Spatially resolved gene regulatory and disease-related vulnerability map of the adult Macaque cortex.

Single cell approaches have increased our knowledge about the cell type composition of the non-human primate (NHP), but a detailed characterization of area-specific regulatory features remains outstanding. We generated single-cell transcriptomic and chromatin accessibility (single-cell ATAC) data of 358,237 cells from prefrontal cortex (PFC), primary motor cortex (M1) and primary visual cortex (V1) of adult female cynomolgus monkey brain, and integrated this dataset with Stereo-seq (spatial enhanced resolution omics-sequencing) of the corresponding cortical areas to assign topographic information to molecular states. We identified area-specific chromatin accessible sites and their targeted genes, including the cell type-specific transcriptional regulatory network associated with excitatory neurons heterogeneity. We reveal calcium ion transport and axon guidance genes related to specialized functions of PFC and M1, identified the similarities and differences between adult macaque and human oligodendrocyte trajectories, and mapped the genetic variants and gene perturbations of human diseases to NHP cortical cells. This resource establishes a transcriptomic and chromatin accessibility combinatory regulatory landscape at a single-cell and spatially resolved resolution in NHP cortex.

Isolation and characterization of two novel serotypes of Tibet orbivirus from Culicoides and sentinel cattle in Yunnan Province of China.

Tibet orbivirus (TIBOV), a new candidate of Orbivirus genus, was initially isolated from mosquitoes in Tibet in 2009 and subsequently from both Culicoides and mosquitoes in several provinces of China and Japan. Little is known about the origin, genetic diversity, dissemination and pathogenicity of TIBOV, although its potential threat to animal health has been acknowledged. In this study, two viruses, V290/YNSZ and V298/YNJH, were isolated from the Culicoides and sentinel cattle in Yunnan Province. Their genome sequences, cell tropism in mammalian and insect cell lines along with pathogenicity in suckling mice were determined. Genome phylogenetic analyses confirmed their classification as TIBOV species; however, OC1 proteins of the V290/YNSZ and V298/YNJH shared maximum sequence identities of 31.5% and 33.9% with other recognized TIBOV serotypes (TIBOV-1 to TIBOV-4) and formed two monophyletic branches in phylogenetic tree, indicating they represented two novel TIBOV serotypes which were tentatively designated as TIBOV-5 and TIBOV-6. The viruses replicated robustly in BHK, Vero and C6/36 cells and triggered overt clinical symptoms in suckling mice after intracerebral inoculation, causing mortality of 100% and 25%. Cross-sectional epidemiology analysis revealed silent circulation of TIBOV in Yunnan Province with overall prevalence of 16.4% (18/110) in cattle, 10.8% (13/120) in goats and 5.5% (6/110) in swine. The prevalence patterns of four investigated TIBOV serotypes (TIBOV-1, -2, -5 and 6) differed from each one another, with their positive rates ranging from 8.2% (9/110) for TIBOV-2 in cattle to 0.9% (1/110) for TIBOV-1 and TIBOV-5 in cattle and swine. Our findings provided new insights for diversity, pathogenicity and epidemiology of TIBOV and formed a basis for future studies addressing the geographical distribution and the zoonotic potential of TIBOV.

Epidemiological investigation and phylogenetic analysis of Classical Swine Fever virus in Yunnan province from 2015 to 2021.

BACKGROUND: Classical swine fever virus (CSFV), the causative agent of classical swine fever (CFS), is a highly contagious disease that poses a serious threat to Chinese pig populations. OBJECTIVES: Many provinces of China, such as Shandong, Henan, Hebei, Heilongjiang, and Liaoning provinces, have reported epidemics of CSFV, while the references to the epidemic of CSFV in Yunnan province are rare. This study examined the epidemic characteristics of the CSFV in Yunnan province. METHODS: In this study, 326 tissue samples were collected from different regions in Yunnan province from 2015 to 2021. A reverse transcription-polymerase chain reaction (RT-PCR), sequences analysis, and phylogenetic analysis were performed for the pathogenic detection and analysis of these 326 clinical specimens. RESULTS: Approximately 3.37% (11/326) of specimens tested positive for the CSFV by RT-PCR, which is lower than that of other regions of China. Sequence analysis of the partial E2 sequences of eleven CSFV strains showed that they shared 89.0-100.0% nucleotide (nt) and 95.0-100.0% amino acid (aa) homology, respectively. Phylogenetic analysis showed that these novel isolates belonged to the subgenotypes 2.1c and 2.1d, with subgenotype 2.1c being predominant. CONCLUSIONS: The CSFV was sporadic in China's Yunnan province from 2015 to 2021. Both 2.1c and 2.1d subgenotypes were found in this region, but 2.1c was dominant.

Full Genome Sequencing of Three Sedoreoviridae Viruses Isolated from Culicoides spp. (Diptera, Ceratopogonidae) in China.

Sedoreoviridae is a family of viruses belonging to the order Reovirales and comprises six genera, two of which, Orbivirus and Seadornavirus, contain arboviruses that cause disease in humans and livestock. Areas such as Yunnan Province in southwestern China, have high arboviral activity due in part to warm and wet summers, which support high populations of biting flies such as mosquitoes and Culicoides. Three viral isolates previously obtained from Culicoides collected at cattle farms in Shizong County of Yunnan Province, China, between 2019 and 2020 were completely sequenced and identified as Banna virus (BAV) genotype A of Seadornavirus and serotypes 1 and 7 of epizootic hemorrhagic disease virus (EHDV) of Orbivirus. These results suggest that Culicoidestainanus and C. orientalis are potential vectors of BAV and EHDV, respectively, and represent the first association of a BAV with C. tainanus and of an arbovirus with C. orientalis. Analysis using VP9 generally agreed with the current groupings within this genus based on VP12, although the classification for some strains should be corrected. Furthermore, the placement of Kadipiro virus (KDV) and Liao ning virus (LNV) in Seadornavirus may need confirmation as phylogenetic analysis placed these viruses as sister to other species in the genus.

Genetic and Pathogenic Characterisation of a Virulent Akabane Virus Isolated from Goats in Yunnan, China.

Introduction: Akabane virus (AKAV) has been detected in a variety of host species in China, but there are only limited records of its occurrence in goats. However, more attention needs to be paid to understanding the diversity of viruses in this species. The aim of the study was to explore the genotype characteristics and variation trend of AKAV and their relationship with virulence in Yunnan, China. Material and Methods: Blood samples were collected from goats during routine surveillance of goat diseases in Yunnan province in 2019. The AKAV CX-01 strain was isolated using BHK-21 cells. To understand pathogenicity, the virus was intraperitoneally (IP) and intracerebrally (IC) inoculated into suckling mice and tissue samples were subsequently analysed histopathologically and immunohistochemically. Results: Akabane virus CX-01 strain induced encephalitis and impairment of the central nervous system with fatal consequences. Phylogenetic analysis based on the ORF sequences of the small segments indicated that the AKAV isolate used was most closely related to the GD18134/2018 Chinese midge and bovine NM BS/1strains, while phylogenetic analysis based on the medium segments showed a close relationship between CX-01 and the Chinese GLXCH01 strain. Conclusion: The CX-01 isolate was related to AKAV genogroup Ia and probably originated from a recombination of different strains.

Epidemiological Investigation and Genetic Analysis of Pseudorabies Virus in Yunnan Province of China from 2017 to 2021.

In recent years, the prevalence of pseudorabies virus (PRV) has caused huge economic losses to the Chinese pig industry. Meanwhile, PRV infection in humans also sounded the alarm about its cross-species transmission from pigs to humans. To study the regional PRV epidemic, serological and epidemiological investigations of PRV in pig populations from Yunnan Province during 2017-2021 were performed. The results showed that 31.37% (6324/20,158, 95% CI 30.73-32.01) of serum samples were positive for PRV glycoprotein E (gE)-specific antibodies via enzyme-linked immunosorbent assay (ELISA). The risk factors, including the breeding scale and development stage, were significantly associated with PRV seroprevalence among pigs in Yunnan Province. Of the 416 tissue samples collected from PRV-suspected pigs in Yunnan Province, 43 (10.33%, 95% CI 7.41-13.26) samples were positive for PRV-gE nucleic acid in which 15 novel PRV strains from these PRV-positive samples were isolated, whose gC and gE sequences were analyzed. Phylogenetic analysis showed that all 15 isolates obtained in this study belonged to the genotype II. Additionally, the gC gene of one isolate (YuN-YL-2017) was genetically closer to variant PRV strains compared with others, while the gE gene was in the same clade with other classical PRV strains, indicating that this isolate might be a recombinant strain generated from the classical and variant strains. The results revealed the severe PRV epidemic in Yunnan Province and indicated that PRV variants are the major genotypes threatening the pig industry development.

Single-cell multiomics reveals heterogeneous cell states linked to metastatic potential in liver cancer cell lines.

Hepatocellular carcinoma (HCC) is the most common liver cancer with a high rate of metastasis. However, the molecular mechanisms that drive metastasis remain unclear. We combined single-cell transcriptomic, proteomic, and chromatin accessibility data to investigate how heterogeneous phenotypes contribute to metastatic potential in five HCC cell lines. We confirmed that the prevalence of a mesenchymal state and levels of cell proliferation are linked to the metastatic potential. We also identified a rare hypoxic subtype that has a higher capacity for glycolysis and exhibits dormant, invasive, and malignant characteristics. This subtype has increased metastatic potential. We further identified a robust 14-gene panel representing this hypoxia signature and this hypoxia signature could serve as a prognostic index. Our data provide a valuable data resource, facilitate a deeper understanding of metastatic mechanisms, and may help diagnosis of metastatic potential in individual patients, thus supporting personalized medicine.

scDPN for High-throughput Single-cell CNV Detection to Uncover Clonal Evolution During HCC Recurrence.

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution. Unfortunately, classical DNA amplification-based methods have low throughput and introduce coverage bias during sample preamplification. We developed a single-cell DNA library preparation method without preamplification in nanolitre scale (scDPN) to address these issues. The method achieved a throughput of up to 1800 cells per run for copy number variation (CNV) detection. Also, our approach demonstrated a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy for cell line and tumor tissue evaluation. We used this approach to profile the tumor clones in paired primary and relapsed tumor samples of hepatocellular carcinoma (HCC). We identified three clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumor containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumor, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.

Bluetongue virus non-structural protein 3 (NS3) and NS4 coordinatively antagonize type â interferon signaling by targeting STAT1.

Previous studies have pointed out that bluetongue virus (BTV) down-regulates the expression levels of type Ⅰ interferon (IFN-Ⅰ) and inhibits IFN-Ⅰ signaling by targeting on the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription protein (STAT) pathway. However, individual viral protein could not effectively block IFN-Ⅰ signaling. There is a need to explore the underlying mechanisms by which viral proteins of BTV coordinate to antagonize the IFN-Ⅰ signaling. We investigated the coordinative role of BTV-1 nonstructural protein 3 (NS3) and NS4 in counteracting IFN-Ⅰ signaling in the JAK-STAT pathway by directly interacting with STAT1. The NS3 and NS4 targeted the SH2 domain of STAT1 to inhibit its phosphorylation, heterodimerization, nuclear translocation, as well as activation of downstream genes of the JAK-STAT pathway. NS3 and NS4 impaired STAT1 phosphorylation induced by IFN-Ⅰ in a dose dependent manner. Overall, this study confirmed that NS3 and NS4 of BTV participate in interfering with IFN-Ⅰ signaling process. Also, a new mechanism employed by BTV to evade host innate immune responses was revealed.

Multiregion single-cell sequencing reveals the transcriptional landscape of the immune microenvironment of colorectal cancer.

The tumor microenvironment is a complex ecosystem formed by distinct and interacting cell populations, and its composition is related to cancer prognosis and response to clinical treatment. In this study, we have taken the advantage of two single-cell RNA sequencing technologies (Smart-seq2 and DNBelab C4) to generate an atlas of 15,115 immune and nonimmune cells from primary tumors and hepatic metastases of 18 colorectal cancer (CRC) patients. We observed extensive changes in the proportions and functional states of T cells and B cells in tumor tissues, compared to those of paired non-tumor tissues. Importantly, we found that B cells from early CRC tumor were identified to be pre-B like expressing tumor suppressors, whereas B cells from advanced CRC tumors tended to be developed into plasma cells. We also identified the association of IgA+ IGLC2+ plasma cells with poor CRC prognosis, and demonstrated a significant interaction between B-cell and myeloid-cell signaling, and found CCL8+ cycling B cells/CCR5+ T-cell interactions as a potential antitumoral mechanism in advanced CRC tumors. Our results provide deeper insights into the immune infiltration within CRC, and a new perspective for the future research in immunotherapies for CRC.

Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high relapse and low 5-year survival rates. Single-cell profiling in relapsed HCC may aid in the design of effective anticancer therapies, including immunotherapies. We profiled the transcriptomes of ∼17,000 cells from 18 primary or early-relapse HCC cases. Early-relapse tumors have reduced levels of regulatory T cells, increased dendritic cells (DCs), and increased infiltrated CD8+ T cells, compared with primary tumors, in two independent cohorts. Remarkably, CD8+ T cells in recurrent tumors overexpressed KLRB1 (CD161) and displayed an innate-like low cytotoxic state, with low clonal expansion, unlike the classical exhausted state observed in primary HCC. The enrichment of these cells was associated with a worse prognosis. Differential gene expression and interaction analyses revealed potential immune evasion mechanisms in recurrent tumor cells that dampen DC antigen presentation and recruit innate-like CD8+ T cells. Our comprehensive picture of the HCC ecosystem provides deeper insights into immune evasion mechanisms associated with tumor relapse.

Comparative analysis of sequencing technologies for single-cell transcriptomics.

Single-cell RNA-seq technologies require library preparation prior to sequencing. Here, we present the first report to compare the cheaper BGISEQ-500 platform to the Illumina HiSeq platform for scRNA-seq. We generate a resource of 468 single cells and 1297 matched single cDNA samples, performing SMARTer and Smart-seq2 protocols on two cell lines with RNA spike-ins. We sequence these libraries on both platforms using single- and paired-end reads. The platforms have comparable sensitivity and accuracy in terms of quantification of gene expression, and low technical variability. Our study provides a standardized scRNA-seq resource to benchmark new scRNA-seq library preparation protocols and sequencing platforms.