Department of Veterinary Medicine Theses and Dissertations

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

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End date: 2019Subject: search.filters.subject.Biology, Veterinary Science

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    Genetics of Avian Paramyxovirus serotype 2
    (2010) Subbiah, Madhuri; Samal, Siba K.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Avian Paramyxovirus (APMV) serotype 2 is one of the nine serotypes of APMV that infect a variety of bird species around the world. In chickens and turkeys, APMV-2 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-2, the complete genome sequences of prototype strain Yucaipa and strains Bangor, England and Kenya were determined. The genome lengths of APMV-2 strains Yucaipa, Bangor, England and Kenya are 14904, 15024, 14904, 14916 nucleotides (nt), respectively. Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Sequence comparison of APMV-2 strains England and Kenya with prototype strain Yucaipa show 94-98% nt and 90-100% aggregate amino acid (aa) identities. However, strain Bangor shares low level of nt and predicted aa sequence identities with the other three strains. The phylogenetic and serological analyses of all four strains indicated the existence of two subgroups: strains Yucaipa, England and Kenya represented one subgroup and strain Bangor represented the other subgroup. All four strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenicity of APMV-2, a reverse genetics system for strain Yucaipa was established in which infectious recombinant APMV-2 was recovered from a cloned APMV-2 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to wild type virus. Recombinant APMV-2 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of mutant viruses by replacing the fusion protein (F) cleavage site of APMV-2 with those of APMV serotypes 1 to 9 demonstrated that the amino acid composition at F protein cleavage site does not affect the pathogenicity of APMV-2. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-2 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-2 would be of considerable utility for introducing defined mutations into the genome of this virus and develop vaccine vector for animal and human pathogens.
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    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.
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    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.
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    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.
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    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.
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    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.
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    EPIDEMIOLOGY OF SALMONELLA CONTAMINATION OF POULTRY MEAT PRODUCTS: KNOWLEDGE GAPS IN THE FARM TO STORE PRODUCTS
    (2004-11-29) Myint, Maung San; Johnson, Yvette J; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The ultimate goal of controlling foodborne hazards is to reduce the risk of disease to consumers, and the economic burden related to foodborne illness. A literature review identified four areas of insufficient data on the epidemiology of Salmonella. A cross-sectional study was used to estimate prevalence of enteric bacterial contamination of plant-based animal feed and milk replacer from Maryland and Northern Virginia. All samples were negative for Campylobacter; 0.6% were positive for Salmonella; 5.7 % for E.coli; and 50.6% for Enterococcus. Samples purchased in summer of 2002 were 38 times more likely to be contaminated with Enterococcus than samples purchased in winter of 2002 (p-value<0.001). Enterococcus positive samples were 8 times more likely to be E.coli positive than Enterococcus negative samples. Another cross-sectional study was used to assess the association between the pattern of airflow and the distribution of fecal coliforms and Salmonella in commercial chicken litter. At moderate relative humidity (about 50%), there was a significant association between regions of reduced airflow and increased coliform and Salmonella contamination within a poultry house. An analysis of a PCR technique to validate sensitivity and specificity relative to culture techniques for detecting Salmonella contamination in retail poultry meat was conducted. When only BPW pre-enrichment was used, the PCR test had a sensitivity of 85%. This increased to 89 - 100% when BPW pre-enrichment was followed by selective enrichment with RV or TT-H broth, respectively when conventional culture is the gold standard. A minimum of 12 hours pre-enrichment and 100 cfu was necessary to achieve 100% sensitivity with PCR. Random poultry meat samples from 10 retail grocery outlets in Maryland were collected in the final cross-sectional study. Overall Salmonella prevalence in poultry meat products was 23% (C.I 15.16 - 30.86). Integrator brand ground chicken meat had an increased Salmonella prevalence compared to non-ground meat products; however this difference was not significant (p=0.0533). Store brand non-ground chicken meat products were 18 times more likely to be contaminated with Salmonella than integrator brands (C.I. 5.41-61.26).
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    Role of Hemagglutinin-Neuraminidase Protein in Newcastle Disease Virus Pathogenesis
    (2003-12-17) Panda, Aruna; Samal, Siba K; Veterinary Medical Science
    The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is an important multifunctional envelope protein, which plays key roles in virus attachment, neuraminidase and fusion promotion activities. Using the established reverse genetics technique for the recovery of infectious NDV from cloned cDNA, the role of the HN protein in NDV pathogenesis was explored. By exchanging the HN gene between an avirulent NDV strain LaSota and a virulent NDV strain Beaudette C (BC), two chimeric viruses were generated. In vitro and in vivo studies done with the chimeras indicated an important role of the HN protein in modulating NDV virulence in hosts. The HN gene of NDV has six glycosylation sites, two of which are not used for addition of carbohydrates. The exact role of the four functional glycosylation sites in NDV pathogenesis is unknown. To understand the importance of these sites in influencing NDV virulence, each site was eliminated individually and in combination on a cDNA clone of NDV strain BC. Four single mutant viruses with each of the sites (1-4) and one double mutant virus with sites 1 and 2 eliminated, were recovered. Results from this study established the key role of glycosylation site 4 and glycosylation sites 1 and 2 in combination, in influencing NDV pathogenesis. A recent crystal structure of the HN protein of NDV gives valuable information about the location of amino acids involved in receptor-binding, neuraminidase and fusion promotion activities of the protein. To study the effect of mutagenesis of such key amino acids on NDV pathogenesis in vivo, five recombinant viruses with mutations in residues at the receptor-binding domains of the HN protein and in residues differing between the LaSota and BC strains of NDV, were generated. In vitro and in vivo studies with these viruses indicated the important role of certain residues in viral infectivity. In summary, the importance of the HN protein in NDV pathogenesis was established. Further studies indicated the role of glycosylation sites in modulating NDV virulence. The relevance of certain key amino acid residues on the HN protein in NDV pathogenesis was also investigated.