The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs

The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes. HIGHLIGHTSO_LINCAP primarily binds structured RNAs. C_LIO_LINCAP prefers multiple RNA structures separated by single-stranded linkers. C_LIO_LINCAP favors binding to long RNAs. C_LI

Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19. One Sentence SummaryAtomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.

Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2Population Bottlenecks and Intra-host Evolution during Human-to-Human Transmission of SARS-CoV-2

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Author summaryIn this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.

Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients

As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

Multiple approaches for massively parallel sequencing of HCoV-19 genomes directly from clinical samples

COVID-19 has caused a major epidemic worldwide, however, much is yet to be known about the epidemiology and evolution of the virus. One reason is that the challenges underneath sequencing HCoV-19 directly from clinical samples have not been completely tackled. Here we illustrate the application of amplicon and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of HCoV-19 from clinical samples covering a range of sample types and viral load. We also examine and compare the bias, sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. This is, to date, the first work systematically implements amplicon and capture approaches in sequencing HCoV-19, as well as the first comparative study across methods. Our work offers practical solutions for genome sequencing and analyses of HCoV-19 and other emerging viruses.

Advances of 18F-FDG PET/CT in the detection of brown adipose tissue / 中华核医学与分子影像杂志

Nowadays, more attentions have been paid on brown adipose tissue (BAT) because BAT plays a great role in individuals with obesity and metabolic diseases. 18F-fluorodeoxyglucose (FDG) PET/CT is noninvasive and sensitive to detect the location, volume, and metabolic activity of BAT. 18F-FDG PET/CT for BAT provides new insight for the prevention and clinical management of obesity and diabetes. The review summarizes the methods to activate BAT, the preparation of patients, 18F-FDG PET/CT imaging techniques and clinical application.

Significance of plasma miRNA-544a expression level in diagnosis and treatment of lung cancer / 肿瘤研究与临床

Objective To investigate the clinical values of detection of plasma miRNA-544a (miR-544a) expression level in the diagnosis and treatment of lung cancer.Methods A total of 110 patients diagnosed with lung cancer in Ⅰ-Ⅱ stage from June 2017 to March 2018 in Shiyan Taihe Hospital were collected,and 35 patients with benign pulmonary nodules and 64 healthy people were also collected as the controls.Realtime quantitative polymerase chain reaction (RT-qPCR) was used to detect the level of miR-S44a in plasma of 64 healthy people,110 patients with lung cancer (50 newly diagnosed patients without anti-tumor treatment,33 patients one week after radical resection,27 patients after one week chemotherapy with the same dose) and 35 patients with benign pulmonary nodules.Of the 50 newly diagnosed patients,42 cases were non-small cell lung cancer and 8 cases were small cell lung cancer.The plasma expression level of miR-544a in each group was compared by using Mann-Whitney U test,and the efficacy of miR-544a in the diagnosis of lung cancer was evaluated by the receiver operationg characteristic (ROC) curve and the area under the curve (AUC).Results The plasma expression levels of miR-544a in the newly diagnosed untreated lung cancer group,one week after operation group,one week chemotherapy group,healthy control group and benign pulmonary nodule group were 1.40 nmol/L (0.55 nmol/L,8.76 nmol/L),33.52 nmol/L (3.64 nmol/L,250.47 nmo/L),8.87 nmol/L (0.68 nmol/L,125.43 nmol/L),0.31 nmol/L (0.17 nmol/L,1.19 nmol/L),1.04 nmol/L (0.31 nmol/L,4.62 nmol/L),respectively,and the differences between untreated lung cancer group and the other 4 groups were statistically significant (Z =-4.483,P ＜ 0.001;Z =-4.274,P ＜ 0.001;Z =-2.562,P =0.01;Z =-2.152,P =0.031).The expression levels of miR-544a in non-small cell lung cancer group and small cell lung cancer group were 1.40 nmol/L (0.66 nmol/L,8.76 nmol/L) and 1.37 nmol/L (0.26 nmol/L,36.97 nmol/L),respectively.The differences between non-small cell lung cancer group and healthy control group and benign pulmonary nodule group were statistically significant (Z =-4.463,P ＜ 0.001;Z =-2.026,P =0.043).Compared with the healthy people,the AUC of miR-544a for diagnosing the lung cancer was 0.841,the sensitivity was 87.5 ％,and the specificity was 68.0 ％.Compared with the benign pulmonary nodule,the AUC for diagnosing lung cancer was 0.638,the sensitive was 45.7 ％,and the specificity was 80.0 ％.Conclusions The plasma expression level of miR-544a has certain significances in the differential diagnosis of early stage lung cancer and benign pulmonary nodules and healthy people,and it can be used as a potential biomarker for diagnosing early stage lung cancer,especially for the non-small cell lung cancer.The plasma expression of miR-544a is increased after surgery or chemotherapy,suggesting that its expression may be related to the occurrence and development of lung cancer,and miR-544a may become a new target for cancer treatment.