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.

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.

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.

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.

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.

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 role of tryptophan metabolism in iNOS transcription and nitric oxide production by chicken macrophage cells upon treatment with interferon gamma.

The influence of de novo synthesis of nicotinamide adenine dinucleotide (NAD) through the kynurenine (KYN) pathway of tryptophan (TRP) degradation on gene transcription of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in chicken interferon gamma (ChIFN-gamma)-stimulated and non-stimulated chicken macrophage cell line HD11 was investigated. Interferon gamma up regulation of iNOS transcription and NO production was dependent on an undisturbed flow through the KYN pathway. Inhibition of indoleamine-2,3-dioxygenase, the rate-limiting enzyme of TRP catabolism, by 1-methyl-l-tryptophan (1-mTRP) down regulated both iNOS gene transcription and NO production. Addition of KYN to 1-mTRP-treated, ChIFN-gamma-stimulated macrophages circumvented the down regulation of iNOS transcription and NO production. Inhibition of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, replication and transcription, which cleaves NAD into nicotinamide and ADP-ribose, down regulated iNOS gene transcription and NO production in ChIFN-gamma-stimulated HD11 cells. Our results suggest that prevention of NAD depletion in HD11 cells by ChIFN-gamma-mediated induction of IDO facilitates iNOS transcription and NO production. This effect is most likely a result of PARP1 automodification in the presence of NAD, known to facilitate transcription by changing chromatin structure and to allow NFkappaB binding to iNOS promoter which is hindered by direct protein-protein interaction between NFkappaB and unmodified PARP1.

Activation of the TLR3 pathway regulates IFNbeta production in chickens.

Toll-like receptors (TLRs) play key roles in the response to pathogens and in mammals the host response to virus critically relies on TLR3 to detect viral-derived dsRNA. However, in chickens there is a paucity of information about this pathway, and in view of the recent concerns with regard to highly pathogenic avian influenza, there is a clear need for understanding these antiviral pathways. Furthermore, TLR3 engagement is important to the outcome of viral infection because of its role in the induction of interferons (IFNs) and the diverse antiviral effects that these molecules induce. With this in mind, we have investigated the role of TLR3 and its impact on the production of IFNs. We show that in the chicken, poly(I:C), a dsRNA analogue, rapidly induces type 1 IFN similar to that seen in mammals. Furthermore, IFN can activate the upregulation of TLR3, which in some cell types induces them to become responsive to dsRNA. These data highlight the similar function that TLR3 plays in chickens and mammals. To determine the role of chicken TLR3 in response to poly(I:C), we used RNAi-mediated gene silencing to show that poly(I:C)-stimulated IFNbeta expression involves TLR3 signalling. The interrelationship between TLR3 and interferon as well as the observed increase in TLR3 and IFNbeta expression during H5N1 avian influenza infection indicates the importance of these molecules in viral infections in chickens.

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.

IFN-gamma enhances immune responses to E. coli infection in the chicken.

Escherichia coli infection of the respiratory system in chickens occurs as a sequel to a variety of environmental stressors or microbial infections, culminating as chronic respiratory disease (CRD) syndrome or colibacillosis. These diseases cause significant production losses in poultry. With the growing concerns about the use of antibiotics in animal production, for diseases such as CRD, alternative natural agents, like cytokines, may be considered for enhancing health by stimulating the immune system. The current study was aimed at understanding the in vivo effects of recombinant chicken interferon-gamma (ChIFN-gamma) treatment on a variety of immunologic parameters during E. coli infection in chickens. Administration of ChIFN-gamma to chickens increased the percentage of phagocytes in lung and blood of E. coli-infected birds. At the phenotypic level, there was an increase in the percentage of cells expressing MHC II in the air sac, with a concomitant reduction in the proportion of these cells in blood. Furthermore, the blood plasma from ChIFN-gamma-treated infected birds showed an increased level of interleukin-6 (IL-6) activity. Cumulatively, these findings are indicative of in vivo enhancement of immune responses due to ChIFN-gamma. However, administration of ChIFN-gamma protein did not mitigate the development of air sac lesions following E. coli infection.

Interleukin-6 expression after infectious bronchitis virus infection in chickens.

Viral infections in chickens pose a major health threat to the poultry industry. Infectious bronchitis virus (IBV) usually causes respiratory disease; however, the disease severity is influenced by the genotype of the chicken and the IBV strain involved. Nephropathogenic strains of IBV, such as the Australian T strain, can cause high mortalities due to kidney failure characterized by mononuclear cell infiltration and inflammation. In a previous study, a line of specific pathogen-free chickens, the S-line, was shown to be susceptible to high mortalities from IBV infection. The cause of these high mortalities is unknown but it is suspected that differential cytokine expression may play a role. With this in mind, we decided to study the role of the proinflammatory cytokine interleukin (IL)-6 during infection to determine its contribution to nephritis and influence on disease susceptibility. To investigate this, we infected the susceptible S-line and the more disease-resilient HWL line with the T strain of IBV and measured their cytokine response levels. In both lines of birds, IL-6 mRNA levels were elevated in the kidneys at 4 d postinfection. However, in S-line chickens, these levels were 20 times higher than those in the HWL chickens. In addition, S-line birds also showed three times higher serum IL-6 levels than HWL birds after IBV infection. These findings suggest that IL-6 may play a role in IBV-induced nephritis and may open an avenue to develop alternative strategies, such as the use of antiinflammatory cytokines, to overcome the nephropathogenic effects of IBV.

Interleukin-2 directly induces activation and proliferation of chicken T cells in vivo.

Cytokines, as immune activators, have been investigated in mammalian systems as natural adjuvants and therapeutics. In particular, interleukin-2 (IL-2) has been studied widely as a vaccine adjuvant and immuno-enhancer because of its role in activating T cell proliferation. We show here that the first nonmammalian IL-2 gene cloned, chicken IL-2 (ChIL-2), exhibits similar biologic activities to those of mammalian IL-2. To assess the activities of ChIL-2 in vivo, we injected birds with recombinant ChIL-2 (rChIL-2) protein. rChIL-2 treatment induced peripheral blood lymphocytes to express cell surface IL-2 receptors (IL-2R) within 48 h and resulted in an increase in the proportion of peripheral blood CD4+ and CD8+ T cells. Using bromodeoxyuridine (BrdU) incorporation as a measurement of cell proliferation, we showed the increase in T cell populations to be due to cell proliferation. The ability of ChIL-2 to cause both activation and proliferation of T cells in vivo indicates that it has the potential to be used as an immune activator.

The emerging role of avian cytokines as immunotherapeutics and vaccine adjuvants.

The use of antibiotic feed additives and chemical antimicrobials in food production animals is a double-edged sword. On one hand, it helps to prevent the outbreak of disease and promotes the growth of animals, but on the other hand, concerns are mounting over the emergence of antibiotic-resistant bacteria. As a consequence, some countries have already banned the use of in-feed antibiotics which has resulted in meat producers urgently seeking environmentally friendly alternative methods to control disease. Cytokines are proteins that control the type and extent of an immune response following infection or vaccination. They therefore represent excellent naturally occurring therapeutics. The use of cytokines in poultry has become more feasible with the discovery of a number of avian cytokine genes. Since the immune system of chickens is similar to that of mammals, they offer an attractive model system to study the effectiveness of cytokine therapy in the control of disease in livestock. This review will focus on the recent advances made in avian cytokines, with a particular focus on their assessment as therapeutic agents and vaccine adjuvants.

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.

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.

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.

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.

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.

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.