Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL28B in hepatitis C virus-associated steatosis.

BACKGROUND & AIMS: Steatosis is a prominent feature of hepatitis C, especially in patients infected with genotype 3. The analysis of genetic polymorphisms influencing steatosis in chronic hepatitis C has been limited by the studies' small sample size, and important single nucleotide polymorphisms (SNPs), such as those in the patatin-like phospholipase family 3 protein (PNPLA3), were never evaluated. METHODS: We analyzed the role of SNPs, from 19 systematically selected candidate genes, on steatosis in 626 Caucasian hepatitis C virus (HCV) infected patients. SNPs were extracted from a genome-wide association-generated dataset. Associations of alleles with the presence and/or different severity of steatosis were evaluated by univariate and multivariate logistic regression, accounting for all relevant covariates. RESULTS: The risk of steatosis was increased by carriage of I148M in PNPLA3, but only in patients with HCV genotypes non-3 (odds ratio [OR]=1.9, 95% confidence interval [CI]=1.6-2.3, p<0.001) and similar, albeit weaker associations were found for SNPs in peroxisome proliferator-activated receptor-γ (PPARG) and interleukin-28B (IL28B). Carriage of a SNP in the microsomal triglyceride transfer protein (MTTP) increased the risk of steatosis, but only in patients with HCV genotype 3 (rs1800803, OR=3.4, 95% CI=2.4-4.9, p=0.001). CONCLUSIONS: The rs738409 SNP in PNPLA3 is associated with an increased risk of steatosis in patients infected with HCV genotypes non-3. Host genes affect steatosis depending on the infecting HCV genotype, suggesting their interaction with viral factors.

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.

Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNbeta-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNbeta and IFNbeta-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1beta. Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.

Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

Expression of the mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) in tobacco leaves leads to phosphate export.

Phosphate homeostasis in multicellular eukaryotes depends on both phosphate influx and efflux. The mammalian Xenotropic Polytropic Virus Receptor 1 (XPR1) shares homology to the Arabidopsis PHO1, a phosphate exporter expressed in roots. However, phosphate export activity of XPR1 has not yet been demonstrated in a heterologous system. Here, wedemonstrate that transient expression in tobacco leaves of XPR1-GFP leads to specific phosphate export. Like PHO1-GFP, XPR1-GFP is localized predominantly to the endomembrane system in tobacco cells. These results show that tobacco leaves are a good heterologous system to study the transport activity of members of the PHO1/XPR1 family.

Long-lasting stem cell-like memory CD8+ T cells with a naïve-like profile upon yellow fever vaccination.

Efficient and persisting immune memory is essential for long-term protection from infectious and malignant diseases. The yellow fever (YF) vaccine is a live attenuated virus that mediates lifelong protection, with recent studies showing that the CD8(+) T cell response is particularly robust. Yet, limited data exist regarding the long-term CD8(+) T cell response, with no studies beyond 5 years after vaccination. We investigated 41 vaccinees, spanning 0.27 to 35 years after vaccination. YF-specific CD8(+) T cells were readily detected in almost all donors (38 of 41), with frequencies decreasing with time. As previously described, effector cells dominated the response early after vaccination. We detected a population of naïve-like YF-specific CD8(+) T cells that was stably maintained for more than 25 years and was capable of self-renewal ex vivo. In-depth analyses of markers and genome-wide mRNA profiling showed that naïve-like YF-specific CD8(+) T cells in vaccinees (i) were distinct from genuine naïve cells in unvaccinated donors, (ii) resembled the recently described stem cell-like memory subset (Tscm), and (iii) among all differentiated subsets, had profiles closest to naïve cells. Our findings reveal that CD8(+) Tscm are efficiently induced by a vaccine in humans, persist for decades, and preserve a naïveness-like profile. These data support YF vaccination as an optimal mechanistic model for the study of long-lasting memory CD8(+) T cells in humans.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED: Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION: Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity. (Hepatology 2015;62:1375-1387).

Measles virus infection – Interplay between virus and host cells

Measles, caused by measles virus (MV), is a highly contagious viral disease causing severe respiratory infection and a typical rash. Despite the availability of a protective vaccine, measles is still the leading vaccine-preventable cause of childhood mortality worldwide. The high mortality associated with the disease is mainly due to an increased susceptibility to secondary infections during the period of immunosuppression that continues for several weeks after recovery. The present study was undertaken to elucidate the role of cytoskeletal components in the regulation of MV infection. The most interesting finding was that MV replication was activated in unstimulated peripheral blood mononuclear cells (PBMC) when globular actin was converted into the filamentous form with jasplakinolide. This provides a new aspect in our understanding of MV infection in PBMC. In the second part of the thesis we investigated MV-induced structural changes of cellular nuclear matrix, which is a proteinaceous framework of the nucleus similar to the cytoskeleton in the cytoplasm. We showed that cleavage of nuclear markers was virusspecific and a general caspase inhibitor rescued MV-infected cells from cell death. Furthermore, we studied MV-induced innate immune mechanisms in lung epithelial and endothelial cells. Our results showed that MV infection resulted in activation of the double stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and toll-like receptor 3 (TLR3) gene expression, followed by high expression of antiviral cytokine mRNA.

Alzheimer's disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression.

BACKGROUND: Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing. RESULTS: The human APP, βCTF and Aβ42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy ((1)H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks. CONCLUSIONS: Altogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, βCTF and Aβ42/Aβ40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.

The VZV/IE63-specific T cell response prevents herpes zoster in fingolimod-treated patients.

OBJECTIVE: To assess longitudinally the antiviral immune response of T cells from patients with multiple sclerosis (MS) treated with fingolimod (FTY) vs other disease-modifying treatments (DMTs). METHODS: We assessed cellular immune responses specific to influenza virus (FLU), JC virus (JCV), and varicella-zoster virus (VZV) using quantification of interferon-γ secretion by enzyme-linked immunospot in patients with MS on FTY (n = 31), including 2 with herpes zoster (HZ), natalizumab (n = 11), and other DMTs (n = 11). We used viral lysates for FLU and VZV and a pool of peptides for FLU, JCV (VP-1), and VZV (IE63). RESULTS: Besides an expected drop of T cells, we found that, proportionally to the number of CD3(+) T cells, only FTY-treated patients with MS exhibited an increased VZV/IE63-specific T cell response peaking 6 months into treatment, a response that returned to baseline after 12 and 24 months. Two FTY-treated patients developed an HZ 6 months into treatment, coinciding with an absent VZV/IE63-specific T cell response. However, cellular immune responses specific to VZV lysate, JCV, and FLU (lysate and pool of peptide epitopes) were similar between all 3 categories (FTY, natalizumab, and other DMTs) of study patients. CONCLUSIONS: FTY-treated patients with MS exhibit an increased VZV/IE63-specific cellular immune response after 6 months of treatment. FTY-treated patients who develop an HZ are not able to mount such a response, suggesting that a T cell response directed against this viral protein may be key in preventing the occurrence of HZ.

The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

UNLABELLED: Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivityin vitro Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection ofTmprss2knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion ofTmprss4alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2(-/-)Tmprss4(-/-)double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virusin vivo IMPORTANCE: Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously thatTmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes,Tmprss2andTmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza virusesin vivo.

Cytopathology of callus cells infected with grapevine leafroll-associated virus 3

The cytopathology of grapevine (Vitis spp.) callus tissue infected with Grapevine leafroll-associated virus 3 (GLRaV-3), genus Vitivirus was studied in order to investigate the usefulness of callus cultures to study grapevine leafroll-associated viruses. Ultrathin sections were made from in vitro callus obtained from stems and shoots of GLRaV-3 infected grapevine plants. Callus was composed of two types of tissue. Translucent, soft callus was formed and composed of large loosely arranged cells, containing big vacuoles and a thin layer of cytoplasm. Other parts of the callus were brown-coloured and composed of small compactly arranged cells, which showed flexuous and rod-shaped closterovirus-like particles, with 10-12 nm in diameter, at higher magnifications. Groups of vesicles formed by a single membrane were also observed, with sizes ranging from 50-200 nm, containing fine fibrillar material, also typical of closterovirus infections. Virus concentration was monitored by Immunosorbent electron microscopy (ISEM) tests, which showed that in vitro culture of callus tissue from grapevine infected plants, could be used to study the GLRaV viruses through many successive generations, despite the decline in virus concentration after repeated transfers. No virus particles were observed in callus tissue obtained from healthy grapevines.

An isolate of Apple stem grooving virus associated with Cleopatra mandarin fruit intumescence

A citrus tatter leaf isolate (CTLV-Cl) of Apple stem grooving virus (ASGV) has been found to be associated with a fruit rind intumescence in Cleopatra mandarin (Citrus reshni) in Limeira (SP). The CTLV-Cl was mechanically transmitted to the main experimental herbaceous hosts of CTLV. Chenopodium quinoa and C. amaranticolor reacted with local lesions and systemic symptoms while other test plants reacted somewhat differently than what is reported for CTLV. A pair of primers designed for specific detection of ASGV and CTLV amplified the expected 801 bp fragment from the CTLV-Cl-infected plants. Typical capillovirus-like particles were observed by the electron microscope in experimentally infected C. quinoa and C. amaranticolor leaves.

Tomato chlorotic spot virus in hydroponically-grown lettuce in São Paulo State, Brazil

In the regions of Campinas and Sumaré, São Paulo, Brazil, hidroponically grown crops of Lettuce (Lactuca sativa) cv. Verônica, which showed virus-like symptoms were examined by electron microscope, biological, serological and molecular tests. Pleomorphic, enveloped particles (80-100 nm in diameter) were always detected in these samples. Experimentally inoculated host plants, including lettuce, reacted with tospoviruses-induced symptoms. Some differences were observed in Gomphrena globosa, which reacted by showing local lesions and systemic mosaic. Two isolates of Tomato chlorotic spot virus (TCSV) were identified by DAS-ELISA and by RT-PCR. The sequencing and alignment of the RT-PCR coat protein amplified fragments have indicated a high degree of homology with the TCSV sequences stored in the GenBank. This is the first report of losses due to a virus from the genus Tospovirus in commercial hydroponic lettuce crops in Brazil. Further epidemiological studies are needed for better understanding the spread of the virus in hydroponic crops, since Tomato spotted wilt virus (TSWV) is reported to spread through the nutritive solution.