Comparative analysis of the complete genome sequence of the California MSW strain of myxoma virus reveals potential host adaptations

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.This work was funded in part by grant R01 AI093804 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. E.C.H. was supported by an NHMRC Australia Fellowship, and D.C.T. was supported by an ARC Future Fellowship

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome

Genome scale evolution of myxoma virus reveals host-pathogen adaptation and rapid geographic spread

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.This work was funded in part by grant R01 AI093804 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. E.C.H. is funded by an NHMRC Australia Fellowship. D.C.T. is funded by an ARC Future Fellowship

Most viral peptides displayed by class I MHC on infected cells are immunogenic

CD8+ T cells are essential effectors in antiviral immunity, recognizing short virus-derived peptides presented by MHC class I (pMHCI) on the surface of infected cells. However, the fraction of viral pMHCI on infected cells that are immunogenic has not been shown for any virus. To approach this fundamental question, we used peptide sequencing by high-resolution mass spectrometry to identify more than 170 vaccinia virus pMHCI presented on infected mouse cells. Next, we screened each peptide for immunogenicity in multiple virus-infected mice, revealing a wide range of immunogenicities. A surprisingly high fraction (>80%) of pMHCI were immunogenic in at least one infected mouse, and nearly 40% were immunogenic across more than half of the mice screened. The high number of peptides found to be immunogenic and the distribution of responses across mice give us insight into the specificity of antiviral CD8+ T cell responses.This work was supported by a Project Grant from the National Health and Medical Research Council Australia (NHMRC) (APP1084283) (to D.C.T., A.W.P., and N.P.C.); an NHMRC Senior Research Fellowship (APP1104329) (to D.C.T.); an NHMRC Principal Research Fellowship (APP1137739) (to A.W.P.); and a Viertel Fellowship, ARC Future Fellowship, and NHMRC Program Grant (APP1071916) (to N.L.L.G.)

Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA

CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens

CD11b+, Ly6G+ Cells Produce Type I Interferon and Exhibit Tissue Protective Properties Following Peripheral Virus Infection

The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b+Ly6C+Ly6G- monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b+Ly6C+Ly6G+ cells. The phenotype of the CD11b+Ly6C+Ly6G+ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b+Ly6C+Ly6G+ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b+Ly6C+Ly6G+ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G+ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b+Ly6C+Ly6G+ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction

Development of a smart livestock farming tool for identifying animal growth using artificial intelligence

Affordable tools with the ability to continuously monitor the growth rate of livestock animals are highly sought after by the livestock industries. This demand is driven by the potential for these tools to assist in improving animal welfare and production efficiency. In a rapidly growing population the demand for meat is escalating, especially in Asia, where the middle class is currently expanding. Meanwhile in the western world there is growing consumer concern surrounding animal husbandry, with certain organisations labelling some of the current husbandry practices cruel or sub-standard. The environmental impacts of livestock farming are also increasingly becoming scrutinised, pressuring researchers to find new methods to increase the efficiency of livestock nutrition, and improve health (disease prevention), reproductive and waste management practices. At the centre of these problems is the ever-changing individual animal as it continuously adapts to its surrounding environment and available resources. Livestock growth is a fundamental measure which can be used for diagnostic purposes in these areas, therefore the main objective of this study was to develop a system to automatically determine the growth of individual and groups of livestock animals (pigs) using welfare friendly and non-invasive methods. A machine vision system was selected to undertake this weight estimation task, whereby pigs’ body measurements are extracted from images and used to estimate their weight without physical interference. Reviews prompted the development of a methodology to determine the weight-estimation equations as a function of not just the animals’ body measurements but also their pose. Subsequently equations were generated from shapes that conformed closely to a specified reference template shape. Thus, to enhance precision during weight estimation the template shape was directly linked to the equation and pose validation aspects of the system. Filters were developed to provide recognition via the confirmation of the characteristic template shape and known body measurement and weight relationships. The shape filter ensured that 94% of weight estimates that passed through to output were within ±5 kg of the actual weight of the pig. Using the shape and limit filter in unison ensured that greater than 97% of the samples which passed had an weight estimate within ±5 kg of the actual weight of the pigs and 68% of the total number of samples were within ±2 kg. Statistical modelling was used to determine the importance of different body measurements in estimating weight. Subsequently a multivariate linear weight estimation equation was created to estimate pigs’ weight using a stepwise selection of variables. The multivariate linear equation estimated 2% more sample weights within ±2 kg error and 3% less sample weights greater than ±5 kg error than the closest non-linear equation. Software was written to automatically recognise pigs inside the field of view (FOV) of the camera and to extract 16 body measurements from the pigs’ body contours. Height was manually recorded from the back of a sample of pigs to determine its strength in weight estimation. Including the pig’s height in the weight-estimation equation did improve predictive performance with a 7.34 % improvement in the number of samples estimated within ±2 kg of the pigs’ actual weight compared to a multivariate equation without the height parameter. Although, this improvement was not significant enough to justify the additional practical development required to collect the height information automatically during the weight estimation process. Both off-line simulation and on-farm experiments were undertaken using data collected from commercial facilities. During an off-line simulation, the shape and dimension filters were applied across a dataset containing over 20,000 frame samples of over 500 pigs. Gut fill was used as a guide to determine a practical error margin for measuring the weight of individual pigs across the course of a day. The machine vision system was found to operate within an acceptable error margin of 50 % of the gut fill according to the equation and average shape template used during off-line simulations. As on average pigs in the weight-range of 45 to 115 kg had their live body weight estimated to within 3.16 % and 2.20 % of their actual live body weight, respectively. For pigs less than 45 kg in weight the piGUI system operated, on average, to within 67% of the weight attributed to gut fill (between ±1.07 and ±1.49 kg error). During off-line simulations, the percentage mean-relative error obtained by the piGUI system was between 5.1 and 3.7% for pigs in the weaner to grower weight range (15 to 45 kg) and less than or equal to 2.5% for grower finisher pigs between 45 and 115 kg. Thus, on average, the system was able to estimate the pig’s body mass with practical precision. The system labelled ‘piGUI’ was installed in pens at commercial facilities which housed pigs in group-sizes of between 10 and 160 pigs. During testing, the system determined the average weight of groups of pigs on a daily basis, tracking the group’s growth rate. In some trials, the pig’s weights were also estimated along with the weight deviation of the group. During a 22 day trial period the system estimated the average weight of a group of finisher pigs within 2.1%, on the seven days when the actual group weight was recorded from an electronic scale. No information was passed between successive days by the system. The diagnostic power of the piGUI system was also tested on-farm. A deflection away from the standard growth curve was recorded during two successive batches of grower pigs after reaching weights greater than ~45 kg. These growth deflections were believed to be caused by stress related directly or indirectly to temperature, as the summer temperatures reached over 38°C during these batches. The level of animal activity recorded by the system, the temperatures leading up to the deflection in growth and figures reported in literature support this theory. The piGUI system was also tested to see whether it could estimate the weight of sows in their early stages of pregnancy and whether it could detect changes in the body measurements of individual sows before and after giving birth. A group of eleven sows between day 71 and day 82 of pregnancy had their group weight estimated to within 0.1 kg of their actual group weight. Eighty-two percent of their individual weights were estimated within a practical range of ±5 kg of their actual weight. The metric body measurements of two Large White × Landrace sows were also recovered by the vision system before and after giving birth. The widths and lengths of the sows’ recorded by the vision system were consistent with those found in literature. Indicating that the device may be used to monitor sow weight and body morphology in future. The developed device was also tested at various locations within the pen environment. Radio Frequency Identification (RFID) was integrated into the system to determine whether bias in group estimates could occur as a result of the sampling region observed within the pen. A layout bias was discovered, caused by certain pigs visiting the FOV (containing the feeder) more frequently or for longer durations than others. Subsequently, feeding behaviour was determined using the RFID information collected and demand for the feeder was calculated for the pigs individually and as a group. The number of social interactions between pigs at the feeder was also determined, thus providing a method to identify social interaction and potentially the competitive nature of pigs automatically. A comparative study was undertaken between a commercial system ‘System-A’ and the piGUI system. System-A failed to correctly estimate the group average weight of the finisher pigs in the trials. It was apparent that necessary conversions were not taking place within System-A’s software to normalise the extracted body measurements to suit weight-estimation equation coefficients. It was found that, System-A’s growth data would require a multiplication by a scalar factor to adjust the growth data to valid weight ranges. Code within the piGUI software performed the necessary conversions automatically during initialisation and was not burdened by this limitation. The piGUI system estimated the group average weight to within 2.1% on each of the seven days when the actual weight of the pigs were determined using the electronic scale. On these days, System-A reported group average weight estimates in excess of 16 kg error of the actual group average weight. It was clear that the distribution of weight data recorded daily by the piGUI system was far more concentrated around a mean estimate value than system-A. The results of this PhD study demonstrate that the average weight of groups of pigs can be calculated with sufficient practical accuracy. The precision achieved during this study was better than reported in the literature and the precision of the system was also favourable compared to a commercially available system. Therefore the developed system can be used for practical purposes on commercial farms to determine the average weight and growth of groups of grower-finisher pigs

Automated camera-based height and flower detection for wheat and chickpea

The National Variety Trials (NVT) involve a yearly coordination of 630 grain trials conducted across 250 locations in Australia. At different stages of the crop season Trial Service Providers visually assess the attributes of the grain plants in each trial-plot to evaluate the growth and development of the different grain varieties. This involves manual measurements related to: (i) plant dimensions (height, canopy size); (ii) different stages of growth (seedling, tillering, jointing, boot and flowering); and (iii) germination rate. However, the availability of personnel to perform this monitoring is likely to be constrained to larger research stations. A camera-based remote crop monitoring system and image analysis algorithms have been developed to automatically capture images of the trial plots, identify flowers and extract plant growth. This camera system would be permanently installed at the trial site to continuously collect images of the plots throughout the crop season. The system will reduce labour requirements and travel costs associated with manual inspection of grain variety trials, and improve timeliness and accuracy of growth stage identification

Remote monitoring and automatic detection of grain crop attributes

This project has developed a proof-of-concept low cost internet-enabled camera-based monitoring and image processing system for National Variety Trials (NVT). The system will reduce labour requirements and travel costs associated with manual inspection of grain variety trials, and improve timeliness and accuracy of growth stage identification. A camera-based system remotely captures images of a plot, automatically detects plant growth and flowers for two grain crop types and displays results on a website. Manual measurements have been compared with image analysis results in collaboration with NVT Trial Service Providers