Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma

Dual Infection and Superinfection Inhibition of Epithelial Skin Cells by Two Alphaherpesviruses Co-Occur in the Natural Host

Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2), better known as Marek’s disease virus (MDV), was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV), with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP) or monomeric RFP (mRFP) fused to the UL47 (VP13/14) herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1) has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be superinfected by wild-type VZV, this could potentiate recombination events of VZV as well

Herpesvirus Telomerase RNA(vTR)-Dependent Lymphoma Formation Does Not Require Interaction of vTR with Telomerase Reverse Transcriptase (TERT)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)n. Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity

Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia

Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells

Effective Treatment of Respiratory Alphaherpesvirus Infection Using RNA Interference

BACKGROUND: Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNA's), delivered intranasally, could prove a valuable alternative for infection control. siRNA's against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model. METHODOLOGY/PRINCIPAL FNDINGS: siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection. CONCLUSIONS/SIGNIFICANCE: siRNA treatment has potential for both prevention and early treatment of EHV-1 infections

Structural characteristics and antiviral activity of multiple peptides derived from MDV glycoproteins B and H

<p>Abstract</p> <p>Background</p> <p>Marek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB.</p> <p>Results</p> <p>Four peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC₅₀) of below 12 μM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 μl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further.</p> <p>Conclusions</p> <p>The five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.</p

Spleen transcriptome response to infection with avian pathogenic Escherichia coli in broiler chickens

<p>Abstract</p> <p>Background</p> <p>Avian pathogenic <it>Escherichia coli </it>(APEC) is detrimental to poultry health and its zoonotic potential is a food safety concern. Regulation of antimicrobials in food-production animals has put greater focus on enhancing host resistance to bacterial infections through genetics. To better define effective mechanism of host resistance, global gene expression in the spleen of chickens, harvested at two times post-infection (PI) with APEC, was measured using microarray technology, in a design that will enable investigation of effects of vaccination, challenge, and pathology level.</p> <p>Results</p> <p>There were 1,101 genes significantly differentially expressed between severely infected and non-infected groups on day 1 PI and 1,723 on day 5 PI. Very little difference was seen between mildly infected and non-infected groups on either time point. Between birds exhibiting mild and severe pathology, there were 2 significantly differentially expressed genes on day 1 PI and 799 on day 5 PI. Groups with greater pathology had more genes with increased expression than decreased expression levels. Several predominate immune pathways, Toll-like receptor, Jak-STAT, and cytokine signaling, were represented between challenged and non-challenged groups. Vaccination had, surprisingly, no detectible effect on gene expression, although it significantly protected the birds from observable gross lesions. Functional characterization of significantly expressed genes revealed unique gene ontology classifications during each time point, with many unique to a particular treatment or class contrast.</p> <p>Conclusions</p> <p>More severe pathology caused by APEC infection was associated with a high level of gene expression differences and increase in gene expression levels. Many of the significantly differentially expressed genes were unique to a particular treatment, pathology level or time point. The present study not only investigates the transcriptomic regulations of APEC infection, but also the degree of pathology associated with that infection. This study will allow for greater discovery into host mechanisms for disease resistance, providing targets for marker assisted selection and advanced drug development.</p

Temporal transcriptome changes induced by MDV in marek's disease-resistant and -susceptible inbred chickens

<p>Abstract</p> <p>Background</p> <p>Marek's disease (MD) is a lymphoproliferative disease in chickens caused by Marek's disease virus (MDV) and characterized by T cell lymphoma and infiltration of lymphoid cells into various organs such as liver, spleen, peripheral nerves and muscle. Resistance to MD and disease risk have long been thought to be influenced both by genetic and environmental factors, the combination of which contributes to the observed outcome in an individual. We hypothesize that after MDV infection, genes related to MD-resistance or -susceptibility may exhibit different trends in transcriptional activity in chicken lines having a varying degree of resistance to MD.</p> <p>Results</p> <p>In order to study the mechanisms of resistance and susceptibility to MD, we performed genome-wide temporal expression analysis in spleen tissues from MD-resistant line 6₃, susceptible line 7₂and recombinant congenic strain M (RCS-M) that has a phenotype intermediate between lines 6₃and 7₂after MDV infection. Three time points of the MDV life cycle in chicken were selected for study: 5 days post infection (dpi), 10dpi and 21dpi, representing the early cytolytic, latent and late cytolytic stages, respectively. We observed similar gene expression profiles at the three time points in line 6₃and RCS-M chickens that are both different from line 7₂. Pathway analysis using Ingenuity Pathway Analysis (IPA) showed that MDV can broadly influence the chickens irrespective of whether they are resistant or susceptible to MD. However, some pathways like cardiac arrhythmia and cardiovascular disease were found to be affected only in line 7₂; while some networks related to cell-mediated immune response and antigen presentation were enriched only in line 6₃and RCS-M. We identified 78 and 30 candidate genes associated with MD resistance, at 10 and 21dpi respectively, by considering genes having the same trend of expression change after MDV infection in lines 6₃and RCS-M. On the other hand, by considering genes with the same trend of expression change after MDV infection in lines 7₂and RCS-M, we identified 78 and 43 genes at 10 and 21dpi, respectively, which may be associated with MD-susceptibility.</p> <p>Conclusions</p> <p>By testing temporal transcriptome changes using three representative chicken lines with different resistance to MD, we identified 108 candidate genes for MD-resistance and 121 candidate genes for MD-susceptibility over the three time points. Genes included in our resistance or susceptibility genes lists that are also involved in more than 5 biofunctions, such as <it>CD8α</it>, <it>IL8</it>, <it>USP18</it>, and <it>CTLA4</it>, are considered to be important genes involved in MD-resistance or -susceptibility. We were also able to identify several biofunctions related with immune response that we believe play an important role in MD-resistance.</p