An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Dynamic peripheral blood microRNA expression landscape during the peri-implantation stage in women with successful pregnancy achieved by single frozen-thawed blastocyst transfer.

STUDY QUESTION: What are the dynamic expression features of plasma microRNAs (miRNAs) during the peri-implantation period in women with successful pregnancy via single frozen-thawed blastocyst transfer? SUMMARY ANSWER: There is a significant change in the plasma miRNA expression profile before and after blastocyst transfer, during the window of implantation. WHAT IS KNOWN ALREADY: The expression of miRNAs in peripheral blood has indicative functions during the peri-implantation period. Nevertheless, the dynamic expression profile of circulating miRNAs during the peri-implantation stage in women with a successful pregnancy has not been studied. STUDY DESIGN SIZE DURATION: Seventy-six women treated for infertility with a single frozen-thawed blastocyst transfer in a natural cycle were included in this study. Among them, 57 women had implantation success and a live birth, while 19 patients experienced implantation failure. Peripheral blood samples were collected at five different time points throughout the peri-implantation period, including D0 (ovulation day), D3, D5, D7, and D9 in this cycle of embryo transfer. The plasma miRNAs in women with blastocyst transfer were isolated, sequenced, and analyzed. PARTICIPANTS/MATERIALS SETTING METHODS: Peripheral blood samples were collected in EDTA tubes and stored at -80°C until further use. miRNAs were isolated from blood, cDNA libraries were constructed, and the resulting sequences were mapped to the human genome. The plasma miRNAs were initially analyzed in a screening cohort (n = 34) with successful pregnancy. Trajectory analysis, including a global test and pairwise comparisons, was performed to detect dynamic differentially expressed (DE) miRNAs. Fuzzy c-means clustering was conducted for all dynamic DE miRNAs. The correlation between DE miRNAs and clinical characteristics of patients was investigated using a linear mixed model. Target genes of the miRNAs were predicted, and functional annotation analysis was performed. The expression of DE miRNAs was also identified in a validation set consisting of women with successful (n = 23) and unsuccessful (n = 19) pregnancies. MAIN RESULTS AND THE ROLE OF CHANCE: Following small RNA sequencing, a total of 2656 miRNAs were determined as valid read values. After trajectory analysis, 26 DE miRNAs (false discovery rate < 0.05) were identified by the global test, while pairwise comparisons in addition identified 20 DE miRNAs. A total of seven distinct clusters representing different temporal patterns of miRNA expression were discovered. Nineteen DE miRNAs were further identified to be associated with at least one clinical trait. Endometrium thickness and progesterone level showed a correlation with multiple DE miRNAs (including two of the same miRNAs, hsa-miR-1-3p and hsa-miR-6741-3p). Moreover, the 19 DE miRNAs were predicted to have 403 gene targets, and there were 51 (12.7%) predicted genes likely involved in both decidualization and embryo implantation. Functional annotation for predicted targets of those clinically related DE miRNAs suggested the involvement of vascular endothelial growth factor and Wnt signaling pathways, as well as responses to hormones, immune responses, and cell adhesion-related signaling pathways during the peri-implantation stage. LARGE SCALE DATA: The raw miRNA sequence data reported in this article have been deposited in the Genome Sequence Archive (GSA-Human: HRA005227) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human/browse/HRA005227. LIMITATIONS REASONS FOR CAUTION: Although the RNA sequencing results revealed the global dynamic changes of miRNA expression, further experiments examining the clinical significance of the identified DE miRNAs in embryo implantation outcome and the relevant regulatory mechanisms involved are warranted. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the dynamic landscape of the miRNA transcriptome could shed light on the physiological mechanisms involved from ovulation to the post-implantation stage, as well as identifying biomarkers that characterize stage-related biological process. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Major clinical research project of Tangdu Hospital (2021LCYJ004) and the Discipline Platform Improvement Plan of Tangdu Hospital (2020XKPT003). The funders had no influence on the study design, data collection, and analysis, decision to publish, or preparation of the article. There are no conflicts of interest to declare.

IL18R1-Related Molecules as Biomarkers for Asthma Severity and Prognostic Markers for Idiopathic Pulmonary Fibrosis.

To determine the role of inflammation-related proteins in predicting asthma severity and outcome, 92 inflammation-related proteins were measured in the asthmatic serum using Olink analysis. Different bioinformatics algorithms were developed to cross analyze with the single-cell or transcriptome data sets from the Gene Expression Omnibus database to explore the role of IL18R1 and related genes in asthma and idiopathic pulmonary fibrosis (IPF). Olink identified 52 differentially expressed proteins in asthma. They were strongly linked to the cytokine-cytokine receptor interaction, TNF, and NF-κB signaling pathway. Seven proteins were found in both single-cell RNA and Olink analyses. Among them, IL18R1 was predominantly expressed in mast cells, and the results suggested enhanced communication between mast cells and CD 8+ T cells. IL18R1 was upregulated in serum and induced sputum and bronchoalveolar lavage fluid of patients with uncontrolled or severe asthma. IL18R1 was positively correlated with TNFSF1 and OSM and S100A12. The diagnostic efficacy of these serum IL18R1-related molecules for asthma ranged from 0.839 to 0.921. Moreover, high levels of IL18R1, TNFSF1, OSM, and S100A12 were significantly associated with shorter survival times and worse lung function. IL18R1-related molecules may serve as biomarkers for monitoring uncontrolled or severe asthma and as prognostic markers for IPF.

Comprehensive analysis of immune-related genes for classification and immune microenvironment of asthma.

OBJECTIVES: To determine the effects of immune-related genes (IRGs) and immune landscape of induced sputum, and develop novel, non-invasive diagnostic molecular therapeutic targets for asthma. METHODS: GSE76262 datasets were used to identify differentially expressed IRGs in asthma. Key IRGs were detected using a protein-protein interaction network. Receiver operating characteristic (ROC) curves were analyzed to investigate the diagnostic value of key IRGs. Gene set enrichment analysis (GSEA) was performed with WebGestalt. Single-sample gene set enrichment analysis and CIBERSORT were used to investigate the immune landscape of induced sputum. RESULTS: A total of 75 potential IRGs were associated with asthma, most of which were involved in the NF-kappa B signaling pathway. ROC analysis showed AUC values for the hub pathway ranging from 0.676-0.767, with moderate diagnostic value for asthma. We also identified IRGs-related cytokines (TNF-α, IL-1ß, IL-8 and IL-6) in 76 asthma and 91 control serum samples to further explore diagnostic efficacy, showing a cumulative AUC of 0.998 for these four related cytokines. Analysis of immune cell infiltration levels showed that follicular helper T cells, activated dendritic cells, activated mast cells and eosinophils were significantly higher and macrophages M0 and macrophages M2 were significantly reduced in sputum from patients with asthma. CONCLUSIONS: IRGs-related cytokines and immune infiltration may contribute to the diagnosis and immune classification of asthma.

Evaluating the Effects of Storage Conditions on Multiple Cell-Free RNAs in Plasma by High-Throughput Sequencing.

Background: Plasma cell-free RNAs (cfRNAs) can serve as noninvasive biomarkers for the diagnosis and monitoring of diseases. However, the delay in blood processing may lead to unreliable results. Therefore, an unbiased evaluation based on the whole transcriptome under different storage conditions is needed. Methods: Here, blood samples were collected in ethylenediaminetetraacetic acid tubes and processed immediately (0 hour), or stored at room temperature (RT) or 4°C for different time intervals (2, 6, and 24 hours) before plasma separation. High-throughput sequencing was applied to assess the effects of storage conditions on the transcript profiles and fragment characteristics of plasma cell-free mRNA, long noncoding RNA (lncRNA), and small RNAs. Results: More genes changed their expression levels with time when blood was stored at RT compared with those at 4°C. Cell-free mRNA and lncRNA were relatively stable in blood preserved at 4°C for 6 hours, while cell-free microRNA (miRNA) and piwi-interacting RNA (piRNA) remained stable at 4°C for 24 hours. After 24 hours, more contamination of the leukocyte-derived RNAs occurred at RT, possibly due to apoptosis. Meanwhile, significant changes were also observed regarding the characteristics of the RNA fragments, including fragment size, the proportion of intron, and the pyrimidine frequency of the fragmented 3' end. Fifteen tissue-enriched genes were detected in the plasma but not expressed in leukocytes. The expression level and fragment length of these genes gradually decreased during storage, suggesting the degradation of the cfRNA and the dilution of leukocyte-derived RNA with other tissue-derived cfRNA. Conclusions: Our results suggest that the contamination of leukocyte-derived RNA and the degradation of original cfRNA contribute to the changes in the cfRNA expression profiles and the fragment characteristics during short-term storage. The storage of blood at 4°C for 6 hours allows plasma cfRNA to remain relatively stable, which will be useful for further studies or clinical applications where adequate quantification or the fragment signature of cfRNA is required.

Whole genome sequences reveal the Xanthomonas perforans population is shaped by the tomato production system.

Modern agricultural practices increase the potential for plant pathogen spread, while the advent of affordable whole genome sequencing enables in-depth studies of pathogen movement. Population genomic studies may decipher pathogen movement and population structure as a result of complex agricultural production systems. We used whole genome sequences of 281 Xanthomonas perforans strains collected within one tomato production season across Florida and southern Georgia fields to test for population genetic structure associated with tomato production system variables. We identified six clusters of X. perforans from core gene SNPs that corresponded with phylogenetic lineages. Using whole genome SNPs, we found genetic structure among farms, transplant facilities, cultivars, seed producers, grower operations, regions, and counties. Overall, grower operations that produced their own transplants were associated with genetically distinct and less diverse populations of strains compared to grower operations that received transplants from multiple sources. The degree of genetic differentiation among components of Florida's tomato production system varied between clusters, suggesting differential dispersal of the strains, such as through seed or contaminated transplants versus local movement within farms. Overall, we showed that the genetic variation of a bacterial plant pathogen is shaped by the structure of the plant production system.

The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood.

The expression of human and microbial genes serves as biomarkers for disease and health. Blood RNA is an important biological resource for precision medicine and translational medicine. However, few studies have assessed the human transcriptome profiles and microbial communities composition and diversity of peripheral blood from different cell isolation methods, which could affect the reproducibility of researches. We collected peripheral blood from three healthy donors and processed it immediately. We used RNA sequencing to investigate the effect of three leukocyte isolation methods including buffy coat (BC) extraction, red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) isolation with the comparison with whole blood (WB), through analyzing the sensitivity of gene detection, the whole transcriptome profiling and microbial composition and diversity. Our data showed that BC extraction with high globin mRNA mapping rate had similar transcriptome profiles with WB, while RBC lysis and PBMC isolation depleted RBCs effectively. With the efficient depletion of RBC and distinct compositions of leukocyte subsets, RNA-seq of RBC lysis and PBMC isolation uniquely detected genes from specific cell types, like granulocytes and NK cells. In addition, we observed that the microbial composition and diversity were more affected by individuals than isolation methods. Our results showed that blood cell isolations could largely influence the sensitivity of detection of human genes and transcriptome profile.

Impact of storage conditions on peripheral leukocytes transcriptome.

Leukocytes reflect the physiological and pathological states of each individual, and transcriptomic data of leukocytes have been used to reflect health conditions. Since the overall impact of ex vivo conditions on the leukocyte transcriptome before RNA stabilization remains unclear, we evaluated the influence of temporary storage conditions on the leukocyte transcriptome through RNA sequencing. We collected peripheral blood with EDTA tubes, which were processed immediately or stored either at 4 °C or room temperature (RT, 18-22 °C) for 2 h, 6 h and 24 h. Total cellular RNA was extracted from 42 leukocyte samples after red blood cells lysis for subsequent RNA sequencing. We applied weighted gene co-expression network analysis to construct co-expression networks of mRNA and lncRNA among the samples, and then performed gene ontology (GO) term enrichment to explore possible biological processes affected by storage conditions. Storage conditions change the gene expression of peripheral leukocytes. Comparing with fresh leukocytes, storage for 24 h at 4 °C and RT affected 1515 (1.51%) and 10,823 (10.82%) genes, respectively. Pathway enrichment analysis identified nucleosome assembly enriched in up-regulated genes at both conditions. When blood was stored at RT for 24 h, genes involved in apoptotic signaling pathway, negative regulation of cell cycle and lymphocyte activation were upregulated, while the relative proportion of neutrophils was significantly decreased. Temporary storage conditions profoundly affect the gene expression profiles of leukocytes and might further change cell viability and state. Storage of blood samples at 4 °C within 6 h largely maintains their original transcriptome.

Assessing Changes and Associations in the Xanthomonas perforans Population Across Florida Commercial Tomato Fields Via a Statewide Survey.

Before 1991, Xanthomonas euvesicatoria was the causal agent of bacterial spot of tomato in Florida but was quickly replaced by X. perforans. The X. perforans population has changed in genotype and phenotype despite lack of a clear selection pressure. To determine the current Xanthomonas population in Florida, we collected 585 Xanthomonas strains from 70 tomato fields, representing 22 farms across eight counties, in the Florida tomato production region. Strains were isolated from 23 cultivars across eight seed producers and were associated with eight transplant facilities during the fall 2017 season. Our collection was phenotypically and genotypically characterized. Only X. perforans was identified, and all strains except one (99.8%) were tolerant to copper sulfate and 25% of strains were resistant to streptomycin sulfate. Most of the strains (99.3%) that were resistant to streptomycin sulfate were sequence type 1. The X. perforans population consisted of tomato races 3 (8%) and 4 (92%) and all three previously reported sequence types, ranging from 22 to 46% frequency. Approximately half of all strains, none of which were sequence type 2, produced bacteriocins against X. euvesicatoria. Effector profiles were highly variable among strains, which could impact the strains' host range. The effector xopJ4, which was previously thought to be conserved in X. perforans tomato pathogens, was absent in 19 strains. Nonmetric multidimensional scaling and network analyses show how strains and strain traits were associated with production system variables, including anonymized farms and transplant facilities. These analyses show that the composition of the Florida X. perforans population is diverse and complex.

Global Cropland Connectivity: A Risk Factor for Invasion and Saturation by Emerging Pathogens and Pests.

The geographic pattern of cropland is an important risk factor for invasion and saturation by crop-specific pathogens and arthropods. Understanding cropland networks supports smart pest sampling and mitigation strategies. We evaluate global networks of cropland connectivity for key vegetatively propagated crops (banana and plantain, cassava, potato, sweet potato, and yam) important for food security in the tropics. For each crop, potential movement between geographic location pairs was evaluated using a gravity model, with associated uncertainty quantification. The highly linked hub and bridge locations in cropland connectivity risk maps are likely priorities for surveillance and management, and for tracing intraregion movement of pathogens and pests. Important locations are identified beyond those locations that simply have high crop density. Cropland connectivity risk maps provide a new risk component for integration with other factors-such as climatic suitability, genetic resistance, and global trade routes-to inform pest risk assessment and mitigation.

DNA methyltransferases have an essential role in female fecundity in brown planthopper, Nilaparvata lugens.

DNA methylation is an ancient epigenetic modification present in all three domains of life. However, the understanding of DNA methylation in insects is limited. Here, we amplified the full-length transcripts of the DNA methyltransferases Nlu-Dnmt1 and Nlu-Dnmt3, indicating that a complete DNA methylation toolkit exists in the brown planthopper, Nilaparvata lugens, a destructive pest in rice production. Nlu-DNMT1 and Nlu-DNMT3 had the conserved motifs and domains of the DNA methyltransferase family. Nlu-Dnmt1 and Nlu-Dnmt3 were highly expressed in the mated and gravid female adults but weakly expressed in larvae, male adults, and virgin female adults. Silencing Nlu-Dnmt1 and Nlu-Dnmt3 in gravid brachypterous female adults led to fewer offspring, suggesting that DNA methylation regulates female fecundity in insects.

Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua.

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1(st) to 5(th) instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4(th) instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited "half-ecdysis". In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.