Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day −55 and −27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day −70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

(1) Background: Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses-and dogs in some instances-and is closely related to hepatitis C virus (HCV) that has infected up to 3% of the world's human population, causing an epidemic of liver cirrhosis and cancer. EqHV also chronically infects the liver of horses, but does not appear to cause serious liver damages. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. (2) Methods: In this retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. (3) Results: Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. (4) Conclusions: As seemingly benign viruses could turn deadly (e.g., Zika flavivirus) and EqHV happens to have infected a significant proportion of the world's horse herds, EqHV infectious cycle should be further clarified.

Like hepatitis C virus (HCV) in humans, the newly identified equine hepacivirus (NPHV) displays a predominating liver tropism that may evolve into chronic infections. The genomes of the two viruses share several organizational and functional features and are phylogenetically closest amongst the Hepacivirus genus. A limited amount of data is available regarding the spread of hepacivirus infections in horses. In this study, we asked whether in a more representative sample the prevalence and distribution of NPHV infections in France would resemble that reported so far in other countries. A total of 1033 horses sera from stud farms throughout France were analysed by qRT-PCR to determine the prevalence of ongoing NPHV infections and viral loads; in positive samples, partial sequences of NPHV's genome (5'UTR, NS3 and NS5B genes) were determined. Serum concentrations of biliary acids, glutamate dehydrogenase (GLDH) and L-gamma-glutamyl transferase (γ-GT) were measured for most horses. We detected NPHV infections in 6.2% of the horses, a prevalence that reached 8.3% in thoroughbreds and was significantly higher than in other breeds. The presence of circulating virus was neither significantly associated with biological disturbances nor with clinical hepatic impairment. Our phylogenetic analysis was based on both neighbour-joining and maximum-likelihood approaches. Its result shows that, like almost everywhere else in the world so far, two major groups of NPHV strains infect French domestic horses. Based on genetic distances, we propose a classification into two separate NPHV subtypes. Viral loads in the serum of horses infected by the main subtype were, in average, four times higher than in those infected by the second subtype. We hypothesize that amino acid substitutions in the palm domain of NS5B between NPHV subtypes could underlie viral phenotypes that explain this result.

AIM To determine if calnexin (CANX), RAB1 and alpha-tubulin were involved in the production of hepatitis C virus (HCV) particles by baby hamster kidney-West Nile virus (BHK-WNV) cells. METHODS Using a siRNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observed in thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model. RESULTS We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome. CONCLUSION Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.

Studying the immunological processes taking place during the initial steps of acute hepatitis C virus (HCV) infection has been a challenge in patients. Shin et al. have recently reported that delayed induction, not impaired recruitment of specific CD8+ T cells, causes the late onset of acute hepatitis C in chimpanzees (Gastroenterology, 2011). However, further elucidation of the underlying mechanisms is difficult in vivo. We made observations consistent with their conclusions in human liver slices inoculated ex vivo with HCV produced in cell culture (HCVcc). Autologous immune cells were purified from blood and differentially stained prior to their incubation with the slices for 2 hours. A two-photon confocal microscopic analysis revealed that many more stained dendritic and T cells contracted interactions within two-day infected slices than non-inoculated ones (p < 0.001). While in the first instance some dendritic and T cells entered into closer interactions, they never did in the latter case. These results suggest that ex vivo infection of human liver slices with HCVcc may be useful for gaining experimental insight regarding the immunological processes taking place at early steps of HCV infections.

Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i. e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes.

Hepatocellular carcinoma cell lines supporting HCV replication in culture produce infectious particles (cell culture-derived HCV) for only a limited number of strains. Mutations in the viral genome resulting from the combined effects of the error-prone HCV RNA polymerase coupled with selection of the best-adapted viral variants generate a drift in cell culture-derived HCV properties, which can result in a total loss of in vivo infectivity in the chimpanzee model. Other systems for producing HCV or HCV-related particles, some of which bypass HCV RNA replication, have been developed to study the biology of this virus, with limitations of their own. This review briefly analyzes the pros and cons of these in vitro models, focusing on recent advances.

CD4+ and CD8+ T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4+CD8+ double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4highCD8low, were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4lowCD8high and CD4highCD8high DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8high cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver.

Reply: We were highly interested in the study by Kottilil et al.,1 which reported the existence of distinct immunologic imprints in peripheral blood mononuclear cells (PBMCs) of patients chronically monoinfected with hepatitis C virus (HCV) and and those chronically coinfected with HCV/human immunodeficiency virus (HIV), compared to HIV-monoinfected or noninfected individuals. In addition, interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) proinflammatory cytokines were found within a cluster of genes significantly up-regulated only in the group of HCV-monoinfected individuals, and were also measured by enzyme-linked immunosorbent assay in the supernatants of cultured PBMCs. We have had the opportunity to study the PBMCs of five patients monoinfected with HCV and five patients coinfected with HIV/HCV at the acute phase of the HCV infection (<4 months from the date of contamination) and before antiviral treatment. The T cell proliferative response to (HCV) NS3 or (HIV) gag (overlapping 15-unit oligomers), CEF (cytomegalovirus, Epstein-Barr virus, and flu virus) peptide mix or tetanus toxoid (TT) was investigated, and the production of cytokines in response to the same antigens as well as staphylococcus enterotoxin B (SEB) was measured in the supernatants of PBMCs in culture. In the T cell proliferation assay, a response to at least one antigen was observed for five patients: four in the HCV group and one in the HIV/HCV-coinfected group. The latter patient only responded to HIV gag peptide pool, i.e., not to HCV NS3. In the HCV group, one patient responded to NS3, and none responded to gag. Interestingly, the production of IL-8 was already high and not responsive to the antigens; however, patterns were identical in monoinfected and coinfected patients (not shown). At variance with IL-8 (Fig. 1), no TNF-α production was detected without antigenic stimulation. An increased TNF-α production by PBMCs of HCV-monoinfected patients was observed in response to NS3, CEF, TT, and SEB, whereas those of HCV/HIV-coinfected ones only responded to gag, CEF, TT, and SEB, i.e., surprisingly not to NS3. Production of TNF-α by PBMCs isolated from patients at the acute phase of HCV infection. After 6-day stimulation2 with the indicated antigens, TNF-α immunoreactivity was measured in culture supernatants using xMAP technology (Luminex, Austin, TX); error bars represent standard error of the mean. The fact that patients coinfected with HIV/HCV failed to respond to the pool of NS3 peptides whereas patients monoinfected with HCV did was a trend also observed with the production of other cytokines, including interferon-γ, interferon-inducible protein-10/chemokin (C-X-C motif) ligand 10 (CXCL10), macrophage inflammatory protein-1α/chemokine (C-C motif) ligand 3, and MIG/CXCL9 (not shown). For several cytokines, productions in response to other antigens were similar in both groups, which did not support the hypothesis that globally impaired immune responses in HIV/HCV-coinfected patients explained the lack of anti-HCV immune response in vitro. These results outline the discrepancies existing between PBMCs at the acute phase of HCV infection (as in the present results) and its chronic stage,1 in both HCV-monoinfected and HCV/HIV-coinfected patients. They additionally suggest that patients with HIV who more rapidly develop liver complications when coinfected with HCV could be the consequence of a specific defect of their cellular response to HCV antigens at the acute phase of infection. Michelina Nascimbeni*, Thomas Montange , Helen K. W. Law , Vincent Mallet* §, Bertrand Saunier*, Yves Rivière , Stanislas Pol* §, * Hepatitis C Virus Laboratory, Department of Immunology, Institut Cochin, Institut National de la Santé et de la Recherche Meédicale U567, Centre National de la Recherche Scientifique (CNRS) UMR8104, Paris-Descartes University, Paris, France, Laboratory of Viral Immune Pathology, CNRS URA3015, Paris, France, Center of Human Immunology, Department of Immunology, Institut Pasteur, Paris, France, § Hepatology Unit, Department of Hepato-Gastroenterology, Cochin Hospital, Paris, France.

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8alphaalpha homodimers. CD8alphaalpha is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8alphaalpha(+) IELs "snatch" TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8alphaalpha IELs with intestinal cells.

ABSTRACT The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.

L'invention concerne des procedes permettant d'obtenir des complexes du virus de l'hepatite C (HCV) et des particules de type HCV renfermant des genes structurels du HCV. Dans un procede, des cellules renfermant des particules de type HCV sont lysees avec de la digitonine, en presence d'inhibiteurs de la protease. Du polyethylene glycol est ajoute lentement au lysat, de maniere a fournir un precipite comprenant des complexes des proteines structurelles HCV associees a des vesicules ou micelles lipidiques et des complexes renfermant des proteines structurelles virales sous la forme d'agregats insolubles. Dans un autre procede, le lysat est centrifuge dans un coussin de sucrose. Le culot est, de preference, soumis ensuite a une ultracentrifugation a equilibre, de maniere a fournir une preparation de particules de type HCV de dimensions heterogenes. Le troisieme procede consiste a soumettre les cellules infectees a un choc hypertonique/hypotonique et a lyser les cellules avec de la digitonine, en presence d'inhibiteurs de la protease. Le lysat est agglomere et fractionne de maniere a fournir une population de particules de type HCV sensiblement homogenes et possedant un diametre moyen d'environ 50 nm. On entend par « sensiblement homogenes » que la forme des particules est similaire et les dimensions des particules varient d'au maximum 10 %. L'invention concerne enfin des procedes d'utilisation des complexes HCV et des particules de type HCV comme outils de criblage et de diagnostic, des compositions immunogeniques, ainsi que des procedes de traitement de patients presentant des symptomes d'une infection au HCV, au moyen de composes ou de substances interferant avec la liaison ou l'internalisation des particules de type HCV selon l'invention dans des recepteurs de l'asialoglycoproteine.

Hepatitis C virus-like particles (HCV-LPs) containing the structural proteins of HCV H77 strain (1a genotype) was used as a model for HCV virion to study virus-cell interaction. HCV-LPs showed a buoyant density of 1.17 to 1.22 g/cm(3) in a sucrose gradient and formed double-shelled particles 35 to 49 nm in diameter. Flow cytometry analysis by an indirect method (detection with anti-E2 antibody) and a direct method (use of dye-labeled HCV-LPs) showed that HCV-LPs binds to several human hepatic (primary hepatocytes, HepG2, HuH7, and NKNT-3) and T-cell (Molt-4) lines. HCV-LPs binding to cells occurred in a dose- and calcium-dependent manner and was not mediated by CD81. Scatchard plot analysis suggests the presence of two binding sites for HCV-LPs with high (K(d) approximately 1 microg/ml) and low (K(d) approximately 50 to 60 microg/ml) affinities of binding. Anti-E1 and -E2 antibodies inhibited HCV-LPs binding to cells. While preincubation of HCV-LPs with very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), or high-density lipoprotein (HDL) blocked its binding to cells, preincubation of cells with VLDL, LDL, HDL, or anti-LDL-R antibody did not. Confocal microscopy analysis showed that, after binding to cells, dye-labeled HCV-LPs were internalized into the cytoplasm. This process could be inhibited with anti-E1 or anti-E2 antibodies, suggesting that E1 and E2 proteins mediate HCV-LPs binding and, subsequently, their entry into cells. Altogether, our results indicate that HCV-LPs can be used to further characterize the mechanisms involved in the early steps of HCV infection.