Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence.

Infection of young foals with Equine Parvovirus-Hepatitis following a fatal non-biologic case of Theiler's disease.

Theiler's disease (TD) is a (sub-)acute hepatitis in adult horses and one of the most common causes of acute hepatic failure. Recent findings indicate that equine parvovirus hepatitis (EqPV-H) likely causes TD and that its transmission occurs via iatrogenic and/or natural routes. Following the death of an EqPV-H positive mare with TD, close-contact mares and foals in the same paddock were monitored to evaluate if there was any evidence of EqPV-H. For this purpose, the serum of close contact horses was examined 6 and 42 days after the mare's death for the presence of EqPV-H DNA and changes in liver-associated serum biochemical parameters. The foals had higher EqPV-H viral loads than the mares. Apart from the mare that was euthanized, none of the horses included in this study showed signs of severe disease and nor did they have particularly elevated liver enzymes. Nucleotide sequence analysis revealed no major differences between the viral DNA detected in the serum of the dead mare and any of the in-contact horses. In conclusion, our data confirmed previous findings that horizontal transmission of EqPV-H may occur through close contact between horses.

Dose-Dependent Hepacivirus Infection Reveals Linkage between Infectious Dose and Immune Response.

More than 70 million people worldwide are still infected with the hepatitis C virus 30 years after its discovery, underscoring the need for a vaccine. To develop an effective prophylactic vaccine, detailed knowledge of the correlates of protection and an immunocompetent surrogate model are needed. In this study, we describe the minimum dose required for robust equine hepacivirus (EqHV) infection in equids and examined how this relates to duration of infection, seroconversion, and transcriptomic responses. To investigate mechanisms of hepaciviral persistence, immune response, and immune-mediated pathology, we inoculated eight EqHV naive horses with doses ranging from 1-2 copies to 1.3 × 106 RNA copies per inoculation. We characterized infection kinetics, pathology, and transcriptomic responses via next generation sequencing. The minimal infectious dose of EqHV in horses was estimated at 13 RNA copies, whereas 6 to 7 copies were insufficient to cause infection. Peak viremia did not correlate with infectious dose, while seroconversion and duration of infection appeared to be affected. Notably, seroconversion was undetectable in the low-dose infections within the surveillance period (40 to 50 days). In addition, transcriptomic analysis revealed a nearly dose-dependent effect, with greater immune activation and inflammatory response observed in high-dose infections than in low-dose infections. Interestingly, inoculation with 6-7 copies of RNA that did not result in productive infection, but was associated with a strong immune response, similar to that observed in the high-dose infections. IMPORTANCE We demonstrate that the EqHV dose of infection plays an important role for inducing immune responses, possibly linked to early clearance in high-dose and prolonged viremia in low-dose infections. In particular, pathways associated with innate and adaptive immune responses, as well as inflammatory responses, were more strongly upregulated in high-dose infections than in lower doses. Hence, inoculation with low doses may enable EqHV to evade strong immune responses in the early phase and therefore promote robust, long-lasting infection.

Experimental cross-species infection of donkeys with equine hepacivirus and analysis of host immune signatures.

BACKGROUND: The Equine Hepacivirus (EqHV) is an equine-specific and liver-tropic virus belonging to the diverse genus of Hepaciviruses. It was recently found in a large donkey (Equus asinus) cohort with a similar seroprevalence (30%), but lower rate of RNA-positive animals (0.3%) compared to horses. These rare infection events indicate either a lack of adaptation to the new host or a predominantly acute course of infection. METHODS: In order to analyze the susceptibility and the course of EqHV infection in donkeys, we inoculated two adult female donkeys and one control horse intravenously with purified EqHV from a naturally infected horse. Liver biopsies were taken before and after inoculation to study changes in the transcriptome. RESULTS: Infection kinetics were similar between the equids. All animals were EqHV PCR-positive from day three. EqHV RNA-levels declined when the animals seroconverted and both donkeys cleared the virus from the blood by week 12. Infection did not have an impact on the clinical findings and no significant histopathological differences were seen. Blood biochemistry revealed a mild increase in GLDH at the time of seroconversion in horses, which was less pronounced in donkeys. Transcriptomic analysis revealed a distinct set of differentially expressed genes, including viral host factors and immune genes. CONCLUSION: To summarize, our findings indicate that donkeys are a natural host of EqHV, due to the almost identical infection kinetics. The different immune responses do however suggest different mechanisms in reacting to hepaciviral infections.

The Human Liver-Expressed Lectin CD302 Restricts Hepatitis C Virus Infection.

C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.

Clinical Course of Infection and Cross-Species Detection of Equine Parvovirus-Hepatitis.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler's disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler's disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013-2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013-2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.