Department of Veterinary Medicine Theses and Dissertations
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Item 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.Item 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.Item VIRAL IMMUNE EVASION OF FCRN FUNCTIONS(2018) LIU, XIAOYANG; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human Cytomegalovirus (HCMV) is known to evade host immunity, allowing it to persistently infect humans. Although the strategies of HCMV to evade cellular immunity is well studied, there is limited understanding on how HCMV antagonizes humoral immunity. The neonatal Fc receptor (FcRn), an MHC class I-related FcγR, plays a critical role in IgG-mediated humoral immunity. Through screening the HCMV proteome, we discovered that US11 specifically captured FcRn in both virally-infected and US11-expressing cells. US11 selectively inhibited the assembly of FcRn with β2m, impaired FcRn IgG binding capacity and blocked FcRn trafficking to the endosome by retention of FcRn in ER. Furthermore, US11 recruited Derlin-1 and E3 ubiquitin ligase TMEM129, to induce degradation of FcRn in US11+ or HCMV-infected cells. This complex led to the dislocation of FcRn from the ER to the cytosol and facilitated its degradation in an ubiquitination and proteasome-dependent manner. The cytosolic interaction between FcRn and Derlin-1 was shown necessary for degrading FcRn. FcRn is widely expressed in most cell susceptible to HCMV infection, including epithelial, endothelial and macrophage. Our data showed that either HCMV infection or recombinant US11 expression significantly inhibited human IgG transcytosis across polarized human primary intestinal epithelial Caco-2 cells, Vascular endothelial HMEC-1 cells and placental trophoblast BeWo cells, and facilitated considerable IgG degradation inside endothelial HMEC-1 cells. Hence, our results show that HCMV exploits the Derlin-1/TMEM129 pathway through US11 to disable FcRn, revealing a novel strategy for viral evasion from antibody mediated-immunity. We also studied whether HCMV viral FcγRs (gp34 and gp68) and US11 work together to facilitate IgG degradation. HCMV vFcγRs has been reported to internalize IgG via endocytosis. Interestingly, we found that in acidic pH (6.0) condition, the IgG binding capacity of gp34 was largely impaired while the IgG binding capacity of gp68 had minimal change. Consequently, in the presence of FcRn, gp34 did not enhance IgG degradation whereas gp68 significantly promoted the IgG degradation. Furthermore, the presence of US11 induced more gp34 and gp68-mediated IgG degradation in FcRn+ cells.Item A NOVEL INTERFERON-INDUCING PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS STRAIN: CHARACTERIZATION AND VACCINE DEVELOPMENT(2018) Ma, Zexu; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Porcine reproductive and respiratory syndrome virus (PRRSV) causes a swine infectious disease characterized by severe reproductive failure in sows and respiratory disease in pigs of all ages. Despite substantial efforts to control PRRS, no production or vaccination regimen has demonstrated sustaining success. Type I interferons (IFNs) are critical to the innate immunity against viral infections and play an important role in activation of the adaptive immune response. PRRSV appears to antagonize induction of type I IFNs. Fortunately, we discovered an atypical PRRSV strain, A2MC2, which induces type I IFNs in cultured cells. A2MC2 elicits earlier onset and higher levels of virus-neutralizing antibodies than the Ingelvac PRRS® MLV in pigs. However, moderate virulence of A2MC2 was observed in infected piglets. The objective of this project was to characterize A2MC2 and explore this unique strain for the development of an improved vaccine against PRRS. First, I attenuated this strain by serial passaging in MARC-145 cells for 90 consecutive passages. The passage 90 virus (A2MC2-P90) was avirulent and retained the capability of IFN induction. The A2MC2-P90 virus induced higher level virus-neutralizing antibodies in pigs. Secondly, I constructed an infectious cDNA clone of A2MC2. The recovered virus from the infectious clone was similar to the parental strain in growth properties and IFN induction. Gene fragment swapping demonstrated that the middle half genome of A2MC2 was essential for its IFN induction. Thirdly, I conducted studies to exam the genetic source of A2MC2 in IFN induction. Comparison of A2MC2 and other closely relevant PRRSV strain identifies five unique non-synonymous nucleotides. These five nucleotides remained unchanged in the A2MC2-P90 virus. Site-directed mutagenesis indicated that one unique nucleotide in A2MC2 genome was critical in the IFN induction as mutation of this nucleotide led to the loss of IFN induction. Together, our data demonstrate that A2MC2 is a novel strain that is worth further exploration for an improved vaccine against PRRS. The infectious clone of A2MC2 will be useful for the development of a marker vaccine by insertion of a marker sequence into the A2MC2 genome.Item IMPROVED NEWCASTLE DISEASE VIRUS VACCINES AND VECTORS(2017) Manoharan, Vinoth Kumar; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Newcastle disease (ND) is an economically important disease of poultry worldwide. The use of vaccines to control ND is necessary because of frequent outbreaks of the disease in enzootic countries. The fusion (F) and hemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are multifunctional proteins that play critical roles during infection. The F protein of NDV is a type I membrane glycoprotein that mediates the fusion of viral envelope to the host cell membrane. The F protein activation initiates a series of conformational changes in the F protein leading to virus-cell membrane fusion, which occurs at the cell surface at neutral pH thus modulating NDV entry and spread. In the present study, we investigated the role of tyrosine to alanine mutation at amino acid position 524 and 527 in the F protein cytoplasmic tail (CT) of NDV strain LaSota by using reverse genetic techniques. Our results suggest that tyrosine residues at 524 and 527 position of F protein CT domain play a major role in fusogenicity and in replication thus modulating NDV infectivity. The F protein is synthesized as an inactive precursor, F0, which is functionally activated after cleavage by host cell proteases into F1 and F2 polypeptides, linked by disulfide bonds. The amino acid sequence surrounding the F protein cleavage site determines the virulence of NDV. We also studied the role of other avian paramyxovirus fusion protein cleavage site sequences in F protein cleavage of NDV strain Banjarmasin. This study has helped us to understand the requirement of F protein cleavage site in proteolytic processing, plaque formation and virus infectivity. Further, the role of these F cleavage site mutant viruses as genotype-matched vaccines for virulent NDV infection has been explored. Reverse genetics has also been used to develop NDV strains as a potential vaccine vectors for various human and animal pathogens, such as highly pathogenic avian influenza (H5N1), human immunodeficiency virus, severe acute respiratory syndrome coronavirus, ebola virus, respiratory syncytial virus and human parainfluenza virus type 3. NDV has several characteristics that makes it a suitable candidate for vaccine vector development. It is safe in humans and animals due to natural host range restriction, expresses foreign protein abundantly, infects via intranasal route, produces both humoral and mucosal immune responses, is antigenically distinct from human and animal pathogens, and lack of preexisting immunity to NDV in humans and animals. In one vaccine trial with non-human primates, the mesogenic NDV strain Beaudette C (BC) replicated to a high titer and induced a substantially higher antibody response compared to the lentogenic strain LaSota, and thus appeared to be more effective. However, NDV strains that have a polybasic cleavage site in the F protein and an intracerebral pathogenicity index (ICPI) >0.7 have been classified as Select Agents. Most mesogenic strains, including strain BC, fall into this category and therefore cannot be handled in BSL-2 conditions. In this study, we constructed a series of recombinant (rNDV) vectors containing the cleavage site sequence of avirulent strain LaSota and other avian paramyxoviruses, together with various regions of the F protein exchanged between NDV strains AKO-18 and BC. We used these modified rNDV vectors to express SIV gp160 envelope protein and immunized guinea pigs. Our results showed that rNDV/SIV vaccines were immunogenic and effectively neutralized SIV mac251 strain in vitro. These results support the idea of the use of NDV as a vaccine vector for expression of SIV immunogens capable of inducing neutralizing antibodies against diverse SIV strains, thus providing an improved vaccine vector platform for ultimately testing the NDV vectored vaccines in non-human primates and humans.Item Identification and Characterization of New Small Molecule Inhibitors of Picornavirus Replication(2016) Siltz, Lauren Ashley Ford; Belov, George A; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Picornaviridae family consists of positive-strand RNA viruses that are the causative agents of a variety of diseases in humans and animals. Few drugs targeting picornaviruses are available, making the discovery of new antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. The antiviral effect of these compounds is not likely related to their known cellular targets because other inhibitors targeting the same pathways did not inhibit viral replication. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited the formation of a functional replication complex, while E5(1) and E7(2) affected replication after the replication complex had formed. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Poliovirus resistant to E7(2) had a single mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target either PI4KIIIβ (major enviroxime-like compounds) or OSBP (minor enviroxime-like compounds), cellular factors involved in lipid metabolism and shown to be important for replication of diverse positive-strand RNA viruses. We classified E7(2) as a minor enviroxime-like compound, because the localization of OSBP changed in the presence of this inhibitor. Interestingly, both E7(2) and major enviroxime-like compound GW5074 interfered with the viral polyprotein processing. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. Studies with these compounds shed light on pathways shared by diverse picornaviruses that could be potential targets for the development of broad-spectrum antiviral drugs.Item 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.Item Interferense of Host Innate Immune Response by Hepatitis E Virus(2014) Nan, Yuchen; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The host antiviral innate immunity mainly relies on host pattern recognition receptors (PRR) and downstream interferon (IFN) signaling. Host PRR for RNA viruses include Toll-like receptors (TLR) and Retinoic acid-inducible gene I (RIG-I) like receptors (RLR). Activation of both TLR and RLR pathways can eventually lead to the secretion of type I IFNs, which can modulate both innate and adaptive immune responses against viral pathogens, including hepatitis E virus (HEV). HEV causes acute hepatitis in humans and has been responsible for several outbreaks of hepatitis across the world. Currently, no commercial vaccine is available for the prevention of HEV infection in any country except China. HEV biology and pathogenesis as well as its responses to host innate immunity are poorly understood, though other hepatitis viruses, including the hepatitis A, B and C viruses, have been much better studied. In this study, how HEV interferes with IFN induction and IFN-activated signaling had been examined. Results showed that the protein encoded by HEV ORF1 can inhibit type I IFN synthesis and downstream JAK/STAT signaling pathway. However, the HEV ORF3 product is able to enhance RIG-I-mediated signaling to a certain extent. These data suggest that HEV proteins interfere with the host innate immune response and may exert the diverse roles depending on the stage and/or context of infection. These studies contribute to a better understanding of HEV pathogenesis and may facilitate a strategy development for the prevention and control of HEV infection.Item IMPROVED INFECTIOUS LARYNGOTRACHEITIS VIRUS VACCINES USING NEWCASTLE DISEASE VIRUS VECTOR(2013) Kanabagatte Basavarajappa, Mallikarjuna; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens for which safe and efficacious vaccines are not available currently. In the present study, we have generated three recombinant Newcastle disease viruses (rNDV's) expressing three major envelope glycoproteins gB, gC and gD of ILTV individually. A single oculonasal inoculation of chickens with rNDV's elicited detectable level of systemic antibodies specific to ILTV. Following challenge with virulent strain of ILTV, chickens immunized with the rNDV's displayed partial protection with reduced clinical signs and shorter duration of disease compared to the control group. Our data suggested that NDV vectored ILTV vaccines are useful against ILTV infection, but might require augmentation by a second dose or require modification of ILTV glycoproteins which allow them to incorporate into the mature rNDV virions for better induction of humoral and cell mediated immune responses.Item Review of the Molecular Biology and Epidemiology of Infectious Laryngotracheitis (Gallid Herpesvirus-1)(2012) Menendez, Kimberly Rae; Tablante, Nathaniel L; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A review of the molecular biology and epidemiology of avian infectious laryngotracheitis (ILT) is conducted due to the outdated state of current ILT review material. The objective of this review is to include updated information on the molecular biology of Gallid herpesvirus 1 (GaHV-1), the causative agent of ILT, and to present the latest information on the molecular epidemiology of ILT. Recent developments in molecular biology specific to GaHV-1 have been made and are highlighted in this review, and the role of current and historical use of live-attenuated vaccines is associated with the global and molecular epidemiology of ILT. Also, target genes for detection and strain differentiation are compiled by region of the world, and the global distribution of ILT is illustrated. Additionally, the field of epigenetics related to virus-host interactions is reviewed, and the molecular, epidemiologic, and epigenetic factors investigated are related to prospects for future eradication of ILT.