Comprehensive genomic analysis of the SARS-CoV-2 Omicron variant BA.2.76 in Jining City, China, 2022

Objective This study aims to analyze the molecular characteristics of the novel coronavirus (SARS-CoV-2) Omicron variant BA.2.76 in Jining City, China. Methods Whole-genome sequencing was performed on 87 cases of SARS-CoV-2 infection. Evolutionary trees were constructed using bioinformatics software to analyze sequence homology, variant sites, N-glycosylation sites, and phosphorylation sites. Results All 87 SARS-CoV-2 whole-genome sequences were classified under the evolutionary branch of the Omicron variant BA.2.76. Their similarity to the reference strain Wuhan-Hu-1 ranged from 99.72 to 99.74%. In comparison to the reference strain Wuhan-Hu-1, the 87 sequences exhibited 77–84 nucleotide differences and 27 nucleotide deletions. A total of 69 amino acid variant sites, 9 amino acid deletions, and 1 stop codon mutation were identified across 18 proteins. Among them, the spike (S) protein exhibited the highest number of variant sites, and the ORF8 protein showed a Q27 stop mutation. Multiple proteins displayed variations in glycosylation and phosphorylation sites. Conclusion SARS-CoV-2 continues to evolve, giving rise to new strains with enhanced transmission, stronger immune evasion capabilities, and reduced pathogenicity. The application of high-throughput sequencing technologies in the epidemic prevention and control of COVID-19 provides crucial insights into the evolutionary and variant characteristics of the virus at the genomic level, thereby holding significant implications for the prevention and control of the COVID-19 pandemic. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10246-w.

The Coronavirus Disease 2019 (COVID- 19) is an acute respiratory infectious disease caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1].Since its outbreak in December 2019, it has posed a serious threat to global public health [2,3].SARS-CoV-2, a single-stranded positive-sense RNA virus, exhibits a high mutation rate, leading to the emergence of multiple variants of concern (VOC) as identified by the World Health Organization, including the Alpha variant, Beta variant, Gamma variant, Delta variant, and the Omicron variant.The Omicron variant, first detected in Africa in November 2021, has since become the dominant strain worldwide due to its significant mutations, contributing to its increased transmissibility [4,5].
With the Omicron variant's continuous evolution, several subvariants have emerged, namely BA.1, BA.2, BA.3, BA.4, and BA.5 [6,7].In August 2022, the BA.2.76 subvariant was identified in Liaoning Province, China, marking a new phase in the virus's adaptation.This subvariant's spread to other regions such as Sichuan, Chongqing, Hebei, and Gansu have underscored the ongoing challenges in managing the COVID-19 epidemic within China.T Against this backdrop, our study focused on the Omicron BA.2.76 subvariant, conducting whole-genome sequencing and genetic evolution analysis on specimens from 87 cases in Jining City in 2022.This investigation aims to deepen our understanding of the SARS-CoV-2's evolutionary dynamics and variant characteristics, thereby enhancing the data available for COVID-19 prevention and control strategies in China.
The emergence of the Omicron variant, particularly the BA.2.76 subvariant, underscores the need for a multifaceted response encompassing vigilant surveillance, continuous research, and adaptive public health strategies.Initial observations suggest that while the Omicron variant demonstrates enhanced transmissibility, its impact on disease severity and the effectiveness of existing vaccines may vary, necessitating ongoing evaluation and possible adjustments to vaccination policies.This context of rapid viral evolution and the emergence of new subvariants highlights the critical importance of localized genomic studies, such as ours, in contributing to the global understanding of COVID-19's trajectory and informing tailored response measures.

Specimen collection
From September 1, 2022, to October 4, 2022, throat swab specimens from suspected cases of early or communitydetected Omicron variant BA.2.76 outbreak infections were collected in Jining City, Shandong Province, China.The swab samples were placed in virus sampling tubes (Jinan Biobio Biotechnology Co., Ltd.), kept at 2-8℃, and transported to the microbiology laboratories of the Jining City Center for Disease Control and Prevention and/or the microbiology laboratories of the Rencheng District Center for Disease Control and Prevention within 2 h.

Nucleic acid detection
Throat swab specimens (200 µL) were obtained and underwent nucleic acid extraction using the qEx-DNA/ RNA Virus Nucleic Acid Extraction or Purification Kit (Xi'an Tianlong Technology Co., Ltd.) and the Libex 96 Fully Automatic Nucleic Acid Extractor (Xi'an Tianlong Technology Co., Ltd.).Nucleic acid testing was conducted using the Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Real-time Fluorescent Quantitative PCR) from Shanghai Bio-Germ Medical Technology Co., Ltd. and the Gentier Real-time Fluorescent Quantitative PCR Instrument (Xi'an Tianlong Technology Co., Ltd.).

Genetic sequencing
For the comprehensive analysis of SARS-CoV-2 nucleic acid in positive specimens, we employed the ULSEN ultra-sensitive coronavirus whole-genome capture kit (Beijing Weimai Future Technology Co., Ltd).This sophisticated kit facilitated the targeted capture and amplification of the entire SARS-CoV-2 genome.Subsequent to amplification, the resultant products underwent purification using the AMpure XP nucleic acid purification kit, also from Beijing Weimai Future Technology Co., Ltd.Library construction followed, leveraging the Nextera XT DNA Library Preparation kit and Nextera XT Index Kit v2 Set A, (Illumina, Inc. (USA)).Postlibrary preparation, the sequencing of the SARS-CoV-2 whole genome was executed utilizing the MiSeq Reagent Kit v2, 300-cycles, and the MiSeq sequencer, manufactured by (Illumina, Inc.).The data presented in the study are deposited in the National Microbiology Data Center (NMDC), accession number list in Supplementary Table S1.

Whole genome sequencing analysis
From September 1 to October 4, 2022, COVID-19 outbreak occurred in Jining City, Shandong Province, China.Whole genome sequencing of SARS-CoV-2 was conducted on specimens collected from infected individuals during this period, resulting in 87 complete SARS-CoV-2 genome sequences.The sequences ranged in length from 29,669 bp to 29,860 bp, and were designated as hCoV-19/Jining/JN01/2022 -hCoV-19/Jining/JN87/2022. The sequence coverage ranged from 99.2 to 99.9%.Analysis using Nextclade v 2.14.1 confirmed that all sequences belong to the Omicron BA.2.76 variant.The nucleotide sequence similarity among the 87 sequences ranged from 99.97 to 100% (Fig. 1A).When compared to the Wuhan-Hu-1 reference sequence, the similarity of the 87 sequences ranged from 99.72 to 99.74% (Fig. 1B).

Genetic evolution analysis
Utilizing 87 Omicron BA.2.76 sequences obtained from Jining City, we conducted a comprehensive genetic evolution analysis by constructing an evolutionary tree.This tree included reference sequences such as Wuhan-Hu-1, early isolates of the novel coronavirus, and variants like Alpha, Beta, Gamma, Delta, Omicron BA.1, Omicron BA.2, Omicron BA.2.76, Omicron BA.3, Omicron BA.4, and Omicron BA.5.The 87 sequences manifested as an independent evolutionary cluster, forming a branch with Omicron BA.2 and a sub-branch with Omicron BA.2.76.The evolution of the SARS-CoV-2 genome in these 87 Omicron BA.2.76 sequences from Jining City consistently corresponds to the Pangolin classification.The resultant evolutionary tree delineates two distinctive branches, implying the plausible existence of two principal transmission chains within the Omicron BA.2.76 lineage in Jining City (Fig. 2).

Non-coding region compilation analysis
In the 87 Omicron BA.2.76 sequences from Jining City, a C241T mutation was observed at the 241 nucleotide position within the stem-loop structure of the 5'UTR SL5B [8].Additionally, one sequence exhibited a G29692A mutation in the hypervariable region (HVR) of the 3'UTR [8].Utilizing RNAfold software, secondary structures for both the 5'UTR and 3'UTR were constructed.The C241T mutation in the 5'UTR and the G29692A mutation in the 3'UTR were identified within loop structures.Both mutation sites are not located in the stem structures of the 5'UTR and 3'UTR, not affecting the nucleotide pairing within these regions, thereby exerting minimal impact on the secondary structures of both areas (Fig. 3).
Within the 87 Omicron BA.2.76 sequences from Jining City, a consistent A28271T mutation was identified in the N translation initiation region.The nucleotide at position 28,271, situated at -3 in the N translation initiation region, underwent a change from A to T due to the  A28271T mutation (Fig. 4A).Upon analysis of SARS-CoV-2 variant sequences downloaded from GISAID, it was observed that the A28271T mutation is present in SARS-CoV-2 Alpha, Delta, and Omicron variants.

General overview
In the 87 Omicron BA.2.76 sequences from Jining City, a total of 69 amino acid residue mutations were observed, along with 9 amino acid deletions at three locations, and one termination mutation.Amino acid mutations occurred in 18 proteins, with the S protein exhibiting the highest mutation frequency-32 amino acid positions were altered, and one location had a deletion of three amino acid residues.Following S protein, the N protein had 5 amino acid mutations, including one location with a deletion of three amino acids (Table 1).

ORF8 protein termination mutation analysis
The open reading frame 8 (ORF8) in all 87 Omicron BA.2.76 sequences from Jining City exhibited a C27972T mutation, resulting in the conversion of the nucleotide coding for the 27th amino acid in the ORF8 protein from CAA to the stop codon TAA.Consequently, the encoded protein underwent a termination mutation, generating a truncated ORF8 protein named ORF8 Q27 Termination Protein.ORF8 Q27 Termination Protein comprises only the first 26 amino acids of the ORF8 protein, including the complete signal peptide's initial 17 amino acids and the first 9 amino acids of the N-terminal immunoglobulin-like domain (Fig. 4B).

N-Glycosylation analysis
Utilizing NetNGlyc-1.0 software to analyze protein N-glycosylation sites, the 87 Omicron BA.2.76 sequences from Jining City exhibited the T24I mutation in the papain-like protease (Plpro), resulting in the alteration of NIT22-24 to NII22-24 and the loss of the N22 glycosylation site.Additionally, the S protein in the 87 sequences had the T19I mutation, leading to the modification of NLT17-19 to NLI17-19 and the loss of the N17 glycosylation site.Furthermore, the Y248N mutation in the S protein resulted in the transformation of YLT248-250 to NLT248-250, introducing an additional N248 glycosylation site (Table 2).

Phosphorylation analysis
Phosphorylation site analysis using NetPhos-3.1 revealed several modifications in the 87 Omicron BA.2.76 sequences from Jining City.Notably, the non-structural protein 1 (NSP1) lost the S135 phosphorylation site, and the papain-like protease (Plpro) lost the T24 phosphorylation site while gaining an S489 phosphorylation site.The NSP6 protein lost the S106 phosphorylation site, the helicase lost the S263 phosphorylation site, and the S protein lost phosphorylation sites at T19, S375, T376, S477, T478, and T547.Conversely, the S protein gained phosphorylation sites at S24, S408, and Y655.Additionally, the ORF3a protein lost the T223 phosphorylation site, and the E protein lost the S55 phosphorylation site.The N protein lost phosphorylation sites at S33 and S413 (Table 2).

Discussion
The Omicron variant, characterized by increased transmissibility and a faster transmission rate, has emerged as the predominant strain of SARS-CoV-2, reshaping the landscape of the pandemic [5][6][7]10].This study's sequences, originating from a COVID-19 outbreak caused by the Omicron BA.2.76 variant in Jining City, Shandong Province, China, exhibit high homogeneity and form an independent cluster within the evolutionary tree.The comparison with the Wuhan-Hu-1 strain revealed 105 nucleotide mutations and 27 deletions, indicating the variant's significant genetic evolution.These findings include mutations in non-coding regions such as C241T in the 5'UTR and G29692A in the 3'UTR, whose impacts on the virus's replication and translation mechanisms warrant further investigation.The − 3 nucleotide position at the eukaryotic translation initiation site is pivotal for translation efficiency, where − 3 A interacts with eukaryotic initiation factor 2α (eIF2α), enhancing the recognition of the translation initiation site (TIS) and protein synthesis [11][12][13][14].Given that viruses utilize the host's translational machinery for protein synthesis, the A28271T mutation, which changes − 3 A to -3T in the N gene's translation initiation region, could impair the translation efficiency of the N gene, potentially leading to diminished N protein levels in host cells [15][16][17][18].The N protein, crucial for SARS-CoV-2 structure, significantly influences cytokine storm induction through the promotion of inflammatory factor production, exacerbating pneumonia in those infected [19][20][21][22].Thus, a reduction in N protein expression might attenuate the inflammatory response, potentially moderating the severity of pneumonia.
Recent findings underscore the Omicron variant's increased transmissibility, yet its impact on disease severity varies markedly across different populations [56].This variation presents a spectrum of clinical outcomes, suggesting less severe symptoms compared to earlier variants.Such variability, influenced by both strain-specific characteristics and vaccination status, underscores the ongoing need for vigilance and targeted research [57,58].Our focused examination of Omicron variant BA.2.76 in Jining City sheds light on this variant's behavior, highlighting the critical role of localized data in understanding the pandemic's evolving dynamics.
Moreover, the response to Omicron's spread has necessitated a reassessment of vaccine efficacy.Preliminary analyses indicate a diminished neutralizing response, prompting a recalibration of vaccination strategies, such as booster doses and potential vaccine formula adjustments, crucial for sustaining global vaccination efforts [59].The reliability of diagnostic tests in the face of Omicron's emergence has sparked rigorous evaluations, with current evidence supporting the continued effectiveness of most PCR and antigen tests [60].This ensures the integrity of diagnostic protocols, essential for effective pandemic management.Simultaneously, the global initiative to sequence the SARS-CoV-2 genome has been pivotal in tracking the variant's spread and evolution, providing invaluable insights into its global distribution.This sequencing effort, by analyzing data from diverse regions, aids in identifying mutation patterns and transmission dynamics, informing public health strategies.Additionally, the advent of Omicron raises significant questions regarding the current therapeutic options' efficacy, including monoclonal antibodies and antiviral drugs.The nuanced impact observed necessitates continuous research into therapeutic strategies, highlighting the importance of adapting treatment approaches to effectively combat the challenges presented by emerging viral strains.
In conclusion, this study reports 87 sequences of the Omicron BA.2.76 variant, offering significant insights into the genomic landscape of the Omicron variant worldwide.These results enhance our grasp of SARS-CoV-2's evolutionary dynamics and offer crucial data for assessing shifts in the virus's pathogenicity.

Table 1
Compares genetic variations in the Omicron BA.2.76 sequence between Jining City and Wuhan-Hu-1 TIR: translation initiation region

Table 2
Analysis of variations in N-glycosylation and phosphorylation sites in the 87 sequences of Omicron BA.2.76 in Jining City, compared with Wuhan-Hu-1