Temperature-induced reactivation of Marek's disease virus-transformed T cells ex vivo.

Marek's disease virus (MDV) establishes latency in chicken T lymphocytes that can lead to T cell transformation and cancer. Transformed Marek's disease chicken cell lines (MDCCs) can be expanded ex vivo and provide a valuable model to study latency, transformation, and reactivation. Here, we developed MDCCs from chickens infected with MDV that fluoresce during lytic replication and reactivation. Sodium butyrate treatment increased fluorescent protein expression as evidenced by fluorescent microscopy, flow cytometry, and western blotting; however, it caused significant apoptosis and necrosis. Treatment of MDCCs by decreasing the temperature resulted in robust MDV reactivation without significant induction of apoptosis and necrosis. Furthermore, MDV reactivation was significantly affected by the time in culture that can affect downstream reactivation analyses. In all, our data show that fluorescent protein expression during reactivation is a robust tool to examine viral replication in live cells ex vivo, and temperature treatment is an efficient technique to induce reactivation without punitive effects on cell viability seen with chemical treatment.

The Conserved Herpesviridae Protein Kinase (CHPK) of Gallid alphaherpesvirus 3 (GaHV3) Is Required for Horizontal Spread and Natural Infection in Chickens.

We have formerly identified the conserved herpesvirus protein kinase (CHPK) as essential for horizontal transmission of Marek's disease virus (MDV). Thus far, it has been confirmed that the mutation of the invariant lysine (K) of CHPKs abrogates kinase activity and that CHPK activity is required for MDV horizontal transmission. Since CHPK is conserved among all members of the Herpesviridae, we hypothesized that CHPK, and specifically its kinase activity, is important for the horizontal transmission of other herpesviruses. To test this hypothesis, we utilized our experimental and natural infection model in chickens with MD vaccine strain 301B/1 of Gallid alphaherpesvirus 3 (GaHV3). First, we mutated the invariant lysine (K) 157 of 301B/1 CHPK to alanine (A) and determined whether it was required for horizontal transmission. To confirm the requirement of 301B/1 CHPK activity for transmission, a rescued virus was generated in which the A157 was changed back to a K (A157K). Despite both the CHPK mutant (K157A) and rescuant (A157K) viruses having replication defects in vivo, only the CHPK mutant (K157A) was unable to spread to contact chickens, while both wild-type and rescuant (A157K) viruses transmitted efficiently, confirming the importance of CHPK activity for horizontal spread. The data confirm that CHPK is required for GaHV3 transmission and suggest that the requirement of avian CHPKs for natural infection is conserved.

The requirement of glycoprotein C (gC) for interindividual spread is a conserved function of gC for avian herpesviruses.

We have formerly shown that glycoprotein C (gC) of Gallid alphaherpesvirus 2, better known as Marek's disease (MD) alphaherpesvirus (MDV), is required for interindividual spread in chickens. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized gC was important for interindividual spread of other alphaherpesviruses. To test this hypothesis, we first generated a fluorescent protein tagged clone of Gallid alphaherpesvirus 3 MD vaccine strain 301B/1 to track virus replication in cell culture and chickens using fluorescent microscopy. Following validation of this system, we removed the open reading frame of 301B/1 gC from the genome and determined whether it was required for interindividual spread using experimental and natural infection studies. Interindividual spread of MD vaccine 301B/1 was abrogated by removal of 301B/1 gC. Rescuent virus in which 301B/1 gC was inserted back into the genome efficiently spread among chickens. To further study the conserved function of gC, we replaced 301B/1 gC with MDV gC and this virus also efficiently spread in chickens. These data suggest the essential function of alphaherpesvirus gC proteins is conserved and can be exploited during the generation of future vaccines against MD that affects the poultry industry worldwide.

Evidence of binaural integration benefits following ARIA training for children and adolescents diagnosed with amblyaudia.

OBJECTIVE: The purpose of this study was to demonstrate the efficacy of Auditory Rehabilitation for Interaural Asymmetry (ARIA) to improve dichotic listening scores in children and adolescents diagnosed with amblyaudia and other binaural integration deficits. DESIGN: The study is a field experiment without randomisation. STUDY: Participants placed into groups based on dichotic listening test scores received four sessions of ARIA training. Baseline scores were compared to performance during the final session of training and to scores obtained 2 or more months after completion of ARIA. SAMPLE: A total of 125 children participated at five different clinical sites. RESULTS: Dichotic listening scores improved across all participants. Post hoc analyses demonstrated highly significant gains in non-dominant ear performance and reductions of interaural asymmetry among participants diagnosed with amblyaudia at both post-ARIA measurements. Participants in other diagnostic groups also showed significant benefits for some post-ARIA measures. CONCLUSIONS: Results demonstrate that ARIA training is an effective method for improving binaural integration skills among children and adolescents identified with dichotic listening weaknesses during assessments for auditory processing disorder (APD), especially for those diagnosed with amblyaudia. Benefits achieved following ARIA training remain stable across several months.

Diagnosis of amblyaudia in children referred for auditory processing assessment.

Children (n = 141) referred to 5 clinical sites for auditory processing disorder assessment were tested with two dichotic listening tests, one with word pairs and the other with pairs of digits, as part of a comprehensive diagnostic battery. Scores from the Randomized Dichotic Digits Test and the Dichotic Words Test were compared to age-appropriate norms and used to place children into one of four diagnostic categories (normal, dichotic dysaudia, amblyaudia, or amblyaudia plus) or to identify them as undiagnosed. Results from the two dichotic tests led to diagnosis of 56% of the children tested, leaving 44% undiagnosed. When results from a third dichotic listening test were used as a tie-breaker among originally undiagnosed children, a total of 79% of the children's scores were placed into diagnostic categories (13% normal, 19% dichotic dysaudia, 35% amblyaudia, 12% amblyaudia plus). Amblyaudia, a binaural integration deficit evident only from dichotic listening test results, was most prevalent (35% + 12% = 47%) in this population of children suspected of auditory processing weaknesses. Since amblyaudia responds to treatment with Auditory Rehabilitation for Interaural Asymmetry (ARIA), clinicians are guided through the protocol for identifying diagnostic categories so that they can make appropriate referrals for rehabilitation.

Telomere maintenance mechanisms in malignant peripheral nerve sheath tumors: expression and prognostic relevance.

This study investigated the prevalence and the prognostic relevance of the 2 known telomere maintenance mechanisms (TMMs), telomerase activity (TA) and alternative lengthening of telomeres (ALT), in malignant peripheral nerve sheath tumors (MPNST). In 57 specimens from 49 patients with MPNST (35 sporadic, 14 neurofibromatosis type 1-related), TA was determined using the telomeric repeat amplification protocol, and ALT was detected by assaying ALT-associated promyelocytic leukemia bodies (APB) and terminal restriction fragment (TRF) length distribution. TA or ALT (defined on the basis of APB) alone was found in 24.6% or 26.3% of the lesions, respectively, whereas 6 cases (10.5%) were TA+/ALT+. A concordance between APB and TRF results in defining the ALT status was observed in 44 of 57 cases (77.2%; P < .0001). TA was more frequently expressed in samples from patients with neurofibromatosis type 1 than in those with sporadic disease (60% vs 29.4%, P = 0.087). In the overall series, TA proved to be prognostic for 5-year disease-specific death (hazard ratio, 3.78; 95% confidence interval [CI], 1.60-8.95; P = .002), even when adjusted for the presence of neurofibromatosis type 1 (hazard ratio, 4.22; 95% CI, 1.804-9.874; P = .001) and margin status after surgery (hazard ratio, 5.78; 95% CI, 2.19-15.26; P < .001). Conversely, ALT did not significantly affect clinical outcome of MPNST using either APB expression (hazard ratio, 1.25; 95% CI 0.54-2.89; P = 0.605) or TRF distribution (hazard ratio, 0.57; 95% CI, 0.17-1.96; P = .375) as the detection approach. Our results indicate for the first time that both TMMs, TA and ALT, are present in MPNST and differentially affect patient prognosis.

TCEAL7 inhibition of c-Myc activity in alternative lengthening of telomeres regulates hTERT expression.

Replicative senescence forms a major barrier to tumor progression. Cancer cells bypass this by using one of the two known telomere maintenance mechanisms: telomerase or the recombination-based alternative lengthening of telomeres (ALT) mechanism. The molecular details of ALT are currently poorly understood. We have previously shown that telomerase is actively repressed through complex networks of kinase, gene expression, and chromatin regulation. In this study, we aimed to gain further understanding of the role of kinases in the regulation of telomerase expression in ALT cells. Using a whole human kinome small interfering RNA (siRNA) screen, we highlighted 106 kinases whose expression is linked to human telomerase reverse transcriptase (hTERT) promoter activity. Network modeling of transcriptional regulation implicated c-Myc as a key regulator of the 106 kinase hits. Given our previous observations of lower c-Myc activity in ALT cells, we further explored its potential to regulate telomerase expression in ALT. We found increased c-Myc binding at the hTERT promoter in telomerase-positive compared with ALT cells, although no expression differences in c-Myc, Mad, or Max were observed between ALT and telomerase-positive cells that could explain decreased c-Myc activity in ALT. Instead, we found increased expression of the c-Myc competitive inhibitor TCEAL7 in ALT cells and tumors and that alteration of TCEAL7 expression levels in ALT and telomerase-positive cells affects hTERT expression. Lower c-Myc activity in ALT may therefore be obtained through TCEAL7 regulation. Thus, TCEAL7 may present an interesting novel target for cancer therapy, which warrants further investigation.

Multiple alternative splicing to exons II and III of viral interleukin-8 (vIL-8) in the Marek's disease virus genome: the importance of vIL-8 exon I.

The Marek's disease virus (MDV) Eco Q (Meq) and the interleukin-8 (IL-8) MDV homologue (vIL-8) genes, and the open reading frames RLORF5a and RLORF4 are encoded within the repeat long (IR(L) and TR(L)) regions of the MDV genome. The recent cloning and characterization of RLORF4 led to the identification of a RLORF4/vIL-8 splice variant using 3' rapid amplification of cDNA ends (RACE). Further characterization of 3'RACE products amplified with primers located within the Meq, RLORF5a, or RLORF4 genes showed the presence of many splice variants. Two novel Meq splice variants were detected, in addition to splice variants encoding portions of RLORF5a and RLORF4 combined with exons II and III of vIL-8 (RLORF5a/vIL-8 and RLORF4/vIL-8, respectively). Analysis of expression in MDV-infected chickens showed that the RLORF5a/vIL-8 and 3 of 4 RLORF4/vIL-8 transcripts were only expressed at 4 days post-infection. Since a number of transcripts encoded vIL-8 exons II and III, this suggested that exon I may be non-essential for vIL-8 function(s). Virus reconstituted from the oncogenic pRB-1B bacterial artificial chromosome with vIL-8 exon I deleted showed decreased early replication and reduced incidence of tumor development, similar to deletion mutants lacking the complete vIL-8 gene.

Impact of deletions within the Bam HI-L fragment of attenuated Marek's disease virus on vIL-8 expression and the newly identified transcript of open reading frame LORF4.

Marek's disease (MD) in chickens is caused by MD herpesvirus (MDV), which induces T cell lymphomas. The early pathogenesis of MDV infection is characterized by a primary infection in B lymphocytes followed by infection of activated T lymphocytes. It has been speculated that a MDV-encoded homologue of interleukin-8 (vIL-8) may be important to attract activated T lymphocytes to infected B lymphocytes. Recently, more virulent strains of MDV have emerged, named very virulent plus (vv+)MDV, that cause earlier and more prolonged cytolytic infections compared to less virulent strains. In this report, it was found that vIL-8 mRNA expression in vivo was increased in very virulent (vv) and vv+MDV strains compared to mild (m) and virulent (v) strains, and could not be detected in two attenuated MDV strains examined using very sensitive real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. In order to identify potential mechanisms for the increased vIL-8 mRNA expression in more virulent strains, and lack thereof in attenuated strains, the vIL-8 gene and putative promoter sequences upstream of the vIL-8 gene were compared from 10 different MDV strains, including attenuated derivatives. Only the JM-16 strain (both non-attenuated and attenuated) and attenuated 584A (584Ap80C) encoded a predicted vIL-8 gene sequence different from all other strains examined. Within the putative vIL-8 gene promoter sequence, there was little difference among the non-attenuated strains; however significant deletions were identified in the attenuated JM-16/p71, Md11 (R2/23), and 584Ap80C strains. Additionally, these deletions were located within a previously hypothetical open reading frame (ORF) named LORF4. Rapid amplification of cDNA ends identified a full-length transcript of LORF4 in the MDV-transformed lymphoblastoid cell line MSB-1, and deletions within this ORF caused truncated predicted proteins in 4 out of 6 attenuated MDV strains examined.