Detection and characterisation of an Endogenous Betaretrovirus in Australian Wild Deer

Endogenous retroviruses (ERVs) are the remnants of past retroviral infections that once invaded the host’s germline and were vertically transmitted. ERV sequences have been reported in mammals, but their distribution and diversity in cervids are unclear. Using next-generation sequencing, we identified a nearly complete genome of an endogenous betaretrovirus in fallow deer (Dama dama). Further genomic analysis showed that this provirus, tentatively named cervid endogenous betaretrovirus 1 (CERV β1), has typical betaretroviral genome features (gag-pro-pol-env) and the betaretrovirus-specific dUTPase domain. In addition, CERV β1 pol sequences were detected by PCR in the six non-native deer species with wild populations in Australia. Phylogenetic analyses demonstrated that CERV β1 sequences from subfamily Cervinae clustered as sister taxa to ERV-like sequences in species of subfamily Muntiacinae. These findings, therefore, suggest that CERV β1 endogenisation occurred after the split of these two subfamilies (between 3.3 and 5 million years ago). Our results provide important insights into the evolution of betaretroviruses in cervids

Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2–5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on “Successfully transitioning from post-doc to lab head”, winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.Karla J. Helbig ... Michael R. Beard ... et al

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

Evaluation of haemoparasite and Sarcocystis infections in Australian wild deer

Wild animals are natural reservoir hosts for a variety of pathogens that can be transmitted to other wildlife, livestock, other domestic animals, and humans. Wild deer (family Cervidae) in Europe, Asia, and North and South America have been reported to be infected with gastrointestinal and vector-borne parasites. In Australia, wild deer populations have expanded considerably in recent years, yet there is little information regarding which pathogens are present and whether these pathogens pose biosecurity threats to humans, wildlife, livestock, or other domestic animals. To address this knowledge gap, PCR-based screening for five parasitic genera was conducted in blood samples (n = 243) sourced from chital deer (Axis axis), fallow deer (Dama dama), rusa deer (Rusa timorensis) and sambar deer (Rusa unicolor) sampled in eastern Australia. These blood samples were tested for the presence of DNA from Plasmodium spp., Trypanosoma spp., Babesia spp., Theileria spp. and Sarcocystis spp. Further, the presence of antibodies against Babesia bovis was investigated in serum samples (n = 105) by immunofluorescence. In this study, neither parasite DNA nor antibodies were detected for any of the five genera investigated. These results indicate that wild deer are not currently host reservoirs for Plasmodium, Trypanosoma, Babesia, Theileria or Sarcocystis parasites in eastern Australia. We conclude that in eastern Australia, wild deer do not currently play a significant role in the transmission of these parasites. This survey represents the first large-scale molecular study of its type in Australian wild deer and provides important baseline information about the parasitic infection status of these animals. The expanding populations of wild deer throughout Australia warrant similar surveys in other parts of the country and surveillance efforts to continually assess the level of threat wild deer could pose to humans, wildlife, livestock and other domestic animals

First evidence of Entamoeba parasites in Australian Wild Deer and assessment of transmission to cattle

Australian wild deer populations have significantly expanded in size and distribution in recent decades. Due to their role in pathogen transmission, these deer populations pose a biosecurity risk to the livestock industry. However, little is known about the infection status of wild deer in Australia. The intestinal parasite Entamoeba bovis has been previously detected in farm and wild ruminants worldwide, but its epidemiology and distribution in wild ruminants remain largely unexplored. To investigate this knowledge gap, faecal samples of wild deer and domestic cattle from south-eastern Australia were collected and analysed for the presence of Entamoeba spp. using PCR and phylogenetic analysis of the conserved 18S rRNA gene. E. bovis parasites were detected at high prevalence in cattle and wild deer hosts, and two distinct Entamoeba ribosomal lineages (RLs), RL1 and RL8, were identified in wild deer. Phylogenetic analysis further revealed the existance of a novel Entamoeba species in sambar deer and a novel Entamoeba RL in fallow deer. While we anticipated cross-species transmission of E. bovis between wild deer and cattle, the data generated in this study demonstrated transmission is yet to occur in Australia. Overall, this study has identified novel variants of Entamoeba and constitutes the first report of Entamoeba in fallow deer and sambar deer, expanding the host range of this parasite. Epidemiological investigations and continued surveillance of Entamoeba parasites in farm ruminants and wild animals will be required to evaluate pathogen emergence and transmission to livestock

Investigation of sphingosine kinase 1 in interferon responses during dengue virus infection

Dengue virus (DENV) regulates sphingosine kinase (SK)-1 activity and chemical inhibition of SK1 reduces DENV infection. In primary murine embryonic fibroblasts (pMEFs) lacking SK1 however, DENV infection is enhanced and this is associated with induction of normal levels of interferon beta (IFN-β) but reduced levels of IFN-stimulated genes (ISGs). We have further investigated this link between SK1 and type I IFN responses. DENV infection downregulates cell-surface IFN-alpha receptor (IFNAR)1 in both wild-type (WT) and SK1-/- pMEF, but, consistent with poor ISG responses, shows reduced induction of phosphorylated (p)-STAT1 and key IFN regulatory factors (IRF)1 and -7 in SK1-/- pMEF. Direct IFN stimulation induced ISGs (viperin, IFIT1), CXCL10, IRF1 and -7 and p-STAT1. Responses, however, were significantly reduced in SK1-/- pMEF, except for IFN-stimulated CXCL10 and IRF7. Poor IFN responses in SK1-/- pMEF were associated with a small reduction in basal cell-surface IFNAR1 and IRF1 mRNA in uninfected SK1-/- compared with WT pMEF. In contrast, treatment of cells with the SK1 inhibitor, SK1-I or expression of an inhibitory SK1 short hairpin RNA (shRNA), both of which reduce DENV infection, does not alter basal IRF1 mRNA or affect type I IFN stimulation of p-STAT1. Thus, cells genetically lacking SK1 can induce many responses normally following DENV infection, but have adaptive changes in IFNAR1 and IRF1 that compromise DENV-induced type I IFN responses. This suggests a biological link between SK1 and IFN-stimulated pathways. Other approaches to reduce SK1 activity, however, do not influence these important antiviral pathways but reduce infection and may be useful antiviral strategies.Amanda L Aloia, Julie K Calvert, Jennifer N Clarke, Lorena T Davies, Karla J Helbig, Stuart M Pitson, and Jillian M Car

Mechanism of interferon stimulated gene induction in HIV-1 infected macrophages

Viruses manipulate the complex interferon and interferon stimulated gene (ISG) system in different ways. We have previously shown that HIV inhibits type I and III interferons in its key target cells but directly stimulates a subset of >20 ISGs in macrophages and dendritic cells many of which are antiviral. Here we examined the mechanism of induction of ISGs and showed this occurred in two phases. The first phase was transient (0-24hpi) induced mainly by extracellular vesicles, and one of its component proteins HSP90α, contained within the HIV inoculum. The second dominant and persistent phase (>48hpi) was induced via newly transcribed HIV RNA and sensed via RIGI, as shown by the reduction in ISG expression after the knock down of the RIGI adaptor, MAVS, by siRNA and the inhibition of both the initiation and elongation of HIV transcription, by shRNA transcriptional silencing. We further defined the induction pathway, showing sequential HIV RNA stimulation via Tat, RIGI, MAVS, IRF1 and IRF7 also identified by siRNA knockdown. IRF1 also plays a key role in the first phase. We also showed that the ISGs, IFITs 1-3 inhibited HIV production, measured as extracellular infectious virus. All induced antiviral ISGs probably lead to restriction of HIV replication in macrophages, contributing to a persistent, non-cytopathic infection while the inhibition of interferon facilitates spread to adjacent cells. Both may influence the size of macrophage HIV reservoirs in vivo. Elucidating the mechanisms of ISG induction may help devise immunotherapeutic strategies to limit the size of these reservoirs. IMPORTANCE HIV, like other viruses, manipulates the antiviral interferon and interferon stimulated gene (ISG) system to facilitate its initial infection and establishment of viral reservoirs. HIV specifically inhibits all type l and lll interferons in its target cells, including, macrophages, dendritic cells and T cells. It also induces a subset of over 20 ISGs of differing composition in each cell target. This occurs in two temporal phases in macrophages. Extracellular vesicles contained within the inoculum induced the first and transient phase of ISGs. Newly transcribed HIV RNA induced the second and dominant ISG phase and here the full induction pathway is defined. Therefore, HIV nucleic acids, which are potent inducers of interferon and ISGs, are initially concealed and antiviral ISGs are not fully induced until replication is well established. Theses antiviral ISGs may contribute to the persistent infection in macrophages and to the establishment of viral reservoirs in vivo.Najla Nasr, Abdullateef A. Alshehri, Thomas K. Wright, Maryam Shahid, Bonnie M. Heiner, Andrew N. Harman, Rachel A. Botting, Karla J. Helbig, Michael R. Beard, Kazuo Suzuki, Anthony D. Kelleher, Paul Hertzog, Anthony L. Cunningha

Viperin binds STING and enhances the type-I interferon response following dsDNA detection

First published: 01 November 2020Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin's broad antiviral activity by demonstrating the protein's ability to synergistically enhance the innate immune dsDNA signalling pathway to limit viral infection. Viperin co-localised with the key signalling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin's necessity to bind the cytosolic iron-sulphur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signalling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signalling pathway; which results in an enhanced antiviral state. We also provide evidence for viperin's radical SAM enzymatic activity to self-limit its immunomodulatory functions. These data further define viperin's role as a positive regulator of innate immune signalling, offering a mechanism of viperin's broad antiviral capacity.Keaton M Crosse, Ebony A Monson, Arti B Dumbrepatil, Monique Smith, Yeu-Yang Tseng, Kylie H Van der Hoe