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The transcriptome of porcine peripheral blood mononuclear cells (PBMC) at single cell (sc) resolution is well described, but little is understood about the cis-regulatory mechanism behind scPBMC gene expression. Here, we profiled the open chromatin landscape of porcine PBMC that define cis-regulatory elements and mechanism contributing to the transcription using single nucleus ATAC sequencing (snATAC-seq). Approximately 22 % of the identified peaks overlapped with annotated transcription start sites (TSS). Using clustering based on open chromatin pattern similarity, we demonstrate that cell type annotations using snATAC-seq are highly concordant to that reported for sc RNA sequencing (scRNA-seq). The differentially accessible peaks (DAPs) for each cell type were characterized and the pattern of accessibility of the DAPs near cell type markers across cell types was similar to that of the average gene expression level of corresponding marker genes. Additionally, we found that peaks identified in snATAC-seq have the potential power to predict the cell type specific transcription starting site (TSS). We identified both transcription factors (TFs) whose binding motif were enriched in cell type DAPs of multiple cell types and cell type specific TFs by conducting transcription factor binding motif (TFBM) analysis. Furthermore, we identified the putative enhancer or promoter regions bound by TFs for each differentially expressed gene (DEG) with a DAP that overlapped with its TSS by generating cis-co-accessibility networks (CCAN). To predict the regulators of such DEGs, TFBM analysis was performed for each CCAN. The regulator TF-target DEG pairs predicted in this way were largely consistent with the results reported in the ENCODE Transcription Factor Targets Dataset (TFTD). This snATAC-seq approach provides insights into the regulation of chromatin accessibility landscape of porcine PBMCs and enables discovery of TFs predicted to control DEG through binding regulatory elements whose chromatin accessibility correlates with the DEG promoter region., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophils are vital components of the immune system for limiting the invasion and proliferation of pathogens in the body. Surprisingly, the functional annotation of porcine neutrophils is still limited. The transcriptomic and epigenetic assessment of porcine neutrophils from healthy pigs was performed by bulk RNA sequencing and transposase accessible chromatin sequencing (ATAC-seq). First, we sequenced and compared the transcriptome of porcine neutrophils with eight other immune cell transcriptomes to identify a neutrophil-enriched gene list within a detected neutrophil co-expression module. Second, we used ATAC-seq analysis to report for the first time the genome-wide chromatin accessible regions of porcine neutrophils. A combined analysis using both transcriptomic and chromatin accessibility data further defined the neutrophil co-expression network controlled by transcription factors likely important for neutrophil lineage commitment and function. We identified chromatin accessible regions around promoters of neutrophil-specific genes that were predicted to be bound by neutrophil-specific transcription factors. Additionally, published DNA methylation data from porcine immune cells including neutrophils were used to link low DNA methylation patterns to accessible chromatin regions and genes with highly enriched expression in porcine neutrophils. In summary, our data provides the first integrative analysis of the accessible chromatin regions and transcriptional status of porcine neutrophils, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the utility of chromatin accessible regions to identify and enrich our understanding of transcriptional networks in a cell type such as neutrophils., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Herrera-Uribe, Lim, Byrne, Daharsh, Liu, Corbett, Marco, Schroyen, Koltes, Loving and Tuggle.)

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Herrera-Uribe, Wiarda, Sivasankaran, Daharsh, Liu, Byrne, Smith, Lunney, Loving and Tuggle.)

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans.  There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax ( n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 10 5 50% tissue culture infective dose(s) (TCID 50 )] D/OK ( n = 6) or D/600 ( n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine ( n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 10 5 TCID 50 ) with IDV D/OK ( n = 6) or D/660 ( n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model. IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections., (Copyright © 2018 American Society for Microbiology.)

The HIV-1 pandemic is a consequence of the cross-species transmission of simian immunodeficiency virus in wild chimpanzees (SIVcpz) to humans. Our previous study demonstrated SIVcpz strains that are closely related to the ancestral viruses of HIV-1 groups M (SIVcpzMB897) and N (SIVcpzEK505) and two SIVcpz lineages that are not associated with any known HIV-1 infections in humans (SIVcpzMT145 and SIVcpzBF1167), all can readily infect and robustly replicate in the humanized-BLT mouse model of humans. However, the comparative pathogenicity of different SIVcpz strains remains unknown. Herein, we compared the pathogenicity of the above four SIVcpz strains with HIV-1 using humanized-BLT mice. Unexpectedly, we found that all four SIVcpz strains were significantly less pathogenic or non-pathogenic compared to HIV-1, manifesting lower degrees of CD4+ T-cell depletion and immune activation. Transcriptome analyses of CD4+ T cells from hu-BLT mice infected with SIVcpz versus HIV-1 revealed enhanced expression of genes related to cell survival and reduced inflammation/immune activation in SIVcpz-infected mice. Together, our study results demonstrate for the first time that SIVcpz is significantly less or non-pathogenic to human immune cells compared to HIV-1. Our findings lay the groundwork for a possible new understanding of the evolutionary origins of HIV-1, where the initial SIVcpz cross-species transmission virus may be initially less pathogenic to humans.

Characterizing the transmitted/founder (T/F) viruses of multi-variant SIV infection may shed new light on the understanding of mucosal transmission. We intrarectally inoculated six Chinese rhesus macaques with a single high dose of SIVmac251 (3.1 × 10 4 TCID 50 ) and obtained 985 full-length env sequences from multiple tissues at 6 and 10 days post-infection by single genome amplification (SGA). All 6 monkeys were infected with a range of 2 to 8 T/F viruses and the dominant variants from the inoculum were still dominant in different tissues from each monkey. Interestingly, our data showed that a cluster of rare T/F viruses was unequally represented in different tissues. This cluster of rare T/F viruses phylogenetically related to the non-dominant SIV variants in the inoculum and was not detected in any rectum tissues, but could be identified in the descending colon, jejunum, spleen, or plasma. In 2 out of 6 macaques, identical SIVmac251 variants belonging to this cluster were detected simultaneously in descending colon/jejunum and the inoculum. We also demonstrated that the average CG dinucleotide frequency of these rare T/F viruses found in tissues, as well as non-dominant variants in the inoculum, was significantly higher than the dominant T/F viruses in tissues and the inoculum. Collectively, these findings suggest that descending colon/jejunum might be more susceptible than rectum to SIV in the very early phase of infection. And host CG suppression, which was previously shown to inhibit HIV replication in vitro , may also contribute to the bottleneck selection during in vivo transmission.