Department of Veterinary Medicine Theses and Dissertations
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Item MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH(2009) Ammayappan, Arun; Vakharia, Vikram N; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Infectious hematopoietic necrosis virus (IHNV) is a pathogen of major economic importance to the aquaculture industry. The long-term goal of our work is to develop a safe and effective recombinant IHNV vaccine and possibly use IHNV as a virus vector to express foreign genes. To achieve this goal, the complete genome of IHNV 220-90 virulent strain was sequenced and characterized. Subsequently, a full-length cDNA clone of IHNV was generated by constructing the full length cDNA clone, between the cytomegalovirus (CMV) promoter and the autocatalytic hammerhead and hepatitis delta virus ribozymes. Transfection of a full-length plasmid, along with the supporting plasmids resulted in the recovery of infectious rIHNV-220-90. Characterization of the rIHNV-220-90 showed that its growth characteristics in tissue culture were comparable to those of the parental virus. The possible role of IHNV proteins in virulence was explored to some extent. For this, the entire genome of attenuated virus (IHNV-61) was sequenced and compared with its virulent strain. The comparative sequencing analysis studies revealed that majority of differences were located in the glycoprotein gene. The M and G genes, and the trailer region between virulent and attenuated viruses were exchanged; recombinant chimeric viruses were recovered and studied for their pathogenicity in rainbow trout. The results obtained from in vivo studies indicate that the glycoprotein plays a major role in IHNV virulence in fish, whereas the M gene and trailer region play a negligible role in virulence of IHNV. The potential of rIHNV to serve as a viral vector was explored by expressing the VP2 protein of IPNV and hemagglutinin-estrase (HE) protein of ISAV. The recovered rIHNV-VP2 and rIHNV-HE viruses stably expressed the VP2 and HE proteins respectively for at least five serial passages and showed characteristics comparable to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively to examine the molecular determinants of virulence, pathogenesis, and new approaches for vaccine development.Item FCRN MEDIATED MUCOSAL IMMUNITY AND SUBUNIT VACCINE DELIVERY(2009) YE, LILIN; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)FcRn, the neonatal Fc receptor, is an MHC class I related molecule, functions as an IgG protector and transporter. Binding of IgG by FcRn exclusively occurrs at acidic pH, in correlation with the fact that FcRn mainly resides in acidic endosomes. Herein, we found an association of FcRn with invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn heavy chain alone or heavy chain-beta-2-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was detected in IFN--treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was detected in the late endosome/lysosome only in the presence of Ii or upon exposure to IFN-. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or Ii chain. Vaccine strategies to prevent invasive mucosal pathogens are being sought due to the fact that 90% of infectious diseases are initiated at mucosal surfaces. However, our ability to deliver an mucosal vaccine antigen for induction of the protective immunity is limited. FcRn mediates the transport of IgG across polarized epithelial cells. Taking advantage of this unique transfer pathway, I sought to delivery of antigens across mucosal barrier using IgG Fc fused proteins. It was demonstrated that intranasal immunization with a model antigen herpes simplex virus type-2 (HSV-2) glycoprotein gD fused with an IgG Fc fragment combination with CpG ODN adjuvant resulted in a complete protection of wild type, but not FcRn knockout mice that were intravaginally challenged with virulent HSV-2 186. The immunization induced efficient mucosal and systemic antibody as well as long lasting memory immune responses. These results are the first to demonstrate that the FcRn-IgG transcellular pathway may represent a novel mucosal vaccine delivery path against mucosal infections.Item FUNCTIONAL CHARACTERIZATION OF THE INTERACTION OF HEPATITIS E VIRUS ORF3 PRODUCT WITH THE CYTOSKELETON(2008) Kannan, Harilakshmi; Zhang, Yan-Jin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hepatitis E virus (HEV) causes several outbreaks of hepatitis in humans. Many aspects of HEV pathogenesis are not well understood. The HEV ORF3 product (henceforth known as vp13) is a multifunctional protein essential for infection of animals. To better understand the vp13 functions, this study was performed. We observed that vp13 protein was associated with the microtubules (MT) in transfected cells. Mutational studies revealed that both hydrophobic domains at the N-terminal region of vp13 are required for the vp13-MT interaction. Our studies also showed that HEV vp13 protein increased the stability of the MT, activated the apoptotic pathway, and, increased the levels of tumor suppressor gene p53 and its downstream effector p21Cip/WAF1 in the transfected cells. However, no noticeable effect on cell survival was observed. These results indicated that HEV vp13 protein may act as a viral regulatory protein.Item Host Molecular Responses in Chickens Infected with an Avian Influenza Virus(2008-11-20) Ramirez-Nieto, Gloria Consuelo; Perez, Daniel R.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian influenza virus has a segmented RNA genome that allows the virus to evolve continuously and generate new strains. Wild birds serve as natural reservoirs of avian influenza virus and provide a potential source for emergence of new viruses, which traverse host barriers and infect new avian or mammalian species. The mechanisms involved in this process are not completely understood. Our main goal is to understand host-pathogen interactions involved in avian influenza pathogenicity. As part of our approach we studied the effect of pre-exposure of chickens to IBDV (infectious bursal disease virus) on host susceptibility to infection, disease progression, and host molecular responses to infection with a mallard H5N2 low pathogenic avian influenza (LPAI) virus. We found that prior exposure of chickens to IBDV led to increased susceptibility to infection with the mallard H5N2 LPAI virus compared to normal chickens. This increased susceptibility allowed us to further adapt the virus to chickens. After 22 passages (P22) in IBDV-pre-exposed chickens, the LPAI virus replicated substantially better than the wild-type (WT) mallard virus in both IBDV-exposed and normal chickens. Interestingly, the P22 virus showed similar levels of replication in the respiratory and intestinal tracts of both groups, although it caused exacerbated signs of disease and severe lesions in the IBDV-pre-exposed group. We suggest that prior IBDV exposure provides a port of entry for avian influenza in an otherwise resistant chicken population. Furthermore, adaptation of avian influenza (AI) in IBDV-exposed chickens may allow for the selection of AI virus strains with expanded tissue tropism. We also studied the effects of host response to H5N2 AI in normal and IBDV-infected birds using high-throughput gene expression analysis. We demonstrated that IBDV-exposed chickens showed less than optimal humoral responses to LPAI infection as well as alterations in local molecular pathways that eventually led to exacerbated disease and death. At the molecular level we found amino acid substitutions in the surface glycoprotein hemagglutinin (HA). Those changes suggest selection for a virus that binds to and replicates more efficiently in chickens. Taken together our results suggest that IBDV-pre-exposure may play a role in exacerbating AI-induced pathogenicity.Item Role of Noncoding Regions in Newcastle Disease Virus Replication and pathogenesis(2008-11-17) Yan, Yongqi; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The roles of the intergenic sequences (IGS) and untranslated regions (UTR) in Newcastle disease virus (NDV) transcription and pathogenesis are not clear. By our established reverse genetics system, we investigated the role of these noncoding regions in NDV life cycle. The infectious recombinant viruses containing increased/decreased length of F-HN and HN-L IGS were recovered and the transcription and pathogenicity of mutants were characterized. Our studies indicated that increased F-HN or HN-L IGS length reduced the downstream gene transcription. Morever, all IGS mutants were attenuated in chickens and the level of attenuation was increased as the length of IGS increased. The mutant viruses with modified 5' and 3' UTR of HN mRNA were also recovered. The transcription, translation and pathogenecity of these recombinant viruses were characterized. Our studies indicated that the UTRs are not essential for NDV replication in vitro. Complete deletions of 5' HN UTR down regulated its transcription, translation levels and incorporation of HN protein into virus particle, therefore, attenuated the pathogenicity of NDV in chickens. Moreover, studies on the HN UTRs replaced with corresponding NP UTRs virus suggested that UTRs can be exchanged between NDV mRNAs without affecting the replication of virus in vitro and in vivo. In summary, my research identifies for the first time the role of noncoding regions in NDV replication and pathogenesis and provides novel methods for the development of attenuated live vaccines for NDV.Item Development of a mouse model for the t(10:11)(p13;q14) chromosomal translocation associated with acute leukemia in humans(2008-08-08) Caudell, David L; Samal, Siba K; Aplan, Peter D; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Acute leukemia is associated with a wide spectrum of gross chromosomal rearrangements. These acquired mutations include balanced and unbalanced chromosomal translocations. The analysis of chromosomal translocations has provided much insight into understanding the biology of hematologic malignancies, leading to improved diagnosis and classification, as well as identification of novel therapeutic targets. The rare but recurring chromosomal translocation [t(10;11)(p13;q21)] results in a CALM-AF10 fusion that occurs in patients with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). CALM-AF10 transgenic mice developed AML with lymphoid features and had Hoxa gene cluster upregulation. In this model, mice developed leukemia after a long latency period with incomplete penetrance. These findings suggest that additional genetic events are needed to complement CALM-AF10 mediated leukemic transformation. Retroviral insertional mutagenesis was used to identify complementary genetic events that might collaborate with CALM-AF10 during leukemic transformation. A cohort of CALM-AF10 mice was infected with the Mol4070LTR retrovirus; by 5.5 months of age, 50% of the transgenic mice developed AML, a clear acceleration of disease onset compared to either wild type littermates injected with the retrovirus or CALM-AF10 mice not injected with the retrovirus. The tumors assayed by Southern blotting for viral integration showed clonal to oligoclonal expansion. Ligation-mediated PCR and sequence analysis of DNA derived from leukemic cells was used to identify potential collaborating genes at the retroviral insertion sites including Evi1, Nf1, kRas, Zeb2, and Mnl. Identification of these genes as a potential collaborating gene with CALM-AF10 supports the emerging paradigm in leukemia biology that predicts that most, if not all leukemic cells must undergo at least two collaborative events to produce a fully transformed cell. One of these events typically leads to impaired differentiation and enhanced renewal of stem cells, whereas the second event leads to increased proliferation and/or decreased apoptosis. It has been shown here that retroviral infection accelerates the onset of acute leukemia, and identified genes that potentially collaborate with the CALM-AF10 fusion gene in the leukemic transformation process. This transgenic murine model serves as a model system for studying leukemogenesis similar to that observed in humans with leukemia.Item Intervention Strategies to Reduce Foodborne Pathogens in Poultry During Grow-out and Processing(2008-04-28) Tan, Rommel Max Tan Sim Lan; Tablante, Nathaniel L; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several foodborne pathogens like Salmonella spp., Campylobacter jejuni and Clostridium perfringens can occasionally be traced to poultry sources. The development of intervention strategies that are applicable to different stages of poultry production can help lessen the level of these pathogens in poultry by-products and hence, reduce the incidence of poultry-borne food poisoning. In the present study, the efficacy of Poultry Litter Treatment® in reducing Clostridium perfringens counts in poultry litter was investigated. The effect of windrow-composting in reducing microbial load in poultry litter was also studied. In addition, a study of bacterial profiles in a poultry processing line was conducted. Finally, the efficacies of two online reprocessing antimicrobials in reducing bacterial pathogen load were compared.Item Regulation of Virulence by BarA-UvrY Two-Component system and LuxS in Extraintestinal Pathogenic Escherichia coli.(2007-12-05) Palaniyandi, Senthilkumar; Mukhopadhyay, Suman; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Pathogenic E. coli cause intestinal or extraintestinal infections in many host species. E. coli strains enter the intestinal tract through food and colonize the intestinal epithelium to cause infections. In animals and humans, E. coli causes gastroenteritis, neonatal meningitis and urinary tract infections. In birds, E. coli causes a complex syndrome called avian colibacillosis. The orthologs of BarA-UvrY two-component (TCS) system is known to regulate a number of phenotypic traits in gamma proteobacteria, although their role in Extraintestinal pathogenic Escherichia coli (ExPEC) virulence is yet to be determined. The barA gene is membrane bound sensor kinase protein and the uvrY gene encodes the cognate response regulator in E. coli. Work in this study has focused how the BarA-UvrY and LuxS system regulates in vivo virulence in uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC) during infection. The main goal of this study is to look at how BarA-UvrY TCS and LuxS regulate virulence in APEC 7122 and UPEC CFT073. In this study, we studied the role of BarA-UvrY TCS system in regulation of virulence in the aforementioned ExPEC strains using animal model and tissue culture system and the role of LuxS in regulation of virulence determination in ExPEC. Our results indicate that BarA-UvrY regulates multiple virulence properties in APEC 7122 and UPEC CFT073 and that LuxS regulates partial virulence properties in APEC 7122 and UPEC CFT073.Item The role of Newcastle disease virus internal proteins in pathogenesis(2007-09-24) Rout, Subrat N; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The internal proteins, nucleocaspid protein (NP), phosphoprotein (P) and large polymerase protein (L) of Newcastle disease virus (NDV), play an important role in transcription and replication of the viral genome. However, their role in NDV pathogenesis has not been explored. In this study, the importance of internal proteins in NDV virulence was evaluated through a chimeric approach using an established reverse genetics technique. The L gene between an avirulent NDV strain LaSota and a moderately virulent NDV strain Beaudette C (BC) was exchanged, recombinant chimeric viruses were recovered and studied for their pathogenicity in the natural host, chicken. The results obtained from in vivo studies indicated that the L gene of NDV modulate role in NDV virulence in chickens. The NP and P genes of NDV were exchanged between BC and LaSota individually as well as in combination; chimeric viruses were recovered, indicating that heterologous NP and P genes were functional. In vitro replication of chimeric NP and P recombinant viruses in DF-1 cells indicated that the exchange of NP or P gene in NDV did not affect the replication of the chimeric viruses. The in vivo studies in chickens showed that the change in pathogenicity of these chimeric viruses was minimal and homotypic interaction between NP and P proteins is necessary for optimum pathogenicity of the virus.Item Molecular Epidemiology and Surveillance of Avian Influenza in Wild and Domestic Birds(2006-05-09) Pascua, Annabelle Morano; Tablante, Nathaniel L; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Surveillance of the existence of avian influenza virus in birds is essential in understanding its epidemiology and potential zoonosis. Point surveillance was made on December 2004 and May to August 2005 in wild birds, domestic poultry and environment. Seven out of 67 samples were positive for avian influenza infection resulting to a 10.4 % isolation rate during the winter. Partial sequencing revealed that all isolates were of H11N3 subtype. In the summer, a total of 584 tracheal, cloacal and environmental swabs were tested in the laboratory through virus isolation, real- time PCR and RT-PCR. All samples were negative. To understand the evolution and ecology of the isolated virus, further sequencing was done for all eight genes of H11N3 and each gene sequence was phylogenetically analyzed with available sequences in the Influenza Sequence Database. Replication and transmission of H11N3 were also investigated through experimental infection of chicken and quail.