Browsing by Subject "PRRSV"
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Item Antibody repertoire dynamics in the changing landscape of infection(2013-05) Schwartz, John CharlesAntibody responses are fundamentally important to effector and memory mechanisms of disease resistance. In order to respond to a nearly infinite array of possible antigens, the antibody repertoire must be suitably diverse. To achieve this necessary high level of diversity, the antibody repertoire has evolved a unique recombinatorial system consisting of a large number of gene segments that can recombine in different combinations to yield an astronomical array of potential antigen-binding structures. Understanding the antibody repertoire of swine (Sus scrofa) can inform about host genetic differences that may affect disease susceptibility and resistance. Also, it may allow identification of antibody molecules that are important in the host immune response against specific pathogens. Such knowledge could potentially be used in the future to develop selective breeding programs for animals that possess desirable immunological traits, and to screen for specific antibody molecules that are of either therapeutic or diagnostic importance. Knowledge of antibody repertoire diversity in swine has heretofore been lacking. While most previous studies have focused heavily on understanding the heavy chain repertoire by analyzing hundreds of cDNA clones, there have been few investigations of the porcine light chain repertoire. This study was designed to characterize the organization and complexity of both the kappa and lambda light chain loci in the pig genome. Findings revealed extensive allelic variation between both homologous pairs of chromosomes in a single sow and suggested non-crossover homologous recombination (i.e. gene conversion) as a potential evolutionary mechanism to explain at least part of that variation. Armed with this new information, and with that from the previously characterized heavy chain locus, antibody variable region amplicon libraries were generated from lymphoid tissues of pigs either infected (n=2) or mock-infected (heavy chain, n=2; light chain, n=3) with the major swine pathogen, porcine reproductive and respiratory syndrome virus (PRRSV). It is hypothesized that the major anti-PRRSV antibody responses would be detectible in infected animals compared to their control counterparts. Approximately a half-million reads for each heavy and light chain library were generated. From this data, diversity of the expressed antibody repertoire was assessed, including gene segment usage and allelic variability, and anti-PRRSV responses. As predicted, due to biological necessity, the heavy and light chain repertoires possessed a rich array of putatively functional antibody transcripts (heavy chain richness estimate, 3x105 molecules; kappa light chain, 1.5x105; lambda light chain, 2.3 x 105), despite being restricted in their germline to a small number of functional D and J gene segments, a single heavy chain V gene segment family and four light chain V gene families. Interestingly, a power-law distribution of antibody abundances was detected similar to what has previously been reported in zebrafish (Danio rerio), whereby a small number of antibody sequences are exceptionally common and the vast majority are exceptionally rare. Substantial allelic variation was also detected, most notably in the lambda locus. Four out of 5 pigs possessed a functional copy of a previously undescribed V gene segment (IGLV3-1-1) which substantially contributed to the expressed repertoire of the animals that possessed a copy. Importantly, a small number of antibody sequences were detected which were incredibly abundant (>1% of the entire repertoire) in PRRSV-infected pigs and rare in uninfected pigs. It is hypothesized that these highly abundant antibody molecules are PRRSV-specific. Using the knowledge obtained from these studies, future investigations will examine the repertoire for specific heavy and light chain pairs from PRRSV-infected pigs that can neutralize PRRSV using an antibody yeast-display system. In addition, specific heavy and light chain pairs identified in our expression analysis and deemed putatively PRRSV-specific are to be tested for epitopic specificity against labeled PRRSV as well as individual PRRSV recombinant antigens. This last method represents a potential novel and non-lethal means of generating antigen-specific recombinant antibodies derived from lymphoid tissue of immunized animals.Item Characterization of Broadly Neutralizing Antibodies to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)(2020-01) Young, JordanPorcine reproductive and respiratory syndrome (PRRS) is an all too common disease with a devastating impact on pork producers in the US and worldwide. The disease is caused by the PRRS virus (PRRSV) which leads to abortions and other forms of reproductive failure in sows and severe respiratory disease in adults and neonates. Unfortunately, current vaccines against PRRSV provide limited protection and often only protects against closely related viruses. Efficacious vaccine-based prevention of infectious disease is based on antigen-specific long-lived memory B and T cells. Protective, or neutralizing, antibodies produced by memory B cells, that are activated in response to virulent pathogen challenge, are critical to this process. Thus, it is of utmost importance to investigate antibodies with neutralizing potential especially broadly neutralizing antibodies (bnAbs), which possess the ability to neutralize distantly related strains. In this manuscript, I take a reverse approach to the classic neutralization study, starting first with pigs we know are broadly neutralizing and then investigating the identity of the responsible B cells, using a novel technique for the isolation of PRRSV neutralizing antibodies. PBMCs were harvested from pigs sequentially exposed to divergent PRRSV isolates and vaccine. Memory B cells were then transduced, with a proprietary retroviral vector developed by AIMM Therapeutics, containing genes highly expressed by germinal center B cells, creating an immortalized B-cell population. B-cells were then sorted by FACS and five PRRSV-specific B cells were isolated. All identified PRRSV-specific antibodies were found to be broadly binding to all PRRSV-2 isolates tested, but not PRRSV-1. Antibodies against GP5 protein, commonly thought to be the PRRSV neutralizing epitope, were found to be highly abundant, as four out five clonal B cells were GP5 specific. Next, an isolated GP5 clone was discovered to be neutralizing against homologous, but not heterologous PRRSV. Sequencing of this clone’s heavy chain variable region and CDR3 revealed a gene that was heavily mutated compared to germline sequence, suggesting somatic hypermutation playing an essential role in generation of broadly neutralizing antibodies. Further investigation of these antibodies, and others, may lead to the elucidation of conserved neutralizing epitopes that can be exploited for improved vaccine design and lays the groundwork for the study of bnAbs against other porcine pathogens.Item Characterization of the multidomain Nsp2 Replicase protein of porcine reproductive and respiratory syndrome virus.(2008-12) Han, JunThis dissertation focused on understanding the biology of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV), the etiological agent of porcine reproductive and respiratory syndrome (PRRS). PRRSV nsp2 is a multidomain protein, containing a putative N-terminal cysteine protease PL2 domain, a 500-700aa middle region of unknown function, a transmembrane domain and a C-terminal tail with uncertainty. In this dissertation, we report the following. (i) PRRSV nsp2 is undergoing rapid evolution in field strains exemplified by viral isolates MN184A and B. (ii) We showed that PRRSV nsp2 hypervariable regions aa12-35 and aa324-813 were not essential for viral replication in MARC-145 cells by using a reverse genetics system based on strain VR-2332. In contrast, deletion of the cysteine protease PL2 core domain, the PL2 downstream flanking sequence (aa181-323), the predicted transmembrane domain and the C-terminal domain were lethal to the virus. (iii) We provided evidence that the nsp2 protein encodes an active PL2 protease and mediates nsp2/3 processing in CHO cells with a substrate preference for the dipeptide G1196|G1197. The PL2 protease possessed both trans- and cis-cleavage activities, which could be distinguished by point mutations. Site-directed mutagenesis studies revealed that mutations that caused a specific loss of trans function of the PL2 protease, but not cis activity, were detrimental to the virus. In addition, we showed that the conserved aspartic acid residues (e.g., Asp89) played an important role in the PL2 trans-cleavage activity. (iv) We investigated the proteolytic processing of nsp2 in MARC-145 cells using recombinant PRRSV expressing foreign epitope-tagged nsp2 protein. We showed the presence of the nsp2 protein as different isoforms in PRRSV-infected cells, which appeared to share the same N terminus but differed in their respective C-termini. The nsp2 species emerged almost simultaneously in the early stage of PRRSV infection, were stable and had low turnover rates. Deletion mutagenesis suggested that the smaller nsp2 species (e.g. nsp2d, e and f) were not essential for viral replication in cell culture. Lastly, a cellular protein, heat shock 70kDa protein 5 (HSPA5), was identified as a coimmunoprecipitate of nsp2.Item Immunological selection as a driver of porcine reproductive and respiratory syndrome virus (PRRSV) evolution(2016-07) Wang, XiongPorcine reproductive and respiratory syndrome (PRRS) is still one of the most devastating swine infectious diseases worldwide since its initial outbreak in the late 1980s. Its etiologic agent, PRRS virus (PRRSV), is a single strand RNA virus that belongs to the order of Nidovirales, family Arteriviridae and genus Arterivirus. PRRSV is small, viral partial size is about 53nm including a RNA genome with the size of ~ 15kb. PRRSV is highly host restricted to porcine monocyte cells. Currently in the field, biosecurity and passive immunization are the major solutions to reducing the impact of PRRSV endemic. Yet two major factors, the rapidly evolution of PRRSV and the incomplete and highly variable cross-protection induced by passive vaccination, heavily contribute to the penetration of PRRSV to swine herds and result in the emergence and re-emergence of virulent PRRSV. Similar to other RNA virus like human immunodeficiency virus 1 (HIV-1), hepatitis C virus (HCV) and foot-and-mouth disease virus (FMDV), the lack of error prone mechanism during viral replication leads to the production of tremendous mutations in PRRSV progeny. The selection pressure of porcine intrinsic, innate and adaptive immunity on PRRSV population helps shape and drive PRRSV mutation direction. Together, they drive PRRSV’s rapid evolution. Yet there is a limitation in systemic understanding how genetic variation is generated and what selection forces drive PRRSV evolution. The overall objective of this dissertation was to characterize PRRSV evolution in intra-population and field level, as well as exploring the driving forces hidden in the porcine monocyte cells by utilizing high- throughput sequencing and bioinformatics. The findings herein built up an optimized standard protocol to assemble PRRSV whole genome from high-throughput sequencing yields, which can be broadly adapted to other highly-mutated RNA virus. PRRSV infectious clones, similar to field isolates, exist as quasispecies, its population diversity was decreasing under consistent selection pressure of permissive cell intrinsic selection yet retaining significant diversified progeny. In this thesis, an emerging virulent PRRSV in vaccinated herds was identified as a recently evolved member of virulent lineage instead of new virulent strain via built comprehensive and standard analysis pipeline. IFNs and IFN-induced ARFs are highly induced in PAMs after PRRSV inoculation; potential causative key pathways were identified in the PAM age-dependent susceptibility difference scenario. Putting together, all the findings and results provided an improved systematic insight of PRRSV evolution and host innate response, which is a vital immunological selection driver. Ultimately, a better understanding of PRRSV evolution and its driver will lead to a more effective disease prevention, control and elimination.Item Mechanisms of immune protection against porcine reproductive and respiratory syndrome virus (PRRSV)(2015-05) Robinson, SallyPorcine reproductive and respiratory virus (PRRSV) is a rapidly evolving and diverse RNA virus that causes significant disease to swine populations globally. There are no specific treatments for PRRSV; biosecurity measures and immunization are the mainstays of PRRSV prevention and control. Although immunization plays a critical role in reducing disease and stabilizing virus-endemic herds, vaccines provide incomplete and unpredictable cross-protection against the diversity of PRRSV strains. A limitation for improving immunization strategies is that mechanisms responsible for immunity against PRRSV and correlates of cross-protection are poorly understood. The objective of this dissertation was to re-evaluate aspects of PRRSV immunity in light of contemporary information for novel clues to identify mechanisms of protection. The approach was to investigate the role of recently discovered PRRSV protein ORF5a in immunity, and to re-evaluate the role of neutralizing antibodies in PRRSV cross-protection. The findings herein demonstrate that ORF5a protein is not important for immune protection against PRRSV but the maintenance of ORF5a in fine evolutionary balance with the GP5 variable glycosylation region suggests it plays a critical role in the virus life cycle. High levels of broadly-neutralizing antibodies to PRRSV were identified from sows in typical commercial settings. Neutralizing antibodies isolated from sows provided cross-protection to naïve animals against diverse strains of PRRSV. This information provides a basis for renewed investigation into the mechanisms of neutralizing antibody-mediated protection against PRRSV and strategies for induction of broadly-neutralizing antibodies. Improved understanding of immune mechanisms to PRRSV will enable more effective control, prevention and ultimately, elimination of the virus.Item The porcine memory B cell in conferring long term adaptive immunity to viral pathogens(2017-06) Rahe, MichaelPorcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen of swine health and well-being worldwide. Discovered nearly thirty years ago, there is still no vaccine capable of producing a broadly protective immune response against the virus. This deficiency is due in large part to a failure to understand how the adaptive immune system responds to vaccination or infection. Specifically, there is little to no knowledge regarding the all-important memory immune response to PRRSV. The objective of this dissertation was to fill in this significant gap in knowledge by identifying and characterizing the memory B cell response to PRRSV vaccination. First, we identified the presence of memory B cells against PRRSV non-structural protein 7 (nsp7) through the use of a novel in vitro B cell culture system. Next, we created and validated a novel reagent, a B cell tetramer, against nsp7 to enhance the speed and sensitivity of memory cell identification. Finally, through the utilization of the nsp7 tetramer, we evaluated the regional specificity and dynamic nsp7 specific B cell response to PRRSV MLV vaccination within select secondary lymphoid organs. These results constitute the first evidence of regional specialization of the B cell response to vaccination in an outbred animal species. Furthermore, the presented methods are a blueprint for the study of antigen specific cellular responses to any significant pathogen of animals important for food or fiber.Item Structural and functional characterization of porcine reproductive and respiratory syndrome virus N-glycans.(2011-12) Li, JuanPorcine reproductive and respiratory syndrome (PRRS) is one of the most severe infectious diseases facing the swine industry worldwide. The etiologic agent, PRRS virus (PRRSV), belongs to the order Nidovirales, family Arteriviridae, genus Arterivirus. It is a positive-sense ssRNA virus and has a smooth spherical envelope embedded with seven proteins, four of which are glycoproteins (GP). Since the major envelope proteins GP5 and matrix (M) have short ectodomains, the broadly distributed viral glycans likely cover the virion surface and stretch out as antennae, thus interacting with host cells and contributing to viral infection. Previous studies suggest potential roles of PRRSV envelope protein-linked glycans in virus assembly, virus attachment to target cells, virus neutralization and antigenicity. In particular, sialic acids on GP5 have been shown to bind sialoadhesin on porcine macrophages, mediating virus attachment and internalization. Nevertheless, the complete profile of GP5-linked glycan compositions and structures, and the role of specific glycan moieties in virus infection have yet to be determined. Herein, we purified the North American prototype PRRSV, VR-2332, and analyzed viral glycans in the aspects of composition, structures and functions in virus infection. Endoglycosidase digestion of virus showed that GP5 was the primary protein substrate among all the four envelope glycoproteins, and that the glycans were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers and terminal sialic acids, which was also confirmed by lectin co-precipitation. Based on the structural information, we further demonstrated that GlcNAc and LacNAc oligomer-specific lectins bound to PRRSV and blocked virus attachment, resulting in reduced infection. However, GlcNAc oligomers and LacNAc did not compete with virus to block infection, suggesting that GlcNAc and LacNAc oligomers are not directly involved in virus entry. Finally, removal or alteration of N-glycans from PRRSV envelope proteins did not affect infection, indicating that envelope protein-linked N-glycans are not required for PRRSV infection. In conclusion, GP5 contains most of the PRRSV glycans, which are primarily complex-type N-glycans. GlcNAc and LacNAc oligomers and sialic acids on the PRRSV envelope are accessible for specific recognition that may reduce infection by steric hindrance. Envelope protein-linked N-glycans are not required for PRRSV infection. Our findings provide a glycan database for molecular structural studies of PRRSV and facilitate a better understanding of molecular host-virus interactions.