GARP and EARP are required for efficient BoHV-1 replication as identified by a genome wide CRISPR knockout screen

The advances in gene editing bring unprecedented opportunities in high throughput functional genomics to animal research. Here we describe a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome. Using it, we conducted genome wide screens during Bovine Herpes Virus type 1 (BoHV-1) replication and compiled a list of pro-viral and anti-viral candidates. These candidates might influence multiple aspects of BoHV-1 biology such as viral entry, genome replication and transcription, viral protein trafficking and virion maturation in the cytoplasm. Some of the most intriguing examples are VPS51, VPS52 and VPS53 that code for subunits of two membrane tethering complexes, the endosome-associated recycling protein (EARP) complex and the Golgi-associated retrograde protein (GARP) complex. These complexes mediate endosomal recycling and retrograde trafficking to the trans Golgi Network (TGN). Simultaneous loss of both complexes in MDBKs resulted in greatly reduced production of infectious BoHV-1 virions. We also found that viruses released by these deficient cells severely lack VP8, the most abundant tegument protein of BoHV-1 that are crucial for its virulence. In combination with previous reports, our data suggest vital roles GARP and EARP play during viral protein packaging and capsid re-envelopment in the cytoplasm. It also contributes to evidence that both the TGN and the recycling endosomes are recruited in this process, mediated by these complexes. The btCRISPRko.v1 library generated here has been controlled for quality and shown to be effective in host gene discovery. We hope it will facilitate efforts in the study of other pathogens and various aspects of cell biology in cattle.</p

Ovine herpesvirus-2 encoded microRNAs target virus genes involved in virus latency

Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is poorly understood. We previously identified eight miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF 20 (cell cycle inhibition), ORF 50 (reactivation) and ORF 73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5’ UTRs of ORF 20 and ORF 73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8 respectively, and the 3’UTR of ORF 50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2 infected bovine T cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF 50 and a smaller but non-significant decrease in ORF 20. However, we were unable to demonstrate a decrease in ORF 73. MCF is a disease of dysregulated lymphocyte proliferation, miRNA inhibition of ORF 20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORFs 50 and 73 play opposing roles in latency, it has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation in conditions that are unfavourable for viral replication, our data would support this hypothesis

The Fontan epidemic: population projections from the Australia and New Zealand Fontan registry

Background: The number and age demographic of the future Fontan population is unknown

Hemoglobin determination with paired emitter detector diode

Two ordinary green light-emitting diodes used as light emitter and detector coupled with simple voltmeter form a complete, cost-effective prototype of a photometric hemoglobinometer. The device has been optimized for cuvette assays of total hemoglobin (Hb) in diluted blood using three different chemical methods recommended for the needs of clinical analysis (namely Drabkin, lauryl sulfate, and dithionite methods). The utility of developed device for real analytics has been validated by the assays of total Hb content in human blood. The results of analysis are fully compatible with those obtained using clinically recommended method and clinical analyzer

Expression of Ovine Herpesvirus -2 Encoded MicroRNAs in an Immortalised Bovine - Cell Line

Ovine herpesvirus-2 (OvHV-2) infects most sheep, where it establishes an asymptomatic, latent infection. Infection of susceptible hosts e.g. cattle and deer results in malignant catarrhal fever, a fatal lymphoproliferative disease characterised by uncontrolled lymphocyte proliferation and non MHC restricted cytotoxicity. The same cell populations are infected in both cattle and sheep but only in cattle does virus infection cause dysregulation of cell function leading to disease. The mechanism by which OvHV-2 induces this uncontrolled proliferation is unknown. A number of herpesviruses have been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. We hypothesised that OvHV-2 encodes miRNAs and that these play a role in pathogenesis. Analysis of massively parallel sequencing data from an OvHV-2 persistently-infected bovine lymphoid cell line (BJ1035) identified forty-five possible virus-encoded miRNAs. We previously confirmed the expression of eight OvHV-2 miRNAs by northern hybridization. In this study we used RT-PCR to confirm the expression of an additional twenty-seven OvHV-2-encoded miRNAs. All thirty-five OvHV-2 miRNAs are expressed from the same virus genome strand and the majority (30) are encoded in an approximately 9 kb region that contains no predicted virus open reading frames. Future identification of the cellular and virus targets of these miRNAs will inform our understanding of MCF pathogenesis