Emerging Microbes & Infections

2021, VOL. 10
https://doi.org/10.1080/22221751.2021.1898291

REVIEW

Lessons learned 1 year after SARS-CoV-2 emergence leading to COVID-19

pandemic
Kelvin Kai-Wang To a,b,c,d, Siddharth Sridhar a,b,c,d, Kelvin Hei-Yeung Chiud, Derek Ling-Lung Hungd,
Xin Lid, Ivan Fan-Ngai Hunge, Anthony Raymond Tame, Tom Wai-Hin Chungd, Jasper Fuk-Woo Chan a,b,c,d,
Anna Jian-Xia Zhang a,b,c, Vincent Chi-Chung Chengd and Kwok-Yung Yuen a,b,c,d
a
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region,
People’s Republic of China; bDepartment of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong
Kong Special Administrative Region, People’s Republic of China; cCarol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The
University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People’s Republic of China; dDepartment of Microbiology,
Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, People’s Republic of China; eDepartment of Medicine, Li Ka
Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People’s Republic of China

ABSTRACT
Without modern medical management and vaccines, the severity of the Coronavirus Disease 2019 (COVID-19) pandemic
caused by severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) might approach the magnitude of 1894-
plague (12 million deaths) and 1918-A(H1N1) influenza (50 million deaths) pandemics. The COVID-19 pandemic was
heralded by the 2003 SARS epidemic which led to the discovery of human and civet SARS-CoV-1, bat SARS-related-CoVs,
Middle East respiratory syndrome (MERS)-related bat CoV HKU4 and HKU5, and other novel animal coronaviruses. The
suspected animal-to-human jumping of 4 betacoronaviruses including the human coronaviruses OC43(1890), SARS-CoV-1
(2003), MERS-CoV(2012), and SARS-CoV-2(2019) indicates their significant pandemic potential. The presence of a large
reservoir of coronaviruses in bats and other wild mammals, culture of mixing and selling them in urban markets with
suboptimal hygiene, habit of eating exotic mammals in highly populated areas, and the rapid and frequent air travels from
these areas are perfect ingredients for brewing rapidly exploding epidemics. The possibility of emergence of a hypothetical
SARS-CoV-3 or other novel viruses from animals or laboratories, and therefore needs for global preparedness should not be
ignored. We reviewed representative publications on the epidemiology, virology, clinical manifestations, pathology,
laboratory diagnostics, treatment, vaccination, and infection control of COVID-19 as of 20 January 2021, which is 1 year
after person-to-person transmission of SARS-CoV-2 was announced. The difficulties of mass testing, labour-intensive contact
tracing, importance of compliance to universal masking, low efficacy of antiviral treatment for severe disease, possibilities of
vaccine or antiviral-resistant virus variants and SARS-CoV-2 becoming another common cold coronavirus are discussed.

ARTICLE HISTORY Received 7 February 2021; Revised 26 February 2021; Accepted 28 February 2021

KEYWORDS Coronavirus; COVID-19; SARS-CoV-2; Pandemic; Pathogenesis; Diagnostics; Treatment; Vaccines

The chronology of the pandemic sequencing (NGS) on bronchoalveolar lavage fluids

of three Wuhan patients [6]. The complete genome
An outbreak of acute community-acquired atypical
sequences of SARS-CoV-2 clustered in a distinct
pneumonia of unknown aetiology was reported in
clade from SARS-CoV within the genus Sarbecovirus.
Wuhan, the capital of Hubei province in central
The draft genome sequence was released on 10 Janu-
China, in December 2019. The initial cluster of cases ary 2020, 10 days after the outbreak was announced.
was related to the Huanan seafood wholesale market As an escalating number of local cases was reported
where wild game animals were also sold [1]. During in Wuhan, a family cluster was identified in Shenzhen,
subsequent investigation, severe acute respiratory syn- a city in southern China 550 miles from Wuhan,
drome (SARS) coronavirus 2 (SARS-CoV-2) was between 10 and 15 January 2020 [7]. Six members of
detected in 33 out of 585 environmental samples this family had returned from a trip to Wuhan
taken from the market [2]. However, 45% of the between 29 December 2019 and 4 January 2020.
cases with onset before 1 January 2020 had no appar- Two of them visited a local hospital where a paediatric
ent link to this market [3]. Retrospective molecular relative was hospitalized for pneumonia. Five of these
clock inference studies using phylogenetic analysis six family members were clinically and/or virologically
suggested that the earliest cases likely emerged diagnosed with Coronavirus Disease 2019 (COVID-
between October and November 2019 [4, 5]. The cul- 19) after returning to Shenzhen. In addition, a seventh
prit virus was identified using next-generation family member, who did not go to Wuhan or visit wet

CONTACT Kelvin Kai-Wang To kelvinto@hku.hk; Kwok-Yung Yuen kyyuen@hku.hk

Supplemental data for this article can be accessed https://doi.org/10.1080/22221751.2021.1898291
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrest-
ricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
508 K. K-W. To et al.

markets in the preceding 14 days, became infected a large polyprotein pp1ab, which is proteolytically
after staying in the same household with infected rela- cleaved into 16 non-structural proteins (Nsps) critical
tives. This familial cluster provided clear evidence of for viral replication [16]. Towards the 3′ end of the
person-to-person transmission and inter-city spread genome, the S, E, M, and N genes encode key struc-
by air travel. Furthermore, the report of an imported tural proteins found in the mature virion [18]. The
case in Thailand on 13 January 2020, and subsequently spike (S) glycoprotein forms trimers on the virion sur-
other countries suggested that global dissemination face and binds to human angiotensin-converting
might have occurred earlier by frequent air travel. enzyme 2 (ACE2) receptor for cell entry [19]. It con-
By the end of January 2020, SARS-CoV-2 was tains two subunits S1 and S2 with a polybasic site
reported in 31 provinces in China, across East and PRRA at the junction, which enables effective cleavage
Southeast Asia, and to Europe and the United States. by furin and other proteases [5]. This multibasic clea-
Community transmission was detected in other vage site appears to be an important virulence factor
Asian countries, a large part of Europe, the Middle which may enhance virus replication and multiple tis-
East and the United States since February 2020 [8– sue tropism as in the case of avian influenza A(H5N1)
11]. Figure 1 gives a detailed account of the unfolding virus [20, 21]. Mutations in this site can attenuate
of the pandemic. By April 2020, the total number of pathogenicity in animal models and may be an attrac-
COVID-19 cases surpassed 1 million as more and tive option for designing live attenuated vaccines [21].
more countries entered partial or nation-wide lock Another cleavage site, called S2’, is located within the
down. The death toll due to COVID-19 reached 1 S2 region, and is cleaved by the transmembrane serine
million on 25 September 2020. By 22 December 2020, protease 2 (TMPRSS2) [22]. S protein contains major
with the Chilean army reporting 36 cases at its research immunogenic epitopes, particularly concentrated in
station in Antarctica, COVID-19 cases have been the N-terminal domain (NTD) and receptor binding
reported to affect all seven continents [12]. As of 4 Feb- domain (RBD) of the S1 subunit, which are targets
ruary 2021, there have been more than 103 million of neutralizing antibodies. The envelope (E) protein
confirmed cases with over 2 million deaths [13]. likely forms a viroporin, which is important for virus
assembly and release, and is also a putative virulence
determinant [23]. The membrane (M) protein is an
Taxonomy, genomic organization, and
abundantly expressed structural protein within the
replication cycle of SARS-CoV-2
lipid envelope that is also important for viral morpho-
SARS-CoV-2 belongs to the Betacoronavirus genus of genesis and interferon suppression [24]. Finally, the
the family Coronaviridae. This genus also includes the nucleocapsid protein (N) stabilizes the RNA genome
human respiratory pathogens SARS-CoV-1, Middle in a helical complex [25] and serves as a key target
East respiratory syndrome (MERS) coronavirus of adaptive immunity. In addition, there are a number
(MERS-CoV), human coronavirus (HCoV)-HKU1, of accessory proteins, the function of some of which
and HCoV-OC43 [14]. Together with the closely remains unknown. ORF3a may function as an inducer
related bat coronavirus RaTG13 and SARS-CoV-1, of apoptosis [26]. Both ORF6 and ORF8 are involved
SARS-CoV-2 is classified as a member of the Sarbecov- in interferon antagonism while ORF7a may be
irus subgenus of SARS-related coronaviruses [15]. involved in inhibiting cellular translation [27–29].
Rapid characterization of SARS-CoV-2 by electron ORF8 can bind to IL-17 receptor A (IL17RA) which
microscopy and NGS confirmed that it shared the may modulate the inflammatory response, and higher
structural features and genomic organization of blood levels of soluble IL17A has been associated with
other betacoronaviruses [6, 7]. It is a pleomorphic milder disease [30]. Interestingly, circulating variants
enveloped virus (size range: 60–140 nm) studded with loss-of-function deletions in SARS-CoV-2
with distinctive surface spikes. The positive-sense ORF3b, ORF7a/7b, and ORF8 have been found, indi-
single-stranded RNA genome of SARS-CoV-2 is cating that these are not absolutely essential for viral
around 29–30 kB in size and is organized as methyl- infection and may be remnants required for infection
capped-5’UTR-ORF1a/b-S-ORF3-E-M-ORF6-ORF7a of an unidentified intermediate host [31–34]. ORF9b,
/b-ORF8-N/ORF9b-ORF10-3’UTR-poly A tail [16] an accessory protein translated from an alternative
(Table 1). One of the earliest published genome, open reading frame within the N gene, interacts with
hCoV-19/Wuhan/IVDC-HB-01/2019 (GISAID acces- the host mitochondrial import receptor protein
sion number (EPI_ISL_402119), has a genome size of TOM70 and suppresses type I interferon response
29,891 bp. A study using ribosome profiling tech- [30, 35]. As for ORF10, it appears dispensable for cel-
niques showed the presence of additional upstream lular infection [36, 37].
and internal open reading frames (ORFs) [17]. The Stages of the replication cycle of SARS-CoV-2 have
genome lacks the hemagglutinin esterase gene found been rapidly inferred from empirical data and extant
in some other betacoronaviruses. ORF1ab, which knowledge of other betacoronaviruses. The first step
comprises two-thirds of the entire genome, encodes in cellular infection by SARS-CoV-2 is the binding
 Emerging Microbes & Infections 509

Figure 1. Chronology of events leading to the COVID-19 pandemic.

of S protein to the host cell surface entry factors such specialized double membrane vesicles (DMV). Within
as the membrane associated and soluble ACE2 recep- the DMV system, the complex operates to replicate
tor [38] which may be preceded by weaker binding of genomic RNA and transcribe subgenomic RNAs,
the S protein to attachment factors such as heparan which are subsequently translated into structural pro-
sulphate [39]. Other entry factors that facilitate attach- teins [50]. Viral assembly occurs within the endoplas-
ment or entry include neuropilin-1 [40, 41], the tyro- mic reticulum, Golgi, and intermediate complex
sine-protein kinase receptor UFO (AXL) [42], CD147 (ERGIC) where membranes studded with viral struc-
[43], high-density lipoprotein (HDL) scavenger recep- tural proteins interact with N-encapsidated viral geno-
tor B type 1 (SR-B1) [44], integrins [45, 46], angioten- mic RNA [51]. Pre-activation of S protein by host
sion II receptor 1 (AT1) and vasopressin receptor 2, furin protease may occur before mature viruses are
but their role in natural infection is currently unclear. released from the cell by exocytosis of secretory ves-
Proteases such as surface TMPRSS2 and endosomal icles. As in other coronaviruses, subgenomic RNAs
cathespsin L [46] cleave the S protein to activate are produced, and most subgenomic RNA consists
SARS-CoV-2 entry by endocytosis and membrane of a leader sequence in the 5’ untranslated region of
fusion [22]. Within the cell, the virus uncoats to the genome and connected to the S gene or other
release its genomic RNA into the cytoplasm for trans- genes in the 3’ end [50]. Hence, translated viral pro-
lation [47]. The translated pp1a and pp1ab polypro- teins are more abundant towards the 3’ end of the gen-
teins are proteolytically cleaved to individual Nsps, ome, which may affect the sensitivity of diagnostic
many of which form the replicase-transcriptase com- assays using them as targets for RT–PCR or antigen
plex [48, 49]. These complexes are localized within detection. One exception is the nsp1, which has been
510 K. K-W. To et al.

Table 1. SARS-CoV-2 gene products.

Gene
product Putative primary function Role in pathogenesis
Nsp1 Inhibit host protein translation; Degradation of host mRNA and Suppression of interferon response
disruption of mRNA export machinery to inhibit host gene gene
expression
Nsp2 Unknown
Nsp3 Polyprotein processing, de-ADP ribosylation, deubiquitination,
interferon antagonist, formation of double membrane vesicles
Nsp4 Formation of double membrane vesicles associated with replication
complexes
Nsp5 3C-like protease domain, polyprotein processing Inhibit interferon signalling
Nsp6 Formation of double membrane vesicles associated with replication Interferon antagonist
complexes
Nsp7 Accessory subunit of RNA-dependent RNA polymerase
Nsp8 Accessory subunit of RNA-dependent RNA polymerase; primase or 3′
terminal adenylyltransferase
Nsp9 RNA-binding protein with a peptide binding site [48]
Nsp10 Co-factor of nsp14 and nsp16 for methyltransferase activity Interacts with NF-κB-repressing factor to facilitate interleukin-8 (IL-8)
induction, which potentially increase IL-8-mediated chemotaxis of
neutrophils and overexuberant host inflammation [49]
Nsp11 Unknown
Nsp12 RNA-dependent RNA polymerase, nucleotidyltransferase
Nsp13 Helicase Potent interferon antagonist
RNA 5′ triphosphatase
Nsp14 Proof-reading exonuclease Potent interferon antagonist
RNA cap formation guanosine N7-methyltransferase
Nsp15 Endoribonuclease Interferon antagonist Potent interferon antagonist
Nsp16 Ribose 2′ -O-Methyltransferase, RNA cap formation
S Binds to host cell receptor
ORF3a Induce apoptosis [26]
ORF3b Interferon antagonist [33]
E Envelope forms a homopentameric cation channel May conduct Ca2+ out of the ERGIC lumen to activate the host
inflammasome [23]
M Membrane Inhibit type 1 and III interferon production by direct interaction with
RIG-I/MDA-5 and impeding downstream signalling [24]
ORF6 Potent interferon antagonist (block STAT1 and STAT2 nuclear
translocation) [29]
ORF7a/b Unknown
ORF8 Downregulation of MHC-1, binds IL-17RA Inhibit interferon pathway
N Viral RNA genome protection and packaging, Virus particle release
ORF9b Interacts with host protein TOM70 [30, 35] Inhibit type I interferon [35]
ORF10 Unknown; suspected membrane protein forming viroporin [36]

shown to be highly expressed and was found to be a coronavirus and the pangolin SARS related corona-
sensitive target for RT–PCR [52]. Direct RNA sequen- virus [55, 56]. However, another study suggested
cing also reveals the presence of non-canonical subge- that recombination may not be involved in the gener-
nomic RNAs in which the 5’ breakpoint is located ation of SARS-CoV-2, but the RBD of SARS-CoV-2
within the ORF1a gene [50]. As for the putative struc- shares the same ancestral trait as bat viruses [57].
tural RNA found in SARS-CoV-2, the 5′ UTR has sev- The divergence date between SARS-CoV-2 and bat
eral stem-loops (SL1–5) which may be involved in sarbecovirus has been estimated to be 1948 [57].
mediating viral replication as in other betacorona- Since its first detection in humans in December
viruses. The ORF1a-ORF1b junction has a pseudo- 2019, many mutations have been found throughout
knotted structure pivotal for programmed ribosomal the SARS-CoV-2 genome [58]. The mutation rate
frameshifting and translation of the ORF1ab polypro- has been estimated to be 1.1 × 10−3 nucleotide substi-
tein. The 3′ UTR has the s2 m motif, conserved octa- tutions per site per year [59]. The time of origin of
nucleotide and many unexpected folds [53]. SARS-CoV-2 was estimated to be late November
2019 [59]. The mutation rate is fastest at the S, N,
ORF1ab, ORF3a, and ORF8 genes [60, 61].
Virus evolution
In addition to inter-host genetic diversity, mixed
The origin of SARS-CoV-2 is still unknown. Recombi- viral populations can be present within an individual
nation is a frequent event for the viral subgenus Sarbe- patient. A variant initially present at low frequency
covirus, which contains SARS-CoV, bat SARS related in an individual can become the predominant viral
CoV, and SARS-CoV-2 [54]. Some studies suggested population during the course of illness. In our pre-
that the bat SARS-CoV-2-like coronaviruses are vious study, we demonstrated the emergence of the S
recombinants of lineages related to SARS-CoV and protein W152L mutation in a patient with severe dis-
SARS-CoV-2, and SARS-CoV-2 may result from ease [62]. In a study analysing the viral genomes from
recombinations between these bat SARS related patients in Austria, an intra-host minor variant was
 Emerging Microbes & Infections 511

found to be transmitted to others, and become the pre- transmission has been documented in Europe [71].
dominant viral variant in another patient [61]. Furthermore, the S N501Y variant can be selected in
virus adaptation experiments using Balb/c mice [72].
Several notable variants have emerged since the
Virus mutations and variants
beginning of the COVID-19 pandemic, including
SARS-CoV-2 has evolved into different clades and B.1.1.7 (VOC-202012/01), B.1.351 (501Y.V2) and P.1
lineages (Figure 2). Currently, there are three major (VOC202101/02) which were first reported from the
nomenclature systems for the different clades or United Kingdom, South Africa and Brazil, respectively
lineages. The GISAID and Nextstrain systems were [73]. These variants usually increase transmissibility
used since the beginning of the pandemic, and the and, thereby, rapidly replace existing lineages. Some
clades or lineages are defined by signature mutations. of these variants share certain critical mutations in
The GISAID clade is currently divided into S, L, and the S protein RBD. For example, N501Y is present
V, and different clades carrying the D614G mutation in the B.1.1.7, B.1.351, and P.1, while E484 K is present
(G, GH, GR, GV), and O. The Nextstrain is divided in the B.1.351 and P.1 in addition to N501Y and
into 19 (A, B) and 20 (A-J) according to the year D614G (Table 2).
and order when the clade emerged. Although GISAID One of the first major variants identified for SARS-
and Nextstrain nomenclatures are useful in under- CoV-2 were deletions at the S protein S1/S2 junction.
standing the virus evolution in a macroscopic scale, These were readily seen during passage in Vero E6
these systems are not able to delineate more detailed cells [21]. S1/S2 junction deletion variants have been
outbreak cluster information. The Pango lineage, found to be less virulent in a hamster model [21]. S1/
first proposed in July 2020, is a dynamic system, S2 junction deleted variants also naturally exist in
which takes into account whether the lineage is patients’ samples before any passage in cell cultures [74].
actively spreading or not [63, 64]. The Pango lineage ORF8 is a unique protein in SARS-CoV-2 [16] and
system has a much finer resolution than GISAID or is found to be immunogenic [75]. However, ORF8-
Nextstrain, and is particularly useful to capture the deleted or truncated mutants have been identified fre-
emergence of novel variants. quently. In a Singapore study, patients infected with
As the virus evolves, many novel variants have been ORF8-deleted mutants have milder disease than
found. The analysis of viral variants helps epidemiolo- those infected with wild-type SARS-CoV-2 [31].
gical investigations. For example, whole viral genome ORF3b deleted mutants have also emerged with the
analysis during the 2020 summer outbreak in Hong D614G mutation. Truncation of ORF3b confers the
Kong showed that the outbreak was most likely linked loss of its function of interferon antagonism [32].
to viral variants imported by travellers [65]. Further- D614G mutation was not reported in the initial out-
more, variant analysis can be used to identify factors break in China, but is now found in almost all strains
that affect transmissibility or virulence of the virus. globally. Several studies have evaluated the impact of
In vitro screening using serial passage or site-directed D614G on the SARS-CoV-2. Collectively, they show
mutagenesis identified mutations in the S protein that that D614G variant replicates to a higher titre in vitro
allow the virus to escape neutralization by convales- and in vivo, and transmits more efficiently, but does
cent plasma or infect cells more efficiently [66, 67]. not affect disease severity or confer a significant change
Long-term SARS-CoV-2 shedding in immunocom- in neutralizing activity of convalescent sera [66, 76–78].
promised individuals with acquired hypogammaglo- Mechanistically, D614 mutation affects the confor-
bulinemia can lead to a long duration of virus mation of the S protein, which allows more efficient
shedding and a larger genetic diversity with continu- binding to the human ACE2 receptor [79].
ous turnover of dominant viral species throughout There have been multiple outbreaks of SARS-CoV-
the course of infection. Deletion H69 and V70 in the 2 infection among mink farms in Europe [80]. Mink-
S protein NTD was reported in a B-cell depleted associated human infections have been identified [71].
patient which may be related to selection by convales- A unique lineage has been found in these mink-associ-
cent plasma therapy [68]. Furthermore, remdesivir ated human cases from Denmark, including 4
failure with D484Y mutation at RNA dependent mutations in the S protein (Δ69-70, Y453F, I692 V,
RNA-polymerase was also reported in a B-cell immu- M1229I). Y453 has been shown to be involved in
nodeficient patient with protracted SARS-CoV-2 receptor binding [66]. Preliminary investigation with
shedding [69]. 9 COVID-19 convalescent serum specimens showed
Viral variants can also emerge during circulation in a statistically significant reduction in neutralizing anti-
animals. Since SARS-CoV-2 can infect many animals body titre [73].
naturally or experimentally [70], there is always a The B.1.1.7 variant was first detected in September
danger of human-to-animal SARS-CoV-2 trans- 2020, spread rapidly in south-eastern England by
mission, followed by the genesis of mutants in animals December, and has become the predominant variant
which then jump back into human. Mink-to-human in the UK. This variant has increased transmissibility
512 K. K-W. To et al.

Figure 2. (A) Whole genome phylogenetic tree of betacoronaviruses. The tree was constructed by maximum likelihood method
with the best-fit substitution model GTR + F+R5 using IQTree2. Bootstrap values were calculated by 500 trees. SARS-CoV-2 are
highlighted in red. Human coronavirus 229E (NC_002645) was used as outgroup. (B) Whole genome phylogenetic analysis show-
ing different clades of SARS-CoV-2. The tree was constructed by maximum likelihood method with the best-fit substitution model
TIM2+F + I using IQTree2. Bootstrap values were calculated by 500 trees. Clade information as inferred by Nextstrain or Pango
lineage are shown. HK1 is the predominant lineage found during the 2020 summer peak in Hong Kong, while W4 is the predo-
minant lineage that is found in almost all local cases in Hong Kong since November 2020. The reference genome Wuhan-Hu-1
(GenBank accession number MN908947.3) is used as the root of the tree.

and is now found worldwide [81]. This variant is N501Y). Monoclonal antibody or nanobody targeting
defined by 17 mutations, including a non-synon- the S protein amino acid positions 417 or 484 showed
ymous S N501Y at RBD, and the P681H mutation reduced binding to the B.1.351 variant [83]. Further-
which is located in the furin cleavage site. However, more, neutralizing antibody against the B.1.351 var-
no change in neutralizing activity by sera of vaccine iant could not be detected in 48% of convalescent
recipients of the BNT162b2 mRNA vaccine was sera of COVID-19 patients [83].
found against pseudoviruses bearing the Wuhan refer- The P.1 variant has 17 unique mutations including
ence strain and the B.1.1.7 variant [82]. RBD E484 K and N501Y mutations has emerged [84].
The B.1.351 variant has rapidly increased in South This is a descendent of the lineage B.1.1.28.1, and now
Africa in late 2020. This variant possesses several known as the P.1 lineage which is mainly limited to
mutations in S protein NTD (L18F, D80A, D215G, Brazil, but has also been reported in Japan, Korea,
Δ242-244, R246I) and RBD (K417N, E484 K, and and Faroe Islands [85].
 Emerging Microbes & Infections 513

Table 2. Amino acid mutations and nucleotide deletions infection. Large quantities of particles, with the
present in each variant. majority of less than 5 microns, can be emitted during
United normal speech, and the amount is positively correlated
Kingdom South
(VOC- Africa Brazil with the loudness of vocalization [96]. Aerobiological
Variant 202012/01) (501Y.V2) (VOC202101/02) study showed that particles produced in the human
Pangolin lineage B.1.1.7 B.1.351 B.1.1.28.1 respiratory tract represent a continuum of sizes
(Lineage P.1)
Number of countries 93 45 15 instead of a sharp distinction into respiratory droplet
reported with (≥5 microns) or airborne aerosol (<5 microns). The
varianta
Genes concentration of respiratory droplets and airborne
orf1ab T1001I K1655N S1118L aerosol carrying SARS-CoV-2 should be inversely pro-
A1708D K1795Q
I2230T
portional to distance from the source patient. Short-
Del:11288:9 Del:11288-9 range airborne spread should be the predominant
Del:21765:6 route of SARS-CoV-2 transmission.
Del:21991:3
S N501Y D80A L18F In addition, contact with frequently touched sur-
A570D D215G T20N faces, shared items, and food that are contaminated
P681H K417N P26S
T716I E484K D138Y by infectious respiratory droplets likely represent
S982A N501Y R190S another route of transmission of SARS-CoV-2 [97].
D1118H A701V K417T
E484K One study found that 5% of the near-patient environ-
N501Y mental samples contained SARS-CoV-2 RNA with a
H655Y
T1027I
median viral load of 9.2 × 102 copies/mL [98], with
Orf3a G174C the highest contamination rates on patients’ mobile
Orf8 Q27* E92K phones, floors, bed rails and air exhaust vents [91–
R52I
Y73C 93, 98]. Though still considered controversial as a por-
E P71L tal of transmission, several outbreaks have been linked
N D3L T205I P80R
S235F to contaminated frozen food, their packaging
* stop codon. materials and storage environments [99]. The half-
a
According to the PANGO lineages website https://cov-lineages.org/ life of SARS-CoV-2 infectivity was 1.7–2.7 days at
global_report.html on 21st February 2021.
20°C, which is reduced to a few hours at 40°C [100].
At the highest viral load excreted by infectious
A key concern about viral variants is whether they
patients, viral particles remained viable for up to 28
increase the risk of reinfection or vaccine failures. In
days at 20°C on common surfaces such as glass, stain-
the first case of reinfection reported in August 2020,
less steel, and polymer banknotes [100]. The relative
the second episode was caused by a D614G variant
humidity also affects the rate of viral decay, which
[86]. In a reinfection case reported from Brazil, the
was most rapid at 65% relative humidity and slower
second episode was caused by the E484 K variant
either at lower (40%) or higher (75%) humidity [101].
[87]. Virus carrying the E484 K was shown to be less
Other routes of transmission, including faecal–oral,
susceptible to neutralization by sera from mRNA vac-
and contact with various body fluids including urine,
cine recipients [88].
tears, and breast milk, have been postulated [102–
106]. Indeed, oral SARS-CoV-2 inoculation can estab-
lish subclinical respiratory infection with virus shed-
Transmission routes
ding in the hamster model [107]. Human vertical or
SARS-CoV-2 is believed to spread predominantly via perinatal transmission from mother to babies is rare
short-range airborne aerosol, respiratory droplets, but possible [108].
and direct or indirect contact with infectious respirat- Vertical transmissions in high rise buildings by fae-
ory droplets. Airborne transmission of SARS-CoV-2 cal aerosols through chimney effect, wake effect and
has been elegantly demonstrated in the hamster minor leaks in sewage, vent pipes, or light wells were
model [89, 90]. Low level of SARS-CoV-2 RNA (con- reported [109]. However, the significance of these
centrations in air up to 3.4 × 103 RNA copies per m3 alternative routes of transmission in driving the com-
air sampled) could be detected in the air samples munity epidemic is still unclear.
obtained from the environment housing COVID-19
patients even in the absence of aerosol-generating pro-
Epidemiological characteristics
cedures [91–94]. Viable SARS-CoV-2 virus could be
isolated from air samples collected as far as 4.8 m The mean incubation period of SARS-CoV-2 infection
away from COVID-19 patients with estimated viral was 4.0–5.2 days, and incubation period of longer than
concentrations of 6–74 TCID50 units/L of air [95], 14 days has been reported [3, 110]. During the early
substantiating the hypothesis that aerosol dissemina- stage of the pandemic, the mean serial interval was
tion of SARS-CoV-2 may serve as a source of 4.0–7.5 days [3, 110, 111], the epidemic doubling
514 K. K-W. To et al.

Table 3. Histopathology and pathogenesis of COVID-19.

Organ Histopathology Features of vascular involvement References
Lung . Diffuse alveolar damage with lymphocytic/ monocytic . Perivascular cuffing by lymphocytes with fibrin/ hyaline [143, 151,
infiltrate together with intra-alveolar fibrinous exudate, thrombi seen within pulmonary vessels and capillaries 153]
hyaline membrane formation at acute stage. . Congested vessels
. Type II pneumocyte hyperplasia with interstitial fibrosis at
late stage
. Increase in pulmonary megakaryocytes

Heart . Small or multifocal lymphocytic infiltrate with dysmorphic . Epicardial capillaries with prominent lymphomonocytic [144, 151]
cardiomyocyte and rare necrosis (milder pathology when endotheliitis
compared with the lung) . Macrovascular or microvascular thrombi
. Eosinophilic myocarditis (rare) . Intraluminal megakaryocytes

Brain . Activation of astrocytes and microglia with infiltration of . Intravascular thrombi with perivascular microhaemorrhages [174-179]
cytotoxic T cell mainly in brainstem and meninges and intramural inflammatory infiltrates
. Occasional expression of viral antigen at cortical neurons . Multiple microscopic ischaemic infarct with or without
antigen expression at endothelium

Kidney . Acute tubular injury . Hemosiderin granules and pigmented casts, together with [143, 145,
. Interstitial fibrosis abundant erythrocyte with obstruction of peritubular 167]
. Podocyte vacuolation capillary lumen with activation of endothelium
. Loss of brush border in proximal tubule
. Focal segmental glomerulosclerosis
. Granulomatous interstitial nephritis

Liver . Histiocytic hyperplasia . Platelet fibrin thrombi in sinusoid, central vein or portal vein [143, 145,
. Focal macrovascular and microvascular steatosis . Megakaryocytes in sinusoid 167]
. Patchy hepatic necrosis in centrilobular and periportal . Sinusoidal congestion
areas . Ischaemic necrosis

Spleen White pulp depletion Splenic infarction [143]

Skin . Parakeratosis, acanthosis, dyskeratotic keratinocytes, . Dermal infiltrate with perivascular and intramural [145, 162]
necrotic keratinocytes, acantholytic clefts, lymphocyte lymphocyte in muscular wall of small vessels
satellitosis and pseudoherpetic of the epidermis . Occasional intravascular hyaline/ fibrin thrombi
. Vascular deposition of C4d by immunohistochemical
staining

Placenta Villous infarction, atherosis and fibrinoid necrosis of maternal [146]

vessels
Testis . Interstitial edema with leukocyte infiltration [147]
. Sertoli cells showed swelling, vacuolation and
cytoplasmic rarefaction, detachment from tubular
basement membranes, and loss and sloughing into
lumens of the intratubular cell mass

time was 6.5–7.4 days [3, 112], and the highly context- infected with SARS-CoV-2 [118, 119]. In ex vivo
dependent basic reproductive number (R0) was 2.2– human lung tissues, SARS-CoV-2 generated 3.2-fold
2.7 [3, 113, 114]. But estimating R0 with precision is more infectious virus particles than did SARS-CoV-
difficult due to the substantial proportion of unde- 1, but did not significantly induce host pro-inflamma-
tected cases and varying testing policies. Literature tory response [120], which explains the high pro-
on transmission heterogeneity is scarce. Heterogeneity portion of asymptomatic or mildly symptomatic
in infectious disease dynamics, where most individuals cases in the COVID-19 pandemic. Moreover, in con-
infect only a few others while a small subset of the trast to SARS-CoV-1 patients whose viral load in
population is responsible for the majority of new nasopharyngeal aspirates peaked at around day 10 of
cases, is commonplace. Retrospective history from symptoms [121], the viral load in the respiratory
135 cases between 21 January and 26 February 2020 samples of COVID-19 patients was highest during
in Tianjin, China, showed significant transmission the first few days of symptom onset [90]. It was esti-
heterogeneity with a coefficient of dispersion of 0.25 mated that presymptomatic transmission accounted
[115]. The estimated overall infection fatality ratio for 4.2–44.4% of secondary COVID-19 cases [122–
(IFR) in China was 0.66% which increased with age 125]. The secondary attack rate within Wuhan house-
[116]. This is similar to the IFR estimate of 0.6% holds was 15.6%, with the presympomatic cases being
inferred using the corrected IFR on the Diamond the most infectious [126]. In addition, the lack of herd
Princess cruise ship [117]. immunity at the early stage of the pandemic adds to
An important reason for the rapid spread of the susceptibility of the general population. The esti-
COVID-19 is the presence of asymptomatic and pre- mated seroprevalence rate in Wuhan was 3.2%−3.9%
symptomatic transmission. Asymptomatic or mildly in March 2020 [127–129], and similar figure of 4.1%
symptomatic cases constitute 30–60% of all patients was recorded in California in April 2020 [130].
 Emerging Microbes & Infections 515

Mask-off activities such as dining, singing, swim- The hyaline membrane was attributed to an increase
ming, and other physical activities are especially in vascular permeability (termed as “bradykinin
dangerous in overcrowded indoor venues with subop- storm”) and accumulation of hyaluronic acid in the
timal ventilation or contaminated frequently-touched alveolar space, leading to trapping of high volume of
surfaces that are poorly sanitized [131]. Thus out- water [154]. Moreover, serum autoantibodies directed
breaks have been reported as clusters in family against many immunomodulatory proteins including
homes, restaurants, bars, markets, religious premises, cytokines, chemokines, complement activation com-
cruises, carriers, construction sites, dancing studio, ponents, and cell surface proteins were found in a
schools, nursing homes, and healthcare facilities high throughput extracellular antigen profiling study
[132]. Several superspreading events have been high- which may add to the tissue damage by immune com-
lighted. A British individual who attended a confer- plex deposition and complement [155]. These auto-
ence in Singapore in January 2020 has spread the antibodies may also impair immune function and
virus across the UK, France, and Japan through the virological control by inhibiting immunoreceptor sig-
exposure at a ski resort, where 13 of the 21 exposed nalling. The presence of these autoantibodies includ-
people eventually tested positive [133]. From late Feb- ing those against interferons is strongly associated
ruary to early March 2020, an outbreak associated with with disease severity [156].
the Sunday worshipping event in a church caused Although SARS-CoV-1 is more virulent based on in
61.3% of the 8162 confirmed COVID-19 in the Repub- vitro studies in terms of replication and cell damage
lic of Korea [134, 135]. The outbreak related to an [142, 148], SARS-CoV-2 appears unique in causing
index patient on the Diamond Princess cruise ship endotheliitis [152, 157], as evident by viral particles
has led to the quarantine of the passengers and cruise in vascular endothelium using electron microscopy
members at the Port of Yokohama in Japan, on which [157]. Soluble endothelial markers such as angiopoie-
696 of the 3711 passengers (18.8%) tested positive for tin-2 level are positively correlated with severity of
SARS-CoV-2 [118]. In fact, using a susceptible– COVID-19 [158]. Furthermore, endotheliitis increases
exposed–infectious–removed (SEIR) model that inte- propensity of thromboembolism and multisystem
grates dynamic mobility networks based on mobile involvement in COVID-19 patients [152, 157, 159,
phone data, a small minority of “superspreader” at 160]. Widespread thrombosis could be related to the
points of interest, most notably full-service restau- hyperinflammatory and hypercoagulopathy states,
rants, was found to account for a large majority of termed as “immune-thrombosis” [160, 161]. Direct
COVID-19 cases [136]. Selective implementation of endothelial injury triggers innate immune response,
specific restrictive measures at these critical control including activation of monocytes and complement
points of interest may be most effective. Hospital out- pathways, leading to deposition of terminal comp-
breaks at wards, dialysis centres, and outpatient clinics lement components C5b-9 (membrane attack com-
[137, 138] fuel the community outbreaks and vice plex), C4d [162], and mannose binding lectin
versa which adds to the burden of infection control. (MBL)-associated serine protease (MASP) in the
The long environmental survival of SARS-CoV-2, microvasculature [163]. Complement and endo-
high proportion of asymptomatic or mildly sympto- thelium activation induce the production of von Will-
matic patients, peaking of viral load before or at pres- ebrand factor (vWF) and factor VIII (FVIII), while
entation and therefore its high transmissibility reducing antithrombin and ADAMTS13 activity
warrants universal masking, diligent hand hygiene, [164]. Activated neutrophils release neutrophil extra-
and stringent social distancing measures for the suc- cellular traps to stabilize microthrombi [165–167].
cessful control before the herd immunity is built up Macro- and micro-vascular thrombosis and intralum-
by vaccination. inal megakaryocyte are more common features than
lymphocytic infiltration of myocardium in patients
with cardiac involvement [168]. In terms of lymphoid
Histopathology and pathogenesis of
organ involvement, T-cell depletion occurred in the
COVID-19
spleen [169]. Necrosis or atrophy in the lymphoid tis-
SARS-CoV-2 can cause infection in multiple organs as sue of lymph nodes and white pulp of the spleen are
shown in both in vitro and in vivo studies [139–150]. commonly observed extrapulmonary pathologies
with common histopathological features summarized [170].
in Table 3. Autopsy showed that pulmonary involve- COVID-19 may affect the central nervous system
ment with diffuse alveolar damage together with due to indirect effects of cytokine storm or suspected
hyaline membrane formation and pulmonary micro- direct virus invasion. The S1 protein can cross the
emboli are the most prominent acute histopathologi- blood brain barrier in a mouse model. Furthermore,
cal findings [151] (Figure 3). These features were intranasally administered S1 also entered the brain
often associated with high inflammatory cytokines with significant uptake at olfactory bulb and hippo-
and increased angiogenesis in fatal cases [152, 153]. campus, although at levels around 10 times lower
516 K. K-W. To et al.

Figure 3. Histology of lung tissue section. (A) Image of hematoxylin and eosin (H&E) stained lung tissue shows diffuse alveolar
exudation and inflammatory infiltration; a medium size blood vessel containing thrombus which almost blocks the entire lumen
(arrow heads). Scale bar = 500 µm. (B) Magnified H&E image shows severe hyaline membrane formation in the alveolar space
(open arrows). Scale bar = 200 µm. (C) Magnified H&E image shows severe mononuclear immune cell infiltration in the alveolar
space (solid arrows). Scale bar = 50 µm. (D) Immunofluorescence stained SARS-CoV-2 nucleocapsid (N) antigen in alveoli (white
arrows); the insert image showing a few N protein expressing cells in a small bronchial lumen. Scale bar = 100 µm.

than that after intravenous administration [171]. The hamsters, suggesting that direct neuronal invasion by
endotheliitis and systemic inflammatory response syn- virus is possible [183].
drome with neuronal activation are also postulated to
explain the neurological manifestations [172, 173].
Immunological profile of patients with
Autopsy studies of the brain showed that ischaemic
COVID-19
infarct with perivascular microhaemorrhage together
with neutrophilic plugs or intravascular microthrombi Innate immunity is the first line of defence against
were common features [174–179]. Similarly, radio- infection. Yet SARS-CoV-2 may evade innate immu-
logical features of vascular inflammation were nity by antagonizing host interferon response. Viral
observed in magnetic resonance imaging (MRI) of proteins that have been shown to antagonize inter-
the brain [180]. T-cell lymphocytic infiltrates were feron response include Nsp1, Nsp3, Nsp12, Nsp13,
commonly seen at perivascular, parenchymal as well Nsp14, Nsp15, ORF3, and ORF6 [27, 142, 184–186].
as leptomeningeal areas with microglial and astrocyte Furthermore, the frequency of dendritic cells, T cells,
activation [167, 169, 175, 178, 179]. Clinical improve- NK cells, and monocytes was significantly reduced in
ment of encephalopathic symptoms with steroid tends the peripheral blood of acute patients when compared
to suggest a dominant role of inflammatory response with healthy donors [187]. In particular, decrease in
[181]. The detection of SARS-CoV-2 in the brain levels of CCR2 expression in dendritic cells may lead
and cerebrospinal fluid by immunohistochemical to poor maturation on stimulation [188], further redu-
staining and RT–PCR yield inconsistent results cing levels of interferons, and hence poor stimulation
[167], as viral RNA detected from brain biopsy may of CD4+ and CD8+ T lymphocytes during acute phase
come from vascular endothelium instead of neurons. of infection [187]. Furthermore, infiltration of mono-
The localization of S antigen and visualization of cytes/macrophages in lungs can increase pro-inflam-
virus-like particles at the endothelium were observed matory cytokines and chemokines such as IL-6 and
in some patients with endotheliitis while only few IP-10, which fuels the cytokine storm [189]. Th2 cyto-
could demonstrate S protein expression in cortical kines, such as IL-5 and IL-13, are elevated in patients
astrocytes [174, 182]. The more consistent finding is with severe COVID-19 [190].
the expression of virus nucleocapsid antigen in olfac- For humoral adaptive immune response, most
tory sustentacular and horizontal basal cells in some recovered patients develop SARS-CoV-2-specific
patients and also in the olfactory neurons in infected IgA, IgG, and IgM response not only against S
 Emerging Microbes & Infections 517

(including RBD) and N but also other non-structural seasonal coronaviruses [210]. SARS-CoV-2-specific T
proteins [191–193]. The peak antibody response lymphocytes (CD4+, CD8+) decreased with half-
appears at around 1 month [194] and is higher lives of 3–5 months [198].
among patients with more severe disease [195]. Most The overall clinical phenotype of COVID-19 is
studies showed a static or slow decline in neutralizing determined by the degree of early control of viral
antibody and IgG response after few months [196, load by innate and adaptive immune responses, the
197], while IgA and IgM declines more rapidly inflammatory and apoptotic damage of cells triggered
[194]. One study estimated the half-life of S protein by the burden of virus, the functional reserve of the
IgG to be 140 days [198]. Notably, some patients affected organs and the compensatory regenerative
who recovered rapidly showed increasing titres over or reparative power of the host tissues.
time [196]. The IgG and IgM levels in saliva correlate
with those of serum [194]. There is also a difference in
Clinical manifestations
antibody response between adults and children. While
adults develop antibody against both N and S proteins COVID-19 is primarily a respiratory disease which
equally well, children have a stronger anti-S antibody can manifest as acute upper or/and lower respiratory
response than anti-N antibody [199]. S-specific mem- tract syndrome of varying severity. The symptom
ory B lymphocytes showed increase in abundance over onset of COVID-19 is more likely to be gradual
time, suggesting that patients can develop rapid anti- than the abrupt onset in influenza. The patient can
body response during reinfection, as was seen in our manifest with asymptomatic virus shedding, or a
previously reported reinfection case [198, 200, 201]. self-limited syndrome of fever, fatigue, myalgia,
The exact duration of detectable serum neutralizing arthralgia, rhinorrhoea, sore throat, and/or conjunc-
antibody titre after natural infection or vaccination tivitis at one end of the spectrum. But it can also
still awaits long-term follow-up study. progress to persistent fever, cough, hemoptysis, silent
Cases of reinfection have been reported [86], and hypoxia, chest discomfort or pain, respiratory failure,
neutralizing antibody could not be detected at pres- or even multiorgan failure [211, 212]. Impairment of
entation of the second episode of infection 5 months smell (hyposmia, anosmia, and parosmia) or taste
after the first episode [201]. Magnitude and duration (dysgeusia) has been recognized as important che-
of persistence of IgG or neutralizing antibody mosensory disturbances in COVID-19 [213]. Non-
correlate with severity of COVID-19 in some studies conductive olfactory dysfunction (OD) may be the
[193, 195]. sole manifestation [214]. Other extrapulmonary
For cell-mediated adaptive immunity, SARS-CoV-2 manifestations include diarrhoea, lymphopenia,
leads to T-cell lymphopenia and functional impair- thrombocytopenia, deranged liver and renal func-
ment of both CD4+ and CD8+ T cells during the tion, rhabdomyolysis, meningoencephalitis, stroke,
acute stage [187, 202]. Total CD4+ and CD8+ T cells seizure, Guillain–Barré syndrome, cardiac arrhyth-
are reduced in both mild and severe diseases, but par- mia or heart block, pancreatitis, Kawasaki disease
ticularly lower among severe cases [203]. SARS-CoV-2 like multisystem vasculitis, skin rash or chilblain-
specific CD4+ and CD8+ T cells can be detected in like lesions, thromboembolism, and acute thyroiditis
about 50% of patients during the acute period and [215–217]. In an analysis of 72314 COVID-19 in
>80% of patients in the convalescent stage [204, China up to 11 February 2020, 81% of the laboratory
205]. The development of SARS-CoV-2 specific T- confirmed patients had mild to moderate illness, 14%
cell response is impaired among patients with severe had severe disease, and 5% were critically ill requir-
COVID-19 [204]. There is a higher frequency of mem- ing intensive care [218].
ory CD4 than CD8 T-cell responses against N and Clinical improvement of mild and moderate cases
RBD [204]. SARS-CoV-2-specific CD8+ memory T- generally occurs around 10 days after symptom
cell responses are directed primarily to the S and M onset which coincides with at least 1 log reduction
proteins especially among those who recovered from of respiratory viral load [219] and the rise of serum
severe COVID-19. The levels of TH17 cells were elev- antibodies against N or S protein [220]. However,
ated among severe cases [206]. The frequency of T fol- clinical deterioration of moderate disease to respirat-
licular helper cells during the convalescent phase is ory failure may also occur at this time with persistent
higher among patients with severe disease than those salivary viral load and increasing lymphopenia in
with milder disease, which correlated with the neutra- these worsening patients [221, 222]. Chest radiograph
lizing antibody titre [207]. SARS-CoV-2 specific T-cell or lung CT scan typically showed bilateral multifocal
immunity can also be found in up to 83% of non- and peripheral ground glass opacities (Figure 4)
COVID-19 individuals which may suggest some which may deteriorate to dense consolidation in pro-
cross reactive T-cell immunity that may or may not gressive disease [223]. The radiological abnormalities
be protective [208–210]. Pre-existing memory CD4+ usually peak by 2 weeks after symptom onset and
T cells are cross reactive for SARS-CoV-2 and other are replaced by fibrosis with recovery [224]. The
518 K. K-W. To et al.

prognosis of COVID-19 is worse in elderly obese Laboratory diagnosis

males or those with comorbidities such as hyperten-
One of the most important aspects in curbing the
sion, diabetes mellitus, atherosclerotic vascular dis-
spread of the virus and improving the prognosis is
eases, vitamin D deficiency, and other chronic
rapid yet accurate diagnosis of infection followed by
medical illness [225]. Patients with X-linked putative
timely isolation, contact tracing and treatment. Mol-
TLR7 loss of function variant, autoantibody against
ecular testing is now the mainstay of diagnosis, sup-
type 1 interferons, defective mutations of IFNAR2 or
plemented by point-of- care antigen testing (POCT)
other interferon signalling genes, antiviral restriction
[243]. Antibody detection aids in assessment of immu-
enzyme activators (OAS), blood group A and the
nity, contact tracing, and disease prevalence in the
associated SNPs found by GWAS are associated with
population. A multitude of diagnostic platforms,
severe disease [226–230]. Acute kidney injury affected
both in-house and on commercial platforms, are
>20% of hospitalized patients and >50% of those
developed to meet these demands [244].
requiring ICU admission, but the rate varies widely
between studies [231]. The overall crude fatality rate
for laboratory confirmed cases is about 2% [232],
Specimen collection
but can be as high as 21.9% in patients over 80 years
of age [233]. Early bacterial and fungal superinfections Viral load in the respiratory tract is highest at or soon
are uncommon but late superinfections, including after symptom onset [222], and it decreases at a rate of
invasive pulmonary aspergillosis, were reported in 1 log10 per week [90]. Testing nasopharyngeal aspirate,
those with prolonged ICU stays and treatment by nasopharyngeal swab, or throat swab is adequate for
immunomodulatory agents [234]. early-stage infection, especially asymptomatic or
Follow-up study at 6 months after COVID-19 mild upper respiratory tract infections. Patients with
symptom onset showed that over 60% of these patients lower respiratory tract symptoms should send sputum
had persistent symptom of fatigue or muscle weakness to enhance sensitivity [245]. Though broncho-alveolar
[235]. Sleep difficulties (26%), anxiety or depression lavage (BAL) showed the highest positive rate among
(23%) were not uncommon [235]. Other symptoms different respiratory specimens, it is only indicated
include smell or taste disorder, palpitations, joint in those with severe lower respiratory tract involve-
pain, dizziness, diarrhoea, vomiting, and chest pain ment when the nasopharyngeal and throat specimens
which constitute a constellation of symptoms termed are tested negative [246]. Posterior oropharyngeal
“post-acute COVID-19 syndrome”. This group of secretion (POS) or deep throat saliva is increasingly
patients are also called “COVID long haulers” [236]. studied as it represents a pooling of posterior naso-
Those with severe disease requiring respiratory sup- pharyngeal, oropharyngeal and lower respiratory
port had lung diffusion impairment [235]. However, secretions during the supine position during sleep,
little objective evidence of post-acute COVID-19 syn- when taken in the early morning before breakfast
drome can be found on investigations which bear and mouth rinsing [90, 247]. It can be self-collected
some similarity to chronic fatigue syndrome or myal- by patients with instructions, reducing patient dis-
gic encephalomyelitis. The cause was speculated to a comfort, circumventing swab shortage, and minimiz-
dysregulated immune system which was activated to ing aerosol exposure for health care professionals.
fight SARS-CoV-2 but failed to dampen down after- The cost of collecting POS could be 2.59-fold lower
wards [237]. The other differential diagnoses are either than nasopharyngeal specimen [248]. The sensitivity
an autoimmune process triggered by SARS-CoV-2 or is comparable with nasopharyngeal swab in properly
a persistent SARS-CoV-2 infection which cannot be collected specimens by cooperative patients [249–
easily detected by conventional testing [238]. The 252] . The sensitivity does not vary much between
relationship between the presence of serum autoanti- early morning and at least 2 h after meal [253].
bodies and the post-acute COVID-19 syndrome For non-airway specimens, viral shedding by RT–
requires further investigations. PCR was found in faecal material in 40.5% of patients
In general, children have a shorter and milder after the first week of symptom onset and could per-
disease than adults [239, 240]. However, a rare but sist for 3 weeks or more [254]. Presence of viral RNA
life-threatening Kawasaki-like disease, known as in the sewage system may provide a cost-effective and
multisystem inflammatory syndrome in children non-invasive way of monitoring the disease spread
(MIS-C) or paediatric inflammatory syndrome tem- within the community and may serve as an early
porally associated with SARS-CoV-2, are seen warning system for population that lacks access to
during the convalescent phase of the illness [97, healthcare [255]. Enveloped virus has affinity to bio-
241]. Children with MIS-C are usually older, has a solids which may allow testing of sludge at sewage
lower lymphocyte and platelet count, and a higher treatment plants with better sensitivity than testing
level of CRP and ferritin than those with Kawasaki influent [256]. Composite sampling is used in most
disease [242]. studies [257]. Sewage sample should be concentrated
 Emerging Microbes & Infections 519

Figure 4. Typical changes of COVID-19 pneumonia on lung computed tomography showing bilateral multifocal patchy ground
glass opacities: (A) transverse view; (B) coronal view.

followed by efficient RNA extraction to prevent inhi- High throughput and automated commercial plat-
bition of molecular assays [256]. Viral RNA can also forms have been developed for molecular SARS-
be detected in the blood in about 30% of severe CoV-2 diagnosis. Molecular POCT enables rapid test-
patients, but the detection rate is much lower in ing near the site of collection in areas with little labora-
milder cases [90, 246]. Even without ocular symp- tory support [249]. To improve diagnostic sensitivities
toms, the conjunctival secretion may contain a of molecular assays, clustered regularly interspaced
small amount of SARS-CoV-2 RNA in around 8% short palindromic repeat (CRISPR)-based technology
of patients [258]. Viral RNA is rarely found in the has been employed by coupling with Cas enzyme
urine [246]. [268]. Target enrichment sequencing by NGS with
To accommodate the large amount of specimen for nanopore or Illumina technology can unravel the
screening asymptomatic population, pooling of clini- entire genome within a few days. Sharing of genetic
cal specimens, up to 5–30 samples per pool [259, data facilitates tracking of disease spread, understand-
260], is an additional strategy to cope with reagent ing of disease transmission route, monitoring of viral
shortages, at the expense of possibly longer processing genome evolution and detecting novel variants.
time and reduced diagnostic sensitivity of the weakly
positive sample [261]. It is efficient only when the
expected positive sample number is low as positive Antigen detection
pool requires individual retesting [262]. Strategic N is abundantly expressed in SARS-CoV-2 and is thus
retesting of a defined group [263] and the use of math- widely used as the target for COVID-19 antigen test
ematical models to stratify pool size by age groups [269]. Detection is achieved by capturing viral antigen
based on their respective disease prevalence may in clinical specimens by monoclonal antibodies fixed
improve efficiency [264]. on a membrane in colorimetric lateral immunoassays.
Though this assay can be delivered as POCT in an out-
patient or even non-healthcare setting, it has low sen-
Molecular testing sitivity when compared with RT–PCR assays
Reverse-transcriptase polymerase chain reaction especially for samples with low viral load. In general,
(RT–PCR) is the most widely used technique. Poten- antigen test is negative when their Ct values on quan-
tial molecular targets for SARS-CoV-2 include struc- titative RT–PCR are more than 25, although the Ct
tural proteins (e.g. S, E, helicase (hel), N, M and values vary with different assays and conditions [270].
non-structural regions such as the RNA-dependent
RNA polymerase region (Rdrp), and other ORF1ab
Antibody detection
targets [52, 265, 266]. There is currently no consen-
sus on which gene confers the best diagnostic per- While antibody testing is generally not useful for acute
formance. Presently, one bat SARS related CoV management, it can be used for retrospective diagnosis
conserved and one SARS-CoV-2 specific target and seroprevalence study to understand herd immunity
regions are recommended to mitigate effect of ran- [271]. Commonly employed techniques are lateral flow,
dom mutation or genetic drift while maintaining chemiluminescent, immunofluorescent, and enzyme-
specificity [265]. However, mutations can affect the linked immunosorbent assays [272, 273]. Median sero-
sensitivity of detection by RT–PCR. For example, conversion times following symptom onset are 11 days
mutations in the S gene of the UK variant B.1.1.7 for total antibodies, 12 and 14 days for IgM and IgG
has led to the failure of some RT–PCR primers tar- respectively [220]. After 14 days, 56–97% of patients
geting the S gene [267]. develop IgM and 91–100% of patients develop IgG
520 K. K-W. To et al.

[274], with no significant time difference between IgM Vero E6 cells which have abundant ACE2
and IgG response [275]. IgM peaks at around 3 weeks expression are commonly used for virus isolation
after symptom onset and falls to baseline level after [142]. Vero E6 cell line that expresses TMRPSS2 can
day 36 [276]. The duration of IgG or neutralizing anti- result in better culture yield and reduce the likelihood
body positivity remains controversial. Some study of in vitro selection of S1/S2 junction site deletion
showed decrease in neutralizing antibody titre within mutant [289, 290]. SARS-CoV-2 also grows in
3 months after symptom onset, while others showed human continuous cell lines such as Calu3 (lung can-
no such decrease [200]. Antibody development against cer), Huh7 (liver cancer) and Caco2 (colonic cancer)
S and N protein is comparable by 1 month after infec- [142]. It grows modestly on U251 (glioblastoma)
tion [277]. Titre of anti-S or anti-S RBD antibody may which is not seen with SARS-CoV-1 [142]. Organoid
better reflect protection against reinfection [277]. systems such as bat and human intestinal organoids
Traditional neutralization assay requires manipu- are susceptible to SARS-CoV-2 and are developed to
lation of live virus and necessitate biosafety level 3 lab- better study tissue tropism, the dynamics of infection
oratories. Pseudovirus neutralization assay using and testing of therapeutic targets. SARS-CoV-2 is suc-
vesicular stomatitis virus (VSV) expressing S protein cessfully cultured in human intestinal organoids from
of SARS-CoV-2 containing the RBD, can be used in a stool specimen with high Ct value of 33.6, demonstrat-
biosafety level 2 facilities [278]. Neutralizing antibodies ing possible enteric infection by oro-faecal route [141].
are directed towards the RBD and NTD. Both sites are
situated at the tip of the S protein. Surrogate virus neu-
Treatment
tralization assay based on antibody-mediated blockage
of RBD-ACE2 interaction has been developed [279]. Except in places where all infected cases are legally
Studies have shown serological cross-reactivity required for mandatory hospital isolation, most patients
between SARS-CoV-2 and SARS-CoV, with decreas- with mild symptoms require only home isolation, moni-
ing frequency of cross-reaction from N protein, S toring, and symptomatic treatment. Those with persist-
protein to RBD domain by enzyme immunoassay ent fever, fatigue, and dyspnoea would require
[52, 280], with no significant cross neutralization admission for full assessment, respiratory support, and
[281]. Cross-reactivity against other seasonal human targeted anticoagulation by low molecular weight
coronaviruses in SARS-CoV-2 infection has been heparin to prevent thromboembolic events. Since the
shown as well, though intensity is not as great as viral load peaks at the time of symptom onset or presen-
that with SARS-CoV [52, 281, 282]. tation [90], antiviral treatment is unlikely to work unless
Antibody test has also been used to assess whether given early when the disease is still mild. Remdesivir has
SARS-CoV-2 has circulated in the population before been shown to shorten the duration of hospitalization by
the isolation of the virus. A study from Italy reported 5 days in a randomized control trial which did not moni-
that anti-RBD antibody could be found in blood samples tor the serial viral load changes after treatment [291].
collected as early as September 2019 [283]. In the United The WHO Solidarity trial, a multinational trial with
States, 106 of 7389 of residual specimens from blood 11,330 adult patients, found that remdesivir, lopinavir-
donors collected between 13 December 2019 and 17 Jan- ritonaivir, interferon β-1a, and hydroxychloroquine,
uary 2020, tested positive for IgG against SARS-CoV-2, have little or no clinical benefit when given as monother-
and neutralizing antibody was detected in 84 of 90 of apy, especially when started at the stage of respiratory
these samples [284]. Though these studies suggest that failure [292]. However, a combination of interferon β-
COVID-19 may have emerged much earlier than the 1b, lopinavir-ritonavir and ribavirin was shown to
first RT–PCR confirmed case, the possibility of EIA shorten the duration of hospitalization and reduce the
cross-reactivity with other coronaviruses cannot be viral load by 2–3 log between day 6 and day 11 after
excluded. symptom onset if given early in a randomized control
trial [293]. Similarly, inhaled interferon β-1a was also
shown to improve symptoms in mild cases in another
Viral culture
randomized control trial without viral load monitoring
Infectiousness of SARS-CoV-2 in clinical specimens [294]. This is not unexpected because while SARS-
can only be demonstrated by cell culture assays in bio- CoV-2 is highly susceptible to interferons in vitro, the
safety level 3 facilities. Furthermore, cell culture is virus was shown to reduce type 1 interferon produced
essential for the evaluation of potential antiviral com- in ex vivo infected lung tissue explant [120, 295]. Fur-
pounds and vaccines [285]. Viral culture turned nega- thermore, about 13% of patients with severe COVID-
tive in 97% of patients by 10 days after symptom onset, 19 were found to have high titres of auto-antibody
coinciding with the time of seroconversion [286]. Dur- against type 1 interferons and especially against inter-
ation of live virus shedding is believed to be even feron-α [156].
shorter in faecal specimen [287]. Shedding is prolonged Though individual or cocktail neutralizing mono-
in severe and immunocompromised cases [288]. clonal antibody treatment has been shown to reduce
 Emerging Microbes & Infections 521

viral load when given early after symptom onset and during the epidemic surge and then disappeared with
before the appearance of serum anti-SARS-CoV-2 successful implementation of epidemiological control
antibody in non-hospitalized patients [296, 297], this measures. Successful epidemic control depends on
approach has not yet been shown to reduce morbidity stopping case importation, minimizing community
and mortality. A clinical trial of a monoclonal anti- dissemination by social distancing measures, early
body, LY-CoV555, did not show clinical benefit detection and isolation of cases by extensive testing,
among hospitalized patients [298]. Similarly, conva- rapid contact tracing and quarantine, and individual
lescent plasma with neutralizing antibody only protection by universal masking and diligent hand
improved clinical status of elderly with mild hygiene. The resulting reduction of case load will pro-
COVID-19 when given within 3 days of symptom tect our hospital and intensive care unit from paralysis
onset and was not effective after hypoxaemia devel- and prevent the burnout of healthcare workers. Control
oped in randomized clinical trials [299]. Additional at the border depends on minimizing the number of
treatment trials are still ongoing or being planned to flights from highly epidemic areas with dangerous
ascertain the clinical effectiveness of clinically virus mutants, and testing all incoming travellers with
approved drugs discovered in drug repurposing no exemption, enclosed transportation and quarantin-
studies such as ivermectin, umifenovir, favipiravir, ing them for 14–21 days till negative surveillance test-
camostat, nafamostat, teicoplanin, and bismuth com- ing. During the severe winter epidemic, city and even
pounds [223, 300, 301]. nation-wide lockdown with curfew to prevent gather-
While currently available antivirals have not ings is useful to enforce social distancing. The alterna-
demonstrated survival benefit, several immunomodu- tive way is to close or reduce the time of opening and
lators have been shown to improve survival. Dexa- occupancy of high-risk premises such as eateries, bars
methasone has been shown to reduce mortality by and fitness clubs where masks are often taken off.
about 30% in patients requiring oxygen supplemen- With sporadic clusters, district closure with mandatory
tation [302, 303]. Baricitinib, an inhibitor of Janus RT–PCR testing of everyone followed by another test-
kinase, was shown to improve survival in patients trea- ing at day 5–14 can be useful in stopping community
ted with remdesivir, with a hazard ratio of death of 0.65 transmission. Universal masking when outside home
[304]. Conflicting or preliminarily positive results is demonstrated to stop the asymptomatic infected
regarding the use of histamine receptor 2 antagonist individual from shedding virus and to prevent suscep-
famotidine, vitamin D, IL6 inhibitor tocilizumab and tible individuals from acquiring infection as hinted by
colchicine were reported [305–307]. Fluvoxamine, a the hamster model [90]. Although surgical masks
selective serotonin reuptake inhibitor with high only have a fairly high effectiveness in blocking aerosols
affinity for σ-1 receptor appeared to prevent clinical in the micron size range [311], it appears to be nearly as
deterioration when given as early treatment for mild effective as N95 respirator [312]. Universal masking is
COVID-19 [308]. An open-labelled randomized trial shown useful in community epidemiological studies
showed that patients treated with recombinant [313, 314]. Every case of unexplained fever or respirat-
human granulocyte colony stimulating factor have a ory symptom should undergo mandatory testing.
lower risk of progressing to acute respiratory distress Repeated testing is indicated if the symptom persists
syndrome, sepsis, or septic shock [309]. Additional as false negative may happen. While asymptomatic
therapeutic approach that may include the manipu- infection does occur, more than 80% of patients
lation of complement, neutrophil trapping function develop symptoms during the course of illness [315].
and TNF function are being discussed. More definitive Moreover, only around 10% of infected persons are
large randomized control treatment trials are needed to responsible for 80% of SARS-CoV-2 transmission.
confirm the usefulness of these immunomodulators. Thus catching this 10% by rapid multilayer contact tra-
Despite respiratory support by non-invasive venti- cing, early testing and quarantine of close contacts may
lation by bilevel positive airway pressure or continu- identify the related asymptomatic or presymptomatic
ous positive airway pressure, some patients will still cases to stop further transmission. Rapid multilayer
deteriorate and necessitate intubation and mechanical contact tracing, including non-close contacts and con-
ventilation. In those who failed positive end expiratory tacts of close contacts going back to more than 2 days
pressure and prone ventilation, extracorporeal mem- before symptom onset, may be value added. Such
brane oxygenation is the last step to support the labour-intensive contact tracing can be facilitated by
patient till spontaneous recovery [310]. a trained team with artificial intelligence analysing
data of mobile phone applications or electronic pay-
ment. But these should be conducted in a manner to
Public health measures
protect individual privacy [316]. Timely risk com-
We have shown that different epidemic waves in Hong munication and education through media and internet
Kong Special Administrative Region were due to differ- are extremely important to secure cooperation from
ent imported lineages of virus which became dominant the public to make epidemic control a success.
522 K. K-W. To et al.

Infection control [332]. Reprocessing of N95 surgical respirator for

reuse in performing aerosol generating procedures
The key measures of infection control against nosoco-
was also supported by the IDSA expert panel [333].
mial outbreaks of COVID-19 include a combination
The methods of reprocessing include the use of vapor-
of active surveillance for early case identification, iso-
ized, plasma, ionized hydrogen peroxide, ultraviolet
lation of suspected and confirmed case in the airborne
radiation, and steam sterilization [333]. Quantitative
infection isolation room (AIIR) with the implemen-
fit test of N95 mask was performed to determine the
tation of standard, contact, droplets, and airborne pre-
maximum frequency of reprocessing [333].
cautions, as well as contact tracing to identify the
Infection control training for proper donning and
potential secondary cases [317–320]. These infection
doffing of PPE is of utmost importance. Directly
control measures which had been proven to be effec-
observed donning and doffing was promoted to maxi-
tive in controlling SARS in 2003 were not as successful
mize the protection and reduce the risk of self-contami-
for COVID-19 [321], because asymptomatic infection
nation [334]. Simulation training has been used to
contributes to a significant part of transmission and
enhance competency and alertness of healthcare
that the viral load peaks around the time of symptom
workers, especially on the performance of high-risk pro-
onset. Thus universal screening of all hospital admis-
cedures such as cardiopulmonary resuscitation [335].
sions or outpatient attendance by RT–PCR is war-
ranted to reduce the risk of healthcare-related
outbreaks. The risk for nosocomial transmission is
Animal models
especially high when asymptomatic COVID-19
patients are placed in non-AIIR rooms, or/and put SARS-CoV-2 probably evolved from an ancestral bat
on high-flow oxygen or non-invasive ventilation virus and jumped to humans via an unknown inter-
[322]. Therefore, universal masking for healthcare mediate host [5]. SARS-CoV-2-related bat corona-
workers and hospitalized patients, if not medically viruses have now been found outside China,
contraindicated, in the clinical areas should be including Cambodia [336], Thailand [337], and Japan
enforced to reduce the risk of COVID-19 transmission [338]. Over the course of the pandemic, it has become
by respiratory droplets and short-range airborne route increasingly clear that SARS-CoV-2 has the potential to
[323]. In fact, universal masking in the clinical areas infect a wide range of animals. Natural human-to-ani-
can achieve zero nosocomial transmission of other mal transmission events involving dogs, cats, lions,
respiratory viruses such as influenza A, influenza B, tigers, and minks have been reported [71, 339–341].
and respiratory syncytial virus [324]. Universal mask- Surrogate entry assays suggest that the S glycoprotein
ing in the community also reduced the incidence of of SARS-CoV-2 has wide tropism for a variety of mam-
COVID-19 in the general population [325]. malian ACE2 receptors [342]. Therefore, it is not sur-
The overwhelming burden of hospitalized COVID- prising that efficient animal models for COVID-19
19 patients is another risk factor of nosocomial out- could be rapidly established [343]. The first of these
breaks. Alternative hospital sites such as temporary was the golden Syrian hamster (Mesocricetus auratus),
shelter hospital and convention halls have been built which was quickly identified as a suitable model based
or re-purposed in mainland China, Hong Kong on molecular docking analysis of its ACE2 with the
Special Administrative Region, the UK, the USA and SARS-CoV-2 RBD [89]. The clinical features of
Singapore as temporary measures to meet sudden COVID-19 in human are well replicated in hamsters,
surge in COVID-19 [326–328]. The infection control which demonstrate a mild-to-moderate disease course
logistics and workflow in these alternative sites should with histopathological evidence of pneumonia. There-
be carefully planned and implemented to minimize fore, hamsters are ideally suited to study the pathogen-
the risk of outbreak [327]. The ventilation system of esis of SARS-CoV-2. Viral load dynamics in infected
these alternative sites, especially the convention hall, hamsters echo those of humans. Hamsters are able to
was difficult to match with the hospital standard of transmit disease to each other via contact or non-con-
6–12 air changes per hour. Another parameter of ven- tact transmission, thereby facilitating transmission
tilation by volume of air per second per person of studies [344]. The key limitation is the relative paucity
around 60 L/s/person was considered acceptable as of specific antibodies for detecting hamster biomarkers.
recommended by World Health Organization [329]. Other small animal models for SARS-CoV-2
Appropriate use of personal protective equipment research include ferrets (Mustela putorius furo) and
(PPE) is associated with a decreased risk of COVID- mice (Mus musculus). Ferrets have a long pedigree
19 [330]. Full PPE includes use of N95 respirator, of use in influenza research and are also susceptible
cap, face shield, gloves, and isolation gown of ASTM to SARS-CoV-2 although the disease phenotype is
levels 1–3 were recommended [331]. However, critical quite mild and predominantly restricted to the upper
shortage of PPE, especially N95 respirator, was a glo- respiratory tract [345, 346]. Given their convenience,
bal problem during the initial phase of pandemic mice models have also been developed, although this
 Emerging Microbes & Infections 523

requires either virus adaptation to mouse ACE2 or adenovirus and human adenovirus 26/5 vectored vac-
humanized ACE2-expressing mice [343]. These have cines also induce high titres of neutralizing antibody
the disadvantage of modifying the disease phenotype, and strong cell-mediated immunity with at least 70%
especially in human-ACE2 transgenic mice which vaccine efficacy [359, 360]. Further analysis of a
have mild respiratory but severe brain disease. phase 3 clinical trial showed that an adenovirus-vec-
Depending on the promotor used, these human tor-based vaccine was more effective if the interval
ACE2-transgenic mice exhibit variable phenotypes, between the first and second dose was 12 weeks or
ranging from mild disease to severe disease with ence- longer [361]. While the phase 3 clinical data from
phalitis and even death [347, 348]. Mice humanized the beta-propiolactone inactivated whole virion vac-
with human ACE2 using CRISPR/Cas9 knockin tech- cine have not yet been published in peer reviewed
nology supports SARS-CoV-2 replication in the respir- journals, the data from phase 2 trials suggested that
atory tract and brain tissues but generally develop only the vaccine is safe and can induce neutralizing anti-
mild to moderate disease [349]. Adenovirus or adeno- bodies, but the data on cell-mediated immunity is lim-
associated virus-transduced mice develop self-limiting ited at this stage [362, 363]. All three kinds of vaccines
viral pneumonia, but has the advantages of being easy are likely to prevent severe symptomatic infection, but
to generate and could be quickly adapted for different may not be able to prevent upper airway infection or
mouse strains [350, 351]. Laboratory rabbits can be transmissions, and are not well tested in children or
infected with asymptomatic virus shedding [352]. pregnancy. The saponin-based recombinant trimeric
As the ultimate origin of SARS-CoV-2 is likely to be spike nanoparticle appears to induce the best serum
from bats, one group has also demonstrated efficient neutralizing antibody and reasonable cell-mediated
infection of a fruit bat model (Rousettus aegyptiacus) immunity but phase 3 clinical trial data have not
with the virus. Fruit bats showed minimal clinical fea- been published [364]. However, there are preliminary
tures of infection, but were capable of transmitting evidence that spike RBD virus mutants from South
infection [353]. Notably, pigs and chickens, which Africa and Brazil with E484 K mutation may reduce
are in close contact with humans, are not able to sup- the neutralizing antibody titres induced by these vac-
port productive infection, thus ruling them out as cines [365, 366]. But as long as these vaccines protect
intermediate hosts [353, 354]. vaccine recipients from severe disease, SARS-CoV-2
Small animal models such as those described above may just become another circulating common cold
are convenient, but definitive evaluation of pathogen- coronavirus when most of the global population has
esis, antivirals and vaccines requires non-human pri- developed herd immunity by natural infections, or
mate models. Rhesus macaques (Macaca mulatta), vaccination against the early Wuhan-related virus
cynomolgus macaques (Macaca fascicularis), African strains. Initial animal studies and phase 3 vaccination
green monkeys (Chlorocebus aethiops), and baboons trials did not reveal any vaccine enhanced disease or
(Papio) are all susceptible to COVID-19 [355, 356]. antibody-dependent disease enhancement [367].
Disease in non-human primates is typically mild, but Instead, vaccination within 3 days before or after
disease severity and viral shedding increases with age virus challenge in hamsters still showed varying degree
as in humans. of protection despite the lack of detectable neutraliz-
ing antibody titre at that juncture [367]. To maximize
protection of the available vaccines, further studies on
Vaccines
the effects of prime and boost approach by different
Over 70 SARS-CoV-2 vaccines developed from differ- combinations of vaccines are warranted. With the
ent vaccine technology platforms including inacti- increasing availability of safe and effective vaccines,
vated whole virion, live attenuated virus, nucleic the battle is to fight misinformation and vaccine hesi-
acid, virus vectors, and recombinant S protein, are tancy by strategic education and risk communication
already in clinical trials. Four vaccine candidates so as to achieve a herd immunity of 70–80%.
have published their phase 3 clinical data. While
they all appear safe in clinical trials, each has its merits
Epilogue
and demerits. The mRNA lipid nanoparticle vaccines
induce good serum neutralizing antibody and cell- Emerging coronaviruses from animals have caused
mediated immunity but requires stringent cold storage SARS in 2002–2003, MERS in 2012, and COVID-19
at −20 to −70°C [357]. Though this is a new technol- in 2019. These viruses have probably originated in
ogy, side effects are generally mild. Rare cases of ana- bats and gone through intermediate wild mammals
phylaxis, possibly due to polyethylene glycol, have before jumping into humans. We predicted in 2007
been reported after millions of doses have been admi- that “the presence of a large reservoir of SARS-CoV-
nistered [358]. Concerns of vaccine exacerbation of like viruses in horseshoe bats, together with the cul-
underlying medical illness in frail elderly aged over ture of eating exotic mammals in southern China, is
80 years are not yet substantiated. The chimpanzee a time bomb. The possibility of the reemergence of
524 K. K-W. To et al.

SARS and other novel viruses from animals or labora- Hong Kong Limited and Luminex Corporation. The other
tories and therefore the need for preparedness should authors declared no conflict of interests. The funding
not be ignored” [368]. Spillover of SARS-CoV-2 from sources had no role in study design, data collection, analysis
or interpretation or writing of the report. The correspond-
animals to humans appears to have happened in 2019. ing authors had full access to all the data in the study and
But unlike the other two highly pathogenic corona- had final responsibility for the decision to submit for
viruses, the highly transmissible SARS-CoV-2 is able publication.
to overwhelm the healthcare system, inflict psycho-
physical morbidities and mortalities, and disrupt our
socioeconomic activities. More extensive and sus- Funding
tained animal surveillance for novel coronaviruses, This study was partly supported by the Consultancy Service
monitoring of their evolution, and assessment of for Enhancing Laboratory Surveillance of Emerging Infec-
their risk of species jumping should be performed to tious Diseases and Research Capability on Antimicrobial
Resistance for Department of Health of the Hong Kong
understand the origin of SARS-CoV-2, the intermedi-
Special Administrative Region Government. The funding
ate animal host, and to prepare for the next epidemic. sources had no role in the study design, data collection,
The functions of many NSPs and ORFs of SARS-CoV- analysis, interpretation, or writing of the report.
2, and their roles in viral life cycle and pathogenesis,
are still uncertain. Unlike SARS which is usually
quite symptomatic, the viral and immunological ORCID
mechanisms underlying the generally milder symp- Kelvin Kai-Wang To http://orcid.org/0000-0002-1921-
toms or lack of symptoms in COVID-19 warrant 5824
more investigations. The types of samples and tests Siddharth Sridhar http://orcid.org/0000-0002-2022-8307
which can provide rapid, inexpensive and accurate Jasper Fuk-Woo Chan http://orcid.org/0000-0001-6336-
6657
diagnosis still need more research and development. Anna Jian-Xia Zhang http://orcid.org/0000-0002-5087-
With the early peaking of viral load, any effective anti- 3614
viral strategy must be able to suppress the viral load Kwok-Yung Yuen http://orcid.org/0000-0002-2083-1552
sharply and coupled with immunomodulatory agents
in order to improve the clinical outcome. Close moni-
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