High-Fidelity Long-Read Sequencing of an Avian Herpesvirus Reveals Extensive Intrapopulation Diversity in Tandem Repeat Regions.

Comparative genomic studies of Marek's disease virus (MDV) have suggested that attenuated and virulent strains share >98% sequence identity. However, these estimates fail to account for variation in regions of the MDV genome harboring tandem repeats. To resolve these loci and enable assessments of intrapopulation diversity, we used a PacBio Sequel II platform to sequence MDV strains CVI988/Rispens (attenuated), HPRS-B14 (virulent), Md5 (very virulent) and 675A (very virulent plus).

Comparative analysis of multiple consensus genomes of the same strain of Marek's disease virus reveals intrastrain variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloging divergent nucleotides between strains with different phenotypes. However, most comparative genomic studies of MDV rely on previously published consensus genomes despite the confirmed existence of MDV strains as mixed viral populations. To assess the reliability of interstrain genomic comparisons relying on published consensus genomes of MDV, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates.

Comparative Analysis of Multiple Consensus Genomes of the Same Strain of Marek's Disease Virus Reveals Intrastrain Variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloguing divergent nucleotides between strains with different phenotypes. However, each MDV strain is typically represented by a single consensus genome despite the confirmed existence of mixed viral populations. To assess the reliability of single-consensus interstrain genomic comparisons, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates.

Marek's Disease in an Indian Peafowl (Pavo cristatus) with Clinical Ocular Disease and Paraparesis.

Marek's disease (MD) is caused by virulent strains of Gallid alphaherpesvirus type 2 (MD virus serotype 1; MDV 1) and frequently causes a lymphoproliferative disorder in poultry and other galliform birds worldwide. However, within the peafowl (Phasianinae) subfamily, there are only rare confirmed reports of MD. Here we report MD in an Indian peafowl (Pavo cristatus), which clinically presented with hindlimb paraparesis and intraocular swelling of the right eye.

Rapid, Sensitive, and Species-Specific Detection of Conventional and Recombinant Herpesvirus of Turkeys Vaccines Using Loop-Mediated Isothermal Amplification Coupled With a Lateral Flow Device Readout.

Marek's disease, an economically important disease of chickens caused by virulent serotype 1 strains of the Marek's disease virus (MDV-1), is effectively controlled in the field by live attenuated vaccine viruses including herpesvirus of turkeys (HVT)-both conventional HVT (strain FC126) and, in recent years, recombinant HVT viruses carrying foreign genes from other avian viruses to protect against both Marek's disease and other avian viral diseases. Testing to monitor and confirm successful vaccination is important, but any such test must differentiate HVT from MDV-1 and MDV-2, as vaccination does not prevent infection with these serotypes. End-point and real-time PCR tests are widely used to detect and differentiate HVT, MDV-1 and MDV-2 but require expensive specialist laboratory equipment and trained operators.

Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System.

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens.

Molecular characterisation and phylogenetic analysis of Marek's disease virus in Turkish layer chickens.

1. There is no current data about the genotypes of Marek's disease virus (MDV) in Turkish poultry flocks; hence, this study was performed to analyse CVI988/Rispens, turkey herpesvirus (HVT) vaccine viruses and MDV field viruses as well as to perform phylogenetic analysis of MDV in Turkish layer chickens. 2.

Balance simplices of 3-species May-Leonard systems.

We investigate the existence of a two-dimensional invariant manifold that attracts all nonzero orbits in 3 species Lotka-Volterra systems with identical linear growth rates. This manifold, which we call the is the common boundary of the basin of repulsion of the origin and the basin of repulsion of infinity. The balance simplex is linked to ecological models where there is 'growth when rare' and competition for finite resources.

Molecular detection of Marek's disease virus in feather and blood samples from young laying hens in Colombia.

Marek's disease virus (MDV) is an immunosuppressive pathogen that can cause low production efficiency and high mortality rates in chickens. There is no current information on the MDV serotypes and pathotypes circulating in vaccinated commercial farms in Colombia where the birds are vaccinated in the incubator with Gallid herpesvirus (GaHV-2) and Meleagrid herpesvirus 1 (MeHV-1). Based on that, the main focus of this study was to understand the MDV's infection dynamics for the three known serotypes and to detect wild-virus pathogenic strains in 4-layer poultry farms in Antioquia.

The balance simplex in non-competitive 2-species scaled Lotka-Volterra systems.

Explicit expressions in terms of Gaussian Hypergeometric functions are found for a 'balance' manifold that connects the non-zero steady states of a 2-species, non-competitive, scaled Lotka-Volterra system by the unique heteroclinic orbits. In this model, the parameters are the interspecific interaction coefficients which affects the form of the solution used. Similar to the carrying simplex of the competitive model, this balance simplex is the common boundary of the basin of repulsion of the origin and infinity, and is smooth except possibly at steady states.

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing.

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT.

Application of CRISPR/Cas9 Gene Editing System on MDV-1 Genome for the Study of Gene Function.

Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek's disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants.

A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system.

Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV.

Industry-Wide Surveillance of Marek's Disease Virus on Commercial Poultry Farms.

Marek's disease virus is a herpesvirus of chickens that costs the worldwide poultry industry more than US$1 billion annually. Two generations of Marek's disease vaccines have shown reduced efficacy over the last half century due to evolution of the virus. Understanding where the virus is present may give insight into whether continued reductions in efficacy are likely.

Molecular characterization of Marek's disease virus in a poultry layer farm from Colombia.

Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective.

Real-time PCR for differential quantification of CVI988 vaccine virus and virulent strains of Marek's disease virus.

CVI988/Rispens vaccine, the 'gold standard' vaccine against Marek's disease in poultry, is not easily distinguishable from virulent strains of Marek's disease herpesvirus (MDV). Accurate differential measurement of CVI988 and virulent MDV is commercially important to confirm successful vaccination, to diagnose Marek's disease, and to investigate causes of vaccine failure. A real-time quantitative PCR assay to distinguish CVI988 and virulent MDV based on a consistent single nucleotide polymorphism in the pp38 gene, was developed, optimised and validated using common primers to amplify both viruses, but differential detection of PCR products using two short probes specific for either CVI988 or virulent MDV.

Mathematical Model of Ammonia Handling in the Rat Renal Medulla.

The kidney is one of the main organs that produces ammonia and release it into the circulation. Under normal conditions, between 30 and 50% of the ammonia produced in the kidney is excreted in the urine, the rest being absorbed into the systemic circulation via the renal vein. In acidosis and in some pathological conditions, the proportion of urinary excretion can increase to 70% of the ammonia produced in the kidney.

Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens.

Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom is that natural selection will remove highly lethal pathogens if host death greatly reduces transmission. Vaccines that keep hosts alive but still allow transmission could thus allow very virulent strains to circulate in a population. Here we show experimentally that immunization of chickens against Marek's disease virus enhances the fitness of more virulent strains, making it possible for hyperpathogenic strains to transmit.

Arterial ammonia levels in cirrhosis are determined by systemic and hepatic hemodynamics, and by organ function: a quantitative modelling study.

Background & Aims: Hyperammonaemia is a common complication of chronic liver failure. Two main factors are thought to underlie this complication: a loss of hepatic detoxification function and the development of portosystemic shunting. However, few studies have tried to quantify the importance of portosystemic shunting.

Relationship between levels of very virulent MDV in poultry dust and in feather tips from vaccinated chickens.

To assess the effect of various vaccine strains on replication and shedding of virulent Marek's disease virus from experimentally infected chickens, quantitative PCR (q-PCR) methods were developed to accurately quantify viral DNA in infected chickens and in the environment in which they were housed. Four groups of 10 chickens, kept in poultry isolators, were vaccinated at 1 day old with one of four vaccines covering each of the three vaccine serotypes, then challenged with very virulent MDV strain Md5 at 8 days of age. At regular time-points, feather tips were collected from each chicken and poultry dust was collected from the air-extract prefilter of each isolator.

Induction of lymphomas by inoculation of Marek's disease virus-derived lymphoblastoid cell lines: prevention by CVI988 vaccination.

Lymphoblastoid cell lines 265(L) and 990(O) are monoclonal lymphomas, derived respectively from liver and ovarian tumours, generated in inbred P-line (MHC B(19)/B(19)) chickens infected with RB-1B strain of Marek's disease virus (MDV) and pRB-1B5 BAC clone respectively. These were inoculated into inbred, MDV-susceptible, P-line chickens by intra-venous or intra-abdominal routes. Additional groups of birds were vaccinated using 1000 plaque-forming units of CVI988 vaccine 8 days prior to inoculation of the cell lines.

Quantifying the likelihood of co-existence for communities with asymmetric competition.

Trade-offs in performance of different ecological functions within a species are commonly offered as an explanation for co-existence in natural communities. Single trade-offs between competitive ability and other life history traits have been shown to support a large number of species, as a result of strong competitive asymmetry. We consider a single competition-fecundity trade-off in a homogeneous environment, and examine the effect of the form of asymmetry on the likelihood of species co-existing.