A suspected case of COVID-19 infection is said to be confirmed if the respiratory tract aspirate or blood samples test positive

for SARS-CoV-2 nucleic acid using RT-PCR or by the identification of SARS- CoV-2 genetic sequence in respiratory tract aspirate or blood
samples (80).The patient will be confirmed as cured when two subsequent oral swab results are negative(153).Recently,the live virus was
detected in the self-collected saliva of patients infected with COVID-19.These findings were confirmative of using saliva as a
noninvasive specimen for the diagnosis of COVID-19 infection in suspected individuals(152).It has also been observed that the
initial screening of COVID-19 patients infected with RT-PCR may give negative results even if they have chest CT findings that are
suggestive of infection. Hence,for the accurate diagnosis of COVID-19,a combination of repeated swab tests using RT-PCR and
CT scanning is required to prevent the possibility of false-negative results during disease screening(154).RT-PCR is the most
widely used test for diagnosing COVID-19.However,it has some significant limitations from the clinical perspective, since it will not
give any clarity regarding disease progression.Droplet digital PCR(ddPCR)can be used for the quantification of viral load in
the samples obtained from lower respiratory tracts.
Coronaviruses in Humans—SARS,MERS,

andCOVID-19

Coronavirus infection in humans is commonly associated with mild to severe respiratory diseases, with high fever,severe
inflammation,cough,and internal organ dysfunction that can even lead to death(92).Most of the identified coronaviruses cause the
common cold in humans.However,this changed when SARS-CoV was identified,paving the way for severe forms of the disease in humans(22).
Our previous experience with the outbreaks of other coronaviruses,like SARS and MERS,suggests that the mode of transmission in COVID-19
as mainly human-to-human transmission via direct contact, droplets,and fomites(25).Recent studies have demonstrated that the virus
could remain viable for hours in aerosols and up to days on surfaces;thus, aerosol and fomite contamination could play potent
roles in the transmission of SARS-CoV-2 (257).

The immune response against coronavirus is vital to control and get rid of the infection.However, maladjusted immune responses may
contribute to the immunopathology of the disease,resulting in impairment of pulmonary gas exchange. Understanding the
interaction between CoVs and
host innate immune systems could enlighten our
INTRODUCTION

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

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

South China Seafood Market in Wuhan(2).CoVs

belong to the family Coronaviridae(subfamily

Coronavirinae),the members of which infect a broad

 Recently,95 full-length genomic sequences of SARAS-CoV-2 strains available in the National Center for Biotechnology
Information and GISAID databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying variations
in the viral genome(260).All the viral strains revealed high homology of 99.99%(99.91% to 100%)at the nucleotide level and
99.99% (99.79%to 100%)at the amino acid level.Overall variation was found to be low in ORF regions,with 13 variation sites
recognized in la,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
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 animal 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
into the host cell.Heptad repeat 1(HR1)and heptad repeat 2(HR2)can interact and form a six-helix bundle that brings the viral 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).
pieces of evidence are available that link NSAID uses with the occurrence of respiratory and cardiovascular adverse effects.
Hence, 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

CONCLUDING REMARKS

Several years after the global SARS epidemic, the current SARS-CoV-2/COVID-19 pandemic has 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.Live- animal 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
and deaths.The COVID-19 outbreak has also been associated with severe economic impacts globally due to the sudden interruption
of global trade and supply chains that forced multinational companies to make decisions that led to significant economic losses(66).The
recent increase in the number of confirmed critically ill patients with COVID-19 has already surpassed the intensive care
supplies, limiting intensive care services to only a small portion of critically ill patients (67).This might also have contributed to the
increased case fatality rate
observed in the COVID-19 outbreak.

Viewpoint on SARS-CoV-2 Transmission,

Spread,and Emergence

The novel coronavirus was identified within 1 month(28 days)of the outbreak.This is impressively fast compared to the time
taken to identify SARS- CoV reported in Foshan,Guangdong Province, China (125 days)(68).Immediately after the confirmation
of viral etiology, the Chinese virologists rapidly released the genomic sequence of SARS-CoV-2,which played a crucial role in
controlling the spread of this newly emerged novel coronavirus to other parts of the world(69).The possible origin of SARS-CoV-2 and
the first mode of
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 welll⁶3.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 dosel⁶4.Regarding inactivated vaccines,a successful phase I/II trial involv- ing
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 productionl⁶5.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.

Futureperspectives
COVID-19 is the third highly pathogenic human coro- navirus disease to date.Although less deadly than SARS and MERS,the rapid
spreading of this highly conta- gious disease has posed the severest threat to global health in this century.The SARS-
CoV-2 outbreak has lasted for more than halfa year now,and it is likely that
and other SARSr-CoVs (FIG.2).Using sequences of five conserved replicative domains in pplab(3C-like protease (3CLpro),nidovirus RNA-
dependent RNA polymerase (RdRp)-associated nucleotidyltransferase(NiRAN), RdRp,zinc-binding domain(ZBD)and
HEL1),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 phyloge- netically related,SARS-CoV-2 is distinct from all 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 pro- teins of most members in the subgenus
Sarbecovirus, sharing amino acid sequence similarities of 76.7-
77.0%with SARS-CoVs from civets and humans,
possible origin of SAl

disease transmission are not yet identified(70). Analysis of the initial cluster of infections suggests that the infected individuals had a
common exposure point,a seafood market in Wuhan,Hubei Province, China( Fig.6).The restaurants of this market are well-known for
providing different types of wild animals for human consumption(71).The Huanan South China Seafood Market also sells live animals,
such as poultry,bats,snakes,and marmots (72).This might be the point where zoonotic (animal-to- human) transmission occurred (71).
Although SARS-CoV-2 is alleged to have originated from an animal host (zoonotic origin)with further human-to-
human transmission (Fig. 6),the likelihood of

foodborne transmission should be ruled out with

further investigations,since it is a latent possibility

(1).Additionally,other potential and expected routes would be associated with transmission,as in other respiratory viruses,by direct
contact,such as shaking contaminated hands,or by direct contact with contaminated surfaces(Fig.6).Still,whether blood transfusion and
organ transplantation(276),as well as transplacental and perinatal routes,are possible routes for SARS-CoV-2 transmission needs to be
determined (Fig.6).
of plasma cytokines,which suggests an immunopatho- logical process caused by a cytokine storm⁰8687.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 ofrespiratory fail- ure,acute cardiac injury and heart failure that led to death,regardless ofa history ofcardiovascular disease*6
(FIG.4).Most patients recovered enough to be released fromhospitalin2weeks°80(FIG.4).
Early transmission of SARS-CoV-2 in Wuhan in December 2019 was initially linked to the Huanan Seafood
Wholesale Market,and it was suggested as the source of the outbreak⁹22,60.However,community transmission might have
happened before that⁸.Later, ongoing human-to-human transmission propagated the outbreak".It is generally accepted that SARS-CoV-
2 is more transmissible than SARS-CoV and MERS-CoV; however,determination of an accurate reproduction number(R0)for
COVID-19 is not possible yet,as many asymptomatic infections cannot be accurately accounted for at this stage³9.An estimated R0 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-CoV⁰.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 set- tings,such as migrant worker communities,slaughter- houses and meat packing plants,indicating the necessity
of isolating infected people⁹,1291-3.Nosocomial transmis- sion was not the main source of transmission in China because of the
implementation of infection control measures in clinical settings₉.By contrast,a high risk of nosocomial transmission was
reported in some other
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 ofthe genome has identified 380 amino acid substitutions between the amino acid sequences of
】
[n e
SARS-CoV-2 and 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 i]n
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
 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 SARS- CoV- 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
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 ofCOVID- 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).
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 (RE(COVERY)trial,a national clin- ical trial
programme in the UK,has stopped treatment with lopinavir andritonavir as no significant beneficial effect was observed in a randomized
trial established in
March2020withatotalof1,596patients45.Nevertheless,
Inhibitionofvirusentry.SARS-CoV-2usesACE2asthe receptor and human proteases as entry activators;sub- sequently it fuses the viral membrane
with the cell mem- brane 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 respira- tory 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 prog- nosis of or accelerate SARS-CoV-2 clearance
inpatients with mild to moderate COVID-19(REFS124,125).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 postoper- ative 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 (wild- type mice infected with a mouse-adapted SARS-CoV strain)l26,127.Recently,a study
revealed that camostat mesylate blocks the entry of SARS-CoV-2 into human lung cells⁴.Thus,it can be a potential antiviral
drug against SARS-CoV-2infection,although so far there are not sufficient clinical data to support its efficacy.
residues for receptor binding(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 identity".The repeated occurrence of SARS-CoV-2-related coronavirus
infec- tions 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 organs⁰.These abnormalities suggest that pangolins
are unlikely to be the reservoir ofthese coronaviruses but more likely acquired the viruses after spillover from the
natural hosts.

An intermediate host usually plays an important role in the outbreak ofbat-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 identi-
cal to the corresponding viruses in humans (more than 99%genome sequenceidentity)'.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 avail-
able 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.
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