SARS-CoV-2 within-host diversity of human hosts and its implications for viral immune evasion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously evolving, bringing great challenges to the control of the virus. In the present study, we investigated the characteristics of SARS-CoV-2 within-host diversity of human hosts and its implications for immune evasion using about 2,00,000 high-depth next-generation genome sequencing data of SARS-CoV-2. A total of 44% of the samples showed within-host variations (iSNVs), and the average number of iSNVs in the samples with iSNV was 1.90. C-to-U is the dominant substitution pattern for iSNVs. C-to-U/G-to-A and A-to-G/U-to-C preferentially occur in 5'-CG-3' and 5'-AU-3' motifs, respectively. In addition, we found that SARS-CoV-2 within-host variations are under negative selection. About 15.6% iSNVs had an impact on the content of the CpG dinucleotide (CpG) in SARS-CoV-2 genomes. We detected signatures of faster loss of CpG-gaining iSNVs, possibly resulting from zinc-finger antiviral protein-mediated antiviral activities targeting CpG, which could be the major reason for CpG depletion in SARS-CoV-2 consensus genomes. The non-synonymous iSNVs in the S gene can largely alter the S protein's antigenic features, and many of these iSNVs are distributed in the amino-terminal domain (NTD) and receptor-binding domain (RBD). These results suggest that SARS-CoV-2 interacts actively with human hosts and attempts to take different evolutionary strategies to escape human innate and adaptive immunity. These new findings further deepen and widen our understanding of the within-host evolutionary features of SARS-CoV-2.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of the coronavirus disease 2019, has evolved rapidly since it was discovered. Recent studies have pointed out that some mutations in the SARS-CoV-2 S protein could confer SARS-CoV-2 the ability to evade the human adaptive immune system. In addition, it is observed that the content of the CpG dinucleotide in SARS-CoV-2 genome sequences has decreased over time, reflecting the adaptation to the human host. The significance of our research is revealing the characteristics of SARS-CoV-2 within-host diversity of human hosts, identifying the causes of CpG depletion in SARS-CoV-2 consensus genomes, and exploring the potential impacts of non-synonymous within-host variations in the S gene on immune escape, which could further deepen and widen our understanding of the evolutionary features of SARS-CoV-2.

AutoVEM: An automated tool to real-time monitor epidemic trends and key mutations in SARS-CoV-2 evolution.

With the global epidemic of SARS-CoV-2, it is important to effectively monitor the variation, haplotype subgroup epidemic trends and key mutations of SARS-CoV-2 over time. This is of great significance to the development of new vaccines, the update of therapeutic drugs, and the improvement of detection methods. The AutoVEM tool developed in the present study could complete all mutations detections, haplotypes classification, haplotype subgroup epidemic trends and candidate key mutations analysis for 131,576 SARS-CoV-2 genome sequences in 18 h on a 1 core CPU and 2 GB RAM computer. Through haplotype subgroup epidemic trends analysis of 131,576 genome sequences, the great significance of the previous 4 specific sites (C241T, C3037T, C14408T and A23403G) was further revealed, and 6 new mutation sites of highly linked (T445C, C6286T, C22227T, G25563T, C26801G and G29645T) were discovered for the first time that might be related to the infectivity, pathogenicity or host adaptability of SARS-CoV-2. In brief, we proposed an integrative method and developed an efficient automated tool to monitor haplotype subgroup epidemic trends and screen for the candidate key mutations in the evolution of SARS-CoV-2 over time for the first time, and all data could be updated quickly to track the prevalence of previous key mutations and new candidate key mutations because of high efficiency of the tool. In addition, the idea of combinatorial analysis in the present study can also provide a reference for the mutation monitoring of other viruses.

AutoVEM2: A flexible automated tool to analyze candidate key mutations and epidemic trends for virus.

In our previous work, we developed an automated tool, AutoVEM, for real-time monitoring the candidate key mutations and epidemic trends of SARS-CoV-2. In this research, we further developed AutoVEM into AutoVEM2. AutoVEM2 is composed of three modules, including call module, analysis module, and plot module, which can be used modularly or as a whole for any virus, as long as the corresponding reference genome is provided. Therefore, it's much more flexible than AutoVEM. Here, we analyzed three existing viruses by AutoVEM2, including SARS-CoV-2, HBV and HPV-16, to show the functions, effectiveness and flexibility of AutoVEM2. We found that the N501Y locus was almost completely linked to the other 16 loci in SARS-CoV-2 genomes from the UK and Europe. Among the 17 loci, 5 loci were on the S protein and all of the five mutations cause amino acid changes, which may influence the epidemic traits of SARS-CoV-2. And some candidate key mutations of HBV and HPV-16, including T350G of HPV-16 and C659T of HBV, were detected. In brief, we developed a flexible automated tool to analyze candidate key mutations and epidemic trends for any virus, which would become a standard process for virus analysis based on genome sequences in the future.

Analyses of Long-Term Epidemic Trends and Evolution Characteristics of Haplotype Subtypes Reveal the Dynamic Selection on SARS-CoV-2.

The scale of SARS-CoV-2 infection and death is so enormous that further study of the molecular and evolutionary characteristics of SARS-CoV-2 will help us better understand and respond to SARS-CoV-2 outbreaks. The present study analyzed the epidemic and evolutionary characteristics of haplotype subtypes or regions based on 1.8 million high-quality SARS-CoV-2 genomic data. The estimated ratio of the rates of non-synonymous to synonymous changes (Ka/Ks) in North America and the United States were always more than 1.0, while the Ka/Ks in other continents and countries showed a sharp decline, then a slow increase to 1.0, and a dramatic increase over time. H1 (B.1) with the highest substitution rate has become the most dominant haplotype subtype since March 2020 and has evolved into multiple haplotype subtypes with smaller substitution rates. Many evolutionary characteristics of early SARS-CoV-2, such as H3 being the only early haplotype subtype that existed for the shortest time, the global prevalence of H1 and H1-5 (B.1.1) within a month after being detected, and many high divergent genome sequences early in February 2020, indicate the missing of early SARS-CoV-2 genomic data. SARS-CoV-2 experienced dynamic selection from December 2019 to August 2021 and has been under strong positive selection since May 2021. Its transmissibility and the ability of immune escape may be greatly enhanced over time. This will bring greater challenges to the control of the pandemic.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10-17 M (approximately 6 copies/µL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10-16 M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

Comprehensive evolution and molecular characteristics of a large number of SARS-CoV-2 genomes reveal its epidemic trends.

OBJECTIVES: To further reveal the phylogenetic evolution and molecular characteristics of the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on a large number of genomes and provide a basis for the prevention and treatment of SARS-CoV-2. METHODS: Various evolution analysis methods were employed. RESULTS: The estimated ratio of the rates of non-synonymous to synonymous changes (Ka/Ks) of SARS-CoV-2 was 1.008 or 1.094 based on 622 or 3624 SARS-CoV-2 genomes and nine key specific sites of high linkage, and four major haplotypes were found: H1, H2, H3 and H4. The results of Ka/Ks, detected population size and development trends of each major haplotype showed that H3 and H4 subgroups were going through a purify evolution and almost disappeared after detection, indicating that they might have existed for a long time. The H1 and H2 subgroups were going through a near neutral or neutral evolution and globally increased with time, and the frequency of H1 was generally high in Europe and correlated with the death rate (r >0.37), suggesting that these two haplotypes might relate to the infectivity or pathogenicity of SARS-CoV-2. CONCLUSIONS: Several key specific sites and haplotypes related to the infectivity or pathogenicity of SARS-CoV-2, and the possible earlier origin time and place of SARS-CoV-2 were indicated based on the evolution and epidemiology of 16,373 SARS-CoV-2 genomes.

Prediction and evolution of B cell epitopes of surface protein in SARS-CoV-2.

BACKGROUND: In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. METHODS: The analyses were related to epitopes on 3 proteins, including spike (S), envelope (E) and membrane (M) proteins, which are located on the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. RESULTS: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. CONCLUSION: The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.