Inferred Causal Mechanisms of Persistent FMDV Infection in Cattle from Differential Gene Expression in the Nasopharyngeal Mucosa.

Foot-and-mouth disease virus (FMDV) can persistently infect pharyngeal epithelia in ruminants but not in pigs. Our previous studies demonstrated that persistent FMDV infection in cattle was associated with under-expression of several chemokines that recruit immune cells. This report focuses on the analysis of differentially expressed genes (DEG) identified during the transitional phase of infection, defined as the period when animals diverge between becoming carriers or terminators. During this phase, Th17-stimulating cytokines (IL6 and IL23A) and Th17-recruiting chemokines (CCL14 and CCL20) were upregulated in animals that were still infected (transitional carriers) compared to those that had recently cleared infection (terminators), whereas chemokines recruiting neutrophils and CD8+ T effector cells (CCL3 and ELR+CXCLs) were downregulated. Upregulated Th17-specific receptor, CCR6, and Th17-associated genes, CD146, MIR155, and ThPOK, suggested increased Th17 cell activity in transitional carriers. However, a complex interplay of the Th17 regulatory axis was indicated by non-significant upregulation of IL17A and downregulation of IL17F, two hallmarks of TH17 activity. Other DEG suggested that transitional carriers had upregulated aryl hydrocarbon receptor (AHR), non-canonical NFκB signaling, and downregulated canonical NFκB signaling. The results described herein provide novel insights into the mechanisms of establishment of FMDV persistence. Additionally, the fact that ruminants, unlike pigs, produce a large amount of AHR ligands suggests a plausible explanation of why FMDV persists in ruminants, but not in pigs.

Serum Neutralizing and Enhancing Effects on African Swine Fever Virus Infectivity in Adherent Pig PBMC.

African swine fever virus (ASFV) causes hemorrhagic fever with mortality rates of up to 100% in domestic pigs. Currently, there are no commercial vaccines for the disease. Only some live-attenuated viruses have been able to protect pigs from ASFV infection. The immune mechanisms involved in the protection are unclear. Immune sera can neutralize ASFV but incompletely. The mechanisms involved are not fully understood. Currently, there is no standardized protocol for ASFV neutralization assays. In this study, a flow cytometry-based ASFV neutralization assay was developed and tested in pig adherent PBMC using a virulent ASFV containing a fluorescent protein gene as a substrate for neutralization. As with previous studies, the percentage of infected macrophages was approximately five time higher than that of infected monocytes, and nearly all infected cells displayed no staining with anti-CD16 antibodies. Sera from naïve pigs and pigs immunized with a live-attenuated ASFV and fully protected against parental virus were used in the assay. The sera displayed incomplete neutralization with MOI-dependent neutralizing efficacies. Extracellular, but not intracellular, virions suspended in naïve serum were more infectious than those in the culture medium, as reported for some enveloped viruses, suggesting a novel mechanism of ASFV infection in macrophages. Both the intracellular and extracellular virions could not be completely neutralized.

Selenium and the 15kDa Selenoprotein Impact Colorectal Tumorigenesis by Modulating Intestinal Barrier Integrity.

Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof's potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing.

Molecular Pathogenesis and Immune Evasion of Vesicular Stomatitis New Jersey Virus Inferred from Genes Expression Changes in Infected Porcine Macrophages.

The molecular mechanisms associated with the pathogenesis of vesicular stomatitis virus (VSV) in livestock remain poorly understood. Several studies have highlighted the relevant role of macrophages in controlling the systemic dissemination of VSV during infection in different animal models, including mice, cattle, and pigs. To gain more insight into the molecular mechanisms used by VSV to impair the immune response in macrophages, we used microarrays to determine the transcriptomic changes produced by VSV infection in primary cultures of porcine macrophages. The results indicated that VSV infection induced the massive expression of multiple anorexic, pyrogenic, proinflammatory, and immunosuppressive genes. Overall, the interferon (IFN) response appeared to be suppressed, leading to the absence of stimulation of interferon-stimulated genes (ISG). Interestingly, VSV infection promoted the expression of several genes known to downregulate the expression of IFNß. This represents an alternate mechanism for VSV control of the IFN response, beyond the recognized mechanisms mediated by the matrix protein. Although there was no significant differential gene expression in macrophages infected with a highly virulent epidemic strain compared to a less virulent endemic strain, the endemic strain consistently induced higher expression of all upregulated cytokines and chemokines. Collectively, this study provides novel insights into VSV molecular pathogenesis and immune evasion that warrant further investigation.

Mechanisms of Maintenance of Foot-and-Mouth Disease Virus Persistence Inferred From Genes Differentially Expressed in Nasopharyngeal Epithelia of Virus Carriers and Non-carriers.

Foot-and-mouth disease virus (FMDV) causes persistent infection of nasopharyngeal epithelial cells in ~50% of infected ruminants. The mechanisms involved are not clear. This study provides a continued investigation of differentially expressed genes (DEG) identified in a previously published transcriptomic study analyzing micro-dissected epithelial samples from FMDV carriers and non-carriers. Pathway analysis of DEG indicated that immune cell trafficking, cell death and hematological system could be affected by the differential gene expression. Further examination of the DEG identified five downregulated (chemerin, CCL23, CXCL15, CXCL16, and CXCL17) and one upregulated (CCL2) chemokines in carriers compared to non-carriers. The differential expression could reduce the recruitment of neutrophils, antigen-experienced T cells and dendritic cells and increase the migration of macrophages and NK cells to the epithelia in carriers, which was supported by DEG expressed in these immune cells. Downregulated chemokine expression could be mainly due to the inhibition of canonical NFκB signaling based on DEG in the signaling pathways and transcription factor binding sites predicted from the proximal promoters. Additionally, upregulated CD69, IL33, and NID1 and downregulated CASP3, IL17RA, NCR3LG1, TP53BP1, TRAF3, and TRAF6 in carriers could inhibit the Th17 response, NK cell cytotoxicity and apoptosis. Based on our findings, we hypothesize that (1) under-expression of chemokines that recruit neutrophils, antigen-experienced T cells and dendritic cells, (2) blocking NK cell binding to target cells and (3) suppression of apoptosis induced by death receptor signaling, viral RNA, and cell-mediated cytotoxicity in the epithelia compromised virus clearance and allowed FMDV to persist. These hypothesized mechanisms provide novel information for further investigation of persistent FMDV infection.

A novel bovine CXCL15 gene in the GRO chemokine gene cluster.

In our previous transcriptomic studies using DNA microarray analysis, a probe designed from an unknown expressed sequence tag (EST) showed significant differential gene expression in the pharyngeal epithelia. The objectives of this study are to annotate the gene sequence and compare the gene transcription levels among different bovine tissues based on our published microarray data. The gene transcribing the EST contains a 90-amino-acid protein sequence. The results of bioinformatic analyses using comparative genetics, multiple sequence alignments, phylogenetic analysis and promoter sequence analysis indicated that this gene is a novel ELR+ CXCL gene orthologous to mouse CXCL15. The gene is highly conserved in ruminants and exists in many other mammals but not in chickens, primates or pigs. Phylogenetic analysis and gene structures showed that CXCL15 is closer to CXCL8 than to other ELR+ CXCLs. Our microarray data show that bovine CXCL15 expression was higher in laser capture micro-dissected bovine pharyngeal epithelia than in the whole pharyngeal tissues, which agrees with the expression in mice. However, unlike the high expression in the mouse lung, our results showed that the bovine nasal turbinate, dorsal nasopharynx, dorsal soft palate and tongue expressed higher levels of CXCL15 than the lung and skins. Promoter analysis showed that ruminants have more immune-related transcription factor binding sites in the proximal promoters of CXCL15 than mouse. CXCL15 has previously only been reported in mice and has neutrophil chemotactic activity. Given the critical roles of neutrophils in innate immunity, this study provides useful information for further characterization of bovine CXCL15.

Phenotypic diversity in an emerging mycoplasmal disease.

The avian pathogen Mycoplasma gallisepticum (MG) is a known pathogen of poultry, and newly emerged pathogen of house finches wherein it is associated with lethal conjunctivitis. Factors present in MG that are known to mediate virulence include cytadherence, sialidase activity, peroxide production, and biofilm formation. We have quantitatively assessed these factors for MG isolates from house finches from a temporal and geographic distribution across the continental United States that show differing capacity for virulence in vivo. Statistically significant (P < 0.05) differences were observed across strains for sialidase activity, cytadherence, and hydrogen peroxide production. Sialidase activity increased over time in geographically static populations, but did not correlate with time overall. All strains were able to bind α-2,6-linked sialic acid. No strains were found to bind α-2,3-linked sialic acid. All strains produced biofilms in vitro; however, no significant differences were observed in the density of biofilms across strains. Quantitative variance in virulence-associated traits is consistent with within-host evolutionary adaptation in response to a change in ecological niche by a parasitic pathogen.

Immunologic Pathways in Protective versus Maladaptive Host Responses to Attenuated and Pathogenic Strains of Mycoplasma gallisepticum.

Mycoplasmas are small bacterial commensals or pathogens that commonly colonize host mucosal tissues and avoid rapid clearance, in part by stimulating inflammatory, immunopathogenic responses. We previously characterized a wide array of transcriptomic perturbations in avian host tracheal mucosae infected with virulent, immunopathologic Mycoplasma gallisepticum; however, mechanisms delineating these from protective responses, such as those induced upon vaccination, have not been thoroughly explored. In this study, host transcriptomic responses to two experimental M. gallisepticum vaccines were assessed during the first 2 days of infection. Relative to virulent infection, host metabolic and immune gene responses to both vaccines were greatly decreased, including early innate immune responses critical to disease development and subsequent adaptive immunity. These data specify host genes and potential mechanisms contributing to maladaptive versus beneficial host responses-information critical for design of vaccines efficacious in both limiting inflammation and enabling pathogen clearance.