Discovery and vertical transmission analysis of Dabieshan Tick Virus in Haemaphysalis longicornis ticks from Chengde, China.

Introduction: Ticks are important blood-sucking ectoparasites that can transmit various pathogens, posing significant threats to the wellbeing of humans and livestock. Dabieshan tick virus (DBTV) was initially discovered in 2015 in Haemaphysalis longicornis ticks from the Dabieshan mountain region in Hubei Province, China. In recent years, DBTV has been discovered in various regions of China, including Shandong, Zhejiang, Liaoning, Hubei, Yunnan, and Guizhou Provinces. However, the researches on tick-borne transmission of DBTV are scarce. Methods: This study utilized the small RNA sequencing (sRNA-seq) method to identify tick-associated viruses in ticks collected from Chengde in Hebei Province and Yongcheng in Henan Province, leading to the discovery of a new DBTV strain in Hebei. The complete coding genome of DBTV Hebei strain was obtained through RNA-seq and Sanger sequencing. Furthermore, the transmission experiment of DBTV in H. longicornis was examined in laboratory for the first time. Results: DBTV was detected in newly molted adult H. longicornis ticks collected in Chengde, Hebei Province. Additionally, DBTV was also detected in both unfed nymphs and engorged females of H. longicornis collected from Chengde, with a positive rate of 20% and 56.25%, respectively. The complete coding genome of DBTV (OP682840 and OP716696) were obtained, and phylogenetic analysis revealed that the DBTV Hebei strain clustered with previously reported DBTV strains. Furthermore, this virus was observed in engorged females, eggs, and larvae of the subsequent generation. Discussion: It is necessary to expand the scope of DBTV investigation, particularly in northern China. This study demonstrated that DBTV can be transmitted from engorged females to larvae of the next generation. Moreover, the detection of DBTV in unfed nymphs and adults (which moulted from engorged nymphs) collected from the filed of Chengde suggests that H. longicornis serves as a potential transmission host and reservoir for DBTV through transstadial and transovarial transmission. However, there remains a lack of research on the isolation and pathogenicity of DBTV, highlighting the need for further studies to mitigate potential harm to the health of animals and humans.

Eimeria falciformis extracellular vesicles differentially express host cell lncRNAs.

Long noncoding RNAs (lncRNAs) are regulatory transcripts during protozoan infections in the host intestinal epithelial cells (IECs). Apicomplexan Eimeria falciformis sporozoite extracellular vesicles (EVs) contain virulence factors that modulate host IECs pro-inflammatory genes and immune responses. In this study, E. falciformis sporozoites were made to interact with inactivated host cells, and the parasite EVs were separated from total secretome by ultracentrifugation and purified on density gradient medium. Dose-dependent bio-activity of E. falciformis EVs was investigated by RNA sequencing, functional annotation and quantitative PCR. It was found that E. falciformis EVs induced mRNA, circRNA, and lncRNA expressions in mouse IECs. Of 38, 217 lncRNAs assembled, 157 and 152 were upwardly and downwardly expressed respectively. Differentially expressed lncRNAs were associated with cytokines, pyroptosis, and immune signaling pathways including FoxO, NF-κB, MAPK, and TGF-ß. In essence, E. falciformis EVs altered host cell RNA expressions during the interaction with host IECs. Also, differentially expressed lncRNAs are potential diagnostic transcripts during Eimeria infections.

Multimodal analysis of cell-free DNA whole-methylome sequencing for cancer detection and localization.

Multimodal epigenetic characterization of cell-free DNA (cfDNA) could improve the performance of blood-based early cancer detection. However, integrative profiling of cfDNA methylome and fragmentome has been technologically challenging. Here, we adapt an enzyme-mediated methylation sequencing method for comprehensive analysis of genome-wide cfDNA methylation, fragmentation, and copy number alteration (CNA) characteristics for enhanced cancer detection. We apply this method to plasma samples of 497 healthy controls and 780 patients of seven cancer types and develop an ensemble classifier by incorporating methylation, fragmentation, and CNA features. In the test cohort, our approach achieves an area under the curve value of 0.966 for overall cancer detection. Detection sensitivity for early-stage patients achieves 73% at 99% specificity. Finally, we demonstrate the feasibility to accurately localize the origin of cancer signals with combined methylation and fragmentation profiling of tissue-specific accessible chromatin regions. Overall, this proof-of-concept study provides a technical platform to utilize multimodal cfDNA features for improved cancer detection.

Quantitative proteomic analysis of local and systemic extracellular vesicles during Eimeria falciformis infectious cycle in the host.

BACKGROUND: Extracellular vesicles (EVs) are membranous structures that are formed during pathophysiology, host-parasite interactions and parasite motility. Typically, apicomplexan-infected host cells secrete EVs which traverse local and systemic strata of the host as the parasites develop. METHODS: Extracellular vesicles were isolated from the caecum and serum of Eimeria falciformis-infected mice during oocyst ingestion (0 h post-infection [0 hpi]), merozont stages 1 and 2 (68 and 116 hpi), oocyst shedding (7 days post-infection [7 dpi]) and host recovery (10 dpi) and subsequently characterized and profiled by tandem mass tag (TMT). RESULTS: With the progression of E. falciformis life stages, subpopulation of EVs bearing EV biomarkers, including CD9, CD82, heat shock protein 70 (HSP70) and major histocompatibility complex (MHC) molecules, increased. A total of 860 and 1024 differentially expressed proteins were identified in serum EVs (sEVs) and caecum EVs (cEVs), respectively. Identified immune-related molecules (such as cytokines, receptors, immunoglobins, complements, hormones, inflammasomes), ion exchange and cell death-associated proteins were significantly expressed, at least during the E. falciformis first and second merozont stages. Bioinformatics assessment indicated that sEV proteins were at all time points implicated in antigen processing and presentation as well as natural killer cell-mediated cytotoxicity (68 hpi), complement activation/blood coagulation (116 hpi/10 dpi) and catabolic activities (7 dpi). In contrast, cEV proteins were involved in catabolic process, ion transport and antigen presentation (68 and 116 hpi). Host response to E. falciformis infection was similar to intestinal bacterium at 7 dpi and cell adhesion and intercellular protein transport at 10 dpi. In both systems, ferroptosis and necroptosis were common across the parasite's infectious cycle while apoptosis occurred at 68 hpi. CONCLUSION: The proteomic data indicate that E. falciformis infection co-opts cellular and humoral responses through EV secretions, and that, host cell death and ionic imbalance are associated with E. falciformis infection. This study offers additional insight into host-parasite interactions and host regulatory EV proteins as potential disease indicators or diagnostic molecules.

Formulation enhanced the stability of Foot-and-mouth virus and prolonged vaccine storage.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects cloven-hoofed animals. Vaccination is the most effective measure to control FMD. However, FMDV particles are prone to dissociation, leading to insufficient potency of vaccine. Based on this characteristic, a combination of twenty percentage trehalose, 500 mM NaCl and 3 mM CuSO4·5H2O was developed to increase viral stability. Heating-resistance test showed that FMDV infectivity was maintained when formulated with formulation. Additionally, the half-life of FMDV inactivation was prolonged remarkably. Sequencing analysis demonstrated that viral genome could not be altered in serial passages. Vaccine stability was monitored for up to 1 year at 4 °C, with a higher level of 146S content remained. This study suggested that the formulation could protect FMDV against massive structural breakdown and extend the shelf life of vaccine. Our findings could provide strategy to develop more solutions for the stabilization of viral vaccine.

Establishment and application of a solid-phase blocking ELISA method for detection of antibodies against classical swine fever virus.

BACKGROUND: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. OBJECTIVES: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. METHODS: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. RESULTS: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. CONCLUSIONS: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

Infection Dynamics of Clostridium perfringens Fingerprinting in Buffalo and Cattle of Punjab Province, Pakistan.

Clostridium perfringens produces core virulence factors that are responsible for causing hemorrhagic abomasitis and enterotoxemia making food, animals, and humans susceptible to its infection. In this study, C. perfringens was isolated from necropsied intestinal content of buffalo and cattle belonging to four major bovine-producing regions in the Punjab Province of Pakistan for the purpose offind out the genetic variation. Out of total 160 bovine samples (n: 160), thirty-three (n: 33) isolates of C. perfringens were obtained from buffalo (Bubales bubalis) and cattle (Bos indicus) that were further subjected to biochemical tests; 16S rRNA based identification and toxinotyping was done using PCR (Polymerase Chain Reaction) and PFGE (Pulse Field Gel Electrophoresis) pulsotypesfor genetic diversity. Occurrence of C. perfringens was found to be maximum in zone-IV (Bhakkar and Dera Ghazi Khan) according to the heatmap. Correlation was found to be significant and positive among the toxinotypes (α-toxin, and ε-toxin). Response surface methodology (RSM) via central composite design (CCD) and Box-Behnken design (BBD) demonstrated substantial frequency of C. perfringens based toxinotypes in all sampling zones. PFGE distinguished all isolates into 26 different pulsotypes using SmaI subtyping. Co-clustering analysis based on PFGE further decoded a diversegenetic relationship among the collected isolates. This study could help us to advance toward disease array of C. perfringens and its probable transmission and control. This study demonstrates PFGE patterns from Pakistan, and typing of C. perfringens by PFGE helps illustrate and mitigate the incidence of running pulsotypes.

Eimeria falciformis secretes extracellular vesicles to modulate proinflammatory response during interaction with mouse intestinal epithelial cells.

BACKGROUND: Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs). METHODS: Eimeria falciformis sporozoites were allowed to interact with inactivated MIECs. Parasite secretions were separated into EV and vesicle-free (VF) fractions by discontinuous centrifugation and ultracentrifugation. Secreted EVs were purified in an iodixanol density gradient medium and the protein composition of both EV and VF fractions were analyzed by liquid chromatoraphy-tandem mass spectroscopy. The inflammatory activities of E. falciformis sporozoite EV on MIECs were then investigated. RESULTS: During the interaction of E. falciformis sporozoites with inactivated MIECs, the parasite secreted VF and vesicle-bound molecules. Eimeria falciformis vesicles are typical pathogenic protozoan EVs with a mean diameter of 264 ± 2 nm, and enclosed heat shock protein (Hsp) 70 as classical EV marker. Refractile body-associated aspartyl proteinase (or eimepsin), GAP45 and aminopeptidase were the main components of E. falciformis sporozoite EVs, while VF proteins include Hsp90, actin, Vps54 and kinases, among others. Proteomic data revealed that E. falciformis EV and VF proteins are aggregates of bioactive, antigenic and immunogenic molecules which act in concert for E. falciformis sporozoite motility, pathogenesis and survival. Moreover, in MIECs, E. falciformis EVs induced upregulation of gene expression and secretion of IL-1ß, IL-6, IL-17, IL-18, MCP1 as well as pyroptosis-dependent caspase 11 and NLRP6 inflammasomes with the concomitant secretion of lactate dehydrogenase. CONCLUSIONS: Eimeria falciformis sporozoite interaction with MIECs triggered the secretion of immunogenic and antigenic proteins. In addition, E. falciformis sporozoite EVs constitute parasite-associated molecular pattern that induced inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs.

In Vitro Analysis of TGF-ß Signaling Modulation of Porcine Alveolar Macrophages in Porcine Circovirus Type 2b Infection.

Porcine circovirus 2 (PCV2) has been recognized as an immunosuppressive pathogen. However, the crosstalk between this virus and its host cells in related signaling pathways remains poorly understood. In this study, the expression profiles of 84 genes involved in transforming growth factor-beta (TGF-ß) signaling pathway were probed in PCV2b-infected primary porcine alveolar macrophages (PAMs) by using an RT2 profiler PCR array system. The protein expression levels of cytokines involved in the TGF-ß signaling pathway were determined with a RayBiotech fluorescent Quantibody® porcine cytokine array system. Results showed that 48, 30, and 42 genes were differentially expressed at 1, 24, and 48 h after infection, respectively. A large number of genes analyzed by a co-expression network and implicated in transcriptional regulation and apoptosis were differentially expressed in PCV2b-infected PAMs. Among these genes, TGF-ß, interleukin-10, CCAAT/enhancer-binding protein beta (C/EBPB), growth arrest, and DNA-damage-inducible 45 beta (GADD45B), and BCL2 were upregulated. By contrast, SMAD family member 1 (smad1) and smad3 were downregulated. These results suggested that the TGF-ß signaling pathway was repressed in PAMs at the early onset of PCV2 infection. The inhibited apoptosis was indicated by the upregulated C/EBPB, GADD45B, and BCL2, and by the downregulated smad1 and smad3, which possibly increased the duration of PCV2 replication-permissive conditions and caused a persistent infection. Our study may provide insights into the underlying antiviral functional changes in the immune system of PCV2-infected pigs.

Eimeria proteins: order amidst disorder.

Apicomplexans are important pathogens that cause severe infections in humans and animals. The biology and pathogeneses of these parasites have shown that proteins are intrinsically modulated during developmental transitions, physiological processes and disease progression. Also, proteins are integral components of parasite structural elements and organelles. Among apicomplexan parasites, Eimeria species are an important disease aetiology for economically important animals wherein identification and characterisation of proteins have been long-winded. Nonetheless, this review seeks to give a comprehensive overview of constitutively expressed Eimeria proteins. These molecules are discussed across developmental stages, organelles and sub-cellular components vis-à-vis their biological functions. In addition, hindsight and suggestions are offered with intention to summarise the existing trend of eimerian protein characterisation and to provide a baseline for future studies.

Clostridium butyricum Supplement Can Ameliorate the Intestinal Barrier Roles in Broiler Chickens Experimentally Infected With Clostridium perfringens.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an economically important disease in the broiler. Among normal flora in the broiler intestinal region, Clostridium butyricum has been identified as a probiotic agent that reduces the susceptibility of broilers to C. perfringens. However, the effects of C. butyricum supplement on broiler intestinal integrity during NE are largely unknown. In this study, we investigated the effects of C. butyricum on the growth performance, intestinal morphology and barrier function, and the functions of immune-related cytokines under NE in broilers. Chickens were divided into five groups: control group (NC), supplement C. butyricum only group (CB), NE-infected group (PC), supplement C. butyricum from Day 14 (NECB1) to Day 22 NE-infected group, and supplement C. butyricum from Day 1 (NECB2) to Day 22 NE-infected group. The results showed that there were significantly decreased average daily weight gain and increased feed conversion rate in the infected group (PC) compared with the C. butyricum-supplemented groups (NECB1 and NECB2) through the diet. Histopathological observation on the Hematoxylin-Eosin staining avian small intestine sections revealed that supplementation of C. butyricum (NECB1 and NECB2) could increase the intestinal villus height/crypt depth and lessen the intestinal damage under NE. ELISA and Limulus test showed that broilers infected with NE (PC) had higher serum IgA and lipopolysaccharide content; however, after C. butyricum supplementation (NECB1 and NECB2), they returned to a normal level. Furthermore, real-time PCR and Western blot results indicated that compared with PC, supplementing C. butyricum (NECB1 and NECB2) could initialize the expressions of genes related to the intestinal barrier-associated molecules (such as CLDN-1, CLDN-3, OCLN, MUC2, ZO-1, and CLDN5), cytokines (such as IL-10, IL-6, and TGFB1), and C. perfringens plc gene expression. Moreover, the results detected by the Ussing chamber suggested that C. butyricum (NECB1 and NECB2) could amend the decrease in conductivity value and short-circuit current value caused by NE. In addition, NECB2 significantly reduced the upregulation of fluorescein isothiocyanate-dextran flux caused by the NE disease. In conclusion, these findings suggest that dietary supplementation of C. butyricum in broilers with NE improved chicken growth performance, intestinal integrity and barrier function, and immunological status. Notably, no statistical difference was observed with the addition of C. butyricum on day 1 or day 14.

Identification of a new cell-penetrating peptide derived from the african swine fever virus CD2v protein.

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV's EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP]3 labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery.

Occurrence and Toxicogenetic Profiling of Clostridium perfringens in Buffalo and Cattle: An Update from Pakistan.

Clostridium perfringens is a Gram-positive bacterium that possess seven toxinotypes (A, B, C, D, E, F, and G) that are responsible for the production of six major toxins, i.e., α, ß, ε, ι, CPE, and NetB. The aim of this study is to find out the occurrence of toxinotypes in buffalo and cattle of Punjab province in Pakistan and their corresponding toxin-encoding genes from the isolated toxinotypes. To accomplish this aim, six districts in Punjab province were selected (i.e., Lahore, Sahiwal, Cheecha Watni, Bhakkar, Dera Ghazi Khan, and Bahawalpur) and a total of 240 buffalo and 240 cattle were selected for the collection of samples. From isolation and molecular analysis (16S rRNA), it was observed that out of seven toxinotypes (A-G), two toxinotypes (A and D) were found at most, whereas other toxinotypes, i.e., B, C, E, F, and G, were not found. The most frequently occurring toxinotype was type A (buffalo: 149/240; cattle: 157/240) whereas type D (buffalo: 8/240 cattle: 7/240) was found to occur the least. Genes encoding toxinotypes A and D were cpa and etx, respectively, whereas genes encoding other toxinotypes were not observed. The occurrence of isolated toxinotypes was studied using response surface methodology, which suggested a considerable occurrence of the isolated toxinotypes (A and D) in both buffalo and cattle. Association between type A and type D was found to be significant among the isolated toxinotypes in both buffalo and cattle (p ≤ 0.05). Correlation was also found to be positive and significant between type A and type D. C. perfringens exhibits a range of toxinotypes that can be diagnosed via genotyping, which is more reliable than classical toxinotyping.

Evaluation and Optimization of Antibiotics Resistance Profile against Clostridium perfringens from Buffalo and Cattle in Pakistan.

Clostridium perfringens is a serious threat to successful bovine farming. It causes severe damage to the buffalo and cattle health causing a drastic reduction in milk and meat production. In Pakistan, C. perfringens is a constant threat, and for its management, antibiotics are mostly used. Most bovine farmers use a single antibiotic to suppress the bacterial infection which in turn, increases the antimicrobial resistance (AMR) against the particular antibiotic. To reduce the resistance, the administration of multiple antibiotics in their standard doses at different times can be a possible remedy to manage the AMR and reduce their viability. This study aims to evaluate the effect of 11 commonly used antibiotics at their standard concentrations for inhibiting 33 strains of C. perfringens from five districts of Punjab province in Pakistan. Based on the zone of inhibition, ciprofloxacin, ampicillin, and cefotaxime (CAC) at their standard concentrations effectively inhibited the bacterium. These antibiotics showed appropriate significance statistically, i.e., correlation, Chi-square test, and cluster analysis. Optimization of these antibiotics using response surface methodology (RSM) revealed that the selected antibiotics from medium to high range not only reduce the bacterial propagation but also their population up to a considerable extent. Hence, the health of milk- and meat-producing large animals could be improved, which will be cost-effective and less harmful to the animal, human health, and the environment. Moreover, optimized administration of the selected antibiotics would reduce the impact of drug-resistant superbugs.

Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots.

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10-7 and 1.35 × 10-8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

Porcine Circovirus Type 2 Induces Single Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) Downregulation to Promote Interleukin-1ß Upregulation in Porcine Alveolar Macrophage.

Multisystemic inflammation in pigs affected by porcine circovirus type 2 (PCV2) indicates the disordered expression of inflammatory cytokines. However, the PCV2-induced expression profile of inflammation cytokines and its regulating mechanism remain poorly understood. In this study, inflammatory cytokines and receptors in porcine alveolar macrophages (PAMs) after PCV2 infection were profiled in vitro by an RT2 ProfilerTM PCR array assay. The regulatory mechanism of interleukin-1ß (IL-1ß) expression was investigated. Results showed that 49 of 84 inflammation cytokines and receptors were differentially expressed (p < 0.05, absolute fold change ≥2) in PAMs at different stages post-PCV2 infection. Moreover, the overexpression of single-immunoglobulin interleukin-1 related receptor (SIGIRR) or the blocking of NF-κB activation by its inhibitor markedly decreased IL-1ß secretion. This finding suggested that PCV2-induced overexpression of IL-1ß was associated with the downregulation of SIGIRR and the activation of NF-κB. Furthermore, the excessive activity of NF-κB in SIGIRR-knockout PAMs cell line, indicating that SIGIRR negatively regulated IL-1ß production by inhibiting the activation of NF-κB. Overall, PCV2-induced downregulation of SIGIRR induction of NF-κB activation is a critical process in enhancing IL-1ß production in PAMs. This study may provide insights into the underlying inflammatory response that occurs in pigs following PCV2 infection.

Mucosal-Associated Invariant T Cells Expressing the TRAV1-TRAJ33 Chain Are Present in Pigs.

Mucosal-associated invariant T (MAIT) cells are a subpopulation of evolutionarily conserved innate-like T lymphocytes bearing invariant or semi-invariant TCRα chains paired with a biased usage of TCRß chains and restricted by highly conserved monomorphic MHC class I-like molecule, MR1. Consistent with their phylogenetically conserved characteristics, MAIT cells have been implicated in host immune responses to microbial infections and non-infectious diseases, such as tuberculosis, typhoid fever, and multiple sclerosis. To date, MAIT cells have been identified in humans, mice, cows, sheep, and several non-human primates, but not in pigs. Here, we cloned porcine MAIT (pMAIT) TCRα sequences from PBMC cDNA, and then analyzed the TCRß usage of pMAIT cells expressing the TRAV1-TRAJ33 chain, finding that pMAIT cells use a limited array of TCRß chains (predominantly TRBV20S and TRBV29S). We estimated the frequency of TRAV1-TRAJ33 transcripts in peripheral blood and tissues, demonstrating that TRAV1-TRAJ33 transcripts are expressed in all tested tissues. Analysis of the expression of TRAV1-TRAJ33 transcripts in three T-cell subpopulations from peripheral blood and tissues showed that TRAV1-TRAJ33 transcripts can be expressed by CD4+CD8-, CD8+CD4-, and CD4-CD8- T cells. Using a single-cell PCR assay, we demonstrated that pMAIT cells with the TRAV1-TRAJ33 chain express cell surface markers IL-18Rα, IL-7Rα, CCR9, CCR5, and/or CXCR6, and transcription factors PLZF, and T-bet and/or RORγt. In conclusion, pMAIT cells expressing the TRAV1-TRAJ33 chain have characteristics similar to human and mouse MAIT cells, further supporting the idea that the pig is an animal model for investigating MAIT cell functions in human disease.

Proteomic analysis of murine bone marrow derived dendritic cells in response to peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) poses a great threat to livestock husbandry, especially goat farming due to its high mortality and morbidity. Dendritic cells (DCs), as the principal stimulators of naive Th cells were widely used in antigen processing and presenting. In the previous study, we tested the effects of PPRV on murine bone marrow derived dendritic cells (BMDCs) including surface markers and cytokines. While the aim of this study is to detect the proteomic profile of BMDCs stimulated with PPRV towards key proteins involved in. Following PPRV stimulation, 110 differentially expressed proteins (DEPs) were identified through iTRAQ labelling with LC-MS/MS approach, of which 94 DEPs were up-regulated and 16 DEPs were down-regulated, respectively. Among them 15 out of 110 DGPs were related to innate immune system, three were involved in cell apoptosis, RPS15a and Smox were related to translation of viral mRNA. Additionally, western blot analysis showed identical results to iTRAQ analysis. There will be profound significance for understanding antigen-presenting of BMDCs after stimulation with PPRV.

Molecular cloning and characterization of the pig MHC class â -related MR1 gene.

Major histocompatibility complex (MHC) class Ⅰ-related protein 1 (MR1), the most highly conserved MHC class Ⅰ molecule among mammals, is the restricting molecule for mucosal-associated invariant T (MAIT) cells. MAIT cells, a novel subset of T cells, play important roles in modulating the immune responses to infectious and non-infectious diseases, and recognize antigens in the context of MR1. MR1 has been identified in many species, including human, mouse, sheep, and cow. Here, we cloned and characterized pig (Sus scrofa) MR1 (pMR1) transcripts, including five unique splice variants, from pig peripheral blood mononuclear cell cDNA. We also examined the tissue distribution of pMR1 and confirmed reactivity of pMR1 using a MR1 specific monoclonal antibody 26.5, demonstrating that the pMR1 gene was expressed in all tested tissues. Finally, we predicted the pMR1 3D structure and analyzed the docking mode of the MR1-5-OP-RU complex, finding that the docking mode of pMR1 with 5-OP-RU is similar to human MR1 docking. Collectively, this description of pMR1 adds to our understanding of the evolution of MHC molecules, and provides a theoretical basis for the subsequent study of pig MAIT cells.

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus.

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.