La diarrhée épidémique porcine (DEP) est apparue en Europe à la fin des années 1970 et est causée par un alpha-coronavirus appelé virus de la diarrhée épidémique porcine (PEDV). Des épizooties sévères ont été rapportées en Asie à partir de 2010 et en Amérique à partir de 2013. Deux génotypes de souches de PEDV, se différenciant par des insertions/délétions dans le gène S, ont été isolés et circulent toujours actuellement dans ces régions. Ils regroupent d’une part les souches « S-InDel » présentes sur tous les continents, notamment en Europe y compris la France, et d’autre part les souches « S-non-InDel » hautement virulentes qui circulent en Asie et sur le continent américain uniquement. Les porcs infectés par le PEDV sont sujets à d’importantes diarrhées aqueuses ainsi qu’à des vomissements parfois accompagnés de signes de déshydratation. Les conséquences de la maladie sont d’autant plus importantes en cas d’infection par une souche « S-non-InDel » avec une mortalité pouvant atteindrLa pathogénicité comparée de deux souches de PEDV a été étudiée dans le cadre de cette thèse : une souche « S-non-InDel » isolée aux Etats-Unis en 2014 et une souche « S-InDel » isolée en France la même année. L’étude de la pathogénicité comparée a consisté à étudier les caractéristiques de transmission horizontale et les conséquences cliniques de l’infection en conditions expérimentales. En outre, l’excrétion dans la semence de ces deux souches a également été explorée. La dernière partie du projet de thèse était consacrée à l’étude des interactions du virus avec l’intestin ainsi que l’immu / Porcine epidemic diarrhea (PED) appeared in Europe at the end of 1970s and is caused by an alpha-coronavirus named porcine epidemic diarrhea virus (PEDV). Severe PED epizootic cases have been reported in Asia from 2010 and in America from 2013. Two genotypes of PEDV strains, differing by insertions/deletions in the S gene, have been described and circulating to date. The first genotype is represented by « S-InDel » strains group present on all continents among which Europe, including France. The second one is represented by the so-called highly virulent « S-non-InDel » strains which circulate in Asia and America only. The pigs infected by PEDV are affected by important aqueous diarrheas and sometimes vomiting accompanied by signs of dehydration. The consequences of the disease are more important in case of infection by an « S-non-InDel » strain with a mortality rate that could reach 100 % in suckling piglets.Uncertainties remained on pathogenicity differences according to the strains of PEDV and on impacts of a potential introduction of « S-non-InDel » PEDV strain in France. This thesis aimed at studying and comparing the pathogenicity of two strains of PEDV, an « S-non-InDel » PEDV strain isolated in the United-States in 2014 and an « S-InDel » PEDV strain isolated in France the same year. To compare pathogenicity between strains, the horizontal transmission and the clinical consequences of the infection in experimental conditions have been studied. Moreover the virus shedding in semen was also explored for the two strains. The last part of the project of the thesis was

Over 80% of the newly emerging infectious diseases are caused by RNA viruses. Major global problems associated with the development of vaccines against the RNA virus are their high genetic and antigenic diversity. Hence, effective control of epidemics with newly emerging RNA viruses require improved vaccines which are either specific to the new strain or broadly effective even when new viral strains emerge. The main focus of this dissertation is to develop epidemic vaccines using these two approaches. Using a newly emerged swine enteric virus called porcine epidemic diarrhea virus (PEDV) as a model, our first goal was to develop a quick and easy method for rapid response vaccines with potential applicability to a range of RNA viruses. We hypothesized that the methods which can disrupt genomic RNA without impacting the structural integrity of the virus would result in attenuated vaccine with minimum replication in the host while inducing immune responses. As hypothesized, developed rapid response PEDV vaccine induced complete protection against the virulent challenge virus, while vaccine viral shedding was not detected in vaccinated pigs. To address the second problem of rapid viral evolution leading to vaccines becoming obsolete, we used swine influenza virus (SIV) as a model to develop and test a universal vaccine composed of peptides encoding conserved antigenic epitopes which are present in most influenza A viruses. Importantly, a novel amphiphilic invertible polymer (AIP) was used to address the well-recognized problem of poor antigenicity of peptides. We hypothesized that peptides encoding conserved epitopes when conjugated with an AIP will induce strong immune responses and protect against challenge virus. While the conserved epitopes were previously tested by others in mice, we were the first to test a combination of these epitopes in pigs. Pigs vaccinated with the peptide polymer vaccine mounted strong antibody responses against the epitopes indicating that the delivery system was effective. However, protection against replication of the challenge virus was delayed. In summary, the methods developed and tested in this body of work significantly contribute to the area of emergency response management in infectious disease outbreaks. / United States Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA) / North Dakota State Agricultural Products Utilization Committee (ND APUC) / North Dakota State Board of Agricultural Research (ND SABRE)

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Master of Science / Grain Science and Industry / Cassandra K. Jones / Feed mill biosecurity is a growing concern for the feed industries, especially since the entry of Porcine Epidemic Diarrhea Virus (PEDV) to the United States. Porcine Epidemic Diarrhea Virus (PEDV) is primarily transmitted by fecal-oral contamination. However, research has confirmed swine feed and ingredients as potential vectors of transmission, so strategies are needed to mitigate PEDV in feed. The objective of the first experiment was to evaluate the effectiveness of various chemical additives to prevent or mitigate PEDV in swine feed and ingredients that had been contaminated post-processing. Time, formaldehyde, medium chain fatty acids, essential oils, and organic acids all enhance the degradation of PEDV RNA in swine feed and ingredients, but their effectiveness varies within matrix. Notably, the medium chain fatty acids were equally as successful at mitigating PEDV as a commercially-available formaldehyde product. Salmonella is also another potential feed safety hazard in animal feed ingredients. Thermal mitigation of Salmonella in ingredients and feed manufacturing is effective, but it does not eliminate the potential for cross contamination. Therefore, the objective of the second experiment was to evaluate the effectiveness of chemicals to mitigate Salmonella cross-contamination in rendered proteins over time. Both chemical treatment and time reduced Salmonella concentrations, but their effectiveness was again matrix dependent. Chemical treatment with medium chain fatty acids or a commercial formaldehyde product was most effective at mitigating Salmonella in rendered protein meals. The final experiment was conducted to evaluate the effectiveness of a dry granular acid, sodium bisulfate (SBS; Jones-Hamilton, Co., Waldridge, OH), to mitigate contamination of Salmonella in poultry feed. A surrogate organism, Enterococcus faecium, was utilized for this research in order to evaluate the effectiveness of SBS. Thermal processing, SBS concentration, and time all impacted biological pathogen levels in poultry diets, and including a dry granular acid may be an effective method to reduce pathogen risk. However, the most significant reduction of Enterococcus faecium was due to thermal mitigation. Notably, pelleting reduced Enterococcus faecium by 2-3 logs on day 0. In summary, both thermal processing and chemical inclusion can be used to reduce the risk of microbial pathogens in feed.

<div>The papain-like protease (PLP) domain of nonstructural protein 3 (nsp3) of the coronavirus (CoV) genome promotes viral replication by processing the CoV polyprotein (protease) and also antagonize innate immune responses by deubiquitinating (DUB) and deISGylating (deISG) host substrates. Selectively removing the DUB/deISG activities of PLP while keeping the protease activity intact is a potential strategy for designing a live attenuated virus. However, it is unclear in the literature the precise mechanism by which PLPs support CoV evasion of the innate immune system. Deciphering the substrate specificity of PLPs for host ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) can therefore help in the design of PLP mutants that selectively lack one activity for evaluating the DUB and deISG mechanism in CoV pathogenesis and replication. </div><div> In this dissertation, we investigate the structure and function of the single PLP (PLpro) from beta-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which are dangerous viral pathogens that emerged from a zoonotic source to cause infectious disease in the human population. Additionally, we translate the knowledge gained to the equivalent PLP2 from alpha-CoV porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV), which cause fatal disease in suckling piglets on industrial pork farms and household cats, respectively. The primary objective of this work is to rationally design PLP mutants across beta- and alpha-CoVs to help attenuate CoV infection, as no antiviral or vaccine exist for human CoVs and the efficacy of PEDV vaccines are an ongoing research topic. </div><div><br></div><div>In Chapter 1, different human, animal, and the bat origin CoV strains are introduced. The CoV life-cycle and virion structure are outlined, along with the replicase complex for viral replication. The multidomain nsp3 from alpha- and beta-CoV genomes are also described with a focus on the PLP domain and its proposed cleavage sites of the viral polyprotein. The discovery of the first viral protease DUB and the multiple activities of PLPs are defined, which includes a proposed model of how DUB versus deISG activities may act in the innate immune response. This leads into the therapeutic potential of PLP for an antiviral or live attenuated vaccine, which is followed by the introduction of live attenuated vaccines and the reverse genetics system. Next, proof of concept studies on PLP2 mutants are described and the introduction is concluded by stating the ultimate goal for the design of PLP mutants.</div><div><br></div><div>In Chapter 2, we hypothesize that the flanking ubiquitin-like (Ubl2) domain of MERS-CoV PLpro is not required for its enzymatic function. We characterize the specific activity, kinetics, substrate specificity, and inhibition of the PLpro enzyme with and without the Ubl2 domain and reveal that the Ubl2 domain does not significantly alter PLpro function. We determine the structure of the core PLpro, smallest catalytic unit to 1.9 Å resolution and observed no structural changes compared to the wild-type. Additionally, we demonstrate that a purported MERS-CoV PLpro inhibitor is nonselective in non-reducing conditions and should not be pursed for therapeutic use. We show that the core PLpro enzyme i.e. without the Ubl2 domain is a stable and robust construct for crystallization and is also thermally stable based on thermal melting studies with utility for structure-based drug design. </div><div><br></div><div>In Chapter 3, we shed light on the specificity of SARS-CoV PLpro towards Ub versus ISG15 by characterizing the specific activity and kinetic parameters of SARS-CoV PLpro mutants. In addition, the structure of SARS-CoV PLpro in complex with the C-terminal domain of ISG15 is determined and compared with the Ub-bound structure. Based on the structure and kinetic results, the altered specificities of SARS-CoV PLpro mutants Arg167Glu, Met209Ala, and Gln233Glu are compared with the wild-type. Arg167Glu mutant exhibits DUB hyperactivity and is expected to adopt a more favorable interaction with the Arg42 of Ub. At the same time, ARG167GLU contains a shorter side-chain that hinders interaction with the unique Trp123 of ISG15 for deISG activity compared to the wild-type. These results aid in the development of SARS-CoV PLpro mutants that have directed shifts in substrate specificity for Ub versus ISG15. </div><div><br></div><div>In Chapter 4, the process and antiviral activity of ISGylation is reviewed and how viruses can modulate host-derived versus virus-derived machineries to counteract ISGylation for viral infection. MERS-CoV PLpro is cross-reactive for Ub, but less is known about its specificity towards ISG15. In this study, we determine the structure of MERS-CoV PLpro bound with ISG15 to 2.3 Å resolution and reveal a small hydrophobic pocket of ISG15 that consists of P130 and W123, which differs from Ub hydrophobic patch. We design and determine the kinetic parameters for 13 PLpro mutants and reveal that MERS-CoV PLpro only has a single ubiquitin recognition (SUb1) site. Kinetic studies show that removing the charge of the R1649 greatly enhances DUB/protease activity while mutating in an Arg near R42 of Ub or ISG15 hydrophobic region is detrimental to both DUB/deISG activities. Kinetic experiments and probe-reactivity assays showed that Val1691Arg, Val1691Lys, and His1652Arg mutants are drastically reduced DUB/deISG activities compared to the wild-type. Overall, MERS-CoV PLpro mutants with alter kinetic profiles will be useful for discovery tools and DUB/deISG deficient mutants are great candidates for removing host cell antagonism activity by PLpro for live attenuated vaccines.</div><div><br></div><div>In Chapter 5, the goal is to translate the knowledge gained in Chapters 2-4 on beta-CoVs PLpro and evaluate the substrate specificity of alpha-CoVs FIPV and PEDV PLP2 for mutagenesis experiments. First, we design and purify the core PLP2 enzymes for kinetics. PLP2s are efficient DUBs that prefer Ub to ISG15 in vitro, and this preference is conserved in beta-CoV MHV PLP2 as well as alpha-CoV NL63 PLP2. We determine the structure of alpha-CoV PEDV PLP2 to 1.95 Å resolution and reveal the unique Zn-finger coordinating Cys3-His arrangement of the alpha-CoV genus that differs from past beta-CoV PLP crystal structures. To determine residues of the SUb1 site, we generate a homology model of FIPV PLP2 and overlay our PLP2 structures with MERS-CoV PLpro bound with Ub. In addition, we create electrostatic surface maps across coronaviral PLP subfamilies to evaluate the charge distribution of the SUb1 for the rational design of several FIPV and PEDV PLP2 mutants. We evaluate the turnover of PLP mutants for FRET-based substrates and reveal that His101ArgFIPV and Asn101ArgPEDV are drastically reduced in Ub-AMC activity while their peptide activities are within 2-fold of the wild-type. These mutants show delayed reactivity for Ub probes and no longer cleave Ub-chains displaying isopeptide bonds compared to the wild-type. Results from this study reveal a hot spot in both alpha- and beta-CoVs that can be used to selectively remove DUB activity of PLPs for generating a DUB deficient PLP enzyme. </div><div><br></div><div>In this dissertation, we investigate the substrate specificity of PLPs across alpha- and beta-CoVs and develop a fingerprint for Ub and also shed light on ISG15 recognition. Specifically, hot spots were identified in the SUb1 site of different PLPs, which recognize R42 and hydrophobic Ile44 of Ub. Position 97-98 of PLPs can be used to remove DUB activity by substituting an Arg, but usually effect protease function. Substituting an Arg at position 101 and 136 of coronaviral PLPs serve as the best strategy to remove DUB function while not hindering active site functionality. The DUB/deISG deficient mutants described will be useful for inhibiting the ability of PLPs to function in the innate immune response. Ultimately, this work provides a guide for identifying attenuating mutants in existing CoVs for live attenuated vaccines and also a blueprint for engineering PLPs from new emerging CoVs. </div>

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two of the most prevalent swine pathogens that have impacted the global swine industry for decades. Both are RNA viruses with increasing heterogeneity over the years, making a vaccine solution ever so challenging. Modified live-attenuated vaccines (MLVs) have been the most common approach, but the long-term safety regarding their potential for pathogenic reversion still needs to be addressed. Subunit based vaccines have been the focus of numerous development studies around the world with renewed interest in their promising prospects in both safety and efficacy. Our lab has developed a unique approach to use hepatitis B virus core capsid protein (HBcAg) as a vaccine delivery vehicle for either PRRSV or PEDV viral epitope antigens. Recombinantly produced HBcAg forms an icosahedral capsid virus-like particle (VLP) that has 240 repeats in a single assembled particle. By inserting different epitope antigens from these porcine pathogens into the particle, we can achieve repetitive antigen presentation to the host's immune system by taking advantage of the polymeric nature of VLP. The first animal study evaluated the efficacy of 4 VLP based vaccine candidates against PRRSV in mice. These 4 vaccines incorporated 2 B-cell epitopes (61QAAIEVYEPGRS72 and 89ELGFVVPPGLSS100) and 2 T-cell epitopes (117LAALICFVIRLAKNC131 and 149KGRLYRWRSPVIIEK163) from PRRSV structural proteins GP3 and GP5 respectively. Candidate GP3-4 was able to stimulate a significant viral neutralizing response in mouse sera against two PRRSV strains, one being heterologous, demonstrating its potential of cross-protection against PRRSV. The second animal study took an optimized VLP vaccine candidate against PEDV from previous development studies in mice, and assessed its efficacy through a comprehensive pregnant gilt vaccination and neonatal piglet challenge model. The vaccine candidate incorporated B-cell epitope 748YSNIGVCK755 from the PEDV spike protein. It was able to elicit significant viral neutralization antibody titer in gilt milk at 3 days post-farrowing (DPF), and provided nursing piglets with clinical relief in terms of morbidity, viral shedding, small intestinal lesions, and 10 days post-challenge (DPC) survival rate. / Doctor of Philosophy / Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are two pathogens that infect pigs, resulting in immense economic losses to the global pork production industry every year. Both viruses have large diversity with various strains due to mutations that have occurred over the years. This makes vaccine development that aims at combating the pathogens even more challenging. One common vaccine strategy has been immunizing animals with modified live viruses with decreased pathogenicity. Naturally, long term safety of this option has been a concern. A much safer vaccine approach that is purely protein based has attracted renewed interest around the world. Protein based vaccines lack genetic materials from the viruses and are not able to replicate inside the host. Our lab has developed a platform that uses protein-based particles (VLPs) originated from the hepatitis B virus (HBV), and incorporates short pieces of proteins from either PRRSV or PEDV to train host's immune system to recognize these pathogens, and hopefully to prevent future infection. For the first animal study, we tested 4 VLP vaccine candidates against PRRSV in mice and discovered that mouse serum from one candidate GP3-4 was able to prevent infection of 2 distinct PRRSV strains in petri dishes, paving the way for further development. For the second animal study, we took an optimized VLP vaccine candidate against PEDV from previous mouse studies, and evaluated its performance in pigs. We immunized pregnant mother pigs with the vaccine before they gave birth, then experimentally infected newborn piglets with the virus. Piglets from the vaccinated mothers showed improved clinical signs and faster recovery from the infection.