P.

101 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 Corona viridae (subfamily Corona virinae), the members of which infect a broad
P.102 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 beta coronaviruses and specific
detection of SARS-CoV-2, developed by the U.S. CDC (Table 1) (258, 259).
P.103residues 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
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 organs 40. 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)'. 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.
P.104 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
The use of
(302).
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
P.105Coronaviruses in Humans-SARS, MERS, and COVID-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
P.106 Recently, 95 full-length genomic sequences of
SARAS-COV-2 strains available in the National Center for Biotechnology Information and
GISAID databases were subjected to multiple-sequence alignment and phylogenetic analyses
for studying variations in the viral genome (260). All the viral strains revealed high homology
of 99.99% (99.91% to 100%) at the nucleotide level and 99.99% (99.79% to 100%) at the
amino acid level. Overall variation was found to be low in ORF regions, with 13 variation sites
recognized in 1a, 1b, S, 3a, M, 8, and N regions. Mutation rates of 30.53% (29/95) and
29.47% (28/95) were observed at nt 28144 (ORF8) and nt 8782 (ORF1a) positions,
respectively. Owing to such selective mutations, a few specific regions of SARS-CoV-2 should
not be considered for designing primers and probes. The SARS-CoV-2 reference sequence
could pave the way to study molecular biology and pathobiology, along with developing
diagnostics and appropriate prevention and control strategies for countering SARS-CoV-2
(260).
Nucleic acids of SARS-CoV-2 can be detected from samples (64) such as bronchoalveolar
lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal
swabs, feces, blood, and urine, with different levels of diagnostic performance (Table 2) (80,
245, 246). The viral loads
P.107 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
P.108 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
P.109 Sampic.
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.
P.110 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).
P.111 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. Some 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
hospitalised 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 analyse even the asymptomatic infections; several
serological tests are in progress for SARS-COV-2.14, 30
P.112CONCLUDING REMARKS to
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 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
P.113•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 well 163. 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 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 production 165. 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
P.114encircled with an
envelope containing viral nucleocapsid. The nucleocapsids in CoVs are arranged in helical
symmetry, which reflects an atypical attribute in positive-sense RNA viruses (30). The
electron micrographs of SARS-CoV-2 revealed a diverging spherical outline with some degree
of pleomorphism, virion diameters varying from 60 to 140 nm, and distinct spikes of 9 to 12
nm, giving the virus the appearance of a solar corona (3). The CoV genome is arranged
linearly as 5'-leader-UTR- replicase-structural genes (S-E-M-N)-3'
UTR-
poly(A) (32). Accessory genes, such as 3a/b, 4a/b, and the hemagglutinin-esterase gene (HE),
are also seen intermingled with the structural genes (30). SARS-COV-2 has also been found
to be arranged similarly and encodes several accessory proteins, although it lacks the HE,
which is characteristic of some beta coronaviruses (31). The positive-sense genome of CoVs
serves as the mRNA and is translated to polyprotein 1a/lab (ppla/lab) (33). A replication-
transcription complex (RTC) is formed in double-membrane vesicles (DMVs) by nonstructural
proteins (nsps), encoded by the polyprotein gene (34). Subsequently, the RTC synthesizes a
nested set of sub genomic RNAs (sgRNAs) via discontinuous transcription (35).
P.115 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).
P.116 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). Recently, it was found that
the anal swabs gave more positive results than oral swabs in the later stages of infection
(153). Hence, clinicians have to be cautious while discharging any COVID-19-infected patient
based on negative oral swab test results due to the possibility of fecal-oral transmission.
Even though the viral loads in stool samples were found to be less than those of respiratory
samples, strict precautionary measures have to be followed while handling stool samples of
COVID-19 suspected or infected patients (151). Children infected with SARS-CoV-2
experience only a mild form of illness and recover immediately after treatment. It was
recently found that stool samples of SARS-CoV-2- infected children that gave negative throat
swab results were positive within ten days of negative results. This could result in the fecal-
oral transmission of SARS-CoV-2 infections, especially in children (290). Hence, to prevent
the fecal-oral transmission of SARS-CoV-2, infected COVID-19 patients should only be
considered negative when they test negative for SARS-CoV-2 in the stool sample.
P.117 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
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
P.118 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
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
P.119 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 mean time 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 major 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
P.120 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 manage COVID- 19 patients include remdesivir,
lopinavir/ritonavir alone or in a blend with interferon beta, convalescent plasma, and
monoclonal antibodies antibodies (MAbs);
hospitalized
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 patients (ChiCTR2000029308) (188). In addition, the use of
hydroxychloroquine and tocilizumab for their for 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
P.121 Based on molecular characterization, SARS-
CoV-2 is considered a new Beta coronavirus
belonging to the subgenus Sarbe covirus (3). A few other critical zoonotic viruses (MERS-
related CoV and SARS-related CoV) belong to the same genus. However, SARS-CoV-2 was
identified as a distinct virus based on the percent identity with other Beta coronavirus;
conserved open reading frame 1a/b (ORF1a/b) is below 90% identity (3). An overall 80%
nucleotide identity was observed between SARS-CoV-2 and the original SARS-CoV, along
with 89% identity with ZC45 and ZXC21 SARS- related CoVs of bats (2, 31, 36). In addition,
82% identity has been observed between SARS-CoV-2 and human SARS-CoV Tor2 and human
SARS-CoV BJ01 2003 (31). A sequence identity of only 51.8% was observed between MERS-
related CoV and the recently emerged SARS-CoV-2 (37). Phylogenetic analysis of the
structural genes also revealed that SARS-CoV-2 is closer to bat SARS-related CoV. Therefore,
SARS-CoV-2 might have originated from bats, while other amplifier hosts might have played
a role in disease transmission to humans (31). Of note, the other two zoonotic CoVs (MERS-
related CoV and SARS-related CoV) also originated from bats (38, 39). Nevertheless, for SARS
and MERS, civet
P.122 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 (pplab), 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 Beta coronavirus. Within this clade, SARS-CoV-2 is
grouped in a distinct lineage together with four horse- shoe bat coronavirus isolates
(RaTG13, RmYN02, ZC45 and ZXC21) as well as novel coronaviruses recently identified in
pangolins, which group parallel to SARS-CoV
P.123 areas. For example, a cohort study in London revea 44% of the frontline health-care
workers from a hosp 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
period. 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 trans- mission was also observed in
the ferret experiments mentioned above. SARS-CoV-2-infected ferrets shed
P.124 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 host 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. In 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 both in vivo and in vitro
P.125 SARS- or MERS-CoV outbreak (120). However, there has been concern regarding the
impact of SARS-CoV-2/COVID-19 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 immediately post birth; however, RT-PCR failed to
confirm 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).
associated with
COVID-19 infection was pneumonia, and some developed acute respiratory distress
syndrome (ARDS). The blood biochemistry indexes, such as albumin, lactate dehydrogenase,
C- reactive protein, lymphocytes (percent), and neutrophils (percent) give an idea about the
disease severity in COVID-19 infection (121). During COVID-19, patients may present
leukocytosis, leukopenia with lymphopenia (244), hypoalbuminemia, and an increase of
lactate dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and,
especially, D-dimer
P.126 between 4 and -70°C. Urine samples must also be collected using a sterile container
and stored in the laboratory at a temperature that ranges between 4 and -70°C,32
7 PREGNANCY
Currently, there is a paucity of knowledge and data related to the consequences of COVID-19
during pregnancy. However, pregnant
40-42
women seem to have a high risk of developing severe infection and complications during the
recent 2019-nCoV outbreak.41-43 This speculation was based on previous available scientific
reports on coronaviruses during pregnancy (SARS-CoV and MERS-CoV) as well as the limited
number of COVID-19 cases.
Analysing the clinical features and outcomes of 10 newborns (including two sets of twins) in
China, whose mothers are confirmed cases of COVID-19, revealed that perinatal infection
with 2019-nCoV may lead to adverse outcomes for the neonates, for example, premature
labour, respiratory distress, thrombocytopenia with 44 abnormal liver function and even
death. It is still unclear whether or not the COVID-19 infection can be transmitted during
pregnancy to the foetus through the transplacental route.42
A recent case series report, which assessed intrauterine vertical transmission of
P.127 8 PREVENTION
The WHO and other agencies such as the CDC have published protective measures to
mitigate the spread of COVID-19. This involves frequent hand washing with handwash
containing 60% of alcohol and soap for at least 20 seconds. Another important measure is
avoiding close contact with sick people and keeping a social distance of 1 metre always to
everyone who is coughing and sneezing. Not touching the nose, eyes and mouth was also
suggested. While coughing or sneezing, covering the mouth and nose with a cloth/tissue or
the bent elbow is advised. Staying at home is recommended for those who are sick, and
wearing a facial mask is advised when going out among people. Furthermore, it is
recommended to clean and sterilise frequently touched surfaces such as phones and
doorknobs on a daily basis. 51, 52 Staying at home as much as possible is
advisable for those who are at higher risk for severe illness, to minimise the risk of exposure
to COVID-19 during outbreaks.53
P.128 6.5 Specimen collection and storage
A Nasopharyngeal and oropharyngeal swab should be collected using Dacron or polyester
flocked swabs. It should be transported to the laboratory at a temperature of 4°C and stored
in the laboratory between 4 and -70°C on the basis of the number of days and, in order to
increase the viral load, both nasopharyngeal and oropharyngeal swabs should be placed in
the same tube. Bronchoalveolar lavage and nasopharyngeal aspirate should be collected in a
sterile container and transported similarly to the laboratory by maintain a temperature of
4°C.
Sputum samples, especially from the lower respiratory tract, should be collected with the
help of a sterile container and stored, whereas tissue from a biopsy or autopsy should be
collected using a sterile container along with saline. However, both should be stored in the
laboratory at a temperature that ranges
between 4 and -70°C. Whole blood for detecting the antigen, particularly in the first week of
illness, should be collected in a collecting tube and stored in the laboratory between 4 and -
70°C. Urine samples must also be collected using a sterile container and stored
P.129turtles, ducks, fish, Siamese crocodiles, and other animal meats without any fear of
COVID-19. The Chinese government is encouraging people to feel they can return to
normalcy. However, this could be a risk, as it has been mentioned in advisories that people
should avoid contact with live-dead animals as much as possible, as SARS-CoV-2 has shown
zoonotic spillover. Additionally, we cannot rule out the possibility of new mutations in the
same virus being closely related to contact with both animals and humans at the market
(284). In January 2020, China imposed a temporary ban on the sale of live- dead animals in
wet markets. However, now hundreds of such wet markets have been reopened without
optimizing standard food safety and sanitation practices (286).
With China being the most populated country in the world and due to its domestic and
international food exportation policies, the whole world is now facing the menace of COVID-
19, including China itself. Wet markets of live-dead animals do not maintain strict food
hygienic practices. Fresh blood splashes are present everywhere, on the floor and tabletops,
and such food customs could encourage many pathogens to adapt, mutate, and jump the
species barrier. As a result, the whole world is suffering from novel SARS-CoV-2, with more
than
P.130 differs from that in SARS-CoV in the five residues critical for ACE2 binding, namely
Y455L, 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.5) (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-CoV and enables better contact with the N-terminal helix of hACE2
(REF.5). Biochemical data confirmed that the structural features of the SARS-CoV-2 RBD has
strengthened its hACE2 binding affinity compared with that of SARS-CoV 50,52,53
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 L and 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 (REFS 56,57). SARS-CoV-2 pseudovirus
entry assays revealed that TMPRSS2 and cathepsin L have cumu- lative effects with furin on
activating virus entry55. Analysis of the cryo-electron microscopy structure of SARS-CoV-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.
P.131 on significance of frequent and good hand hygiene and sanitation practices 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 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
P.132 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. Hence, 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
P.133 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
P.134 World TS
species barrier. AS a result, the WHOTC suffering from novel SARS-CoV-2, with more than
4,170,424 cases and 287,399 deaths across the globe. There is an urgent need for a rational
international campaign against the unhealthy food practices of China to encourage the
sellers to increase hygienic food practices or close the crude live-dead animal wet markets.
There is a need to modify food policies at national and international levels to avoid further
life threats and economic consequences from any emerging or reemerging pandemic due to
close animal-human interaction (285).
Even though individuals of all ages and sexes are susceptible to COVID-19, older people with
an underlying chronic disease are more likely to become severely infected (80). Recently,
individuals with asymptomatic infection were also found to act as a source of infection to
susceptible individuals (81). Both the asymptomatic and symptomatic patients secrete
similar viral loads, which indicates that the transmission capacity of asymptomatic or
minimally symptomatic patients is very high. Thus, SARS-CoV-2 transmission can happen
early in the course of infection (82). Atypical clinical manifestations have also been reported
in COVID-19 in which the only reporting symptom was fatigue. Such patients may lack
respiratory signs, such as fever, cough, and sputum (83). Hence, the clinicians
P.135 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 may benefit from the use of timely anti-
inflammation treatment. 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
P.135 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 may benefit from the use of timely anti-
inflammation treatment. 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
P.136Animal Models and Cell Cultures
For evaluating the potential of vaccines and therapeutics against CoVs, including SARS-CoV,
MERS-CoVs, and the presently emerging SARS- CoV-2, suitable animal models that can mimic
the clinical disease are needed (211, 212). Various animal models were assessed for SARS-
and MERS- CoVs, 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 has 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 mouse DPP4 altered with
human DPP4 (hDPP4), hDPP4-transduced mice, and hDPP4-Tg mice (transgenic for
expressing
P.137 and chest discomfort, and in severe cases dyspnea and bilateral lung infiltration67.
Among the first 27 docu- mented hospitalized patients, most cases were epidemi- ologically
linked to Huanan Seafood Wholesale Market a wet market located in downtown Wuhan,
which sells not only seafood but also live animals, including poultry and wildlife48.
According to a retrospective study, the onset of the first known case dates back to 8
December 2019 (REF). On 31 December, Wuhan Municipal Health Commission notified the
public of a pneumonia out- break of unidentified cause and informed the World Health
Organization (WHO) (FIG. 1).
By metagenomic RNA sequencing and virus isola tion from bronchoalveolar lavage fluid
samples from patients with severe pneumonia, independent teams of Chinese scientists
identified that the causative agent of this emerging disease is a betacoronavirus that had
never been seen before6,10,11. On 9 January 2020, the result of this etiological
identification was publicly announced (FIG. 1). The first genome sequence of the novel coro-
navirus was published on the Virological website on 10 January, and more nearly complete
genome sequences determined by different research institutes were then released via the
GISAID database on 12 January Later, more patients with no history of exposure to Huanan
Seafood Wholesale Market were identified. Several familial clusters of infection were
reported and nosocomial infection also occurred in health-care facilities. All these cases
provided clear evidence for human-to-human transmission of the new virus4,12-14 As the
outbreak coincided with the approach of the lunar New Year, travel between cities before
the festival facilitated virus transmission in China. This novel coro- navirus pneumonia soon
spread to other cities in Hube province and to other parts of China. Within 1 month.
P.138 health emergency on 31 January 2020; subsequently, on 11 March 2020, they declared
it a pandemic situation. At present, we are not in a position to effectively treat COVID-19,
since neither approved vaccines nor specific antiviral drugs for treating human CoV
infections are available (7-9). Most nations are currently making efforts to prevent the
further spreading of this potentially deadly virus by implementing preventive and control
strategies.
In domestic animals, infections with CoVs are associated with a broad spectrum of
pathological conditions. Apart from infectious bronchitis virus, canine respiratory CoV, and
mouse hepatitis virus, CoVs are predominantly associated with gastrointestinal diseases (10).
The emergence of novel CoVs may have become possible because of multiple CoVs being
maintained in their natural host, which could have favored the probability of genetic
recombination (10). High genetic diversity and the ability to infect multiple host species are a
result of high-frequency mutations in CoVs, which occur due to the instability of RNA-
dependent RNA polymerases along with higher rates of homologous RNA recombination (10,
11). Identifying the origin of SARS-CoV-2 and the pathogen's evolution will be helpful for
disease surveillance (12), development of
P.139with SARS and MERS (117).
SARS-COV-2 invades the lung parenchyma, resulting in severe interstitial inflammation of the
lungs. This is evident on computed tomography (CT) images as ground-glass opacity in the
lungs. This lesion initially involves a single lobe but later expands to multiple lung lobes
(118). The histological assessment of lung biopsy samples obtained from COVID-19-infected
patients revealed diffuse alveolar damage, cellular fibromyxoid exudates, hyaline membrane
formation, and desquamation of pneumocytes, indicative of acute respiratory distress
syndrome (119). It was also found that the SARS-CoV-2-infected patients often have
lymphocytopenia with or without leukocyte abnormalities. The degree of lymphocytopenia
gives an idea about disease prognosis, as it is found to be positively correlated with disease
severity (118). Pregnant women are considered to have a higher risk of getting infected by
COVID-19. The coronaviruses can cause adverse outcomes for the fetus, such as intrauterine
growth restriction, spontaneous abortion, preterm delivery, and perinatal death.
Nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical
transmission) of CoVs is low and was not seen during either the SARS- or MERS-CoV
outbreak (120). However,
P.140 The comprehensive sequence analysis of the SARS-COV-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
involvement 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 high 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