Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells.

Exogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14-15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.

Marker counter-selection via CRISPR/Cas9 co-targeting for efficient generation of genome edited avian cell lines and germ cells.

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells.

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.

In Vivo Inhibition of Marek's Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4.

Marek's disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Transgenesis and web resources in quail.

Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (Coturnix coturnix japonica) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.

Potential benefits of gene editing for the future of poultry farming.

The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Immunological Comparison of Native and Recombinant Hen's Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis.

Chicken serum albumin (CSA) is a hen's egg yolk allergen causing IgE-mediated allergy. The objective of this study was to produce a recombinant version of CSA and compare its IgE reactivity to natural CSA (nCSA). CSA was cloned and expressed as a soluble fraction in the yeast Kluyveromyces lactis (K. lactis) protein expression system. The gene encoding CSA was amplified with a C-terminal hemagglutinin epitope tag by polymerase chain reaction (PCR) and cloned into the pKLAC2 expression vector prior to transforming into K. lactis. Recombinant CSA (rCSA) was purified by immunoprecipitation. Twenty-one patients allergic to hen's egg white were examined for sensitisation against nCSA. 38% of patients were found to be sensitised to CSA based on Western immunoassay. Immunoglobulin E (IgE) binding capacity of rCSA and nCSA was analysed by ELISA using sera from patients sensitised to CSA. Levels of IgE-binding were similar for both the recombinant and the natural CSA, indicating the existence of similar epitopes. rCSA produced in this study is a potential candidate to be used in component-resolved diagnosis (CRD) of egg yolk allergy. The usefulness of rCSA in CRD of egg yolk allergy warrants further characterisation using sera from patients with allergy to hen's egg yolk in future studies.

Innovative approaches to genome editing in avian species.

The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal. In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells (PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing (STAGE). The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.

shRNAs targeting either the glycoprotein or polymerase genes inhibit Viral haemorrhagic septicaemia virus replication in zebrafish ZF4 cells.

Viral haemorrhagic septicaemia virus (VHSV) represents an important disease of finfish. To explore the potential of shRNAs to combat this disease nucleotide sequences of either the VHSV glycoprotein (G) or polymerase (L) gene were targeted. To test their function, shRNAs were expressed in zebrafish epithelial ZF-4 cells utilizing the zebrafish U6-2 promoter. Five of the six shRNA molecules successfully reduced VHSV replication by between 2 and 4 logs in titre relative to an irrelevant control shRNA at all MOIs and also reduced viral CPE at the highest MOI. To ensure that observed reductions in viral titre were dependent on shRNA silencing, potential non-specific antiviral responses were assessed. Only the ineffective shRNA, which formed an improper hairpin when analysed in silico, induced an antiviral response as measured by induction of interferon (ifnphi1) and Mx (MxA) genes. These results represent an important preliminary step in the generation of transgenic zebrafish resistant to VHSV.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

Advances in genetic engineering of the avian genome: "Realising the promise".

This review provides an historic perspective of the key steps from those reported at the 1st Transgenic Animal Research Conference in 1997 through to the very latest developments in avian transgenesis. Eighteen years later, on the occasion of the 10th conference in this series, we have seen breakthrough advances in the use of viral vectors and transposons to transform the germline via the direct manipulation of the chicken embryo, through to the establishment of PGC cultures allowing in vitro modification, expansion into populations to analyse the genetic modifications and then injection of these cells into embryos to create germline chimeras. We have now reached an unprecedented time in the history of chicken transgenic research where we have the technology to introduce precise, targeted modifications into the chicken genome, ranging from; new transgenes that provide improved phenotypes such as increased resilience to economically important diseases; the targeted disruption of immunoglobulin genes and replacement with human sequences to generate transgenic chickens that express "humanised" antibodies for biopharming; and the deletion of specific nucleotides to generate targeted gene knockout chickens for functional genomics. The impact of these advances is set to be realised through applications in chickens, and other bird species as models in scientific research, for novel biotechnology and to protect and improve agricultural productivity.

Overexpression of Anti-Müllerian Hormone Disrupts Gonadal Sex Differentiation, Blocks Sex Hormone Synthesis, and Supports Cell Autonomous Sex Development in the Chicken.

The primary role of Anti-Müllerian hormone (AMH) during mammalian development is the regression of Müllerian ducts in males. This highly conserved function is retained in birds and is supported by the high levels of AMH expression in developing testes. Mammalian AMH expression is regulated by a combination of transcription factors, the most important being Sry-type high-mobility-group box transcription factor-9 (SOX9). In the chicken embryo, however, AMH mRNA expression precedes that of SOX9, leading to the view that AMH may play a more central role in avian testicular development. To define its role in chicken gonadal development, AMH was overexpressed using the RCASBP viral vector. AMH caused the gonads of both sexes to develop as small and undeveloped structures at both embryonic and adult stages. Molecular analysis revealed that although female gonads developed testis-like cords, gonads lacked Sertoli cells and were incapable of steroidogenesis. A similar gonadal phenotype was also observed in males, with a complete loss of both Sertoli cells, disrupted SOX9 expression and gonadal steroidogenesis. At sexual maturity both sexes showed a female external phenotype but retained sexually dimorphic body weights that matched their genetic sexes. These data suggest that AMH does not operate as an early testis activator in the chicken but can affect downstream events, such as sex steroid hormone production. In addition, this study provides a unique opportunity to assess chicken sexual development in an environment of sex hormone deficiency, demonstrating the importance of both hormonal signaling and direct cell autonomous factors for somatic sex identity in birds.

Transgenic Chickens Overexpressing Aromatase Have High Estrogen Levels but Maintain a Predominantly Male Phenotype.

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterized steroidogenic pathway, which is a multistep process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. Ectopic overexpression of aromatase in male chicken embryos induces gonadal sex reversal, and male embryos treated with estradiol become feminized; however, this is not permanent. To test whether a continuous supply of estrogen in adult chickens could induce stable male to female sex reversal, 2 transgenic male chickens overexpressing aromatase were generated using the Tol2/transposase system. These birds had robust ectopic aromatase expression, which resulted in the production of high serum levels of estradiol. Transgenic males had female-like wattle and comb growth and feathering, but they retained male weights, displayed leg spurs, and developed testes. Despite the small sample size, this data strongly suggests that high levels of circulating estrogen are insufficient to maintain a female gonadal phenotype in adult birds. Previous observations of gynandromorph birds and embryos with mixed sex chimeric gonads have highlighted the role of cell autonomous sex identity in chickens. This might imply that in the study described here, direct genetic effects of the male chromosomes largely prevailed over the hormonal profile of the aromatase transgenic birds. This data therefore support the emerging view of at least partial cell autonomous sex development in birds. However, a larger study will confirm this intriguing observation.

Anti-Müllerian Hormone Is Required for Chicken Embryonic Urogenital System Growth but Not Sexual Differentiation.

In mammals, the primary role of anti-Müllerian hormone (AMH) during development is the regression of Müllerian ducts in males. These structures otherwise develop into fallopian tubes, oviducts, and upper vagina, as in females. This highly conserved function is retained in birds and is supported by the high levels of AMH expression in developing testes. In mammals, AMH expression is controlled partly by the transcription factor, SOX9. However, in the chicken, AMH mRNA expression precedes that of SOX9 , leading to the view that AMH may lie upstream of SOX9 and play a more central role in avian testicular development. To help define the role of AMH in chicken gonad development, we suppressed AMH expression in chicken embryos using RNA interference. In males, AMH knockdown did not affect the expression of key testis pathway genes, and testis cords developed normally. However, a reduction in the size of the mesonephros and gonads was observed, a phenotype that was evident in both sexes. This growth defect occurred as a result of the reduced proliferative capacity of the cells of these tissues, and male gonads also had a significant reduction in germ cell numbers. These data suggest that although AMH does not directly contribute to testicular or ovarian differentiation, it is required in a sex-independent manner for proper cell proliferation and urogenital system growth.

Overexpression of aromatase alone is sufficient for ovarian development in genetically male chicken embryos.

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.

Trop2 regulates motility and lamellipodia formation in cultured fetal lung fibroblasts.

Proliferation and migration of fibroblasts are vital for fetal lung development. However, the regulatory mechanisms are poorly understood. We have previously shown that TROP2 gene expression is closely associated with fetal lung cell proliferation in vivo and that TROP2 knockdown decreases proliferation of fetal lung fibroblasts in culture. We hypothesized that the Trop2 protein also regulates the morphology and motility of fetal lung fibroblasts. Fibroblasts isolated from fetal rat lungs (gestational age embryonic day 19) adopted a myofibroblast-like morphology in culture. Trop2 protein was localized to lamellipodia. TROP2 siRNA significantly decreased: TROP2 mRNA levels by 77%, the proportion of cells containing Trop2 protein by 70%, and cell proliferation by 50%. TROP2 siRNA also decreased the degree of motility as determined by the number of gridlines that cells moved across (2.2 ± 0.2 vs. 3.2 ± 0.2; P < 0.001). TROP2 knockdown altered cell morphology, causing a notable absence of lamellipodia and abnormal localization of components of the cell migration apparatus, and it reduced phosphorylated ERK1 and ERK2 levels. In contrast, TROP2 overexpression significantly increased: TROP2 mRNA levels by 40-fold, cell proliferation by 40%, the proportion of cells that were motile by 20%, and the number of gridlines that cells moved across (2.1 ± 0.2 vs. 1.6 ± 0.1; P < 0.001). Our data suggest that Trop2 regulates cell proliferation and motility and that it does so by regulating the ERK pathway and several critical components of the cell migration apparatus.

A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells.

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.

Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi) based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs) targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.