Differentially Expressed Genes Induced by Erythropoietin Receptor Overexpression in Rat Mammary Adenocarcinoma RAMA 37-28 Cells.

The erythropoietin receptor (EPOR) is a transmembrane type I receptor with an essential role in the proliferation and differentiation of erythroid progenitors. Besides its function during erythropoiesis, EPOR is expressed and has protective effect in various non-hematopoietic tissues, including tumors. Currently, the advantageous aspect of EPOR related to different cellular events is still under scientific investigation. Besides its well-known effect on cell proliferation, apoptosis and differentiation, our integrative functional study revealed its possible associations with metabolic processes, transport of small molecules, signal transduction and tumorigenesis. Comparative transcriptome analysis (RNA-seq) identified 233 differentially expressed genes (DEGs) in EPOR overexpressed RAMA 37-28 cells compared to parental RAMA 37 cells, whereas 145 genes were downregulated and 88 upregulated. Of these, for example, GPC4, RAP2C, STK26, ZFP955A, KIT, GAS6, PTPRF and CXCR4 were downregulated and CDH13, NR0B1, OCM2, GPM6B, TM7SF3, PARVB, VEGFD and STAT5A were upregulated. Surprisingly, two ephrin receptors, EPHA4 and EPHB3, and EFNB1 ligand were found to be upregulated as well. Our study is the first demonstrating robust differentially expressed genes evoked by simple EPOR overexpression without the addition of erythropoietin ligand in a manner which remains to be elucidated.

Differential transcriptome response of blood brain barrier spheroids to neuroinvasive Neisseria and Borrelia.

Background: The blood-brain barrier (BBB), a highly regulated interface between the blood and the brain, prevents blood-borne substances and pathogens from entering the CNS. Nevertheless, pathogens like Neisseria meningitidis and Borrelia bavariensis can breach the BBB and infect the brain parenchyma. The self-assembling BBB-spheroids can simulate the cross talk occurring between the cells of the barrier and neuroinvasive pathogens. Methods: BBB spheroids were generated by co-culturing human brain microvascular endothelial cells (hBMECs), pericytes and astrocytes. The BBB attributes of spheroids were confirmed by mapping the localization of cells, observing permeability of angiopep2 and non-permeability of dextran. Fluorescent Neisseria, Borrelia or E. coli (non-neuroinvasive) were incubated with spheroids to observe the adherence, invasion and spheroid integrity. Transcriptome analysis with NGS was employed to investigate the response of BBB cells to infections. Results: hBMECs were localized throughout the spheroids, whereas pericytes and astrocytes were concentrated around the core. Within 1 hr of exposure, Neisseria and Borrelia adhered to spheroids, and their microcolonization increased from 5 to 24 hrs. Integrity of spheroids was compromised by both Neisseria and Borrelia, but not by E. coli infection. Transcriptome analysis revealed a significant change in the expression of 781 genes (467 up and 314 down regulated) in spheroids infected with Neisseria, while Borrelia altered the expression of 621 genes (225 up and 396 down regulated). The differentially expressed genes could be clustered into various biological pathways like cell adhesion, extracellular matrix related, metallothionines, members of TGF beta, WNT signaling, and immune response. Among the differentially expressed genes, 455 (48%) genes were inversely expressed during Neisseria and Borrelia infection. Conclusion: The self-assembling spheroids were used to perceive the BBB response to neuroinvasive pathogens - Neisseria and Borrelia. Compromised integrity of spheroids during Neisseria and Borrelia infection as opposed to its intactness and non-adherence of E. coli (non-neuroinvasive) denotes the pathogen dependent fate of BBB. Genes categorized into various biological functions indicated weakened barrier properties of BBB and heightened innate immune response. Inverse expression of 48% genes commonly identified during Neisseria and Borrelia infection exemplifies unique response of BBB to varying neuropathogens.

Signaling events evoked by domain III of envelop glycoprotein of tick-borne encephalitis virus and West Nile virus in human brain microvascular endothelial cells.

Tick-borne encephalitis virus and West Nile virus can cross the blood-brain barrier via hematogenous route. The attachment of a virion to the cells of a neurovascular unit, which is mediated by domain III of glycoprotein E, initiates a series of events that may aid viral entry. Thus, we sought to uncover the post-attachment biological events elicited in brain microvascular endothelial cells by domain III. RNA sequencing of cells treated with DIII of TBEV and WNV showed significant alteration in the expression of 309 and 1076 genes, respectively. Pathway analysis revealed activation of the TAM receptor pathway. Several genes that regulate tight-junction integrity were also activated, including pro-inflammatory cytokines and chemokines, cell-adhesion molecules, claudins, and matrix metalloprotease (mainly ADAM17). Results also indicate activation of a pro-apoptotic pathway. TLR2 was upregulated in both cases, but MyD88 was not. In the case of TBEV DIII, a MyD88 independent pathway was activated. Furthermore, both cases showed dramatic dysregulation of IFN and IFN-induced genes. Results strongly suggest that the virus contact to the cell surface emanates a series of events namely viral attachment and diffusion, breakdown of tight junctions, induction of virus uptake, apoptosis, reorganization of the extracellular-matrix, and activation of the innate immune system.

Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein.

West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIIIL297-S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1G299-K307 and DIII-2V371-R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1G299-K307 and DIII-2V371-R388 on a dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC50) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAbA10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAbA10 targeting DIII-2V371-R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV-induced neuropathogenesis.

Simple and rapid pipeline for the production of cyclic and linear small-sized peptides in E. coli.

Small and medium-sized peptides are gaining popularity in biomedical applications, including therapeutic target development. As an alternative to chemical synthesis, we describe a complete pipeline for the production of linear as well as structurally constrained cyclic peptides in an E. coli expression system in this study. A plasmid vector containing a novel N terminal HOE tag (28 amino acids in length) that fuses with the peptide was created. The HOE tag contains sites for both chemical (CNBr) and enzymatic (enterokinase) cleavage, making it easy to isolate the peptide after production. A total of 21 peptides (17 cyclic and 4 linear) were synthesized, and the HOE tag was successfully removed using either CNBr (9 peptides) or enterokinase (12 peptides). The presence of a disulfide bond was confirmed in six representative cyclic peptides. In this study we have provided detailed instructions on primers design strategy, overexpression and purification of HOE tagged peptides, chemical and enzymatic cleavage, and confirmation of the cyclic form of peptides. We are confident that this pipeline will assist researchers in producing multiple recombinant peptides in a cost-effective and time-efficient manner.

Comprehensive Mapping of the Cell Response to Borrelia bavariensis in the Brain Microvascular Endothelial Cells in vitro Using RNA-Seq.

Borrelia bavariensis can invade the central nervous system (CNS) by crossing the blood-brain barrier (BBB). It is predicted that B. bavariensis evokes numerous signaling cascades in the human brain microvascular endothelial cells (hBMECs) and exploits them to traverse across the BBB. The complete picture of signaling events in hBMECs induced by B. bavariensis remains uncovered. Using RNA sequencing, we mapped 11,398 genes and identified 295 differentially expressed genes (DEGs, 251 upregulated genes and 44 downregulated genes) in B. bavariensis challenged hBMECs. The results obtained from RNA-seq were validated with qPCR. Gene ontology analysis revealed the participation of DEGs in a number of biological processes like cell communication, organization of the extracellular matrix, vesicle-mediated transport, cell response triggered by pattern recognition receptors, antigen processing via MHC class I, cellular stress, metabolism, signal transduction, etc. The expression of several non-protein coding genes was also evoked. In this manuscript, we discuss in detail the correlation between several signaling cascades elicited and the translocation of BBB by B. bavariensis. The data revealed here may contribute to a better understanding of the mechanisms employed by B. bavariensis to cross the BBB.

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection.

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.

Transcriptomic analysis of human brain microvascular endothelial cells exposed to laminin binding protein (adhesion lipoprotein) and Streptococcus pneumoniae.

Streptococcus pneumoniae invades the CNS and triggers a strong cellular response. To date, signaling events that occur in the human brain microvascular endothelial cells (hBMECs), in response to pneumococci or its surface adhesins are not mapped comprehensively. We evaluated the response of hBMECs to the adhesion lipoprotein (a laminin binding protein-Lbp) or live pneumococci. Lbp is a surface adhesin recently identified as a potential ligand, which binds to the hBMECs. Transcriptomic analysis was performed by RNA-seq of three independent biological replicates and validated with qRT-PCR using 11 genes. In total 350 differentially expressed genes (DEGs) were identified after infection with S. pneumoniae, whereas 443 DEGs when challenged with Lbp. Total 231 DEGs were common in both treatments. Integrative functional analysis revealed participation of DEGs in cytokine, chemokine, TNF signaling pathways and phagosome formation. Moreover, Lbp induced cell senescence and breakdown, and remodeling of ECM. This is the first report which maps complete picture of cell signaling events in the hBMECs triggered against S. pneumoniae and Lbp. The data obtained here could contribute in a better understanding of the invasion of pneumococci across BBB and underscores role of Lbp adhesin in evoking the gene expression in neurovascular unit.

Identification of the proteins of Borrelia garinii interacting with human brain microvascular endothelial cells.

Lyme borreliosis is one of the major tick-borne diseases in Europe. Events of the translocation of Borrelia across the blood-brain barrier (BBB) involve multiple interactions between borrelial surface proteins and receptors on the brain microvascular endothelial cells (hBMECs). In this study, we aimed to identify proteins of Borrelia that plausibly interact with hBMECs. The surface proteome of live Borrelia (a neuroinvasive strain of B. garinii) was crosslinked with biotin prior to its incubation with hBMECs. The interacting proteins were recovered by affinity purification, followed by SWATH-MS. Twenty-four interacting candidates were grouped into outer membrane proteins (n = 12) and inner membrane proteins (n = 12) based on the subcellular location as per the predictions of LocateP. Other algorithms like TMHMM 2.0 and LipoP, ontology search and literature review were subsequently applied to each of the identified protein candidates to shortlist the most probable interactors. Six proteins namely, LysM domain protein, BESBP-5, Antigen S1, CRASP-1 (Bg071), Erp23 protein and Mlp family Lipoprotein were selected to produce their recombinant forms and experimentally validate their interaction with hBMECs. All the recombinant proteins interacted with hBMECs, in ELISA and immunocytochemistry. We present here a high-throughput approach of generating a dataset of plausible borrelial ligands followed by a systematic bioinformatic pipeline to categorize the proteins for experimental validation.

A simple and rapid pipeline for identification of receptor-binding sites on the surface proteins of pathogens.

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells.

Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A33 to K69) and the first coiled-coil domain (L121 to K158). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gdA33-K69 or NadA-ccL121-K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHHF3-binding NadA-gdA33-K69 and VHHG9-binding NadA-ccL121-K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.

Transcriptome analysis of human brain microvascular endothelial cells response to Neisseria meningitidis and its antigen MafA using RNA-seq.

Interaction of Neisseria meningitidis (NM) with human brain microvascular endothelial cells (hBMECs) initiates of multiple cellular processes, which allow bacterial translocation across the blood-brain barrier (BBB). NM is equipped with several antigens, which interacts with the host cell receptors. Recently we have shown that adhesin MafA (UniProtKB-X5EG71), relatively less studied protein, is one of those surface exposed antigens that adhere to hBMECs. The present study was designed to comprehensively map the undergoing biological processes in hBMECs challenged with NM or MafA using RNA sequencing. 708 and 726 differentially expressed genes (DEGs) were identified in hBMECs exposed to NM and MafA, respectively. Gene ontology analysis of the DEGs revealed that several biological processes, which may alter the permeability of BBB, were activated. Comparative analysis of DEGs revealed that MafA, alike NM, might provoke TLR-dependent pathway and augment cytokine response. Moreover, both MafA and NM were able to induce genes involved in cell surface modifications, endocytosis, extracellular matrix remodulation and anoikis/apoptosis. In conclusion, this study for the first time describes effect of NM on the global gene expression in hBMECs using high-throughput RNA-seq. It also presents ability of MafA to induce gene expression, which might aid NM in breaching the BBB.

Proteomic and structural differences in lumpfish skin among the dorsal, caudal and ventral regions.

Fish skin is a vital organ that serves a multitude of functions including mechanical protection, homeostasis, osmoregulation and protection against diseases. The expression of skin proteins changes under different physiological conditions. However, little is known about differences in protein expression among various body sites in naïve fish. The objectives of this work is to study potential differences in protein and gene expression among dorsal, caudal and ventral regions of lumpfish skin employing 2D gel based proteomics and real-time PCR and to assess structural differences between these regions by using Alcian blue and Periodic acid Schiff stained skin sections. The proteins collagen alfa-1, collagen alfa-2, heat shock cognate 71 kDa, histone H4, parvalbumin, natterin-2, 40S ribosomal protein S12, topoisomerase A and topoisomerase B were differentially expressed among the three regions. mRNA expression of apoa1, hspa8 and hist1h2b showed significant differences between regions. Skin photomicrographs showed differences in epidermal thickness and goblet cell counts. The ventral region showed relatively high protein expression, goblet cell count and epidermal thickness compared to dorsal and caudal regions. Overall, this study provides an important benchmark for comparative analysis of skin proteins and structure between different parts of the lumpfish body.

Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells.

Neisseria meningitidis is able to translocate the blood-brain barrier and cause meningitis. Bacterial translocation is a crucial step in the onset of neuroinvasion that involves interactions between pathogen surface proteins and host cells receptors. In this study, we applied a systematic workflow to recover and identify proteins of N. meningitidis that may interact with human brain microvascular endothelial cells (hBMECs). Biotinylated proteome of N. meningitidis was incubated with hBMECs, interacting proteins were recovered by affinity purification and identified by SWATH-MS. Interactome of N. meningitidis comprised of 41 potentially surface exposed proteins. These were assigned into groups based on their probability to interact with hBMECs: high priority candidates (21 outer membrane proteins), medium priority candidates (14 inner membrane proteins) and low priority candidates (six secretory proteins). Ontology analysis provided information for 17 out of 41 surface proteins. Based on the series of bioinformatic analyses and literature review, five surface proteins (adhesin MafA1, major outer membrane protein P.IB, putative adhesin/invasion, putative lipoprotein and membrane lipoprotein) were selected and their recombinant forms were produced for experimental validation of interaction with hBMECs by ELISA and immunocytochemistry. All candidates showed interaction with hBMECs. In this study, we present a high-throughput approach to generate a dataset of plausible meningococcal ligands followed by systematic bioinformatic pipeline to categorize the proteins for experimental validation.

Proteomic and bioinformatic pipeline to screen the ligands of S. pneumoniae interacting with human brain microvascular endothelial cells.

The mechanisms by which Streptococcus pneumoniae penetrates the blood-brain barrier (BBB), reach the CNS and causes meningitis are not fully understood. Adhesion of bacterial cells on the brain microvascular endothelial cells (BMECs), mediated through protein-protein interactions, is one of the crucial steps in translocation of bacteria across BBB. In this work, we proposed a systematic workflow for identification of cell wall associated ligands of pneumococcus that might adhere to the human BMECs. The proteome of S. pneumoniae was biotinylated and incubated with BMECs. Interacting proteins were recovered by affinity purification and identified by data independent acquisition (DIA). A total of 44 proteins were identified from which 22 were found to be surface-exposed. Based on the subcellular location, ontology, protein interactive analysis and literature review, five ligands (adhesion lipoprotein, endo-ß-N-acetylglucosaminidase, PhtA and two hypothetical proteins, Spr0777 and Spr1730) were selected to validate experimentally (ELISA and immunocytochemistry) the ligand-BMECs interaction. In this study, we proposed a high-throughput approach to generate a dataset of plausible bacterial ligands followed by systematic bioinformatics pipeline to categorize the protein candidates for experimental validation. The approach proposed here could contribute in the fast and reliable screening of ligands that interact with host cells.

The Challenges and Advances in Diagnosis of Vector-Borne Diseases: Where Do We Stand?

Vector-borne diseases (VBD) are of major importance to human and animal health. In recent years, VBD have been emerging or re-emerging in many geographical areas, alarming new disease threats and economic losses. The precise diagnosis of many of these diseases still remains a major challenge because of the lack of comprehensive data available on accurate and reliable diagnostic methods. Here, we conducted a systematic and in-depth review of the former, current, and upcoming techniques employed for the diagnosis of VBD.

Joining the in vitro immunization of alpaca lymphocytes and phage display: rapid and cost effective pipeline for sdAb synthesis.

BACKGROUND: Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). RESULTS: In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry. CONCLUSION: A proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

Detection of West Nile virus and tick-borne encephalitis virus in birds in Slovakia, using a universal primer set.

West Nile virus (WNV) is a mosquito-borne neurotropic pathogen that presents a major public health concern. Information on WNV prevalence and circulation in Slovakia is insufficient. Oral and cloacal swabs and bird brain samples were tested for flavivirus RNA by RT-PCR using newly designed generic primers. The species designation was confirmed by sequencing. WNV was detected in swab and brain samples, whereas one brain sample was positive for tick-borne encephalitis virus (TBEV). The WNV sequences clustered with lineages 1 and 2. These results confirm the circulation of WNV in birds in Slovakia and emphasize the risk of infection of humans and horses.

Exploitation of complement regulatory proteins by Borrelia and Francisella.

Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

Dynamics of antibody response and viraemia following natural infection of porcine circovirus 2 (PCV-2) in a conventional pig herd.

Porcine circovirus 2 (PCV-2) is a primary agent of post-weaning multi-systemic wasting syndrome (PMWS), ubiquitous in pig herds. The course of viraemia and seroconversion in naturally infected pigs were investigated in piglets from the 2nd week of their life. Piglets were divided into seropositive (Ab(+)) and seronegative (Ab(-)) groups. Subsequently, after vaccination against PCV-2 (Ingelvac(®) CIRCOFLEX™, Böehringer Ingelheim), they were further divided into non-vaccinated seronegative (NVAC/Ab(-)) and seropositive (NVAC/Ab(+)), and vaccinated seronegative (VAC/Ab(-)) and seropositive (VAC/Ab(+)). PCV-2 colostral antibodies failed to prevent development of natural PCV-2 infection in conventional piglets; however, this occurred at a higher age in comparison with seronegative pigs. Neither colostral nor post-infection antibodies prevented development of viraemia, which persisted up to the end of the study (the 19th week), but without clinical signs of PMWS. Vaccination failed to prevent development of natural PCV-2 infection, but viraemia was limited to between the 8th and 10th week. The presence of colostral anti-PCV-2 antibodies did not show any untoward effect to vaccination; on the contrary, VAC/Ab(+) animals showed the lowest titre of viraemia.