Expanding the host range of hepatitis C virus through viral adaptation

Hepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytes in vivo and in vitro Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytes in vivo in the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV's ability to enter murine hepatocytes in vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C. IMPORTANCE: At least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV

Inactivation and survival of hepatitis C virus on inanimate surfaces.

Hepatitis C virus (HCV) cross-contamination from inanimate surfaces or objects has been implicated in transmission of HCV in health-care settings and among injection drug users. We established HCV-based carrier and drug transmission assays that simulate practical conditions to study inactivation and survival of HCV on inanimate surfaces

Thermostability of seven hepatitis C virus genotypes in vitro and in vivo

Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2days was observed at 37 degrees C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures

Different features of Vδ2 T and NK cells in fatal and non-fatal human Ebola infections

Background: Human Ebola infection is characterized by a paralysis of the immune system. A signature of αβ T cells in fatal Ebola infection has been recently proposed, while the involvement of innate immune cells in the protection/pathogenesis of Ebola infection is unknown. Aim of this study was to analyze γδ T and NK cells in patients from the Ebola outbreak of 2014–2015 occurred in West Africa, and to assess their association with the clinical outcome. Methodology/Principal findings: Nineteen Ebola-infected patients were enrolled at the time of admission to the Ebola Treatment Centre in Guinea. Patients were divided in two groups on the basis of the clinical outcome. The analysis was performed by using multiparametric flow cytometry established by the European Mobile Laboratory in the field. A low frequency of Vδ2 T-cells was observed during Ebola infection, independently from the clinical outcome. Moreover, Vδ2 T-cells from Ebola patients massively expressed CD95 apoptotic marker, suggesting the involvement of apoptotic mechanisms in Vδ2 T-cell loss. Interestingly, Vδ2 T-cells from survivors expressed an effector phenotype and presented a lower expression of the CTLA-4 exhaustion marker than fatalities, suggesting a role of effector Vδ2 T-cells in the protection. Furthermore, patients with fatal Ebola infection were characterized by a lower NK cell frequency than patients with non fatal infection. In particular, both CD56bright and CD56dim NK frequency were very low both in fatal and non fatal infections, while a higher frequency of CD56neg NK cells was associated to non-fatal infections. Finally, NK activation and expression of NKp46 and CD158a were independent from clinical outcome. Conclusions/Significances: Altogether, the data suggest that both effector Vδ2 T-cells and NK cells may play a role in the complex network of protective response to EBOV infection. Further studies are required to characterize the protective effector functions of Vδ2 and NK cells

Ion-channel function and cross-species determinants in viral assembly of nonprimate hepacivirus p7

Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though both viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 by using various NMR structures were generated highlighting conserved residues important for ion-channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited similar liposome permeability features than HCV p7 indicative of similar ion-channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar sub-cellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N-terminus and transmembrane domains were non-exchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C-terminus of NPHV p7 were readily exchangeable allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins including liposome permeability and broadly acting determinants were identified which modulate hepaciviral virion assembly and contribute to the host-species barrier

Clinical course of infection and viral tissue tropism of hepatitis C virus-like nonprimate hepaciviruses in horses

Hepatitis C virus (HCV) has a very narrow species and tissue tropism and efficiently replicates only in humans and the chimpanzee. Recently, several studies identified close relatives to HCV in different animal species. Among these novel viruses, the nonprimate hepaciviruses (NPHV) that infect horses are the closest relatives of HCV described to date. In this study, we analyzed the NPHV prevalence in northern Germany and characterized the clinical course of infection and viral tissue tropism to explore the relevance of HCV-related horse viruses as a model for HCV infection. We found that approximately 31.4% of 433 horses were seropositive for antibodies (Abs) against NPHV and approximately 2.5% carried viral RNA. Liver function analyses revealed no indication for hepatic impairment in 7 of 11 horses. However, serum gamma-glutamyl transferase (GGT) concentrations were mildly elevated in 3 horses, and 1 horse displayed even highly elevated GGT levels. Furthermore, we observed that NPHV infection could be cleared in individual horses with a simultaneous emergence of nonstructural (NS)3-specific Abs and transient elevation of serum levels of liver-specific enzymes indicative for a hepatic inflammation. In other individual horses, chronic infections could be observed with the copresence of viral RNA and NS3-specific Abs for over 6 months. For the determination of viral tissue tropism, we analyzed different organs and tissues of 1 NPHV-positive horse using quantitative real-time polymerase chain reaction and fluorescent in situ hydridization and detected NPHV RNA mainly in the liver and at lower amounts in other organs.; Similar to HCV infections in humans, this work demonstrates acute and chronic stages of NPHV infection in horses with viral RNA detectable predominantly within the liver