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.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liverresident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the setup of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis. Video Link The video component of this article can be found at https://www.jove.com/video/58333/ 10,11. This offers the unique opportunity to evaluate cell/cell interactions in a complex 3D microenvironment. Additionally, the extended culture period of this platform facilitates the evaluation of sequential drug treatments and their impact on HBV persistence, which are not possible using conventional hepatocyte culture systems. This protocol describes how 3D liver-on-a-chip cultures are generated, either for monocultures of PHH or for cocultures of PHH with Kupffer cells. Furthermore, we describe the production of purified HBV for low-multiplicity-of-infection studies, as well as the subsequent analysis of host and viral responses.

Summary Traumatic injury initiates a large and complex immune response in the minutes after the initial insult, comprising of simultaneous pro‐ and anti‐inflammatory responses. In patients that survive the initial injury, these immune responses are believed to contribute towards complications such as the development of sepsis and multiple organ dysfunction syndrome. These post‐traumatic complications affect a significant proportion of patients and are a major contributing factor for poor outcomes and an increased burden on healthcare systems. Therefore, understanding the immune responses to trauma is crucial for improving patient outcomes through the development of novel therapeutics and refining resuscitation strategies. In order to do this, preclinical animal models must mimic human immune responses as much as possible, and as such, we need to understand the constraints of each species in the context of trauma. A number of species have been used in this field; however, these models are limited by their genetic background and their capacity for recapitulating human immune function. This review provides a brief overview of the immune response in critically injured human patients and discusses the most commonly used species for modelling trauma, focusing on how their immune response to serious injury and haemorrhage compares to that of humans.

HCV vaccine development is stymied by the high genetic diversity of the virus and the variability of the envelope glycoproteins. One strategy to overcome this is to identify conserved, functionally important regions—such as the epitopes of broadly neutralizing antibodies (bNAbs)—and use these as a basis for structure-based vaccine design. Here, we report an anti-idiotype approach that has generated an antibody that mimics a highly conserved neutralizing epitope on HCV E2. Crucially, a mutagenesis screen was used to identify the antibody, designated B2.1 A, whose binding characteristics to the bNAb AP33 closely resemble those of the original antigen. Protein crystallography confirmed that B2.1 A is a structural mimic of the AP33 epitope. When used as an immunogen B2.1 A induced antibodies that recognized the same epitope and E2 residues as AP33 and most importantly protected against HCV challenge in a mouse model.