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.

A path towards SARS-CoV-2 attenuation: metabolic pressure on CTP synthesis rules the virus evolution

Fighting the COVID-19 epidemic summons deep understanding of the way SARS-CoV-2 taps into its host cell metabolic resources. We describe here the singular metabolic background that creates a bottleneck constraining coronaviruses to evolve towards likely attenuation in the long term. Cytidine triphosphate (CTP) is at the crossroad of the biosynthetic processes that allow the virus to multiply. This is because CTP is in demand for three essential steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope and, finally, it is a critical building block of the host transfer RNAs synthesis. The CCA 3-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate (UTP) that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. We also highlight and provide a raison detre to viperin, an enzyme of innate antiviral immunity, which synthesizes 3-deoxy-3',4-didehydro-CTP (ddhCTP) as an extremely efficient antiviral nucleotide.

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.

Effect and mechanism of fasudil on proliferation and differentiation of neural stem cells / 中国组织工程研究

BACKGROUND: The self-repairing ability of damaged brain and spinal cord is limited. It is important to search for potential therapeutics to promote the proliferation and differentiation of neural stem cells. OBJECTIVE: To investigate the effect of fasudil on the proliferation and differentiation of primary cultured neural stem cells and its potential mechanism. METHODS: Neural stem cells were obtained from the brain tissue of 15-day-old fetal rats in vitro. The expression of Nestin in the cells was detected by immunofluorescence. After treatment with fasudil at different concentrations (50, 100, 200 μmol/L) for 24, 48 and 72 hours, the proliferation rate of neural stem cells was detected by MTT, and the apoptosis rate was detected by flow cytometry. After further treatment with autophagy inhibitor, necrosis inhibitor, apoptosis inducer and ferroptosis inducer, the proliferation rates of neural stem cells were detected by MTT and the levels of malondialdehyde were detected by biochemical method. The expression of Nestin, doublecortin, microtubuleassociated protein and glial fibrillary acidic protein in neural stem cells were detected by western blot and immunofluorescence after treatment with 200 μmol/L fasudil for 10 days. RESULTS AND CONCLUSION: The positive expression of Nestin protein in primary cultured neural stem cells was observed. The proliferation rate of neural stem cells increased gradually with the increase of fasudil concentration as well as with the prolongation of action time (P < 0.05). Both apoptosis inhibitor and ferroptosis inhibitor can increase the proliferation rate of neural stem cells (P < 0.05). Fasudil increased the proliferation rate of neural stem cells treated by apoptosis inducer and ferroptosis inducer (P < 0.05). Fasudil and ferroptosis inhibitors both decreased the level of malondialdehyde in neural stem cells, while ferroptosis inducers increased the level of malondialdehyde in neural stem cells (P < 0.05). After treatment with fasudil, the expression of doublecortin and glial fibrillary acidic protein protein in neural stem cells increased, and the expression of Nestin decreased (P < 0.05). To conclude, fasudil can improve the survival of neural stem cells by inhibiting apoptosis and ferroptosis, and moreover, it can promote the proliferation and differentiation of neural stem cells into neuron-like cells and glial cells.

Effect and mechanism of fasudil on proliferation and differentiation of neural stem cells / 中国组织工程研究

BACKGROUND: The self-repairing ability of damaged brain and spinal cord is limited. It is important to search for potential therapeutics to promote the proliferation and differentiation of neural stem cells. OBJECTIVE: To investigate the effect of fasudil on the proliferation and differentiation of primary cultured neural stem cells and its potential mechanism. METHODS: Neural stem cells were obtained from the brain tissue of 15-day-old fetal rats in vitro. The expression of Nestin in the cells was detected by immunofluorescence. After treatment with fasudil at different concentrations (50, 100, 200 μmol/L) for 24, 48 and 72 hours, the proliferation rate of neural stem cells was detected by MTT, and the apoptosis rate was detected by flow cytometry. After further treatment with autophagy inhibitor, necrosis inhibitor, apoptosis inducer and ferroptosis inducer, the proliferation rates of neural stem cells were detected by MTT and the levels of malondialdehyde were detected by biochemical method. The expression of Nestin, doublecortin, microtubuleassociated protein and glial fibrillary acidic protein in neural stem cells were detected by western blot and immunofluorescence after treatment with 200 μmol/L fasudil for 10 days. RESULTS AND CONCLUSION: The positive expression of Nestin protein in primary cultured neural stem cells was observed. The proliferation rate of neural stem cells increased gradually with the increase of fasudil concentration as well as with the prolongation of action time (P < 0.05). Both apoptosis inhibitor and ferroptosis inhibitor can increase the proliferation rate of neural stem cells (P < 0.05). Fasudil increased the proliferation rate of neural stem cells treated by apoptosis inducer and ferroptosis inducer (P < 0.05). Fasudil and ferroptosis inhibitors both decreased the level of malondialdehyde in neural stem cells, while ferroptosis inducers increased the level of malondialdehyde in neural stem cells (P < 0.05). After treatment with fasudil, the expression of doublecortin and glial fibrillary acidic protein protein in neural stem cells increased, and the expression of Nestin decreased (P < 0.05). To conclude, fasudil can improve the survival of neural stem cells by inhibiting apoptosis and ferroptosis, and moreover, it can promote the proliferation and differentiation of neural stem cells into neuron-like cells and glial cells.