Immunological studies of fragments of herpes simplex virus type 2 glycoprotein B expressed in Escherichia coli

The herpes simplex virus (HSV) envelope glycoprotein gB is known to be highly immunogenic. Related glycoproteins, with conserved amino acid sequence, occur in members representative of all the herpesvirus subfamilies. In this work the immunogenicity of HSV-2 gB has been studied using molecular cloning techniques and in vivo and in vitro immuno-assays. A 4.7kbp fragment of HSV-2 representing 0.345-0.376 MU was shown to hybridise to an HSV-2 messenger RNA of 3.2kb. This translated in vitro to a polypeptide of 92kDa which comigrated on SDS PAGE with immunoprecipitated gB-2. A restriction map of the 4.7kbp DNA was subsequently shown to correspond to a published gB-2 gene sequence. Fragments of this DNA were expressed fused to β-galactosidase in E.coli by random cloning into the vector pXY460. A number of HSV-2 serologically positive clones were characterised by DNA sequencing. The gB-2 specific sequences expressed by three clones were unambiguously determined. Their respective fusion proteins were immunopurified by anti-β-galactosidase affinity chromatography. These antigens were assayed in vitro by lymphoproliferative responses and in vivo by delayed-type hypersensitivity and protection studies. Ag59, representing gB-2 codons 339-394, was the most antigenic and immunogenic of the recombinant antigens and provided 36% protection in a 10x LD50 lethal challenge

Genome editing in poultry - opportunities and impacts

Poultry products (meat and eggs) are a major source of animal protein on which the world is increasingly reliant to feed a rapidly growing population. Improved breeds and advances in farm management practices have had a large impact on the poultry industry. For example, using current genetic stock and production practices, broiler chickens can weigh 2 kg in about 34 days. Forty-five years ago it would have typically taken over 60 days. These impressive advances have been made using traditional selective breeding methods and more recently by using genomics. Now, with the availability of precision genome engineering tools there are new opportunities to improve poultry production above and beyond those achievable by traditional means. One major opportunity is disease resilience, particularly for viral diseases such as avian influenza that has devastating impacts on the poultry industry. Resilience to specific diseases can be a notoriously difficult trait to select for using traditional breeding and the latest technologies that precisely edit the genome have created new ways to address this challenge

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

Bioprospecting for biological control agents for invasive tilapia in Australia

Originating in Africa, tilapia (Pisces, Cichlidae) now have a worldwide distribution and are both a prime model system for evolutionary biology and an important aquaculture species in over 135 countries. In contrast, Mozambique tilapia (Oreochromis mossambicus) is also listed in the top 100 of the world's worst invasive alien species and has been documented to have severe impacts on freshwater ecosystems primarily through displacement of native species and habitat alteration. In Australia, both O. mossambicus and the lesser-known spotted tilapia (Tilapia mariae) have established significant populations within Queensland waters, and recent incursions into northern New South Wales are of great concern. Eradication attempts using a combination of electrofishing and piscicide (poison) are rarely successful in open waterways, and given their invasive nature, there is a lack of demonstrated broad-scale effective control mechanisms for tilapia. Biological control (biocontrol), where it is feasible can be a cost-effective, a safe (species specific) and practical solution to managing invasive species because it does not require reapplication of chemicals or poisons, and once established should be self-sustaining. Based on the development of previous viral biocontrol strategies for rabbits and carp, we used a robust assessment framework for bioprospecting of biocontrol agents and found that tilapia lake virus (TiLV), and possibly tilapia parvovirus (TiPV), may offer the potential for biocontrol for invasive tilapia in Australia. TiLV causes high mortality in wild and cultured tilapia, but not in other species, and spreads through a waterborne route - an important transmission pathway for a successful viral biocontrol of fish. However, safety and efficacy, two major concerns for a successful biocontrol virus, need to be taken into consideration before the use of any exotic biocontrol virus is considered. Herein, we describe a systematic approach to assess known pathogens for their suitability as potential agents for biological control of tilapia and outline the possible next steps to further investigate the top candidates

miRNA_targets : a database for miRNA target predictions in coding and non-coding regions of mRNAs

AbstractMicroRNAs (miRNAs) are small non-coding RNAs that play a role in post-transcriptional regulation of gene expression in most eukaryotes. They help in fine-tuning gene expression by targeting messenger RNAs (mRNA). The interactions of miRNAs and mRNAs are sequence specific and computational tools have been developed to predict miRNA target sites on mRNAs, but miRNA research has been mainly focused on target sites within 3′ untranslated regions (UTRs) of genes. There is a need for an easily accessible repository of genome wide full length mRNA — miRNA target predictions with versatile search capabilities and visualization tools. We have created a web accessible database of miRNA target predictions for human, mouse, cow, chicken, Zebra fish, fruit fly and Caenorhabditis elegans using two different target prediction algorithms, The database has target predictions for miRNA's on 5′ UTRs, coding region and 3′ UTRs of all mRNAs. This database can be freely accessed at http://mamsap.it.deakin.edu.au/mirna_targets/

Identifying knowledge gaps for gene drive research to control invasive animal species: The next CRISPR step

Invasive animals have been linked to the extinctions of native wildlife, and to significant agricultural financial losses or impacts. Current approaches to control invasive species require ongoing resources and management over large geographic scales, and often result in the short-term suppression of populations. New and innovative approaches are warranted. Recently, the RNA guided gene drive system based on CRISPR/Cas9 is being proposed as a potential gene editing tool that could be used by wildlife managers as a non-lethal addition or alternative to help reduce pest animal populations. While regulatory control and social acceptance are crucial issues that must be addressed, there is an opportunity now to identify the knowledge and research gaps that exist for some important invasive species. Here we systematically determine the knowledge gaps for pest species for which gene drives could potentially be applied. We apply a conceptual ecological risk framework within the gene drive context within an Australian environment to identify key requirements for undertaking work on seven exemplar invasive species in Australia. This framework allows an evaluation of the potential research on an invasive species of interest and within a gene drive and risk context. We consider the currently available biological, genetic and ecological information for the house mouse, European red fox, feral cat, European rabbit, cane toad, black rat and European starling to evaluate knowledge gaps and identify candidate species for future research. We discuss these findings in the context of future thematic areas of research worth pursuing in preparation for a more formal assessment of the use of gene drives as a novel strategy for the control of these and other invasive species. Keywords: Invasive species, Gene drive, CRISPR, Pest management, Island