| The Corona Virus Disease 2019（COVID-19）emerged in late 2019 in Wuhan,rapidly evolving into a global pandemic.The ensuing crisis featured recurring waves,primarily driven by the emergence of SARS-CoV-2 variants such as Alpha,Beta,Delta,and Omicron.As of September 21,2023,the global tally stood at 770,778,396confirmed COVID-19 cases and 6,958,499 fatalities.This pandemic has etched its significance as one of the most formidable infectious outbreaks of the 21st century.As population immunity strengthened,mortality rates have steadily declined,alleviating the strain on healthcare systems.The current global trajectory indicates a declining trend,allowing nations to regain elements of pre-pandemic normalcy.On May 5,2023,the World Health Organization declassified COVID-19 as a"Public Health Emergency of International Concern".Nevertheless,it is imperative to recognize that the threat to global health from COVID-19 endures.SARS-CoV-2,the etiological agent of the COVID-19 pandemic,represents the latest in a series of coronavirus outbreaks in recent history.Preceding this global crisis,the world had grappled with two other major coronavirus outbreaks in the span of two decades,both bearing substantial consequences.The emergence of these three significant coronavirus epidemics underscores the remarkable mutability of coronaviruses and the substantial threat they pose to human society.Over time,genetic mutations can significantly alter their transmissibility,virulence,and susceptibility to existing treatments and vaccines.Research endeavors directed toward unraveling the mutation and evolutionary dynamics of SARS-CoV-2 have proven pivotal in comprehending its attributes,forecasting potential ramifications,and devising efficacious prevention and control measures.High-throughput sequencing and genomics represent indispensable tools in this pursuit,facilitating swift acquisition of genomic data from viruses and other pathogens.In-depth analysis of these genomic sequences empowers researchers to swiftly detect SARS-CoV-2 in the nascent stages of an outbreak,monitor its real-time global dissemination,and delve deeply into the virus’s evolutionary trajectories.This investigation is centered on the novel coronavirus and employs high-throughput sequencing,comparative genomics,and population genetics methodologies.The research encompasses three distinct domains:（1）Development of High-Throughput Sequencing Strategies and Protocols Tailored to the Novel CoronavirusThis segment of the study entails the establishment of high-throughput sequencing strategies and methodologies customized for two distinct sample types:raw specimens sourced from clinical or environmental origins and cell culture products cultivated within the laboratory environment.For cell culture product samples with a heightened viral load,a direct RNA extraction and sequencing approach was adopted,employing the MGISEQ-2000sequencing platform.Corresponding data analysis workflows were devised to generate comprehensive genomic sequences.In contrast,for raw samples characterized by lower viral concentrations,we employed Ampli Seq amplification technology for targeted sample amplification and subsequent sequencing,in conjunction with the Ion Gene StudioTMS5 Plus sequencing platform.A specialized data analysis process was developed for this sequencing strategy and platform to acquire the genomic sequences.To validate the established high-throughput sequencing methods and analysis procedures for SARS-CoV-2,82 raw samples and 183 cell culture product samples were meticulously examined.Consequently,we successfully obtained SARS-CoV-2sequences from all 183 cell culture product samples,whereas 37 of the 82 raw samples yielded high-quality full-length genomic sequences.Furthermore,in recognition of the urgent need for expeditious SARS-CoV-2 detection,this study optimized the sequencing system,enhancing detection speed while maintaining the precision of diagnostic outcomes.（2）Evolutionary Dynamics of SARS-CoV-2:Insights from Natural Outbreak SamplesThis doctoral thesis investigates the evolutionary dynamics of SARS-CoV-2,focusing on samples collected during the initial outbreaks in Wuhan and Beijing’s Xinfadi market.The study delves into the mutations and evolutionary trajectories of the virus under natural outbreak and transmission conditions.Notably,a new wave of local transmission emerged in Beijing’s Xinfadi market on June 11,2020,following 56consecutive days of zero local new cases.To trace the source of this outbreak,we collected 82 samples from COVID-19 patients and the environment at the Xinfadi market.Leveraging the high-throughput sequencing method developed for raw samples,we successfully obtained high-quality full-length genomic sequences for 37 of these samples,subjecting them to single nucleotide polymorphism analysis and phylogenetic analysis.Our findings revealed that the genomic sequences of the Xinfadi outbreak exhibited common mutation sites corresponding to the specific mutation points of the B.1.1 lineage prevalent in Europe at that time,placing both on the same phylogenetic branch in the evolutionary tree.Comparative genomics and population genetics were then employed to analyze transmission patterns and evolutionary dynamics in both Wuhan and Beijing.Phylogenetic analysis indicated that the Xinfadi virus sequence belonged to the B.1.1 branch,distinct from the lineage branch in Wuhan.This suggests that the Xinfadi outbreak resulted from an imported case originating in Europe.Despite their lineage differences,both regions exhibited similar transmission patterns,featuring dense primary transmission centers.However,Wuhan displayed a broader spread,hinting at multiple transmission events,while secondary transmission in Beijing was less evident,likely due to a swift and effective outbreak response curtailing further spread.Furthermore,Bayesian evolutionary analysis estimated that the ancestral virus of the Xinfadi outbreak in Beijing emerged in early May 2020.In comparison to the average evolutionary rate of SARS-CoV-2,the early-stage evolutionary rate in Wuhan and the Xinfadi market was 8.073×10-4 subs/site/year,lower than the average evolutionary rate of SARS-CoV-2,which stands at 2.4×10-3 subs/site/year.This implies that the outbreaks in both regions represent a relatively stable phase in the early evolution of SARS-CoV-2,characterized by adaptation to its new host environment.As the pandemic spread and the virus encountered increased opportunities for replication and mutation,especially in densely populated areas,its rate of evolution may have accelerated.This comparative study enhances our comprehension of SARS-CoV-2transmission patterns,offering valuable insights for tailoring preventive and control measures to various transmission scenarios.Additionally,it serves as a reference for future infectious disease outbreaks,enabling the prompt and accurate identification of pathogens and containment of their spread.（3）Exploring SARS-CoV-2 Mutation Patterns through Controlled Laboratory PassagingIn the evolving landscape of the COVID-19 pandemic,Variants of Concern（VOC）of SARS-CoV-2 have arisen,posing new challenges.This research investigates the mutation patterns and evolutionary trends of SARS-CoV-2 variants,with a particular focus on the primary VOCs-Alpha,Beta,and Delta.The study employs co-infection and continuous passaging experiments,observing the wild-type SARS-CoV-2 strain and these variants in diverse susceptible cell lines.The aim is to understand their competitiveness and adaptability,especially in response to varying antibody pressure.High-throughput deep sequencing analysis reveals marked differences in the replicative adaptability of the wild-type and the three VOCs across different cell types and under varying antibody conditions.For instance,the Beta variant demonstrates superior adaptability in Caco-2 cells,while the wild-type and Alpha variant exhibit enhanced adaptability in Vero E6 cells.The study postulates that these distinctions may be associated with specific cellular characteristics.Moreover,it underscores the significant influence of antibody pressure on the virus’s adaptability.Introduction of high-concentration neutralizing antibodies intensifies competition between variants,leading to alterations in their adaptability advantages.Notably,the Beta variant,which initially displayed reduced adaptability in Vero E6 cells under normal conditions,becomes the dominant strain in the presence of high-concentration neutralizing antibodies.These findings underscore the complex role of antibodies in the transmission and mutation of the coronavirus.Monoclonal purification demonstrates that co-infection and continuous passaging yield homologous recombination events,primarily occurring between nucleotide positions 22995-28866 of the viral genome,suggesting a potential hotspot region for viral genomic recombination.Furthermore,the study identifies the suppressive effect of immune pressure on the frequency of these recombination events.This research provides novel insights into the adaptability and competitive dynamics of SARS-CoV-2 variants in various cellular environments.Moreover,it offers the first direct experimental evidence of homologous recombination between SARS-CoV-2 variants.These findings enhance our understanding of the virus’s transmission mechanisms and mutation trends,contributing to the scientific foundation for COVID-19 vaccine development and public health intervention strategies.Conclusion:This study leveraged the SARS-CoV-2 virus as a model to establish and optimize a SARS-CoV-2 detection and analysis system based on high-throughput sequencing platforms.This system facilitated the swift identification and source tracing of virus samples.Utilizing this platform,we conducted comparative genomic analyses on virus strains associated with the Wuhan and Beijing outbreaks.The findings highlighted distinctions in the transmission patterns between the two outbreaks,with the Wuhan outbreak exhibiting more pronounced secondary spread.Moreover,sequences from the Xinfadi market outbreak bore a striking resemblance to the B.1.1 lineage prevalent in Europe at that time,indicating an external origin for the Xinfadi outbreak.In delving deeper into the mutation mechanisms of SARS-CoV-2,we conducted a series of laboratory experiments involving continuous passaging,co-infection,sequencing analysis,and more,focusing on the primary variant strains.This comprehensive approach allowed us to investigate the genetic mutation patterns and evolutionary dynamics of the virus.Notably,our findings revealed that the wild-type and Alpha variant dominated in Vero E6 cells,while the Beta variant exhibited dominance in Caco-2 cells.Remarkably,the adaptability of the Beta variant in cells was significantly enhanced under immune pressure.Monoclonal purification of passaged samples uncovered the existence of seven recombinant strains resulting from interactions between these four virus strains.Notably,our research successfully replicated SARS-CoV-2 recombination in vitro,thereby establishing a model for simulating viral recombination phenomena.In general,this doctoral thesis presents a comprehensive examination of strategies and methodologies with potential applicability in the analysis of pathogenic features during future viral outbreaks.The research outcomes significantly enhance our insights into the evolution,attributes,and transmission dynamics of the SARS-CoV-2 virus.These findings not only lay a robust scientific groundwork for the enhancement of pandemic containment measures and advancements in viral infection treatments but also offer valuable guidance for the continued development and refinement of public health safety protocols. |
