1.Research and application of the SARS-CoV-2 vaccine based on adenovirus vector technology platform.
Ying ZHANG ; Wen Zhou YU ; Zun Dong YIN ; Tong Zhan WANG ; Xiao Dong SUN ; Ai Qiang XU
Chinese Journal of Preventive Medicine 2023;57(7):1082-1095
During the global efforts to prevent and control the COVID-19 pandemic, extensive research and development of SARS-CoV-2 vaccines using various technical approaches have taken place. Among these, vaccines based on adenovirus vector have gained substantial knowledge and experience in effectively combating potential emerging infectious diseases, while also providing novel ideas and methodologies for vaccine research and development (R&D). This comprehensive review focuses on the adenovirus vector technology platform in vaccine R&D, emphasizing the importance of mucosal immunity induced by adenoviral vector-based vaccine for COVID-19 prevention. Furthermore, it analyzes the key technical challenges and obstacles encountered in the development of vaccines based on the adenovirus vector technology platform, with the aim of providing valuable insights and references for researchers and professionals in related fields.
Humans
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COVID-19 Vaccines
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Pandemics/prevention & control*
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COVID-19/prevention & control*
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SARS-CoV-2/genetics*
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Viral Vaccines/genetics*
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Adenoviridae/genetics*
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Technology
2.Establishment and preliminary application of quantitative real-time PCR assay for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA.
Xiao Juan ZHU ; Yin CHEN ; Bin WU ; Yi Yue GE ; Tao WU ; Qiao QIAO ; Kang Chen ZHAO ; Lun Biao CUI
Chinese Journal of Preventive Medicine 2023;57(2):268-272
Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×102 copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.
Humans
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SARS-CoV-2/genetics*
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COVID-19/diagnosis*
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Subgenomic RNA
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Real-Time Polymerase Chain Reaction/methods*
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RNA, Viral/genetics*
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Sensitivity and Specificity
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Nucleocapsid/chemistry*
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COVID-19 Testing
3.Assessment of SARS-CoV-2 RNA shedding in semen of 36 males with symptomatic, asymptomatic, and convalescent infection during the first and second wave of COVID-19 pandemic in Italy.
Carlo PAVONE ; Giovanni M GIAMMANCO ; Andrea Pio CASCINO ; Davide BAIAMONTE ; Mirko PINELLI ; Enrico CANGELOSI ; Chiara FILIZZOLO ; Giuseppa SCIORTINO ; Simona De GRAZIA ; Floriana BONURA
Asian Journal of Andrology 2022;24(2):135-138
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), has been identified in the human testis, but the risk of transmission of SARS-CoV-2 through sexual intercourse still needs to be defined. The goal of our study was to determine if SARS-CoV-2 is detectable in the semen of patients suffering or recovering from coronavirus disease-19 (COVID-19), still testing positive at nasopharyngeal swabs but showing mild or no symptoms at the time of sampling. Detection of SARS-CoV-2 RNA in semen was performed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR targeting open reading frame (ORF) 1ab. Medical history of the enrolled patients was taken, including COVID-19-correlated symptoms, both at the time of diagnosis and at the time of interview. Results of real-time RT-PCR and nested PCR in semen showed no evidence of SARS-CoV-2 RNA in the 36 patients suffering or recovering from COVID-19 but still positive in a nasopharyngeal swab, from over 116 patients enrolled in the study. SARS-CoV-2 detection and persistence in semen would have an impact on both clinical practice and public health strategies, but our results would suggest that SARS-CoV-2 is not present in the semen of men recovering from COVID-19.
COVID-19/epidemiology*
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Humans
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Male
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Pandemics
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RNA, Viral/genetics*
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SARS-CoV-2/genetics*
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Semen
4.Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.
Chun-Chun GAO ; Man LI ; Wei DENG ; Chun-Hui MA ; Yu-Sheng CHEN ; Yong-Qiao SUN ; Tingfu DU ; Qian-Lan LIU ; Wen-Jie LI ; Bing ZHANG ; Lihong SUN ; Si-Meng LIU ; Fengli LI ; Feifei QI ; Yajin QU ; Xinyang GE ; Jiangning LIU ; Peng WANG ; Yamei NIU ; Zhiyong LIANG ; Yong-Liang ZHAO ; Bo HUANG ; Xiao-Zhong PENG ; Ying YANG ; Chuan QIN ; Wei-Min TONG ; Yun-Gui YANG
Protein & Cell 2022;13(12):920-939
SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.
Animals
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COVID-19/genetics*
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Macaca mulatta
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SARS-CoV-2/genetics*
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Transcriptome
5.mRNA vaccines for infectious diseases: research progress and applications.
Fengming QIN ; Ning REN ; Wenyu CHENG ; Heng WEI
Chinese Journal of Biotechnology 2023;39(10):3966-3984
Messenger RNA (mRNA) vaccines emerge as promising vaccines to prevent infectious diseases. Compared with traditional vaccines, mRNA vaccines present numerous advantages, such as high potency, safe administration, rapid production potentials, and cost-effective manufacturing. In 2020, two COVID-19 vaccines (BNT162b2 and mRNA-1273) were approved by the Food and Drug Administration (FDA). The two vaccines showed high efficiency in combating COVID-19, which indicates the great advantages of mRNA technology in developing vaccines against emergent infectious diseases. Here, we summarize the type, immune mechanisms, modification methods of mRNA vaccines, and their applications in preventing infectious diseases. Current challenges and future perspectives in developing mRNA vaccines are also discussed.
United States
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Humans
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mRNA Vaccines
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BNT162 Vaccine
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COVID-19 Vaccines/genetics*
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Communicable Diseases
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RNA, Messenger/genetics*
6.CRISPR-based molecular diagnostics: a review.
Wenjun SUN ; Xingxu HUANG ; Xinjie WANG
Chinese Journal of Biotechnology 2023;39(1):60-73
Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.
Humans
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CRISPR-Cas Systems/genetics*
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COVID-19/genetics*
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Pandemics
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Artificial Intelligence
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Nucleic Acids
7.Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay.
Yunan MA ; Lirong ZOU ; Yuanhao LIANG ; Quanxun LIU ; Qian SUN ; Yulian PANG ; Hongqing LIN ; Xiaoling DENG ; Shixing TANG
Journal of Southern Medical University 2023;43(4):516-526
OBJECTIVE:
To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.
METHODS:
We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.
RESULTS:
This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.
CONCLUSION
By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Humans
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COVID-19
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CRISPR-Cas Systems
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Genotype
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Reproducibility of Results
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Reverse Transcriptase Polymerase Chain Reaction
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SARS-CoV-2/genetics*
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RNA
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COVID-19 Testing
8.PathogenTrack and Yeskit: tools for identifying intracellular pathogens from single-cell RNA-sequencing datasets as illustrated by application to COVID-19.
Wei ZHANG ; Xiaoguang XU ; Ziyu FU ; Jian CHEN ; Saijuan CHEN ; Yun TAN
Frontiers of Medicine 2022;16(2):251-262
Pathogenic microbes can induce cellular dysfunction, immune response, and cause infectious disease and other diseases including cancers. However, the cellular distributions of pathogens and their impact on host cells remain rarely explored due to the limited methods. Taking advantage of single-cell RNA-sequencing (scRNA-seq) analysis, we can assess the transcriptomic features at the single-cell level. Still, the tools used to interpret pathogens (such as viruses, bacteria, and fungi) at the single-cell level remain to be explored. Here, we introduced PathogenTrack, a python-based computational pipeline that uses unmapped scRNA-seq data to identify intracellular pathogens at the single-cell level. In addition, we established an R package named Yeskit to import, integrate, analyze, and interpret pathogen abundance and transcriptomic features in host cells. Robustness of these tools has been tested on various real and simulated scRNA-seq datasets. PathogenTrack is competitive to the state-of-the-art tools such as Viral-Track, and the first tools for identifying bacteria at the single-cell level. Using the raw data of bronchoalveolar lavage fluid samples (BALF) from COVID-19 patients in the SRA database, we found the SARS-CoV-2 virus exists in multiple cell types including epithelial cells and macrophages. SARS-CoV-2-positive neutrophils showed increased expression of genes related to type I interferon pathway and antigen presenting module. Additionally, we observed the Haemophilus parahaemolyticus in some macrophage and epithelial cells, indicating a co-infection of the bacterium in some severe cases of COVID-19. The PathogenTrack pipeline and the Yeskit package are publicly available at GitHub.
COVID-19
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Humans
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RNA
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SARS-CoV-2/genetics*
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Single-Cell Analysis/methods*
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Transcriptome
9.SARS -CoV -2 Omicron BA.5 infection recurrence after Paxlovid treatment: The first case report in Hunan Province.
Meiping CHEN ; Ting GUO ; Zhiguo ZHOU ; Dong CHEN ; Haibo ZHOU
Journal of Central South University(Medical Sciences) 2022;47(12):1775-1780
We report a case of coronavirus disease 2019 (COVID-19) patient who was cured by oral administration of nirmatrelvir/ritonavir (Paxlovid). The patient was treated with Paxlovid after being first infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.5 variant. On the 11th day after SARS-CoV-2 nucleic acid test turned negative, SARS-CoV-2 nucleic acid test was positive again, and the threshold of nucleic acid cycle number was equivalent to that of the first infection. The results of two whole gene sequencing showed that it was the same virus strain infection, suggesting that the case was re-positive. Without specific treatment, SARS-CoV-2 nucleic acid detection in nasopharyngeal swab turned negative. It is not uncommon for Paxlovid to recover after treating COVID-19, and most of the patients can recover without specific treatment. However, it is necessary to further study the mechanism that may lead to the recovery of SARS-CoV-2.
Humans
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COVID-19
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SARS-CoV-2/genetics*
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Administration, Oral
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Nucleic Acids
10.Genomic Epidemiology of Imported Cases of COVID-19 in Guangdong Province, China, October 2020 - May 2021.
Dan LIANG ; Tao WANG ; Jiao Jiao LI ; Da Wei GUAN ; Guan Ting ZHANG ; Yu Feng LIANG ; An An LI ; Wen Shan HONG ; Li WANG ; Meng Lin CHEN ; Xiao Ling DENG ; Feng Juan CHEN ; Xing Fei PAN ; Hong Ling JIA ; Chun Liang LEI ; Chang Wen KE
Biomedical and Environmental Sciences 2022;35(5):393-401
Objective:
The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been engendering enormous hazards to the world. We obtained the complete genome sequences of SARS-CoV-2 from imported cases admitted to the Guangzhou Eighth People's Hospital, which was appointed by the Guangdong provincial government to treat coronavirus disease 2019 (COVID-19). The SARS-CoV-2 diversity was analyzed, and the mutation characteristics, time, and regional trend of variant emergence were evaluated.
Methods:
In total, 177 throat swab samples were obtained from COVID-19 patients (from October 2020 to May 2021). High-throughput sequencing technology was used to detect the viral sequences of patients infected with SARS-CoV-2. Phylogenetic and molecular evolutionary analyses were used to evaluate the mutation characteristics and the time and regional trends of variants.
Results:
We observed that the imported cases mainly occurred after January 2021, peaking in May 2021, with the highest proportion observed from cases originating from the United States. The main lineages were found in Europe, Africa, and North America, and B.1.1.7 and B.1.351 were the two major sublineages. Sublineage B.1.618 was the Asian lineage (Indian) found in this study, and B.1.1.228 was not included in the lineage list of the Pangolin web. A reasonably high homology was observed among all samples. The total frequency of mutations showed that the open reading frame 1a (ORF1a) protein had the highest mutation density at the nucleotide level, and the D614G mutation in the spike protein was the commonest at the amino acid level. Most importantly, we identified some amino acid mutations in positions S, ORF7b, and ORF9b, and they have neither been reported on the Global Initiative of Sharing All Influenza Data nor published in PubMed among all missense mutations.
Conclusion
These results suggested the diversity of lineages and sublineages and the high homology at the amino acid level among imported cases infected with SARS-CoV-2 in Guangdong Province, China.
Amino Acids
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COVID-19/epidemiology*
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Genomics
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Humans
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Mutation
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Phylogeny
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SARS-CoV-2/genetics*