Online ISSN: 2515-8260

Volume 2, Issue 4

Volume 2, Issue 4, Summer 2015

Signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) are key expression Markers in cardiac specific precursor selection from hADSCs

Vinod Kumar Verma; Syed Sultan Beevi; Usha Shalini; Suguna Ratnakar Kamaraju; Lakshmi Kumari Kona; Yamuna Mohanram; Lakshmi Kiran Chelluri

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 93-101

Cardiomyocyte enrichment strategies so far have not yielded scalable cardiac specific cell type. More so, the current data is restricted to embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs), wherein the use of viral vectors is fraught with increased risk during clinical use. Herein, we profiled time-dependent gene/protein expression patterns across the cardiac ectoderm, endoderm, and mesoderm for isolating cardiac precursors from human adipose derived stem cells (hADSC). Methods Direct cardiac differentiation of hADSCs was carried out with 5-azacytidine and basic fibroblast growth factor (bFGF) in a one month long culture. The cells were periodically harvested, analyzed for unique persistent markers and their inherent regulation using quantitative polymerase chain reaction (qPCR), flow cytometry, immunoblot and immunocytochemistry assays. The identified markers were super paramagnetic iron oxide nanoparticle (SPION) tagged for segregation by magnetic activated cell sorting (MACS) and further evaluated their differentiation potential and checked for the purity by flow cytometry.
The results demonstrated pronounced up-regulation of mesodermal and mature cardiac lineage markers at three weeks, while there was a down-regulation of pluripotent stem cell markers. This perhaps could be attributed to de-differentiation in maintaining the cardiac phenotype. However, signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) persisted all through the culture period of one month, making them the most relevant and reliable cardiac specific markers. Dual labeling of these markers to SPION for cardiomyocyte enrichment by MACS column yielded cardiomyogenic-like cells in differentiation cultures with several functional positive markers. Conclusions Thus, SIRPA and KDR together provide cues in the enhancement and up-scaling of cardiomyocyte production in the cell replacement therapy. Focal points • Benchside Identification of specific cell phenotypic markers to identify cardiac precursors in any tissue source with minimal cell manipulation is a novel process development tool in clinical translation. • Bedside A product developed in a closed system would minimize extraneous contaminants in long term cultures and development of such procedures minimizes culture failure rates from bench side. • Industry This unique identification of cell-specific marker would enable a tissue-specific translational plan and immensely help in the cardiac regeneration. • Government Financial investment and support from the government is vital in the optimization and validation for better health care and would contribute in reducing the disease burden. • Regulatory The stringent regulatory guidelines worldwide on minimal cell manipulation for entering into stem cell based clinical trials preclude the need to develop alternate approaches in product and process developmental technology, which can be easily translated in a clinical setup.

Bardet–Biedl syndrome: A model for translational research in rare diseases

Robert M. Haws; Anthony D. Krentz; Rachel V. Stankowski; Robert D. Steiner

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 102-109

Bardet–Biedl syndrome (BBS) is a rare, multisystemic, genetic disease and member of a group of disorders called ciliopathies. This syndrome provides a mechanistic model for ciliopathies that may also extend to common disorders with complex inheritance patterns, including diabetes mellitus and obesity. Dysregulation of signaling pathways altering the cellular response to the extracellular environment is primary to the ciliopathies and characteristic of BBS. As BBS-centered translational research moves forward, innovative advances provide opportunities to improve the care of individuals with BBS and other rare diseases as well as common related conditions. This review aims to highlight the current understanding of the mechanisms underlying BBS and opportunities for advancing the care of individuals with rare diseases. Focal points: Bedside: understanding the multi-dimensional manifestations of ciliopathies, specifically Bardet– Biedl Syndrome (BBS) as a model ciliopathy, will accelerate research into therapeutic targets for ciliopathies, allowing for improved therapies for individuals with these debilitating disorders. Benchside: elucidating the molecular mechanisms of BBS is likely to increase the chance of discovering novel therapeutic approaches that may be generalizable to other ciliopathies and perhaps to common related disorders, such as obesity and diabetes mellitus. Industry: application of known drugs to new indications, or drug repositioning, and development of novel therapeutics, including gene therapies in BBS, may open new avenues for therapeutic discovery and development. Community: rare diseases affect millions of individuals throughout the world with significant impact on quality of life and longevity. The development of multidisciplinary clinics for BBS and effective implementation of a rare disease registry provides a model for advancing the care of individuals with rare diseases. Government and Regulatory Agencies: the importance of rare disease research and the impact of that research on common disorders should be supported with adequate funding and resources. Understanding the molecular pathways underlying ciliopathies, such as BBS, and advancement of translational medicine in ciliopathies will have far reaching societal benefits

Development of a decision-making biomarker for CRTH2 antagonism in clinical studies

Daniel S. Strasser; Herve Farine; Martin Holdener; Jochen Zisowsky; Rene Roscher; Julie Hoerner; Martine Gehin; Patricia N. Sidharta; Jasper Dingemanse; Peter M.A. Groenen

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 118-125

Biomarkers have shown to improve success rates in the development of novel drugs, providing essential information in the early phases of clinical development for decision-making. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is pursued as a drug target for a number of inflammatory diseases. CRTH2 antagonists block the activation and migration of key inflammatory cells such as eosinophils, basophils, and Th2 cells. The mechanism of action of CRTH2 antagonists was established in cells isolated from human blood. Biomarkers derived from these experiments were included in clinical studies to investigate the mechanism of action and potency of CRTH2 antagonists in human. For clinical phase I studies with the CRTH2 antagonist ACT-453859, a follow-up molecule of setipiprant, inclusion of the most precise and robust pharmacodynamic (PD) biomarker with a clinically relevant target effect was desired to aid phase II dose selection. Candidate biomarkers such as IL-13 secretion from Th2 cells and CRTH2, CD11b and CD203 modulation on basophils and eosinophils in whole blood were compared in terms of signal intensity and variability. Blockade of CRTH2 receptor internalization was finally chosen as PD biomarker and rigorously tested in a feasibility study. The assay showed excellent robustness, an intra-assay precision of 5% and inter-subject variability smaller than 15%. Based on phase II clinical study results with setipiprant, 90% CRTH2 receptor blockade was defined as clinically relevant PD effect. This target PD effect provides the means to take decisions based on the data generated in the phase I clinical studies with ACT-453859. Focal points • Bedside Biomarkers offer a great potential to influence decisions taken during early clinical development. For clinical phase I studies with the CRTH2 antagonist ACT-453859, a follow-up molecule of setipiprant, inclusion of a biomarker was desired to aid phase II dose selection. In order to facilitate decision-making, we developed a biomarker that delivers high quality data under clinical circumstance and defined a relevant target biomarker effect. • Benchside In-vitro experiments with human whole blood identified CRTH2 receptor internalization on basophils and eosinophils as the most precise and robust biomarker. Clinical results obtained with setipiprant in a seasonal allergic rhinitis study were used to define the clinically relevant target biomarker effect of 90% CRTH2 receptor blockade. Proof for the chosen target biomarker effect remains to be demonstrated in phase II clinical studies with ACT-453859.

The 14th Annual Meeting of the Rocky Mountain Virology Association: Current Advances in Virology and Prion Biology in the Rocky Mountain region

J. Rovnak; Randall J. Cohrs

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

The 14th annual meeting of the Rocky Mountain Virology Association was held October 3–5, 2014 at the Colorado State University Mountain Campus in Pingree Park, Colorado. We had classic mountain weather with clear cold starry skies at night and bright warm days. Approximately 80 students, post-docs, and early-stage and established investigators, who perform basic and clinical research, were in attendance. The aims of the program are to bring together regional and national virologists and prion biologists to share and discuss their scientific data and ideas, to foster graduate student education and postdoctoral training, and to help early stage investigators in their career development. This year we instituted a peer review program for student and post-doc presentations in order to provide constructive, written feedback. Poster presenters also had an opportunity to provide 3- minute, single-slide, oral introductions to their work. Our guest speakers, Rich Condit from the University of Florida, Lee Fortunato from the University of Idaho, Cathy Miller from Iowa State University, and Nicole Garneau from the Denver Museum of Nature and Science, all provided inspirational presentations of their research efforts and raised the bar on critical participation in all aspects of the meeting.

Simian varicella virus is present in skin tissue of rhesus macaques after experimental reactivation

Miller A; Traina-Dorge V; Blackmon Aa; Wellish Ma; Deharo E; Gilden Da; Mahalingam R

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Varicella zoster virus (VZV) causes varicella (chickenpox), establishes latency in ganglia and reactivates decades later to produce zoster in the elderly. Clinical, pathological, immunological and virological features of simian varicella virus (SVV) infection of primates parallel human VZV infection. Primary SVV infection of primates, cause varicella, after which virus becomes latent in ganglionic neurons and reactivates upon social and environmental stress. Five rhesus macaques were infected intrabronchially with 4.0x105 pfu of SVV. Two weeks later, the monkeys developed varicella rash. Twenty months later four of the monkeys were treated once with a 50 mg/kg of anti-CD4 antibody. All 5 monkeys developed zoster rash, 7–55 days after the treatment. Punch biopsies of the skin rash were analyzed for the presence of SVV antigens by immunohistochemistry and immunofluorescence. SVV ORF 63 protein and glycoproteins gH and L were detected in sweat glands in skin from all 5 monkeys.

Interferon gamma allows long-term maintenance of VZV-infected neurons in vitro

Baird NL; Bowlin JL; Cohrs RJ; Gilden D

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 126-127

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus. During primary infection, VZV causes varicella (chicken pox), after which the virus go latent in ganglionic neurons along the entire neuraxis before reactivating decades later to cause zoster (shingles). Interferon gamma (IFNγ), produced during viral infection, stimulates transcription of genes that mediate antiviral responses. Herein, it was tested whether IFNγ treatment of human neurons inhibits VZV infection of human neurons in vitro. Infected neurons not treated with IFNγ developed a cytopathic effect in 4 weeks, during which time VZV DNA increased 7-fold and viral RNA accumulated. Infected neurons cultured in the presence of IFNγ for 8 weeks or infected neurons cultured in IFNγ for 4 weeks followed by cytokine removal for an additional 4 weeks had only a 2.8- and 3.6-fold increase of viral DNA, respectively in the 8 weeks post-infection. Furthermore, levels of VZV transcripts did not increase between 4 and 8 weeks post-infection when IFNγ was removed at 4 weeks post-infection, and even began to decrease when the cultures were maintained in IFNγ for the entire 8 weeks. In accordance with reduced DNA accumulation and mRNA levels when infected neurons were maintained in IFNγ, less CPE was evident at 8 weeks post-infection compared to cultures which had IFNγ removed at 4 weeks post-infection.

The Retroviral Cyclin Controls CDK8-mediated Transcription Elongation and Reinitiation

Claire H.BirkenheuerConnie D.BrewsterSandra L.QuackenbushJoelRovnak

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Walleye dermal sarcoma virus is a complex retrovirus that causes seasonal tumors in walleye fish. RV-cyclin is one accessory protein encoded by the virus, and is one of only two viral proteins expressed during tumor development. Therefore, role of RV-cyclin in tumor development was explored. RV-cyclin interacts with host cyclin dependent kinase8 (CDK8). CDK8 has oncogenic like properties in colon cancer and melanoma, and one target of CDK8 kinase activity is the carboxy terminal domain of RNA Pol II. Here qRT-PCR analysis demonstrates RV-cyclin’s direct interaction with CDK8 increases transcript levels of another set of oncogenes—the serum-response genes (Fos, EGR1, and Jun). Nuclear run-on experiments, and chromatin immunoprecipitation experiments with an antibody to RNA Pol II, show that RV-cyclin enhances transcription elongation along the EGR1 gene locus. This enhancement correlates with increased recruitment of CDK8 to the EGR1 gene locus. In addition to increasing CDK8 occupancy at the EGR1 gene, in vitro kinase experiments demonstrate RV-cyclin increases the amount of CDK8-phosphorylation on the CTD of RNA Pol II. In conclusion, not only does RV-cyclin direct CDK8 to specific genes during tumor development, RV-cyclin enhances CDK8 kinase activity while it is there. The end result of the CDK8-RV-cyclin interaction is a rise in the mRNA levels of another pool of oncogenes, the serum-response genes. This is one mechanism by which RV-cyclin could contribute to the development of walleye dermal sarcoma.

Predicting Prion Propensity of Human Proteins

Cascarina S; Ross E.

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

In humans only a single prion-forming protein named PrPc (for “cellular prion protein”) is currently known, yet many more neurodegenerative disorders involve aberrant protein aggregation. The classical model for these diseases has involved cell-autonomous aggregation, assuming that aggregation occurs independently in each cell within a diseased patient. However, more recent models have proposed a non-cell-autonomous progression of disease in which aggregates formed in one cell may be transmitted to neighboring cells. These aggregate seeds then cause aggregation of the soluble protein in the “infected” cells, similar to the prion diseases. Within the past few years, a number of proteins that exhibit prion-like aggregation and spread to neighboring tissues have been discovered in patients with Amyotrophic Lateral Sclerosis (ALS). Although ALS has been studied for a number of decades, these proteins were only recently linked to ALS by chance. This demonstrates a clear need for an accurate method to systematically identify additional proteins that may play a pathological role in neurodegenerative disorders.

Ivermectin for the Control of West Nile Virus Transmission

Nguyen C; Burton T; Kuklinski W; Gray M; Foy BD

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Presently there are limited options for controlling the transmission of West Nile virus (WNV), including the use of larvicides and adulticides to target the mosquito vector. However, these methods are poorly-targeted, restricted to wealthy semi-urban and urban areas that are able to fund the efforts, and opposed in some communities due to toxicity concerns. This study evaluated the use of endectocide-treated bird feed to control WNV transmission by targeting the primary vector in Colorado, Culex tarsalis. Ivermectin susceptibility in C. tarsalis was first measured through ivermectin-spiked bloodmeals fed using membrane feeders, and the LC50 was determined to be 49.94 ng/ml (39.71-59.93 95% CI, n=988). Chickens were then fed ivermectin-treated feed to examine its safety and palatability, and mosquitoes were blood fed directly on the chickens to assess in vivo effects. Finally, ivermectin pharmokinetics were analyzed using vein blood from chickens as well the C. tarsalis that bloodfed on the chickens. A mixture of 200 mg ivermectin/kg of bird feed was determined to be a palatable and safe dose on which chickens would feed while also being effective in killing C. tarsalis in bioassays. Pharmacokinetic data from the in vivo tests produced conflicting results compared to in vitro blood feeds but drug was detected in chicken blood at concentrations that may be expected to affect C. tarsalis.

Bovine herpesvirus 4 not detected in free-ranging domestic cats from California, Colorado, and Florida

Chiu E; Troyer R; VandeWoude S

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 127-128

Previous studies have reported that domestic cats can be naturally infected with bovine herpesvirus 4 (BHV4), and experimental inoculations have been linked to feline urolithiasis. It has been difficult to recapitulate initial diagnostic and experimental observations, thus here we have initiated a study to evaluate BHV4 presence in a large cohort of cats at risk for exposure to circulating feline viruses using a sensitive and specific assay. Domestic cat blood DNA samples (n=101) collected from California, Colorado, and Florida were screened for BHV4 using sensitive real time PCR. In contrast to BHV4 containing tissue culture extracts, all domestic cat blood samples were negative for BHV4.

Longitudinal analysis of blood-borne prion infection

Alan M. Elder; Davin M. Henderson; Amy V. Nalls; AnthonyE. Kincaid; Edward A. Hoover; Jason C. Bartz; CandaceK. Mathiason

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Transmissible spongiform encephalopathies (TSEs), or prion diseases, affecting human and animal species can be transmitted from TSE-infected individuals to naïve susceptible hosts during the long asymptomatic (years to decades) and symptomatic disease stages. The presence of infectious hematogenous prions in asymptomatic TSE-infected hosts demonstrates the highly infectious nature of blood-borne prions in hosts lacking overt clinical symptoms. It is currently unknown when and how infectious prions first enter the blood following initial exposure. We have previously shown that the whole-blood real-time quaking-induced conversion assay (wbRT-QuIC) possesses 100% specificity and >92% sensitivity, making it an ideal tool to address this question. Here, we use wbRT-QuIC to analyze whole blood collected from oral, extranasal or aerosol TSE-exposed hosts for blood-borne prions. Our results demonstrate that conversion competent prions in the inoculum are capable of crossing mucosal surfaces and entering the circulatory system within 30 min—no matter the route of exposure.

Dengue virus requires the unsaturated fatty acid biosynthesis pathway for its infection in the mammalian host

Rebekah C. Gullberg; Richard J. Kuhn; Rushika Perera

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Dengue virus (DENV) infection is a significant global health concern with over 40% of the world’s population at risk and currently no therapeutics or vaccines available. Understanding host viral interactions is key to developing novel therapeutic options. Dengue virus is a positive sense RNA virus that induces the formation of invaginations in the endoplasmic reticulum to replicate its genome. Increased phospholipid biosynthesis is key to the formation of these replication compartments as well as viral maturation and release. It is now evident that viral proteins mediate this change in the cellular phospholipid repertoire, but the precise mechanisms are unknown. We have shown that siRNA mediated knockdown as well as pharmacological inhibition of key enzymes in the unsaturated phospholipid biosynthesis pathway reduces DENV replication. Unsaturated fatty acids, when incorporated into membrane phospholipids are a key mechanism for providing fluidity and curvature of membranes enhancing the assembly and function of membrane bound enzymes.

Chikungunya virus in non-mammalian species: a possible new reservoir

Airn Hartwig; Angela Bosco-Lauth; Richard Bowen

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Chikungunya virus (CHIKV) is an arbovirus distributed widely in tropical regions of the world that causes a febrile and often painful disease in adults and children. Recent outbreaks of CHIKV infection in the Caribbean have raised concerns about establishment of this virus in North America. A significant question about the transmission cycle of CHIKV is whether non-human reservoir hosts are important in maintenance or transmission of the virus. We conducted experimental infections with CHIKV and discovered that several reptiles and amphibians developed viremia of sufficient magnitude to possibly serve as reservoir hosts. One or two strains of CHIKV were inoculated into a variety of ball pythons, Burmese pythons, Northern garter snakes, American alligators, green iguanas, painted turtles, leopard frogs, Bufo species toads and cane toads. Viremia was not detected in alligators or cane toads but all other species developed viremia at variable magnitude. Peak viremia in the other species varied from 2.8 (Burmese pythons) to 4.7 (leopard frogs) log10 pfu/ml. We also conducted experiment to evaluate the effect of ambient temperature changes to monitor the “over wintering” capabilities of CHIKV in snakes. Northern garter snakes were inoculated a South African strain of CHIKV at temperatures of 16 C versus 26 C and tested for viremia.

Experimental Modoc virus infection of deer mice (Peromyscus maniculatus)

Hume G; Hawkinson A; Aboellail T; Schountz T

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 128-129

Modoc virus (MODV) is a flavivirus that was first isolated from deer mice (Peromyscus maniculatus) in Modoc County, California during a 1958 surveillance study for novel viruses. Although many flaviviruses are arthropod-borne, MODV has no known intermediate. Subsequent to its initial isolation, MODV was detected in deer mice found in other regions of the United States, including northeastern Colorado. These findings suggested that deer mice may be a reservoir host of MODV. We intramuscularly inoculated 18 deer mice with 105 TCID50 of MODV strain M544 for susceptibility testing. Groups of three deer mice were euthanized for necropsy and tissue collection on days 2, 4, 7, 11, 21, and 31. No conspicuous signs of disease occurred in the deer mice; however, minor pulmonary multifocal vasculitis and hemorrhages, multifocal portal hepatitis and splenic lymphoid hyperplasia with hemosiderosis were detected in several deer mice. No virus was detected in sera, suggesting viremia did not occur. Neutralizing antibody was detected as early as day 7-post inoculation, and thereafter all deer were seropositive. MODV RNA was detected by PCR in organs of deer mice euthanized between days 2 and 4, with lung tissue of one deer mouse euthanized on day 7 also indicating the presence of MODV RNA. Viral RNA was detected in most spleens but less frequently in the kidneys and hearts.

Assessing Mother to Offspring Transmission of Chronic Wasting Disease Using Transgenic Mouse Models

Willingham K; McNulty E; Anderson K; Hayes-Klug J; Nalls A; Mathiason C

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy (TSE), or prion disease, of free-ranging and captive cervids (deer, elk and moose). The presence of sufficient infectious prions in the tissues, bodily fluids (urine, saliva, and blood) and environments of clinical and preclinical CWD-infected animals is thought to account for its high transmission efficiency. Recently it has been recognized that transmission from mother to offspring may contribute to the facile transmission of some TSEs. Although the mechanism of maternal transmission has yet to be elucidated, the extended asymptomatic TSE carrier phase, lasting years to decades, suggests that maternal transmission may have implications in the spread of prions. Placental trafficking and/or secretion in milk are two means by which maternal prion transmission may occur. In these studies we explore CWD maternal transmission during early and late CWD infection using a transgenic mouse model (TgCerPRP) expressing cervid prion protein. Naïve and CWD-infected dams were bred during early (45 dpi) and late (120 dpi) infection and were allowed to bear and raise their offspring. Milk was collected from the dams for prion analysis, and the offspring were observed for TSE disease progression. Terminal tissues harvested from these dams and offspring were analyzed for prions. We have demonstrated: 1) that CWD-infected TgCerPRP females successfully breed and bear offspring, 2) the presence of PrPCWD in reproductive and mammary tissue harvested from CWD-infected dams, and 3) clinical disease progression in offspring born to CWD-infected dams.

Serological evidence that Tacaribe virus is circulating among bats in Trinidad and Tobago

Malmlov A; Seetahal J; Carrington C; Ramkisson V; Foster J; Munster V; Quackenbush S; Schountz T

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Tacaribe virus (TCRV) is a bisegmented, ambisense, RNA virus within the genus Arenavirus. Arenaviruses are grouped into Old World lymphocytic choriomeningitis-Lassa virus complex and the New World Tacaribe complex viruses. TCRV is placed within the Tacaribe complex along with the South American hemorrhagic fever viruses: Chapare, Guanarito, Junin, Machupo, and Sabia viruses. The only isolates of TCRV were from 11 artibeus bats collected by investigators at the Trinidad Regional Virology Laboratory in the Republic of Trinidad in the 1950 s. TCRV has not been isolated since, although serological data from the 1970 s suggested it may circulate among Caribbean bats. Only one isolate remains, TRVL-11573, and it has been passaged in suckling mice and Vero cells. We sought to determine if TCRV is still circulating in bat populations in Trinidad through serological investigation. We developed an ELISA and western blot assay using His-tagged recombinant TCRV nucleocapsid antigen. Serum from Artibeus jamaicensis that had been experimentally infected with TCRV was used as a positive control, and serum collected from an uninfected A. jamaicensis used as a negative control. ELISA screen of bloods from 84 bats of various species captured in Trinidad identified several, mostly artibeus bats, as seropositive for antibodies to TCRV.

Assessing Milk from CWD-Lactating Deer for Infectious Prions

Willingham K; McNulty E; Anderson K; Hayes-Klug J; Nalls A; Mathiason C

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Transmissible spongiform encephalopathies (TSEs), or prions, cause a fatal neurodegenerative disease affecting mammals including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, variant Creutzfeldt-Jakob disease in humans and chronic wasting disease (CWD) in deer, elk and moose. CWD, the only prion disease to infect a native free-ranging population, has now been detected in 22 American states, 2 Canadian provinces and South Korea. While horizontal transmission is credited for much of the spread of CWD, few studies have monitored the potential for vertical/maternal transmission with an emphasis on lactation. Using a small, polyestrous cervid— the Reeves’ muntjac deer— we are addressing this issue by supplementing naïve Reeve’s muntjac fawns (n=5) with milk collected from CWD-inoculated, pre-clinical and clinical muntjac doe. Blood, saliva, feces, urine and lymphoid biopsies will be collected from milk-exposed fawns at 10d, 21d, 40d, 3mo, 6mo, 12 and 18 mo pi to aid in CWD diagnosis. Similar samples, with the addition of mammary biopsy, will be collected from each mother doe at 3 months intervals to monitor CWD status. CWD fawn and mother doe CWD status will be monitored by immunohistochemistry, real time quaking induced conversion assay (RT-QuIC), protein misfolding cyclic amplification (PMCA) and clinical disease progression

Impact of Dengue Virus Infection on Global Metabolic Alterations in the Aedes aegypti Mosquito Vector

Nunya Chotiwan; Irma Sanchez-Vargus; Jeffrey M. Grabowski; Amber Hopf-jannasch; Victoria Hedrick; Erik Gough; Ernesto Nakayasu; Devika Sirohi; Catherine A. Hill; Richard J. Kuhn; Rushika Perera

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Aedes aegypti mosquitoes are the primary vectors transmitting dengue virus (DENV), one of the most aggressive re-emerging pathogens worldwide causing more than 390 million infections per year. The spread of the virus is greatly dependent upon successful replication within both the human host and mosquito vector. Much effort has been placed in understanding the dynamics of virus transmission and replication in both organisms, but little is known about the global impact of DENV on metabolic pathways. Previous studies have demonstrated perturbations in human and Aedes albopictus cellular metabolic environments during DENV infection. Some of these perturbations include increasing the production of membranous lipids that had the capability to induce membrane curvature and permeability, as well as visibly altering both human and mosquito intracellular membrane architecture to support DENV replication. In this study, we have explored metabolic changes in Aedes aegypti midgut and salivary glands upon DENV (serotype 2) infection.

Alphavirus Infection of the CNS: Entry, Dissemination, and Neurodegeneration

Phillips AT; Rico AB; Aboellail TA; Olson KE

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Alphaviruses most often associated with neuroinvasive disease are limited to the Americas and include strains of EEEV, VEEV, and WEEV. The process of alphavirus entry into the CNS of infected vertebrates following challenge is not well-understood. It is thought that virus entry into the CNS depends on the inoculation route. It is well-established that olfactory sensory neurons provide access to the CNS following challenge with airborne virus. However, less knowledge is available regarding virus entry into the CNS following peripheral, non-olfactory infection, which appears to rely on some form of hematogenous spread. We sought to determine the precise route of CNS entry following footpad inoculation by using a combination of in vivo/ex vivo bioluminescence imaging and traditional histological examination methods. We found a consistent pattern in the spatiotemporal distribution of virus among the imaged brains, none of which involved the olfactory bulb.

Sterilization and Disposal of Agricultural Quarantine Waste

Laura Pulscher; Erin McNulty; Amy V. Nalls; Craig Ramsey; Candace K. Mathiason

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Approximately 150 million people and almost $40 billion worth of agricultural commodities go through U.S. international ports annually. Ports seize animal and plant products potentially contaminated with high risk diseases that then must be decontaminated before entering the waste stream. Currently, there are only 3 methods of decontamination accepted by the Animal Plant and Health Inspection Service at U.S. ports and borders including incineration, high temperature cooking, and discharge of ground waste as sewage. In this study we assess the efficacy of a relatively new decontamination technology, alkaline digestion, to mitigate infectious agents. Transmissible Spongiform Encephalopathies (TSEs), a member of the protein misfolding diseases (ex: Alzheimer’s and Parkinson’s Diseases), were chosen as the infectious agent for this study because they rank as the hardest to kill microbe/pathogen, affect both human and animal species worldwide and are shed by infected hosts into the environment establishing highly infectious biota. Chronic wasting disease (CWD), an emerging TSE of cervid species (deer, elk, moose) in North America, has recently been spotlighted as a potential concern for European countries, and recapitulates human and animal TSE pathogenesis and shedding. For these reasons CWD is ideal for mitigation studies. We processed CWD positive and negative materials by alkaline digestion under standard temperature and pressure at time intervals of 2, 4, and 6 h. Samples were retrieved after digestion, were neutralized and inoculated intracerebrally into transgenic mice expressing the cervid protein to determine remaining prion infectivity. In addition, the samples (pre and post alkaline digestion) were tested for amplification competent prions by Protein Misfolding Cyclic Amplification (PMCA). Preliminary results suggest a lack of amplification competent prions in samples processed by alkaline digestion at 2, 4, and 6 h cycles as compared to nondigested samples.

Alphavirus E1 Glycoprotein-Liposome-Nucleic Acid Complexes Protect Mice from Lethal Challenge with Multiple Alphaviruses

Rico A; Phillips A; Schountz T; Toth A; Jarvis D; Powers A; Olson K

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 130-131

Alphaviruses are globally distributed, mosquito borne pathogens that cause death and disease in vertebrates, including humans. Therapeutics to combat alphaviral disease are non-existant and only a handful of IND status vaccines are available. Of the available vaccines most are associated with a poor immunological response and a high rate of reactivity, and none protects against more than a single alphavirus species. We designed and tested novel alphavirus vaccines comprised of the E1 glycoproteins of western equine encephalitis virus (WEEV) or Venezuelan equine encephalitis virus (VEEV). Immunization with cationic lipid nucleic acid complexes (CLNCs) and alphavirus E1ecto mixture (lipid-antigen-nucleic acid complexes:LANACs) provided significant protection in mice challenged with either WEEV, VEEV or eastern equine encephalitis virus (EEEV) regardless of challenge route. LANAC immunized mice mount a strong humoral immune response lacking neutralizing antibody. Passive transfer of immune sera from LANAC immunized mice to non-immunized mice confers protection to challenge, indicating that non-neutralizing antibody is sufficient for protection.

Efficient replication and shedding of MERS CoV from the upper respiratory tract of experimentally infected dromedary camels

Adney DR; Brown VR; van Doremalen N; Bushmaker T; Scott D; de Wit E; Munster VA; Bowen RA

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

The Middle East respiratory syndrome coronavirus (MERS CoV) is a novel coronavirus first recognized in 2012 and is associated with severe respiratory disease in humans. Virus has been isolated from dromedary camels in endemic areas, and many camels also have neutralizing antibodies against the virus, suggesting that they are likely a reservoir host. In order to better understand the role of camels in virus transmission we experimentally infected 3 adult, male dromedary camels with a human isolate of MERS CoV. All animals developed a transient, upper respiratory tract infection associated with very minor clinical disease.

Predicting New Prion Candidates in Yeast

Shattuck J; Waetcher A; Ross E.

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Prions are infectious proteins capable of self-propagating and transmitting between organisms. Even though there is no homolog to the mammalian prion protein in yeast, several soluble proteins can form heritable aggregates de novo. These proteins provide a model system to investigate the nucleation, aggregation and propagation steps involved in the formation of a prion fibril. Several prion prediction algorithms have been developed to predict yeast proteins that have the propensity to form prions. One of these algorithms was previously developed in our laboratory (Prion Aggregation Prediction Algorithm, PAPA, Toombs et al., 2012). Therefore, we used PAPA to scan the yeast proteome to extract proteins that contain domains predicted to have prion activity (prion-like domains). These prion-like domains will be tested in four prion activity assays to assess their activity in vivo as well as in vitro. Here we provide preliminary evidence that we are successful at predicting yeast proteins that present prion activity in vivo. Following characterization of these prion-like domains, we will test the respective full-length proteins for prion activity using microscopy as well as developing phenotypic assays.

Detection of Immunodominant Proteins of Felis catus Gammaherpesvirus 1

Stutzman-Rodriguez K; Troyer R; VandeWoude S

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

We recently identified and sequenced a novel herpesvirus of domestic cats, Felis catus gammaherpesvirus 1 (FcaGHV1). FcaGHV1 is a member of the gammaherpesvirus subfamily which also includes the human cancer-associated herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). As a first step toward developing a serologic assay to detect exposure to FcaGHV1, we are seeking to determine which viral proteins elicit an antibody response in naturally occurring domestic cat infections. We cloned selected FcaGHV1 genes into a mammalian expression vector and performed transfections of a feline cell line for expression of recombinant FcaGHV1 proteins. We fixed cells with paraformaldehyde and methanol-acetone and tested reactivity to serum from cats naturally infected with FcaGHV1 using immunofluorescent antibody staining. An FIV immunoflouresence test was developed as a positive control for transfection and assay function. Serum from specific pathogen-free laboratory cats served as negative controls. Preliminary data from 9 cats with FcaGHV1 infection indicates that capsid protein ORF 65 and tegument protein ORF38 may elicit antibodies during naturally occurring FcaGHV1 infection. Results of this study will suggest which FcaGHV1 proteins are immunodominant during natural infection.

Further Characterization of Rio Grande Virus and Potential for Serological Cross Reactivity with other Phleboviruses

Szymczak M; Reeves W; Miller M

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 131-132

Members of the genus Phlebovirus (family Bunyaviridae) are new and emerging disease pathogens of humans and animals. Newly identified viruses include Heartland virus (HRTV), Lone Star virus in the USA, and Severe Fever with Thrombocytopenia Syndrome virus in Asia. Assays to support surveillance, epidemiologic studies, and diagnosis of these viruses may also detect related viruses within the genus, confounding interpretation. Rio Grande virus (RGV) was isolated in 1973 from southern plains woodrats (Neotoma micropus) in the United States and has been preliminarily identified as a phlebovirus transmitted by the sand fly Lutzomyia anthophora. RGV is not known to cause disease in humans, but it could be detected by assays designed for HRTV or other phleboviruses. The goal of this study was to determine antigenic cross-reaction between RGV and other phleboviruses. A commercially available ELISA based sand fly fever antigen detection kit was tested for the ability to detect RGV and other New and Old-World phleboviruses, including attenuated Rift valley fever virus (RVFV) strain MP12, Punta Toro virus (PTV), Toscana virus, Aguacate virus, Anhanga virus, Arumowot virus, and Chagres virus. Immunocytochemistry and Western blotting were used to detect cross reactions between RGV, MP12, and PTV using rabbit anti-RVFV nucleocapsid protein and glycoproteins GC and GN, mouse monoclonal anti-PTV, and sheep polyclonal anti-MP12.

The Intrathecal Antibody Response in Multiple Sclerosis Brain Does Not React Against Measles Virus

Deandra L Walker; Mark P Burgoon

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) and is the most common disabling neurological disease of young adults. Although the cause of MS is unknown, genetic and epidemiological studies indicate that MS may be triggered by an environmental agent. The presence of intrathecally produced antibodies, which produce oligoclonal Ig bands in the CNS of MS patients, provides tools for investigating the target of the inflammatory response. In most of the CNS conditions with oligoclonal bands the target is known and the antibody is directed against an infectious, causative agent. For example, in subacute sclerosing panencephalitis, a measles virus (MV) infection of the brain, the oligoclonal bands and intrathecal antibodies are primarily directed against MV. In earlier studies of MS, we demonstrated that the intrathecal antibody response in MS brain does not react to varicella zoster or Epstein-Barr virus. The current study investigates the reactivity of the intrathecal antibody response in MS brain to MV. We isolated individual CD38(+) plasma cells from MS brain to produce recombinant antibodies (rAbs). These rAbs likely represent oligoclonal bands and were used to immunostain MV-infected or uninfected monkey kidney (Vero) cells.

Poliovirus and Group C Enteroviruses: Knowledge Gaps Relevant to Eradication

Barton DJ; Kempf BJ; Cooper DA.

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Poliovirus eradication is one of the most challenging public health endeavors in modern times ( Social, political, economic & scientific factors have made this goal elusive. When eradication goals were first established in 1988, there was little appreciation of viral RNA recombination, enterovirus species groups and their relevance to eradication. Now, it is clear that RNA recombination between live-attenuated vaccine strains of poliovirus and non-polio group C enteroviruses results in circulating vaccine-derived polioviruses (cVDPV) and corresponding outbreaks of paralytic disease, a significant obstacle to eradication. By understanding enterovirus species groups, it becomes clear that poliovirus capsid proteins can be eradicated; however, the remainder of poliovirus RNA genomes will survive indefinitely in other group C enteroviruses. To help address these obstacles to eradication, the Barton lab studies molecular features of 3Dpol involved in viral RNA replication and recombination. A dsRNA clamp of 3Dpol that holds RNA products of replication as they exit the polymerase plays important roles in the polyadenylation of viral RNA, the fidelity of RNA replication, ribavirin sensitivity and viral RNA recombination. In other experiments, we identified a group C enterovirus RNA involved in the inhibition of ribonuclease L, an antiviral endoribonuclease. The RNase L ciRNA plays important but largely unexplored roles in pathogenesis. Using novel deep sequencing methods,

Current Disease and Epidemiologic Problems: What’s Hot?

Calisher CH

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Included among the many current world problems are viral diseases of considerable significance and threat. This talk could not possible cover them all but will discuss the emergence of Middle East respiratory syndrome in Saudi Arabia and nearby countries,

Transcriptome markers of viral persistence in naturally-infected Andes Hantavirus (Bunyaviridae) seropositive rice rats

Corey L. Campbell; Fernando Torres-Perez; Mariana AcunaRetamar; Tony Schountz

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 132-133

The long-tailed pygmy rice rat (Oligoryzomys longicaudatus) is the reservoir host of Andes (ANDV) and Oran hantaviruses (Bunyaviridae). To examine transcriptome features of persistently infected rice rats, spleens from ANDV sero-positive wild-caught rice rats were assessed. RNA-seq analysis, de novo reference-independent assembly and stringent orthology assignments produced 17,756 unique coding and non-coding RNAs. Differential expression analysis of persistently-infected seropositive rice rat spleens revealed 18 differentially expressed transcripts from 16 unique genes. A three-pronged effect on the immune response were observed in 1) suppression of the JAK-STAT pathway at Stat5b and Ccnot1, as well as 2) a bias toward a TH2 response in the enrichment of caspase-1 and 3) stimulation of RIG-I pathway factors Ppp1cc and MFF. Two of these differentially expressed transcripts, caspase-1 and STAT5b, code for proteins expected to stimulate T helper follicular (TFH) cell development, a phenomenon that has also been described for hantavirus-infected P. maniculatus. Differential expression of a single seropositive rice rat with a higher viral load revealed a robust response of 243 differentially expressed transcripts, suggesting an acute infection.

Animation of VZV DNA

Randall. J. Cohrs; J. Rovnak

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Varicella zoster virus (VZV) is a ubiquitous neurotropic alphaherpesvirus that typically causes childhood varicella (chickenpox) on primary infection and zoster (shingles) after reactivation. During latency most of the ~70 virus genes are transcriptionally silent; however, analysis of latent VZV gene transcription in its natural setting requires analysis of human ganglia removed at autopsy. Recognizing the problems associated with such samples, we have observed that as the post-mortem time interval increases, so do the number of VZV genes transcribed. Based on our data and recent similar findings concerning reactivation of HSV-1,

Viralign: A tool for uncovering functional viral elements

Frietze S; R.J. Cohrs; Kaufer B

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

The availability of broad epigenomic profiles of human tissues provides an opportunity to uncover viral sequences and their corresponding functional regulatory elements in otherwise overlooked datasets. We developed Viralign, a throughput screening method to discover and interpret viral functional information in existing short read archive data. Using a comprehensive reference database, Viralign scans sequence data for known viral sequences and generates an alignment report with read information and genome coverage. Viralign analyzes functional datasets for regulatory elements and provides coordinate and visualization files that can be viewed in a genome browser. Additionally, this method searches for potential integration sites and variants by genome assembly. In a pilot study, we performed H3K27me3 ChIP-seq in monocytes of an HHV6 infected individual and compared this to U2OS cells infected with HHV6A and HHV6B and use Viralign to detect HHV6 insertion loci and H3K27me3 enriched regions.

Autophagic flux without a block differentiates varicella from herpes simplex virus infection

Charles Grose

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Varicella-zoster virus (VZV) is a herpesvirus that causes a characteristic vesicular exanthem in humans with primary infection (varicella) or reactivation (zoster). We have previously observed that vesicular cells are filled with autophagosomes that are easily detectable by confocal microscopy after immunolabeling for the LC3 protein. Through a 3D imaging software program called Imaris we have quantitated autophagosomes as greater than 100 per cell. Similarly, we have assessed autophagy in VZV-infected monolayers after inoculation by the traditional method with infected cells at a ratio of one infected to 8 uninfected cells. Again, autophagosomes are easily detected, but their count is lower than that observed in human skin cells. As an additional control, we enumerated the autophagosomes in the Severe Combined Immuno-Deficient (SCID) Mouse model of VZV infection. In this model, human skin is inserted under the skin of the mouse and subsequently inoculated with VZV-infected cells. Again, autophagy was abundant in the VZV-infected skin and minimal in the mock-infected skin sample. Subsequently, we investigated autophagy following infection with sonically prepared cell free virus in cultured cells. After cell free virus inoculation, autophagy was detected in a majority of infected cells at all time points, but was less than that seen after an infected-cell inoculum. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid);

Recombination, reassortment, and many-to-one genotypes in natural arenavirus infections

Stenglein M; Jacobson E; DeRisi J

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 133-134

Mutation, recombination, and reassortment generate virus particles with variable genotypes, some of which may be better adapted to infect a new host, resist drug treatment, or escape immune pressure. The arenaviruses are a family of viruses that package a large (L) and small (S) genome segment. Arenaviruses infect mammals and snakes and are associated with fatal disease in both groups of animals. Although recombination and reassortment are well documented in some virus families, neither process has been observed in natural arenavirus infections. In this study, we documented a surprising degree of genetic diversity in arenavirus-infected snakes. Instead of one or two viral species or quasispecies, individual animals harbored complex populations of viral genotypes composed of up to 5 S and 11 L genotypes, which replicated as stable ensembles in culture. S and L segment genotype accumulation was not balanced and a particular S segment genotype dominated, both in individual animals and at a population level. Numerous instances of recombination and reassortment were detected. Some recombinant segments had unusual organizations with 2 intergenic regions.

Roche Research Portfolio: Trusted Performance, Efficient Workflow Solutions

Konet D

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Along with Roche Pharmaceuticals, Roche Diagnostics is an important part of the foundation that modern healthcare builds upon. Our broad range of innovative diagnostic tests and systems play a pivotal role in the groundbreaking area of integrated healthcare solutions and cover the early detection, targeted screening, evaluation and monitoring of disease.

Preemption, the Virus-Serum-Toxin Act, and the USDA: a case study using iatrogenic abortion due to BoHV-1 vaccines in pregnant cows

O’Toole D; Miller MM

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Three major vaccine manufacturers in the United States currently sell multivalent vaccines containing modified live bovine herpesvirus 1 (BoHV-1) for use in pregnant cattle. The first of these products entered the US market in 2003. Yet it has been known since the early 1960 s that vaccinal BoHV-1 causes abortion in cattle. The products became popular as they can be used year-round, regardless of pregnancy status in herds. Abortifacient effects have been considered to be minimal, provided initial vaccination is done during the previous 12 months using specific vaccine products and in accordance with label directions. Single nucleotide polymorphisms (SNPs) in BoHV-1 can be used to resolve whether post-vaccination outbreaks of abortion in cattle herds are iatrogenic (Fulton et al.; Vaccine. 2013; 31(11):1471-1479). We tested tissues from 10 abortion episodes (2010–2014) where an apparent association existed between recent use of modified live BoHV-1 and abortion 1–3 months later. Products were used on or off label in individual outbreaks. All 10 episodes had SNP patterns consistent with those of commonly-used modified live BoHV-1 strains (O’Toole et al.; Vet Pathol. 2014, In press). In spite of this, it is likely such products will remain on the market. This is due the absence of meaningful post-marketing surveillance of suspect adverse reactions in animals by the USDA, compounded by the courts’ interpretation of the Virus-Serum-Toxin Act of 1913 [Lynnbrook Farms v. SmithKline Beecham Corp.,

Structure-based Engineering of Sabin 2 Poliovirus Polymerase to Alter Replication Fidelity

Keith A; Campagnola S; Peersen O.

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

Picornaviruses cause a wide range of ailments, including myocarditis, poliomyelitis, and vesicular lesion type diseases. Excellent vaccines exist for several of them, and the development of the live-attenuated oral polio vaccine (OPV) provided an efficient and cost-effective avenue for successful poliovirus eradication in the majority of the world. However, one hurdle for developing successful live-attenuated vaccines lies with the viral RNA-dependent-RNA-polymerase (RdRP) enzyme whose low replication fidelity allows for reversion of attenuated viruses to disease causing variants. Improving the replication fidelity of RdRPs is an attractive avenue for virus attenuation because it may curtail such reversion issues. We have previously solved the crystal structures of several picornaviral polymerase-RNA complexes that show the structural changes taking place within these polymerases during active site closure and catalysis (Gong et al., 2010, 2013). Based on this, we engineered a panel of fidelity variant coxsackievirus B3 polymerases that caused reduced infectivity and attenuated virus growth in mice (Gnädig et al., PNAS, 2012). We hypothesize that such modulation of polymerase fidelity via structure based protein engineering can provide an effective platform to improve the design of live-attenuated vaccines. To investigate this further we have generated over a dozen mutations in the poliovirus Sabin 2 strain polymerase and carried out in vitro biochemical assays to show that these can either increase or decrease polymerase fidelity while having minor effects on elongation rates and processivity. The fidelity modulation can arise from single point mutations, multi-site mutations that replace entire groups of interacting residues, or from grafting in structurally homologous sequences from related polymerases.

Generating new prions by targeted mutation or segment duplication

Hendrich C; Paul KR; Waechter A; Harman M; Ross ED.

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages 134-135

Prions are infectious agents composed entirely of protein. Prion activity results from the conversion of soluble proteins into an insoluble, self-templating amyloid form. Nine different amyloid-based prions have been identified in yeast. All but one contain a glutamine/asparagine (Q/N) rich region that is responsible for prion activity. Similar Q/N-rich regions are over-represented in eukaryotic genomes. In humans, aggregation-causing mutations in Q/N-rich proteins have been linked to various degenerative diseases, including ALS. Our lab previously developed a prediction algorithm, PAPA (Prion Aggregation Prediction Algorithm), to predict the aggregation propensity of Q/N-rich proteins, and to predict the effects of mutations on aggregation propensity. Here, we tested the ability of PAPA to design mutations to turn non-prion proteins into prions. We identified four yeast Q/N-rich protein fragments that lacked any detectable aggregation or prion activity. In each case, a small number of designed mutations were sufficient to cause these domains to aggregate, and in two cases, to create bona fide prion activity. We then tested whether simply generating tandem repeats of short, aggregation-prone segments within these domains would likewise be sufficient to create prion activity. We found that such segment duplications consistently increased prion activity in a length-dependent manner.

Interferon response in a hamster model of arenavirus hemorrhagic disease

Schountz T; A. Phillips; A Rico; C Campbell; T Aboellail; A McGuire; S Quackenbush; K Olsen; C H Calishe

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 4, Pages -

A difficulty in studying hemorrhagic arenavirus pathogenesis is a lack of animal models that recapitulate human disease and which can be manipulated at BSL-3 or lower. Pirital virus (PIRV), a South American arenavirus hosted by Alston’s cotton rats (Sigmodon alstoni), has not been shown to cause disease in humans and is considered a BSL-3 agent. Infection of Syrian golden hamsters (Mesocricetus auratus) causes a disease that shares many features of the South American hemorrhagic fevers and Lassa fever. Significantly, neurological manifestations occur, a feature commonly found in Argentine hemorrhagic fever and which is absent in most other models of arenavirus disease. We examined the pathogenesis of PIRV infection by clinical and molecular methods, with a focus on gene expression of the antiviral type I interferon response using RNA-Seq.