Center for Viral Pathogenesis Research Symposium November 29, 2017 - - PDF document

Center for Viral Pathogenesis Research Symposium November 29, 2017 – University of Kansas Medical Center PRESENTATION ABSTRACTS Kevin Ault, MD – University of Kansas Medical Center Nicholas Wallace, PhD – Kansas State University High Risk α-Papillomavirus Oncogenes Induce Aberrant Translesion Synthesis High risk α-papillomaviruses (HR α-HPV) cause anogenital and oropharyngeal tumors. These malignancies are dependent on HR α-HPV E6 and E7 and associated with destabilized genomes. Because HR α-HPV E6 and E7 are known to alter cellular signaling pathways, we hypothesize that the genomic instability in HR α-HPV+ cancers could be attributable to aberrant responses to DNA damage. To evaluate our hypothesis, we analyzed gene expression in 86 cervical carcinomas and 58 control samples. We found a significant upregulation of genes involved in replication in the tumors and predicted increased replication would require an enhance ability to tolerate damage during S-

• phase. The translesion synthesis (TLS) pathway prevents deleterious replication fork collapse by allowing the fork to

bypass any DNA lesion that it encounters. Consistent with this hypothesis TLS gene transcripts were more abundant in cervical cancers with the notable exception of the TLS-specific polymerase (Pol η) that is required for lesion

• bypass. We have confirmed these results and shown that they are driven by HR α-HPV E6 and E7. Further, our data

demonstrate that HR α-HPV E6 and E7 prevent the induction of pol η in response to crosslinked DNA and sensitize cells to DNA crosslinking agents. We speculate that inhibition of TLS by HPV oncogenes may explain the

• bservation that HPV-associated malignancies are acutely sensitive to DNA crosslinking drugs.

Rollie Clem, PhD – Kansas State University Unraveling the Arbovirus Midgut Escape Barrier in Mosquito Vectors When an arbovirus is acquired by a mosquito in a blood meal, available evidence indicates that the first tissue that is infected is the midgut. Subsequently, in order to for transmission to occur, the arbovirus must replicate in the midgut epithelium and then escape from the midgut, presumably by crossing the midgut basal lamina. Disseminated infection is required for transmission because the virus must eventually replicate in salivary glands in order to be injected along with saliva in a subsequent blood meal. However, there are known arbovirus/vector combinations where the virus is able to infect and replicate in the midgut epithelium, but is not able to efficiently disseminate to the rest of the mosquito. This has led to the concept of the midgut escape barrier, but there is almost nothing known about the molecular basis for this barrier. I will discuss our recent work on trying to understand the mechanisms by which the alphavirus Sindbis virus escapes from the midgut of the mosquito Aedes aegypti.

• A. Sally Davis, DVM, PhD – Kansas State University

Viral Pathogenesis through the Lens of an Investigative Veterinary Pathologist Investigative or experimental veterinary pathology is defined as a branch of veterinary medicine dealing with the essential nature of disease, especially changes in animal tissues and organs that cause or are caused by disease. Add to this a focus on infectious diseases and you have a unique perspective from which to look at host-pathogen interactions, a core component of viral pathogenesis. The laboratory of investigative pathology currently focuses on emerging and zoonotic viral diseases such as Rift Valley fever and influenza from a primary hypothesis driven research point of view with a small focus area in Pneumocystis research. However, equally important is our focus on investigative pathology techniques development. We work closely with the K-State Veterinary Diagnostic Laboratory's (VDL) Histology Lab to identify assays of mutual interest and co-develop, validate and optimize these for use in our laboratory and as a service by the VDL. Examples of recent project-based work in histochemistry, immunohistochemistry, immunofluorescence, digital microscopy and molecular diagnostic assays using formalin- fixed, paraffin-embedded tissues as their starting point will be shared. Additionally, we are actively involved in the development of new biosample handling techniques running the spectrum from biosample acquisition through fixation, inactivation, decalcification and further preparation of samples for optimal use in downstream assays. This focus leads us to tackle issues related to visualization of markers in tissue, improved tissue treatments for better molecular biology endpoints and maximizing archival materials for retrospective studies.

Brandon DeKosky, PhD – University of Kansas - Lawrence High-Throughput Characterization of Anti-Viral Human Antibody Responses Antibodies are a critical feature of our adaptive immune system for viral protection, and each antibody is comprised

• f a unique heavy and a light chain peptide sequence that is encoded by a single B cell. The DeKosky laboratory

applies a custom-developed technology to characterize the repertoire of paired antibody heavy and light chain sequences from over one million single B cells at a time. This suite of technologies includes RT-PCR-based DNA molecular engineering, massively parallel emulsion droplet reactions, Next Generation DNA sequencing, and high- throughput computational analysis. We are applying this unique workflow to characterize the response to vaccines and diseases in human patients to better understand the mechanisms and features of adaptive immune protection against viral pathogens. Navneet Dhillon, PhD – University of Kansas Medical Center HIV-1, Illicit Drugs and Pulmonary Vascular Remodeling The prolonged survival of human immuno-deficiency virus (HIV-1)-infected patients with the use of antiretroviral therapy has resulted in an increase in the incidence of non-infectious cardio-pulmonary complications including HIV-related pulmonary arterial hypertension (HRPAH). Given that intravenous drug use (IVDU) has been found to be one of the most common risk factor of HRPAH, the research focus of the talk will be on how HIV-1 and drugs of abuse interact and contribute to this enhanced pulmonary vascular remodeling. Ying Fang, PhD – Kansas State University A Naturally Occurring Cross Order Recombinant of Enterovirus and Torovirus Enteroviruses comprise a highly diversified group of viruses, which are implicated in a wide range of diseases in human and animals. Genetic recombination has been considered as a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we identified a special case of cross-order recombination between enterovirus G (order picornavirales) and Torovirus (order Nidovirales). This naturally occurring recombinant event may have broad implications for other picornaviral and/or nidoviral species. Importantly, we demonstrated that the exogenous ToV-PLP gene that inserted into the EVG genome encodes a deubiquitinase/deISGylase and potentially suppresses host cellular innate immune responses. Our results provide insights on how gain of function through genetic recombination, in particular cross-order recombination, may improve the ability of a virus to evade host immunity. William Groutas, PhD – Wichita State University Drug Discovery and Development of Antiviral Therapeutics Viruses belonging to the Picornaviridae, Caliciviridae and Coronaviridae families include several medically- important pathogens. These include the causative agents of outbreaks of acute gastroenteritis (noroviruses), high- mortality systemic diseases (SARS-CoV and MERS-CoV) and hand, foot, and mouth disease (enteroviruses). There are currently no effective vaccines or antiviral agents against these viruses. The aforementioned viruses possess 3C

• r 3C-like proteases that are essential for virus replication, consequently, agents that inhibit these proteases are of

potential therapeutic value. Using a structure-guided approach, the design of an array of novel inhibitors of norovirus with in vivo efficacy was accomplished. We have furthermore demonstrated for the first time in clinical trials that inhibition of feline infectious peritonitis virus (FIPV) 3CL protease reverses the progression of fatal infectious peritonitis (FIP). Since FIP disease progression is quite rapid and its pathogenesis primarily immune-mediated, features shared by MERS-CoV and SARS-CoV, we hypothesize that judiciously-designed 3CL protease inhibitors may reverse the pathogenesis of MERS-CoV in affected hosts. An overview of the scope and current status of our drug discovery program will be presented. Ferdaus Hassan, PhD – Children’s Mercy Hospital Host-Response to Enterovirus-D68 infection in Children: From Bench-to-Bed Side Enterovirus-D68 (EV-D68) is a relatively unknown virus. In the summer of 2014, EV-D68 caused a nationwide

• utbreak, effecting mainly school-aged children and Kansas City was one the most effected regions. During that

summer, among all the children that were tested, almost 60% were positive for EV-D68 infection. Many of them required hospitalization and intensive care. To understand the mechanism of pathogenesis, EV-D68 was isolated, infected in lung epithelial cell A-549 and RNA-seq was done to determine the pattern of gene expression and further analyzed by Ingenuity pathway. In addition, clinical respiratory samples were obtained from children with or without

EV-D68 infection and host innate immune response was determined by multiplex ELISA. Our data suggests that EV-D68 infection suppresses the immune response, providing insight into a potential mechanism contributing to severe disease progression. Maria Kalamvoki, PhD – University of Kansas Medical Center Strategies that HSV-1 Has Evolved to Counteract Host Antiviral Responses Herpes simplex viruses 1 and 2 are significant human pathogens causing diseases ranging in severity from benign cold sores to lethal encephalitis. Following primary infection HSV-1 and HSV-2 establish latent reservoirs in sensory ganglia and persists for the life of the individual. Reactivation of these viruses in sensory neurons facilitates progeny virus production and virus dissemination. Innate immunity plays a pivotal role in controlling HSV replication, spread, latency, and the severity of HSV diseases. My laboratory focuses on mechanisms through which herpes simplex viruses counteract innate immune responses. Recently, we observed that during HSV infection innate immune factors are packaged in extracellular vesicles and delivered to uninfected cells restricting virus replication. This appears to be a mechanism that controls the dissemination of the virus promoting viral persistence. The properties and functions of extracellular vesicles released from HSV infected cells are currently under investigation in my laboratory. Carole McArthur, MD, PhD – University of Missouri - Kansas City 20 years of HIV/TB-related Bioprospecting on the African Continent The impact of HIV on human health has been profound. HIV is the pandemic of the 21st Century and is a major driver of diagnostic technology. The epicenter of the HIV/AIDS epidemic is the Congo basin where ancestors of HIV-1 Group M and SIVcpz were transmitted from Chimpanzees and Gorillas to man. Advances in technology development resulting from our HIV diversity studies on unique non-B HIV subtypes at our laboratory in Cameroon and Congo from 1998 until the present will be described. The development of sensitive NGS using Illumina and Luminex Flexmap serology to archival samples will be summarized. Advances in our understanding of HIV diversity add further support to the origin of HIV in the Congo basin and emphasize the need for continuing diagnostic surveillance. Christophe Nicot, PhD – University of Kansas Medical Center Director, Center for Viral Pathogenesis Human T-cell Leukemia Virus Type I: 35 Years Later In 1977, epidemiological studies revealed the presence of unusual clusters of adult T-cell leukemia/lymphoma (ATLL) in some areas of Japan, suggesting a transmissible agent may be involved in the disease. The first description of HTLV-I came after the discovery of the human T-cell growth factor interleukin-2, allowing long-term in vitro culture of T cells and establishment of T-cell lines from a patient with a cutaneous T-cell lymphoma (CTCL). HTLV-I infects more than 25 million people worldwide and is endemic in Japan, central Africa, South America and the Caribbean basin. The end of the HTLV-I provirus genome contains open reading frames encoding for regulatory proteins involved in virus replication, immune escape and pathogenesis. The viral protein Tax is a transcriptional activator of virus expression. Tax plays a critical role in T-cell transformation by activating cellular signaling pathways (NF-kB, AKT, Wnt and Notch), inactivating cell cycle checkpoints, inhibiting DNA repair pathways and tumor suppressors (p53, RB1 and p16ink4), inhibiting apoptosis and reactivating telomerase expression leading to uncontrolled cellular proliferation. The four-year survival rates are 5.0% for the acute type, 5.7% for the lymphoma type, 26.9% for the chronic type, and 62.8% for the smoldering type. Therapeutic options for ATLL are limited. High-dose radiotherapy or chemotherapy regimens, by themselves or in combination, are ineffective in ATL patients. An antiretroviral therapy regimen comprised of AZT and IFN has shown promising results as a first-line therapy in chronic or smoldering ATL but not for lymphoma or acute ATLL patients. This presentation will present and discuss past and recent contributions made by my laboratory in the search of therapy response markers and development of more effective treatments for ATLL.

• A. Townsend Peterson, PhD – University of Kansas – Lawrence

Ecology and Geography of Transmission of Viral Diseases My work focuses on aspects of the geography of biodiversity, with a focus on distributional ecology and disease transmission risk mapping. In the latter field, my work has touched numerous disease systems, including Chagas disease, malaria, dengue, leishmaniasis, and ebola and Marburg disease. In general, this work recasts disease geography in a context of ecology and biogeography, and is collaborative in nature and usually involves geographers, computer scientists, and biologists. Juergen Richt, DVM, PhD – Kansas State University Jean-Paul J. Gonzalez, MD, PhD – Kansas State University Research Progress at the Center of Excellence for Emerging and Zoonotic Animal Diseases The Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD) at Kansas State University was established in 2010 to help protect the nation’s agricultural and public health sectors against high-consequence foreign animal, emerging and zoonotic disease threats. CEEZAD has four principal missions:

• Development of novel, safe, efficacious and DIVA-compatible vaccines for prevention and control of high-

impact emerging and zoonotic diseases that can be manufactured in the U.S.

• Development and expansion of technologies and platforms for laboratory and point-of-need pathogen

detection.

• Development of models to predict high-consequence disease behavior in the U.S. to aid prevention or
• utbreak control.
• Development of education and training programs for students, veterinarians, first responders and researchers

in high-impact animal diseases and animal emergencies. Rangaraj Selvarangan, PhD – Children’s Mercy Hospital Human Parechovirus Central Nervous System Infection in Children The human parechoviruses (HPeVs) are part of a newly classified genus, Parechovirus, in the family Picornaviridae. In 1956, HPeV type 1 and HPeV type 2 were first isolated by Wigand and Sabin from the stool samples of 2 children with diarrhea in Ohio, and since then 16 HPeV serotypes have been identified. HPeV1 is the most common HPeV identified to date and is primarily associated with asymptomatic seroconversion, or mild respiratory or gastrointestinal infection, and infrequently with CNS infection. HPeV1 infection mostly occurs in children aged <1 year, and almost all children become infected by age 5. Even though severe disease, including flaccid paralysis, myocarditis and encephalitis, were initially reported with HPeV1, recent reports show only mild gastrointestinal and respiratory symptoms. HPeV-2 infections occur very rarely, presenting as mild acute gastroenteritis. HPeV3 is the second most prevalent and perhaps the most important HPeV type. It was first identified in the stool of a 1-year-old Japanese girl with fever, diarrhea, and transient paralysis. Since then, HPeV3 has been associated with sepsis and fever in young infants, is the most common HPeV type recovered from cerebrospinal fluid (CSF), and, based on limited data from other countries, appears to have a biannual cycle. We have found a variable yearly prevalence of up to 17% HPeV in CSF samples tested, and all were HPeV3 in the study years (2006-2014). Most of our patients were 1-2 months old and presented as sepsis-like illness with fever and suspected meningitis. Researchers have described white matter abnormalities and long-term sequelae in 10 infants < 1 month of age with HPeV CNS

• infection. The severity of the imaging abnormalities correlated with later neurologic injury. Clinical outcomes

varied, with normal neurodevelopment confirmed in 6/10 cases and the remaining 4 infants developed cerebral palsy, epilepsy, cerebral visual impairment, learning disabilities, and/or mild distal hypertonia. Another study reported a 3-week-old with confirmed HPeV-3 infection, presenting as a sepsis-like illness that developed hepatitis and coagulopathy. Long-term neurodevelopmental follow-up has been suggested to be important for those < 1 month

• f age with HPeV CNS infection because of these reported sequelae. The proposed talk will help in better

understanding of HPeV-associated infections, clinical manifestations and eventually better patient management practices.