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

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. In summary, our LANAC vaccine has both therapeutic and prophylactic potential and is able to offer protection against distinct alphavirus species irrespective of the route of infection.

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. Ultimately, we may identify new prion candidates in yeast, which will contribute information about the parameters necessary for prion formation and insight into the functions prions play in yeast. In addition, by confirming PAPA's ability to predict prion proteins from the yeast proteome, it allows the possibility to apply this methodology to other proteomes. 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 cancerassociated herpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcomaassociated 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. With this information we plan to develop a serologic assay and further evaluate FcaGHV1 as a model for EBV and KSHV. 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 ELISA test detected cross reactivity for all phleboviruses excluding RGV, but Western blotting detected the presumed RGV nucleocapsid protein (N) using rabbit anti-RVFV-N serum, RGV-infected cells were also identified when labeled with this antibody. Our findings demonstrate assay specific antigenic cross reactivity between these phleboviruses, thus further characterization of the molecular targets of the cross-reaction is required for proper interpretation of serological assays. http://dx.doi.org/10.1016/j.nhtm.2015.07.023

The Intrathecal Antibody Response in Multiple Sclerosis Brain Does Not React Against Measles Virus
Deandra L Walker, Mark P Burgoon

Department of Neurology, University of Colorado School of Medicine, Aurora, CO
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( þ) 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. Although many of the rAbs from MS brain reacted against auto-antigens in several mouse and human tissues, none of fifteen MS rAbs reacted against MV-infected cells. These results indicate that measles virus is not a disease-relevant antigen in MS. http://dx.doi.org/10.1016/j.nhtm.2015.07.024

Poliovirus and Group C Enteroviruses: Knowledge Gaps Relevant to Eradication
Barton DJ, Kempf BJ, Cooper DA.

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO
Poliovirus eradication is one of the most challenging public health endeavors in modern times (www.polioeradication.org). 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 liveattenuated 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 3D pol involved in viral RNA replication and recombination. A dsRNA clamp of 3D pol 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, we found that RNase L targets viral RNA encoding neutralizing epitopes of capsid proteins, sparing most other regions of viral RNA. These data suggest an important interplay between neutralizing antibodies, neutralization escape mutations and antiviral endoribonucleases. A better understanding of viral RNA recombination, enterovirus species groups and antiviral endoribonucleases should help achieve and maintain poliovirus eradication. 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, ebolavirus hemorrhagic fever in West Africa, chikungunya in the Western Hemisphere, and the reemergence of poliomyelitis.