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In comparison to the embryo, very little work has been carried out on the expression and role of Hox genes in the adult animal. An expression profile of all 39 vertebrate Hox genes on a select panel of adult human tissues reveals that in fact these genes are widely expressed throughout the adult human and a colinear pattern of expression is displayed similar to that of the developing embryo. Of particular interest is the abundance of Hox genes that are expressed within the adult adrenal gland. Adrenal cortical cells are continuously renewed to sustain production of zonal steroids. Cell proliferation occurs at the periphery of the cortex and cells are then displaced centripetally, phenotypically switching as they migrate through the gland before undergoing apoptosis at the zona reticularis/medullary boundary. It is still unclear which mechanisms cause the cells to differentiate as they cross the zonal boundaries and we hypothesise that Hox genes may be involved in the phenotypic switching of the adrenocortical cells. In situ hybridisation experiments were carried out on adult rat adrenal gland sections and Hox gene expression was localized within the zonal borders, coinciding with the localization of cells that undergo phenotypic differentiation, and thus supporting our hypothesis that Hox genes may be involved in the phenotypic switching of the adrenocortical cells. As in the developing embryo, the genes display colinear expression with the 3' Hox genes being expressed within the outer gland and the 5' genes within the inner zones. [ABSTRACT FROM AUTHOR]

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia, and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis than from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease. [ABSTRACT FROM PUBLISHER]

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 balance simplex, 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. By including alternative food sources for predators we cater for predator-prey type models. In the case that the model is competitive, the balance simplex coincides with the carrying simplex which is an unordered manifold (no two points may be ordered componentwise), but for non-competitive models the balance simplex need not be unordered. The balance simplex of our models contains all limit sets and is the graph of a piecewise analytic function over the unit probability simplex.

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. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

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.

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. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

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. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

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. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines., (Copyright © 2018 The Pirbright Institute. Published by Elsevier Ltd.. All rights reserved.)

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. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo., (© 2016 Poultry Science Association Inc.)

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. Mechanisms regulating the balance between urinary excretion and renal vein release are not fully understood. We developed a mathematical model that reflects current thinking about renal ammonia handling in order to investigate the role of each tubular segment and identify some of the components which might control this balance. The model treats the movements of water, sodium chloride, urea, NH3 and [Formula: see text], and non-reabsorbable solute in an idealized renal medulla of the rat at steady state. A parameter study was performed to identify the transport parameters and microenvironmental conditions that most affect the rate of urinary ammonia excretion. Our results suggest that urinary ammonia excretion is mainly determined by those parameters that affect ammonia recycling in the loops of Henle. In particular, our results suggest a critical role for interstitial pH in the outer medulla and for luminal pH along the inner medullary collecting ducts.

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. Here, we used a theoretical approach to test the hypothesis that the development of portosystemic shunting is sufficient to cause hyperammonaemia in cirrhosis., Methods: Two mathematical models are developed. The first one describes the main vascular resistances of the circulation and is used to provide scenarios for the distributions of organ blood flow in cirrhosis, which are necessary to run the second model. The second model predicts arterial ammonia levels resulting from ammonia metabolism in gut, liver, kidney, muscle and brain, and the distribution of organ blood flow., Results: The fraction of gastrointestinal blood flow shunted through collaterals was estimated to be 41% in mild cirrhosis, 69% in moderate and 85% in severe cases. In the second model, the redistribution of organ blood flow associated with severe cirrhosis was sufficient to cause hyperammonaemia, even when the hepatic detoxification function and the ammonia production were set to normal., Conclusions: The model indicates that the development of portosystemic shunting in cirrhosis is sufficient to cause hyperammonaemia. Interventions that reduce the fraction of shunting may be future targets of therapy to control severity of hyperammonaemia., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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. We find conditions that allow co-existence of two species for a general competition function, and show that (1) two species can only co-exist if the competition function is sufficiently steep when the species are similar; (2) when competition is determined by a linear function, no more than two species can co-exist; (3) when the competition between two individuals is determined by a discontinuous step function, this single trade-off can support an arbitrarily large number of species. Further, we show analytically that as the degree of asymmetry in competition increases, the probability of a given number of species co-existing also increases, but note that even in the most favourable conditions, large numbers of species co-existing along a single trade-off is highly unlikely. On this basis, we suggest it is unlikely that single trade-offs are able to support high levels of bio-diversity without interacting other processes.

The 'gold standard' vaccine against Marek's disease in poultry is the CVI988/Rispens virus, which is not easily distinguishable, antigenically or genetically, from virulent Marek's disease herpesvirus. Accurate differential measurement of the CVI988 vaccine and virulent viruses is important to investigate mechanisms of vaccinal protection. Minimal sequence differences between CVI988 and virulent MDV strains restrict the application of molecular diagnostic methods such as real-time PCR to distinguish between these viruses. The use of bacterial-artificial-chromosome (BAC) cloned CVI988 virus, which carries the BAC vector sequences in place of the U(s)2 gene, allows its differential quantification from virulent strains using real-time PCR assays that target the BAC vector sequence and the U(S)2 gene respectively. These novel assays allowed investigation of replication of both serotype-1 vaccine virus (cloned CVI988) and challenge virus (RB-1B strain) in tissues of individual chickens in an experimental vaccination-challenge model of Marek's disease., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A widely used vaccine against Marek's disease (MD) in poultry is the virus SB-1, which is antigenically-related to the causative agent, Marek's disease herpesvirus. We recently cloned the SB-1 genome as an infectious bacterial artificial chromosome, BAC, (pSB-1). The protective efficacies and replication kinetics of pSB-1 and the parent strain (SB-1) were compared in an experimental model of MD induced by a virulent strain, RB-1B. Although vaccine virus replication and shedding was lower for pSB-1 than for SB-1, both vaccines reduced replication and shedding of RB-1B, and were equally effective in protecting chickens against MD. With the cloning of pSB-1, we have now generated full length genomic clones of MD vaccine virus strains belonging to each of the three serotypes. Vaccine viruses derived from each of these clones demonstrated protective efficacies at levels similar to those produced by the respective parent viruses, demonstrating their suitability to be used as vaccine candidates., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Marek's disease virus serotype 2 (Gallid herpesvirus 3) is a non-pathogenic alphaherpesvirus belonging to the Mardivirus genus, used widely in live vaccines against Marek's disease. Although the complete genome sequence of the MDV-2 strain HPRS-24 has been published, very little is known about the gene functions. As a first step for carrying out functional genomic analysis of MDV-2, the full-length genome of the MDV-2 vaccine strain SB-1 was cloned as an infectious bacterial artificial chromosome (BAC) clone pSB-1. Virus reconstituted from the pSB-1 clone showed morphological and growth characteristics in cell culture very similar to the parent virus. Generation of SB-1 constructs deleted in glycoprotein E and viruses expressing Citrine-UL35 fusion protein by the application of different BAC mutagenesis techniques demonstrated the amenability of the pSB-1 clone for reverse genetics approaches to identify molecular determinants associated with different biological features of this virus. The generation of replication-competent infectious clones of SB-1, together with those of CVI988 and herpesvirus of turkey strains described previously, completes the portfolio of generating infectious BAC clones of the MD vaccine strains belonging to all the three serotypes, paving the way for the application of reverse genetics for functional analysis of immunogenic determinants of these vaccines as well as for developing novel recombinant vectors.

In this study, we have identified an internal ribosome entry site (IRES) from the highly infectious herpesvirus Marek's disease virus (MDV). The IRES was mapped to the intercistronic region (ICR) of a bicistronic mRNA that we cloned from the MDV-transformed CD4(+) T-cell line MSB-1. The transcript is a member of a family of mRNAs expressed as immediate-early genes with two open reading frames (ORF). The first ORF encodes a 14-kDa polypeptide with two N-terminal splice variants, whereas the second ORF is contained entirely within a single exon and encodes a 12-kDa protein also known as RLORF9. We have shown that the ICR that separates the two ORFs functions as an IRES that controls the translation of RLORF9 when cap-dependent translation is inhibited. Deletion analysis revealed that there are two potential IRES elements within the ICR. Reverse genetic experiments with the oncogenic strain of MDV type 1 indicated that deletion of IRES-controlled RLORF9 does not significantly affect viral replication or MDV-induced mortality.

Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40-cre cassette within the loxP sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo. Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen.