Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing.

The monkeypox virus (MPXV) has triggered a current outbreak globally. Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control. It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads, as it is one of the DNA viruses with the largest genome and the most AT-biased, and has a significant number of tandem repeats. Here we evaluated the performance of metagenomic and amplicon sequencing techniques, and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland. We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens. Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes. Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences. Besides, several intra-host single nucleotide variations were identified in the first imported mpox case. This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens. The findings of this study will significantly enhance the surveillance of MPXV.

Differentiation and characterization of rhesus monkey atrial and ventricular cardiomyocytes from induced pluripotent stem cells.

The combination of non-human primate animals and their induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provides not only transplantation models for cell-based therapy of heart diseases, but also opportunities for heart-related drug research on both cellular and animal levels. However, the subtypes and electrophysiology properties of non-human primate iPSC-CMs hadn't been detailed characterized. In this study, we generated rhesus monkey induced pluripotent stem cells (riPSCs), and efficiently differentiated them into ventricular or atrial cardiomyocytes by modulating retinoic acid (RA) pathways. Our results revealed that the electrophysiological characteristics and response to canonical drugs of riPSC-CMs were similar with those of human pluripotent stem cell derived CMs. Therefore, rhesus monkeys and their iPSC-CMs provide a powerful and practicable system for heart related biomedical research.

Efficient Differentiation of TBX18+/WT1+ Epicardial-Like Cells from Human Pluripotent Stem Cells Using Small Molecular Compounds.

The epicardium promotes neovascularization and cardiomyocyte regeneration by generating vascular smooth muscle cells (SMCs) and producing regenerative factors after adult heart infarction. It is therefore a potential cell resource for repair of the injured heart. However, the epicardium also participates in fibrosis and scarring of the injured heart, complicating its use in regenerative medicine. In this study, we report coexpression of TBX18 and WT1 in the majority of epicardial cells during mouse embryonic epicardial development. Furthermore, we describe a convenient chemically defined, immunogen-free, small molecule-based method for generating TBX18+/WT1+ epicardial-like cell populations with 80% homogeneity from human pluripotent stem cells by modulation of the WNT and retinoic acid signaling pathways. These epicardial-like cells exhibited characteristic epicardial cell morphology following passaging and differentiation into functional SMCs or cardiac fibroblast-like cells. Our findings add to existing understanding of human epicardial development and provide an efficient and stable method for generating both human epicardial-like cells and SMCs.