Kristie Jenkins

Cytokines as adjuvants for avian vaccines

The worldwide trend towards a reduced reliance on in‐feed antibiotics has increased the pressure to develop alternative strategies to manage infectious diseases in poultry. With this in mind, there is a great emphasis on vaccine use and the enhancement of existing vaccines to provide long‐term protection. Currently existing adjuvants for poultry can have deleterious side‐effects, such as inflammation, resulting in the down‐grading of meat quality and a subsequent reduction in profits. Therefore, to enhance the use of vaccination, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants in poultry is attracting considerable attention, and their potential role as such has been addressed by several studies. The recent identification of a number of chicken cytokine genes has provided the possibility to study their effectiveness in enhancing the immune response during infection and vaccination. This review focuses on the recent studies involving the assessment of cytokines as vaccine adjuvants.

Promotion of Hendra Virus Replication by MicroRNA 146a

ABSTRACT 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.

Mitochondrially localised MUL1 is a novel modulator of antiviral signaling

The innate immune response to virus must be balanced to eliminate infection yet limit damaging inflammation. A critical arm of the antiviral response is launched by the retinoic acid‐inducible‐gene I (RIG‐I) protein. RIG‐I is activated by viral RNA then associates with the mitochondrial antiviral signaling (MAVS) protein to subsequently induce potent inflammatory cytokines. Here, we demonstrate the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) is a crucial moderator of RIG‐I signaling. MUL1 is localized to the mitochondria where it interacts with MAVS and catalyzes RIG‐I post‐translational modifications that inhibit RIG‐I‐dependent cell signaling. Accordingly, depletion of MUL1 potentiated RIG‐I mediated nuclear factor‐kappa B (NF‐κB) and interferon (IFN) β reporter activity. Moreover, depletion of MUL1 boosted the antiviral response and increased proinflammatory cytokines following challenge with the RNA mimetic poly I:C and Sendai virus. We therefore submit that MUL1 is a novel regulator of the RIG‐I‐like receptor‐dependent antiviral response, that otherwise functions to limit inflammation.

Characterisation of novel microRNAs in the Black flying fox (Pteropus alecto) by deep sequencing

BackgroundBats are a major source of new and emerging viral diseases. Despite the fact that bats carry and shed highly pathogenic viruses including Ebola, Nipah and SARS, they rarely display clinical symptoms of infection. Host factors influencing viral replication are poorly understood in bats and are likely to include both pre- and post-transcriptional regulatory mechanisms. MicroRNAs are a major mechanism of post-transcriptional gene regulation, however very little is known about them in bats.ResultsThis study describes 399 microRNAs identified by deep sequencing of small RNA isolated from tissues of the Black flying fox, Pteropus alecto, a confirmed natural reservoir of the human pathogens Hendra virus and Australian bat lyssavirus. Of the microRNAs identified, more than 100 are unique amongst vertebrates, including a subset containing mutations in critical seed regions. Clusters of rapidly-evolving microRNAs were identified, as well as microRNAs predicted to target genes involved in antiviral immunity, the DNA damage response, apoptosis and autophagy. Closer inspection of the predicted targets for several highly supported novel miRNA candidates suggests putative roles in host-virus interaction.ConclusionsMicroRNAs are likely to play major roles in regulating virus-host interaction in bats, via dampening of inflammatory responses (limiting the effects of immunopathology), and directly limiting the extent of viral replication, either through restricting the availability of essential factors or by controlling apoptosis. Characterisation of the bat microRNA repertoire is an essential step towards understanding transcriptional regulation during viral infection, and will assist in the identification of mechanisms that enable bats to act as natural virus reservoirs. This in turn will facilitate the development of antiviral strategies for use in humans and other species.

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.

miRNA modulation of SOCS1 using an influenza A virus delivery system

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-β cytokines.

Visualising single molecules of HIV-1 and miRNA nucleic acids.

BackgroundThe scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length.ResultsWe used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.ConclusionsTaken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.

Sex selection in layer chickens

The ability to detect and remove male chicks pre-hatch would be a big step forward to the egg-laying and related industries. The current practice of culling male chicks post-hatch creates a major ethical dilemma for many countries. Hatching out and growing male layer chicks is not a sustainable option for farmers. A genetic based in ovo sex selection application would effectively negate the need to cull or grow out male chickens and would contribute to a more sustainable industry with a view to future food security. Recent advancements in avian gene technology allow specific marking of the sex-determining chromosome in chickens so that the males can be identified before hatching and removed before incubation. This provides a simple solution to meet a pressing need for the industry and a leading opportunity for the adoption of biotechnology in animal agriculture.

UC Davis Transgenic Animal Research Conference X (TARC X): Tahoe City, CA, USA, August 9-12, 2015.

The 2015 Transgenic Animal Research Conference was the tenth conference in the series of biannual gatherings hosted by the University of California, Davis at beautiful Lake Tahoe. This conference was originally designed to bring together researchers from all over the world working on genome engineering in livestock species, and has continued with this goal for 18 years. To mark this milestone the structure of TARC X was modified from its typical format. Original research was still presented, however the conference also featured a series of reviews spanning the advances made in the field from the first TARC conference in 1997 up till the present. This prospective allowed participants to appreciate how many and how varied the technical barriers we as scientists have overcome. However a common theme that emerged was that no matter how much the technology has advanced commercial applications have floundered. With the AquAdvantage salmon still not receiving approval from the FDA and very few transgenic animals with agricultural applications heading into the regulatory pipeline there was a general feeling of frustration with the essentially dysfunctional regulatory system that is currently in place. In contrast there was a great deal of optimism and excitement generated by the multiple groups successfully employing gene editor technologies like CRISPR/Cas9 and TALENs. There was an enthusiasm for the possibilities presented by the technology as well as a focus on how to differentiate gene editing technology from transgenic technology in the minds of the public as well in the regulatory system. The research presentations at TARC X highlighted some impressive technological advancements. Editor technology is now pervasive and being successfully applied to multiple species including sheep, pigs, cows, and chickens in labs all over the world (B. Teluga, T. Doran, M. Tizard, M. Liu). In the area of stem cell research Pablo Ross presented very promising results on the derivation of bovine ESCs, which has long been a goal of many labs in the field. Another breakthrough was in chimeric interspecies complementation which has been done successfully to generate mouse/rat chimeric models and there is work in progress in pigs with the aim of 1 day growing functional human organs in pigs (J. Wu, H. Nakauchi). There was a breadth of applications that either transgenic or editor technologies were being employed to achieve. Staying true to the original proposed purpose of genetically engineered agricultural species there were talks about improved production traits and improved animal products (M. Liu, L. C. Cooper (&) K. Jenkins CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia e-mail: caitlin.cooper@csiro.au