Browsing by Author "VandeWoude, Susan, advisor"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Open Access Applications of feline immunodeficiency virus as a model to study HIV pathogenesis(Colorado State University. Libraries, 2018) Miller, Craig Andrew, author; VandeWoude, Susan, advisor; Hoover, Edward, committee member; Han, Sushan, committee member; Webb, Craig, committee memberFeline immunodeficiency virus (FIV) is a naturally-occurring retrovirus that infects domestic and non-domestic feline species, and produces progressive immune depletion that eventually results in an acquired immunodeficiency syndrome (AIDS). While it is accepted that FIV is primarily transmitted by biting, few studies have evaluated FIV oral infection kinetics and transmission mechanisms over the last 20 years. Modern quantitative analyses applied to natural FIV oral infection could significantly further our understanding of lentiviral oral disease and transmission. In this Chapter 1 of this dissertation, I characterized FIV salivary viral kinetics and antibody secretions to more fully document oral viral pathogenesis. The results of this research demonstrate that (i) oral lymphoid tissues serve as a site for enhanced FIV replication, resulting in accumulation of FIV particles and FIV-infected cells in saliva, and (ii) failure to induce a virus-specific oral mucosal antibody response, and/or viral capability to overcome inhibitory components in saliva may perpetuate chronic oral cavity infection. Most importantly, these results provide a model of oral FIV pathogenesis and suggest alternative diagnostic modalities and translational approaches to study oral HIV infection. Feline immunodeficiency virus and human immunodeficiency virus (HIV) utilize parallel modes of receptor-mediated entry. The FIV surface glycoprotein (SU) is an important vaccine target for induction of virus neutralizing antibodies, and autoantibodies to the FIV binding receptor (CD134) block FIV infection ex vivo; highlighting the potential for immunotherapies which utilize anti-receptor antibodies to block viral infection. In Chapter 2 of this dissertation, I immunized cats with soluble CD134, recombinant FIV-SU protein, and/or CD134+SU complexes prior to challenge with FIV to determine if vaccination with CD134-SU complexes could induce protection against FIV infection. Immunization induced production of anti-CD134 and anti-SU antibodies in vaccinated cats, and purified anti-CD134 and anti-SU antibodies significantly inhibited FIV infection in vitro. However, no vaccine combination protected cats from FIV infection in vivo and vaccination induced high titers of antibodies directed at vaccine by-products relative to target antigens. The results of this research reinforce the need to monitor components of vaccine preparations, and emphasize that vaccination may induce proliferation of susceptible target cells and enhancement of heat-labile serum components that counteract neutralizing antibodies. Feline immunodeficiency virus induces lifelong infection in cats and may result in a spectrum of immunodeficiency-related diseases. Both prednisolone and cyclosporine A (CsA) are commonly used clinically to treat lymphoproliferative and immune-mediated diseases in cats, but the impact of these compounds on FIV infection has not been well documented, and their understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective use of these therapies in FIV infected cats. In Chapter 3 of this dissertation, I administered immunosuppressive doses of prednisolone or CsA to cats chronically infected with FIV and monitored alterations in hematological parameters and FIV viral/proviral loads in response to therapy. Interestingly, both treatments caused (i) acute increases in CD4+ lymphocytes, (ii) increased FIV viremia, and (iii) significant alterations in cytokine expression that favored a shift toward a Th2 response. The results of this research highlight the potential for immunosuppressive drug-induced perturbation of FIV replication and underscores the need for consideration of chronic viral infection status when prescribing immunomodulatory medications. Mucosal immune dysfunction, bacterial translocation, systemic immune activation, and chronic inflammation are well-documented features of chronic HIV infection. Despite the success of combinational antiretroviral therapy (cART) in diminishing HIV viral replication and prolonging immune function, a multitude of systemic and local manifestations of HIV infection persist, including the development of chronic inflammation (periodontitis and gingivitis). Commonly used animal models for studying HIV pathogenesis, including SIV/SHIV infections of non-human primates (NHPs) or HIV infections in humanized mice, do not reliably incite oral lesions. In contrast, gingivitis and periodontitis are primary clinical signs associated with untreated natural and experimental FIV-infection, and are principal attributes of this model that may be exploited to investigate pathogenic mechanisms involved in the perturbation of the oral immune system and microbial environment. Therefore, in Chapter 4, I outline the future directions and research goals for my career, and I present preliminary research results obtained thus far in my studies of the pathogenic mechanisms of HIV-induced oral disease. By assessing FIV-associated changes in clinical status, oral microbiota, local and systemic viral burden, and immune profile under such treatment protocols, future studies implementing the feline model of lentiviral-induced oral disease may provide a cornerstone to expand our understanding of the complex interactions between HIV infection, oral immune dysfunction, and the perturbations to the oral microbiota that occur in the context of HIV infection.Item Open Access Investigating molecular determinants of FIV pathogenesis(Colorado State University. Libraries, 2009) Thompson, Jesse Alan, author; VandeWoude, Susan, advisorFive FIV clades have been identified and are distinguished by envelope sequence. Two isolates, FIV-PPR and FIV-CPG (molecular clone FIV-C36), belonging to clades A and C, respectively, are variable with regard to disease potential. Chimeric viruses constructed between phenotypically distinct strains of FIV are potentially useful tools to identify molecular determinants of virulence. Several chimeric constructs were therefore developed by exchanging elements between FIV-C36 and FIV-PPR. FIV-PCenv and FIV-PC3'LTR were two resulting chimeras that were capable of competent in vitro replication. Studies in Chapter One aimed to characterize these viruses in the domestic cat model and to test the hypothesis that elements surrounding the env region contribute to in vivo pathology. FIV-PC3'LTR, containing FIV-C36 rev2 and 3' LTR was infectious, although attenuated compared to parental constructs. FIV-PCenv, containing FIV-C36 vif, otfA, env, and the first exon of rev, displayed a phenotype intermediate to parental viruses with regard to replication kinetics, and CD4+ T-cell and neutrophil declines, but peak viral load and development of clinical disease was delayed by three weeks compared to FIV-PPR, FIV-PC3'LTR, and FIV-C36. Studies in Chapter Two evaluate potential mechanisms for the delayed phenotype of FIV-PCenv. To test the hypothesis that delayed kinetics observed during FIV-PCenv infections was due to adaptation of the construct during first round infections, pooled plasma from the in vivo study described in Chapter One was used to inoculate a second cohort of cats. Passaged FIV-PCenv again displayed intermediate phenotype in terms of viral replication and immunopathology, but onset of acute viremia was no longer delayed. To further pinpoint particular genes that contribute to FIV pathogenesis, three additional chimeras were generated using PCR-driven overlap extension as described in Chapter Three. Overlapping-PCR was utilized to produce chimeras with specifically substituted ORF genes encoding the FIV-C36 regulatory proteins Vif and OrfA for those from FIV-PPR; chimeras FIV-PCvif, FIV-PCvif/orfA, and FIV-PCorfA were successfully constructed. Upon infection of the feline MYA-1 T-cell line, all three chimeras produced measurable RT activity. Further, in vitro analysis of FIV-PCvif/orfA demonstrated that this construct had replication properties equal to those of FIV-C36 as measured by viral capsid and RNA genome production.Item Open Access Seroanalysis of Felis catus gammaherpesvirus 1 infection in domestic cats(Colorado State University. Libraries, 2015) Stutzman-Rodriguez, Kathryn, author; VandeWoude, Susan, advisor; Rovnak, Joel, committee member; Duval, Dawn, committee memberWe recently described 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). To determine FcaGHV1 antigens that elicit a detectable humoral-immune response in naturally-infected domestic cats, I chose to evaluate seven FcaGHV1 proteins. These proteins are conserved across the subfamily and antigenic in other gammaherpesvirus infections. I amplified and cloned each of the seven FcaGHV1 genes into a mammalian expression vector and transfected intact clones into Crandell Rees feline kidney (CRFK) cells. I developed an immunofluorescent antibody test using transfected cells exposed to sera from nine shelter cats diagnosed as FcaGHV1-positive by quantitative PCR (qPCR) of blood-cell DNA. This analysis indicated that tegument proteins ORF52 and ORF38 reacted most consistently with serum from cats with positive FcaGHV1-qPCR reactions. Based on these results, recombinant antigens were used to develop two optimized indirect ELISAs. Genes for ORF52 and ORF38 were cloned into a mammalian expression vector. Antigens were produced in a transient transfection system and purified using immunoprecipitation. Indirect ELISA conditions were optimized using known positive and negative controls. Using the two optimized ELISAs, I screened sera from 133 shelter cats that had been previously tested by FcaGHV1-qPCR. Seroprevalence of FcaGHV1 reactive antibodies was 32%, compared to the previously published 16% prevalence evaluated by qPCR. Nineteen of twenty qPCR positive cats were also seroreactive against one or both antigens on ELISA. Sera from 24 cats were seropositive based upon ELISA testing, but negative using qPCR analysis. Risk factors identified in previous publications were confirmed by ELISA, namely geographic location, male sex, adult age, and association of FcaGHV1 with several co-infections. Based on our knowledge of gammaherpesvirus latency, this ELISA provides evidence of viral exposure, while qPCR viral DNA detection likely represents reactivation from latency or primary infection. The addition of serologic analysis as a measurement of FcaGHV1 exposure will aid in determining association of FcaGHV1 with disease and routes of transmission.