Search

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals. Electronic supplementary material The online version of this article (10.1007/s12250-020-00242-1) contains supplementary material, which is available to authorized users.

RNA-remodeling proteins, including RNA helicases and chaperones, play vital roles in the remodeling of structured RNAs. During viral replication, viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements. Coxsackieviruses B3 (CVB3) and Coxsackieviruses B5 (CVB5), belonging to the genus Enterovirus in the family Picornaviridae, have been reported to cause various infectious diseases such as hand-foot-and-mouth disease, aseptic meningitis, and viral myocarditis. However, little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins. In this study, we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A, B, and C motifs, and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner, but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP. In addition, we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions. Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.

Immune evasion and inhibition of apoptosis are required for successful virus infection. However, inhibition of apoptosis can increase antiviral immune responses, which can then clear viral infections. Here we show that human cytomegalovirus (HCMV)-encoded UL37 exon-1 protein (UL37x1) not only inhibits apoptosis but also suppresses the cGAS-STING immune pathway. Using co-immunoprecipitation assays, we found that UL37x1 binds to TBK1 to abrogate the TBK1-STING-IRF3 interaction. Although the anti-apoptosis function of UL37x1 increases immune signalling, the immunosuppressive role of UL37x1 counteracts this undesirable side-effect. Furthermore, we used mutational analyses to show that the loss of either immunosuppressive or anti-apoptotic function of UL37x1 significantly reduced HCMV replication in human primary foreskin fibroblasts and humanized mice by over twofold. Finally, loss of both functions resulted in over fourfold reduction of HCMV replication in the same cell type and mouse model, showing that both UL37x1 functions are crucial for HCMV infection. We conclude that this sophisticated mechanism enables HCMV to control innate immunity and apoptosis to ensure efficient infection.

Rationale: Children usually develop less severe symptoms responding to Coronavirus Disease 2019 (COVID-19) than adults. However, little is known about the molecular alterations and pathogenesis of COVID-19 in children. Methods: We conducted plasma proteomic and metabolomic profilings of the blood samples of a cohort containing 18 COVID-19-children with mild symptoms and 12 healthy children, which were enrolled from hospital admissions and outpatients, respectively. Statistical analyses were performed to identify molecules specifically altered in COVID-19-children. We also developed a machine learning-based pipeline named inference of biomolecular combinations with minimal bias (iBM) to prioritize proteins and metabolites strongly altered in COVID-19-children, and experimentally validated the predictions. Results: By comparing to the multi-omic data in adults, we identified 44 proteins and 249 metabolites differentially altered in COVID-19-children against healthy children or COVID-19-adults. Further analyses demonstrated that both deteriorative immune response/inflammation processes and protective antioxidant or anti-inflammatory processes were markedly induced in COVID-19-children. Using iBM, we prioritized two combinations that contained 5 proteins and 5 metabolites, respectively, each exhibiting a total area under curve (AUC) value of 100% to accurately distinguish COVID-19-children from healthy children or COVID-19-adults. Further experiments validated that all the 5 proteins were up-regulated upon coronavirus infection. Interestingly, we found that the prioritized metabolites inhibited the expression of pro-inflammatory factors, and two of them, methylmalonic acid (MMA) and mannitol, also suppressed coronaviral replication, implying a protective role of these metabolites in COVID-19-children. Conclusion: The finding of a strong antagonism of deteriorative and protective effects provided new insights on the mechanism and pathogenesis of COVID-19 in children that mostly underwent mild symptoms. The identified metabolites strongly altered in COVID-19-children could serve as potential therapeutic agents of COVID-19.

Background: Children usually develop less severe disease responding to COVID-19 than adults. However, little is known about the detailed mechanism and pathogenesis of children with COVID-19 (CC), and its difference to adults with COVID-19 (AC). Methods: We conducted a plasma proteomic and metabolomic profiling, using the blood samples of 30 children including 18 CC cases and 12 healthy children (HC). By comparing the multi-omic data of AC, standard statistical tests were used to identify differentially expressed proteins (DEPs) and metabolites (DEMs) exclusively altered in CC. Enrichment analyses were conducted to identify biological processes/pathways specifically enriched in CC. To identify potential CC-specific biomarkers, we developed a new machine learning-based method named inference of biomarker combinations with minimal bias (iBM). Further experiments were conducted to validate the predicted metabolic markers. Findings: By quantifying 757 proteins and 1174 metabolites, we identified 44 DEPs and 249 DEMs exclusively altered in CC. Enrichment analyses demonstrated that in CC both deleterious immune response/inflammation processes and protective anti-inflammatory processes were strongly induced in the proteomic level, whereas protective anabolism-related processes were enriched in the metabolomic level. Using iBM, we prioritized two CC-specific biomarker combinations that contained 5 proteins and 5 metabolites, respectively, each exhibiting a total area under curve (AUC) value of 100% to accurately distinguish CC from HC or AC. Further experiments showed that the identified metabolites not only inhibited the expression of pro-inflammatory factors, but also suppressed coronaviral replication, implying that these factors played key roles in protecting pediatric patients from both viral infection and infection-induced inflammation. Interpretation: The finding of a strong antagonism of deleterious and protective effects provided new insights on the mechanism and pathogenesis of CC cases that mostly undergo mild symptoms. The identified CC-specific biomarkers could serve as candidate drug targets or therapeutic agents of COVID-19. Funding Statement: This work was supported by the Strategic Priority Research Program of CAS (XDB29010300 to X.Z.), the National Science and Technology Major Project (2018ZX10101004 to X.Z.), National Natural Science Foundation of China (81873964 to Y.Q., 31930021, 31970633 and 34671360 to Y.X., and 31670161 to X.Z.), Grant from the CAS Youth Innovation Promotion Association (2020332 to Y.Q.), the program for HUST Academic Frontier Youth Team (Y.X.). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All work performed in this study was approved by the Guangzhou Women and Children's Medical Center Ethics Committee and Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious diseases.

The pandemic of the coronavirus disease 2019 (COVID-19) has become a global public health crisis. The symptoms of COVID-19 range from mild to severe, but the physiological changes associated with COVID-19 are barely understood. In this study, we performed targeted metabolomic and lipidomic analyses of plasma from a cohort of patients with COVID-19 who had experienced different symptoms. We found that metabolite and lipid alterations exhibit apparent correlation with the course of disease in these patients, indicating that the development of COVID-19 affected their whole-body metabolism. In particular, malic acid of the TCA cycle and carbamoyl phosphate of the urea cycle result in altered energy metabolism and hepatic dysfunction, respectively. It should be noted that carbamoyl phosphate is profoundly down-regulated in patients who died compared with patients with mild symptoms. And, more importantly, guanosine monophosphate (GMP), which is mediated not only by GMP synthase but also by CD39 and CD73, is significantly changed between healthy subjects and patients with COVID-19, as well as between the mild and fatal cases. In addition, dyslipidemia was observed in patients with COVID-19. Overall, the disturbed metabolic patterns have been found to align with the progress and severity of COVID-19. This work provides valuable knowledge about plasma biomarkers associated with COVID-19 and potential therapeutic targets, as well as an important resource for further studies of the pathogenesis of COVID-19.

We investigate a possible role of the Hukou allocation system (China’s household registration system) in influencing regional productivity through a firm’s strategic behavior with respect to the retention of workers. We show that the level of regional productivity can be low due to inefficiency arising from the retention of mismatched workers when Hukou are allocated to workers through their employer rather than being allocated to them directly. This paper offers a new explanation for an observed difference in firm productivity between Beijing and Shanghai in China, and demonstrates that the way of allocating Hukou is a source of productivity differences across regions.