Hypoxia and HIF-1 Trigger Marek's Disease Virus Reactivation in Lymphoma-Derived Latently Infected T Lymphocytes.

Latency is a hallmark of herpesviruses, allowing them to persist in their host without virion production. Acute exposure to hypoxia (below 3% O2) was identified as a trigger of latent-to-lytic switch (reactivation) for human oncogenic gammaherpesviruses (Kaposi's sarcoma-associated virus [KSHV] and Epstein-Barr virus [EBV]). Therefore, we hypothesized that hypoxia could also induce reactivation of Marek's disease virus (MDV), which shares biological properties with EBV and KSHV (notably oncogenic properties), in lymphocytes. Acute exposure to hypoxia (1% O2) of two MDV-latently infected cell lines derived from MD tumors (3867K and MSB-1) induced MDV reactivation. A bioinformatic analysis of the RB-1B MDV genome revealed 214 putative hypoxia response element consensus sequences on 119 open reading frames. Reverse transcriptase quantitative PCR (RT-qPCR) analysis showed five MDV genes strongly upregulated early after hypoxia. In 3867K cells under normoxia, pharmacological agents mimicking hypoxia (MLN4924 and CoCl2) increased MDV reactivation, but to a lower level than real hypoxia. Overexpression of wild-type or stabilized human hypoxia inducible factor 1α (HIF-1α) in MSB-1 cells in normoxia also promoted MDV reactivation. Under such conditions, the lytic cycle was detected in cells with a sustainable HIF-1α expression but also in HIF-1α-negative cells, indicating that MDV reactivation is mediated by HIF-1 in a direct and/or indirect manner. Lastly, we demonstrated by a reporter assay that HIF-1α overexpression induced the transactivation of two viral promoters, shown to be upregulated in hypoxia. These results suggest that hypoxia may play a crucial role in the late lytic replication phase observed in vivo in MDV-infected chickens exhibiting tumors, since a hypoxic microenvironment is a hallmark of most solid tumors. IMPORTANCE Latent-to-lytic switch of herpesviruses (also known as reactivation) is responsible for pathology recurrences and/or viral shedding. Studying physiological triggers of reactivation is therefore important for health to limit lesions and viral transmission. Marek's disease virus (MDV) is a potent oncogenic alphaherpesvirus establishing latency in T lymphocytes and causing lethal T lymphomas in chickens. In vivo, a second lytic phase is observed during the tumoral stage. Hypoxia being a hallmark of tumors, we wondered whether hypoxia induces MDV reactivation in latently infected T lymphocytes, like previously shown for EBV and KSHV in B lymphocytes. In this study, we demonstrated that acute hypoxia (1% O2) triggers MDV reactivation in two MDV transformed T-cell lines. We provide some molecular basis of this reactivation by showing that hypoxia inducible factor 1 (HIF-1) overexpression induces MDV reactivation to an extent similar to that of hypoxia after 24 h. Hypoxia is therefore a reactivation stimulus shared by mammalian and avian oncogenic herpesviruses of different genera.

Differentiated Cells in Prolonged Hypoxia Produce Highly Infectious Native-Like Hepatitis C Virus Particles.

BACKGROUND AND AIMS: Standard hepatitis C virus (HCV) cell-culture models present an altered lipid metabolism and thus produce lipid-poor lipoviral particles (LVPs). These models are thereby weakly adapted to explore the complete natural viral life cycle. APPROACH AND RESULTS: To overcome these limitations, we used an HCV cell-culture model based on both cellular differentiation and sustained hypoxia to better mimic the host-cell environment. The long-term exposure of Huh7.5 cells to DMSO and hypoxia (1% O2 ) significantly enhanced the expression of major differentiation markers and the cellular hypoxia adaptive response by contrast with undifferentiated and normoxic (21% O2 ) standard conditions. Because hepatocyte-like differentiation and hypoxia are key regulators of intracellular lipid metabolism, we characterized the distribution of lipid droplets (LDs) and demonstrated that experimental cells significantly accumulate larger and more numerous LDs relative to standard cell-culture conditions. An immunocapture (IC) and transmission electron microscopy (TEM) method showed that differentiated and hypoxic Huh7.5 cells produced lipoproteins significantly larger than those produced by standard Huh7.5 cell cultures. The experimental cell culture model is permissive to HCV-Japanese fulminant hepatitis (JFH1) infection and produces very-low-buoyant-density LVPs that are 6-fold more infectious than LVPs formed by standard JFH1-infected Huh7.5 cells. Finally, the IC-TEM approach and antibody-neutralization experiments revealed that LVPs were highly lipidated, had a global ultrastructure and a conformation of the envelope glycoprotein complex E1E2 close to that of the ones circulating in infected individuals. CONCLUSIONS: This relevant HCV cell culture model thus mimics the complete native intracellular HCV life cycle and, by extension, can be proposed as a model of choice for studies of other hepatotropic viruses.

The envelope protein of Zika virus interacts with apolipoprotein E early in the infectious cycle and this interaction is conserved on the secreted viral particles.

BACKGROUND: Zika virus (ZIKV), a member of the Flaviviridae family, has caused massive outbreaks of infection in tropical areas over the last decade and has now begun spreading to temperate countries. Little is currently known about the specific host factors involved in the intracellular life cycle of ZIKV. Flaviviridae viruses interact closely with host-cell lipid metabolism and associated secretory pathways. Another Flaviviridae, hepatitis C virus, is highly dependent on apolipoprotein E (ApoE) for the completion of its infectious cycle. We therefore investigated whether ZIKV also interacted with this protein. METHODS: ZIKV infections were performed on both liver and microglia derived cell lines in order to proceed to colocalization analysis and immunoprecipitation assays of ApoE and Zika envelope glycoprotein (Zika E). Transmission electron microscopy combined to immunogold labeling was also performed on the infected cells and related supernatant to study the association of ApoE and Zika E protein in the virus-induced membrane rearrangements and secreted particles, respectively. Finally, the potential of neutralization of anti-ApoE antibodies on ZIKV particles was studied. RESULT: We demonstrated an interaction between ApoE and the Zika E protein. This specific interaction was observed in virus-induced host-cell membrane rearrangements, but also on newly formed intracellular particles. The partial neutralizing effect of anti-ApoE antibody and the immunogold labeling of the two proteins on secreted virions indicates that this interaction is conserved during ZIKV intracellular trafficking and release. CONCLUSIONS: These data suggest that another member of the Flaviviridae also interacts with ApoE, indicating that this could be a common mechanism for the viruses from this family.

Potentiation of doxorubicin efficacy in hepatocellular carcinoma by the DNA repair inhibitor DT01 in preclinical models.

OBJECTIVE: This study aimed to explore the antitumour effect of the DNA repair inhibitor, DT01 (the cholesterol conjugated form of Dbait), as an adjunct treatment to enhance the therapeutic efficacy of transarterial chemoembolization (TACE) in pre-clinical models of hepatocellular carcinoma (HCC). METHODS: A rabbit model bearing liver tumours was either left untreated or treated with TACE or with a combination of TACE+DT01. Tumour growth was monitored by ultrasound. These results were further confirmed in mice grafted with an intrahepatic human HCC model treated with doxorubicin (DOX) alone or DOX+DT01. RESULTS: The combination of DT01 with TACE in a rabbit liver model led to a significant decrease in tumour volume (p=0.03). Colour Doppler and immunohistochemical staining revealed a strong decrease in vascularization in the DT01+TACE-treated group preventing the tumour growth restart observed after TACE alone. Similarly, the DT01 combination with DOX led to significant anti-tumour efficacy compared to DOX alone (p=0.02) in the human HCC model. In addition, a significant decrease in vascularization in the group receiving combination DT01 and DOX treatment was observed. CONCLUSIONS: DT01 is well tolerated and may potentiate HCC treatment by enhancing the DNA-damaging and anti-vascularization effect of TACE with doxorubicin. KEY POINTS: • DT01 combined with TACE leads to significant anti-tumour efficacy without additional toxicity. • A potential anti-angiogenic role of DT01 was identified in preclinical models. • DT01 may potentiate HCC treatment by enhancing the efficacy of TACE.

Human hepatic stellate cells are not permissive for hepatitis C virus entry and replication.

BACKGROUND: Chronic HCV infection is associated with the development of hepatic fibrosis. The direct role of HCV in the fibrogenic process is unknown. Specifically, whether HCV is able to infect hepatic stellate cells (HSCs) is debated. OBJECTIVE: To assess whether human HSCs are susceptible to HCV infection. DESIGN: We combined a set of original HCV models, including the infectious genotype 2a JFH1 model (HCVcc), retroviral pseudoparticles expressing the folded HCV genotype 1b envelope glycoproteins (HCVpp) and a subgenomic genotype 1b HCV replicon, and two relevant cellular models, primary human HSCs from different patients and the LX-2 cell line, to assess whether HCV can infect/replicate in HSCs. RESULTS: In contrast with the hepatocyte cell line Huh-7, neither infectious HCVcc nor HCVpp infected primary human HSCs or LX-2 cells. The cellular expression of host cellular factors required for HCV entry was high in Huh-7 cells but low in HSCs and LX-2 cells, with the exception of CD81. Finally, replication of a genotype 2a full-length RNA genome and a genotype 1b subgenomic replicon was impaired in primary human HSCs and LX-2 cells, which expressed low levels of cellular factors known to play a key role in the HCV life-cycle, suggesting that human HSCs are not permissive for HCV replication. CONCLUSIONS: Human HSCs are refractory to HCV infection. Both HCV entry and replication are deficient in these cells, regardless of the HCV genotype and origin of the cells. Thus, HCV infection of HSCs does not play a role in liver fibrosis. These results do not rule out a direct role of HCV infection of hepatocytes in the fibrogenic process.

'Liver let die': oxidative DNA damage and hepatotropic viruses.

Chronic infections by the hepatotropic viruses hepatitis B virus (HBV) and hepatitis C virus (HCV) are major risk factors for the development of hepatocellular carcinoma (HCC). It is estimated that more than 700,000 individuals per year die from HCC, and around 80 % of HCC is attributable to HBV or HCV infection. Despite the clear clinical importance of virus-associated HCC, the underlying molecular mechanisms remain largely elusive. Oxidative stress, in particular DNA lesions associated with oxidative damage, play a major contributory role in carcinogenesis, and are strongly linked to the development of many cancers, including HCC. A large body of evidence demonstrates that both HBV and HCV induce hepatic oxidative stress, with increased oxidative DNA damage being observed both in infected individuals and in murine models of infection. Here, we review the impact of HBV and HCV on the incidence and repair of oxidative DNA damage. We begin by giving a brief overview of oxidative stress and the repair of DNA lesions induced by oxidative stress. We then review in detail the evidence surrounding the mechanisms by which both viruses stimulate oxidative stress, before focusing on how the viral proteins themselves may perturb the cellular response to oxidative DNA damage, impacting upon genome stability and thus hepatocarcinogenesis.

Hepatitis C virus (HCV) protein expression enhances hepatic fibrosis in HCV transgenic mice exposed to a fibrogenic agent.

BACKGROUND & AIMS: During chronic HCV infection, activation of fibrogenesis appears to be principally related to local inflammation. However, the direct role of hepatic HCV protein expression in fibrogenesis remains unknown. METHODS: We used transgenic mice expressing the full length HCV open reading frame exposed to a 'second hit' of the fibrogenic agent carbon tetrachloride (CCl(4)). Both acute and chronic liver injuries were induced in these mice by CCl(4) injections. Liver injury, expression of matrix re-modeling genes, reactive oxygen species (ROS), inflammation, hepatocyte proliferation, ductular reaction and hepatic progenitor cells (HPC) expansion were examined. RESULTS: After CCl(4) treatment, HCV transgenic mice exhibited enhanced liver fibrosis, significant changes in matrix re-modeling genes and increased ROS production compared to wild type littermates despite no differences in the degree of local inflammation. This increase was accompanied by a decrease in hepatocyte proliferation, which appeared to be due to delayed hepatocyte entry into the S phase. A prominent ductular reaction and hepatic progenitor cell compartment expansion were observed in transgenic animals. These observations closely mirror those previously made in HCV-infected individuals. CONCLUSIONS: Together, these results demonstrate that expression of the HCV proteins in hepatocytes contributes to the development of hepatic fibrosis in the presence of other fibrogenic agents. In the presence of CCl(4), HCV transgenic mice display an intra-hepatic re-organization of several key cellular actors in the fibrogenic process.

Morphological changes in intracellular lipid droplets induced by different hepatitis C virus genotype core sequences and relationship with steatosis.

UNLABELLED: Hepatocellular steatosis is common in patients with chronic hepatitis C. Steatosis can be considered as a true cytopathic lesion induced by hepatitis C virus (HCV) genotype 3, suggesting that one or more viral proteins produced during genotype 3 infection are involved in the steatogenic process, while the same proteins produced during infection by other genotypes are not. We examined in vitro interactions between lipid droplets and full-length core protein isolated from patients with HCV genotype 3a infection, with and without steatosis, and from steatosis-free patients infected by HCV genotype 1b. We also examined morphological changes in the lipid droplets according to the HCV genotype and the presence of steatosis in vivo. Core protein processing by signal peptide peptidase was not affected by sequence differences between the variants. We showed that the core protein of both HCV genotypes 1b and 3a binds tightly to the surface of intracellular lipid droplets. However, cells transfected with genotype 3a contain more neutral lipids in lipid droplets, and more large lipid droplets, than cells transfected with genotype 1b sequences. This suggests that HCV core protein-lipid droplet interaction could play a role in virus-induced steatosis. Importantly, we found no genetic or functional differences between genotype 3a core proteins from patients with and without HCV-induced steatosis. CONCLUSION: This suggests that other viral proteins and/or host factors are involved in the development of hepatocellular steatosis in patients infected by HCV genotype 3a.

Influences of the season and the habitat structure on the foraging ecology of two coua species in the western dry forest of Madagascar.

I studied the foraging ecology of Coquerel's Coua (Coua coquereli) and Giant Coua (Coua gigas), which occur in the dry forest in west Madagascar. This kind of forest is characterised by an alternating of a dry and a rainy season. The foraging behaviour was described in several dimensions: i.e. height and proportion of perching, rate of capture (estimating the food availability), foraging techniques, substrates used, type and size of the captured prey. Their foraging behaviour was different according to the season and to the proximity of water. Habitat structure was important to take in consideration to study their foraging behaviour. They tended to use the same pattern of techniques and substrates, but differed by the proportions they used these variables and also by the possibility to climb into the dense understorey vegetation. Seasonal variation has probably an important effect on the prey availability and the nature of prey captured. The diet of both species is also discussed. I suggest that change in habitat structure and resources levels could be important to take in consideration for the conservation of these forest birds.

The impacts of logging on the microhabitats used by two species of couas in the western forest of Madagascar.

Habitat structure is important to consider in all ecological studies considering the relationships between animals and their environment. Habitat structure can be studied at different scales, from landscape to microhabitat. I studied here the response of two endemic terrestrial birds living in the dry forest of Madagascar. These birds belong to the genus Coua. The study is made at the microhabitat scale in a gallery forest, which has been logged selectively in order to limit the degradation of the forest. Selective logging is promoted to be respectful of the environment by allowing us to exploit the forest without destroying it and the wildlife encountered here. At the microhabitat scale, I underline that selective logging does not affect the Coquerel's coua, which can exploit new microhabitats and increase its density. On the other hand, the giant coua was affected by the restriction of optimal microhabitats for foraging. This species could be adapted to the new habitat by modifying its favourite foraging sites, but by decreasing also the population density. This species was affected by forest degradation, even considered as not destroying. At last, I considered how Coquerel's coua could be used as umbrella species for the endangered mesite.

Habitat selection and density of couas in Madagascar: implication for their conservation.

The forests in Madagascar are threatened by logging and burning. Because of their importance for biodiversity conservation, monitoring their animal populations is also important. We worked at two stations in the western dry forest where we studied three species of terrestrial couas in several forest plots, which differed in their degradation state. Some were burnt; others were logged. By measuring vegetation characteristics and bird densities, it was possible to indicate which characteristics were important for these birds and which conservation measures would be necessary to apply for this forest.

Local extinction in the bird assemblage in the greater Beijing area from 1877 to 2006.

Recent growth in industrialization and the modernization of agricultural activities, combined with human population growth, has greatly modified China's natural environment, particularly in the vicinity of large cities. We compared avifauna checklists made between 1877 and 1938 with current checklists to determine the extent of local bird extinctions during the last century in the greater Beijing area. Our study shows that of the 411 bird species recorded from 1877-1938, 45 (10.9%) were no longer recorded from 2004-2006. Birds recorded as 'rare' in 1938 were more likely to have disappeared in subsequent years. Migrant status also influenced the probability of local bird extinction with winter migrants being the most affected class. Moreover, larger birds were more likely to have disappeared than smaller ones, potentially explained by differential ecological requirements and anthropogenic exploitation. Although our habitat descriptions and diet classification were not predictors of local bird extinction, the ecological processes driving local bird extinction are discussed in the light of historical changes that have impacted this region since the end of the 1930 s. Our results are of importance to the broader conservation of bird wildlife.

Impact of logging on the foraging behaviour of two sympatric species of Couas (Coua coquereli and Coua gigas) in the western dry forest of Madagascar.

Two ground-dwelling couas species, Coquerel's Coua Coua coquereli and Giant Coua Coua gigas, live in sympatry in the dry forest of Madagascar. These birds are typically insectivorous and mainly feed at ground level. The two species differ by size but have the same morphology, suggesting they have the same physical attributes for foraging and prey capture. To test if the two species have the same foraging behaviour, and also to know how habitat disturbance due to logging could affect their foraging behaviour, I compared and analysed the foraging strategies of both species in two different dry forest habitats: unlogged and logged. The two species differed in their foraging behaviour between the two habitats, mainly by the ability to climb in the vegetation, and by the technique used by both species. Coquerel's Coua used more often gleaning and probing in the unlogged forest, while Giant Coua used lunge more often in this habitat. The giant Coua used also more often leaves as a substrate in the logged forest. Some modifications in the diet have been recorded too. These results suggest that anthropogenic disturbance of forest does influence the foraging behaviour of the terrestrial couas species living in the dry forest in Madagascar.

The nonstructural 5A protein of hepatitis C virus genotype 1b does not contain an interferon sensitivity-determining region.

BACKGROUND: The nonstructural (NS) 5A protein of hepatitis C virus (HCV) has been suggested to contain an interferon (IFN) sensitivity-determining region (ISDR). METHODS: We studied whether the degree of viral decline on day 1 is associated with differences in NS5A amino acid sequences among patients receiving IFN- alpha. RESULTS: Phylogenetic analyses of the full-length protein and of functional domains showed no relationship between the baseline protein sequence and the antiviral response. NS5A quasispecies sequences showed no differences in the number of mutations in the putative ISDR relative to a prototype sequence between responders and nonresponders or according to IFN- alpha antiviral efficacy. No relationship was found between antiviral efficacy at 24 h and the baseline sequence of any NS5A region. Amino acid changes were observed in a few cases at 24 h in both responders and nonresponders, but no consistent pattern of amino acid shifts was observed, ruling out the possibility that IFN- alpha selected IFN-resistant variants. CONCLUSION: Our findings show that there is no ISDR in the HCV genotype 1 NS5A protein and that the NS5A sequence does not influence the capacity of IFN- alpha to block viral replication. The findings do not rule out a role for NS5A in subsequent viral clearance.

Hepatitis C virus-induced hepatocellular steatosis.

Two distinct forms of hepatocellular steatosis can be seen in patients with chronic hepatitis C virus (HCV) infection. Classical metabolic risk factors for hepatocellular steatosis account for the vast majority of cases of steatosis in patients infected by non-genotype 3 HCV strains. In contrast, in patients infected by HCV genotype 3, steatosis is generally induced by the virus itself through a direct cytopathic effect, the mechanisms of which remain debated. Mixed forms of steatosis can also be seen in HCV genotype 3-infected patients with metabolic risk factors. Hepatocellular steatosis appears to be associated with more rapid progression of hepatic fibrosis. However, it is unclear whether this association is due to steatosis itself, or rather to metabolic and host factors that promote steatosis and fibrosis concomitantly. This review discusses current knowledge of HCV-induced steatosis and its relation to chronic HCV-associated liver disease.