Whole genome sequence analysis of SARS-CoV-2 from 40 imported cases of confirmed COVID-19 in Sichuan

Objective: To understand the genomic characteristics of SARS-CoV-2 from 40 imported cases with confirmed COVID-19 in Sichuan during January and March 2022. Methods: Total viral RNA was extracted from respiratory samples of 182 confirmed COVID-19 cases who entered China through Chendu International Airport from January to March 2022. Mutation nucleic acid detection kit was used to identify the mutant strains and Illumina sequencing platform was applied for whole genome sequence(WGS) of virus. SARS-CoV-2 reference sequences were downloaded from NCBI database for genetic evolution and antigen variation analysis. The Nextclade and Pangolin online virus analysis platform were used to determine the virus family and type, and to analyze the mutation loci of the virus. The phylogenetic tree was constructed, along with the epidemiological data of cases to analyze the source and correlation of viruses. Results: Among 182 imported COVID-19 cases,B.1.617.2 mutations were identified in 3 cases and B.1.1.529 mutations were detected in 57 cases.A total of 40 SARS-CoV-2 whole genome sequences with coverage>95% were obtained in this study. Nextclade typing analysis showed that 3 sequences belonged to 21J(Delta),5 sequences belonged to 21K(Omicron)and the remaining 32 sequences belonged to 21L(Omicron). Pangolin typing analysis showed that the 3 sequences of 21J(Delta)belonged to AY.4,AY.109and B.1.617.2, the 5sequences of 21K(Omicron)all belonged to BA.1.1, and the remaining 32 sequences of 21L(Omicron)belonged to BA.2. Our sequence results were99.7% consistency with the Omicron variants sequences in current GISAID database. Compared with the reference sequence strain Wuhan-Hu-1(NC_045512.2),45,47and 42nucleotide variation sites and 36,25 and 36amino acid variation sites were found in the 3 sequences of 21J(Delta). There were average 59(26-64)nucleotide mutation sites and 48(10-53)amino acid mutation sites in the 5sequences of 21K(Omicron). The median number of nucleotide mutation sites of 71(66-76)and amino acid mutation sites of 53(40-56)were identified in the 32sequences of 21L(Omicron). Phylogenetic tree analysis showed that 40SARS-CoV-2WGSs were all related to the current variants of concern(VOC). Conclusions Continuous: sequencing of SARS-CoV-2whole genome from imported cases with confirmed COVID-19is of great significance for the prevention and control of the outbreak and prevalence of local epidemic caused by imported viruses in Sichuan.

Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection.

In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5'-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.

Whole genome sequence analysis of SARS-CoV-2 from 40 imported cases of confirmed COVID-19 in Sichuan

Objective To understand the genomic characteristics of SARS-CoV-2 from 40 imported cases with confirmed COVID-19 in Sichuan during January and March 2022. Methods Total viral RNA was extracted from respiratory samples of 182 confirmed COVID-19 cases who entered China through Chendu International Airport from January to March 2022.Mutation nucleic acid detection kit was used to identify the mutant strains and Illumina sequencing platform was applied for whole genome sequence（WGS） of virus.SARS-CoV-2 reference sequences were downloaded from NCBI database for genetic evolution and antigen variation analysis.The Nextclade and Pangolin online virus analysis platform were used to determine the virus family and type,and to analyze the mutation loci of the virus.The phylogenetic tree was constructed,along with the epidemiological data of cases to analyze the source and correlation of viruses. Results Among 182 imported COVID-19 cases,B.1.617.2 mutations were identified in 3 cases and B.1.1.529 mutations were detected in 57 cases.A total of 40 SARS-CoV-2 whole genome sequences with coverage>95% were obtained in this study.Nextclade typing analysis showed that 3 sequences belonged to 21J（Delta）,5 sequences belonged to 21K（Omicron）and the remaining 32 sequences belonged to 21L（Omicron）.Pangolin typing analysis showed that the 3 sequences of 21J（Delta）belonged to AY.4,AY.109and B.1.617.2,the 5sequences of 21K（Omicron）all belonged to BA.1.1,and the remaining 32 sequences of 21L（Omicron）belonged to BA.2.Our sequence results were99.7% consistency with the Omicron variants sequences in current GISAID database.Compared with the reference sequence strain Wuhan-Hu-1（NC＿045512.2）,45,47and 42nucleotide variation sites and 36,25 and 36amino acid variation sites were found in the 3 sequences of 21J（Delta）.There were average 59（26-64）nucleotide mutation sites and 48（10-53）amino acid mutation sites in the 5sequences of 21K（Omicron）.The median number of nucleotide mutation sites of 71（66-76）and amino acid mutation sites of 53（40-56）were identified in the 32sequences of 21L（Omicron）.Phylogenetic tree analysis showed that 40SARS-CoV-2WGSs were all related to the current variants of concern（VOC）. Conclusions Continuous sequencing of SARS-CoV-2whole genome from imported cases with confirmed COVID-19is of great significance for the prevention and control of the outbreak and prevalence of local epidemic caused by imported viruses in Sichuan.

SARS-CoV-2 Seroprevalence and Profiles Among Convalescents in Sichuan Province, China.

Objectives: To explore and understand the SARS-CoV-2 seroprevalence of convalescents, the association between antibody levels and demographic factors, and the seroepidemiology of convalescents of COVID-19 till March 2021. Methods: We recruited 517 voluntary COVID-19 convalescents in Sichuan Province and collected 1,707 serum samples till March 2021. Then we reported the seroprevalence and analyzed the associated factors. Results: Recent travel history was associated with IgM levels. Convalescents who had recent travel history were less likely to be IgM antibody negative [OR = 0.232, 95% CI: (0.128, 0.420)]. Asymptomatic cases had, approximately, twice the odds of being IgM antibody negative compared with symptomatic cases [OR = 2.583, 95% CI: (1.554, 4.293)]. Participants without symptoms were less likely to be IgG seronegative than those with symptoms [OR = 0.511, 95% CI: (0.293, 0.891)]. Convalescents aged 40-59 were less likely to be IgG seronegative than those aged below 20 [OR = 0.364, 95% CI: (0.138, 0.959)]. The duration of positive IgM antibodies persisted 365 days while the IgG persisted more than 399 days. Conclusions: Our findings suggested that recent travel history might be associated with the antibody levels of IgM, while age could be associated with the antibody levels of IgG. Infection type could be associated with both antibody levels of IgM and IgG that declined quicker in asymptomatic cases.

On-Site Detection of SARS-CoV-2 Antigen by Deep Learning-Based Surface-Enhanced Raman Spectroscopy and Its Biochemical Foundations.

A rapid, on-site, and accurate SARS-CoV-2 detection method is crucial for the prevention and control of the COVID-19 epidemic. However, such an ideal screening technology has not yet been developed for the diagnosis of SARS-CoV-2. Here, we have developed a deep learning-based surface-enhanced Raman spectroscopy technique for the sensitive, rapid, and on-site detection of the SARS-CoV-2 antigen in the throat swabs or sputum from 30 confirmed COVID-19 patients. A Raman database based on the spike protein of SARS-CoV-2 was established from experiments and theoretical calculations. The corresponding biochemical foundation for this method is also discussed. The deep learning model could predict the SARS-CoV-2 antigen with an identification accuracy of 87.7%. These results suggested that this method has great potential for the diagnosis, monitoring, and control of SARS-CoV-2 worldwide.

Excretion of SARS-CoV-2 through faecal specimens.

Coronavirus disease 2019 (COVID-19) has become a pandemic with increasing numbers of cases worldwide. SARS-CoV-2, the causative virus of COVID-19, is mainly transmitted through respiratory droplets or through direct and indirect contact with an infected person. The possibility of potential faecal-oral transmission was investigated in this study. We collected 258 faecal specimens from nine provinces in China and detected the nucleic acid of SARS-CoV-2 using real-time RT-PCR. Vero cells were used to isolate the virus from SARS-CoV-2 nucleic acid positive samples, after which sequencing of Spike gene in eight samples was performed. In all, 93 of 258 (36%) stool samples were positive for SARS-CoV-2 RNA. The positive rates of critical, severe, moderate, and mild patients were 54.4%, 56.1%, 30.8%, and 33.3%, respectively. The content of nucleic acid increased within 2 weeks after the onset of the disease. From the perspective of clinical typing, the nucleic acid can be detected in the faeces of critical patients within two weeks and until four to five weeks in the faeces of severe and mild patients. SARS-CoV-2 was isolated from stool specimens of two severe patients. Four non-synonymous mutations in Spike gene were newly detected in three stool samples. A small number of patients had strong faecal detoxification ability. The live virus in faeces could be an important source of contamination, which may lead to infection and further spread in areas with poor sanitary conditions. The findings of this study have public health significance and they should be considered when formulating disease control strategies.