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