Photocrosslinked gelatin/chondroitin sulfate/chitosan-based composites with tunable multifunctionality for bone tissue regeneration.

Novel hydrogel-based multifunctional systems prepared utilizing photocrosslinking and freeze-drying processes (PhotoCross/Freeze-dried) dedicated for bone tissue regeneration are presented. Fabricated materials, composed of methacrylated gelatin, chitosan, and chondroitin sulfate, possess interesting features including bioactivity, biocompatibility, as well as antibacterial activity. Importantly, their degradation and swellability might be easily tuned by playing with the biopolymeric content in the photocrosllinked systems. To broaden the potential application and deliver the therapeutic features, mesoporous silica particles functionalized with methacrylate moieties decorated with hydroxyapatite and loaded with the antiosteoporotic drug, alendronate, (MSP-MA-HAp-ALN) were dispersed within the biopolymeric sol and photocrosslinked. It was demonstrated that the obtained composites are characterized by a significantly extended degradation time, ensuring optimal conditions for balancing hybrids removal with the deposition of fresh bone. We have shown that attachment of MSP-MA-HAp-ALN to the polymeric matrix minimizes the initial burst effect and provides a prolonged release of ALN (up to 22 days). Moreover, the biological evaluation in vitro suggested the capability of the resulted systems to promote bone remodeling. Developed materials might potentially serve as scaffolds that after implantation will fill up bone defects of various origin (osteoporosis, tumour resection, accidents) providing the favourable conditions for bone regeneration and supporting the infections' treatment.

Towards Controlling the Local Bone Tissue Remodeling-Multifunctional Injectable Composites for Osteoporosis Treatment.

Alendronate (ALN) is the most commonly prescribed oral nitrogen-containing bisphosphonate for osteoporosis therapy. However, its administration is associated with serious side effects. Therefore, the drug delivery systems (DDS) enabling local administration and localized action of that drug are still of great importance. Herein, a novel multifunctional DDS system based on the hydroxyapatite-decorated mesoporous silica particles (MSP-NH2-HAp-ALN) embedded into collagen/chitosan/chondroitin sulfate hydrogel for simultaneous osteoporosis treatment and bone regeneration is proposed. In such a system, the hydrogel serves as a carrier for the controlled delivery of ALN at the site of implantation, thus limiting potential adverse effects. The involvement of MSP-NH2-HAp-ALN in the crosslinking process was established, as well as the ability of hybrids to be used as injectable systems. We have shown that the attachment of MSP-NH2-HAp-ALN to the polymeric matrix provides a prolonged ALN release (up to 20 days) and minimizes the initial burst effect. It was revealed that obtained composites are effective osteoconductive materials capable of supporting the osteoblast-like cell (MG-63) functions and inhibiting osteoclast-like cell (J7741.A) proliferation in vitro. The purposely selected biomimetic composition of these materials (biopolymer hydrogel enriched with the mineral phase) allows their biointegration (in vitro study in the simulated body fluid) and delivers the desired physicochemical features (mechanical, wettability, swellability). Furthermore, the antibacterial activity of the composites in in vitro experiments was also demonstrated.

Effect of Serial in Vivo Passages on The Adaptation of H1N1 Avian Influenza Virus To Pigs.

Introduction: The lack of proofreading activity of the viral polymerase and the segmented nature of the influenza A virus (IAV) genome are responsible for the genetic diversity of IAVs and for their ability to adapt to a new host. We tried to adapt avian IAV (avIAV) to the pig by serial passages in vivo and assessed the occurrence of point mutations and their influence on viral fitness in the pig's body. Material and Methods: A total of 25 in vivo avIAV passages of the A/duck/Bavaria/77 strain were performed by inoculation of 50 piglets, and after predetermined numbers of passages 20 uninoculated piglets were exposed to the virus through contact with inoculated animals. Clinical signs of swine influenza were assessed daily. Nasal swabs and lung tissue were used to detect IAV RNA by real-time RT-PCR and isolates from selected passages were sequenced. Results: Apart from a rise in rectal temperature and a sporadic cough, no typical clinical signs were observed in infected pigs. The original strain required 20 passages to improve its replication ability noticeably. A total of 29 amino-acid substitutions were identified. Eighteen of them were detected in the first sequenced isolate, of which 16 were also in all other analysed strains. Additional mutations were detected with more passages. One substitution, threonine (T) 135 to serine (S) in neuraminidase (NA), was only detected in an IAV isolate from a contact-exposed piglet. Conclusion: Passaging 25 times allowed us to obtain a partially swine-adapted IAV. The improvement in isolate replication ability was most likely related to S654 to glycine (G) substitution in the basic protein (PB) 1 as well as to aspartic acid (D) 701 to asparagine (N) and arginine (R) 477 to G in PB2, glutamic acid (E) 204 to D and G239E in haemagglutinin and T135S in NA.

Dehydration of methanol and ethanol over ferrierite originated layered zeolites - the role of acidity and porous structure.

Ferrierites and their delaminated (ITQ-6) and silica intercalated (ITQ-36) forms, with the intended molar Si/Al ratios of zeolite layers of 30 and 50, were synthesized and tested as catalysts of methanol to dimethyl ether (DME) as well as ethanol to diethyl ether (DEE) and ethylene dehydration. It was shown that increased content of acid sites, especially of Brønsted type, resulted in more active catalysts of alcohol dehydration. Brønsted acid sites dominate in ferrierites and their delaminated forms (ITQ-6). Contribution of the Lewis type of acid sites increased in silica pillared ferrierites (ITQ-36) possibly by deposition of aluminium species on the surface of amorphous silica. Conversion of methanol to DME was not limited by internal diffusion of reactants in narrow pores of ferrierite. Such limitation was observed for synthesis of larger DEE molecules over ferrierites. The ITQ-6 catalysts with the opened interlayer structure presented better efficiency in ethanol to DEE conversion due to overcoming these diffusional restrictions. Moreover, selectivity to DEE over ITQ-6 was higher than in the presence of three-dimensional ferrierite.

Catalysts Based on Strontium Titanate Doped with Ni/Co/Cu for Dry Reforming of Methane.

Two series of strontium titanates doped with Ni, Co, or Cu with general formula of SrTi1-xMexO3 for Sr-stoichiometric and Sr0.95Ti1-xMexO3 for Sr-non-stoichiometric materials (where Me = Ni, Co or Cu and x were 0.02 and 0.06) were obtained by the wet chemical method. The samples were calcinated at 900, 950, and 1050 °C and characterized in terms of their structural properties (XRD), the possibility of undergoing the reduction and oxidation reactions (TPR/TPOx), and catalytic properties. All obtained materials were multiphase and although the XRD analysis does not confirm the presence of Ni, Co, and Cu oxides (with one exception for Cu-doped sample), the TPR/TPOx profiles show reduction peaks that can be attributed to the reduction of these oxides which may at first appear in an amorphous form. Catalytic tests in dry reforming of methane reaction showed that the highest catalytic activity was achieved for Ni-doped materials (up to 90% of CH4 conversion) while Co and Cu-doped samples showed only a very slight catalytic effect. Additionally, the decrease in methane conversion with an increasing calcination temperature was observed for Ni-doped strontium titanates.

Fas/FasL Contributes to HSV-1 Brain Infection and Neuroinflammation.

The Fas/FasL pathway plays a key role in immune homeostasis and immune surveillance. In the central nervous system (CNS) Fas/FasL is involved in axonal outgrowth and adult neurogenesis. However, little is known about the role of the Fas/FasL pathway in herpes encephalitis. In this study, we used a neuropathogenic clinical strain of herpes simplex virus type 1 (HSV-1) to explore infection-induced inflammation and immune responses in the mouse brain and the role of Fas/FasL in antiviral CNS immunity. HSV-1 CNS infection induced the infiltration of Fas- FasL-bearing monocytes and T cells in the brain and also to an up-regulation of Fas and FasL expression on resident astrocytes and microglia within infected sites. Upon infection, Fas- and FasL-deficient mice (lpr and gld) were partially protected from encephalitis with a decreased morbidity and mortality compared to WT mice. Fas/FasL deficiency promoted cell-mediated immunity within the CNS. Fas receptor stimulation abrogated HSV-1 induced activation and inflammatory reactions in microglia from WT mice, while lack of Fas or FasL led to a more pronounced activation of monocytes and microglia and also to an enhanced differentiation of these cells into a pro-inflammatory M1 phenotype. Furthermore, the specific immune system was more efficient in Fas- and FasL-deficient mice with significantly higher numbers of infiltrating HSV-1-specific cytotoxic T cells in the brain. Our data indicate that the Fas/FasL pathway leads to excessive neuroinflammation during HSV-1 infection, which is associated with a diminished anti-viral response and an excessive neuroinflammation.

Exfoliated Ferrierite-Related Unilamellar Nanosheets in Solution and Their Use for Preparation of Mixed Zeolite Hierarchical Structures.

Direct exfoliation of layered zeolites into solutions of monolayers has remained unresolved since the 1990s. Recently, zeolite MCM-56 with the MWW topology (layers denoted mww) has been exfoliated directly in high yield by soft-chemical treatment with tetrabutylammonium hydroxide (TBAOH). This has enabled preparation of zeolite-based hierarchical materials and intimate composites with other active species that are unimaginable via the conventional solid-state routes. The extension to other frameworks, which provides broader benefits, diversified activity, and functionality, is not routine and requires finding suitable synthesis formulations, viz. compositions and conditions, of the layered zeolites themselves. This article reports exfoliation and characterization of layers with ferrierite-related structure, denoted bifer, having rectangular lattice constants like those of the FER and CDO zeolites, and thickness of approximately 2 nm, which is twice that of the so-called fer layer. Several techniques were combined to prove the exfoliation, supported by simulations: AFM; in-plane, in situ, and powder X-ray diffraction; TEM; and SAED. The results confirmed (i) the structure and crystallinity of the layers without unequivocal differentiation between the FER and CDO topologies and (ii) uniform thickness in solution (monodispersity), ruling out significant multilayered particles and other impurities. The bifer layers are zeolitic with Brønsted acid sites, demonstrated catalytic activity in the alkylation of mesitylene with benzyl alcohol, and intralayer pores visible in TEM. The practical benefits are demonstrated by the preparation of unprecedented intimately mixed zeolite composites with the mww, with activity greater than the sum of the components despite high content of inert silica as pillars.

Spherical Al-MCM-41 Doped with Copper by Modified TIE Method as Effective Catalyst for Low-Temperature NH3-SCR.

Aluminum containing silica spherical MCM-41 was synthesized and modified with copper by the template ion-exchange method (TIE) and its modified version, including treatment of the samples with ammonia solution directly after template ion-exchange (TIE-NH3). The obtained samples were characterized with respect to their chemical composition (ICP-OES), structure (XRD), texture (low temperature N2 sorption), morphology (SEM-EDS), form and aggregation of deposited copper species (UV-vis DRS), reducibility of copper species (H2-TPR), and surface acidity (NH3-TPD). The deposition of copper by the TIE-NH3 method resulted in much better dispersion of this metal on the MCM-41 surface comparing to copper introduced by TIE method. It was shown that such highly dispersed copper species, mainly monomeric Cu2+ cations, deposited on aluminum containing silica spheres of MCM-41, are significantly more catalytically effective in the NH3-SCR process than analogous catalysts containing aggregated copper oxide species. The catalysts obtained by the TIE-NH3 method effectively operated in much broader temperature and were less active in the side process of direct ammonia oxidation by oxygen.

Titanium-silicon ferrierites and their delaminated forms modified with copper as effective catalysts for low-temperature NH3-SCR.

Titanium-silicon ferrierites with different Si/Ti ratios and their delaminated forms were modified with copper by ion-exchange. The obtained samples were characterized with respect to their chemical composition (ICP-OES), structure (XRD), texture (N2 sorption), morphology (SEM), form and aggregation of titanium and copper species (UV-vis-DRS), reducibility of deposited copper species (H2-TPR) and surface acidity (NH3-TPD). The porous structure of the zeolitic samples strongly influenced the form and aggregation of deposited copper species. In the case of the three dimensional microporous structure of ferrierites (Ti-FER), copper was deposited mainly in the form of aggregated copper oxide species, in contrast to the open micro- and mesoporous structure of delaminated ferrierites (Ti-ITQ-6), where mainly copper in the form of monomeric cations was identified. It was shown that monomeric copper cations are more catalytically active in NO to NO2 oxidation than aggregated copper oxide species and, therefore, for the low-temperature conversion of nitrogen oxides the fast SCR reaction pathway is more effective for delaminated ferrierites modified with copper (Cu-Ti-ITQ-6) than for microporous three dimensional ferrierite catalysts (Cu-Ti-FER).

Catalytic Performance of Spherical MCM-41 Modified with Copper and Iron as Catalysts of NH3-SCR Process.

Spherical MCM-41 with various copper and iron loadings was prepared by surfactant directed co-condensation method. The obtained samples were characterized with respect to their structure (X-ray diffraction, XRD), texture (N2 sorption), morphology (scanning electron microscopy, SEM), chemical composition (inductively coupled plasma optical emission spectrometry, ICP-OES), surface acidity (temperature programmed desorption of ammonia, NH3-TPD), form, and aggregation of iron and copper species (diffuse reflectance UV-Vis spectroscopy, UV-Vis DRS) as well as their reducibility (temperature programmed reduction with hydrogen, H2-TPR). The spherical MCM-41 samples modified with transition metals were tested as catalysts of selective catalytic reduction of NO with ammonia (NH3-SCR). Copper containing catalysts presented high catalytic activity at low-temperature NH3-SCR with a very high selectivity to nitrogen, which is desired reaction products. Similar results were obtained for iron containing catalysts, however in this case the loadings and forms of iron incorporated into silica samples very strongly influenced catalytic performance of the studied samples. The efficiency of the NH3-SCR process at higher temperatures was significantly limited by the side reaction of direct ammonia oxidation. The reactivity of ammonia molecules chemisorbed on the catalysts surface in NO reduction (NH3-SCR) and their selective oxidation (NH3-SCO) was verified by temperature-programmed surface reactions.

Ferrierite and Its Delaminated Forms Modified with Copper as Effective Catalysts for NH3-SCO Process.

Ferrierites and their delaminated forms (ITQ-6), containing aluminum or titanium in the zeolite framework, were synthetized and modified with copper by an ion-exchange method. The obtained samples were characterized with respect to their chemical composition (ICP-OES), structure (XRD, UV-Vis DRS), textural parameters (N2-sorption), surface acidity (NH3-TPD), form and reducibility of deposited copper species (UV-Vis DRS and H2-TPR). Ferrierites and delaminated ITQ-6 zeolites modified with copper were studied as catalysts for the selective catalytic oxidation of ammonia to dinitrogen (NH3-SCO). It was shown that aggregated copper oxide species, which were preferentially formed on Ti-zeolites, were catalytically active in direct low-temperature ammonia oxidation to NO, while copper introduced into Al-zeolites was present mainly in the form of monomeric copper cations catalytically active in selective reduction of NO by ammonia to dinitrogen. It was postulated that ammonia oxidation in the presence of the studied catalysts proceeds according to the internal-selective catalytic reduction mechanism (i-SCR) and therefore the suitable ratio between aggregated copper oxide species and monomeric copper cations is necessary to obtain active and selective catalysts for the NH3-SCO process. Cu/Al-ITQ-6 presented the best catalytic properties possibly due to the most optimal ratio of these copper species.

Detemplated and Pillared 2-Dimensional Zeolite ZSM-55 with Ferrierite Layer Topology as a Carrier for Drugs.

The present studies were conducted to show the potential of 2D zeolites as effective and non-toxic carriers of drugs. Layered zeolites exhibit adjustable interlayer porosity which can be exploited for controlled drug delivery allowing detailed investigation of the drug release because the structure of the carrier is known exactly. This study was conducted with model drugs ciprofloxacin and piracetam, and ZSM-55 with ca 1 nm thick layers, in detemplated and pillared forms. The release profiles differed from the commercial, crystalline forms of drugs-the release rate increased for ciprofloxacin and decreased for piracetam. To understand the dissolution mechanisms the release data were fitted to Korsmeyer-Peppas equation, showing Fickian (for pillared) and anomalous (for detemplated sample) transport. FT-IR studies showed that strong interaction carrier-drug may be responsible for the modified, slowed down release of piracetam while better solubility and faster release of ciprofloxacin was attributed to formation of the protonated form resulting in weaker interaction with the zeolite than in the pure crystalline form. Two independent tests on L929 mice fibroblasts (ToxiLight and PrestoBlue) showed that ZSM-55, in moderate concentrations may be safely used as a carrier of drug molecules, not having negative effect on the cells viability or proliferation rate.

MCM-22, MCM-36, and ITQ-2 Zeolites with Different Si/Al Molar Ratios as Effective Catalysts of Methanol and Ethanol Dehydration.

MCM-22, MCM-36, and ITQ-2 zeolites with the intended Si/Al molar ratios of 15, 25, and 50 were synthetized and tested as catalysts for dehydration of methanol to dimethyl ether and dehydration of ethanol to diethyl ether and ethylene. The surface concentration of acid sites was regulated by the synthesis of zeolite precursors with different aluminum content in the zeolite framework, while the influence of porous structure on the overall efficiency of alcohol conversion was analyzed by application of zeolitic materials with different types of porosity-microporous MCM-22 as well as microporous-mesoporous MCM-36 and ITQ-2. The zeolitic samples were characterized with respect to their: chemical composition (ICP-OES), structure (XRD, FT-IR), texture (N2 sorption), and surface acidity (NH3-TPD). Comparison of the catalytic activity of the studied zeolitic catalysts with other reported catalytic systems, including zeolites with the similar Si/Al ratio as well as γ-Al2O3 (one of the commercial catalysts for methanol dehydration), shows a great potential of MCM-22, MCM-36, and ITQ-2 in the reactions of alcohols dehydration.

Catalytic and photocatalytic oxidation of diphenyl sulphide to diphenyl sulfoxide over titanium dioxide doped with vanadium, zinc, and tin.

Samples of TiO2 (P25) doped with zinc, tin, and vanadium, thermally treated at 550 °C for 6 h, were tested as catalysts and photocatalysts for the oxidation of diphenyl sulphide to diphenyl sulfoxide and sulfone, using hydrogen peroxide as an oxidation agent. Thermal treatment of pure TiO2 and its vanadium-doped forms resulted in a decrease of anatase and an increase of rutile content. The opposite effect was observed for TiO2 doped with zinc or tin, where thermal treatment resulted in the rutile to anatase phase transition. The role of V, Zn, and Sn admixtures as TiO2 phase-composition controllers was postulated. The catalytic and photocatalytic activity was found to be influenced more by the rutile and anatase contents of the samples than the presence of admixtures. The rutile-containing samples, TiO2 and V-TiO2, presented much better activity in the catalytic oxidation of diphenyl sulphide compared with the catalysts that only contained the anatase phase, Sn-TiO2 and Zn-TiO2. The reaction efficiency was significantly improved under UV radiation. In this case, the best photocatalytic activity was found for calcined TiO2, containing both anatase and rutile components. An increase in rutile content, observed in the vanadium-doped TiO2, decreased the efficiency of the photocatalytic diphenyl sulphide oxidation. Thus, the presence of both anatase and rutile phases, with their favourable contributions, typical for P25, is necessary for the effective oxidation of Ph2S to Ph2SO. Moreover, it was shown that for the second oxidation stage, Ph2SO to Ph2SO2, the presence of the rutile phase is very important.

Nitric Oxide Influences HSV-1-Induced Neuroinflammation.

Herpes simplex virus type 1 (HSV-1) has the ability to replicate in neurons and glial cells and to produce encephalitis leading to neurodegeneration. Accumulated evidence suggests that nitric oxide (NO) is a key molecule in the pathogenesis of neurotropic virus infections. NO can exert both cytoprotective as well as cytotoxic effects in the central nervous system (CNS) depending on its concentration, time course exposure, and site of action. In this study, we used an in vitro model of HSV-1-infected primary neuronal and mixed glial cultures as well as an intranasal model of HSV-1 in BALB/c mice to elucidate the role of NO and nonapoptotic Fas signalling in neuroinflammation and neurodegeneration. We found that low, nontoxic concentration of NO decreased HSV-1 replication in neuronal cultures together with production of IFN-alpha and proinflammatory chemokines. However, in HSV-1-infected glial cultures, low concentrations of NO supported virus replication and production of IFN-alpha and proinflammatory chemokines. HSV-1-infected microglia downregulated Fas expression and upregulated its ligand, FasL. Fas signalling led to production of proinflammatory cytokines and chemokines as well as induced iNOS in uninfected bystander glial cells. On the contrary, NO reduced production of IFN-alpha and CXCL10 through nonapoptotic Fas signalling in HSV-1-infected neuronal cultures. Here, we also observed colocalization of NO production with the accumulation of ß-amyloid peptide in HSV-1-infected neurons both in vitro and in vivo. Low levels of the NO donor increased accumulation of ß-amyloid in uninfected primary neuronal cultures, while the NO inhibitor decreased its accumulation in HSV-1-infected neuronal cultures. This study shows for the first time the existence of a link between NO and Fas signalling during HSV-1-induced neuroinflammation and neurodegeneration.

Antiviral Activity of Tannic Acid Modified Silver Nanoparticles: Potential to Activate Immune Response in Herpes Genitalis.

(1) Background: Tannic acid is a plant-derived polyphenol showing antiviral activity mainly because of an interference with the viral adsorption. In this work, we tested whether the modification of silver nanoparticles with tannic acid (TA-AgNPs) can provide a microbicide with additional adjuvant properties to treat genital herpes infection. (2) Methods: The mouse model of the vaginal herpes simplex virus 2 (HSV-2) infection was used to test immune responses after treatment of the primary infection with TA-AgNPs, and later, after a re-challenge with the virus. (3) Results: The mice treated intravaginally with TA-AgNPs showed better clinical scores and lower virus titers in the vaginal tissues soon after treatment. Following a re-challenge, the vaginal tissues treated with TA-AgNPs showed a significant increase in the percentages of IFN-gamma+ CD8+ T-cells, activated B cells, and plasma cells, while the spleens contained significantly higher percentages of IFN-gamma+ NK cells and effector-memory CD8+ T cells in comparison to NaCl-treated group. TA-AgNPs-treated animals also showed significantly better titers of anti-HSV-2 neutralization antibodies in sera; and (4) Conclusions: Our findings suggest that TA-AgNPs sized 33 nm can be an effective anti-viral microbicide to be applied upon the mucosal tissues with additional adjuvant properties enhancing an anti-HSV-2 immune response following secondary challenge.

Characterization of Co and Fe-MCM-56 catalysts for NH3-SCR and N2O decomposition: An in situ FTIR study.

Two-step preparation of iron and cobalt-containing MCM-56 zeolites has been undertaken to evaluate the influence of their physicochemical properties in the selective catalytic reduction (NH3-SCR or DeNOx) of NO using NH3 as a reductant. Zeolites were prepared by the selective leaching of the framework cations by concentrated HNO3 solution and NH4F/HF mixture and consecutively, introduction of Co and Fe heteroatoms, in quantities below 1wt%. Further calcination allowed to obtain highly dispersed active species. Their evaluation and speciation was realized by adsorption of pyridine and NO, followed by FTIR spectroscopy. Both Fe-MCM-56 zeolites showed excellent activities (maximum NO conversion 92%) with high selectivity to dinitrogen (above 99%) in the high temperature NH3-SCR process. High catalytic activity of Fe-MCM-56 zeolites was assigned to the formation of stable nitrates, delivering NO to react with NH3 at higher temperatures and suppressing the direct NO oxidation. It was found that more nitrates was formed in Fe-MCM-56 (HNO3) than in Fe-MCM-56 (HF/NH4F) and that could compensate for the lower Fe loading, resulting in very similar catalytic activity of both catalysts. At the same time both Co-and Fe-MCM-56 zeolites were moderately active in direct N2O decomposition, with maximum N2O conversion not higher than 80% and activity window starting at 500°C. This phenomenon was expected since both types of catalysts contained well dispersed active centers, not beneficial for this reaction.

Reassortment process after co-infection of pigs with avian H1N1 and swine H3N2 influenza viruses.

BACKGROUND: The influenza A virus is highly variable, which, to some degree, is caused by the reassortment of viral genetic material. This process plays a major role in the generation of novel influenza virus strains that can emerge in a new host population. Due to the susceptibility of pigs to infections with avian, swine and human influenza viruses, they are considered intermediate hosts for the adaptation of the avian influenza virus to humans. In order to test the reassortment process in pigs, they were co-infected with H3N2 A/swine/Gent/172/2008 (Gent/08) and H1N1 A/duck/Italy/1447/2005 (Italy/05) and co-housed with a group of naïve piglets. RESULTS: The Gent/08 strains dominated over Italy/05, but reassortment occurred. The reassortant strains of the H1N1 subtype (12.5%) with one gene (NP or M) of swine-origin were identified in the nasal discharge of the contact-exposed piglets. CONCLUSIONS: These results demonstrate that despite their low efficiency, genotypically and phenotypically different influenza A viruses can undergo genetic exchange during co-infection of pigs.

Kinetics of single and dual infection of pigs with swine influenza virus and Actinobacillus pleuropneumoniae.

Porcine respiratory disease complex (PRDC) is a common problem in modern pork production worldwide. Pathogens that are amongst other pathogens frequently involved in PRDC etiology are swine influenza virus (SIV) and A. pleuropneumoniae. The effect of dual infection with mentioned pathogens has not been investigated to date. The aim of the present study was to evaluate the kinetics of single and dual infection of pigs with SIV and A. pleuropneumoniae with regard to clinical course, pathogens shedding, lung lesions and early immune response. The most severe symptoms were observed in co-inoculated piglets. The AUC value for SIV shedding was lower in pigs single inoculated with SIV as compared to co-inoculated animals. In contrast, no significant differences were found between A. pleuropneumoniae shedding in single or dual inoculated pigs. Three out of 5 co-inoculated piglets euthanized at 10 dpi were positive against serotype 2 A. pleuropneumonie. All piglets inoculated with SIV developed specific HI antibodies at 10 dpi. In pigs dual inoculated the specific humoral response against SIV was observed earlier, at 7 dpi. The SIV-like lung lesions were more severe in co-inoculated pigs. In the groups inoculated with A. pleuropneumoniae (single or dual) the acute phase protein response was generally stronger than in SIV-single infected group. Co-infection with SIV and A. pleuropneumoniae potentiated the severity of lung lesions caused by SIV and enhanced virus replication in the lung and nasal SIV shedding. Enhanced SIV replication contributed to a more severe clinical course of the disease as well as earlier and higher magnitude immune response (acute phase proteins, HI antibodies) compared to single inoculated pigs.

Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars.

The study reports the development of a polymerase cross-linking spiral reaction (PCLSR) for the detection of African swine fever virus (ASFV) DNA in blood collected from infected pigs and wild boars. The method uses 3 specifically designed primers. Two outer-spiral primers comprising of 3' sequences complementary to ASFV p72 gene sequence and 5'end sequences complementary to exogenous gene of black widow alpha-latrotoxin as well as additional ASFV specific cross-linking primer. The method is specific exclusively to ASFV DNA without cross-reactions with cDNA of classical swine fever virus (CSFV), porcine reproductive respiratory syndrome (PRRSV) or porcine epidemic diarrhea virus (PEDV). The sensitivity of this technique reached 7.2 × 102 copies per µl-1 of plasmid containing p72 gene. The PCLSR was conducted at 65 °C creating cross-linked complex structures. The results of PCLSR were visualized using SYBR Green I dye, gel electrophoresis while the reaction progress was traced using real-time PCR system that resulted in registration of fluorescent curves and melting peaks at 85.3 °C. The developed PCLSR was examined using blood or tissue samples collected from selected 17 ASF cases from infected wild boars and 3 outbreaks in pigs. Further tests have been also conducted using 55 tissue samples from 23 outbreaks and 22 cases. These results showed that PCLSR might be further used for preliminary and cost-effective detection and surveillance of ASFV.