With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.

BackgroundVector arthropods control arbovirus replication and spread through antiviral innate immune responses including RNA interference (RNAi) pathways. Arbovirus infections have been shown to induce the exogenous small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) pathways, but direct antiviral activity by these host responses in mosquito cells has only been demonstrated against a limited number of positive-strand RNA arboviruses. For bunyaviruses in general, the relative contribution of small RNA pathways in antiviral defences is unknown.Methodology/Principal FindingsThe genus Orthobunyavirus in the Bunyaviridae family harbours a diverse range of mosquito-, midge- and tick-borne arboviruses. We hypothesized that differences in the antiviral RNAi response in vector versus non-vector cells may exist and that could influence viral host range. Using Aedes aegypti-derived mosquito cells, mosquito-borne orthobunyaviruses and midge-borne orthobunyaviruses we showed that bunyavirus infection commonly induced the production of small RNAs and the effects of the small RNA pathways on individual viruses differ in specific vector-arbovirus interactions.Conclusions/SignificanceThese findings have important implications for our understanding of antiviral RNAi pathways and orthobunyavirus-vector interactions and tropism.

SummaryHumanized mice are increasingly appreciated as an incredibly powerful platform for infectious disease research. The often very narrow species tropism of many viral infections, coupled with the sometimes misleading results from preclinical studies in animal models further emphasize the need for more predictive model systems based on human cells rather than surrogates. Humanized mice represent such a model and have been greatly enhanced with regards to their immune system reconstitution as well as immune functionality in the past years, resulting in their recommendation as a preclinical model by the US Food and Drug Administration. This review aims to give a detailed summary of the generation of human peripheral blood lymphocyte‐, CD34+ haematopoietic stem cell‐ and bone marrow/liver/thymus‐reconstituted mice and available improved models (e.g. myeloid‐ or T‐cell‐only mice, MISTRG, NSG‐SGM3). Additionally, we summarize human‐tropic viral infections, for which humanized mice offer a novel approach for the study of disease pathogenesis as well as future perspectives for their use in biomedical, drug and vaccine research.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.

Zika virus (ZIKV) Infection has several outcomes from asymptomatic exposure to rash, conjunctivitis, Guillain-Barré syndrome or congenital Zika syndrome. Analysis of ZIKV immunity is confounded by the fact that several related Flaviviruses infect humans, including Dengue virus 1–4, West Nile virus and Yellow Fever virus. HLA class II restricted T cell cross-reactivity between ZIKV and other Flaviviruses infection(s) or vaccination may contribute to protection or to enhanced immunopathology. We mapped immunodominant, HLA class II restricted, CD4 epitopes from ZIKV Envelope (Env), and Non-structural (NS) NS1, NS3 and NS5 antigens in HLA class II transgenic mice. In several cases, ZIKV primed CD4 cells responded to homologous sequences from other viruses, including DENV1–4, WNV or YFV. However, cross-reactive responses could confer immune deviation - the response to the Env DENV4 p1 epitope in HLA-DR1 resulted in IL-17A immunity, often associated with exacerbated immunopathogenesis. This conservation of recognition across Flaviviruses, may encompass protective and/or pathogenic components and poses challenges to characterization of ZIKV protective immunity.