A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein

Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 perilesional/intact skin biopsies, and 1 serum. Tissue extracts from sarcoid-free equines served as controls. IC/PCR scored positive in 14/24 (58.3%) specimens obtained from sarcoid-patients, but negative for controls. Quantitative IC/PCR demonstrated <125 immunoprecipitable viral genomes/50 microl extract for the majority of specimens. Moreover, full-length BPV-1 genomes were detected in a complex with L1 proteins. These complexes may correspond to virion precursors or intact virions.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

DNA-based analysis of protein variants reveals different genetic variability of the paralogous equine ß-lactoglobulin genes LGB1 and LGB2

The genetic variability of milk protein genes may influence the nutritive value or processing and functional properties of the milk. While numerous protein variants are known in ruminants, knowledge about milk protein variability in horses is still limited. Mare's milk is, however, produced for human consumption in many countries. Beta-lactoglobulin belonging to the protein family of lipocalins, which are known as common food- and airborne allergens, is a major whey protein. It is absent from human milk and thus a key agent in provoking cow's milk protein allergy. Mare's milk is, however, usually better tolerated by most affected people. Several functions of β-lactoglobulin have been discussed, but its ultimate physiological role remains unclear. In the current study, the open reading frames of the two equine β-lactoglobulin paralogues LGB1 and LGB2 were re-sequenced in 249 horses belonging to 14 different breeds in order to predict the existence of protein variants at the DNA-level. Thereby, only a single signal peptide variant of LGB1, but 10 different putative protein variants of LGB2 were identified. In horses, both genes are expressed and in such this is a striking previously unknown difference in genetic variability between the two genes. It can be assumed that LGB1 is the ancestral paralogue, which has an essential function causing a high selection pressure. As horses have very low milk fat content this unknown function might well be related to vitamin-uptake. Further studies are, however, needed, to elucidate the properties of the different gene products.

Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease

Eosinophil extracellular traps (EETs) are part of the innate immune response and are seen in multiple infectious, allergic, and autoimmune eosinophilic diseases. EETs are composed of a meshwork of DNA fibers and eosinophil granule proteins, such as major basic protein (MBP) and eosinophil cationic protein (ECP). Interestingly, the DNA within the EETs appears to have its origin in the mitochondria of eosinophils, which had released most their mitochondrial DNA, but were still viable, exhibiting no evidence of a reduced life span. Multiple eosinophil activation mechanisms are represented, whereby toll-like, cytokine, chemokine, and adhesion receptors can all initiate transmembrane signal transduction processes leading to the formation of EETs. One of the key signaling events required for DNA release is the activation of the NADPH oxidase. Here, we review recent progress made in the understanding the molecular mechanisms involved in DNA and granule protein release, discuss the presence of EETs in disease, speculate on their potential role(s) in pathogenesis, and compare available data on other DNA-releasing cells, particularly neutrophils.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.

Short communication: duplication in the 5'-flanking region of the beta-lactoglobulin gene is linked to the BLG A allele

beta-Lactoglobulin (beta-LG) is the major whey protein in the milk of cows and other ruminants. It is well established that the predominant genetic variants beta-LG A and B are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants. However, the genetic basis for the differentially expressed BLG A and B alleles is still elusive. In this study additional genetic variation further upstream in the 5'-flanking region of the BLG gene was identified, including 6 single nucleotide substitutions, a single nucleotide deletion, and a 7-bp duplication. Comparison of DNA sequences showed that the investigated 5'-flanking region is highly conserved between ruminants, and the duplication g.-1885_-1879dupCTCTCGC and the substitution g.-1888A>G are only found in the BLG A and D alleles in cattle. The cytosine at position g.-1957 and the thymines at positions g.-2008 and g.-2049 are only found in BLG B alleles of cattle. It is suggested that the described genetic variability contributes to the differential allelic expression of the BLG gene.

CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients >or=60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients.

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses

Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5 untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.

Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei

Different life-cycle stages of Trypanosoma brucei are characterized by stage-specific glycoprotein coats. GPEET procyclin, the major surface protein of early procyclic (insect midgut) forms, is transcribed in the nucleolus by RNA polymerase I as part of a polycistronic precursor that is processed to monocistronic mRNAs. In culture, when differentiation to late procyclic forms is triggered by removal of glycerol, the precursor is still transcribed, but accumulation of GPEET mRNA is prevented by a glycerol-responsive element in the 3' UTR. A genome-wide RNAi screen for persistent expression of GPEET in glycerol-free medium identified a novel protein, NRG1 (Nucleolar Regulator of GPEET 1), as a negative regulator. NRG1 associates with GPEET mRNA and with several nucleolar proteins. These include two PUF proteins, TbPUF7 and TbPUF10, and BOP1, a protein required for rRNA processing in other organisms. RNAi against each of these components prolonged or even increased GPEET expression in the absence of glycerol as well as causing a significant reduction in 5.8S rRNA and its immediate precursor. These results indicate that components of a complex used for rRNA maturation can have an additional role in regulating mRNAs that originate in the nucleolus.

Revealing the elusive molecular biology of the vault RNA

Recently several novel and previously reported non-protein-coding RNAs (ncRNAs) have been identified to be upregulated upon Epstein-Barr virus (EBV) infection in human B-lymphocytes. A group of these significantly upregulated ncRNAs are called vault RNAs (vtRNAs). ,b Only about 5% of the total cellular vtRNAs are connected to the vault particle, the largest known ribonucleoprotein particle (RNP) in eukaryotic cells. However the function of this ncRNA family and moreover of the vault particle remains still rather unclear. Our previous findings suggest a link between EBV infection and vtRNA expression. Consequently we are interested which part of the viral genome is responsible for the upregulation and moreover which function the vtRNAs might possess during virus propagation. To address this question we have separately overexpressed specific EBV-encoded, latently expressed proteins in BL2-cells to determine the influence on the vault RNA levels. Thereby we identified one EBV-encoded protein, called Latent Membrane Protein 1 (LMP1), which significantly contributes to the vtRNA upregulation. We used LMP1 mutants to characterize the region of the protein and the responsible pathway for triggering the elevated vtRNA expression. Our results suggest that the NFkB- pathway might be involved in this process. To investigate a possible functional connection between the vtRNA and EBV infection, we have overexpressed vtRNA1-1 in BL41, a cell line usually not expressing this vault RNA. We show that overexpression of vtRNA1-1 leads to a better viral establishment and markedly protects cells from undergoing apoptosis. Knock-down of the major vault protein, the main component of the vault particle, had no effect on EBV infection and apoptosis resistance. Thus these results support the view that the observed phenotype is caused by the vtRNA rather than the vault particle.