Masson Pine pollen (Pinus massoniana) activate HD11 chicken macrophages invitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Masson Pine pollen (Pinus massoniana; MP) are used in Traditional Chinese Medicine to treat gut conditions. Early in vivo work supports this claim and suggests interaction of the material with the gastrointestinal immune system. AIM OF THE STUDY: The present study tested if and how MP material activates HD11 chicken macrophages in vitro using material from different production sites and harvest years. MATERIAL & METHODS: We applied twelve batches of MP from different Chinese production sites and harvest years. Materials were subjected to LAL tests (endotoxic activity), GC-MS (fatty acid analysis), and plate techniques (microbiological background, antimicrobial activity). Furthermore, HD11 chicken macrophages were challenged (6 h, 37 °C) with MP or LPS (E. coli O111:B4), respectively, to quantify nitric oxide (NO) production and immune gene expression (RT-qPCR). RESULTS: MP material promoted strong signals in LAL tests and contained significant amounts of 3-hydroxydodecanoic acid and 3-hydroxymyristic acid, irrespective of processing, harvest year, or origin. The pollen material activated HD11 chicken macrophages, which was confirmed by spikes of NO release and k-means cluster analysis of TLR-signaling pathway gene expression data. Response of NO production to Log2-titration of MP and LPS-treated media was in any case linear and significant. The response was reduced by polymyxin-B (PMB) and the inhibition was twice as strong for LPS than MP. No or minor microbiological background was detected on the majority of MP samples. Three samples showed presence of spoilage microorganisms and Gram-negative bacteria, but this did not correlate to LAL data or bacterial DNA counts. No antimicrobial activity of MP was evident. CONCLUSION: Pollen of the Masson Pine activated HD11 chicken macrophages in vitro, which is likely partially due to a background of bacterial LPS associated with the pollen material. However, as most of the effect (appr. 80%) could not be blocked by PMB this is certainly due to other stimuli. We hypothesize that polysaccharides and oligosaccharides of the pollen matrix have the potential to interact with certain immune receptors presented on the plasma membrane of chicken macrophages.

Nociception in Chicken Embryos, Part I: Analysis of Cardiovascular Responses to a Mechanical Noxious Stimulus.

Although it is assumed that chicken embryos acquire the capacity for nociception while developing in the egg, an exact time point has not yet been specified. The present research was an exploratory study aiming to determine when the capacity of nociception emerges during embryonic development in chickens. Changes in blood pressure and heart rate (HR) in response to a noxious mechanical stimulus at the base of the beak versus a light touch on the beak were examined in chicken embryos between embryonic days (EDs) 7 and 18. Mean arterial pressure (MAP) was the most sensitive parameter for assessing cardiovascular responses. Significant changes in MAP in response to a noxious stimulus were detected in embryos at ED16 to ED18, whereas significant changes in HR were observed at ED17 and ED18. Infiltration anesthesia with the local anesthetic lidocaine significantly reduced the response of MAP on ED18, so the measured cardiovascular changes may be interpreted as nociceptive responses.

Candidate genes associated with heat stress and breeding strategies to relieve its effects in dairy cattle: a deeper insight into the genetic architecture and immune response to heat stress.

The need for food products of animal origin is increasing worldwide. Satisfying these needs in a way that has minimal impact on the environment requires cutting-edge technologies and techniques to enhance the genetic quality of cattle. Heat stress (HS), in particular, is affecting dairy cattle with increasing frequency and severity. As future climatic challenges become more evident, identifying dairy cows that are more tolerant to HS will be important for breeding dairy herds that are better adapted to future environmental conditions and for supporting the sustainability of dairy farming. While research into the genetics of HS in the context of the effect of global warming on dairy cattle is gaining momentum, the specific genomic regions involved in heat tolerance are still not well documented. Advances in omics information, QTL mapping, transcriptome profiling and genome-wide association studies (GWAS) have identified genomic regions and variants associated with tolerance to HS. Such studies could provide deeper insights into the genetic basis for response to HS and make an important contribution to future breeding for heat tolerance, which will help to offset the adverse effects of HS in dairy cattle. Overall, there is a great interest in identifying candidate genes and the proportion of genetic variation associated with heat tolerance in dairy cattle, and this area of research is currently very active worldwide. This review provides comprehensive information pertaining to some of the notable recent studies on the genetic architecture of HS in dairy cattle, with particular emphasis on the identified candidate genes associated with heat tolerance in dairy cattle. Since effective breeding programs require optimal knowledge of the impaired immunity and associated health complications caused by HS, the underlying mechanisms by which HS modulates the immune response and renders animals susceptible to various health disorders are explained. In addition, future breeding strategies to relieve HS in dairy cattle and improve their welfare while maintaining milk production are discussed.

Loss of αß but not γδ T cells in chickens causes a severe phenotype.

The availability of genetically modified mice has facilitated the study of mammalian T cells. No model has yet been developed to study these cells in chickens, an important livestock species with a high availability of γδ T cells. To investigate the role of γδ and αß T cell populations in birds, we generated chickens lacking these T cell populations. This was achieved by genomic deletion of the constant region of the T cell receptor γ or ß chain, leading to a complete loss of either γδ or αß T cells. Our results show that a deletion of αß T cells but not γδ T cells resulted in a severe phenotype in KO chickens. The αß T cell KO chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of αß T cell KO chickens revealed a significant increase in monocytes and expectedly the absence of CD4+ T cells including FoxP3+ regulatory T cells. Surprisingly there was no increase of γδ T cells. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of T cell knockouts in chickens and provide new insights into their function in vertebrates.

Nociception in Chicken Embryos, Part II: Embryonal Development of Electroencephalic Neuronal Activity In Ovo as a Prerequisite for Nociception.

Chicken culling has been forbidden in Germany since 2022; male/female selection and male elimination must be brought to an embryonic status prior to the onset of nociception. The present study evaluated the ontogenetic point at which noxious stimuli could potentially be perceived/processed in the brain in ovo. EEG recordings from randomized hyperpallial brain sites were recorded in ovo and noxious stimuli were applied. Temporal and spectral analyses of the EEG were performed. The onset of physiological neuronal signals could be determined at developmental day 13. ERP/ERSP/ITC analysis did not reveal phase-locked nociceptive responses. Although no central nociceptive responses were documented, adequate EEG responses to noxious stimuli from other brain areas cannot be excluded. The extreme stress impact on the embryo during the recording may overwrite the perception of noniceptive stimuli. The results suggest developmental day 13 as the earliest embryonal stage being able to receive and process nociceptive stimuli.

Nociception in Chicken Embryos, Part III: Analysis of Movements before and after Application of a Noxious Stimulus.

Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore essential and in the interest of animal welfare to be able to precisely define the point at which a chicken embryo is capable of nociception in ovo. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to a noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied in a randomized order. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the mechanical stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The presented results indicate that noxious mechanical stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15.

miRNA Profiles of Canine Intestinal Carcinomas, Lymphomas and Enteritis Analysed by Digital Droplet PCR from FFPE Material.

Most canine intestinal tumours are B-cell or T-cell lymphomas or carcinomas. They have to be distinguished from cases of enteritis. Non-invasive biomarkers such as miRNAs would be a step towards faster diagnosis. The aim of this study was to investigate shifts in miRNA expression in tissue samples collected from cases of enteritis, carcinoma and lymphoma of the small and large intestine to better understand the potential of miRNA as biomarkers for tumour diagnosis and classification. We selected two oncogenic miRNAs (miR-18b and 20b), two tumour suppressive miRNAs (miR-192 and 194) and two potential biomarkers for neoplasms (miR-126 and 214). They were isolated from FFPE material, quantified by ddPCR, normalised with RNU6B and compared with normal tissue values. Our results confirmed that ddPCR is a suitable method for quantifying miRNA from FFPE material. Expression of miR-18b and miR-192 was higher in carcinomas of the small intestine than in those of the large intestine. Specific miRNA patterns were observed in cases of enteritis, B-cell and T-cell lymphoma and carcinoma. However, oncogenic miR-18b and 20b were not elevated in any group and miR-126 and 214 were down-regulated in T-cell and B-cell lymphoma, as well as in carcinomas and lymphoplasmacytic enteritis of the small intestine.

Pathological Findings in Gastrointestinal Neoplasms and Polyps in 860 Cats and a Pilot Study on miRNA Analyses.

Background: Gastrointestinal masses in cats are of clinical relevance, but pathological studies with larger case numbers are lacking. Biomarkers such as miRNA have not yet been investigated in feline intestinal neoplasms. Methods: A retrospective analysis of pathology reports included 860 feline gastrointestinal masses. Immunohistochemistry was performed on 91 lymphomas, 10 sarcomas and 7 mast cell tumours (MCT). Analyses of miRNA-20b and miRNA-192 were performed on 11 lymphomas, 5 carcinomas and 5 control tissues by ddPCR. Results: The pathological diagnosis identified 679 lymphomas, 122 carcinomas, 28 sarcomas, 23 polyps, 7 MCT and 1 leiomyoma. Carcinomas and polyps were most commonly found in the large intestine, lymphomas were most commonly found in the stomach and small intestine and MCT only occurred in the small intestine. Besides the well-described small-cell, mitotic count <2 T-cell lymphomas and the large-cell B-cell lymphomas with a high mitotic count, several variants of lymphomas were identified. The values of miRNA-20b were found to be up-regulated in samples of all types of cancer, whereas miRNA-192 was only up-regulated in carcinomas and B-cell lymphomas. Conclusions: The histopathological and immunohistochemical (sub-)classification of feline intestinal masses confirmed the occurrence of different tumour types, with lymphoma being the most frequent neoplasm. Novel biomarkers such as miRNA-20b and miRNA-192 might have diagnostic potential in feline intestinal neoplasms and should be further investigated.

The Discovery of Chicken Foxp3 Demands Redefinition of Avian Regulatory T Cells.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3 We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4-CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology.

AP-2δ Expression Kinetics in Multimodal Networks in the Developing Chicken Midbrain.

AP-2 is a family of transcription factors involved in many aspects of development, cell differentiation, and regulation of cell growth and death. AP-2δ is a member of this group and specific gene expression patterns are required in the adult mouse brain for the development of parts of the inferior colliculus (IC), as well as the cortex, dorsal thalamus, and superior colliculus. The midbrain is one of the central areas in the brain where multimodal integration, i.e., integration of information from different senses, occurs. Previous data showed that AP-2δ-deficient mice are viable but due to increased apoptosis at the end of embryogenesis, lack part of the posterior midbrain. Despite the absence of the IC in AP-2δ-deficient mice, these animals retain at least some higher auditory functions. Neuronal responses to tones in the neocortex suggest an alternative auditory pathway that bypasses the IC. While sufficient data are available in mammals, little is known about AP-2δ in chickens, an avian model for the localization of sounds and the development of auditory circuits in the brain. Here, we identified and localized AP-2δ expression in the chicken midbrain during embryogenesis. Our data confirmed the presence of AP-2δ in the inferior colliculus and optic tectum (TeO), specifically in shepherd's crook neurons, which are an essential component of the midbrain isthmic network and involved in multimodal integration. AP-2δ expression in the chicken midbrain may be related to the integration of both auditory and visual afferents in these neurons. In the future, these insights may allow for a more detailed study of circuitry and computational rules of auditory and multimodal networks.

A Genetically Engineered Commercial Chicken Line Is Resistant to Highly Pathogenic Avian Leukosis Virus Subgroup J.

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na+/H+ exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field.

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Abrogation of Marek's disease virus replication using CRISPR/Cas9.

Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.

Acquiring Resistance Against a Retroviral Infection via CRISPR/Cas9 Targeted Genome Editing in a Commercial Chicken Line.

Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na+/H+ exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry.

The Long Pentraxin PTX3 Is of Major Importance Among Acute Phase Proteins in Chickens.

The expression level of acute phase proteins (APPs) mirrors the health status of an individual. In human medicine, C-reactive protein (CRP), and other members of the pentraxin family are of significant relevance for assessing disease severity and prognosis. In chickens, however, which represent the most common livestock species around the world, no such marker has yet gained general acceptance. The aim of this study was therefore, to characterize chicken pentraxin 3 (chPTX3) and to evaluate its applicability as a general marker for inflammatory conditions. The mammalian and chicken PTX3 proteins were predicted to be similar in sequence, domain organization and polymeric structure. Nevertheless, some characteristics like certain sequence sections, which have varied during the evolution of mammals, and species-specific glycosylation patterns, suggest distinct biological functions. ChPTX3 is constitutively expressed in various tissues but, interestingly, could not be found in splenic tissue samples without stimulation. However, upon treatment with lipopolysaccharide (LPS), PTX3 expression in chicken spleens increased to 95-fold within hours. A search for PTX3 reads in various publicly available RNA-seq data sets of chicken spleen and bursa of Fabricius also showed that PTX3 expression increases within days after experimental infection with viral and bacterial pathogens. An experimental infection with avian pathogenic E.coli and qPCR analysis of spleen samples further established a challenge dose-dependent significant up-regulation of chPTX3 in subclinically infected birds of up to over 150-fold as compared to untreated controls. Our results indicate the potential of chPTX3 as an APP marker to monitor inflammatory conditions in poultry flocks.

Blocking of the CXCR4-CXCL12 Interaction Inhibits the Migration of Chicken B Cells Into the Bursa of Fabricius.

B cells have first been described in chickens as antibody producing cells and were named after the Bursa of Fabricius, a unique organ supporting their development. Understanding different factors mediating the early migration of B cells into the bursa of Fabricius is crucial for the study of B cell biology. While CXCL12 (stromal derived factor 1) was found to play an important role in B lymphocyte trafficking in mammals, its role in the chicken is still unknown. Previous studies indicated that chicken CXCL12 and its receptor CXCR4 are simultaneously expressed during bursal development. In this study, we investigated whether the CXCR4/CXCL12 interaction mediates B cell migration in chicken embryo. We used the CRISPR/Cas9 system to induce a CXCR4 knockout in chicken B cells which led to chemotaxis inhibition toward CXCL12. This was confirmed by adoptive cell transfer and inhibition of the CXCR4/CXCL12 interaction by blocking with the small inhibitor AMD3100. In addition, we found that the chicken exhibits similarities to mice when it comes to CXCR4 being dependent on B cell receptor expression. B cells lacking the B cell receptor failed to migrate toward CXCL12 and showed no response upon CXCL12 stimulation. Overall, we demonstrated the significance of CXCR4/CXCL12 in chicken B cell development in vivo and the importance of the B cell receptor in CXCR4 dependent signaling.

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+ and CD8+ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

Applications of Gene Editing in Chickens: A New Era Is on the Horizon.

The chicken represents a valuable model for research in the area of immunology, infectious diseases as well as developmental biology. Although it was the first livestock species to have its genome sequenced, there was no reverse genetic technology available to help understanding specific gene functions. Recently, homologous recombination was used to knockout the chicken immunoglobulin genes. Subsequent studies using immunoglobulin knockout birds helped to understand different aspects related to B cell development and antibody production. Furthermore, the latest advances in the field of genome editing including the CRISPR/Cas9 system allowed the introduction of site specific gene modifications in various animal species. Thus, it may provide a powerful tool for the generation of genetically modified chickens carrying resistance for certain pathogens. This was previously demonstrated by targeting the Trp38 region which was shown to be effective in the control of avian leukosis virus in chicken DF-1 cells. Herein we review the current and future prospects of gene editing and how it possibly contributes to the development of resistant chickens against infectious diseases.

Characterization of Chicken Tumor Necrosis Factor-α, a Long Missed Cytokine in Birds.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-α (chTNF-α) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-α were identified in the genomes of 12 additional avian species including Palaeognathae and Neognathae, and the synteny of the closely adjacent loci with mammalian TNF-α orthologs was demonstrated in the crow (Corvus cornix) genome. In addition to chTNF-α, we obtained full sequences for homologs of TNF-α receptors 1 and 2 (TNFR1, TNFR2). chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-α expression in CD4+ but not in CD8+ cells. To gain insights into its biological activity, we generated recombinant chTNF-α in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NFκB-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-α/TNF-α receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems.

HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. METHODS: PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. RESULTS: HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. CONCLUSIONS: In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.