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. All 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 atleast 3 d and have two consecutive negative
molecular tests at 1 d sampling interval. This recommendation is different from pandemic flu where patients
were
 Allclinicians should keep themselves updated about recent developments including global spread of
the disease.
Non-essentialinternational 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 he made to 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 RBD specific 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 SARS CoV) 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 SARSCoV-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 n e e d t o b e a s s e s s e d c l i n i c a l l y. T he 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 a therapeutic agent
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 SARSCoV-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
51 subunit is considered the immunodominant region of the porcine delta corona virus 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 SARS CoV 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 clinical trials in different phases are still
ongoing elsewhere. Immunomodulatory agents. SARS-CoV-2 triggers a strong immune response which
may cause cytokine storm syndrome'''. 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 support'''. Tocilizumab and sarilumab, two types of interleukin-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 trial'''. 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 C5. 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 (REF.'48). another study, the average reproductive number of
COVID-19 was found to be 3.28, which is significantly higher than the initial WHO estimate of 1.4 to
2.5 (77). It is too early to obtain the exact Ro value, since there is a possibility of bias due to insufficient
data. The higher Ro value is indicative of the more significant potential of SARS-CoV-2 transmission in a
susceptible population. This is not the first time where the culinary practices of China have been blamed for the
origin of novel coronavirus infection in humans. Previously, the animals present in the live-animal market were
identified to be the intermediate hosts of the SARS outbreak in China (78). Several wildlife species were
found to harbor potentially evolving coronavirus strains that can overcome the species barrier (79). One of
the main principles of Chinese food culture is that live-slaughtered animals are considered more nutritious
(5). After 4 months of struggle that lasted from December 2019 to March 2020, the COVID-19
situation now seems under control in China. The wet animal markets have reopened, and people have started
buying bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins (scaly anteaters), minks, soup from
palm civet, ostriches, hamsters, snapping turtles, ducks, fish, Siamese crocodiles, and other been used based
on the experience with SARS and MERS. In a historical control study in patients with SARS, patients treated
with lopinavirritonavir 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%,
noninvasive 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 lopinavirritonavir was given to 75% of the patients. The duration of non-
invasive ventilation was 4-22 d [median 9 d] 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 extenaea to caner
cities or nunel province. Cases of COVID-19 in countries outside China were reported in those with no
history of travel to China suggesting that local human-tohuman 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 all people symptomatic or otherwise in isolation for 14
d and tested them for the virus. Cases continued to increase exponentially and modelling studies fever,
cough, and sputum (64 Hence, the clinician, must be on the look-out for the possible occurrence of atypical
clinical manifestations to avoid the possibility of missed diagnosis. The early transmission ability of
SARS-CoV-2 was found to be similar to or slightly higher than that of SARS-CoV, reflecting that it could be
controlled despite moderate to high transmissibility (84). Increasing reports of SARS-CoV-2 in sewage and
w as tew ater w arrants the need for further investigation due to the possibility of fecal-oral transmission.
SARS-CoV-2 present in environmental compartments such as soil and water will finally end up in the wastewater
and sewage sludge of treatment plants (328). Therefore, we have to reevaluate the current wastewater and
sewage sludge treatment procedures and introduce advanced techniques that are specific and effective
against SARS-CoV-2. Since there is active shedding of SARS-CoV-2 in the stool, the prevalence of
infections in a large population can be studied using wastewater-based epidemiology. Recently, reverse
transcription-quantitative PCR (RT-qPCR) was used to enumerate the copies of SARS-CoV-2 RNA
concentrated from wastewater collected from a wastewater treatment plant (327). The calculated viral RNA copy
numbers determine the number of infected individuals. The
13 CONVALESCENT PLASMA THERAPY
Guo Yanhong, an official with the National Health Commission (NHC), stated that convalescent plasma
therapy is a significant method for treating severe COVID-19 patients. Among the COVID-19 patients
currently receiving convalescent plasma therapy in the virus-hit Wuhan, one has been discharged from hospital,
as reported by Chinese science authorities on Monday, 17th February 2020 in Beijing. The first dose
convalescent plasma from a COVID-19 patient was collected on 1st and 9th February 2020 from a severely
ill patient who was given treatment at a hospital in jiangxia District in Wuhan. The presence of the virus in
patients is minimised by the antibodies in the convalescent plasma. Guiqiang stated that donating plasma
may cause minimal harm to the donor and that there is nothing to be worried about. Plasma donors must be
cured patients and discharged from hospital. Only plasma is used, whereas red blood cells (RBC), white blood
cells (WBC) and blood platelets are transfused back into the donor's body. Wang alleged that donor's plasma
will totally improve to its initial state after one or 2 weeks from the day of plasma donation of around 200 to
300 millilitres. 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. Some 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 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. 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-to moderate 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 (173, 174). Hence, knowledge and understanding of S protein-based vaccine development in SARS-
CoV will help to identify potential S protein vaccine candidates in SARS-CoV-2. Therefore, vaccine
strategies based on the whole S protein, S protein subunits, or specific potential epitopes of S protein appear to
be the most promising vaccine candidates against coronaviruses. The RBD of the 51 subunit of S protein has
a superior capacity to induce neutralizing antibodies. This property of the RBD can be utilized for
designing potential SARS CoV vaccines either by using RBD-containing recombinant proteins or
recombinant vectors that encode RBD (175). Hence, the superior genetic similarity existing between SARS-
CoV-2 and SARS CoV can be utilized to repurpose vaccines that have proven in vitro efficacy against SARS-
CoV to be utilized for SARS-CoV-2. The possibility of cross-protection in COVID-19 was evaluated
by comparing the S protein sequences of SARS-CoV-2 with that of SARS CoV. The comparative analysis
confirmed that the variable residues were found concentrated on the 51 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
including 1L2, 1L7, 1L10, (CSF, 1P10, MCP1, MIP1A, and TNF a [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 2530% 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 wk. The median duration of hospital stay 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 system (30). Bovine
coronaviruses (Bo CoVs) are known to infect several domestic and wild ruminants (126). Bo CoV inflicts
neonatal calf diarrhea in adult cattle, leading to bloody diarrhea (winter dysentery) and respiratory disease
complex (shipping fever) in cattle of all age groups (126). Bo CoV-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 Beta corona virus, affecting the enteric and respiratory tract,
respectively (129, 130). IBV, under Gamma corona virus, causes diseases of respiratory, urinary, and
reproductive systems, with substantial economic losses in chickens (131, 132). In small laboratory
animals, mouse hepatitis virus, rat sialoda cryoadenitis coronavirus, and guinea pig and rabbit
coronaviruses are the major CoVs associated with disease manifestations like enteritis, hepatitis, and
respiratory infections (10, 133). Swine acute diarrhea syndrome coronavirus this emerging virus will
establish a niche in humans and coexist with us for a long time'. 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
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]. As per 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
(Arp..) ac thn vrarcaltrIr thrniirrh which 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 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
only a matter of time before another zoonotic coronavirus results in an epidemic by jumping the so-called
species barrier (287). The host spectrum of coronavirus increased when a novel coronavirus, namely, SW1,
was recognized in the liver tissue of a captive beluga whale (Delphinapterus leucas) (138). In recent
decades, several novel coronaviruses were identified from different animal species. Bats can harbor
these viruses without manifesting any clinical disease but are persistently infected (30). They are the only
mammals with the capacity for self-powered flight, which enables them to migrate long distances, unlike land
mammals. Bats are distributed worldwide and also account for about a fifth of all mammalian species (6).
This makes them the ideal reservoir host for many viral agents and also the source of novel coronaviruses that
have yet to be identified. It has become a necessity to study the diversity of coronavirus in the bat
population to prevent future outbreaks that could jeopardize livestock and public health. The repeated outbreaks
caused by bat-origin coronaviruses calls for the development of efficient molecular surveillance strategies for
studying Beta corona virus among animals (12), especially in the Rhinolophus bat family (86). Chinese bats
have high commercial value, since they are used in
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 hydroxy chloroquine
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 48
h 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 pharmacokinetic 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
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 COVID19-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). 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 neutrophils (percent)
give an idea about the disease severity in COVID-19 infection (121). During COVID-19, patients
may present leukocytosis, l e u k o p e n i a w i t h l y m p h o p e n i a ( 2 4 4 ) , hypoalbuminemia, and an
increase of lactate dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and,
especially, D-dimer 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 nr those InTith confirmed or even die, whereas most young people
and children have only mild diseases (non-pneumonia or mild pneumonia) or are asymptomatic'''''.
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 case"'". On infection, the most common symptoms are fever, fatigue and dry cough' '"°'8" Less
common symptoms include sputum production, headache, haemoptysis, diarrhoea, anorexia, sore throat, chest
pain, chills and nausea and vomiting in studies of patients in China''""'8"'81. Self-reported olfactory and taste
disorders were also reported by patients in Italy''. Most people showed signs of diseases after an incubation
period of 1-14 days (most commonly around 5 days), and dyspnoea and pneumonia developed within a
median time of 8 days from illness onset'.
In a report of 72,314 cases in China, 81% of the cases were classified as mild, 14% were severe cases that
required ventilation in an intensive care unit (ICU) and a 5% were critical (that is, the patients had
respiratory failure, septic shock and/or multiple organ dysfunction or failure)`''". On admission, ground-glass
opacity was the most common radiologic finding on chest computed tomography (CT)' 3'60'80'81. Most patients also
developed marked lymphopenia, similar to what was observed in patients with SARS and MERS, and non-
survivors developed severer lymphopenia over time 13'60'"1. Compared with non-ICU patients, ICU patients had
higher levels 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 in Yunnan. This novel bat virus, denoted `RmYNO2', 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 (REF"). In addition to RaTG13 and RmYNO2, phyloge netic 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 Sarbecovirus3" (FIG. 2). The discovery of diverse bat
coronaviruses closely related to SARS-CoV-2 suggests that bats are possible reservoirs of SARS-CoV-2
(REF.''). 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. 8). Beyond bats, pangolins are another wildlife host probablylinked with SARS-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 sublineages 39-4'. The Guangdong strains,
which were isolated or sequenced by different research groups from smuggled pangolins, have 99.8%
sequence identity with each other'. 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 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 recovered patients and used for plasma transfusion twice in a
volume of 200 to 250 ml 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 Si 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 these therapeutic approaches have revealed
OriginandSpreadofCOVID-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 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 with COVID-19 showed typical features on initial CT, including bilateral multilobar
ground-glass opacities with a peripheral or posterior distribution" 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 screening'''. 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 studies"6°'20°'21. 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 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
epidemic progresses, commercial tests will become available. Other laboratory investigations are usually non
specific. 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 and chest discomfort, and in severe cases
dyspnea and bilateral lung infiltration'". Among the first 27 documented hospitalized patients, most cases were
epidemiologically linked to Huanan Seafood Wholesale Market. a wet market located in downtown Wuhan,
which sell not only seafood but also live animals, including poultry and wildlife4'8. 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 outbreak of unidentified cause and
informed the World Health Organization (WHO) 9 (FIG. 1). By metagenomic RNA sequencing and virus
isolation 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 beta corona virus
that had never been seen before'''". 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 tc Huanan Seafood Wholesale Market were identified. Several familial clusters of
infection were reported. and nosocomial infection also occurred in health-cam facilities. All these cases
provided clear evidence for human-to-human transmission of the new virus -1'12-14. 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 coronavirus pneumonia soon spread to other cities in Hubei province and to other parts of
China. Within 1 month. such instance was in 2002-2003 when a new coronavirus of the 13 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 andSpread ofCOVID-19 [1, 2, 6]
In December 2019, adults in Wuhan, capital city of Hubei province and a [median 17 d]. In the case series
of children discussed earlier, all 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
Chinese guidelines [21]. 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 lower
respiratory tracts. Acute viral interstitial pneumonia and humoral and cellular immune responses were
observed'''. Moreover, prolonged virus shedding peaked early in the course of infection in asymptomatic
macaques`'`', and old monkeys showed severer interstitial pneumonia than young monkeys', which is
similar to what is seen in patients with COVID-19. In human ACE2-transgenic mice infected with SARS-
CoV-2, 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 infection'''. In wide-type mice, a SARS-CoV-2 mouse-adapted strain with the N501Y alteration
in the RBD of the S protein was generated at passage 6. Interstitial pneumonia and inflammatory responses
were found in both young and aged mice after infection with the mouse-adapted strain'. Golden hamsters
also showed typical symptoms after being infected with SARS-CoV-2 (REF.77). In other animal models,
including cats and ferrets, SARS-CoV-2 could efficiently replicate in the upper respiratory tract but did not
induce severe clinical symptoms'''. 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-'). Animal models offer important information for understanding the pathogenesis of SARS-
CoV-2 infection and the transmission dynamics of SARSCoV-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 years. 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
article gives a pira'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 0C43 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 beta corona viruses to humans has resulted in severe disease. The
first such instance was in 2002-2003 when a (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 wks 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
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 is also 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 SARSCoV-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).
mask and practice cough hygiene. Caregivers should be asked to wear a surgical mask when in the same
room as patient and use hand hygiene every 15-20 min. The greatest risk in COVID-19 is transmission to
healthcare workers. In the SARS outbreak of 2002, 21% of those affected were healthcare workers [31].
Till date, almost 1500 healthcare workers in China have been infected with 6 deaths. The doctor who
first warned about the virus has died too. It is important to protect healthcare workers to ensure
continuity of care and to prevent transmission of infection to other patients. While COVID-19
transmits as a droplet pathogen and is placed in Category B of infectious agents (highly pathogenic H5N1
and SARS), by the China National Health Commission, infection control measures recommended are those
for 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 hydroxy chloroquine
azithromycin in the treatment of COVID19 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 48 h 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 pharmacokinetic 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
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 COVID19-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 postbirth; 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).
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 neutrophils (percent) give an idea about the disease
severity in COVID-19 infection (121). During COVID-19, patients may present leukocytosis,
l e u k o p e n i a w i t h l y m p h o p e n i a ( 2 4 4 ) , hypoalbuminemia, and an increase of lactate
dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and, especially, D-dimer 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 nr those
In Tith confirmed or even die, whereas most young people and children have only mild diseases (non-
pneumonia or mild pneumonia) or are asymptomatic. 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 case. On infection, the most common symptoms are
fever, fatigue and dry cough' '"°'8" Less common symptoms include sputum production, headache,
haemoptysis, diarrhoea, anorexia, sore throat, chest pain, chills and nausea and vomiting in studies of patients
in China. Self-reported olfactory and taste disorders were also reported by patients in Italy''. Most people
showed signs of diseases after an incubation period of 1-14 days (most commonly around 5 days), and
dyspnoea and pneumonia developed within a median time of 8 days from illness onset'.
In a report of 72,314 cases in China, 81% of the cases were classified as mild, 14% were severe cases that
required ventilation in an intensive care unit (ICU) and a 5% were critical (that is, the patients had
respiratory failure, septic shock and/or multiple organ dysfunction or failure). On admission, ground-glass
opacity was the most common radiologic finding on chest computed tomography (CT). Most patients also
developed marked lymphopenia, similar to what was observed in patients with SARS and MERS, and non-
survivors developed severer lymphopenia over time. Compared with non-ICU patients, ICU patients had
higher levels Based on molecule characterization, SARS- CoV-2 is considered a new Beta corona
virus belonging to the subgenus Sarbecovirus (3). A few other critical zoonotic viruses (MERS-
related CoV and SARS-related CoV) belong to the same genus. However, SARS-CoV-2 was
identified as a distinct virus based on the percent identity with other Beta corona virus; conserved
open reading frame 1a/b (ORF 1 alb) 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 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 diverges The replicase gene
covers two thirds of the 5' genome, and encodes a large polyprotein (pplab),which is proteolytically cleaved
into16 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-CoT'.
The phylogenetic analysis for the whole genome shows that SARS-CoV-2 is clustered with SARS
CoV and SARS-related coronaviruses (SAR Sr 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 ZXC2 ) as
well as novel coronaviruses recently identified in pangolins, which group parallel to SARS CoV areas. For
example, a cohort study in London reveo 44% of the frontline health-care workers from a hosp
were infected with SARS-CoV-2 (REEL' I).The high transmissibility of BARS-CoV-2 may be
attributed to the unique virological features of BARS-CoV-2. Transmission of .SAILS- CoV
occurred mainly after illness onset and peaked following disease severity'. However, the BARS-
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. 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 perio.
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 else9'-'°°.
Airborne transmission was also observed in the ferret experiments mentioned above. SARS-CoV-2-
infected ferrets shed
5 PATHOGENESIS
Coronaviruses are tremendously precise and mature in most of the airway epithelial cells as
observed through both in vivo and in vitro wrought havoc in China and caused a
pandemic situation in the worldwide population, leading to disease outbreaks that have not
been controlled to date, although extensive efforts are being put in place to counter this virus
(25). This virus has been proposed to be designated/named severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV),
which determined the virus belongs to the Severeacute respiratory syndrome-related coronavirus
category and found this virus is related to SARS-CoVs (26). SARS-CoV-2 is a member of the
order Nidovirales family Coronaviridae, subfamily Orthocoronavirinae, which is subdivided
Alphacoronavirus and Beta corona virus originate from bats while Gamma corona virus and Delta
corona virus have evolved from bird and swine gene pools (24, 28, 29, 275)Corona viruses possess
an unsegmented, single stranded, positive-sense RNA genome of around 30 kb, enclosed by a 5'-cap
and 3'-poly(A) tail (30). The genome of SARS-CoV-2 is 29,891 by long, with a GEC content of
38% (31). These viruses are encircled with an envelope containing viral 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-1 9
during pregnancy.40-42 However, pregnant
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.41-43
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 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
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 mini mise the risk of exposure to COVID-19 during outbreaks.53 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 might be lower. Further genetic analysis is required between SARS-CoV-2
and different strains of SARS-CoV and SARS-like (SL} CoVs to evaluate the
possibility of repurposed vaccines against COVID-19. This strategy will be helpful
in the scenario of an outbreak, since much time can be saved, because preliminary
evaluation, including in vitro studies, already would be completed for such vaccine
candidates. Multiepitope subunit vaccines can be considered a promising preventive
strategy against the ongoing COVID-19 pandemic. In silico and advanced
immunoinformatic tools can be used to develop multiepitope subunit vaccines. The
vaccines that are engineered by this technique can be further evaluated using docking
studies and, if found effective, then can be further evaluated in animal models (365).
Identifying epitopes that have the potential to become a vaccine candidate is critical to
developing an effective vaccine against COVID-19. The immunoin form atics
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,
turtles, 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 differs from that in SARS-CoV in the
five residues critical for ACE2 binding, namely Y45511486FN493Q D494S and T501N.
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 (REE'''). Biochemical data
confirmed that the structural features of the SARS-CoV-2 RBD has strengthened its
hACE2 binding affinity compared with that of SARS-CoV5°-52. 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 furin47'5455. 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
pseudo virus entry assays revealed that TMPRSS2 and cathepsin L have cumulative
effects with fur in on activating virus entry' 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 con
formational states5° A lying-down conformation of the SARS-CoV-2 S protein may not be
in fav our of receptor binding but is helpful for immune evasion". 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 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 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 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 out
break 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 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 COV1D-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, JAI(
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
Animal 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-T mice
trans • enic for ex ressin
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. Hand 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, hand hygiene is
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

with SARS and MERS (117).

SARS-CoV-2 invades the lung parenchyma, resulting in severe interstitial
inflammation of the lungs. This is evident on computed tomography (CT) images as
ground-glass opacity in the lungs. This lesion initially involves a single lobe but
later
expands to multiple lung lobes (118). The
histological assessment of lung biopsy samples obtained from COVID-19-infected
patients revealed diffuse alveolar damage, cellular fibromyxoid exudates,
hyaline membrane formation, and desquamation of pneumocytes, indicative of
acute respiratory distress syndrome (119). It was also found that the SARS-CoV-2-
infected patients often have lymphocytopenia with or without leukocyte
abnormalities. The degree of lymphocytopenia gives an idea about disease prognosis, as
it is found to be positively correlated with disease severity (118). Pregnant women
are considered to have a higher risk of getting infected by COVID-19. The coronaviruses
can cause adverse outcomes for the fetus, such as intrauterine growth restriction,
spontaneous abortion, preterm delivery, and perinatal death.
Nevertheless, the possibility of intrauterine maternal-fetal transmission
(vertical transmission) of CoVs is low and was not seen during either the SARS-
or MERS-CoV outbreak (120). However,
I Ile comprenensive seciLo.,111,‘, a11a1y313 ul LAIA, 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
helicase activity.
Among the evaluated compounds, 4-(cyclopent-
1 -en-3 -ylamino)-5- [244-
iodophenyl)hydrazinyl] -4H-1,2,4-triazole-3 -thiol and 4-(cyclopent-1 -en-3 -ylamino)-5 -
[244-
chlorophenyphydrazinyl] -thiol
 were found to be the most potent. These compounds were used for in silico
studies, and molecular docking was accomplished into the active binding site of
MERS-CoV helicase nsp13 (21). Further studies are required for evaluating the
therapeutic potential of these newly identified compounds in the management of
COVID-19 infection.

Passive Immunization/Antibody Therapy/MAb

Monoclonal antibodies (MAbs) may be helpful in the intervention of disease
in CoV-exposed individuals. Patients recovering from SARS showed robust
neutralizing antibodies against this CoV infection (164). A set of MAbs aimed at
the MERS-
CoV S protein-specific domains, comprising six specific epitope groups
interacting with receptor-
binding, membrane fusion, and sialic acid-binding sites, make up crucial entry tasks
of S protein (198, 199). Passive immunization employing weaker and
strongly neutralizing antibodies provided
considerable protection in mice against a MERS-
as an entry receptor while exhibiting an KED similar
to that of SARS-CoV (17, 87, 254, 255). Several countries
have provided recommendations to their people traveling to China (88, 89).
Compared to the previous coronavirus outbreaks caused by SARS-
CoV and VIERS-CoV, the efficiency of SARS-CoV-
2 human-to-human transmission was thought to be less. This assumption was based
on the finding that health workers were affected less than they were in previous
outbreaks of fatal coronaviruses (2). Superspreading events are considered the
main culprit for the extensive transmission of SARS and MERS (90, 91). Almost
half of the VIERS-CoV cases reported in Saudi Arabia are of secondary origin
that occurred through contact with infected asymptomatic or symptomatic
individuals through human-to-human transmission (92). The occurrence of
superspreading events in the COVID-19 outbreak cannot be ruled out until its
possibility is evaluated. Like SARS and MERS, COVID-19 can also infect the lower
respiratory tract, with milder symptoms
(27). The basic reproduction number of COVID-19 has been found to be in the range
of 2.8 to 3.3 based on real-time reports and 3.2 to 3.9 based on predicted infected cases
(84).
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 almost
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
assessed intrauterine vertical transmission of
 COVID-19 infection in nine infants born to
infected mothers, found that none of the
infants tested positive for the virus.45 Likewise, there was no evidence
of intrauterine infection caused by vertical transmission in the SARS
and MERS epidemics.43

The CDC asserts that infants born to mothers

with confirmed COVID-19 are considered
persons under investigation (PUI) and should be temporarily separated
from the mother and
isolated.46

7.1 Breastfeeding and infant care

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 millions 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

We assessed the nucleotide percent similarity using the MegAlign

software program, where the similarity between the novel SARS-CoV-2 isolates
was in the range of 99.4% to 100%. Among the other Serbecovirus CoV sequences,
the novel SARS-CoV-
2 sequences revealed the highest similarity to bat-
SL-CoV, with nucleotide percent identity ranges
between 88.12 and 89.65%. Meanwhile, earlier
reported SARS-CoVs showed 70.6 to 74.9%
similarity to SARS-CoV-2 at the nucleotide level. Further, the nucleotide
percent similarity was 55.4%,
45.5% to 47.9%, 46.2% to 46.6%, and 45.0% to
46.3% to the other four subgenera, namely, Hibecovirus, Nobecovirus,
Merbecovirus, and Embecovirus, respectively. The percent similarity index of
current outbreak isolates indicates a close relationship between SARS-CoV-2
isolates and bat-
SL-CoV, indicating a common origin. However, particular pieces of
evidence based on further complete genomic analysis of current isolates are
necessary to draw any conclusions, although it was ascertained that the current
novel SARS-CoV-2 isolates belong to the subgenus Sarbecovirtis in the diverse
range of betacoronaviruses. Their possible ancestor was hypothesized to be
from bat CoV strains, wherein bats might have played a crucial role in harboring
this class of viruses.

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 palm 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 transmission: 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-adapted virus, during their subsequent
exposure to humans at live markets, promoted further adaptations that resulted in
the epidemic strain (104). Transmission can also
aminotransterase, bilirubin, and, especially, D-ciimer
(244). Middle-aged and elderly patients with primary chronic diseases, especially high
blood pressure and diabetes, were found to be more susceptible to respiratory
failure and, therefore, had poorer prognoses. Providing respiratory support at
early stages improved the disease prognosis and facilitated recovery (18). The ARDS in
COVID-19 is due to the occurrence of cytokine storms that results in
exaggerated immune response, immune regulatory network imbalance, and,
finally, multiple-organ failure (122). In addition to the exaggerated
inflammatory response seen in patients with COVID-19 pneumonia, the bile
duct epithelial cell-
derived hepatocytes upregulate ACE2 expression in liver tissue by compensatory
proliferation that might result in hepatic tissue injury (123).

CORONAVIRUSES IN ANIMALS AND

ZOONOTIC LINKS A BRIEF
VIEWPOINT
Coronavirus can cause disease in several species of domestic and wild animals,
as well as humans
(23). The different animal species that are infected with CoV include horses, camels,
cattle, swine, dogs, cats, rodents, birds, ferrets, minks, bats, rabbits, snakes, and
various other wild animals (20, 30, 79,

(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 comparison 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, which 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

involved in the COVID-19 outbreak is of great importance, because the strain on

their mental well-
being will affect their attention, concentration, and decision-making capacity. Hence,
for control of the COVID-19 outbreak, rapid steps should be taken to protect the mental
health of medical workers (229).
Since the living mammals sold in the wet market are suspected to be the
intermediate host of SARS-
CoV-2, there is a need for strengthening the regulatory mechanism for wild
animal trade (13). The total number of COVID-19 confirmed cases is on a
continuous rise and the cure rate is relatively low, making disease control very
difficult to achieve. The Chinese government is making continuous efforts to
contain the disease by taking emergency control and prevention measures. They have
already built a hospital for patients affected by this virus and
are currently building several more for
accommodating the continuously increasing infected population (230). The effective
control of SARS-
CoV-2/COVID-19 requires high-level interventions like intensive contact tracing,
as well as the quarantine of people with suspected infection and the isolation of
infected individuals. The implementation of rigorous control and preventive measures
together might control the R0 number and reduce the transmission risk (228).
Considering the zoonotic

route warrants the introduction of negative fecal viral nucleic acid test results as
one of the additional discharge criteria in laboratory-confirmed cases of
COVID-19 (326).
The COVID-19 pandemic does not have any novel factors, other than
the genetically unique pathogen and a further possible reservoir. The cause and
the likely future outcome are just repetitions of our previous interactions with
fatal coronaviruses. The only difference is the time of occurrence and the genetic
distinctness of the pathogen involved. Mutations on the RBD of CoVs
facilitated their capability of infecting newer hosts, thereby expanding
their reach to all corners of the world
(85). This is a potential threat to the health of both animals and humans.
Advanced studies using Bayesian phylogeographic reconstruction identified
the most probable origin of SARS-CoV-2 as the bat
SARS-like coronavirus, circulating in the
Rhinolophtts bat family (86).
Phylogenetic analysis of 10 whole-genome
sequences of SARS-CoV-2 showed that they are related to two CoVs of bat
origin, namely, bat-SL-
CoVZC4S and bat-SL-CoVZXC21, which were reported during 2018 in
China (17). It was reported that SARS-CoV-2 had been confirmed to use ACE2
as an entry receptor while exhibiting an RBD similar
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-1 9 patients. Further, the
outcome is reinforced by azithromycin. The role of lopinavir and
ritonavir in the treatment of COVID-1 9 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
hospitalisation. Interim
recommendations on home management of patients with COVID-19
can be found on

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

14 ANTIVIRAL THERAP

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 (MERS-
CoV), 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-1 9.
There has been a proposal to use the combination of protease inhibitors lopinavir-
 ritonavir for
treating the patients affected by COVID-19.62
infected by human beings. However, evidence of cat-
to-human transmission is lacking and requires further studies (332). Rather
than waiting for firmer
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 studying disease
transmission (337). Experimental inoculation was also done in other animal
species and found that the dogs have low susceptibility, while the chickens,
performance (Table 2) (80, 245, 246). The viral loads of SARS-CoV-2 were measured
using N-gene-
specific quantitative RT-PCR in throat swab and sputum samples collected from
COVID-19-infected individuals. The results indicated that the viral load peaked at
around 5 to 6 days following the onset of symptoms, and it ranged from 104 to 107
copies/ml during this time (151). In another study, the viral load was found to be
higher in the nasal swabs than the throat swabs obtained from COVID-19
symptomatic patients (82). Although initially it was thought that viral load would be
associated with poor outcomes, some case reports have shown asymptomatic
individuals with high viral loads (247). Recently, the viral load in nasal and
throat swabs of 17 symptomatic patients was determined, and higher viral loads were
recorded soon after the onset of symptoms, particularly in the nose compared
to the throat. The pattern of viral nucleic acid shedding of SARS-CoV-2-infected
patients was similar to that of influenza patients but seemed to be different from that of
SARS-CoV patients. The viral load detected in asymptomatic patients resembled that
of symptomatic patients as studied in China, which reflects the transmission
perspective of asymptomatic or symptomatic patients having minimum signs
and symptoms (82). Another study,

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 W. Till 05/03/2020 around 96,000 cases of
 coronavirus disease 2019
(COVID-19) 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 eve view
about

vaccine, and Ii-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 Ii-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.

10 RECOMBINANT SUBUNIT
VACCINE

Clover Biopharmaceuticals is producing a recombinant subunit

vaccine based on the trimeric S-protein of COVID-1 9.55 The oral
recombinant vaccine is being expanded by Vaxart in tablet
formulation, using its
proprietary oral vaccine platform.

11 CLINICAL MANAGEMENT
AND TREATMENT

In severe COVID-19 cases, treatment should be given to support vital

organ functions. People who think they may have been exposed to
COVID-19 should contact their healthcare
provider immediately. Healthcare personnel should care for patients in
an Airborne Infection Isolation Room (AIIR). Precautions must be
taken by the healthcare professional, such as contact precautions and
airborne precautions with eye protection.56

Individuals with a mild clinical presentation may not require primary

hospitalisation. Close
monitoring is needed for the persons infected with COVID-19. Elderly
patients and those with prevailing chronic medical conditions such as
(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 Ii-Key peptide
COVID-19 vaccine are
The results of the studies related to SARS-CoV-2 viral loads reflect active replication
of this virus in the upper respiratory tract and prolonged viral shedding after
symptoms disappear, including via stool. Thus, the current case definition needs to be
updated along with a reassessment of the strategies to be adopted for restraining the
SARS-CoV-2 outbreak spread (248). In some cases, the viral load studies of SARS-CoV-
2 have also been useful to recommend precautionary measures when handling specific
samples, e.g., feces. In a recent survey from
17 confirmed cases of SARS-CoV-2 infection with available data (representing days 0 to
13 after onset), stool samples from nine cases (53%; days 0 to 11 after onset) were
positive on RT-PCR analysis. Although the viral loads were lower than those of
respiratory samples (range, 550 copies per ml to
1.21 x 105 copies per ml), this has essential biosafety implications (151).
The samples from 18 SARS-CoV-2-positive patients in
Singapore who had traveled from Wuhan to Singapore showed the presence of viral RNA
in stool and whole blood but not in urine by real-time
RT-PCR (288). Further, novel SARS-CoV-2
infections have been detected in a variety of clinical specimens, like bronchoalveolar
lavage fluid,
wearing a facemask and practising hand
hygiene before feeding the baby. In addition, it is advisable that breast pumps
are cleaned properly after each use and, if possible, a
healthy individual is available to feed the
expressed breast milk to the infant,42

7.2 Children and elderly

population

On the basis of the available reports, COVID-19 among children accounted for 1-
5% of the
confirmed cases, and this population does not seem to be at higher risk for the
disease than adults. There is no difference in the COVID-19 symptoms between
adults and children.
However, the available evidence indicated that children diagnosed with COVID-
19 have milder symptoms than the adults, with a low mortality rate.", 49 On the
contrary, older people who are above the age of 65 years are at higher risk for a
severe course of disease. In the United Stated, approximately 31-59% of those
with confirmed COVID-19 between the ages of 65 and 84 years old required
hospitalisation, 11-31% of them required admission to the intensive care unit, and
4-11% died.5°
assays offer high accuracy in the diagnosis of SARS-
CoV-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 IgG 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
6.3 Serological testing

Serological surveys are also considered to be one of the most effective ones
in facilitating
outbreak investigation and it also helps us to derive a retrospective
assessment of the
disease by estimating the attack rate.32
According to the recent literature, paired serum samples can also help
clinicians to diagnose
COVID-19 in case of false negative results in
NAAT essays.37 The literature also declared that the commercial and non-
commercial serological tests are under consideration in order to
support the practising clinicians by assisting them in diagnosis. Similarly,
there are studies published on COVID-19 which are comprised of the
serological data on clinical samples.38, 39
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. In 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

In 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
In 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
216 countries and regions from all six continents had reported more than 20
million cases of COVID-19, and more than 733,000 patients had died'. High
mortality occurred especially when health-care resources were
overwhelmed. The USA is the country with the largest number of cases so far.
Although genetic evidence suggests that SARS-CoV-2 is a natural virus that
likely originated in animals, there is no conclusion yet about when and where
the virus first entered humans. As some of the first reported cases in
Wuhan had no epidemiological link to the seafood market--, it has been
suggested that the market may not be the initial source of human infection with
SARS-CoV-2. One study from France detected SARS-CoV-2 by PCR in a
stored sample from a patient who had pneumonia at the end of 2019,
suggesting SARS-CoV-2 might have spread there much earlier than the
generally known starting time of the outbreak in France- However, this
individual early report cannot give a solid answer to the origin of SARS-CoV-
2 and contamination, and thus a false positive result cannot be excluded. To
address this highly controversial issue, further retrospective inves-
tigations involving a larger number of banked samples from patients,
animals and environments need to be conducted worldwide with well-
validated assays.

Genomics, phylogeny and taxonomy

As a novel betacoronavirus, SARS-CoV-2 shares 79% genome
sequence identity with SARS-CoV and 50% with MERS-CoV-1. Its
genome organization is shared with other betacoronaviruses. The six
functional open reading frames (ORFs) are arranged in order from 5' to 3':
replicase (ORF1a/ORF1b), spike (S), envelope
(E), membrane (M) and nucleocapsid (N). In addition, seven putative ORFs
encoding accessory proteins are interspersed between the structural
genes25. Most of the proteins encoded by SARS-CoV-2 have a similar
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 animals (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
human beings. However, evidence of cat-

cat and camels, respectively, act as amplifier hosts (40, 41).

Coronavirus genomes and subgenomes encode six ORFs (31). The majority
of the 5' end is occupied by ORF1a/b, which produces 16 nsps. The two
polyproteins, pp la and pp lab, are initially produced from ORF1a/b by a —1
frameshift between ORF1 a and ORF lb (32). The virus-encoded proteases cleave
polyproteins into individual lisps (main protease
[Mpro], chymotrypsin-like protease [3CLpro], and
papain-like proteases [PLPs]) (42). SARS-CoV-2
also encodes these nsps, and their functions have been elucidated recently
(31). Remarkably, a difference between SARS-CoV-2 and other CoVs is the
identification of a novel short putative protein within the ORF3 band, a
secreted protein with an alpha helix and beta-sheet with six strands encoded by
ORF8 (31).
Coronaviruses encode four major structural proteins, namely, spike (S),
membrane (M), envelope
(E), and nucleocapsid (N), which are described in detail below.

S Glycoprotein
Coronavirus S protein is a large, multifunctional class I viral transmembrane
protein. The size of this

responsible for MFRS-CoV and SARS-CoV (3). The

newly emerged SARS-CoV-2 is a group 2B
coronavirus (2). The genome sequences of SARS-
CoV-2 obtained from patients share 79.5% sequence
similarity to the sequence of SARS-CoV (63).
As of 13 May 2020, a total of 4,170,424
confirmed cases of COVID-19 (with 287,399 deaths)
have been reported in more than 210 affected
countries worldwide (WHO Situation Report 114
fatigue. individuals with asymptomatic and atypical 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
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, SARS-
CoV-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 MFRS-CoV (204). Passive
immunotherapy using convalescent plasma is another strategy that can be
used for treating COVID-19-infected, critically ill patients (205).
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 alveo-
lar 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 res-
piratory distress syndrome and respiratory failure, which is considered the
main cause of death in patients with COVID-19 (REFS'"'). Patients of older
age (>60 years) and with serious pre-existing diseases have a greater risk of
developing acute respiratory distress syndrome and
death"2- 7
4 (FIG. 4). Multiple organ failure has also been
 reported in some COVID-19 cases9'13'65.
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
lungs 13,G°,66,6'
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 A M -
transgenic or human A M-knock-in mice),
ferrets and golden hamsters43'48'68-74. In non-
human primate animal mod-
els, 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
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 ORF lb 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-