Introduction:

Over the preceding two decades, coronaviruses (CoVs) have been linked to substantial disease
outbreaks in East Asia and the Middle East. The emergence of severe acute respiratory syndrome
(SARS) occurred in 2002 and 2012, with Middle East Respiratory Syndrome (MERS). Recently,
a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), has
given rise to the coronavirus disease 2019 (COVID-19), presenting a global health threat and
instigating an ongoing pandemic across numerous countries and territories. Health professionals
worldwide are actively engaged in concerted efforts to mitigate and control the further spread of
the disease caused by this novel CoV, first identified in Wuhan City, Hubei Province, China, on
December 12, 2019.
On February 11, 2020, the World Health Organization (WHO) officially designated the current
CoV-associated disease as Covid-19, attributable to SARS-CoV-2. The primary cluster of
patients was found to be associated with the Huanan South China Seafood Market in Wuhan (2).
Coronaviruses (CoVs), belonging to the family Coronaviridae (subfamily Coronavirinae), are
known to infect a broad range of hosts.
Recently, structural analyses of the Spike (S) proteins of the COVID-19 virus have identified 27
amino acid substitutions within a 1,273-amino-acid stretch (16). Specifically, six substitutions
were identified in the Receptor-Binding Domain (RBD) (amino acids 357 to 528), and four
substitutions occurred in the Receptor-Binding Motif (RBM) at the Carboxy-Terminal Domain
(CTD) of the S1 domain (16). It is noteworthy that no amino acid changes were observed in the
RBM, crucial for binding directly to the angiotensin-converting enzyme-2 (ACE2) receptor, as
seen in the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) (16, 46). The focus
now lies on understanding how many genetic differences are necessary to alter the host tropism.
Sequence comparison revealed 17 nonsynonymous changes between the early SARS-CoV-2
sequence and later isolates of SARS-CoV. These changes were distributed across the viral
genome, encompassing ORFlab, ORF8, the spike gene, and ORF7a (4).
Significantly, identical nonsynonymous changes were observed in a familial cluster, indicating
viral evolution during person-to-person transmission (4, 47). Such adaptive evolution events are
frequent and represent an ongoing process once the virus spreads among new hosts (47). While
these evolutionary events may not induce functional changes in the virus, vigilant monitoring of
viral traits is essential. The absence of specific proteins can be associated with altered virulence
in coronaviruses, affecting morphology and tropism (54). For instance, the Envelope (E) protein,
consisting of three domains—a short hydrophilic amino terminal, a large hydrophobic
transmembrane domain, and an efficient C-terminal domain—plays a crucial role. The SARS-
CoV-2 E protein displays a similar amino acid composition without any substitutions (16).
To emphasize on the Nucleocapsid (N) protein, it serves multiple functions, including complex
formation with the viral genome, facilitation of M protein interaction during virion assembly,
and enhancement of virus transcription efficiency (55, 56). Comprising three highly conserved
domains—an N-terminal domain (NTD), an RNA-binding domain or linker region (LKR), and a
C-terminal domain (CTD)—the N protein plays a pivotal role in viral replication. The NTD
binds with the 3' end of the viral genome, potentially through electrostatic interactions, and
exhibits
significant diversity in both length and sequence (58). The charged LKR, also known as the serine
and arginine-rich (SR) domain, is involved in direct interaction with RNA and modulates host
antiviral responses, acting as an antagonist for interferon (60, 61).

The SARS-CoV-2 genome consists of 15 nsps (nspl to nsn10 and nsp12 to nsp16) and 8
accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and ORF14), in addition to important structural
proteins. Each of these proteins has a specific role in viral replication. Unlike SARS-CoV,
SARS-CoV-2 does not contain the 8a protein, and its 8b protein is longer while the 3b protein is
shorter. The nsp7, nspl3, envelope, matrix, p6, and 8b accessory proteins have shown no amino
acid substitutions compared to other coronaviruses.
The virus structure of SARS-CoV-2 is depicted in Fig. 2.

The SARS-CoV-2 pandemic initially surfaced in China and was associated with a significant
number of fatalities. As of May 13, 2020, China reported 84,458 laboratory-confirmed cases and
4,644 deaths (Fig. 4). Presently, SARS-CoV-2 confirmed cases have been reported in more than
210 countries except for Antarctica (Fig. 3 and 4) (WHO Situation Report 114) (25, 64).
COVID- 19 has been reported on all continents except Antarctica. Italy was the epicenter of
concerns for many weeks due to the high number of cases, with 221,216 cases and 30,911
deaths, but currently, the United States has the largest number of cases, 1,322,054, and 79,634
deaths. The United Kingdom has even more cases (226,471) and deaths (32,692) than Italy. A
Johns Hopkins University web platform offers daily updates on the basic epidemiology of the
COVID-19 outbreak.
It has been confirmed that COVID-19 has been detected on numerous cruise ships globally,
including the Diamond Princess, which was quarantined in the Port of Yokohama in Japan. A
total of 239 cruise ships have reported cases of the virus (as shown in Fig. 3). A timeline
outlining significant events relating to the outbreak of the SARS-CoV-2/COVID-19 virus from
8th December 2019 to 13 May 2020 is presented in Fig. 5.
China initially bore the brunt of the COVID-19 outbreak in terms of disease morbidity and
mortality. However, the virus eventually spread to Europe, particularly Italy and Spain, and now
the United States has the highest number of confirmed cases.
Another study found that the average reproductive number of COVID-19 is 3.28, significantly
higher than the initial WHO estimate of 1.4 to 2.5. It is still too early to determine the exact R0
value due to insufficient data, but the higher R0 value suggests a greater potential for SARS-
CoV- 2 transmission in a susceptible population. This is not the first time that Chinese culinary
practices have been blamed for the origin of novel coronavirus infections in humans. Previously,
animals presented in live-animal markets were identified as intermediate hosts of the SARS
outbreak in China. Several wildlife species were found to harbor potentially evolving
coronavirus strains that can overcome the species barrier. One of the fundamental principles of
Chinese food culture is that live-slaughtered animals are considered more nutritious.
Following a four-month struggle spanning from December 2019 to March 2020, the state of
COVID-19 in China appears to be under control. Wet animal markets, previously implicated in
the outbreak's origin, have resumed operations. Consequently, there has been a resurgence in the
trade of various animals, including bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins
(scaly anteaters), minks, soup from palm civets, ostriches, hamsters, snapping turtles, ducks, fish,
Siamese crocodiles, and others. These animals serve as potential entry receptors, exhibiting a
Receptor-Binding Domain (RBD) similar to that of SARS-CoV (17, 87, 254, 255).
Numerous countries have issued recommendations to their citizens traveling to China (88, 89). In
contrast to previous coronavirus outbreaks caused by SARS-CoV and MERS-CoV, SARS-CoV-
2 was initially believed to demonstrate less efficiency in human-to-human transmission.
This
assumption was based on observations that health workers were less affected compared to
previous fatal coronavirus outbreaks (2).
Superspreading events, recognized as the primary driver of extensive transmission in SARS and
MERS, remain a potential factor in the COVID-19 outbreak (90, 91). Nearly half of the MERS-
CoV cases reported in Saudi Arabia were of secondary origin, resulting from contact with
asymptomatic or symptomatic infected individuals through human-to-human transmission (92).
The possibility of superspreading events contributing to the COVID-19 outbreak cannot be
dismissed without thorough evaluation.
Similar to SARS and MERS, COVID-19 exhibits the capability to infect the lower respiratory
tract, often presenting with milder symptoms (27). The basic reproduction number (R0) for
COVID-19 has been reported in the range of 2.8 to 3.3 based on real-time data and 3.2 to 3.9
based on predicted infected cases (84). This parameter is a crucial metric in assessing the
potential for further transmission within a population.
The COVID-19 pandemic is caused by a genetically unique pathogen that has the potential to
infect both animals and humans. It is not a novel factor except for the time of occurrence and the
genetic distinctness of the pathogen involved. Mutations on the Receptor Binding Domain
(RBD) of Coronaviruses (CoVs) have facilitated their ability to infect new hosts, expanding their
reach globally and posing a potential threat to the health of both animals and humans. Advanced
studies using Bayesian phylogeographic reconstruction have identified the bat SARS-like
coronavirus, circulating in the Rhinolophus bat family, as the most probable origin of SARS-
CoV-2.
Phylogenetic analysis of ten whole-genome sequences of SARS-CoV-2 reveals their relationship
to two CoVs of bat origin - bat-SL-CoVZC45 and bat-SL-CoVZXC21 - which were previously
identified in China in 2018. It has been confirmed that SARS-CoV-2 uses ACE2 as an entry
receptor and exhibits an RBD like fever, cough, and sputum. Therefore, clinicians must remain
vigilant and watch out for 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, indicating that it can be controlled despite moderate to high
transmissibility.
Increasing reports of SARS-CoV-2 in sewage and wastewater warrants the need for further
investigation due to the possibility of fecal-oral transmission. SARS-CoV-2 present in
environmental compartments such as soil and water will finally end up in the wastewater and
sewage sludge of treatment plants (328). Therefore, we have to reevaluate the current wastewater
and sewage sludge treatment procedures and introduce advanced techniques that are specific and
effective against SARS-CoV-2. Since there is active shedding of SARS-CoV-2 in the stool, the
prevalence of infections in a large population can be studied using wastewater-based
epidemiology. Recently, reverse transcription- quantitative PCR (RT-qPCR) was used to
enumerate the copies of SARS-CoV-2 RNA concentrated from wastewater collected from a
wastewater treatment plant (327). The calculated viral RNA copy numbers determine the number
of infected individuals.
The species barrier. As a result, the whole world is suffering from novel SARS-CoV-2, with
more than 4,170,424 cases and 287,399 deaths across the globe. There is an There has been an
increasing number of reports of SARS-CoV-2 being found in sewage and wastewater, which
suggests that there may be a possibility of fecal-oral transmission. This means that we need to
investigate further and reevaluate our current wastewater and sewage sludge treatment
procedures, as SARS-CoV-2 can end up in these treatment plants. We need to introduce new and
effective techniques that specifically target SARS-CoV-2. Moreover, since SARS-CoV-2 is
actively shed in stool, studying the prevalence of infections in a large population can be done
through wastewater-based epidemiology. This method involves using reverse transcription-
quantitative PCR (RT-qPCR) to enumerate the copies of SARS-CoV-2 RNA, which is
concentrated from wastewater collected from a wastewater treatment plant. The calculated viral
RNA copy numbers determine the number of infected individuals. The whole world is currently
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, which has resulted in the emergence of this virus. To encourage the sellers to
increase hygienic food practices or close the crude live-dead animal wet markets, food policies at
national and international levels need to be modified. This will help avoid further life-threatening
and economic consequences from any emerging or reemerging pandemic due to close animal-
human interaction. 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).
It is imperative to comprehend that COVID-19 can affect individuals of all ages and genders.
However, elderly individuals who have pre-existing chronic illnesses are more susceptible to
experiencing severe symptoms if infected. Recent studies have discovered that even individuals
with asymptomatic infections can spread the virus to other susceptible individuals. Notably,
asymptomatic patients can transmit viral loads similar to those of symptomatic patients,
indicating a high transmission capacity. As a result, SARS-CoV-2 transmission can occur early
in the course of the infection. Furthermore, COVID-19 can present atypically, whereby only
fatigue is reported, without the typical respiratory signs such as fever, cough, or sputum.
Medical professionals are consuming meat from animals, such as turtles, ducks, fish, and
Siamese crocodiles, without fearing the transmission of COVID-19. The Chinese government is
urging citizens to return to their normal activities, but this may pose a risk as advisories have
cautioned against contact with live or dead animals due to the potential zoonotic spillover of
SARS-CoV-2. Furthermore, there is a possibility of new mutations of the virus resulting from
contact between animals and humans at the market. In January 2020, China temporarily banned
the sale of live or dead animals in wet markets, yet hundreds of such markets have reopened
without adhering to standard food safety and sanitation practices.
Due to China's large population and its domestic and international food export policies, the entire
world is currently facing the threat of COVID-19, including China itself. Wet markets that sell
live and dead animals often do not adhere to strict food hygiene practices. Fresh blood can be
found on the floor and tabletops, and such food customs can encourage pathogens to adapt,
mutate, and cross the species barrier. Consequently, the whole world is now suffering from the
novel SARS- CoV-2 virus, with more than.
In comparison to other emerging viruses such as the Ebola virus, avian H7N9, SARS-CoV, and
MERS-CoV, the SARS-CoV-2 virus exhibits lower pathogenicity and moderate transmissibility.
The risk of mortality among individuals who contract COVID-19 was calculated using the
infection fatality risk (IFR), which was found to range between 0.3% to 0.6%. This IFR is
comparable to that of the previous Asian influenza pandemic that occurred between 1957 to
1958.
It has been proposed that the COVID-19 pandemic curve indicates a significant level of human-
to-human transmission, as opposed to animal-to-human transmission. The initial infection may
have originated from human-to-human transmission at the Wuhan seafood market. However, due
to the zoonotic spillover in COVID-19, it is too early to fully endorse this idea. Following the
initial infection, human-to-human transmission has been observed with a preliminary
reproduction number (Rq) estimate of 1.4 to 2.5, and more recently, estimated to be 2.24 to 3.58.
The average reproductive number of the possible origin of SARS-CoV-2 and the first mode of
disease transmission are yet to be identified. Analysis of the initial cluster of infections suggests
that the infected individuals had a common exposure point, i.e. a seafood market in Wuhan,
Hubei Province, China. The restaurants of this market are known to provide different types of
wild animals for human consumption, including live animals such as poultry, bats, snakes, and
marmots. This may have been the point where zoonotic (animal-to-human) transmission
occurred. However, it is important to rule out the possibility of foodborne transmission with
further investigations. In addition, other potential and expected routes of transmission, such as
direct contact through shaking contaminated hands or touching contaminated surfaces, are also to
be considered. Nevertheless, further research is required to determine whether blood transfusion
and organ transplantation, as well as transplacental and perinatal routes, are possible routes for
SARS- CoV-2 transmission determined in (Fig. 6).
The COVID-19 virus is caused by a coronavirus known as SARS-CoV-2, which belongs to a
different subfamily than the viruses that cause MERS-CoV and SARS-CoV. SARS-CoV-2 is
classified as a group 2B coronavirus and shares 79.5% genetic similarity with SARS-CoV. As of
May 13, 2020, there have been 4,170,424 confirmed cases of COVID-19, resulting in 287,399
deaths across over 210 countries. The global economy has also been severely impacted due to the
disruption of trade and supply chains, leading to significant financial losses for multinational
corporations. The recent surge in critically ill COVID-19 patients has exceeded the capacity of
intensive care facilities, making it difficult to provide adequate care to everyone who needs it.
This, in turn, has contributed to an increase in the case fatality rate observed during the outbreak.
Viewpoint on SARS-CoV-2 Transmission, Spread, and Emergence:
The novel coronavirus was identified within 1 month (28 days) of the outbreak. This is
impressively fast compared to the time taken to identify SARS-CoV reported in Foshan,
Guangdong Province, China (125 days) (68). Immediately after the confirmation of viral
etiology, the Chinese virologists rapidly released the genomic sequence of SARS-CoV-2, which
played a crucial role in controlling the spread of this newly emerged novel coronavirus to other
parts of the world (69). The possible origin of SARS-CoV-2 and the first mode of Splits Tree
phylogeny analysis.
In the unrooted phylogenetic tree of different betacoronaviruses based on the S protein, virus
sequences from different subgenera are grouped into separate clusters. SARS-CoV-2 sequences
from Wuhan and other countries exhibited a close relationship and appeared in a single cluster
(Fig. 1). The Co Vs from the subgenus Sarbecovirus appeared jointly in SplitsTree and divided
into three subclusters, namely, SARS-CoV-2, bat-SARS-like- CoV (bat-SL-CoV), and
SARSCoV (Fig. 1). In the case of other subgenera, like Merbecovirus, all of the sequences
grouped in a single cluster, whereas in Embecovirus, different species, comprised of canine
respiratory CoVs, bovine CoVs, equine CoVs, and human CoV strain (OC43), grouped in a
common cluster. Isolates in the subgenera Nobecovorus and Hibecovirus were found to be
placed separately away from other reported SARS-CoVs but shared a bat origin.
The swift identification of the novel coronavirus within one month (28 days) of the outbreak
represents a remarkable achievement, particularly when compared to the extended timeline of
125 days taken to identify SARS-CoV in Foshan, Guangdong Province, China (68). Following
the confirmation of the viral etiology, Chinese virologists promptly released the genomic
sequence of SARS-CoV-2. This proactive measure played a pivotal role in effectively controlling
the spread of the newly emerged novel coronavirus to various parts of the world (69).
Exploring the potential origin of SARS-CoV-2 involved the initial application of Splits Tree
phylogeny analysis. The unrooted phylogenetic tree constructed based on the S protein,
delineated clusters grouping virus sequences from different subgenera. Notably, SARS-CoV-2
sequences from Wuhan and other nations exhibited a close relationship, forming a cohesive
cluster (Fig. 1). Within the subgenus Sarbecovirus, the Splits Tree analysis revealed a
subdivision into three distinct subclusters: SARS-CoV-2, bat-SARS-like-CoV (bat-SL-CoV),
and SARS-CoV (Fig. 1). In contrast, other subgenera like Merbecovirus showcased all sequences
grouping into a singular cluster. Embecovirus, encompassing various species such as canine
respiratory CoVs, bovine CoVs, equine CoVs, and the human CoV strain (OC43), exhibited
clustering within a common group. Isolates in the subgenera Nobecovorus and Hibecovirus,
while distinct from other reported SARS-CoVs, shared a common bat origin.
CURRENT WORLDWIDE SCENARIO OF SARS-CoV-2
This novel virus, SARS-CoV-2, comes under the subgenus Sarbecovirus of the
Orthocoronavirinae subfamily and is entirely different from the viruses.
The assessment of nucleotide percent similarity was conducted utilizing the MegAlign software
program, revealing a remarkably high degree of similarity among novel SARS-CoV-2 isolates,
ranging from 99.4% to 100%. When compared to other Serbecovirus CoV sequences, the novel
SARS-CoV-2 sequences exhibited the highest similarity to bat-SL-CoV, with nucleotide percent
identity ranging between 88.12% and 89.65%. In contrast, previously reported SARS-CoVs
demonstrated a lower degree of similarity, ranging from 70.6% to 74.9% at the nucleotide level
in comparison to SARS-CoV-2.
Further analysis indicated nucleotide percent similarity indices of 55.4%, 45.5% to 47.9%,
46.2% to 46.6%, and 45.0% to 46.3% when compared to sequences from the other four
subgenera, namely Hibecovirus, Nobecovirus, Merbecovirus, and Embecovirus, respectively.
The percent similarity index of the current outbreak isolates suggests a close relationship
between SARS-CoV-2 isolates and bat-SL-CoV, implying a common origin. However, it is
imperative to note that conclusive evidence based on comprehensive genomic analysis of current
isolates is essential before drawing definitive conclusions. Notwithstanding, it has been
established that the novel SARS-CoV-2 isolates belong to the subgenus Sarbecovirus within the
diverse spectrum of betacoronaviruses. The hypothesis regarding their potential ancestor
originating from bat CoV strains underscores the pivotal role that bats may have played in
harboring this class of viruses.
The N protein
The N protein (Nucleocapsid protein) of coronaviruses serves a multifaceted role, encompassing
various functions critical to viral processes. It participates in complex formation with the viral
genome, facilitates interactions with the M protein during virion assembly, and augments the
transcription efficiency of the virus (55, 56). Structurally, the N protein comprises three highly
conserved and distinct domains: the N-terminal domain (NTD), an RNA-binding domain or
linker region (LKR), and a C-terminal domain (CTD) (57).
The NTD is implicated in binding to the 3' end of the viral genome, potentially through
electrostatic interactions, exhibiting notable divergence in both length and sequence (58). The
charged LKR, recognized for its serine and arginine richness and denoted as the SR (serine and
arginine) domain, engages in direct interaction with RNA in vitro, playing a crucial role in cell
signaling (60, 61). Furthermore, the LKR functions as an antagonist for interferon (IFN) and
RNA interference (62), modulating the host's antiviral response.
Compared to the N protein of SARS-CoV, the N protein of SARS-CoV-2 displays five amino
acid mutations. Two of these mutations are in the intrinsically dispersed region (IDR) at
positions 25 and 26, while the remaining mutations are found in the NTD (position 103), LKR
(position 217), and CTD (position 334) (16). These subtle differences in the amino acid
composition of the N protein contribute to the unique characteristics of SARS-CoV-2,
highlighting the significance of understanding such variations in the context of viral pathogenesis
and host interactions.
M protein
The M protein, the most abundant viral protein within the virion particle, plays a crucial role in
shaping the viral envelope, providing it with a distinct structure (48). Its primary function
involves
binding to the nucleocapsid and serving as a central organizer in the assembly of coronaviruses
(49). Although coronavirus M proteins exhibit considerable diversity in amino acid composition,
they maintain an overall structural similarity across different genera (50).
Structurally, the M protein is characterized by three transmembrane domains, flanked by a short
amino terminus outside the virion and a longer carboxy terminus inside the virion (50). The
interaction between M proteins (M-M interaction) is pivotal for maintaining the overall viral
scaffold. Notably, in the case of SARS-CoV-2, there is no amino acid substitution observed in
the M protein compared to that of SARS-CoV, indicating a conservation of this critical
component across different strains (16). This preservation underscores the significance of the M
protein in the structural integrity and assembly of coronaviruses, highlighting its essential role in
the viral life cycle.
E protein
The coronavirus E protein stands as the most enigmatic and smallest among the major structural
proteins (51), playing a multifunctional role in the pathogenesis, assembly, and release of the
virus
(52). This integral membrane polypeptide is characterized by its small size and acts as a
viroporin, functioning as an ion channel (53). The inactivation or disruption of the E protein is
associated with altered virulence in coronaviruses, leading to changes in morphology and
tropism (54).
On the other hand, the coronavirus S protein, a large and multifunctional class I viral
transmembrane protein, varies in size among different strains. Ranging from 1,160 amino acids
(as observed in IBV, infectious bronchitis virus, in poultry) to 1,400 amino acids (as seen in
FCoV, feline coronavirus) (43), the S protein forms a trimer on the virion surface, contributing to
the characteristic corona or crown-like appearance. Functionally, it is indispensable for the entry
of infectious virion particles into host cells through interaction with various host cellular
receptors
(44). This intricate process highlights the pivotal role of the S protein in mediating viral entry
and underscores its significance in the overall viral life cycle.
Additionally, the coronavirus S protein serves as a crucial determinant for tissue tropism and
host range (45). It holds significance as one of the vital immunodominant proteins of
coronaviruses, capable of eliciting robust host immune responses (45). The ectodomains of all
coronavirus S proteins share a common structural organization, divided into two subunits, SI and
S2 (43). SI, the first subunit, is involved in host receptor binding, while S2 accounts for fusion.
SI is further subdivided into two receptor-binding domains, namely the N-terminal domain
(NTD) and C- terminal domain (CTD), both efficiently interacting with various host receptors
(45). The SI CTD contains the receptor-binding motif (RBM). In each coronavirus spike protein,
the trimeric SI is positioned atop the trimeric S2 (38, 39). Notably, for SARS and MERS, civet
cats and camels respectively serve as amplifier hosts (40, 41).
Coronavirus genomes and sub genomes encode six open reading frames (ORFs) (31).
Predominantly, the 5' end is occupied by ORFla/b, producing 16 non-structural proteins (nsps).
Two polyproteins, ppla and pplab, are initially generated from ORFla/b through a -1 frameshift
between ORFla and ORFlb (32). Virus-encoded proteases cleave polyproteins into individual
nsps, including the main protease (Mpro), chymotrypsin-like protease (3CLpro), and
papain-like
proteases (PLPs) (42). SARS-CoV-2 also encodes these nsps, and recent elucidations have shed
light on their functions (31). Notably, a distinctive feature of SARS-CoV-2 compared to other
coronaviruses is the identification of a novel short putative protein within the ORF3 region—a
secreted protein with an alpha helix and beta-sheet with six strands encoded by ORF8 (31).
In conclusion, coronaviruses encode four major structural proteins—spike (S), membrane (M),
envelope (E), and nucleocapsid (N). The details of each of these proteins have been expounded
upon above.
The S Glycoprotein
The S Glycoprotein, or spike protein, of coronaviruses is a large and multifunctional class I viral
transmembrane protein. Molecular characterization categorizes SARS-CoV-2 as a new Beta
coronavirus within the subgenus Sarbecovirus. Distinct from other Betacoronaviruses, the
conserved open reading frame la/b (ORFla/b) exhibits less than 90% identity, firmly establishing
SARS-CoV-2 as a unique virus (3). Comparative analysis reveals an 80% nucleotide identity
between SARS-CoV-2 and the original SARS-CoV, with 89% identity observed with ZC45 and
ZXC21 SARS-related CoVs found in bats (2, 31, 36). Furthermore, SARS-CoV-2 demonstrates
an 82% identity with human SARS-CoV Tor2 and human SARS-CoV BJ01 2003 (31). In
contrast, a lower sequence identity of 51.8% is observed between MERS-related CoV and the
recently emerged SARS-CoV-2 (37).
Phylogenetic analysis of structural genes indicates SARS-CoV-2's closer relation to bat SARS-
related CoV, suggesting a potential bat origin. Amplifier hosts may have played a role in disease
transmission to humans, analogous to the zoonotic origins of other CoVs (31). Notably, SARS-
CoV-2 shares its origin with bats, similar to other zoonotic CoVs, such as MERS-related CoV
and SARS-related CoV (38, 39).
The SARS-CoV-2 genome follows a linear arrangement of 5'-leader-UTR-replicase-structural
genes (S-E-M-N)-3' UTR-poly(A) (32). Accessory genes, including 3a/b, 4a/b, and the
hemagglutinin-esterase gene (HE), are interspersed with structural genes (30). Although SARS-
CoV-2 encodes several accessory proteins, it lacks HE, a characteristic feature of some
betacoronaviruses (31). The positive-sense genome serves as the mRNA, translated into
polyprotein la/lab (ppla/lab) (33). A replication-transcription complex (RTC) is formed in
double- membrane vesicles (DMVs) by nonstructural proteins (nsps), encoded by the
polyprotein gene
(34). The RTC then synthesizes a nested set of sub genomic RNAs (sgRNAs) via discontinuous
transcription (35).
The designation of the virus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
by the International Committee on Taxonomy of Viruses (ICTV) places it in the Severe acute
respiratory syndrome-related coronavirus category. It is a member of the family Coronaviridae,
subfamily Orthocoronavirinae, and belongs to the order Nidovirales, subdivided into four genera:
Alphacoronavirus, Beta coronavirus, Gammacoronavirus, and Deltacoronavirus (3, 27). The
genera Alphacoronavirus and Beta coronavirus originate from bats, while Gamma coronavirus
and Delta coronavirus have evolved from bird and swine gene pools (24, 28, 29, 275).
Coronaviruses possess a single-stranded, positive-sense RNA genome of around 30 kb, enclosed
by a 5'-cap and 3'-poly(A) tail. The genome of SARS-CoV-2 is 29,891 bp long with a G+C
content of 38% (31). These viruses are enveloped, with an outer membrane containing viral
nucleocapsid. The electron micrographs of SARS-CoV-2 reveal a spherical outline with
pleomorphism, virion diameters varying from 60 to 140 nm, and distinctive spikes of 9 to 12 nm,
giving the virus a solar corona-like appearance (3).
While in vitro studies suggest efficacy of some therapeutic options for treating COVID-19,
randomized animal or human clinical trials are lacking, limiting their practical applicability in
the current pandemic (7, 9, 19-21).
This comprehensive review discusses the various characteristics of SARS-CoV-2/COVID-19
that are causing the current disease outbreaks, as well as the latest developments in diagnosis,
vaccine development, and therapeutics. It also briefly compares this virus to earlier SARS and
MERS CoVs, provides a veterinary perspective on CoVs and this new pathogen, and evaluates
the zoonotic potential of similar CoVs to provide feasible One Health strategies for managing
this fatal virus (22-367).
THE VIRUS (SARS-CoV-2)
SARS-CoV-2, the causative agent of COVID-19, belongs to the family of positive-sense RNA
viruses with a wide range of natural hosts, affecting multiple systems (23, 24). These viruses can
lead to clinical diseases in humans, ranging from the common cold to severe respiratory
conditions like SARS and MERS (17, 279). The recent emergence of SARS-CoV-2 has triggered
a global pandemic, caused widespread havoc and necessitating urgent attention.
SARS-CoV-2 exhibits genetic distinctiveness from both SARS-CoV (79% similarity) and
MERS- CoV (nearly 50%) (17). COVID-19 primarily affects the lungs, with characteristic chest
computer tomography findings showing dense, ground-glass opaque structures and
consolidation shadows
(18). The disease has led to diverse symptoms, with fever, cough, dyspnea, expectoration,
headache, myalgia, or fatigue being the most common.
Less common signs at hospital admission include diarrhea, hemoptysis, and shortness of breath
(14). Asymptomatic individuals have also been implicated in transmission, adding complexity to
disease dynamics (1). In-depth knowledge about the virus is crucial, considering it is a novel
agent, necessitating further comprehensive studies.
Comparative genomic analysis between SARS-CoV-2 and closely related CoVs reveals 27
amino acid substitutions in the sequence coding for the spike protein, with six substitutions in the
receptor-binding domain (RBD) and another six in the underpinning subdomain (SD) (16).
Phylogenetic analysis indicates SARS-CoV-2's close relation (88% similarity) to two bat SARS-
like CoVs (bat-SL-CoVZC45 and bat-SL-CoVZXC21) (Fig. 1).
Coronaviruses exhibit a broad host range, causing symptoms from the common cold to severe
diseases like SARS, MERS, and COVID-19. SARS-CoV-2 is one of the seven CoVs known to
infect humans and belongs to the same lineage as SARS-CoV but is genetically distinct. The
emergence of novel CoVs poses a significant threat to global public health, with COVID-19
being
the third CoV outbreak in humans in the past two decades. Efforts are ongoing to prevent the
further spread of the virus through preventive and control strategies, as specific antiviral drugs or
approved vaccines for human CoV infections are currently unavailable (7-9).
In domestic animals, CoV infections are associated with various pathological conditions,
primarily gastrointestinal diseases. The emergence of novel CoVs may be facilitated by the
maintenance of multiple CoVs in their natural hosts, increasing the probability of genetic
recombination. The high genetic diversity and the ability to infect multiple host species result
from high-frequency mutations in CoVs, driven by RNA-dependent RNA polymerase instability
and higher rates of homologous RNA recombination. Understanding the origin and evolution of
SARS-CoV-2 is crucial for disease surveillance and the development of targeted drugs,
prevention strategies, and potential vaccines.
Coronaviruses in Humans—SARS, MERS, and COVID-19
Coronavirus infection in humans usually causes respiratory diseases ranging from mild to severe.
The symptoms include high fever, severe inflammation, cough, and internal organ dysfunction,
which may lead to death. Most coronaviruses cause the common cold in humans, but the
discovery of SARS-CoV led to severe forms of respiratory disease in humans. Previous
outbreaks of other coronaviruses, such as SARS and MERS, suggest that COVID-19 is mainly
transmitted through human-to-human contact via direct contact, droplets, and fomites. Recent
studies show that the virus can survive for hours in aerosols and for days on surfaces, making
aerosol and fomite contamination potent factors in the transmission of SARS-CoV-2. The
immune response against coronavirus is essential in controlling and eliminating the infection, but
an imbalanced immune response can contribute to the immunopathology of the disease, leading
to impaired pulmonary gas exchange. Understanding the interaction between CoVs and the host's
innate immune systems can help us understand the lung inflammation associated with this
infection.
SARS is a viral respiratory disease caused by an animal CoV that originated from wet markets in
southern China. It adapted to the human host, enabling transmission between humans. The SARS
outbreak reported in 2002 to 2003 affected the Asia Pacific region severely, with 8,098
confirmed cases and 774 total deaths (9.6%). Although the case fatality rate (CFR) of SARS-
CoV-2 (COVID- 19) is lower than that of SARS-CoV, its epidemiological similarity to influenza
viruses is concerning. This can overwhelm the public health system, resulting in a pandemic.
MERS is another respiratory disease first reported in Saudi Arabia in 2012, with a CFR of
around 35%. Available data suggest that the incubation period of SARS-CoV-2, SARS-CoV, and
MERS- CoV is almost the same, with the longest predicted incubation time of SARS-CoV-2
being 14 days. Suspected individuals are isolated for 14 days to prevent further spread. Although
there is a high degree of similarity between the genome sequence of the new coronavirus (SARS-
CoV-2) and SARS-like CoVs, a comparative analysis identified a furin-like cleavage site in the
SARS-CoV-2 S protein that is absent from other SARS-like CoVs. The furin-like cleavage site is
expected to play a role in the virus's life cycle and disease pathogenicity and may act as a
therapeutic target for furin inhibitors. The highly contagious nature of SARS-CoV-2 compared
to its predecessors
may be due to a stabilizing mutation that occurred in the endosome-associated-protein-like
domain of the nsp2 protein.
It has been observed that the destabilizing mutation near the phosphatase domain of nsp3
proteins in SARS-CoV-2 could be a potential mechanism that sets it apart from other CoVs
(100). Although the case fatality rate (CFR) reported for COVID-19 is lower compared to those
of the previous SARS and MERS outbreaks, it has caused more deaths than both combined
(101). The recent discovery of an 832-nucleotide (nt) deletion in ORF8 may be related to the
virus's pathogenesis, which appears to decrease its replicative fitness and lead to attenuated
phenotypes of SARS-CoV- 2 (256).
The coronavirus is the most notable example of a zoonotic disease that has resulted from the
adaptation of viruses to new hosts. The bat coronavirus, which is currently circulating, has spike
proteins that allow for human infection without any adaptation or mutation (105). Bats carry a
significant number of c
Coronaviruses worldwide, and the high plasticity in receptor usage, coupled with the possibility
of adaptive mutation and recombination, can result in frequent interspecies transmission of
coronaviruses from bats to animals and humans (106). However, the pathogenesis of most bat
coronaviruses is unknown as they have not been isolated and studied (4). The hedgehog
coronavirus HKU31 has been identified from amur hedgehogs in China, and studies suggest that
hedgehogs are the reservoir of Betacoronavirus, with evidence of recombination (107).
The current scientific findings on MERS infection suggest that the significant reservoir host and
animal source of MERS infection in humans is the dromedary camels (97). Infected dromedary
camels may not show any visible signs of infection, making it difficult to control populations. In
vitro and in vivo studies on the isolated virus confirmed that there is a potential risk of SARS-
CoV reemergence from viruses that are currently circulating in the bat population (105).
Clinical Pathology of SARS-CoV-2 (COVID-19)
SARS-CoV-2, also known as severe specific contagious pneumonia (SSCP), Wuhan pneumonia,
or COVID-19, exhibits less severe pathogenesis compared to SARS-CoV but demonstrates
superior transmission capability, leading to a rapid increase in cases (reference 110, 111). The
incubation period in familial clusters ranges from 3 to 6 days, with a mean incubation period of
6.4 days and symptoms appearing within 2.1 to 11.1 days (references 112, 113). The median age
of affected individuals is 59 years, with a higher prevalence among males, and severity increases
in age groups above 50 years, similar to SARS and MERS (references 114, 2, 115).
Common symptoms include fever, cough, myalgia or fatigue, and less frequently, headache,
hemoptysis, and diarrhea (references 116, 282). In the early stages, COVID-19 cases transmitted
through human-to-human contact presented milder symptoms compared to those in Wuhan
(reference 14). Mortality rates initially appeared lower than previous SARS outbreaks (reference
101).
In-depth genomic analysis revealed 380 amino acid substitutions between SARS-CoV-2 and
other coronaviruses, potentially contributing to differences in pathogenicity (reference 16).
Research is needed to understand the implications of these amino acid sequence variations on
tropism, pathogenesis, and transmission dynamics. The zoonotic nature of coronaviruses poses
challenges in identifying animal reservoirs actively shedding the virus. In the case of MERS-
CoV, dromedary camels are significant reservoirs, with potential transmission through various
bodily fluids (reference 108). Studies indicate susceptibility in llamas and pigs, suggesting
possible circulation in diverse animal species (reference 109).
Past outbreaks, such as SARS, revealed the possibility of interspecies transmission from animals
in live-animal markets, highlighting the importance of surveillance (reference 78). Bats, known
for harboring coronaviruses without clinical signs, present a significant reservoir due to their
global distribution and large species diversity (references 30, 6). The need for efficient molecular
surveillance, particularly in the Rhinolophus bat family, is emphasized to prevent future
outbreaks (references 12, 86). Studies on SARS-CoV-2 transmission dynamics indicate
differences in viral kinetics compared to other coronaviruses, necessitating unique diagnostic and
isolation strategies (references 253, 82). The ability of asymptomatic or minimally symptomatic
patients to shed the virus for extended periods underscores the importance of comprehensive data
for updating screening procedures in clinics (reference 82).
The importance of frequent and effective hand hygiene and sanitation practices needs to be
emphasized significantly (249-252). Further exploratory research is needed to study the fecal-
oral transmission of SARS-CoV-2, along with environmental investigations to determine
whether the virus can remain viable in environments that facilitate such potent transmission
routes. The correlation between fecal concentrations of viral RNA and disease severity needs to
be established, along with an assessment of gastrointestinal symptoms and the possibility of
detecting fecal SARS- CoV-2 RNA during the COVID-19 incubation period or convalescence
phases (249-252).
Lower respiratory tract sampling techniques, such as bronchoalveolar lavage fluid aspirate, are
considered ideal clinical materials, rather than throat swabs, due to their higher positive rate on
nucleic acid tests (148). Although the diagnosis of COVID-19 can be made using upper
respiratory tract specimens collected using nasopharyngeal and oropharyngeal swabs, these
techniques pose unnecessary risks to healthcare workers due to close contact with patients (152).
It has been reported that a single patient with a high viral load contaminated an entire endoscopy
room by shedding the virus, which may remain viable for a while. A suspected case of COVID-
19 infection is confirmed if respiratory tract aspirate or blood samples test positive for SARS-
CoV-2 nucleic acid using RT-PCR or by identifying SARS-CoV-2 genetic sequence in
respiratory tract aspirate or blood samples (80). The patient will be confirmed as cured when two
subsequent oral swab results are negative (153).
Recent studies have confirmed that live virus can be detected in self-collected saliva of patients
infected with COVID-19. These findings suggest that saliva can be used as a noninvasive
specimen for the diagnosis of COVID-19 in suspected individuals (152). However, initial
screening of COVID-19 patients using RT-PCR may give negative results even if they have
chest CT findings
that suggest infection. Therefore, a combination of repeated swab tests using RT-PCR and CT
scanning is required for the accurate diagnosis of COVID-19, to prevent the possibility of false-
negative results during the disease screening process (154).
RT-PCR is the most widely used test for diagnosing COVID-19. However, it has significant
limitations from a clinical perspective as it does not provide clarity on disease progression.
Droplet digital PCR (ddPCR) can be used to quantify viral load in samples obtained from the
lower respiratory tracts.
The persistence of the virus on surfaces, with a viability lasting at least three days, poses a
significant risk to both uninfected patients and healthcare workers (289). Notably, recent findings
indicate that anal swabs yield more positive results than oral swabs during the later stages of
infection (153). Consequently, caution must be exercised by clinicians when discharging
COVID- 19-infected patients solely based on negative oral swab test results due to the potential
for fecal- oral transmission.Despite lower viral loads in stool samples compared to respiratory
samples, stringent precautionary measures must be observed when handling stool samples from
suspected or confirmed COVID-19 cases (151). In the case of children infected with SARS-
CoV-2, who typically experience mild illness and prompt recovery, there is a noteworthy
discovery that stool samples from infected children may test positive within ten days of negative
throat swab results, emphasizing the risk of fecal-oral transmission, especially in children (290).
Consequently, to mitigate the risk of fecal-oral transmission, COVID-19 patients should only be
considered negative when testing negative for SARS-CoV-2 in stool samples.
Various specimens, including bronchoalveolar lavage fluid, sputum, nasal swabs,
fibrobronchoscope brush biopsy specimens, pharyngeal swabs, feces, and blood, are susceptible
to SARS-CoV-2 transmission (246). The presence of the virus in fecal samples raises serious
public health concerns, as it adds another dimension to transmission routes beyond droplets from
sneezing and coughing. Fecal excretion, documented in previous coronaviruses, suggests the
potential for viable transmission through fecal-oral routes. Thus, in regions with low hygiene
standards and poor sanitation, fecal-oral transmission of SARS-CoV-2 could have severe
consequences, necessitating heightened precautions.Effective measures, such as the use of
ethanol and disinfectants containing chlorine or bleach, are crucial in combating coronaviruses
(249-252). Strict precautions should be followed when handling stools from SARS-CoV-2-
infected patients, and proper disinfection and disposal of biowaste materials and hospital sewage
are imperative. Emphasizing frequent and thorough hand hygiene becomes paramount in
controlling the spread of the virus, especially given the active replication of SARS-CoV-2 in the
upper respiratory tract and prolonged viral shedding through stool even after symptom
disappearance (248).
As viral load studies play a role in recommending precautionary measures for specific samples,
such as feces, updates to the current case definition and a reassessment of strategies to curb the
SARS-CoV-2 outbreak's spread are warranted (248). A recent survey underscores the importance
of such precautions, revealing positive RT-PCR results in stool samples from confirmed cases,
with lower viral loads than respiratory samples but with essential biosafety implications (151).
The examination of samples from 18 individuals who tested positive for SARS-CoV-2 in
Singapore, having traveled from Wuhan, revealed the presence of viral RNA in stool and whole
blood, while urine showed no such presence, as determined by real-time RT-PCR (288).
Additionally, diverse clinical specimens, including bronchoalveolar lavage fluid, exhibited novel
SARS-CoV-2 infections, as outlined in Table 2 (80, 245, 246).
Quantitative RT-PCR targeting the N gene was employed to measure the viral loads of SARS-
CoV-2 in throat swab and sputum samples from COVID-19-infected individuals. Results
indicated a peak viral load approximately 5 to 6 days post-symptom onset, ranging from 10^4 to
10^7 copies/ml during this period (151). Notably, nasal swabs demonstrated higher viral loads
compared to throat swabs in symptomatic COVID-19 patients (82). Contrary to initial
assumptions associating viral load with poor outcomes, some case reports have identified
asymptomatic individuals with elevated viral loads (247). In a recent study involving 17
symptomatic patients, higher viral loads were observed in nasal swabs than in throat swabs
shortly after symptom onset, resembling the shedding pattern of influenza patients but differing
from that of SARS-CoV patients. Furthermore, the viral load in asymptomatic patients closely
mirrored that of symptomatic patients, emphasizing the transmission potential of individuals with
minimal signs and symptoms (82).
A comprehensive analysis of 95 full-length genomic sequences of SARAS-CoV-2 strains from
the National Center for Biotechnology Information and GISAID databases revealed high
homology of 99.99% at the nucleotide level and 99.99% at the amino acid level. Notably, low
overall variation was observed in ORF regions, with 13 variation sites identified in la, lb, S, 3a,
M, 8, and N regions. Mutation rates of 30.53% (29/95) and 29.47% (28/95) were noted at
positions nt 28144 (ORF8) and nt 8782 (ORFla), respectively. These selective mutations suggest
caution in using specific regions for primer and probe design. The SARS-CoV-2 reference
sequence is instrumental in studying molecular biology and pathobiology, facilitating the
development of diagnostics and effective prevention and control strategies against SARS-CoV-2
(260).
Detection of SARS-CoV-2 nucleic acids is possible in various samples, such as bronchoalveolar
lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimens, pharyngeal
swabs, feces, blood, and urine, each exhibiting varying levels of diagnostic performance, as
summarized in Table 2 (80, 245, 246).
DIAGNOSIS OF SARS-CoV-2 (COVID- 19)
RNA tests, such as real-time RT-PCR or next-generation sequencing, can confirm cases of
SARS- CoV-2 (COVID-19). Currently, nucleic acid detection techniques, like RT-PCR, are
considered the most effective method for confirming the diagnosis of COVID-19 cases. Several
companies worldwide are developing and marketing SARS-CoV-2-specific nucleic acid
detection kits, while multiple laboratories are creating their own in-house RT-PCR tests. For
instance, the Shuoshi Biotechnology's SARS-CoV-2 nucleic acid detection kit uses the double
fluorescence PCR method.
As of March 30th, 2020, the U.S. Food and Drug Administration (FDA) had granted Emergency
Use Authorizations (EUAs) to 22 in vitro diagnostics, including the RT-PCR diagnostic panel for
the universal detection of SARS-like betacoronaviruses and specific detection of SARS-CoV-2,
developed by the U.S. CDC.
Dogs have low susceptibility to SARS-CoV-2, while chickens, ducks, and pigs are not
susceptible at all. However, the National Veterinary Services Laboratories of the USDA have
reported COVID-19 in tigers and lions that showed respiratory symptoms like dry cough and
wheezing. These zoo animals are thought to have been infected by an asymptomatic zookeeper.
The number of COVID-19-positive cases in humans is increasing, creating ideal conditions for
viral spillover to other species, such as pigs. Evidence obtained from SARS-CoV suggests that
pigs can get infected with SARS-CoV-2. However, experimental inoculation with SARS-CoV-2
failed to infect pigs. Further studies are required to identify possible animal reservoirs of SARS-
CoV-2 and the seasonal variation in the circulation of these viruses in the animal population.
Collaboration between human and animal health sectors is necessary to evaluate and identify
possible risk factors of transmission between animals and humans. This cooperation will help
devise efficient strategies for managing emerging zoonotic diseases.
While evidence of cat-to-human transmission is lacking, necessary preventive measures are
advised, and social distancing should be practiced among companion animals of different
households. IDEXX, one of the leading veterinary diagnostic companies, has conducted large-
scale testing for COVID-19 in specimens collected from dogs and cats. None of the tested
animals were positive.
A study conducted to investigate different animal species' potential to act as intermediate hosts of
SARS-CoV-2 found that ferrets and cats can be infected by experimental inoculation. Infected
cats also efficiently transmitted the disease to naive cats. SARS-CoV-2 infection and
transmission in ferrets recapitulate the clinical aspects of COVID-19 in humans. Infected ferrets
shed the virus via multiple routes, such as saliva, nasal washes, feces, and urine, making them an
ideal animal model for studying disease transmission. Experimental inoculation was also done in
other animal species, and it was found that dogs have low susceptibility, while chickens, ducks,
and pigs are not at all susceptible to the virus.
The significance of these findings may only be fully realized in the event of a substantial
outbreak caused by a virus like SARS-CoV-2. Reports of COVID-19 in both companion and
wild animals worldwide have been steadily increasing, warranting further studies to assess the
potential of animals, especially companion animals, as efficient reservoir hosts that could
potentially alter the dynamics of human-to-human transmission (330). Presently, there have been
confirmed cases of SARS-CoV-2 in two pet dogs (Hong Kong) and four pet cats (one each from
Belgium and Hong Kong, two from the United States) (335). The World Organization for
Animal Health (OIE) has acknowledged the diagnosis of COVID-19 in dogs and cats due to
human-to-animal transmission
(331). The confirmation of the similarity in gene sequences between SARS-CoV-2 in an infected
pet owner and their dog further supports the occurrence of human-to-animal transmission (333).
While there is evidence that cats can be susceptible to SARS-CoV-2 and can get infected by
human beings, there are currently no reports of SARS-CoV-2 transmission from felines to
humans.
The comprehensive analysis of 95 full-length genomic sequences of SARAS-CoV-2 strains
indicates high homology, with 99.99% at the nucleotide level and 99.99% at the amino acid
level. The potential of pangolins as an intermediate host for SARS-CoV-2 is suggested by the
close similarity in the RBD of the S protein between pangolin-CoV and SARS-CoV-2, along
with evidence of recombination between pangolin-CoV-like viruses and bat-CoV-RaTG 13-like
virus
(145). Human-wildlife interactions, particularly in the context of climate change, are considered
high-risk for the emergence of zoonotic diseases such as SARS-CoV-2 (142). To prevent future
zoonotic spillover events, coordinated efforts are needed to identify high-risk pathogens in wild
animal populations, conduct surveillance among susceptible individuals, and enhance biosecurity
measures related to wildlife trade (146).
The sequence analysis of the SARS-CoV-2 RNA genome reveals it as a recombinant virus from
bat and another coronavirus of unknown origin, with snake identified as the probable animal
reservoir (143). Another analysis suggests 96% identity with bat coronavirus, implying a bat
origin
(63). The involvement of bat-derived materials in causing outbreaks is not ruled out, particularly
in the production of traditional Chinese medicine (TCM) involving wild bats (139). Pangolins,
known carriers of various viruses, including coronaviruses, also pose a risk, as indicated by their
high amino acid identity with COVID-19 (144).
Due to the potential role of farm and wild animals in SARS-CoV-2 infection, the WHO
recommends avoiding unprotected contact with both farm and wild animals (25). Live-animal
markets, like the one in Guangdong, China, provide a setting for animal coronaviruses to amplify
and transmit to new hosts, such as humans, making them critical in the origin of novel zoonotic
diseases (78). Bats, being reservoirs for several viruses, may also play a role in the current
outbreak
(140). A qualitative study in southern China identified frequent human-animal interactions and
low levels of environmental biosecurity as significant risks for the emergence of zoonotic
diseases in local communities (141, 142).
The Swine Acute Diarrhea Syndrome Coronavirus (SADS-CoV) was first identified in suckling
piglets that had severe enteritis and belongs to the Alphacoronavirus genus. The outbreak
resulted in the death of 24,693 piglets across four farms in China. The virus found in 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. The SADS-CoV outbreak
started in Guangdong province, near the location of the SARS pandemic origin. Pigs were not
known to be infected with bat-origin coronaviruses before this outbreak, indicating that the bat-
origin coronavirus jumped to pig by breaking the species barrier. Pigs are considered the mixing
vessel for influenza A viruses, and they may also act as the mixing vessel for coronaviruses since
they are in frequent contact with both humans and multiple wildlife species. Pigs are also
susceptible to infection with human SARS-CoV and MERS-CoV, making this scenario a
nightmare.
Bovine coronaviruses (BoCoVs) are known to infect several domestic and wild ruminants.
BoCoV 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. BoCoV-
like viruses have been noted in humans, suggesting their zoonotic potential as well.
Feline enteric and feline infectious peritonitis (FIP) viruses are the two major feline CoVs. Feline
CoVs can affect the gastrointestinal tract, abdominal cavity (peritonitis), respiratory tract, and
central nervous system. Canines are also affected by CoVs that fall under different genera,
namely, canine enteric coronavirus in Alphacoronavirus and canine respiratory coronavirus in
Betacoronavirus, affecting the enteric and respiratory tract, respectively.
IBV, under Gammacoronavirus, causes diseases of respiratory, urinary, and reproductive
systems, with substantial economic losses in chickens. In small laboratory animals, mouse
hepatitis virus, rat sialodacryoadenitis coronavirus, and guinea pig and rabbit coronaviruses are
the major CoVs associated with disease manifestations like enteritis, hepatitis, and respiratory
infections.
Coronavirus infection is linked to different kinds of clinical manifestations, varying from
enteritis in cows and pigs to upper respiratory disease in chickens and fatal respiratory infections
in humans. Among the CoV genera, Alphacoronavirus and Betacoronavirus infect mammals,
while Gammacoronavirus and Deltacoronavirus mainly infect birds, fishes, and sometimes
mammals. Several novel coronaviruses that come under the genus Deltacoronavirus have been
discovered in the past from birds, as well as from pigs (porcine Coronavirus HKU15).
Transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and
porcine hemagglutinating encephalomyelitis virus (PHEV) are some of the coronaviruses of
swine. Among them, TGEV and PEDV are responsible for causing severe gastroenteritis in
young piglets with noteworthy morbidity and mortality. Infection with PHEV also causes enteric
infection but can cause encephalitis due to its ability to infect the nervous aminotransferase,
bilirubin, and especially D-dimer.
Middle-aged and elderly patients with primary chronic diseases, especially high blood pressure
and diabetes, are found to be more susceptible to respiratory failure and, therefore, have poorer
prognoses. Providing respiratory support at early stages has improved the disease prognosis and
facilitated recovery. The ARDS in COVID-19 is due to cytokine storms that result in
exaggerated immune response, immune regulatory network imbalance, and, finally, multiple-
organ failure. 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.
Exploring Coronavirus Infections in Animals and Their Zoonotic Connections: A Brief Overview
Coronaviruses, known to afflict various domestic and wild animals alongside humans, exhibit a
wide host range including horses, camels, cattle, swine, dogs, cats, rodents, birds, ferrets, minks,
bats, rabbits, snakes, and other wildlife species. Notably, they have been implicated in outbreaks
such as SARS and MERS (23).
- Impact on Pregnancy and Vertical Transmission:
While concerns exist regarding the impact of SARS-CoV-2/COVID-19 on pregnancy, there is
inconclusive evidence of in utero transmission. Although certain cases indicate preterm delivery
and associated consequences, doubts persist about vertical transmission (240-243).
- Clinical Manifestations and Severity:
COVID-19 is associated with pneumonia, and severe cases may develop acute respiratory
distress syndrome (ARDS). Blood biochemistry indexes serve as indicators of disease severity,
including albumin, lactate dehydrogenase, C-reactive protein, and lymphocyte and neutrophil
percentages
(121). Lymphocytopenia is notably correlated with disease severity (118).
- Lung Involvement and Histological Findings:
SARS-CoV-2 invades the lung parenchyma, leading to severe interstitial inflammation observed
as ground-glass opacity in computed tomography (CT) images. Histological assessments reveal
characteristics of acute respiratory distress syndrome (ARDS), including diffuse alveolar
damage, fibromyxoid exudates, hyaline membrane formation, and pneumocyte desquamation
(118, 119).
- Zoonotic Transmission:
Coronaviruses, exemplified by SARS and MERS, have crossed the species barrier from animals
to humans. Bats, recognized as natural reservoir hosts, and intermediary hosts like palm civets
(SARS-CoV) and dromedary camels (MERS-CoV) have played roles in zoonotic transmission
(79, 102).
- Diagnostic Approaches:
Various diagnostic methods, including RT-PCR, isolation, culturing, and sequencing, are
employed to confirm SARS-CoV-2 infection. Rapid and accurate diagnosis is crucial for
outbreak control, with advancements in PCR assays aiding swift detection (155).
- Therapeutic Strategies:
Effective treatment is a challenge, and supportive care is primarily provided to hospitalized
patients. Repurposed antiviral drugs, such as lopinavir/ritonavir and interferon-ß, have shown
promise against CoV infections, though specific therapeutic agents for SARS-CoV-2 are
currently lacking (9, 165).
- Vaccine Development:
Efforts to develop a vaccine against SARS-CoV-2 are underway globally. Several vaccine
candidates, employing various platforms such as mRNA, DNA, and viral vectors, are in different
stages of clinical trials. Collaboration and innovative strategies, like multiepitope subunit
vaccines, hold promise in preventing the ongoing pandemic (168, 169, 365).
- Future Directions and Challenges:
Genetic analysis and repurposing of vaccines based on the genetic similarity between SARS-
CoV and SARS-CoV-2 are avenues for further exploration. Additionally, the identification of
potential
therapeutic compounds and their evaluation for COVID-19 management remain crucial aspects
of ongoing research (150, 173-174).
Monoclonal Antibodies in the Intervention of Coronavirus Diseases: A Comprehensive Overview
Monoclonal antibodies (MAbs) emerge as potential agents for disease intervention in individuals
exposed to coronaviruses (CoVs). Robust neutralizing antibodies against CoV infection were
observed in patients recovering from Severe Acute Respiratory Syndrome (SARS) (164). A
collection of MAbs targeting specific domains of the Middle East Respiratory Syndrome
Coronavirus (MERS-CoV) S protein, involving critical entry tasks, demonstrated considerable
protection in mice against MERS (198, 199).
Ivermectin and Hydroxychloroquine: A Promising Combination?
Ivermectin, though exhibiting a host-directed approach, demonstrated in vitro anti-SARS-CoV-2
activity. While concerns exist regarding its cytotoxicity, combination therapy with
hydroxychloroquine is speculated to have a synergistic effect. Further studies, including in vivo
investigations and randomized clinical control trials, are essential to decipher the mechanism and
clinical utility of this potential treatment (339, 341).
Nafamostat and Newly Synthesized Compounds: Seeking Inhibitory Action:
Nafamostat, a potent MERS-CoV inhibitor, lacks inhibitory action against SARS-CoV-2.
Recently synthesized halogenated triazole compounds were evaluated for their potential to
inhibit viral helicase. However, further studies are required to ascertain their therapeutic potential
in managing COVID-19 infection (194).
Repurposed Drugs and Antiviral Agents: Unraveling Efficacy:
Repurposed drugs, including chloroquine and remdesivir, exhibit antiviral activity against CoVs.
While chloroquine interferes with virus-cell fusion and receptor binding, remdesivir, a nucleotide
analog prodrug, shows promising antiviral activity. Tilorone, an overlooked antiviral drug, may
hold potential against SARS-CoV-2 and warrants further investigation (195, 306).
Therapeutic Challenges and Clinical Trials: Navigating Uncertainties:
Despite the potential of various therapeutic agents, challenges persist in determining their
efficacy. Clinical trials using drugs like lopinavir/ritonavir and traditional Chinese medicine in
combination therapy show initial promise. However, conclusive evidence requires randomized
clinical control studies with larger study populations to guide effective therapeutic management
of COVID-19 (193, 299).
Antiviral Drugs
Several classes of routinely used antiviral drugs, including oseltamivir (neuraminidase inhibitor),
acyclovir, ganciclovir, and ribavirin, have demonstrated no efficacy against COVID-19 and are
therefore not recommended (187). Although oseltamivir, a neuraminidase inhibitor, has been
investigated in Chinese hospitals for suspected COVID-19 cases, its proven efficacy against
SARS-CoV-2 is still lacking (7).
Evidence linking the use of nonsteroidal anti-inflammatory drugs (NSAIDs) to respiratory and
cardiovascular adverse effects has led to a cautious approach. It is recommended to consider
NSAIDs as the first-line option for managing COVID-19 symptoms (302). The use of
corticosteroids in COVID-19 patients remains controversial, necessitating further systematic
clinical studies. Guidelines for managing critically ill adults suggest the use of systemic
corticosteroids in mechanically ventilated adults with acute respiratory distress syndrome
(ARDS)
(303). Generalized use of corticosteroids is not indicated in COVID-19 due to concerns
associated with viral pneumonia. Stem cell therapy using mesenchymal stem cells (MSCs) is a
hopeful strategy with potential immunomodulatory capacity, particularly in attenuating the
cytokine storm observed in severe cases of SARS-CoV-2 infection, thereby reducing mortality.
Among various MSC types, expanded umbilical cord MSCs are considered a potential
therapeutic agent, requiring further validation for managing critically ill COVID-19 patients
(304).
Repurposed Broad-Spectrum Antiviral Drugs
Symptomatic treatment, along with oxygen therapy, is provided to COVID-19 patients showing
severe signs. In cases progressing to respiratory failure and refractory to oxygen therapy,
mechanical ventilation becomes necessary. Adequate hemodynamic support is administered for
COVID-19-induced septic shock (299). Several drug classes are being evaluated for potential
therapeutic action against SARS-CoV-2, broadly classified into three categories: drugs blocking
virus entry, those inhibiting viral replication and survival within the host cell, and those
attenuating the exaggerated host immune response (300). Anti-inflammatory treatment using
drugs like glucocorticoids, cytokine inhibitors, JAK inhibitors, chloroquine, and
hydroxychloroquine may benefit critically ill COVID-19 patients, but careful analysis of the
risk/benefit ratio is essential
(301). While no studies have specifically addressed the application of nonsteroidal anti-
inflammatory drugs (NSAIDs) to COVID-19-infected patients, evidence links NSAIDs and SARS,
necessitating precautionary measures due to the novel virus's unknown nature (36, 189).
Current mainstays of treatment for severely affected SARS-CoV-2 patients in hospitals include
mechanical ventilation, intensive care unit (ICU) admission, and symptomatic and supportive
therapies. Additional therapeutic options being explored include 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). However, ongoing clinical trials are essential to evaluate their safety
and efficacy (7). The design and development of effective drugs, therapeutics, and vaccines
against COVID-19, with adequate evaluation and approval from regulatory bodies, may take
months to a year(s). Continuous efforts are necessary to identify and assess viable drugs and
immunotherapeutic regimens that have proven potency against other viral agents similar to
SARS- CoV-2 (9).
Therapeutics and Drugs
There is no currently licensed specific antiviral treatment for MERS- and SARS-CoV infections,
with the main focus in clinical settings on lessening clinical signs and providing supportive care
(183-186). Effective drugs for managing COVID-19 patients include remdesivir,
lopinavir/ritonavir alone or in a blend with interferon beta, convalescent plasma, and monoclonal
antibodies (MAbs). However, efficacy and safety issues of these drugs require additional clinical
trials (187, 281). A controlled trial of ritonavir-boosted lopinavir and interferon alpha 2b
treatment was performed on hospitalized COVID-19 patients (ChiCTR2000029308) (188).
Hydroxychloroquine and tocilizumab have been proposed for their potential role in modulating
inflammatory responses and antiviral effects, but fool-proof clinical trials are yet to be published
(194, 196, 197, 261-272). A recent clinical trial on adult patients suffering from severe COVID-
19 revealed no benefit of lopinavir-ritonavir treatment over standard care (273).
Efforts to control SARS-CoV-2 infection involve strategies similar to those against MERS and
SARS, along with adopting and strengthening measures based on the risk regions. BCG
vaccination is being explored for its protective role against SARS-CoV-2. Clinical trials are
ongoing to evaluate the impact of childhood BCG vaccination on COVID-19 PCR positivity
rates. Population genetic studies have identified two major types of the SARS-CoV-2 virus, L
and S, with L expected to be the most prevalent (70%), followed by S (30%). The genetic
differences between L and S types are small, but further variations leading to the emergence of
new strains can be expected (363, 364, 366, 367).
Developing a vaccine that can produce cross-reactive antibodies is crucial for recurring
coronavirus outbreaks. The success of such a vaccine depends on its ability to provide protection
against present and future virus versions. Identifying antibodies that can recognize conserved
epitopes, even after considerable variations, is key (362). While numerous vaccine clinical trials
are ongoing worldwide, pregnant women have been excluded from these studies.
Vaccines, Therapeutics, and Drugs
Emerging viruses like Zika, Ebola, and Nipah have prompted a race to design advanced
vaccines, prophylactics, therapeutics, and drug regimens to counteract new threats. Rapid and
colorimetric detection methods, such as RT-LAMP, have been developed for SARS-CoV-2. An
interactive web-based dashboard, smartphone-integrated home-based point-of-care testing
(POCT), and CRISPR-based diagnostic tools, including SHERLOCK and a lateral flow assay,
offer alternatives for rapid detection. Artificial intelligence, using three-dimensional deep-
learning models, aids in diagnosing COVID-19 through CT images.
Tracking and mapping rising incidence rates, disease outbreaks, and community spread are
crucial for effective control strategies. Chest CT, with its superior sensitivity compared to X-ray
screening, is an ideal diagnostic tool for viral pneumonia associated with COVID-19. Serological
assays, such as ELISA, targeting COVID-19 IgM and IgG antibodies, can serve as high-
throughput alternatives. The challenge lies in the availability of diagnostic assay kits, especially
as the number of COVID-19 cases rises.

Antibody-based therapies show promise, with cross-neutralizing SARS-CoV RBD-specific

monoclonal antibodies (MAbs) explored for their effectiveness against COVID-19. Combination
therapies, such as MAbs with the drug remdesivir, are suggested as ideal therapeutic options.
Regeneron is actively working on recognizing potent and specific MAbs to combat COVID-19.
Implementing vigorous prevention and control strategies is essential for controlling the spread of
SARS-CoV-2. Mental health protection for medical workers is crucial, and strengthening the
regulatory mechanism for wild animal trade is recommended. Large-scale screening programs
and effective interventions based on data analysis are essential for disease control. Travel
restrictions and a temporary ban on wildlife trade have contributed significantly to preventing the
global spread of SARS-CoV-2.
Understanding the zoonotic aspects of the disease, particularly in bats, is crucial. In vitro and in
vivo studies should evaluate the risk of future epidemics. Although licensed antiviral drugs or
vaccines against SARS-CoV, MERS-CoV, and SARS-CoV-2 are currently lacking, advances in
designing therapeutic agents against other emerging diseases will expedite the development of
suitable treatments for COVID-19. Until then, reliance on control and prevention measures
remains essential.
Virology
Coronaviruses belong to the nidovirus superfamily, classified into alpha-, beta-, gamma-, and
deltacoronaviruses. Human coronaviruses (HCoVs) include alpha (HCoV-229E, HCoV-NL63)
and beta (HCoV-HKU1, HCoV-OC43, MERS-CoV, SARS-CoV) species. SARS-CoV-2,
causing COVID-19, is a beta-coronavirus with genetic similarities to SARS-CoV but exists as a
distinct variant. The virus's spike protein plays a crucial role in viral entry, exhibiting structural
rearrangements to facilitate membrane fusion with the host cell.
ACE enzyme functions as a receptor for SARS-CoV-2 to enter human cells. The virus's genetic
sequences are vital for diagnostic tests, and laboratories worldwide are developing antibody-
based diagnostic tests. Chest CT is highly specific for COVID-19 pneumonia, revealing ground-
glass opacities. Diagnostic kits detecting genetic sequences face challenges in availability.
Antibody profiles of COVID-19 patients indicate potential for specific diagnostic tests.
Research on cross-neutralizing SARS-CoV RBD-specific MAbs and combination therapies with
remdesivir holds promise for COVID-19 treatment. Control and prevention strategies, including
travel restrictions and a temporary wildlife trade ban, are effective in containing SARS-CoV-2.
Vigorous prevention measures are necessary for controlling the outbreak. Mental health
protection for medical workers and strengthening regulatory mechanisms for wild animal trade
are recommended. Understanding zoonotic aspects and conducting in vitro and in vivo studies
are crucial for preventing future epidemics.
5.1 Mode of Transmission
The origin of COVID-19 transmission was initially associated with a seafood market, where
patients had either worked or visited. However, human-to-human transmission has become
widespread globally, similar to previous epidemics like SARS and MERS. Respiratory droplets
are the primary carriers of coronavirus transmission, entering the lungs through inhaled air or
staying in the nose and mouth. Transmission can also occur through touching infected surfaces
or objects. Airborne safety measures are proposed due to limited awareness of transmission
systems. Asymptomatic individuals or those in the incubation period can act as carriers,
contributing to transmission.
6.1 Laboratory Testing for COVID-19
Assessing COVID-19 patients is based on clinical features and epidemiological factors.
Screening protocols and testing of specimens using nucleic acid amplification tests like RT-PCR
are crucial for outbreak control. If testing facilities are unavailable, specimens should be sent to
reference laboratories suggested by WHO. Suspected patients are also recommended to undergo
routine laboratory investigations for other respiratory pathogens to differentiate from influenza,
parainfluenza, adenovirus, respiratory syncytial virus, and rhinovirus.
6 CLINICAL DIAGNOSIS
COVID-19 symptoms range from mild rhinitis to septic shock, similar to past respiratory
epidemics like SARS and MERS. Some patients may experience intestinal disturbances,
although COVID-19 lacks such symptoms. Radiological examinations reveal unilateral or
bilateral involvement consistent with viral pneumonia, with severe clinical courses observed in
comorbid patients. Diagnosis involves a complete travel and contact history, coupled with
laboratory testing.
Serological screening is preferred for its ability to analyze asymptomatic infections, and several
serological tests for SARS-CoV-2 are in progress.
4.2 Viral Replication
Coronavirus replication occurs within the cytoplasm, closely associated with the endoplasmic
reticulum and other cellular membrane organelles. Different receptors facilitate the invasion of
host cells by human coronaviruses. After uncoating, the genome undergoes transcription and
translation. Replication is characterized by mRNA forming an enclosed group with typical 3'
ends, with about 7 mRNAs produced. These mRNAs express the synthesis of various genome
segments. The proteins are collected at the cell membrane, and genomic RNA forms mature
particles by budding from internal cell membranes.
5 PATHOGENESIS
Coronaviruses show high precision and maturity in most airway epithelial cells, leading to
enhanced nasal secretion, local edema, and damage to host cells. This damage stimulates the
synthesis of inflammatory mediators, causing symptoms like sneezing, difficulty breathing, and
elevated mucosal temperature. The virus primarily affects the lower respiratory tract and other
cells, inducing T-cell apoptosis and immune system collapse. The understanding of COVID-19
transmission risk is incomplete, with respiratory droplets being the major carriers. Human-to-
human transmission is confirmed, and asymptomatic individuals can act as carriers. The
incubation period is typically 3 to 7 days but can be prolonged up to 2 weeks.