Department of Veterinary Medicine Theses and Dissertations

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Antagonizing JAK-STAT signaling by porcine reproductive and respiratory syndrome virus
(2018) Yang, Liping; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by numerous cytokines. JAK-STAT pathways involve in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of their significance in immune response, they are often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and severe respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One possible reason for the weak protective immune response is that PRRSV interferes with innate immunity and modulates cytokine signaling, including JAK-STAT pathways. The objective of this project was to elucidate the mechanisms of PRRSV interference with JAK-STAT2 and JAK-STAT3 signaling. This study demonstrates that PRRSV antagonizes interferon (IFN)-activated JAK-STAT2 signaling and oncostatin M (OSM)-activated JAK-STAT3 pathway via inducing STAT2 and STAT3 degradation. Mechanistically, PRRSV non-structural protein 11 (nsp11) and nsp5 induce the degradation of STAT2 and STAT3, respectively, via the ubiquitin-proteasome pathway. Notably, PRRSV manipulates karyopherin alpha 6 (KPNA6), an importin that is responsible for STAT3 nuclear translocation in the JAK-STAT signaling, to facilitate viral replication. Knockdown of KPNA6 expression led to significant reduction in PRRSV replication. These data demonstrate that PRRSV interferes with different JAK-STAT pathways to evade host antiviral response while harnessing cellular factors for its own replication. These findings provide new insights into PRRSV-cell interactions and its molecular pathogenesis in interference with the host immune response, and facilitate the development of novel antiviral therapeutics.
AVIAN PARAMYXOVIRUS-VECTORED VACCINES AGAINST INFECTIOUS BRONCHITIS VIRUS AND HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS
(2019) Shirvani, Edris; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Highly pathogenic avian influenza (HPAI), infectious bronchitis (IB), and Newcastle disease (ND) are highly contagious and economically important diseases in poultry. Vaccination is the major strategy which is implemented to combat highly pathogenic avian influenza virus (HPAIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV), worldwide. However, among these viruses, some NDV strains are naturally avirulent and have been used as highly safe vaccines for more than 60 years. Live attenuated IBV vaccines that are produced by passaging virulent strains in eggs have safety concerns and are genetically unstable. Inactivated IBV and HPAIV vaccines also are less efficacious and affordable. Therefore, development of alternative vaccines against IBV and HPAIV is highly needed. In this multistep study, we have employed NDV vector and other novel avian paramyxovirus (APMV) vectors to develop improved IBV and HPAIV vaccines. Firstly, we conducted a study to investigate the contributions of the S1, S2, and S proteins of IBV in protection against virulent IBV, and to develop a safe and efficacious recombinant NDV-vectored IBV vaccine. We generated recombinant (rNDV) strain LaSota viruses expressing S1, S2 or S protein of IBV using reverse genetics. We evaluated the protective efficacies of rNDVs against virulent IBV and NDV challenges. Our results showed that the S protein, which contains the S1 and S2 neutralizing epitopes in correct confirmation is the best protective antigen of IBV. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. Secondly, besides rNDV strain LaSota vector, we employed a novel chimeric rNDV/avian paramyxovirus serotype-2 (rNDV/APMV-2) vector that replicates less efficiently and a modified NDV strain LaSota (rLaSota-527) vector that replicates more efficiently to develop a likely improved viral vectored vaccine against IBV. We generated rNDV/APMV-2 or rLaSota-527 virus expressing the best protective protein of IBV (S protein), which was found in the first study. The protective efficacies of rNDV/APMV-2 or rLaSota-527 virus expressing the S protein was evaluated against IBV in chickens. Our results showed that immunization of chickens with either chimeric rNDV/APMV-2 expressing the S protein, which is a better candidate for in ovo vaccination, or rLaSota virus expressing the S protein provided protection against IBV. Most importantly, compared to prime-boost vaccination or vaccination with rLaSota-527 virus expressing the S protein, single immunization of chickens with rLaSota virus expressing the S protein induced better immune responses against IBV. Thirdly, we conducted a study to evaluate the contributions of HA1 and HA2 subunits of HPAIV HA protein in the induction of neutralizing antibodies and protection in chickens, using rNDV strain LaSota vector. Our results showed that the HA1 and HA2 subunits when expressed separately, neither provided protection nor induced neutralizing antibodies. To be effective the HA protein must be incorporated into a vaccine as an intact protein. These results also highlight the importance of using chickens in HPAIV vaccine studies as they are susceptible natural hosts. Finally, we employed APMV-3 strain Netherlands as a vaccine vector, for its high efficiency replication in multiorgans of host, to generate an improved vaccine against HPAIV. Our results showed that immunization of chickens with either rAPMV-3 expressing the HA protein (rAPMV-3/HA) or rNDV expressing the HA protein (rNDV/HA) provided complete protection against HPAIV challenge. However, the immunization of chickens with rAPMV-3/HA induced higher levels of neutralizing antibodies than that induced by rNDV/HA. These results suggest that mass-vaccination with a rAPMV-3/HA might provide better protection against H5N1 HPAIV in field conditions. In conclusion, the individual subunits of the S protein of IBV or the HA protein of HPAIV when expressed separately, neither provided protection nor induced neutralizing antibodies. To provide protective efficacy, the intact HA or S protein must be incorporated into vaccine. The rNDV expressing the S protein is a safe and efficacious bivalent vaccine against IBV and NDV. Other than rNDV strain LaSota, rNDV/APMV-2 and rAPMV-3 are promising vaccine vectors for development vaccines against IBV and HPAIV, respectively.
Biological Significance of selected Ixodes scapularis Transcription Factors regulating Tick Hematophagy and Development
(2023) Antara, Kazi Rifat; Pal, Utpal Professor; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Lyme disease is one of the most prominent vector-borne diseases, which is transmitted by the Ixodes scapularis tick and related species, and the causative agent is the bacterial pathogen Borrelia burgdorferi. Besides I. scapularis, many other tick species are also prolific vectors of several bacterial, viral, or eukaryotic pathogens affecting humans and animals. I. scapularis possess a large genome of 2.26 Gbp, predominantly featuring repetitive DNA or transposomal elements. Although many orthologous genes are present in other arthropods and blood-borne vectors, the genome also encodes numerous unique tick-specific genes. Despite many advances in Ixodes biology and genomics, the molecular basis of their hematophagy and development remains unknown. During feeding on the host, a major tick organ like the gut undergoes remarkable yet poorly understood episodes of cell division and differentiation, accommodating a huge blood meal that can be up to 100-fold greater than their body weight. The gut, therefore, plays a vital role in blood meal acquisition, digestion, and storage, supporting the long-term survival of ticks during prolonged off-host periods of nutrient deprivation. Understanding the molecular mechanism of gut physiology, including cell division and differentiation, is an essential area of research. As transcription factors are central to the biology and development of metazoan organisms yet remain largely uncharacterized in ticks, the goal of this dissertation is to decipher the biological significance of representative groups of major development-associated transcription factors in I. scapularis that are expressed in the gut, especially during blood meal engorgement process. Among them, two of the highly upregulated transcription factors in the gut were chosen for further characterization. We show that both transcription factors, Immunoglobin-fold transcription factor (SuH) and POU domain transcription factor (Nubbin), play essential roles in tick physiology, as their knockdowns impart phenotypic defects, impacting tick feeding, development and life cycle. The latter part of the dissertation will highlight the molecular mechanism of their functions. A fundamental understanding of the molecular basis of tick biology, hematophagy, and development may contribute to developing novel strategies to curb the spread of tick-infection.
The C proteins of human parainfluenza virus type 1 exert broad control over the host innate immune response
(2010) Boonyaratanakornkit, Jim; Samal, Siba; Collins, Peter; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Human parainfluenza virus type 1 (HPIV1) is an important pediatric respiratory pathogen, and its virulence in vivo can be attenuated by introducing mutations into the C gene, a strategy that has been used to design live attenuated candidate vaccines. By tracking gene expression over time, we found that a HPIV1 mutant with a single point mutation in the C gene, referred to as C(F170S), and a HPIV1 mutant with complete deletion of the C gene, referred to as P(C-), altered the expression of over 1000 genes, in sharp contrast to wild-type (WT) HPIV1. Using functional bioinformatics, we found that binding sites for the IRF and NF-kB family of transcription factors were over-represented in many of the C protein targeted pathways. By examining the activation of the major components of the type I interferon (IFN) enhanceosome, we found that the C mutant viruses, but not WT HPIV1, activated IRF3 phosphorylation and IkBB degradation, steps integral to the formation of the interferon enhanceosome. To investigate the basis for the observed antagonism of the host response by the WT C proteins, which are expressed from the C open reading frame as a nested set of carboxy-coterminal proteins, we searched for but were unable to identify C-interacting proteins among members of the RIG-I/MDA5 pathway. Furthermore, we also found that the WT C proteins supplied in trans could block IFNB induced by P(C-) HPIV1 infection but not by heterologous inducers of RIG-I and MDA5, namely RSV and poly I:C, respectively. These two lines of evidence suggested that the HPIV1 C proteins do not directly block type I IFN production, such as by interacting with a host factor of the RIG-I/MDA5 pathway. Using knockout mouse embryonic fibroblasts, we found that HPIV1-induced IFNB production relied mainly on MDA5. Consistent with this observation, a striking amount of intracellular dsRNA was detected during infection with the C mutant HPIV1 viruses but not with WT HPIV1. A marked increase in viral genome, antigenome, and mRNA as well as a decrease in viral protein accumulation provided compelling evidence for dysregulated viral RNA synthesis and an inhibition of viral protein synthesis in the absence of WT C proteins. We suggest that this resulted in an imbalance in the N protein-to-genomic RNA ratio, leading to incomplete encapsidation and an intracellular environment permissive for the generation of dsRNA. PKR activation followed the kinetics of dsRNA accumulation and contributed to both IFNB induction and the reduction in viral protein levels. This study establishes the profound effects that the C proteins of wild-type HPIV1 play in evading the host response to HPIV1 infection, and it extends our current understanding of the innate immune response to HPIV1 infection.
CD23 MEDIATED IGE TRANSCYTOSIS IN AIRWAY INFLAMMATION
(2012) Palaniyandi, Senthilkumar; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
CD23 (FceRII), a C-type lectin type II membrane glycoprotein, plays an important role in IgE homeostasis and development of allergic inflammation. I showed that CD23 was constitutively expressed in the established or primary human airway epithelial cells and its expression was significantly up-regulated by IL-4 stimulation. In a transcytosis assay, human IgE or IgE derived immune complex was transported and enhanced by IL-4 stimulation across a polarized Calu-3 monolayer. A CD23 specific antibody or soluble CD23 significantly reduced the transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed antigen-IgE complexes were competent in inducing degranulation of the cultured human mast cells. This study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation. To verify the above results in a mouse model, CD23 expression was detected in epithelial cells lining mouse airway and enhanced by IL-4 exposure as well as in ovalbumin (OVA) sensitized mouse. I showed that CD23 transported IgE and OVA-IgE derived immune complex across airway epithelial cells in wild-type, but not CD23 knockout (KO), mice. The chimeric CD23KO mice repopulated with wild-type myeloid cells, sensitized and challenged with OVA showed significant reduction in siglec-F+ cells, eosinophils, macrophages and IL-4 in bronchoalveolar lavage fluid recovered 24 hours later compared to the wild-type mice. Our finding of CD23-mediated IgE transport in airway epithelial cells suggest a possibility of CD23 transporting an IgE Fc-fused protein for immunotherapy. CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) which competitively binds CD80 and CD86 expressed on antigen presenting cells and inhibits CD28 mediated co-stimulation of T cell activation. A CTLA4-Fc (IgE) fusion protein produced in Chinese hamster ovary cells was intranasally administrated into mouse airway for assessing its specific transport by CD23. The effect of this fusion protein on the development of allergic inflammation is being fully investigated in wild-type, CD23-KO, and chimeric mouse model.
CHARACTERIZATION AND DEVELOPMENT OF REVERSE GENETICS SYSTEM FOR AVIAN PARAMYXOVIRUS TYPE-3 AND ITS EVALUATION AS A LIVE VIRAL VECTOR
(2010) Kumar, Sachin; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avian Paramyxovirus (APMV) serotype 3 is one of the nine serotypes of APMV that infect a variety of avian species around the world. In chickens and turkeys, APMV-3 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-3, the complete genome sequences of prototype strain Netherlands and strain Wisconsin were determined. The genome length of APMV-3 strain Netherlands is 16,272 and for strain Wisconsin is 16,181 nucleotides (nt). 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. Comparison of the APMV-3 strain Wisconsin nt and the aggregate predicted amino acid (aa) sequences with those of APMV-3 strain Netherlands revealed 67 and 78%, identity, respectively. The phylogenetic and serological analyses of APMV-3 strains Netherlands and Wisconsin indicated the existence of two subgroups within the same serotype. Both the strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenesis of APMV-3, a reverse genetics system for strain Netherlands was established in which infectious recombinant APMV-3 was recovered from a cloned APMV-3 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to parental virus. A recombinant APMV-3 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of recombinant APMV-3 expressing Newcastle disease virus (NDV) F and HN proteins demonstrated that the F protein plays a major role in protection against virulent NDV challenge. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-3 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-3 would be of considerable utility for introducing defined mutations into the genome of this virus and developing a vaccine vector for animal and human pathogens.
CHARACTERIZATION OF BORRELIA BURGDORFERI GENE PRODUCTS CRITICAL FOR PATHOGEN PERSISTENCE AND TRANSMISSION
(2010) KUMAR, MANISH; PAL, UTPAL; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The pathogen of Lyme disease, Borrelia burgdorferi, persists in a natural tick-rodent infection cycle. An assessment of microbial transcriptome in vivo encoding selected membrane proteins shows that bba52 is upregulated during pathogen transmission. Deletion of bba52 in infectious B. burgdorferi did not alter the outcome of murine inflammation or long-term pathogen persistence in mice or ticks. However, the bba52 mutant was impaired for transmission between feeding ticks and mice, phenotypic defects that could be rescued when bba52 was genetically restored to the original genomic locus. I show that BBA52 is a surface-exposed outer membrane protein. As BBA52 is highly immunogenic, I next assessed whether BBA52 could serve as a potential candidate for transmission-blocking vaccines against spirochete infection. Passive transfer of BBA52 antibodies into the ticks did not interfere with microbial persistence in unfed ticks but blocked pathogen transmission from feeding ticks to murine hosts. More importantly, active immunization of mice with recombinant BBA52 protein significantly blocked B. burgdorferi transmission from ticks to naïve murine hosts. As BBA52 antibodies lacked detectable borreliacidal activities, their interference with spirochete survival in vivo could result from the inhibition of BBA52 function, such as vector-pathogen interaction. By far-western analysis using tick gut proteins, I found that BBA52 interacts with a ~ 35 kDa tick gut protein. I also show that BBA52 forms distinct homo-oligomer in borrelial cells and also interacts with two proteins of B. burgdorferi. These BBA52 interacting proteins possess approximate molecular weight of 33 kDa and 25 kDa and are located in the outer membrane and protoplasm, respectively. Taken together, these studies suggested the remarkable existence of B. burgdorferi surface antigens that are differentially expressed in vivo and support microbial transitions between hosts and the vector. Identification and characterization of novel vector-specific and feeding induced borrelial antigens could contribute to the development of transmission-blocking vaccines against Lyme disease.
Characterization of the GBF1-Arf1 axis in enterovirus RNA replication
(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.
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.
DETERMINATION OF GENETIC FACTORS INVOLVED IN THE VIRULENCE OF NEWCASTLE DISEASE VIRUS
(2012) Paldurai, Anandan; SAMAL, SIBA K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Newcastle disease is economically the most important disease of poultry. The causative agent Newcastle disease virus (NDV) is a large, enveloped virus containing single stranded non-segmented negative-sense RNA genome. The genome of NDV contains six genes in the order of 3'Leader-N-P-M-F-HN-L-5'Trailer. NDV has at least three different genome size categories: 15,186, 15,192 and 15,198 nucleotides (nt) in length. The virulence of NDV is considered to be contributed by multiple genes. The importance of genome lengths and the roles of individual genes in virulence of NDV in its natural host, chickens, have not been determined. In this study, the effects of naturally occurring nucleotide insertions in NDV genome and roles of individual genes in the virulence of NDV in chickens were determined. To achieve this goal, reverse genetic systems for two strains of NDV were established for a highly virulent strain Texas GB (GBT) and a moderately virulent strain Beaudette C (BC). Both GBT and BC are isolated from chickens and belong to genotype II of class II NDV strains and have the genome length of 15,186 nt. The 6- and 12-nt insertions in the backbones of rBC and rGBT showed little attenuation in virus replication and in pathogenicity of the parental recombinant viruses. The reciprocal swap between NDV strains BC and GBT for the genes, nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large polymerase (L) protein genes, showed that F protein gene is most important for NDV virulence, followed by the L protein gene. M, HN, N and P genes appeared not to affect the pathotypes of their parental recombinant viruses in chickens. The observations of the present study paves the way for future directions: to use the naturally occurring insertion site in the coding region of the phosphoprotein gene for insertion of potential marker sequences; to determine the amino acid residues important in fusion protein and polymerase protein for replication and pathogenesis of NDV.
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.
Development of a Multilocus Sequence Typing Scheme for Avibacterium paragallinarum
(2023) Harris, Alyssa Meihua; Ghanem, Mostafa; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avibacterium paragallinarum (A. paragallinarum), the causative agent of the respiratory disease Infectious Coryza (IC) in chickens, has seen a rising incidence in the United States. Current strain differentiation is inadequate for detailed epidemiological analysis. The objective of this study was to develop a Multilocus sequence typing (MLST) scheme for A. paragallinarum for outbreak investigations and to offer a better tool for strain differentiation. By evaluating whole genome sequences and clinical samples, we designed PCR amplicons for eighteen gene segments, selected six genes for their nucleotide diversity and discrimination potential. The MLST was used to differentiate seventy-five samples. Our MLST showed greater discriminatory power than existing HPG2-based methods, aligning closely with adhoc core genome MLST in 75 tested sample. Our newly developed MLST scheme enables more accurate strain differentiation, allowing for better understanding of A. paragallinarum epidemiology and population structure to help prevention and control efforts worldwide.
Development of avian paramyxovirus 3 as a vaccine vector against infectious bursal disease in one-day-old specific pathogen free chickens
(2021) Varghese, Berin Parambethu; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
ABSTRACTInfectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease affecting young chickens, resulting in substantial economic losses to the poultry industry worldwide. Although strict hygienic measures and various vaccination strategies have been adopted, IBD remains a major problem for the poultry industry. The economic significance of this disease is exhibited in two ways. First, the disease can cause high morbidity and mortality. Second, severe prolonged immunosuppression of chickens when infected at an early age leads to susceptibility to other diseases and vaccination failure. Therefore, chicks should be protected from early infection, usually achieved by vaccinating breeder flocks and active immunization of the newly hatched chicks. Vaccines include live-attenuated, viral vectored recombinant, subunit, and inactivated vaccines currently available to control infectious bursal disease (IBD). The major flaws of available vaccines are the reversion to virulence, the generation of new variant viruses, inability to replicate in the presence of maternal antibodies, lack of proper delivery systems, recombination and integration into the host genome, and inadequate systemic immune response. To overcome these obstacles, we have evaluated avian paramyxoviruses (APMVs) as vaccine vectors to generate vaccines against IBD. In the present study, we constructed recombinant Newcastle disease virus (NDV) strain LaSota (rLaSota/VP2) and recombinant avian paramyxovirus-3 (APMV-3) strain Netherlands expressing VP2 protein (rAPMV-3/VP2), the immunogenic protein of IBDV, and to evaluate their protective efficacies following immunization of one-day-old specific pathogen-free (SPF) chicks. Our results showed that both recombinant viruses stably express the VP2 protein, and their in vitro growth characteristics were almost similar to their respective parental viruses. Immunization of one-day-old SPF chicks demonstrated that rAPMV-3/VP2 protein elicited IBDV specific neutralizing antibodies and provided complete protection against the IBDV STC challenge. In addition, the rAPMV-3/VP2 protects chickens from clinical signs, gross lesions, and histopathology even at lower vaccine doses. Moreover, rAPMV-3/VP2 provides slightly better protection than the commercial vaccine from histopathology lesion against IBD at four weeks of age. This study suggests that recombinant APMV-3 expressing VP2 protein could be used as a potential vaccine against IBD in field conditions where maternal antibodies exist.
DEVELOPMENT OF AVIAN PARAMYXOVIRUS VECTORED VACCINES
(2022) Elbehairy, Mohamed Adel; Belov, George A; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avian Avulaviruses (formerly Avian Paramyxoviruses, APMV) are important pathogens of avian species and have been used as viral vectors for more than two decades. Among all APMVs, Newcastle disease virus (NDV or APMV-1) has been most extensively used as a vaccine vector for protection against avian and animal diseases, and as an oncolytic agent. For poultry vaccination, the preexisting maternal antibodies against NDV can neutralize APMV-1 vectors resulting in vaccination failure. Hence, there is a need to develop new vaccine vectors that would escape neutralization by the maternal antibodies. In the first part of my study, I created a reverse genetics system for Avian paramyxovirus-3 strain Wisconsin (APMV-3 Wisc.) which was proven to be nonpathogenic for day-old chicks and embryonated chicken eggs. The virus was used as a vector to express the enhanced green fluorescent protein (GFP) as a heterologous antigen. The recombinant APMV-3 Wisc. expressing GFP was compared with similarly constructed APMV-1 strain LaSota and APMV-3 strain Netherlands-based vectors for GFP expression and growth kinetics in vitro, and for immunogenicity, safety, and tissue tropism in day-old specific pathogen-free (SPF) chicks. APMV-3 strain Netherlands (APMV-3 Neth.) showed the highest growth rate and GFP expression in chicken fibroblast DF-1 cells, followed by APMV-1 LaSota and APMV-3 Wisconsin. In day-old chicks, APMV-3 Neth. spread to different organs, decreased feed intake and caused stunted growth. APMV-3 Wisc. and APMV-1 LaSota were confined to the respiratory tract and did not induce any pathogenic effects. All three constructs induced seroconversion of the vaccinated chicks for the vector antigens. Thus, the reverse genetics system created in this study for APMV-3 Wisc. allows the development of safe APMV vector antigenically different from NDV that can be used for day-old chicks vaccination. In addition, it provides a tool to study the molecular basis of APMV3 pathogenesis. In the second part of my study, I explored a novel approach for the expression of a foreign gene as an uninterrupted open reading frame (ORF) with a cognate gene of NDV vector. This approach is expected to promote the foreign gene expression stability. Avian influenza virus (AIV) hemagglutinin (HA) protein-coding sequence was fused in-frame with various proteins of NDV vector, with a 2A self-cleaving peptide, a furin cleavage site, or both, placed between the AIV and NDV sequences for separation of the two proteins. Among different constructs tested, we only recovered viable viruses with AIV HA fused C-terminally to the NDV HN gene. These viruses demonstrated a higher expression level of AIV HA than the vector constructed according to a traditional scheme of expressing the transgene as a separate transcriptional unit. Also, they showed increased stability of the transgene expression over multiple passages in embryonated chicken eggs. Our results demonstrate the advantages and limitations of this novel method of foreign gene expression that need to be considered for the development of NDV-based vaccine or therapeutic vectors.
Development of improved recombinant NDV-vectored vaccines against highly pathogenic avian influenza virus (HPAIV)
(2023) Roy Chowdhury, Ishita; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Highly pathogenic avian influenza viruses (HPAIV) are highly contagious and economically devastating poultry pathogens with a documented transmission to humans causing severe human infections with high mortality. Circulation of these viruses is of public health concern as they have the pandemic potential to mutate to increase transmissibility among humans. The diversity of zoonotic influenza viruses causing human infections is alarming and effective vaccination is needed to control these viruses. Influenza viruses particularly with H7 and H5 subtypes of HA can naturally switch to a highly pathogenic phenotype through different mechanisms. Currently available vaccines are not satisfactory as they are mostly inactivated vaccines that require labor-intensive administration methods and provide suboptimal protection of vaccinated birds. Viral vectors offer crucial advantages over traditional vaccines, including induction of outstanding antibody and cytotoxic lymphocyte responses which is important for the control of viral infections. Newcastle Disease virus (NDV) is a promising vaccine vector for HPAIV since it is highly restricted for replication in the respiratory tract of poultry, it can be easily administered, and it induces both local and systemic immune responses. H7 influenza viruses are classified into two major genetic lineages, American and Eurasian. To develop a universal anti-H7 vaccine, we generated NDV vectors expressing chimeric HA sequences covering both North American and Asian isolates. In the first project, we designed NDV-vectored vaccines against HPAI H7N8 infection. The Hemagglutinin (HA) protein of influenza viruses is responsible for virus attachment to host cell and is the major target of the humoral immune response. Accordingly, we developed vaccines against HPAIV by generating recombinant NDV vectored H7 serotype-specific vaccines expressing HA protein. We also evaluated the protective efficacy of these recombinant vaccines against highly virulent H7 challenges in both broiler chickens and turkeys and the results were promising for broiler chickens, but for turkeys the vaccination design and scheme need to be further modified. In the second part of the study, we designed some recombinant NDV-vectored vaccines with an increased level of expression of H5HA antigen. The transcriptional unit of NDV contains a major open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) followed by conserved transcriptional initiation and termination control sequences. Previous studies have shown that the addition of UTRs of P, M, and F genes positively modulated foreign gene expression. Hence, we hypothesized that cognate NDV mRNA UTRs would improve the expression of a protective antigen by an NDV-vectored vaccine. We generated recombinant NDVs where the HA of the HPAIV strain H5N1 is flanked by 5’ and 3’UTRs of NDV genes and determined the growth characteristics of these recombinant viruses, their stability, the level of HA expression and their transcription and translation modulation. Both studies aimed for the advancement of NDV-vectored vaccines emphasizing the fact of better expression of the protective antigen and improved immunogenicity for avian influenza virus considering two important strains of H5 and H7.
Ecology and Molecular Epidemiology of Avian and Swine Influenza A viruses in Guatemala
(2015) Gonzalez Reiche, Ana Silvia; Perez, Daniel R.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The greatest diversity of Influenza A viruses (IAV) is found in waterfowl species from numerous geographic locations. In addition, multiple IAV are, and continue to be, perpetuated in swine populations around the globe. Due to the zoonotic potential of IAV and to respond more effectively to potential agricultural and public health threats, there is a need to increase surveillance in avian and swine hosts in understudied geographical regions. In Latin America, avian influenza surveillance has been scarce, localized only to places where outbreaks in poultry have occurred. Similarly, active swine influenza surveillance was implemented only after the emergence of the 2009 pandemic strain (pH1N1). The project presented here was aimed at investigating the circulation of IAV in wild birds and pigs in Guatemala. Over 2200 birds were sampled during six consecutive migration seasons from 2007 to 2013 in different locations. Virus prevalence detected by rRT-PCR in positive species ranged from 5.2% to 38%. Preliminary data indicates temporal variation of IAV prevalence in migratory waterfowl. Eighty-three viruses were recovered with 22 different subtype combinations. Through phylogenetic inferences and the analysis of virus genotypes and gene constellations of 60 fully sequenced genomes, we provide a detailed description of the genetic structure of avian IAV circulating in Guatemala. Our results suggest that the virus diversity in this location is sourced from multiple migration flyways from North America. Overlap of these flyways, in a natural geographical bottleneck such as the Neotropics, may contribute to the patterns of extensive genetic reassortment observed at a continental scale. In addition, the results from two nationwide multistage random surveys in pigs demonstrated circulation of swine influenza in commercial and peridomestic herds in Guatemala. Herd prevalence of IAV was 36.3% in 2010 and 34.6% in 2011. Viruses of the H1N1 and H3N2 subtypes and antibodies against viruses of distinct genetic lineages of these subtypes were detected. Our results indicate that human-animal contact likely plays a role in the IAV epidemiology in local swine populations. The findings from this research constitute the most abundant data on the ecology and epidemiology of animal influenza currently available for Central America.
Epidemiological Analysis of Biosecurity Practices and Associated Prevalence of Diseases in Non-Commercial Poultry Flocks
(2012) Madsen, Jennifer Marie; Tablante, Nathaniel L.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
A cross-sectional study was conducted in backyard poultry flocks among nine counties of Maryland from May 2011 to August 2011. The objective of this study was to obtain baseline data from a survey on biosecurity practices and investigate risk factors associated with positive findings of avian influenza (AI), Newcastle disease (ND), infectious laryngotracheitis (ILT), Mycoplasma gallisepticum (MG), and Salmonella Enteritidis (SE). Serum, tracheal, and cloacal swabs were randomly collected from 262 birds among 39 registered premises. Analysis revealed flock prevalence and seroprevalence respectively for the following: AI (0%, 23%), ND (0%, 23%), ILT (26%, 49%), MG (3%, 13%), SE (0%, ND). Vaccine status could not be confirmed for ND, ILT, or MG. Premises positives were identified by partial nucleotide sequencing. No statistically significant associations were identified, however, AI seroprevalence was positively associated with exposure to waterfowl (Relative Risk [RR] = 3.14, 95% confidence interval [CI] 1.1-8.9) and absence of pest control (RR=2.5; 95% CI, 0.6-10.4).
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).
EXPLORING IL-7R-ALPHA DEVELOPMENTAL EFFECTS AND ONCOGENIC COLLABORATIONS
(2017) Cramer, Sarah Delia; Samal, Siba; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Acute lymphoblastic leukemia is the most common cancer of children. Individual cases of leukemia may have multiple genetic lesions, and identifying those that drive leukemogenesis will be important in the development of targeted therapy. Approximately 10% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases have a mutation in IL-7Rα. These mutations are thought to be oncogenic, but little is known about the effects of the mutation on T-cell development. In addition, the mutation does not seem to induce leukemia in the absence of other genetic lesions, suggesting that collaborative mutations are required for leukemogenesis. Based on patient data, potential collaborators include TLX3 expression, HOXA gene cluster overexpression, and NRAS mutation. Given the current state of knowledge regarding mutant IL-7Rα, this project was developed with two specific aims. The first was to investigate the effects of mutant IL-7Rα gain-of-function (IL-7Rα-GOF) on T-cell development in vitro and in vivo. The second was to determine whether candidate collaborative genetic lesions would drive T-ALL formation when combined with mutated IL-7Rα. To address these aims, immature murine thymocytes were cultured on an OP9-DL4 stromal cell system, transduced with retroviral vectors, and injected into sub-lethally irradiated Rag1-/- mice. Resultant diseases were analyzed using a variety of techniques including flow cytometry, histology, immunohistochemistry, ligation-mediated PCR, TCRβ clonality assessment, RNA-sequencing, serial passage, and limiting dilution assay. Studies showed that IL-7Rα-GOF mutation caused an increase of CD8+ cells in vitro. When thymocytes transduced with IL-7Rα-GOF mutation were injected into mice, animals developed a multi-systemic inflammatory disease. This inflammation was not due to imbalance in populations of Treg and Th17 cells, as had been hypothesized. Assessing collaborations with TLX3 expression, HOXA overexpression, and NRAS mutation showed that combination of these genetic lesions with IL-7Rα-GOF mutation caused different neoplastic diseases. The combination of IL-7Rα-GOF mutation and TLX3 expression caused low-penetrance, late-onset T-cell lymphoma. Thymocytes overexpressing the HOXA gene cluster and transduced with IL-7Rα-GOF mutation caused a rapid-onset myeloid leukemia. Combination of IL-7Rα-GOF mutation with mutant NRAS yielded rapid-onset, full-penetrance T-cell lymphoblastic leukemia, suggesting that this combination of mutations was sufficient to induce T-ALL. These experimental results may help to lay the foundation for the development of targeted therapy for pediatric T-ALL.
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.