Influenza C and D Viruses Demonstrated a Differential Respiratory Tissue Tropism in a Comparative Pathogenesis Study in Guinea Pigs.

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism.

Bovine rhinitis B virus is highly prevalent in acute bovine respiratory disease and causes upper respiratory tract infection in calves.

Bovine respiratory disease (BRD) is the most significant cause of cattle morbidity and mortality worldwide. This multifactorial disease has a complex aetiology. Dogma posits a primary viral infection followed by secondary bacterial pneumonia. Bovine rhinitis B virus (BRBV) is an established aetiological agent of BRD, but little is known regarding its pathogenesis. Here, a BRD PCR panel identified 18/153 (11.8 %) lung samples and 20/49 (40.8 %) nasal swabs collected from cattle with respiratory signs as positive for BRBV, which was the most prevalent virus in nasal swabs. Primary bovine tracheal epithelial cells were used to isolate BRBV that was phylogenetically related to contemporary sequences from the USA and Mexico and genetically divergent from the previous sole BRBV isolate. To investigate virus pathogenesis, 1-week-old colostrum-deprived dairy calves were inoculated intranasally with 7.0 log10 TCID50 BRBV. Virus was isolated from nasal swabs, nasal turbinates, trachea and the brain of the challenged animals. Neutralizing antibodies were detected beginning 7 days post-inoculation and peaked at day 14. In situ hybridization (ISH) localized BRBV infection in the upper respiratory ciliated epithelial and goblet cells, occasionally associated with small defects of the superficial cilia lining. Sporadically, pinpoint ISH signals were also detected in cells resembling glial cells in the cerebrum in one calf. Together, these results demonstrate the BRBV infection is highly prevalent in acute BRD samples and while the pathogenicity of BRBV is minimal with infection largely limited to the upper respiratory tract, further research is needed to elucidate a possible initiatory role in BRD.

Development and characterization of an inducible assay system to measure Zika virus capsid interactions.

The global spread of the mosquito-borne Zika virus (ZIKV) infection and its complications including Guillain-Barré syndrome and fetus microcephaly in 2015 have made ZIKV as a significant public health threat. The capsid protein plays crucial roles in ZIKV replication and thus represents an attractive therapeutic target. However, inhibitors of ZIKV capsid assembly have not been rigorously identified due to the lack of a target-based screening system. In this study, we developed a novel ZIKV capsid interaction method based on a split-luciferase complementation assay, which can be used to measure and quantify ZIKV capsid-capsid (C-C) interaction by the restored luciferase signal when capsid proteins interact with each other. Furthermore, a Tet-on inducible stable cell line was generated to screen inhibitors of capsid dimerization. By using of this system, peptides (Pep.15-24 in the N-terminal region of ZIKV capsid protein and Pep.44-58 in the α2 helix of ZIKV capsid protein) were identified to inhibit ZIKV C-C interaction. Overall, this study developed a novel inducible assay system to measure ZIKV capsid interaction and identify ZIKV capsid multimerization inhibitors, which will be applied for future discovery of ZIKV assembly inhibitors.

Toll-like Receptor-4 (TLR4) Agonist-Based Intranasal Nanovaccine Delivery System for Inducing Systemic and Mucosal Immunity.

Eliciting a robust immune response at mucosal sites is critical in preventing the entry of mucosal pathogens such as influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This task is challenging to achieve without the inclusion of a strong and safe mucosal adjuvant. Previously, inulin acetate (InAc), a plant-based polymer, is shown to activate toll-like receptor-4 (TLR4) and elicit a robust systemic immune response as a vaccine adjuvant. This study investigates the potential of nanoparticles prepared with InAc (InAc-NPs) as an intranasal vaccine delivery system to generate both mucosal and systemic immune responses. InAc-NPs (∼250 nm in diameter) activated wild-type (WT) macrophages but failed to activate macrophages from TLR4 knockout mice or WT macrophages when pretreated with a TLR4 antagonist (lipopolysaccharide-RS (LPS-RS)), which indicates the selective nature of a InAc-based nanodelivery system as a TLR4 agonist. Intranasal immunization using antigen-loaded InAc-NPs generated ∼65-fold and 19-fold higher serum IgG1 and IgG2a titers against the antigen, respectively, as compared to PLGA-NPs as a delivery system. InAc-NPs have also stimulated the secretion of sIgA at various mucosal sites, including nasal-associated lymphoid tissues (NALTs), lungs, and intestine, and produced a strong memory response indicative of both humoral and cellular immune activation. Overall, by stimulating both systemic and mucosal immunity, InAc-NPs laid a basis for a potential intranasal delivery system for mucosal vaccination.

Genetic and antigenic characteristics of a human influenza C virus clinical isolate.

Unlike influenza A and B viruses that infect humans and cause severe diseases in seasonal epidemics, influenza C virus (ICV) is a ubiquitous childhood pathogen typically causing mild respiratory symptoms. ICV infections are rarely diagnosed and less research has been performed on it despite the virus being capable of causing severe disease in infants. Here we report on the isolation of a human ICV from a child with acute respiratory disease, provisionally designated C/Victoria/2/2012 (C/Vic). The full-length genome sequence and phylogenetic analysis revealed that the hemagglutinin-esterase-fusion (HEF) gene of C/Vic was derived from C/Sao Paulo lineage, while its PB2 and P3 genes evolved separately from all characterized historical ICV isolates. Furthermore, antigenic analysis using the hemagglutination inhibition (HI) assay found that 1947 C/Taylor virus (C/Taylor lineage) was antigenically more divergent from1966 C/Johannesburg (C/Aichi lineage) than from 2012 C/Vic. Structure modeling of the HEF protein identified two mutations in the 170-loop of the HEF protein around the receptor-binding pocket as a possible antigenic determinant responsible for the discrepant HI results. Taken together, results of our studies reveal novel insights into the genetic and antigenic evolution of ICV and provide a framework for further investigation of its molecular determinants of antigenic property and replication.

Food Protein Based Core-Shell Nanocarriers for Oral Drug Delivery: Effect of Shell Composition on in Vitro and in Vivo Functional Performance of Zein Nanocarriers.

The study was aimed at systematically investigating the influence of shell composition on the particle size, stability, release, cell uptake, permeability, and in vivo gastrointestinal distribution of food protein based nanocarriers for oral delivery applications. Three different core-shell nanocarriers were prepared using food-grade biopolymers including zein-casein (ZC) nanoparticles, zein-lactoferrin (ZLF), nanoparticles and zein-PEG (ZPEG) micelles. Nile red was used as a model hydrophobic dye for in vitro studies. The nanocarriers had negative, positive, and neutral charge, respectively. All three nanocarriers had a particle size of less than 200 nm and a low polydispersity index. The nanoparticles were stable at gastrointestinal pH (2-9) and ionic strength (10-200 mM). The nanocarriers sustained the release of Nile red in simulated gastric and intestinal fluids. ZC nanoparticles showed the slowest release followed by ZLF nanoparticles and ZPEG micelles. The nanocarriers were taken up by endocytosis in Caco-2 cells. ZPEG micelles showed the highest cell uptake and transepithelial permeability followed by ZLF and ZC nanoparticles. ZPEG micelles also showed P-gp inhibitory activity. All three nanocarriers showed bioadhesive properties. Cy 5.5, a near IR dye, was used to study the in vivo biodistribution of the nanocarriers. The nanocarriers showed longer retention in the rat gastrointestinal tract compared to the free dye. Among the three formulations, ZC nanoparticles was retained the longest in the rat gastrointestinal tract (≥24 h). Overall, the outcomes from this study demonstrate the structure-function relationship of core-shell protein nanocarriers. The findings from this study can be used to develop food protein based oral drug delivery systems with specific functional attributes.

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model.

UNLABELLED: Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. IMPORTANCE: Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV.

Evaluation of inhibition of F4ac positive Escherichia coli attachment with xanthine dehydrogenase, butyrophilin, lactadherin and fatty acid binding protein.

BACKGROUND: Neonatal and post-weaning colibacillosis caused by enterotoxigenic E. coli is responsible for substantial economic losses encountered by the pork industry. Intestinal colonization of young piglets by E. coli depends on the efficiency of bacterial attachment to host gastrointestinal epithelium that is mediated by fimbriae. We tested the effect of porcine individual milk fat globule membrane (MFGM) proteins on F4ac positive E. coli attachment to porcine enterocytes in vitro. RESULTS: Butyrophilin, lactadherin and fatty acid binding protein inhibited fimbriae-dependent adherence of E. coli to enterocytes in vitro, while xanthine dehydrogenase did not. The inhibiting activity was dose-dependent for all three proteins, but the inhibiting efficiency was different. CONCLUSIONS: The results indicate that MFGM proteins may interfere with attachment of E. coli to porcine neonatal intestinal mucosa.

The Involvement of Neutrophil in the Immune Dysfunction Associated with BVDV Infection.

Bovine viral diarrhea virus (BVDV) induces immune dysfunction that often results in a secondary bacterial infection in the infected animals. The underlying mechanism of BVDV-induced immune dysfunction is not well understood. The role of BVDV-infected macrophage-secreted factors was investigated. BVDV-infected monocyte-derived macrophage (MDM) supernatants down-regulated the expression of neutrophil L-selectin and CD18. Regardless of the biotype, phagocytic activity and oxidative burst were downregulated by BVDV-infected MDM supernatants. However, only supernatants from cytopathic (cp) BVDV down-regulated nitric oxide production and neutrophil extracellular traps (NET) induction. Our data suggested that BVDV-induced macrophage-secreted factors caused immune dysfunction in neutrophils. Unlike lymphocyte depletion, the negative impact on neutrophils seems to be specific to cp BVDV biotype. Interestingly the majority of modified live BVDV vaccines are based on cp strain of BVDV.

A recombinant chimeric influenza virus vaccine expressing the consensus H3 hemagglutinin elicits broad hemagglutination inhibition antibodies against divergent swine H3N2 influenza viruses.

The global distribution and ongoing evolution of type A swine influenza virus (IAV-S) continue to pose significant challenges against developing broadly protective vaccines to control swine influenza. This study focuses on the hemagglutinin (HA) consensus-based approach towards developing a more broadly protective swine influenza vaccine against various H3 strains circulating in domestic pig populations. By computationally analyzing >1000 swine H3 full-length HA sequences, we generated a consensus H3 and expressed it in the context of influenza A WSN/33 reverse genetics system. The derived recombinant chimeric swine influenza virus with the consensus H3 was inactivated and further evaluated as a potential universal vaccine in pigs. The consensus H3 vaccine elicited broadly active hemagglutination inhibition (HI) antibodies against divergent swine H3N2 influenza viruses including human H3N2 variant of concern, and strains belong to genetic clusters IV, IV-A, IV-B, IV-C, IV-D and IV-F. Importantly, vaccinated pigs were completely protected against challenge with a clinical swine H3N2 isolate in that neither viral shedding nor replication in lungs of vaccinated pigs were observed. These findings warrant further study of the consensus H3 vaccine platform for broad protection against diverse swine influenza viruses.

Characterization of a porcine intestinal epithelial cell line for influenza virus production.

We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log(10) TCID(50) ml(-1). The SD-PJEC cell line was further tested as a potential alternative cell line to Madin-Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log(10) TCID(50) ml(-1), respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.

Microtubule S-glutathionylation as a potential approach for antimitotic agents.

BACKGROUND: Microtubules have been one of the most effective targets for the development of anticancer agents. Cancer cells treated by these agents are characterized by cell arrest at G2/M phase. Microtubule-targeting drugs are, therefore, referred to as antimitotic agents. However, the clinical application of the current antimitotic drugs is hampered by emerging drug resistance which is the major cause of cancer treatment failure. The clinical success of antimitotic drugs and emerging drug resistance has prompted a search for new antimitotic agents, especially those with novel mechanisms of action. The aim of this study was to determine whether microtubules can be S-glutathionylated in cancer cells and whether the glutathionylation will lead to microtubule dysfunction and cell growth inhibition. The study will determine whether microtubule S-glutathionylation can be a novel approach for antimitotic agents. METHODS: 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanyl]propionic acid (2-AAPA) was used as a tool to induce microtubule S-glutathionylation. UACC-62 cells, a human melanoma cell line, were used as a cancer cell model. A pull-down assay with glutathione S-transferase (GST)-agarose beads followed by Western blot analysis was employed to confirm microtubule S-glutathionylation. Immunofluorescence microscopy using a mouse monoclonal anti-α-tubulin-FITC was used to study the effect of the S-glutathionylation on microtubule function; mainly polymerization and depolymerization. Flow cytometry was employed to examine the effect of the S-glutathionylation on cell cycle distribution and apoptosis. Cell morphological change was followed through the use of a Zeiss AXIO Observer A1 microscope. Cancer cell growth inhibition by 2-AAPA was investigated with ten human cancer cell lines. RESULTS: Our investigation demonstrated that cell morphology was changed and microtubules were S-glutathionylated in the presence of 2-AAPA in UACC-62 cells. Accordingly, microtubules were found depolymerized and cells were arrested at G2/M phase. The affected cells were found to undergo apoptosis. Cancer growth inhibition experiments demonstrated that the concentrations of 2-AAPA required to produce the effects on microtubules were compatible to the concentrations producing cancer cell growth inhibition. CONCLUSIONS: The data from this investigation confirms that microtubule S-glutathionylation leads to microtubule dysfunction and cell growth inhibition and can be a novel approach for developing antimitotic agents.

Dose-response on the chemopreventive effects of sarcophine-diol on UVB-induced skin tumor development in SKH-1 hairless mice.

Sarcophine-diol (SD) is a lactone ring-opened analogue of sarcophine. It has shown chemopreventive effects on chemically-induced skin tumor development in female CD-1 mice, as well as in a UVB-induced skin tumor development model in hairless SKH-1 mice at a dose of 30 µg SD applied topically and 180 mJ/cm(2) UVB. The objective of this study was to determine the dose-response on the chemopreventive effects of SD on SKH-1 hairless mice when exposed to a UVB radiation dose of 30 mJ/cm(2). This UVB dose better represents chronic human skin exposure to sunlight leading to skin cancer than previous studies applying much higher UVB doses. Carcinogenesis was initiated and promoted by UVB radiation. Female hairless SKH-1 mice were divided into five groups. The control group was topically treated with 200 µL of acetone (vehicle), and the SD treatment groups were topically treated with SD (30 µg, 45 µg, and 60 µg dissolved in 200 µL of acetone) 1 h before UVB radiation (30 mJ/cm(2)). The last group of animals received 60 µg SD/200 µL acetone without UVB exposure. These treatments were continued for 27 weeks. Tumor multiplicity and tumor volumes were recorded on a weekly basis for 27 weeks. Weight gain and any signs of toxicity were also closely monitored. Histological characteristics and the proliferating cell nuclear antigen (PCNA) were evaluated in the mice skin collected at the end of the experiment. The dose-response study proved a modest increase in chemopreventive effects with the increase in SD dose. SD reduced the number of cells positively stained with PCNA proliferation marker in mice skin. The study also showed that SD application without UVB exposure has no effect on the structure of skin. The results from this study suggest that broader range doses of SD are necessary to improve the chemopreventive effects.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action.

BACKGROUND: Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. METHODS: UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. RESULTS: Magnolol pretreated groups (30, 60 µ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. CONCLUSIONS: Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

Isolation and development of bovine primary respiratory cells as model to study influenza D virus infection.

Influenza D virus (IDV) is a novel type of influenza virus that infects and causes respiratory illness in bovines. Lack of host-specific in vitro model that can recapitulate morphology and physiology of in vivo airway epithelial cells has impeded the study of IDV infection. Here, we established and characterized bovine primary respiratory epithelial cells from nasal turbinate, soft palate, and trachea of the same calf. All three cell types showed characteristics peculiar of epithelial cells, polarized into apical-basolateral membrane, and formed tight junctions. Furthermore, these cells expressed both α-2,3- and α-2,6-linked sialic acids with α-2,3 linkage being more abundant. IDV strains replicated to high titers in these cells, while influenza A and B viruses exhibited moderate to low titers, with influenza C virus replication not detected. These findings suggest that bovine primary airway epithelial cells can be utilized to model infection biology and pathophysiology of IDV and other respiratory pathogens.

Functional study of a role of N-terminal HA stem region of swine influenza A virus in virus replication.

Swine influenza A virus (SIV) is both a pathogen of economic significance to the swine industry and a potential zoonotic organism that may be transmitted to humans. We described here the detailed characterization of a role of N-terminal B-loop and CD helix of HA2 in swine influenza A virus replication. Results of our experiments demonstrated that Hemagglutinin (HA) protein of swine influenza virus could tolerate some mutations in functionally conserved B-loop and CD helix. These mutations, however, have substantially attenuated influenza virus replication in both cell lines and porcine primary tracheal epithelial cells. Significantly, we found that some B-loop or CD helix mutations generated virus mutants that replicated in MDCK and ST cell lines but failed to replicate in primary tracheal epithelial cells, thereby suggesting that swine HA protein may function as a viral virulence and pathogenesis factor. The described mutations may be further explored as attenuated vaccine candidates that can effectively prevent or eliminate the spread of influenza virus within and between swine herds.

Linear polysaccharides reduce production of inflammatory cytokines by LPS-stimulated bovine fibroblasts.

Chronic lesions in the limbs of farm animals cause lameness due to chronic infection and inflammation. Exploratory treatments for chronic wounds in humans may be suitable for adaptation into the field of animal care. Specifically, antimicrobial linear polysaccharides like oxidized regenerated cellulose (ORC) and chitin/chitosan are biodegradable hemostats that are being explored to promote healing of chronic wounds but have not been directly compared using the same biological specimen. Despite their current use in humans, linear polysaccharides possess features that may preclude their use as biodegradable bandages. For example, ORC promotes inflammation when it remains in vivo and chitin/chitosan stimulate size-dependent proinflammatory responses. In order to assess the use of these materials to treat chronic wounds we have compared their effects on cellular toxicity and in stimulating the production of proinflammatory cytokines by bovine epidermal fibroblasts. While neither polysaccharide increased cell mortality, on average, they caused minor alterations in expression of proinflammatory cytokines from cells isolated from different animals. Both polysaccharides reduced expression of proinflammatory cytokines stimulated by microbial lipopolysaccharide. We conclude that the polysaccharides used in this study are relatively inert and may improve healing of chronic epidermal wounds in farm animals.

Characterization of bovine ileal epithelial cell line for lectin binding, susceptibility to enteric pathogens, and TLR mediated immune responses.

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.

Piglet immunization with a spike subunit vaccine enhances disease by porcine epidemic diarrhea virus.

Immunization with an insect cell lysate/baculovirus mixture containing recombinant porcine epidemic diarrhea virus (PEDV) spike protein induced high levels of neutralizing antibodies in both mice and piglets. However, immunization of piglets with this vaccine resulted in enhancement of disease symptoms and virus replication in vaccine recipients exposed to PEDV challenge. Thus, these observations demonstrate a previously unrecognized challenge of PEDV vaccine research, which has important implications for coronavirus vaccine development.

Detection and characterization of influenza A virus PA-PB2 interaction through a bimolecular fluorescence complementation assay.

The influenza virus polymerase complex, consisting of the PA, PB1, and PB2 subunits, is required for the transcription and replication of the influenza A viral genome. Previous studies have shown that PB1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In the current study, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB1 interactions in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies reveal a previously unknown PA-PB2 interaction and provide a framework for further investigation of the biological relevance of the PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus.