Department of Veterinary Medicine Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2762

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Delivery of DNA and Recombinant Infectious Bursal Disease Virus Vaccines in Ovo
(2004-03-16) Moura, Lenita de Cassia; Vakharia, Vikram N; Veterinary Medical Science
Infectious bursal disease virus (IBDV) remains a serious problem for commercial broiler producers throughout the world. An in ovo delivery system for plasmid DNA vaccines was evaluated by studying parameters, such as the route of delivery (air cell vs amniotic cavity), transfection reagent (IFA+DMSO vs polyethylenimine), dose of plasmid DNA (1 to 100 µg/egg), and the nature of humoral immune responses. An optimal response was detected when embryos were inoculated with 60 µg of plasmid DNA. This system for in ovo delivery was used to determine the efficacy of a plasmid DNA vaccine against IBDV in 18-day-old embryos. The DNA vaccine expresses the polyprotein VP2-VP4-VP3 of IBDV. SPF and fertile broiler eggs with maternal antibodies were vaccinated and challenged against IBDV-STC. Two groups of birds (SPF and broilers) received a booster immunization with baculovirus expressed-proteins of IBDV. The DNA vaccine had no detrimental effect on hatchability or first week post-hatch survival. In ovo vaccination generated detectable humoral immune responses as measured by ELISA. Antibody response was significantly enhanced two weeks post the IBDV-protein boost. Broilers vaccinated with plasmid DNA or IBDV-protein boost exhibited partial protection against IBDV-STC strain, whereas, vaccinated SPF chicks were not protected and exhibited severe microscopic lesions after challenge. A second approach in the control of IBDV used a recombinant attenuated vaccine administered in ovo to 18-day-old embryos. The vaccine was genetically tailored to protect from challenges in the field against classic and variant strains of IBDV. SPF and fertile broiler eggs were vaccinated and used to evaluate protection against IBDV-STC challenge. A full dose of the vaccine consisting of 5.6x103 pfu was administered to SPF and commercial broiler embryos. In addition, a half dose containing 2.3x103 pfu was injected in SPF embryos. The vaccine generated high antibody titers in chickens vaccinated with either dosage. All vaccinated groups were protected against mortality. The vaccine did not cause bursal damage and fully protected SPF chicks vaccinated in ovo with 2.3x103 pfu and broiler embryos that received a full dose of the recombinant vaccine. The vaccine had no effect on hatchability or first week survival in either broilers or SPF birds, even when high doses were administered.
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.
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.
REVERSE GENETICS OF AVIAN METAPNEUMOVIRUS
(2005-12-06) Dhanasekaran, Govindarajan; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses to the US turkey industry. With a long-term goal of developing a better vaccine for controlling AMPV in the US, a reverse genetics system to produce infectious AMPV entirely form cloned cDNA was established. To achieve this, the unpublished sequences of the G gene, the L gene, the leader and trailer region were first determined to complete the entire genome sequence of AMPV subgroup C strain Colorado (AMPV/CO). Our results showed that the full-length AMPV/CO genome was 14,150 nucleotides (nt) in length, denoting that AMPV/CO possessed the longest genome among known metapneumoviruses. Subsequently, a cDNA clone encoding the entire 14,150 nt genome was generated by assembling 5 cDNA fragments, representing the entire genome, between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a low-copy number transcription plasmid pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1 and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. The recovered virus was observed to contain the genetic markers that were artificially introduced during cloning. Characterization of the recombinant AMPV/CO showed that its growth characteristics in tissue culture were similar to those of the parental virus. These results demonstrate that infectious AMPV can be generated entirely from cloned DNA using reverse genetics techniques. The potential of AMPV/CO to serve as a viral-vector was examined using green fluorescent protein (GFP) as a reporter. The recovered rAMPV/CO-GFP virus stably expressed GFP for at least five serial passages and showed characteristics similar 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 for various studies involving AMPV molecular biology, pathogenesis and vaccine development.
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.