Introduction
During the current pandemic, the resurgence of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) cases was due to the development of
novel variants of concern.1 Continuous viral
transmission globally has indeed enabled the virus to fine-tune its more
efficient adaptation in human populations, enabling its sustained
transmission.2,3 Genomic surveillance is thus
essential to monitor the continuous evolution of SARS-CoV-2 and to track
the emergence of novel variants or sub-variants. Since January 2020, a
collaborative network of the World Health Organization (WHO), national
authorities, and research institution have been tracking the evolution
of SARS-CoV-2.4
The Spike (S) gene of SARS-CoV-2 is under intense selective
pressure and thus, it is highly mutated. These mutations may result in
increased infectivity, transmissibility, and more resistant to
neutralizing antibodies of the novel variants.5Consistently, the emergence of variants of interest (VOI) or concern
(VOC) was characterized by unique sets of mutations in the Spikegene.2 Currently, the only VOC is the Omicron variant,
first identified in South Africa in early November
2021.4 In fact, the emergence of the Omicron variant
that successfully replaced the previously dominant Delta variant was
also marked by a number of unique mutations in the Spikegene.1,6,7
Multiple mutations and recombination events in the Spike gene
have led to the diversification of the Omicron variant into various
sub-lineages or sub-variants with possibly different phenotypic
characteristics.8,9 Few subvariants, including BQ.1
and XBB, may spread quickly across countries and evolve to be the most
resistant variant against neutralizing
antibodies.10,11 Possible recombination events between
Omicron and Delta variants have also been
identified.12
In addition to the Spike gene, mutations in the gene encoding the
non-structural proteins also play an essential role in SARS-CoV-2
evolution and adaptation.13 As a positive-sense RNA
virus, SARS-CoV-2 encodes its own enzyme to replicate the genome. The
primary RNA polymerase, namely RNA-dependent RNA polymerase (RdRp), is
encoded by nsp12 gene14 which forms the core
polymerase complex with nsp7 and nsp8 proteins.15 Thensp12 gene of SARS-CoV-2 contains both RdRp and N-terminal
nidovirus RdRp-associated nucleotidyltransferase (NiRAN)
domain.16 Several mutations have been identified in
the nsp12 gene, including S6L, P323L, and P323F, although the
effects of these mutations are currently unclear.13,17However, it has been shown that the P323L mutation may increase the
mutation rate of SARS-CoV-2.18 RdRp has also been the
target of various SARS-CoV-2 inhibitors, such as remdesivir, although
comprehensive analyses of the global SARS-CoV-2 genomic dataset showed
that potential remdesivir-escape mutations were very rare, indicating
its little selective pressure.19 Indeed, the RdRp
region demonstrated strong negative (purifying) selection during the
pandemic.20
Other two important proteins for the SARS-CoV-2 replication cycle are
proteases, encoded by nsp3 (papain-like protease, PLpro) andnsp5 genes (chymotrypsin-like main protease, 3CLpro; also known
as main protease, Mpro).21 PLpro and 3CLpro have been
shown to modulate the host innate immune responses by inhibiting
interferon regulatory factor 3 (IRF3) that resulted in attenuated
interferon (IFN) response.22 Both proteins have also
been identified as the targets of IgM antibodies and were associated
with the survival of critical Covid-19 patients.23 In
addition, both PLpro and 3CLpro are potential targets for antiviral drug
development.24 A systematic mutational scanning has
identified several residues within the 3CLpro protein which are critical
for its functionality. These residues are mutation-sensitive and could
serve as promising targets for inhibitors with low likelihood of
resistant development.25,26 Nirmatrelvir (PF-07321332)
is an orally available antiviral drug with selective binding to the
active site of 3CLpro27 and has shown benefits for
treating Covid-19 patients.28 We and others have
identified various mutations in PLpro and 3CLpro
regions17,29, which may pose challenge for antiviral
drug development.
Since the early Covid-19 pandemic, we and others have successfully
sequenced the full-genome of SARS-CoV-2 circulating in several regions
in Indonesia and performed genetic analysis of the isolated
viruses.30-40 These studies demonstrated that
B.1.466.2 lineage predominantly circulated in Indonesia during early
pandemic and only few isolates belonged to the B.1.319
lineage.35,38 The Delta variant emerged in April 2021,
subsequently replaced B.1.466.2 variant, and posed serious challenge to
public health control.35,37 Our previous study showed
that SARS-CoV-2 isolates detected during early pandemic had prominent
mutations in particular gene, including Spike, nsp3, andnsp12 genes.31,38
Our current study aims to analyse the evolutionary pattern and mutation
rate of the Spike, nsp12, nsp3, and nsp5 genes of various
SARS-CoV-2 variants circulating in Yogyakarta and Central Java
provinces, Indonesia. We also performed selective pressure analysis to
investigate which amino acid changes that are potentially maintained by
positive selection to provide insight of the importance of these sites
in virus evolution. This study provides more insights on genetic
variability of SARS-CoV-2 Delta and Omicron variants, particularly those
circulating in our regions (Yogyakarta and Central Java, Indonesia). To
our limited knowledge, our study is the first comprehensive and
comparative evolutionary analysis of Delta and Omicron variants
circulating in Indonesia.
Materials and Methods