Strategies to enable the adoption of animal biotechnology to sustainably improve global food safety and security.

The ability to generate transgenic animals has existed for over 30 years, and from those early days many predicted that the technology would have beneficial applications in agriculture. Numerous transgenic agricultural animals now exist, however to date only one product from a transgenic animal has been approved for the food chain, due in part to cumbersome regulations. Recently, new techniques such as precision breeding have emerged, which enables the introduction of desired traits without the use of transgenes. The rapidly growing human population, environmental degradation, and concerns related to zoonotic and pandemic diseases have increased pressure on the animal agriculture sector to provide a safe, secure and sustainable food supply. There is a clear need to adopt transgenic technologies as well as new methods such as gene editing and precision breeding to meet these challenges and the rising demand for animal products. To achieve this goal, cooperation, education, and communication between multiple stakeholders-including scientists, industry, farmers, governments, trade organizations, NGOs and the public-is necessary. This report is the culmination of concepts first discussed at an OECD sponsored conference and aims to identify the main barriers to the adoption of animal biotechnology, tactics for navigating those barriers, strategies to improve public perception and trust, as well as industry engagement, and actions for governments and trade organizations including the OECD to harmonize regulations and trade agreements. Specifically, the report focuses on animal biotechnologies that are intended to improve breeding and genetics and currently are not routinely used in commercial animal agriculture. We put forward recommendations on how scientists, regulators, and trade organizations can work together to ensure that the potential benefits of animal biotechnology can be realized to meet the future needs of agriculture to feed the world.

Overview of the CSIRO Australian Animal Health Laboratory.

Emerging infectious diseases arising from livestock and wildlife pose serious threats to global human health, as shown by a series of continuous outbreaks involving highly pathogenic influenza, SARS, Ebola and MERS. The risk of pandemics and bioterrorism threats is ever present and growing, but our ability to combat them is limited by the lack of available vaccines, therapeutics and rapid diagnostics. The use of high bio-containment facilities, such as the CSIRO Australian Animal Health Laboratory, plays a key role studying these dangerous pathogens and facilitates the development of countermeasures. To combat diseases like MERS, we must take a holistic approach that involves the development of early biomarkers of infection, a suite of treatment options (vaccines, anti-viral drugs and antibody therapeutics) and appropriate animal models to test the safety and efficacy of candidate treatments.

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

Studying immunity to zoonotic diseases in the natural host - keeping it real.

Zoonotic viruses that emerge from wildlife and domesticated animals pose a serious threat to human and animal health. In many instances, mouse models have improved our understanding of the human immune response to infection; however, when dealing with emerging zoonotic diseases, they may be of limited use. This is particularly the case when the model fails to reproduce the disease status that is seen in the natural reservoir, transmission species or human host. In this Review, we discuss how researchers are placing more emphasis on the study of the immune response to zoonotic infections in the natural reservoir hosts and spillover species. Such studies will not only lead to a greater understanding of how these infections induce variable disease and immune responses in distinct species but also offer important insights into the evolution of mammalian immune systems.

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.

Chicken interferons, their receptors and interferon-stimulated genes.

The prevalence of pathogenic viruses is a serious issue as they pose a constant threat to both the poultry industry and to human health. To prevent these viral infections an understanding of the host-virus response is critical, especially for the development of novel therapeutics. One approach in the control of viral infections would be to boost the immune response through administration of cytokines, such as interferons. However, the innate immune response in chickens is poorly characterised, particularly concerning the interferon pathway. This review will provide an overview of our current understanding of the interferon system of chickens, including their cognate receptors and known interferon-stimulated gene products.

The chicken TH1 response: potential therapeutic applications of ChIFN-γ.

The outcomes of viral infections are costly in terms of human and animal health and welfare worldwide. The observed increase in the virulence of some viruses and failure of many vaccines to stop these infections has lead to the apparent need to develop new anti-viral strategies. One approach to dealing with viral infection may be to employ the therapeutic administration of recombinant cytokines to act as 'immune boosters' to assist in augmenting the host response to virus. With this in mind, a greater understanding of the immune response, particularly cell mediated T-helper-1 (TH1) type responses, is imperative to the development of new anti-viral and vaccination strategies. Following the release of the chicken genome, a number of TH1-type cytokines have been identified, including chicken interleukin-12 (ChIL-12), ChIL-18 and interferon-γ ChIFN-γ), highlighting the nature of the TH1-type response in this non-mammalian vertebrate. To date a detailed analysis of the in vivo biological function of these cytokines has been somewhat hampered by access to large scale production techniques. This review describes the role of TH-1 cytokines in immune responses to viruses and explores their potential use in enhancing anti-viral treatment strategies in chickens. Furthermore, this review focuses on the example of ChIFN-γ treatment of Chicken Anemia Virus (CAV) infection. CAV causes amongst other things thymocyte depletion and thymus atrophy, as well as immunosuppression in chickens. However, due to vaccination, clinical disease appears less often, nevertheless, the subclinical form of the disease is often associated with secondary complicating infections due to an immunocompromised state. Since CAV-induced immunosuppression can cause a marked decrease in the immune response against other pathogens, understanding this aspect of the disease is critically important, as well as providing insights into developing new control approaches. With increasing emphasis on developing alternative control programs for poultry diseases, novel therapeutic strategies provide one approach. We show here that the in ovo administration of ChIFN-γ impacts the depletion of T-cell precursors during CAV infection. Therefore, it appears that ChIFN-γ may have the potential to be used as a novel therapeutic reagent to impact virus infection and alter immunosuppression caused by CAV and potentially other pathogens.

Promotion of Hendra virus replication by microRNA 146a.

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

Ontogeny of the interferon system in chickens.

Newborn vertebrates may be susceptible to infection because the immature status of their immune system results in an inability to make an effective immune response. Consequently, newly hatched chicks appear to be more susceptible to infections than mature chickens. In particular, poultry susceptibility to virus infection may be related to poor expression of innate immune elements involved in antiviral responses. Therefore, in this study we assessed the relative development of the interferon (IFN) system: a protective system against virus infection. We investigated the age-related expression of the elements involved in the IFN response including IFN gene expression, their associated receptors and the pattern recognition receptors (PRR) involved in the regulation of IFNs. We observed that the IFN system is somewhat inadequately expressed in embryos and develops over time, just prior to and after hatching, and therefore chicks may be more susceptible to virus than mature birds because of an immature IFN network.

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks.

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens.

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.

Identifying innate immune pathways of the chicken may lead to new antiviral therapies.

Zoonotic viruses, such as highly pathogenic avian influenza (HPAI), present a significant threat to both the poultry industry and public health. The present method of controlling avian influenza (AI) relies on good farming practice with limited use of vaccination in some countries. However, new ways to control disease outbreaks might be possible with additional knowledge of the natural host response to virus. Moreover, manipulation of the innate immune system in mammals improves the outcomes following viral infection. A similar approach might be applied to the chicken, nevertheless, a greater knowledge of the chicken innate immune system is required. This review outlines important mammalian antiviral mechanisms that have been modulated to strengthen viral immunity and highlights the potential application of these strategies in the chicken, especially in regards, to AI.

Toll-like receptor 7 ligands inhibit influenza A infection in chickens.

Avian influenza virus is endemic in many regions around the world and remains a pandemic threat, a scenario tied closely to outbreaks of the virus in poultry. The innate immune system, in particular the nucleic acid-sensing toll-like receptors (TLRs) -3, -7, -8, and -9, play a major role in coordinating antiviral immune responses. In this study we have investigated the use of TLR ligands as antivirals against influenza A in chickens. The TLR7 ligand poly-C inhibited low-path influenza A growth in the chicken macrophage cell line HD-11 more effectively than poly(I:C), which acts via TLR3. The TLR7 ligand 7-allyl-8-oxoguanosine (loxoribine) inhibited influenza A replication in vitro and in ovo in a dose-dependent manner. Treatment of primary chicken splenocytes with loxoribine resulted in the induction of interferons-α, -ß, and -λ, and interferon-stimulated genes PKR and Mx. These results demonstrate that nucleic acid-sensing TLR ligands show considerable potential as antivirals in chickens and could be incorporated into antiviral strategies.

Immunostimulatory motifs enhance antiviral siRNAs targeting highly pathogenic avian influenza H5N1.

Highly pathogenic avian influenza (HPAI) H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi)-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN) production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (si)RNAs in chicken cells (i) mode of synthesis and (ii) nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5'-UGUGU-3' is a key determinant in inducing high levels of expression of IFN-α, -ß, -λ and interleukin 1-ß in chicken cells. Positioning of this 5'-UGUGU-3' motif at the 5'-end of the sense strand of siRNAs, but not the 3'-end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257) tagged with 5'-UGUGU-3' induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics.

Characterization of chicken Mda5 activity: regulation of IFN-ß in the absence of RIG-I functionality.

In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I-like function may contribute to the chicken's susceptibility to highly pathogenic influenza.

Increased inducible nitric oxide synthase expression in organs is associated with a higher severity of H5N1 influenza virus infection.

BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.

Highly pathogenic (H5N1) avian influenza induces an inflammatory T helper type 1 cytokine response in the chicken.

To better understand the immune response to highly pathogenic avian influenza virus, we compared expression of cytokines in chickens infected with avian influenza virus (A/Vietnam/1203/04) to that in uninfected chickens. Gene expression analyses revealed that influenza disseminated to multiple organs where immune responses could be identified. Among those cytokines influenced by influenza infection were the T helper type (Th)1-associated cytokines interleukin (IL)12 and interferon γ. In addition, a corresponding downregulation of the intracytoplasmic factor GATA3 was identified, whereas the Th2 cytokines IL4 and IL10 did not appear to be impacted by the infection. The inflammatory cytokine IL6 also appeared to be highly upregulated along with type 1 and type 3 interferon. Together, these data indicate that a strong inflammatory and Th1 response occurs after highly pathogenic avian influenza infection in the chicken that has implications for strategies that target the immune system for improving resistance to avian influenza.

Prebiotics modulate immune responses in the gut-associated lymphoid tissue of chickens.

The recent European Union ban on the prophylactic use of in-feed antibiotics has escalated the search for alternatives for use within the poultry industry. When evaluating the efficacy of potential antibiotic alternatives on bird health and productivity, it is important to analyze the competence of the immune cells in the gut-associated lymphoid tissue (GALT), because it is routinely involved in the surveillance of colonizing microbes as well as in interacting with the ingested feed antigens. Therefore, we studied the effect of the prebiotics mannan-oligosaccharide (MOS) and fructo-oligosaccharide (FOS) on the phenotypic and functional competence of immune cells in cecal tonsil (CT), which is a major GALT. Day-old Cobb 500 male broilers were randomized to 4 groups. Control chickens were fed the basal diet only. Chickens in experimental groups received 0.05 g/kg zinc bacitracin or 5 g/kg of either FOS or MOS in addition to basal diet. At the end of 25 d, our comparison of the experimental groups with controls revealed that the addition of prebiotics to diet resulted in a significant reduction in the proportion of B cells and in mitogen responsiveness of lymphocytes in CT. Furthermore, FOS treatment significantly enhanced the IgM and IgG antibody titers in plasma. These findings emphasize the need for the analyses of the gut immune function following treatment with novel feed additives. The knowledge obtained from such analyses may aid in understanding the mechanisms underlying the immune competence of the birds, which needs consideration when selecting and optimizing new feed additives instead of antibiotics for poultry production.

The in vitro and in ovo responses of chickens to TLR9 subfamily ligands.

Although Toll-like receptors (TLRs) have been well characterised in mammals, less work has been carried out in non-mammalian species, such as chickens. In this study the response of chicken cells to the TLR9 subfamily of ligands was characterised in vitro and in ovo. It was found that even though chickens appear to have only one functional receptor to represent the TLR9 subfamily, stimulation of chicken splenocytes with TLR7 and TLR9 ligands induced proinflammatory cytokine production and cell proliferation, similar to that observed when the homologous mammalian receptors are stimulated. Furthermore, we demonstrated that the in ovo administration of these TLR ligands elicits a response, such as cytokine production, that can be detected post-hatch. The current knowledge of the action of TLR ligands in mammals, in conjunction with their immunomodulating ability shown in this study, draws attention to their potential use as therapeutic agents for the poultry industry.

Expression, purification and characterisation of recombinant Escherichia coli derived chicken interleukin-12.

Zoonotic viruses, such as H5N1 Avian Influenza, pose major threats to both animals and humans, and with this in mind there is a need for the development of new anti-viral strategies. The cytokine interleukin-12 (IL-12) is known to play a pivotal regulatory role in the anti-viral response due to its role in the induction of the key anti-viral cytokine IFN-gamma. Therefore, strategies which provide a means for the production of therapeutic quantities of IL-12 may be of major benefit. Here we describe the development of biologically active Escherichia coli (E. coli) derived chicken IL-12 (ChIL-12). The single chain ChIL-12 gene was cloned into the pET32b expression vector, transformed into the BL-21 E. coli strain and expression induced with IPTG. Over expressed protein was solubilised with zwittergent detergent and isolated utilising Nickel ion affinity chromatography. Biological activity was determined as ChIL-12 stimulated proliferation of pre-treated T-cells in vitro. This study is the first example of a biologically active E. coli derived IL-12 from a non-mammalian vertebrate subsequently providing a means for testing the anti-viral therapeutic potential of ChIL-12 in an in vivo model.