A novel dynamic proteomics approach for the measurement of broiler chicken protein fractional synthesis rate.

RATIONALE: The study of protein synthesis in farm animals is uncommon despite its potential to increase knowledge about metabolism and discover new biomarkers of health and growth status. The present study describes a novel dynamic proteomics approach for the measurement of protein fractional synthesis rate (FSR) in broiler chickens. METHODS: Chickens received a 10 g/kg oral dose of 2 H2 O at day 21 of their life. Body water 2 H abundance was measured in plasma samples using a portable Fourier transform infrared spectrometer. Free and protein-bound amino acids (AAs) were isolated and had their 2 H enrichment measured by gas chromatography with mass spectrometry (GC/MS). Peptide 2 H enrichment was measured by proteomics analysis of plasma and muscle samples. Albumin, fibrinogen and muscle protein FSR were calculated from GC/MS and proteomics data. RESULTS: Ala appeared to be more enriched at the site of protein synthesis than in the AA free pools. Glu was found to be the AA closest to isotopic equilibrium between the different AA pools. Glu was used as an anchor to calculate n(AA) values necessary for chicken protein FSR calculation in dynamic proteomics studies. FSR values calculated using proteomics data and GC/MS data showed good agreement as evidenced by a Bland-Altman residual plot. CONCLUSIONS: A new dynamic proteomics approach for the measurement of broiler chicken individual protein FSR based on the administration of a single 2 H2 O oral bolus has been developed and validated. The proposed approach could facilitate new immunological and nutritional studies on free-living animals.

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.

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.

Pathophysiology of the choroid plexus in brain diseases.

The choroid plexus, located in the ventricular system of the central nervous system (CNS), obtains numerous roles critical for the proper development and operating of the CNS. The functions range from the best-known ones of the barrier and cerebrospinal fluid (CSF) producer, through participation in immune answer, 'nourishment, detoxification and reparation of the rest of the CNS. Increase number of studies point out the association between choroid plexus dysfunction, characterized by alterations in secretory, transport and barrier capabilities, and the broad spectrum of clinical conditions, as well as physiological aging. We present a brief overview of pathological states known or speculated to be connected to choroid plexus dysfunction, ranging from neurodevelopmental, to autoimmune and neurodegenerative diseases. We also cover the topic of choroid plexus tumors, as well explained involvement of the choroid plexus in pathogen invasion of the CNS, also referring to the currently actual SARS-CoV-2 infection. Finally, we have also touched conducted studies on the choroid plexus regenerative potential. With the information provided in the review we want to point out the importance and call for further research on the role of the choroid plexus in the sustainability of central nervous system health.

The first serological evidence of Anaplasma phagocytophilum in horses in Slovakia.

Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis. It affects humans and several wild and domesticated mammals, including horses. The aim of our study was a preliminary survey of the occurrence of these re-emerging pathogens in horses in Slovakia. The sera from 200 animals of different ages and both sexes were tested for the presence of A. phagocytophilum antibodies by indirect immunofluorescence assay. Subsequently, detection of the 16S rRNA gene fragment of A. phagocytophilum was attempted by polymerase chain reaction (PCR) in each blood sample. Our results confirmed the presence of specific antibodies in 85 out of 200 individuals (42.5%), but no significant changes were found between the animals of different ages and sexes. However, the PCR analysis did not detect any positive animals. Our data represent one of the highest values of seropositivity to A. phagocytophilum in horses in Central Europe. These results may contribute to a better understanding of the circulation of A. phagocytophilum in this region, thus indicating a potential risk to other susceptible species.

Comprehensive mapping of the cell response to E. coli infection in porcine intestinal epithelial cells pretreated with exopolysaccharide derived from Lactobacillus reuteri.

Bacterial exopolysaccharides (EPSs) are known to modulate immunity. To date, a plethora of studies have reported the effect of EPSs on intestinal cells; however few works have revealed a complete picture of the signalling events in intestinal epithelial cells induced by bacterial EPSs. Here, using transcriptomics, we comprehensively mapped the biological processes in porcine intestinal epithelial cells challenged with EPS derived from Lactobacillus reuteri alone, enterotoxigenic Escherichia coli (ETEC) or ETEC after pretreatment with EPS. The Gene Ontology analysis of differentially expressed genes (DEGs) showed that ETEC is able to evoke biological processes specifically involved in cell junction reorganization, extracellular matrix degradation, and activation of the innate immune response through the activation of pattern recognition receptors, such as TLRs and CTRs. A total of 495 DEGs were induced in ETEC-challenged cells. On the other hand, EPS pretreatment was able to attenuate overexpression of the genes induced by ETEC infection. The most relevant finding of this study is that EPS has a suppressive effect on the inflammatory response evoked by ETEC infection. On the basis of high-throughput RNA-seq, this report is the first to describe the effects of EPSs derived from L. reuteri used as a pretreatment of global gene expression in porcine epithelial cells.

Pursuit of proteomic excellence and the excitement in Kosice, Slovakia, at the 11th Central and Eastern European Proteomic Conference (CEEPC).

The Central and Eastern European Proteomic Conference (CEEPC) successfully launched its second decade of proteomics in Kosice, Slovakia with a program of systems biology, cellular, clinical, veterinary and sports proteomics. Whilst many conferences are struggling to attract participants, CEEPC with its outstanding track record and unique 'family - feel' packaged with excellent ambiance is thriving and bringing together proteomics experts from academia, industry, scientific specialties, clinics and precision medicine communities interested in resolving mysteries about protein functionalities in health and disease. CEEPC is also renowned for addressing humanitarian global healthcare issues, may it be ageing, chronic diseases or global epidemics. This year CEEPC intertwined with Kosice Peace Marathon's pursuit of excellence in sports and initiatives including sports medicine and global peace.

Subunit analysis of mitochondrial cytochrome c oxidase and cytochrome bc1 by reversed-phase high-performance liquid chromatography.

A rapid separation of the ten nuclearly-encoded subunits of mitochondrial cytochrome c oxidase, and ten out of the eleven subunits of cytochrome bc1, was achieved using a short, 50 mm C18-reversed-phase column. The short column decreased the elution time 4-7 fold while maintaining the same resolution quality. Elution was similar to a previously published protocol, i.e., a water/acetonitrile elution gradient containing trifluoroacetic acid. Isolated subunits were identified by MALDI-TOF. The rapidity of the described method makes it extremely useful for determining the subunit composition of isolated mitochondrial complexes. The method can be used for both analytical and micro-preparative purposes.

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.

Identification of B-cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage-displayed gene fragment library.

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B-cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti-OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N-terminus (OspC E1 aa19-27, OspC E2 aa38-53, OspC E3 aa62-66) and three at the C-terminal end (OspC E4 aa155-163, OspC E5 aa184-190 and OspC E6 aa201-207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B-cell epitopes may provide fundamental data for the development of multi-epitope-based diagnostic tools for Lyme disease.

Deciphering the protein interaction in adhesion of Francisella tularensis subsp. holarctica to the endothelial cells.

Extracellular form of Francisella is able to cross various cell barriers and invade multiple organs, such as skin, liver, lung and central nervous system. Transient adhesion of Francisella to endothelial cells may trigger the process of translocation. In this report, we showed that Francisella tularensis subsp. holarctica (Fth) is able to adhere to the endothelial cells, while ICAM-1 may serve as an adhesion molecule for Fth. Pull down and affinity ligand binding assays indicated that the PilE4 could be the probable ligand for ICAM-1. Further deciphering of this ligand:receptor interaction revealed that PilE4 interacts with Ig-like C2-type 1 domain of ICAM-1. To corroborate the role of PilE4 and ICAM-1 interaction in adhesion of extracellular form of Fth to endothelial cells, ICAM-1 was blocked with monoclonal anti-ICAM-1 antibody prior to the incubation with Fth and numbers of adherent bacteria were counted. Blocking of the ICAM-1 significantly reduced (500-fold, P < 0.05) number of adherent Fth compared to unblocked cells. PilE4:ICAM-1 interaction unfolded here may provide a new perspective on molecules involved in the adhesion of extracellular form of Francisella to endothelial cells and probably its translocation across endothelial barriers.

Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast.

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

Complement sensitivity and factor H binding of European Francisella tularensis ssp. holarctica strains in selected animal species.

Francisella tularensis is a Gram-negative bacterium, the causative agent of the zoonotic disease tularaemia. The bacterium has developed several extracellular and intracellular strategies to evade the hosts' innate and adaptive immune responses. The aims of the study were to examine complement sensitivity of wild and attenuated F. tularensis ssp. holarctica strains in animal hosts of distinct sensitivity to the bacterium, to compare the complement-evading ability of wild strains of different phylogeographic background, and to examine the role of factor H in the host-pathogen interactions. Complement sensitivity assays were carried out on various F. tularensis ssp. holarctica wild strains and on the attenuated live vaccine strain (LVS) with sera of the highly sensitive house mouse (Mus musculus), the moderately sensitive European brown hare (Lepus europaeus) and the relatively resistant cattle (Bos taurus). Specific binding of complement regulator factor H to bacterial membrane proteins was examined by Western blot assays. All wild strains interacted with the hosts' complement system and showed no significant differences in their survivability. The attenuated LVS was resistant to serum killing in mouse, but was lysed in the sera of hare and cattle. Direct binding of factor H to F. tularensis membrane proteins was not detected.

Effect of an immune challenge and two feed supplements on broiler chicken individual breast muscle protein synthesis rate.

Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context where antimicrobial growth promoters use is being restricted, it is important to find alternatives as well as to characterize the effect of immunological stress on broiler chicken's growth. Despite its importance, research on broiler chicken muscle protein dynamics has mostly been limited to the study of mixed protein turnover. The present study aims to characterize the effect of a bacterial challenge and the feed supplementation of citrus and cucumber extracts on broiler chicken individual breast muscle proteins fractional synthesis rates (FSR) using a recently developed dynamic proteomics pipeline. Twenty-one day-old broiler chickens were administered a single 2H2O dose before being culled at different timepoints. A total of 60 breast muscle protein extracts from five experimental groups (Unchallenged, Challenged, Control Diet, Diet 1 and Diet 2) were analysed using a DDA proteomics approach. Proteomics data was filtered in order to reliably calculate multiple proteins FSR making use of a newly developed bioinformatics pipeline. Broiler breast muscle proteins FSR uniformly decreased following a bacterial challenge, this change was judged significant for 15 individual proteins, the two major functional clusters identified as well as for mixed breast muscle protein. Citrus or cucumber extract feed supplementation did not show any effect on the breast muscle protein FSR of immunologically challenged broilers. The present study has identified potential predictive markers of breast muscle growth and provided new information on broiler chicken breast muscle protein synthesis which could be essential for improving the efficiency of broiler chicken meat production. SIGNIFICANCE: The present study constitutes the first dynamic proteomics study conducted in a farm animal species which has characterized FSR in a large number of proteins, establishing a precedent for biomarker discovery and assessment of health and growth status. Moreover, it has been evidenced that the decrease in broiler chicken breast muscle protein following an immune challenge is a coordinated event which seems to be the main cause of the decreased growth observed in these animals.

Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain.

BACKGROUND: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds. METHOD: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain-heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood-brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised. RESULTS: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group). CONCLUSION: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.

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.

Immunomodulatory Effect of Lactobacillus reuteri (Limosilactobacillus reuteri) and Its Exopolysaccharides Investigated on Epithelial Cell Line IPEC-J2 Challenged with Salmonella Typhimurium.

The gastrointestinal tract is the largest and most complex component of the immune system. Each component influences the production and regulation of cytokines secreted by intestinal epithelial cells. The aim of this study was to see how the probiotic strain Limosilactobacillus reuteri L26 and its exopolysaccharide (EPS) affect porcine intestinal-epithelial cells IPEC-J2 infected with Salmonella Typhimurium. The results revealed that Salmonella infection up-regulated all studied pro-inflammatory cytokines such as TNF-α, IL-8, IL-6 and TLR4, TLR5 signaling pathways, while decreasing the expression of TGF-ß. An immunosuppressive activity was found in EPS-treated wells, since the transcriptional levels of the studied pro-inflammatory cytokines were not increased, and the pretreatment with EPS was even able to attenuate up-regulated pro-inflammatory genes induced by Salmonella infection. However, there was a significant increase in the expression of mRNA levels of IL-8 and TNF-α in L26-treated cells, although this up-regulation was suppressed in the case of pretreatment. The immunoregulatory function of L. reuteri was also confirmed by the increased level of mRNA expression for TGF-ß, a known immunosuppressive mediator. The most relevant finding of this ex vivo study was a case of immunity modulation, where the probiotic strain L. reuteri stimulated the innate immune-cell response which displayed both anti- and pro-inflammatory activities, and modulated the expression of TLRs in the IPEC-J2 cell line. Our findings also revealed that the pretreatment of cells with either EPS or live lactobacilli prior to infection has a suppressive effect on the inflammatory response induced by Salmonella Typhimurium.

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.

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.