Reinhard Ertl

Phylogenetic analysis suggests independent introduction of feline immunodeficiency virus clades A and B to Central Europe and identifies diverse variants of clade B.

Phylogenetic analysis of European feline immunodeficiency virus sequences confirms a highly distinct pattern in the occurrence of FIV-subtypes within Germany and Austria, indicating little mixing and at least two independent introductions of FIV. There is also evidence for a North to South gradient of FIV subtype distribution in Europe, with subtype A being more common in the North, while subtype B is prevalent in the South of Europe. In addition, among Austrian subtype B sequences, considerable differences from classical subtype B sequences were detected by bootscanning, which either suggests recombination with so far unrecognized subtypes, or a long period of independent evolution.

Dietary calcium concentration and cereals differentially affect mineral balance and tight junction proteins expression in jejunum of weaned pigs

Ca plays an essential role in bone development; however, little is known about its effect on intestinal gene expression in juvenile animals. In the present study, thirty-two weaned pigs (9·5 (SEM 0·11) kg) were assigned to four diets that differed in Ca concentration (adequate v. high) and cereal composition (wheat-barley v. maize) to assess the jejunal and colonic gene expression of nutrient transporters, tight junction proteins, cytokines and pathogen-associated molecular patterns, nutrient digestibility, Ca balance and serum acute-phase response. To estimate the impact of mucosal bacteria on colonic gene expression, Spearmans correlations between colonic gene expression and bacterial abundance were computed. Faecal Ca excretion indicated that more Ca was available along the intestinal tract of the pigs fed high Ca diets as compared to the pigs fed adequate Ca diets (P> 0.05). High Ca diets decreased jejunal zonula occludens 1 (ZO1) and occludin (OCLN) expression, up-regulated jejunal expression of toll-like receptor 2 (TLR2) and down-regulated colonic GLUT2 expression as compared to the adequate Ca diets (P< 0.05). Dietary cereal composition up-regulated jejunal TLR2 expression and interacted (P= 0.021) with dietary Ca on colonic IL1B expression; high Ca concentration up-regulated IL1B expression with wheat-barley diets and down-regulated it with maize diets. Spearmans correlations (r> 0·35; P< 0·05) indicated an association between operational taxonomic units assigned to the phyla Bacteroidetes, Firmicutes and Proteobacteria and bacterial metabolites and mucosal gene expression in the colon. The present results indicate that high Ca diets have the potential to modify the jejunal and colonic mucosal gene expression response which, in turn, interacts with the composition of the basal diet and mucosa-associated bacteria in weaned pigs.

Gag-specific immune enhancement of lentiviral infection after vaccination with an adenoviral vector in an animal model of AIDS

The evaluation of vaccine strategies in animal models is essential for the development of a vaccine against HIV. In efficacy trials conducted in non-human primate models of AIDS, vaccines based on adenoviruses compared favourably with other vaccine vectors. To determine whether this strategy could be transposed to another animal model, and by extension, to humans, we have evaluated the efficacy of adenoviral vectors in a natural model of AIDS, infection of the cat by the feline immunodeficiency virus (FIV). Recombinant canine adenoviruses expressing the envelope glycoproteins or the Gag protein of a primary strain of FIV were constructed. Three groups of six cats were immunised twice with vectors expressing FIV antigens or with a vector expressing an irrelevant antigen, green fluorescent protein, by intramuscular and subcutaneous routes. Humoral responses were elicited against the transgene product in 6/6, 3/6 and 0/6 cats after immunisation against green fluorescent protein, Gag or the envelope glycoproteins, respectively. Six weeks after the second administration, cats were challenged by the intraperitoneal route with the homologous strain, and viral burden in plasma was followed by quantitative RT-PCR. Immunisation with FIV antigens did not afford protection. Rather, viral RNA was detected at earlier time points in cats immunised against Gag than in cats immunised with a vector expressing an irrelevant antigen. Such immune-mediated enhancement did not appear to have a long-range impact on viral set point or inversion of the CD4(+)/CD8(+) ratio. Thus, in the feline AIDS model pre-existing immunity against a viral antigen exacerbated acute phase infection.

Viral transcriptome analysis of feline immunodeficiency virus infected cells using second generation sequencing technology

Feline immunodeficiency virus (FIV) is a widespread pathogen causing immunodeficiency in domestic cats and related wild cat species. The virus genome includes the main structural genes common to all retroviruses as well as accessory genes displaying essential functions during the viral life cycle. Expression of viral genes involves transcription of provirus genomes into full-length transcripts, which are partially processed into several spliced mRNA variants for the translation of particular proteins. Among several FIV isolates derived from domestic cats, notable differences in pathogenicity could be observed leading to identification of low and high pathogenic virus isolates. This study investigates the viral transcriptome of two differentially virulent FIV strains using second generation sequencing (SGS) technology. The expression levels of viral genes as detected by SGS were additionally determined by reverse transcription quantitative PCR analysis in order to compare two methods of mRNA quantification. The different properties of both methods, especially regarding normalization between samples, had to be considered when comparing the resulting data. SGS turned out to be a suitable technique for comparing mRNA transcription between both FIV infected cell lines and the identification of spliced viral transcripts. In contrast to this, the quantification of these spliced isoforms using SGS data was impeded by the short length of sequencing reads. In summary, SGS analysis revealed very consistent mRNA levels for the majority of viral genes between the low pathogenic Petaluma and the more highly pathogenic Glasgow 8 isolate. Notable differences among the two FIV strains could be observed in the viral mRNA splicing where Glasgow 8 displays similarities to the transcription pattern seen in the early stages of natural lentivirus infections. Thus, divergences in the regulation of post-transcriptional RNA processing might represent an additional contributor to the diverse pathogenic effects of individual FIV isolates. Taken together, this study aims to investigate the viral transcriptome as one part of the complex network of virus-host interactions, which will contribute to gaining deeper insights into FIV pathogenesis.

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection.

Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNA-Seq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies.

The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data

Background Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0). Results We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively. Conclusions The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

Expression of Progesterone Receptor Membrane Component 1 (PGRMC1), Progestin and AdipoQ Receptor 7 (PAQPR7), and Plasminogen Activator Inhibitor 1 RNA-Binding Protein (PAIRBP1) in Glioma Spheroids In Vitro

Objective. Some effects of progesterone on glioma cells can be explained through the slow, genomic mediated response via nuclear receptors; the other effects suggest potential role of a fast, nongenomic action mediated by membrane-associated progesterone receptors. Methods. The effects of progesterone treatment on the expression levels of progesterone receptor membrane component 1 (PGRMC1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1), and progestin and adipoQ receptor 7 (PAQR7) on both mRNA and protein levels were investigated in spheroids derived from human glioma cell lines U-87 MG and LN-229. Results. The only significant alteration at the transcript level was the decrease in PGRMC1 mRNA observed in LN-229 spheroids treated with 30 ng/mL of progesterone. No visible alterations at the protein levels were observed using immunohistochemical analysis. Stimulation of U-87 MG spheroids resulted in an increase of PGRMC1 but a decrease of PAIRBP1 protein. Double immunofluorescent detection of PGRMC1 and PAIRBP1 identified the two proteins to be partially colocalized in the cells. Western blot analysis revealed the expected bands for PGRMC1 and PAIRBP1, whereas two bands were detected for PAQR7. Conclusion. The progesterone action is supposed to be mediated via membrane-associated progesterone receptors as the nuclear progesterone receptor was absent in tested spheroids.

Decreased expression of endogenous feline leukemia virus in cat lymphomas: a case control study

BackgroundCats infected with exogenous feline leukemia virus (exFeLV) have a higher chance of lymphoma development than uninfected cats. Furthermore, an increased exFeLV transcription has been detected in lymphomas compared to non-malignant tissues. The possible mechanisms of lymphoma development by exFeLV are insertional mutagenesis or persistent stimulation of host immune cells by viral antigens, bringing them at risk for malignant transformation. Vaccination of cats against exFeLV has in recent years decreased the overall infection rate in most countries. Nevertheless, an increasing number of lymphomas have been diagnosed among exFeLV-negative cats. Endogenous feline leukemia virus (enFeLV) is another retrovirus for which transcription has been observed in cat lymphomas. EnFeLV provirus elements are present in the germline of various cat species and share a high sequence similarity with exFeLV but, due to mutations, are incapable of producing infectious viral particles. However, recombination between exFeLV and enFeLV could produce infectious particles.ResultsWe examined the FeLV expression in cats that have developed malignant lymphomas and discussed the possible mechanisms that could have induced malignant transformation. For expression analysis we used next-generation RNA-sequencing (RNA-Seq) and for validation reverse transcription quantitative PCR (RT-qPCR). First, we showed that there was no expression of exFeLV in all samples, which eliminates the possibility of recombination between exFeLV and enFeLV. Next, we analyzed the difference in expression of three enFeLV genes between control and lymphoma samples. Our analysis showed an average of 3.40-fold decreased viral expression for the three genes in lymphoma compared to control samples. The results were confirmed by RT-qPCR.ConclusionsThere is a decreased expression of enFeLV genes in lymphomas versus control samples, which contradicts previous observations for the exFeLV. Our results suggest that a persistent stimulation of host immune cells is not an appropriate mechanism responsible for malignant transformation caused by feline endogenous retroviruses.

Differences in intestinal size, structure, and function contributing to feed efficiency in broiler chickens reared at geographically distant locations

Abstract The contribution of the intestinal tract to differences in residual feed intake (RFI) has been inconclusively studied in chickens so far. It is also not clear if RFI‐related differences in intestinal function are similar in chickens raised in different environments. The objective was to investigate differences in nutrient retention, visceral organ size, intestinal morphology, jejunal permeability and expression of genes related to barrier function, and innate immune response in chickens of diverging RFI raised at 2 locations (L1: Austria; L2: UK). The experimental protocol was similar, and the same dietary formulation was fed at the 2 locations. Individual BW and feed intake (FI) of chickens (Cobb 500FF) were recorded from d 7 of life. At 5 wk of life, chickens (L1, n = 157; L2 = 192) were ranked according to their RFI, and low, medium, and high RFI chickens were selected (n = 9/RFI group, sex, and location). RFI values were similar between locations within the same RFI group and increased by 446 and 464 g from low to high RFI in females and males, respectively. Location, but not RFI rank, affected growth, nutrient retention, size of the intestine, and jejunal disaccharidase activity. Chickens from L2 had lower total body weight gain and mucosal enzyme activity but higher nutrient retention and longer intestines than chickens at L1. Parameters determined only at L1 showed increased crypt depth in the duodenum and jejunum and enhanced paracellular permeability in low vs. high RFI females. Jejunal expression of IL1B was lower in low vs. high RFI females at L2, whereas that of TLR4 at L1 and MCT1 at both locations was higher in low vs. high RFI males. Correlation analysis between intestinal parameters and feed efficiency metrics indicated that feed conversion ratio was more correlated to intestinal size and function than was RFI. In conclusion, the rearing environment greatly affected intestinal size and function, thereby contributing to the variation in chicken RFI observed across locations.

Cytokine-induced interleukin-1 receptor antagonist protein expression in genetically engineered equine mesenchymal stem cells for osteoarthritis treatment

A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease‐induced substances is highly desirable.