After the emergence of Zika virus (ZIKV) in the Americas in 2015, ZIKV infection was associated for the first time since its discovery with severe symptoms in both adults and congenital cases, including neurological, ocular, and developmental manifestations. Previous ZIKV circulation in Africa and Southeast Asia has been characterized by mild symptoms and small-scale case-counts. It is unclear whether the unprecedented size and severity of the ZIKV outbreak in the Americas are the consequence of a change in the virus, different background flaviviral immunity in the population, or a reporting issue. In addition, ZIKV has been shown to be transmitted through sexual contact, and the shedding of ZIKV from various bodily fluids in both humans and in vivo models suggests that other potential routes of transmission exist. We present here two mouse models that can be used to further investigate ZIKV pathogenesis, transmission, and immune response. Mice lacking interferon regulatory factors 3 and 7 (IRF 3/7 DKO) supported robust infection with the prototype MR766 strain from Uganda, while maintaining a 72% survival rate, and recapitulated symptoms and tissue lesions associated to infection with American ZIKV isolates in humans and other in vivo models. The MR766 strain was capable of causing retinal lesions and viral RNA shedding from the conjunctival fluid, hitherto unreported to be caused by an African strain. Further, ZIKV was visualized in the seminal fluid co-localized with infected epithelial epididymal cells, suggesting a possible cellular component of sexual transmission. Immunocompetent C57BL/6 mice, although not susceptible to ZIKV, were capable of mounting a robust antibody response that strongly neutralized ZIKV and was also able to cross-neutralize DENV2. We further report that homologous re-exposure with ZIKV in C57BL/6 mice reduced the DENV2 cross-neutralizing capability of the antibody population, while at the same time increasing enhancement of DENV2 infection. We conclude that ZIKV strains of the African and Asian lineages share a similar pathogenesis, suggesting that the increased severity of symptoms is unlikely to be due to a change in the virus. We also show that re-exposure to ZIKV can alter the antibody response to increase the risk of heterologous infection.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that circulates predominantly in tropical and subtropical regions. Infection results in severe debilitating polyarthralgia during the acute phase of disease, and reports suggest that chronic arthralgia lasting months to years after the initial infection can occur. More severe and prolonged disease has been associated with pre-existing joint disease, though this has not been experimentally examined. In the research presented herein, two established mouse models (adult IRF 3/7 -/- -/- and wild-type C57BL/6J mice) were utilized to characterize CHIKV-associated bone and joint disease and evaluate its impact on the progression of pre-existing osteoarthritis (OA) utilizing histopathology, μCT, and serology. During acute stages of the disease, CHIKV infection resulted in synovitis, cartilage necrosis, and periosteal necrosis or periostitis. Additionally, IRF mice had severe ischemic bone marrow necrosis, and C57BL/6J mice developed periosteal new bone proliferation. During chronic stages of disease (90 DPI), there was ongoing and progressive synovitis, tendonitis, enthesitis, and cartilage damage, though periostitis and periosteal bone proliferation had resolved. Infection with CHIKV in mice with pre-existing OA had no significant impact on total synovitis scores or chondrocyte cell death, but caused a decrease in volume of osteophytes and subchondral bone, as compared to OA alone. Serology results in both the footpad and intra-articular C57BL/6J models indicated there were alterations in RANKL and OPG associated with CHIKV infection, demonstrating potential changes in bone dynamics. The current experiments demonstrated novel lesions of CHIKV-associated bone and joint disease that have important ramifications for the treatment and prevention of disease. Periosteal bone proliferation associated with CHIKV is a potentially painful but reversible process, whereas articular cartilage damage is progressive and represents a potential mechanism for chronic CHIKV-associated joint disease. Additionally, the alterations in bone associated with CHIKV infection and pre-existing OA, in conjunction with changes in the RANKL-OPG pathways, could have significance in clinical monitoring of OA and treatment choices in individuals with concurrent diseases. Future studies would help determine if RANKL and OPG levels could be useful for tracking disease progression in people and establish the long-term effects of CHIKV infection on OA progression.

Gastrointestinal nematode (GIN) infections affect production systems and exotic hoofstock in zoos, particularly giraffe (Giraffa camelopardalis). Anthelmintic resistance (AR) prevalence is increasing in production systems and zoos. To combat the AR that compounds GIN problems, alternative control methods are used. One such alternative is copper oxide wire particles (COWP), which control the abomasal nematode Haemonchus contortus. COWP was given to seven adult giraffe at Busch Gardens Tampa, at descending dosages: 25 g, 12.5 g, 6.3 g. Treatment administration time was determined by fecal egg count (FEC) being above 600 eggs per gram (EPG), and therefore varied with each animal. FEC following 25 g COWP treatment significantly (p < 0.05) decreased between Week 0 and Weeks 2-5. FEC following 12.5 g COWP treatment significantly (p < 0.05) decreased between Week 0 and Week 2. FEC following 6.3 g COWP treatment showed no reduction between Week 0 and all PT samples. A 25 g COWP treatment was an acceptable part of a GIN control program for treatment of Haemonchus in giraffe, a 12.5 g COWP treatment was minimally acceptable, and a 6.3 g COWP treatment was ineffective. To determine the mechanism of action for COWP, 11 Suffolk lambs were abomasally fistulated and inoculated with H. contortus larvae. Abomasal contents, nematodes, feces, and blood were collected at 0, 12, 36, 60, and 84 hours post-treatment (HPT). pH was measured and nematodes were viewed using scanning (SEM) and transmission (TEM) electron microscopy and energy dispersive x-ray spectroscopy (EDS), and copper content analysis was performed. FEC and packed cell volume (PCV) were determined for each time period. pH was inconclusive. FEC decreased throughout the study and up to 33 days PT; no statistics were performed due to removal of nematodes. PCV had no substantial change, but the study time period was too short for a significant change. SEM showed no obvious changes between pre- and post-treatment. TEM showed cuticle damage present in PT samples. EDS showed copper present in 10 of 11 spots on PT samples. Copper content of nematodes was maximum at 60 HPT. Evidence suggests COWP has a direct effect on H. contortus.

Herpes simplex virus type 1 (HSV-1) is a DNA virus that is a ubiquitous pathogen of humans. The hallmark of the HSV-lifecycle is infection of a mucosal surface with spread to sensory neurons where the virus establishes a latent infection with periodic recurrences for the life of the host. The most common course of symptomatic disease with HSV-1 is the typical mucocutaneous lesion that is self-limiting. HSV-1 can also cause acute encephalitis and ocular pathology on reactivation. Both of these manifestations of the disease have severe consequences. Although ocular infection is less frequent, the extensive prevalence of HSV makes it one of the most common infectious causes of blindness in developed countries. Herpetic encephalitis causes significant morbidity and mortality, particularly in the immunosuppressed or untreated individual. HSV-1 specifies at least 11 glycoproteins that are expressed in the infected cells. The viral glycoproteins are the first interface of the virus with a host cell and are therefore vitally important to virus pathogenesis. Glycoproteins B, C, D, H, and L have been extensively studied and found to be involved in initial attachment, fusion, and entry of the virus into a host cell. Recent work in this laboratory has shown that glycoprotein K (gK) is also involved in the fusion machinery of the virus, however, little was known about the role of gK in the pathogenesis of the virus. To assess the role of gK in pathogenesis, mutant viruses were used an experimental infection in a mouse eye model. In this investigation, gK was found to have a critical role in the pathogenesis of the virus in the eye as well as in spread of the infection to neurons and subsequent development of latency. Following this work, a primary neuronal culture system was established to further define the role that gK played in neuronal infection and transport. Using this system, gK was found to be a critical determinant for neuronal transport and pathology. Since gK deficient viruses appear unable to infect neurons, defining this phenotype may assist in the eventual development of a successful vaccine strain of the virus.

Arthropod-borne viruses (arboviruses) are among the most common agents of human febrile illnesses worldwide. As crucially important emerging pathogens, they have caused multiple, notable epidemics of human disease and unnoticed epizootics over recent decades. Despite the public health relevance, very little is known about the geographic distribution of the agents and vectors, relative impact, and risk factors associated to the arboviral infection in many regions of the world and in the tropics in particular. Presented in this dissertation is an experimental study that explores the serology screening of serum samples from 151 patients whom were diagnosed with undifferentiated febrile illness in Sierra Leone, after ruling out endemic malaria and Lassa fever. Related to the laboratory results of the testing, three exploratory experiments on Koutango virus were developed. The experiment directed special interest into the vector mosquito Aedes aegypti and its ability to uptake, disseminate, and transmit the virus. The study of the early events occurring during the interaction between the virus and cells performed in the laboratory was another area of interest with the objective to predict the disease outcome. In order to explore the vertebrate viremia profile, we attempted to develop a suitable animal model for the Koutango virus study in the laboratory. The overall hypothesis of this research is that arboviruses circulating in West Africa are the cause of undiagnosed febrile illnesses. To investigate the hypothesis, this research explores the poorly understood epidemiological features and geographic range of certain endemic arboviruses, particularly the Koutango virus, and whether or not they circulate in the region with the Aedes aegypti mosquito being the competent vector that transmits the virus. The research in this dissertation contributes to the understanding of the epidemiological features and the actual expanding geographical range of many arboviruses. It describes the gold standard laboratory technique for the serology diagnostic of diverse arboviral diseases. In addition, it explores novel laboratory research techniques that may serve as an important tool for the implementation of effective surveillance programs necessary to explore and control the circulation of diverse arboviruses, particularly those associated with human illnesses in West Africa.

West Nile virus (WNV) is one of several flaviruses known to infect mammalian species, including humans. There were 15,257 horse cases reported in 2002 and 1,086 in 2006 in United States. Recently, significant increases in equine and human cases have been reported in United States. Domain III of the WNV envelope protein binds to cellular receptors, and induces a significant portion of the neutralizing antibody response against the virus. CD40 Ligand (CD40L, CD154) enhances productive interactions between T cells and APC and has been shown to function as a potential adjuvant. In this study, we constructed and expressed a fusion protein consisting of the Domain III of WNV envelope protein fused in-frame with the soluble portion of the equine CD40L. Immunizations of rabbits revealed that the recombinant protein induced antibody that specifically reacted with the WNV and neutralized the virus. Similar experiments were performed with horses. Western immunoblots confirmed that vaccinated horses produced antibodies that specifically reacted with the recombinant WNV E DIII proteins. The recombinant DIII protein with TiterMax or CD40L or both as adjuvant(s) induced significantly higher anti-WNV E DIII antibody activities than control and DIII alone groups after first vaccination. The recombinant DIII-CD40L protein vaccine continually induced the anti-WNV E DIII antibody activities without the adjuvant TiterMax. Moreover, the groups immunized with DIII-CD40L+TiterMax and DIII-CD40L showed stronger neutralization activities from week 8 than the other groups, and they maintained the high titers for at least 10 weeks. The results showed that healthy horses vaccinated with recombinant WNV E DIII protein with equine CD40L demonstrated an antigen specific humoral immune response. The responses were enhanced by booster vaccination. Vaccination with this recombinant WNV E DIII-CD40L protein induced a WNV specific immunity in healthy horses that might contribute to protection from WNV-associated disease. CD40L could be utilized as a non-toxic, alternative adjuvant to boost the immunogenicity of subunit vaccines in horses.

Bacterial pneumonia and sepsis are two important causes of mortality in the world. Emergence of multidrug resistant bacteria has necessitated the development of new treatment and/or prevention strategies to augment host immune defense. In this context, the innate host defense is critical in clearing pathogenic bacteria from the host. Early neutrophil recruitment is a critical step in a multistep requence leading to bacterial clearance. Pattern recognition receptors (PRRs) play a critical role in the innate immune system. Receptor interacting protein 2 (RIP-2) is an adaptor for the nod-like receptors (NLR) NOD1 and NOD2. Nucleotide oligomerisation domain 2 (NOD2) is an intracellular PRR that is shown to be important for host defense against intracellular bacterial pathogens. However, the role of NOD2 and RIP-2 during Gram-negative bacterial pneumonia and polymicrobial sepsis has not been explored. Thus, we hypothesize that the NOD2/RIP-2 axis is critical for host defense during bacterial pneumonia and sepsis/septic peritonitis. To test this hypothesis, we infected NOD2(NOD2-/-), RIP-2(RIP-2-/-) deficient mice intratracheally (i.t) with E. coli (106 CFUs/mouse) and Klebsiella pneumoniae (103 CFUs/mouse). We observed that NOD2/RIP2 signaling is critical for the host defense during gram-negative pneumonia and poly microbial sepsis. The NOD2/RIP2 axis regulates neutrophil recruitment via IL-17A production. We also found that NOD2/RIP-2 signaling is essential for the production of IL-6 and activation of STAT3. We demonstrated that RIP-2 regulates inflammasome activity that is independent of NOD2 signaling. Taken together, these data demonstrate that the NOD2/RIP-2 axis plays a critical role in neutrophil-mediated host defense through IL-17A production and by inflammasome activation. In cecal ligation puncture (CLP) induced sepsis, RIP2-/- mice show increased mortality with higher bacterial burden in the peritoneum and systemic organs compared to WT controls. We found reduced neutrophil influx IL-17A and IL-1beta levels in the peritoneum of RIP2-/- mice after CLP. Furthermore, we also observed increased systemic inflammation accompanied by vital organ damage in the knockout mice. As a whole our data suggest a critical role of RIP2 in neutrophil recruitment, along with IL-17A and IL-1beta during sepsis.

Spotted fever group (SFG) Rickettsia are obligate intracellular bacteria that are carried by ticks. One such tick, Dermacentor variabilis is a vector for the etiologic agent of Rocky Mountain spotted fever, R. rickettsii. These ticks also carry a non-pathogenic R. montanensis, the agent used in this study. Interestingly, field data collected from infected D. variabilis throughout the United States revealed that the majority of Rickettsia in ticks are non-pathogenic species such as R. montanensis. Although ticks serve as both vector and reservoir hosts for SFG Rickettsia, many questions regarding tick-Rickettsia interaction remain unresolved. Therefore, the overall goal of this research was to study the relationship between ticks and Rickettsia, specifically examining the molecular mechanisms of rickettsial infection of tick host. As SFG Rickettsia can move between vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for invasion of both types of host cell. Biochemical inhibition assays revealed that the tick molecules, PI 3-kinase, protein tyrosine kinases, Src, FAK, Rho GTPase Rac1, N-WASP, Arp2/3 complex, actin, and V-ATPase are important for R. montanensis invasion. Further studies were executed to molecularly and functionally characterize the tick molecules, Arp2/3 complex and V-ATPase, which are central to rickettsial internalization. Full length cDNA of Arp2/3 complex subunits and V-ATPase from D. variabilis were isolated. Transcriptional profiles of Arp2/3 complex subunits and V-ATPase showed greater expression of the mRNA in tick ovaries compared to midgut and salivary glands. In addition, to gain insight into rickettsial invasion in nature, Arp2/3 complex inhibition assays were performed in tick tissues. The results demonstrated the involvement of Arp2/3 complex in rickettsial entry into midgut, ovary, and salivary glands. The tick molecules identified in this study may provide novel points of intervention in the transmission of tick-borne rickettsial diseases.

The host-parasite interaction during early filarial nematode migration is poorly understood. The objective of this study was to develop a model of early cutaneous filarid migration using Brugia pahangi in the jird (gerbil) host and measure the histologic and cytokine responses during this period. Male gerbils were intradermally inoculated in the left hindlimb with 100 B. pahangi L3 then necropsied at 3 hours, 24 hours, 3 days, 7 days, and 28 days post-infection. Larvae were recovered and tissues collected for histology and cytokine measurement. At 3 hours, most larvae (96.3%) were recovered from tissues associated with the infection site. Migration away from the infection site occurred within 24 hours. By 7 days, larvae were dispersed throughout the lymphatic system, including the spermatic cord lymphatics. Larvae were identified on histologic exam at all time points and were located in the dermis, muscle, lymphatic vessels, and lymph nodes. Predominantly neutrophilic inflammation was frequently present around larvae in the dermis and muscle at 3 and 24 hours. Levels of the cytokines IL-6, TNF, IFN-gamma, and IL-4 were measured in the spleen and popliteal and renal lymph nodes. IL-6 and TNF both showed a peak at 3 hours followed by consistent decline in all tissues. No clear increase in expression was appreciated for IFN-gamma. IL-4 remained low through 7 days and rose by 28 days in all tissues. These results indicate the ability of filarid L3 to rapidly migrate through host tissue and they support intradermal gerbil infection as a model for early filariasis. Cytokine analysis and histology indicated an acute host inflammatory response following initial infection, with Th2 polarization occurring later in the course of infection.

Summer pasture-associated obstructive pulmonary disease (SPAOPD), a naturally occurring airway disease of horses, is characterized by clinical exacerbation associated with exposure to pasture environment during the summer. Aeroallergens are believed to trigger exacerbation of SPAOPD, cytokines are likely associated with the anamnestic response to aeroallergens, and endothelin (ET)-1 is a potential mediator of airway obstruction. The goal of this dissertation was to describe and explore the interaction of aeroallergens triggering inflammation and T lymphocytes cytokine profile with the recruitment and activation of neutrophils and synthesis of ET-1 by mononuclear leukocytes and airway epithelial cells. The temporal pattern of clinical exacerbation was associated with hot and humid conditions and with increases in grass pollen and mold spore counts. Circulating concentrations of ET-1 were increased during clinical exacerbation of SPAOPD compared with remission and controls. Gene expression of ET-1 and cytokines, interleukin (IL)-8 and IL-4, but not interferon (IFN)-γ, tended to be greater in lungs of SPAOPD-affected than non-affected horses. The immunoreactive ET-1 distribution tended to be greater in airway tissues of affected horses. The putative aeroallergens, grass pollen & mold spores, and ET-1 induced neutrophil activation and chemotaxis in vitro. Putative aeroallergens induced IL-4 and IFN-γ expression and up-regulation of ET-1 release in mononuclear leukocytes. Cultures of airway epithelial cells were established under air-liquid interface and microgravity conditions and evaluated for differentiation. Cytokines IL-4 and tumor necrosis factor (TNF)-α induced up-regulation and directional (basolateral) release of ET-1 by differentiated airway epithelial cells, grown under air-liquid interface.