Latent gammaherpesvirus infection enhances type I IFN response and reduces virus spread in an influenza A virus co-infection model.

Infections with persistent or latent viruses alter host immune homeostasis and have potential to affect the outcome of concomitant acute viral infections such as influenza A virus (IAV). Gammaherpesviruses establish life-long infections and require an on-going immune response to control reactivation. We have used a murine model of co-infection to investigate the response to IAV infection in mice latently infected with the gammaherpesvirus MHV-68. Over the course of infection, latently infected BALB/c mice showed less weight loss, clinical signs, pulmonary cellular infiltration and expression of inflammatory mediators than naïve mice infected with IAV and had significantly more activated CD8+ T cells in the lungs. Four days after IAV infection, virus spread in the lungs of latently infected animals was significantly lower than in naïve animals. By 7 days after IAV infection latently infected lungs express elevated levels of cytokines and chemokines indicating they are primed to respond to the secondary infection. Investigation at an early time point showed that 24 h after IAV infection co-infected animals had higher expression of IFNß and Ddx58 (RIG-I) and a range of ISGs than mice infected with IAV alone suggesting that the type I IFN response plays a role in the protective effect. This effect was mouse strain dependent and did not occur in 129/Sv/Ev mice. These results offer insight into innate immune mechanisms that could be utilized to protect against IAV infection and highlight on-going and persistent viral infections as a significant factor impacting the severity of acute respiratory infections.

Radiographic Evaluation of Carpal Mechanics and the Scapholunate Angle in a Clenched Fist with Dynamic Computed Tomography Imaging.

Purpose: The long-term consequences of injuries to the scapholunate joint can severely limit hand function, and the potential for posttraumatic deformity makes early recognition of these injuries important. The purpose of this study was to evaluate the motion of the scapholunate joint in normal wrists through the radial and ulnar deviation using novel dynamic computed tomography (CT) imaging. Methods: Fifteen participants consented to have their uninjured wrists scanned. A protocol was designed to ensure adequate time, yet limited exposure, for volunteers. Participants began with the hand in a relaxed fist position and then proceeded to clench the hand in a full fist and relax. Once relaxed again, the wrist was maximally ulnarly deviated and then maximally radially deviated in a fluid motion. Dynamic CT imaging was captured throughout the range of motion. Results: The scapholunate angle was measured on dynamic wrist images. The mean range of the scapholunate angle that the wrists moved through was 37.2°-45.9°, and the mean midpoint angle was 41.2° ± 0.4°. All wrists had small, measurable differences in the scapholunate angle when moving from the maximum ulnar deviation to the maximal radial deviation. The average maximum angle change through the range is 11.7°, whereas the average minimum angle change was 0.9°. Conclusions: In this study, scapholunate angle calculations using dynamic wrist CT scans were within the range of accepted normal for the angle in uninjured wrists. With the increased focus on dynamic imaging for wrist motion, it may be possible to derive a standardized protocol for mapping the carpal motion that is clinically applicable and reproducible. Type of study/level of evidence: Diagnostic IV.

Regulation of Ov2 by virus encoded microRNAs.

Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is still poorly understood. We previously identified thirty five miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and are investigating the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. Analysis, using RNAHybrid predicted that two OvHV-2 encoded miRNAs, ovhv2-miR-17-10 and ovhv2-miR-61-1, target transcripts coding for the OvHV-2 bZIP protein Ov2. In other herpesvirus bZIP proteins are known to play important roles in lytic virus replication. Here we show by Flow cytometry and western blotting that ovhv2-miR-17-10 and ovhv2-miR-61-1, reduce the expression of Ov2 protein. The predicted target sites for both miRNAs within the Ov2 gene were disrupted whilst retaining the Ov2 coding sequence. Mutation of the ovhv2-miR-61-1 target sequence restored Ov2 protein expression levels to control levels confirming the identity of its target site. However, it was not possible to determine the binding site of ovhv2-miR-17-10 possibly due to potential G:U pairing introduced during the mutation process. The targeting of Ov2 by two virus-encoded miRNAs suggests an important regulatory role for Ov2 in OvHV-2 replication or reactivation.

Ov2 is a modulator of OvHV-2 RTA mediated gene expression.

Ovine herpesvirus-2 (OvHV-2) is the causative agent of the sheep-associated form of malignant catarrhal fever, a usually fatal lymphoproliferative disease of bison, deer and cattle. Malignant catarrhal fever is a major cause of cattle loss in Africa with approximately 7% affected annually; and in North America has significant impact on bison farming. Research into the mechanisms by which OvHV-2 induces disease in susceptible species has been hampered by a lack of a cell culture system for the virus. Ov2 is a bZIP protein encoded by OvHV-2. Proteins with bZIP domains in other herpesviruses, such as the Kaposi's sarcoma-associated herpesvirus K8 protein and the BZLF1 protein of Epstein-Barr virus are known to play important roles in lytic virus replication. Using a reporter based system, we demonstrate that Ov2 can modulate the activity of the major virus transactivator (Replication and Transcriptional Activator protein, RTA) to 1) drive expression of viral genes predicted to be required for efficient reactivation of the virus, including ORF49; and 2) differentially regulate the expression of the two virus encoded Bcl-2 homologues Ov4.5 and Ov9.

Gas-Phase Folding of a Prototypical Protonated Pentapeptide: Spectroscopic Evidence for Formation of a Charge-Stabilized ß-Hairpin.

Ultraviolet and infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy has been carried out in a tandem mass spectrometer to determine the three-dimensional structure of cryogenically cooled protonated C-terminally methyl esterified leucine enkephalin [YGGFL-OMe+H](+). By comparing the experimental IR spectrum of the dominant conformer with the predictions of DFT M05-2X/6-31+G(d) calculations, a backbone structure was assigned that is analogous to that previously assigned by our group for the unmodified peptide [ Burke, N.L.; et al. Int. J. Mass Spectrom. 2015 , 378 , 196 ], despite the loss of a C-terminal OH binding site that was thought to play an important role in its stabilization. Both structures are characterized by a type II' ß-turn around Gly(3)-Phe(4) and a γ-turn around Gly(2), providing spectroscopic evidence for the formation of a ß-hairpin hydrogen bonding pattern. Rather than disrupting the peptide backbone structure, the protonated N-terminus serves to stabilize the ß-hairpin by positioning itself in a pocket above the turn where it can form H-bonds to the Gly(3) and C-terminus C═O groups. This ß-hairpin type structure has been previously proposed as the biologically active conformation of leucine enkephalin and its methyl ester in the nonpolar cell membrane environment [ Naito, A.; Nishimura, K. Curr. Top. Med. Chem. 2004 , 4 , 135 - 143 ].

Transcriptome analysis of CNS immediately before and after the detection of PrP(Sc) in SSBP/1 sheep scrapie.

Sheep scrapie is a transmissible spongiform encephalopathy (TSE), progressive and fatal neurodegenerative diseases of the central nervous system (CNS) linked to the accumulation of misfolded prion protein, PrP(Sc). New Zealand Cheviot sheep, homozygous for the VRQ genotype of the PRNP gene are most susceptible with an incubation period of 193 days with SSBP/1 scrapie. However, the earliest time point that PrP(Sc) can be detected in the CNS is 125 days (D125). The aim of this study was to quantify changes to the transcriptome of the thalamus and obex (medulla) at times immediately before (D75) and after (D125) PrP(Sc) was detected. Affymetrix gene arrays were used to quantify gene expression in the thalamus and Illumina DGE-tag profiling for obex. Ingenuity Pathway Analysis was used to help describe the biological processes of scrapie pathology. Neurological disease and Cancer were common Bio Functions in each tissue at D75; inflammation and cell death were major processes at D125. Several neurological receptors were significantly increased at D75 (e.g. CHRNA6, GRM1, HCN2), which might be clues to the molecular basis of psychiatric changes associated with TSEs. No genes were significantly differentially expressed at both D75 and D125 and there was no progression of events from earlier to later time points. This implies that there is no simple linear progression of pathological or molecular events. There seems to be a step-change between D75 and D125, correlating with the detection of PrP(Sc), resulting in the involvement of different pathological processes in later TSE disease.

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.

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

Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (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. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (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 ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3' UTR of ORF50 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 ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 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 under conditions that are unfavourable for viral replication and our data supported this hypothesis.

The PrP(C) C1 fragment derived from the ovine A136R154R171PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrP(C) protein in vitro.

Expression of the cellular prion protein (PrP(C)) is crucial for the development of prion diseases. Resistance to prion diseases can result from reduced availability of the prion protein or from amino acid changes in the prion protein sequence. We propose here that increased production of a natural PrP α-cleavage fragment, C1, is also associated with resistance to disease. We show, in brain tissue, that ARR homozygous sheep, associated with resistance to disease, produced PrP(C) comprised of 25% more C1 fragment than PrP(C) from the disease-susceptible ARQ homozygous and highly susceptible VRQ homozygous animals. Only the C1 fragment derived from the ARR allele inhibits in-vitro fibrillisation of other allelic PrP(C) variants. We propose that the increased α-cleavage of ovine ARR PrP(C) contributes to a dominant negative effect of this polymorphism on disease susceptibility. Furthermore, the significant reduction in PrP(C) ß-cleavage product C2 in sheep of the ARR/ARR genotype compared to ARQ/ARQ and VRQ/VRQ genotypes, may add to the complexity of genetic determinants of prion disease susceptibility.

Expression of sheep interleukin 23 (IL23A, alpha subunit p19) in two distinct gastrointestinal diseases.

This paper reports the sequence of sheep interleukin 23A (p19), and shows that it shares 98% identity with bovine IL23A, 85% with human and 76% with mouse IL23A. It also reports the existence of two allelic variants that differ largely within the region encoding the amino terminal polypeptide signal sequence. An optimized RT-qPCR assay was used to quantify IL23A transcripts in sheep infected with two common gastrointestinal pathogens, the intracellular bacterium Mycobacterium avium subspecies paratuberculosis and the parasitic nematode Teladorsagia circumcincta. No differential expression of IL23A was detected in the mesenteric lymph node of sheep with the different pathogenic forms of paratuberculosis, however significantly high levels of IL23A were detected in the ileal mucosa of the paucibacillary form in comparison with the asymptomatic or multibacillary forms. Similarly, significantly high levels were present in the gastric lymph node draining T. circumcincta-infected abomasum in susceptible sheep. High levels of IL23A seem to be associated with lymphocytic infiltration and inflammation in both diseases but not with the macrophage infiltrate of multibacillary paratuberculosis.

Novel virus-encoded microRNA molecules expressed by ovine herpesvirus 2-immortalized bovine T-cells.

A number of herpesviruses have now been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever, a fatal lymphoproliferative disease of cattle. Using massively parallel sequencing and Northern hybridization we have identified eight putative miRNAs encoded by OvHV-2 expressed in an OvHV-2-immortalized bovine lymphocyte cell line. These eight miRNAs are encoded in two areas of the OvHV-2 genome that contain no predicted protein coding regions and show no sequence similarity with other herpesvirus or cellular miRNAs. This represents the first report of the expression of virally encoded miRNAs in the genus Macavirus of herpesviruses.

Expressed gene sequences of two variants of sheep interleukin-25.

This report describes the cloning and characterization of sheep interleukin-25 (IL25) expressed gene sequences and shows that, like humans, sheep express two transcript variants of IL25. Transcript variant 1 (IL25v1) has a 510 bp open reading frame encoding a 169 amino acid polypeptide with a calculated M(r) 19,200. The 498 bp IL25v2 encodes a 165 amino acid polypeptide with a calculated M(r) 18,710; both with an isoelectric point equal to 8.0307. The additional 12 bp of IL-25 isoform 1 are at the 5' end and encode an MYQA peptide, otherwise their sequences are identical. Phylogenetic analysis shows that both sheep IL-25 isoforms are most closely related to cattle and pig IL-25.

Measuring primary care patients' attitudes about dementia screening.

OBJECTIVES: To develop a questionnaire that will capture patients' attitudes about dementia screening in primary care. METHODS: Cross-sectional study of 315 patients aged 65 and older attending urban and rural primary care clinics in Indianapolis and North Carolina. The Perceptions Regarding Investigational Screening for Memory in Primary Care (PRISM-PC) questionnaire was administered via face-to-face or telephone interview. RESULTS: The PRISM-PC questionnaire consists of two separate scales: the patient's acceptance of dementia screening scale and the patient's perceived harms and benefits of dementia screening scale. The face validity of the PRISM-PC questionnaire was based on a systematic literature review and the opinions of 16 clinician-investigators with experience in screening for dementia. Exploratory factor analyses for the acceptance scale revealed the presence of two dimensions: knowledge about dementia risk and testing for dementia. For the benefits and harms scale, exploratory factor analyses identified four dimensions: perceived benefits of screening, stigma of screening, suffering from screening, and impact of screening on patients' independence. The internal consistency of each of the above subscales was good with Cronbach's alpha ranging from 0.58-0.85. CONCLUSION: The PRISM-PC questionnaire captures primary care patients' acceptance, perceived harms, and perceived benefits of dementia screening.

Oil A induces apoptosis of pancreatic cancer cells via caspase activation, redistribution of cell cycle and GADD expression.

AIM: To explore the mechanisms of effects of oil A on apoptosis of human pancreatic cancer cells. METHODS: Cellular DNA content was analyzed by flow cytometry. Western blotting was used for caspase-3 and PARP, caspase-7, caspase-9, cytochrome c, Bcl-2, Bax, Mcl-1, cyclinA, cyclin B1, cyclin D1, cyclin E, CDK2, CDK4, CDK6, P21, P27, GADD45, GADD153. RESULTS: The caspase-3, caspase-7, and caspase-9 activities were significantly increased as well as the cleavage of caspase-3, downstream substrate poly-ADP ribose polymerase (PARP) was induced. The amount of cytochrome c in the cytosolic fraction was increased, while the amount of cytochrome c in the mitochondrial fraction was decreased after oil A treatment. The anti-apoptosis proteins Bcl-2 and Mcl-1 were decreased in parallel and Bax increased, indicating that Bcl-2 family proteins-mitochondria-caspase cascade was responsible for oil-induced apoptosis. The proportion of cells in the G0/G1 decreased in MiaPaCa-2 and AsPC-1 cells after the treatment of oil A for 24 hours. The number of cells in S phase was increased in two cancer cell lines at 24 hours. Therefore, cells were significantly accumulated in G2/M phase. The cells with a sub-G0/G1 DNA content, a hallmark of apoptosis, were seen at 24 hours both in MiaPaCa-2 and AsPC-1 cells following exposure to oil A. The expression of cyclin A and cyclin B1 was slightly decreased and cyclin D1 levels were markedly lowered in MiaPaCa-2 cells. The expression of cyclin A and cyclin B1 was markedly decreased and cyclin D1 levels were slightly lowered in AsPC-1 cells, while cyclin E was not affected and the levels of CDK2, CDK4, and CDK6 were unchanged in MiaPaCa-2 and AsPC-1 cells. In response to oil A, P21 expression was increased, but P27 expression was not affected. The expression of both GADD45 and GADD153 was increased in two cell lines following oil A treatment. CONCLUSION: Oil A induces apoptosis of pancreatic cancer cells via activating caspase cascade, modifying cell cycle progress and changing cell cycle-regulating proteins and GADD expression.

Cerebrospinal fluid shunt survival and etiology of failures: a seven-year institutional experience.

BACKGROUND: Innovations in shunt technology and neuroendoscopy have been increasingly applied to shunt management. However, the relative life span of shunts and the etiology of shunt failure have not been characterized recently. METHODS: We reviewed the records of all shunting procedures at our institution between January 1992 and December 1998. Independent predictors of shunt failure were analyzed via multivariate Cox regression analysis in 836 shunting procedures. Independent predictors of the etiology of failure (infection, proximal obstruction, distal malfunction) were analyzed via multivariate logistic regression analysis in the 383 shunts which failed. RESULTS: A total of 353 pediatric patients underwent 308 shunt placements and 528 revisions. The risk (hazard ratio; HR) of shunt failure decreased as a function of time in both primary placements and revised shunts. In failed shunts, the odds of infection decreased 4-fold per year of shunt function, while the odds of distal malfunction increased 1.45-fold per year. Increasing number of shunt revisions (HR 1.31, p < 0.05), decreasing patient age in years (HR 1.04, p < 0.001), gestational age <40 weeks (HR 2.15, p < 0.001) but not the etiology of hydrocephalus were associated with an increased risk of shunt failure. Revisions versus primary placements, Dandy-Walker cysts and gestational age <40 weeks were independently associated with proximal, distal and infectious causes of failure, respectively. CONCLUSIONS: The long-term shunt revision rates observed here are similar to those reported over the past 2 decades. Shunt life span remains poorer in shunt revisions and in younger patients. Patient characteristics may suggest a specific risk and mechanism of failure, aiding in the long-term management of shunted hydrocephalus.