109, 137). It is 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. lt bas 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 1nolecular surveillance strategies for studying Betacoronavirus among animais
(12), especially in the Rhinolophus bat family (86). Chinese bats havehigh commercial value,
since they are used in the comprehensive sequence analysis of the SARS-C OV-2 RNA
genome identified that the CoV from Wuhan is a recombinant virus of the bat coronavirus
and another coronavirus of unknown origin. The recombination was found to have happened
within the viral spike glycoprotein, which recognizes the cell surface receptor. Further
analysis of the genome based on codon usage identified the snake as the most probable
animal reservoir of SARS-CoV-2 (143). Contrary to these findings, another genome analysis
proposed that the genome of SARS-CoV-2 is 96% identical to bat coronavirus, reflecting its
origin from bats (63). The involvenent of bat-derived materials in causing the current
outbreak cannot be ruled out. High risk is involved in the production of bat-derived materials for
TCM practices involving the handling of wild bats. The use of bats for TCM practices will remain a
severe risk for the occurrence of zoonotic coronavirus epidemics in the future (139).
Furthermore, the pangolins are an endangered species of animals that harbor a wide variety of
viruses, including coronaviruses (144). The coronavirus isolated from Malayan pangolins (Manis
javanica) showed a very higb amino acid identity with COVID-19 at E (100%), M (98.2%), N
(96.7%), and S genes (90.4%). The RBD of S protein in CoV isolated from pangolin was almost
identical (one amino acid difference) to that of SARS-CoV-2.

A co1nparison of the genomes suggests recombination between pangolin-CoV-like viruses with the
bat-CoV-RaTG13-like virus. All this suggests the potential of pangolins to act as the intermediate
host of SARS-CoV-2 (145).

Human-wildlife interactions, wbich are increasing in the context of climate change (142),
are further considered high risk and responsible for the emergence of SARS-CoV. COVID-19
is also suspected of having a similar mode of origin. Hence, to prevent the occurrence of
another zoonotic spillover (1), exhaustive coordinated efforts are needed to identify the high-risk
pathogens harbored by wild animal populations, conducting surveillance among the people who are
susceptible to zoonotic spillover events (12), and to improve the biosecurity measures associated
with the wildlife trade (146).

The serological surveillance studies conducted in people living in proximity to bat caves had
earlier identified the serological confirmation of SARS related CoVs in humans. People living at
the wildlife-human interface, mainly in rural China, are regularly exposed to SARS-related CoVs (147).
These findings will not have any significance until a significant outbreak occurs due to a virus-like
SARS-CoV-2.

There is a steady increase in the reports of COVID-19 in companion and wild animals around the
world. Further studies are required to evaluate the potential of ani1nals (especially companion
animals) to serve as an efficient reservoir host that can further alter the dynamics of human-to-
human transmission (330). To date, two pet dogs (Hong Kong) and four pet cats (one each from
Belgium and Hong Kong, two from the United States) have tested positive for SARS-CoV-2 (335).
The World Organization for Animal Health (OIE) has confirmed the diagnosis of COVID-19 in both
dogs and cats due to human-to-animal transmission (331). The similarity observed in the gene
sequence of SARS CoV-2 from an infected pet owner and his dog further confirms the
occurrence of human-to-animal transmission (333). Even though asymptomatic, feline species
should be considered a potential transmission route from animals to humans (326).

However, currently, there are no reports of SARS CoV-2 transmission from felines to human
beings.

Based on the current evidence, we can conclude that cats are susceptible to SARS-CoV-2 and can
get infected by hrunan beings. However, evidence of cat-to-human transmission is lacking and
requires further studies (332). Rather than waiting for frrmer evidence on animal-to-human
transmission, necessary preventive measures are advised, as well as following social distancing
practices among companion animals of different households (331).

One of the leading veterinary diagnostic companies, IDEXX, has conducted large-scale testing for
COVID-19 in specimens collected from dogs and cats. However, none of the tests turned out to
be positive (334).

In a study conducted to investigate the potential of different animal species to act as the
intermediate host of SARS-CoV-2, it was found that both ferrets and cats can be infected via
experimental inoculation of the virus. In addition, infected cats efficiently transmitted the
 disease to naive cats (329). SARS CoV-2 infection and subsequent transmission in ferrets were
found to recapitulate the clinical aspects of COVID-19 in humans. The infected ferrets also
shed virus via multiple routes, such as saliva, nasal washes, feces, and urine, postinfection,
making them an ideal animal model for study ing disease transmission (337). Experimental

inoculation was also done in other animal species and found that the dogs have low
susceptibility, while the chickens, ducks, and pigs are not at ail susceptible to SARS CoV-2 (329).

Similarly, the National Veterinary Services Laboratories of the USDA have reported

COVID-19 in tigers and lions that exhibited respiratory signs like dry cough and wheezing.
The zoo animals are suspected to have been infected by an asymptomatic zookeeper (335). The
total number of COVID-19- positive cases in human beings is increasing at a high rate,
thereby creating ideal conditions for viral spillover to other species, such as pigs.
However, experimental inoculation with SARS-CoV-2 failed to infect pigs (329).

Further studies are required to identify the possible animal reservoirs of SARS-CoV-2 and the
seasonal variation in the circulation of these viruses in the animal population. Research
collaboration between human and animal health sectors is becoming a necessity to evaluate and
identify the possible risk factors of transmission between animals and humans. Such cooperation
will help to devise efficient strategies for the management of emerging zoonotic diseases (12).

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).

Reccntly, 95 full-length genomic seqences 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 (ORFla) 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 asymptomatic of symptomatic patients having minimum signs and
symptoms (82). Another study, conducted in South Korea, related to SARS-CoV-2 viral load, opined
that SARS-CoV-2 kinetics were significantly different from those of earlier reported CoV infections,
including SARS-CoV (253). SARS CoV-2 transmission can occur early in the viral infection phase ;
thus, diagnosing cases and isolation attempts for this virus warrant different strategies than those
needed to counter SARS-CoV. Studies are required to establish any correlation between SARS CoV-
2 viral load and cultivable virus. Recognizing patients with fewer or no symptoms, along with
having modest detectable viral RNA in the oropharynx for 5 days, indicates the requirement of
data for assessing SARS-CoV-2 transmission dynamics and updating the screening procedures in
the clinics (82).

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 O to 13 after onset), stool samples from
nine cases (53% ; days O 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
5
1.21 x 10 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, significance of frequent
and good hand hygiene and sanitation pratices needs to be given due emphasis (249-252).
Future explorative research needs to be conducted with regard to the fecal-oral transmission
of SARS-CoV-2, along with focusing on environmental investigations to find out if this virus
 could stay viable in situations and atmospheres facilitating such potent routes of
transmission. The correlation of fecal concentrations of viral RNA with disease severity needs
to be determined, along with assessing the gastrointestinal symptoms and the possibility of
fecal SARS-CoV-2 RNA detection during the COVID-19 incubation period or convalescence
phases of the disease (249- 252).
The lower respiratory tract sampling techniques, like bronchoalveolar lavage fluid aspirate, are
considered the ideal clinical materials, rather than the throat swab, due to their higher positive
rate on the nucleic acid test (148). The diagnosis of COVID-19 can be made by using upper
respiratory-tract specimens collected using nasopharyngeal and oropharyngeal swabs. However,
these techniques are associated with unnecessary risks to health care workers due to close
contact with patients (152).

Similarly, a single patient with a high viral load was reported to contaminate an entire
endoscopy room by shedding the virus, which may remain viable for at least 3 days and is
considered a great risk for uninfected patients and health care workers (289).