Characterization of an H3N2 triple reassortant influenza virus with a mutation at the receptor binding domain (D190A) that occurred upon virus transmission from turkeys to pigs

The hemagglutinin (HA) protein of influenza virus mediates essential viral functions including the binding to host receptor and virus entry. It also has the antigenic sites required for virus neutralization by host antibodies. Here, we characterized an H3N2 triple reassortant (TR) influenza virus (A/turkey/Ohio/313053/04) with a mutation at the receptor binding domain (Asp190Ala) that occurred upon virus transmission from turkeys to pigs in an experimental infection study. The mutant virus replicated less efficiently than the parental virus in human, pig and turkey primary tracheal/bronchial epithelial cells, with more than 3-log10 difference in virus titer at 72 hours post infection. In addition, the mutant virus demonstrated lower binding efficiency to plasma membrane preparations from all three cell types compared to the parental virus. Antisera raised against the parental virus reacted equally to both homologous and heterlogous viruses, however, antisera raised against the mutant virus showed 4-8 folds lower reactivity to the parental virus

Differential modulation of cytokine, chemokine and Toll like receptor expression in chickens infected with classical and variant infectious bursal disease virus

Infectious bursal disease (IBD) is an important immunosuppressive disease of chickens. The causative agent, infectious bursal disease virus (IBDV), consists of two serotypes, 1 and 2. Serotype 1 consists of classic IBDV (cIBDV) and variant IBDV (vIBDV). Both of these strains vary in antigenicity and pathogenesis. The goal of this study was to compare the immunopathogenesis of cIBDV and vIBDV. Three-week-old specific pathogen free chickens were inoculated intraocularly with standard challenge strain (STC) (cIBDV) and a variant strain Indiana (IN) (vIBDV). The cIBDV produced more pronounced bursal damage, inflammatory response and infiltration of T cells as compared to vIBDV. There were significant differences in the expression of innate (IFN-α and IFN-β), proinflammatory cytokine and mediator (IL-6 and iNOS) in cIBDV- and vIBDV-infected bursas. The expression of chemokines genes, IL-8 and MIP-α was also higher in cIBDV-infected chickens during the early phase of infection. The expression of Toll like receptor 3 (TLR3) was downregulated at post inoculation days (PIDs) 3, 5, and 7 in the bursas of vIBDV-infected chickens whereas TLR3 was upregulated at PIDs 3 and 5 in cIBDV-infected bursas. In vIBDV-infected bursa, TLR7 expression was downregulated at PIDs 3 and 5 and upregulated at PID 7. However, TLR7 was upregulated at PIDs 3 and 7 in cIBDV-infected bursas. The expression of MyD88 was downregulated whereas TRIF gene expression was upregulated in cIBDV- and vIBDV-infected bursa. These findings demonstrate the critical differences in bursal lesions, infiltration of T cells, expression of cytokines, chemokines and TLRs in the bursa of cIBDV-and vIBDV-infected chickens

Epithelial Cells Derived from Swine Bone Marrow Express Stem Cell Markers and Support Influenza Virus Replication In Vitro

The bone marrow contains heterogeneous population of cells that are involved in the regeneration and repair of diseased organs, including the lungs. In this study, we isolated and characterized progenitor epithelial cells from the bone marrow of 4- to 5-week old germ-free pigs. Microscopically, the cultured cells showed epithelial-like morphology. Phenotypically, these cells expressed the stem cell markers octamer-binding transcription factor (Oct4) and stage-specific embryonic antigen-1 (SSEA-1), the alveolar stem cell marker Clara cell secretory protein (Ccsp), and the epithelial cell markers pan-cytokeratin (Pan-K), cytokeratin-18 (K-18), and occludin. When cultured in epithelial cell growth medium, the progenitor epithelial cells expressed type I and type II pneumocyte markers. Next, we examined the susceptibility of these cells to influenza virus. Progenitor epithelial cells expressed sialic acid receptors utilized by avian and mammalian influenza viruses and were targets for influenza virus replication. Additionally, differentiated type II but not type I pneumocytes supported the replication of influenza virus. Our data indicate that we have identified a unique population of progenitor epithelial cells in the bone marrow that might have airway reconstitution potential and may be a useful model for cell-based therapies for infectious and non-infectious lung diseases

Characterization of progenitor epithelial cells isolated from pig bone marrow.

<p>(<b>A</b>) Colony morphology of progenitor epithelial cells derived from bone marrow. (<b>B</b>) Expression of stem cells markers on progenitor epithelial cells. Epithelial colony cells from primary cell cultures were examined for the expression of lung and other stem cell markers by using specific antibodies directed against Ccsp, Oct4 and SSEA-1. (<b>C</b>) Expression of epithelial cell markers on progenitor epithelial cells. Primary cell cultures were examined for the expression of specific epithelial cell markers: Pan-K, K-18, and occludin. (<b>D–F</b>) Absence of non-specific binding by progenitor epithelial cells. The cells were incubated with FITC-labeled secondary antibodies (D) goat anti-rabbit (E) goat anti-mouse (F) donkey anti-goat antibodies. Nuclei were stained by DAPI.</p

Differentiation potential of progenitor epithelial cells.

<p>Epithelial colony cells were sub-cultured onto collagen I-coated plates and cultured in epithelial cell differentiation medium; on day 5 the cells were examined for (<b>A</b>) morphology of control and differentiated cells and (<b>B</b>) the expression of alveolar cell markers, Aqua5 (a type I pneumocyte marker) and SPC (a type II pneumocyte marker).</p

Replication of influenza virus in progenitor epithelial cells.

<p>(<b>A</b>) Progenitor epithelial cells express α-2,3- and α-2,6-linked sialic acid receptors. Progenitor epithelial cells were examined by flow cytometry for the expression of α-2,3- and α-2,6-linked sialic acid receptors. The cells were stained with FITC-labelled <i>Maackia amurensis</i> lectin II (MAA) specific for α-2,3-linked sialic acid receptors and <i>Sambucus niagra</i> agglutinin (SNA), specific for α-2,6-linked sialic acid receptors. MDCK cells were included as positive controls. Compared to unstained cells (black), cells stained with lectins (red) showed right shift indicating positive staining. BM cells (progenitor bone marrow epithelial cells), MDCK (MDCK cells) (<b>B</b>) Progenitor epithelial cells were infected with SwIV, AvIV or HuIV at a MOI of 1. All virus types tested induced cell cytotoxicity. (<b>C</b>) Virus replication kinetics in primary culture of progenitor epithelial cells after virus infection at 1 MOI. Virus production in infected culture supernatants was measured by titration in MDCK cells. The values are averages ± S.D. of two separate experiments using progenitor epithelial cells from two different pigs in each experiment (n = 4).</p

Replication of influenza virus in differentiated pneumocytes:

<p>Primary cells were subcultured onto collagen I-coated plates in epithelial cell differentiation medium; on day 5 differentiated cells were infected with SwIV and examined for the presence of viral proteins at 24 h after infection. Type II pneumocytes, positive for SPC marker (green) supported the replication of SwIV virus as indicated by expression of viral NP protein (red). Cell nuclei were stained with DAPI (blue). No viral proteins were detected in Aqua5 expressing (green) type I pneumocytes.</p

Respiratory proteins contribute differentially to <it>Campylobacter jejuni</it>’s survival and in vitro interaction with hosts’ intestinal cells

Abstract Background The genetic features that facilitate Campylobacter jejuni’s adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni’s adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni’s i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens). Results C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA’s motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H2O2), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H2O2 was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division. Conclusions We conclude that the RPs contribute to C. jejuni’s motility, H2O2 resistance, biofilm formation, and in vitro interactions with hosts’ intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen’s remarkable potential for adaptation.</p