Characterization of Ovine A3Z1 Restriction Properties against Small Ruminant Lentiviruses (SRLVs)

Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motifWe sincerely acknowledge Sandra Hervás-Stubbs from CIMA for her fruitful help. We also acknowledge Greg Towers, University College London for plasmids and protocols. Funded by CICYT (AGL2010-22341-C04-01) and Navarra’s Government (IIQ010449.RI1, IIQ14064.RI1 and PI042-LENTIMOL). Ramsés Reina was supported by the Spanish Ministry of Science and Innovation “Ramón y Cajal” contract. We acknowledge support of the publication fee by the Public University of Navarra and CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Small ruminant macrophage polarization may play a pivotal role on lentiviral infection

Small ruminant lentiviruses (SRLV) infect the monocyte/macrophage lineage inducing a long-lasting infection affecting body condition, production and welfare of sheep and goats all over the world. Macrophages play a pivotal role on the host's innate and adaptative immune responses against parasites by becoming differentially activated. Macrophage heterogeneity can tentatively be classified into classically differentiated macrophages (M1) through stimulation with IFN-gamma displaying an inflammatory profile, or can be alternatively differentiated by stimulation with IL-4/IL-13 into M2 macrophages with homeostatic functions. Since infection by SRLV can modulate macrophage functions we explored here whether ovine and caprine macrophages can be segregated into M1 and M2 populations and whether this differential polarization represents differential susceptibility to SRLV infection. We found that like in human and mouse systems, ovine and caprine macrophages can be differentiated with particular stimuli into M1/M2 subpopulations displaying specific markers. In addition, small ruminant macrophages are plastic since M1 differentiated macrophages can express M2 markers when the stimulus changes from IFN-gamma to IL-4. SRLV replication was restricted in M1 macrophages and increased in M2 differentiated macrophages respectively according to viral production. Identification of the infection pathways in macrophage populations may provide new targets for eliciting appropriate immune responses against SRLV infection.This work was funded by grants from CICYT (no. AGL2010-22341-C04-01), and the Government of Navarra (no. IIQ14064.RI1). We acknowledge the Public University of Navarra and CSIC for fellowships and the JAE-contract (HC and RR)

Diagnosing infection with small ruminant lentiviruses of genotypes A and B by combining synthetic peptides in ELISA

The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status.The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping.Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1R) and Navarra's Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI-fellow of the Spanish MINECO and R. Reina had a contract of the Public University of Navarra.Peer Reviewe

Identification of the ovine mannose receptor and its possible role in Visna/Maedi virus infection

This study aims to characterize the mannose receptor (MR) gene in sheep and its role in ovine visna/maedi virus (VMV) infection. The deduced amino acid sequence of ovine MR was compatible with a transmembrane protein having a cysteine-rich ricin-type amino-terminal region, a fibronectin type II repeat, eight tandem C-type lectin carbohydrate-recognition domains (CRD), a transmembrane region, and a cytoplasmic carboxy-terminal tail. The ovine and bovine MR sequences were closer to each other compared to human or swine MR. Concanavalin A (ConA) inhibited VMV productive infection, which was restored by mannan totally in ovine skin fibroblasts (OSF) and partially in blood monocyte-derived macrophages (BMDM), suggesting the involvement of mannosylated residues of the VMV ENV protein in the process. ConA impaired also syncytium formation in OSF transfected with an ENV-encoding pN3-plasmid. MR transcripts were found in two common SRLV targets, BMDM and synovial membrane (GSM) cells, but not in OSF. Viral infection of BMDM and especially GSM cells was inhibited by mannan, strongly suggesting that in these cells the MR is an important route of infection involving VMV Env mannosylated residues. Thus, at least three patterns of viral entry into SRLV-target cells can be proposed, involving mainly MR in GSM cells (target in SRLV-induced arthritis), MR in addition to an alternative route in BMDM (target in SRLV infections), and an alternative route excluding MR in OSF (target in cell culture). Different routes of SRLV infection may thus coexist related to the involvement of MR differential expression

Mannose receptor may be involved in small ruminant lentivirus pathogenesis

Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV) of known genotype (A or B), and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR) expression (transcript levels) and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints). Control sheep were SRLV-seropositive asymptomatic (n = 3), seronegative (n = 3) or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case). MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively). The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.Funded by grants from CICYT AGL2010-22341-C04-01 and Gobierno de Navarra IIQ14064.RI1. We acknowledge the Public University of Navarra and CSIC for fellowships and the JAE-contract (HC and RR)

Study of compartmentalization in the visna clinical form of small ruminant lentivirus infection in sheep

<p>Abstract</p> <p>Background</p> <p>A central nervous system (CNS) disease outbreak caused by small ruminant lentiviruses (SRLV) has triggered interest in Spain due to the rapid onset of clinical signs and relevant production losses. In a previous study on this outbreak, the role of LTR in tropism was unclear and <it>env </it>encoded sequences, likely involved in tropism, were not investigated. This study aimed to analyze heterogeneity of SRLV Env regions - TM amino terminal and SU V4, C4 and V5 segments - in order to assess virus compartmentalization in CNS.</p> <p>Results</p> <p>Eight Visna (neurologically) affected sheep of the outbreak were used. Of the 350 clones obtained after PCR amplification, 142 corresponded to CNS samples (spinal cord and choroid plexus) and the remaining to mammary gland, blood cells, bronchoalveolar lavage cells and/or lung. The diversity of the <it>env </it>sequences from CNS was 11.1-16.1% between animals and 0.35-11.6% within each animal, except in one animal presenting two sequence types (30% diversity) in the CNS (one grouping with those of the outbreak), indicative of CNS virus sequence heterogeneity. Outbreak sequences were of genotype A, clustering per animal and compartmentalizing in the animal tissues. No CNS specific signature patterns were found.</p> <p>Conclusions</p> <p>Bayesian approach inferences suggested that proviruses from broncoalveolar lavage cells and peripheral blood mononuclear cells represented the common ancestors (infecting viruses) in the animal and that neuroinvasion in the outbreak involved microevolution after initial infection with an A-type strain. This study demonstrates virus compartmentalization in the CNS and other body tissues in sheep presenting the neurological form of SRLV infection.</p

In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [18F]fluoro-deoxyglucose–MicroPET in a mouse model

A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [18F]fluoro-deoxyglucose–MicroPET ([18F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [18F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [18F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches.This work was supported by grants from Gobierno de Navarra “IIM13002.RI1” and MICINN “CIT-010000-2009-32”