Porcine Reproductive and Respiratory Syndrome virus (PRRSV) causes the disease - Porcine Reproductive and Respiratory Syndrome (PRRS) which is one of the most economically important diseases for pig farmers. Since it was discovered in the United States and Europe, it has quickly affected the swine industry all over the world. Studying and controlling PRRSV has become an important issue in swine industry and scientific community, and has raised the concerns of governments like US and China. By using different bioinformatics and phylogenetics tools, scientists could understand the epidemiology and evolution of PRRSV from genomic data. However, a well-designed database for PRRSV sequence and relevant meta-information are generally required for the tools to produce insightful results. Therefore, I would like to introduce an easily accessible web platform for PRRSV analysis - PRRSV-Webtool. The core component of PRRSV-Webtool is phylogenetic reconstruction. Instead of using traditional phylogenetic reconstruction, a new method of reconstruction was introduced - Reconstruction by Addition of Taxon (RAT). RAT could build a phylogenetic tree from known existing phylogeny. Simulation tests were performed to evaluate the accuracies of RAT using PRRSV dataset. The percentages of correct branch reconstruction are 73.81% for type 1 PRRSV dataset and 80.68% for type 2 PRRSV dataset. Another important function of PRRSV-Webtool is genotyping. RAT could correctly identify the genotype of all sequences in the testing datasets. PRRSV-Webtool combined three main components: database, phylogenetic tool and World Wide Web. By using PRRSV-Webtool, the users can study their own PRRSV genome data easily via the web browser. Tools in PRRSV-Webtool can allow users to know more about their PRRSV isolates related to other field samples. With our PRRSV-Webtool, scientists and veterinaries can help to improve their understanding of PRRSV and help to control the virus by accelerating the process of virus surveillance and field sampling. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Bibliography: leaf 141-157.

Acute Respiratory Distress Syndrome (ARDS) is an inflammatory disease characterized by pulmonary edema, stiff lungs and hypoxemia. Patients with ARDS are more susceptible to VILI (ventilator induced lung injury). Under mechanical ventilation, lung stress and strain are the main determinants of VILI and in patients with muscle effort patient-ventilator asynchrony may enhance this phenomenon. Ventilation modes PCV and VCV with auto-flow can minimize patient-ventilator asynchrony, but then can liberate the offer of flow and tidal volume, compromising the protective ventilatory strategy in ARDS. This study aimed to evaluate the influence of muscle effort and patient-ventilator asynchrony on pulmonary stress and strain in a mechanic lung model of acute respiratory distress syndrome. An experimental bench study was performed, using a lung simulator, ASL 5000TM, in which was configured a lung model with restrictive respiratory mechanics with complacency of 25ml/cmH2O and resistance of 10 cmH2O/L/sec. Muscle effort was adjusted in three situations: no muscular effort (Pmus = 0), with inspiratory muscle effort (Pmus = -5 cmH2O) and inspiratory and expiratory effort (Pmus = -5/+5 cmH2O), all with breathe rate (b) of 20 bpm. Five ventilators were connected to the simulator through and endotracheal tube No 8.0 mm and adjusted on VCV, VCV with Auto-flowTM (in the ventilator in which it was available) and PCV modes, all with tidal volume (VT): 420 ml, PEEP: 10 cmH2O and breath rate set in two situations: b = 15 bpm (lower than b of the respiratory muscle effort) and b = 25 bpm (higher than b of the respiratory muscle effort). Variables analyzed were: maximum VT, alveolar pressure at the end of inspiration, effective PEEP, driving pressure, transpulmonary pressure at the end of inspiration and expiration, average transpulmonary pressure, inspiratory peak flow and analysis of mechanic curves. In the studied lung model the b of the ventilator adjusted higher of the b of the patient and not the muscle effort was the main determinant for the development of patient-ventilator asynchrony, causing large variations of the VT and pulmonary pressures, intensifying the lung stress and strain. The ventilatory modes had similar behavior, although VCV Auto-flowTM and PCV have presented slightly higher values of VT and pulmonary pressures. Thus it is concluded that the proper adjustment of the programed breath rate in the assisted/controlled modes can minimize patient-ventilator asynchrony, reducing lung stress and strain. / A SÃndrome da AngÃstia RespiratÃria Aguda (SARA) à uma doenÃa inflamatÃria caracterizada por edema pulmonar, pulmÃes rÃgidos e hipoxemia. Pacientes com SARA estÃo mais suscetÃveis à VILI (ventilator induced lung injury). Sob ventilaÃÃo mecÃnica, o stress e o strain pulmonares sÃo os principais determinantes da VILI e nos pacientes com esforÃo muscular a assincronia paciente-ventilador pode potencializar este fenÃmeno. Os modos ventilatÃrios PCV e VCV com AutoFlow podem minimizar a assincronia paciente-ventilador, mas por outro lado podem liberar a oferta de fluxo e volume corrente, comprometendo a estratÃgia ventilatÃria protetora na SARA. Objetivou-se avaliar as influÃncias do esforÃo muscular e da assincronia paciente-ventilador sobre o âstrainâ e o âstressâ pulmonares em modelo pulmonar mecÃnico de sÃndrome da angÃstia respiratÃria aguda. Foi realizado um estudo experimental de bancada, utilizando um simulador de pulmÃo, ASL 5000 no qual foi configurado um modelo pulmonar com mecÃnica respiratÃria restritiva, com complacÃncia de 25ml/cmH2O e resistÃncia de 10 cmH2O/L/sec. O esforÃo muscular foi ajustado em trÃs situaÃÃes: sem esforÃo muscular (Pmus=0), com esforÃo muscular inspiratÃrio (Pmus= -5cmH2O) e esforÃo inspiratÃrio e expiratÃrio (Pmus= -5/+5 cmH2O), todos com frequÃncia respiratÃria (f) de 20rpm. Ao simulador foram conectados cinco ventiladores atravÃs de um tubo orotraqueal n 8,0 mm e ajustados nos modos VCV, VCV com sistema AutoFlow (no ventilador que tinha o sistema disponÃvel) e PCV, todos com volume corrente (VC): 420 ml, PEEP: 10 cmH2O e frequÃncia respiratÃria programada em duas situaÃÃes: f=15rpm (< que a f de esforÃo muscular respiratÃrio) e f=25rpm (> que a f de esforÃo muscular respiratÃrio). As variÃveis analisadas foram: VC mÃximo, a pressÃo alveolar no final da inspiraÃÃo, PEEP efetiva, driving pressure, pressÃo transpulmonar no final da inspiraÃÃo e expiraÃÃo, pressÃo transpulmonar mÃdia, pico de fluxo inspiratÃrio e anÃlise das curvas de mecÃnica. No modelo pulmonar estudado a f do ventilador pulmonar ajustada acima da f do paciente e nÃo o esforÃo muscular o principal determinante para o desenvolvimento de assincronia paciente ventilador, causando grandes variaÃÃes de VC e pressÃes pulmonares, o que intensificou o stress e strain pulmonares. Os modos ventilatÃrios tiveram comportamento semelhante, embora os modos VCV AutoFlow e PCV tenham apresentado valores discretamente maiores de VC e pressÃes pulmonares. Desta forma conclui-se que o ajuste adequado da frequÃncia programada nos modos assistido/controlado podem pode minimizar a assincronia paciente ventilador reduzindo o stress e strain pulmonares. Palavras-

Masters of Science / The Coronaviridae family consists of RNA viruses within the order Nidovirales. The family is classified into two genera, namely the corona- and toroviruses. Coronaviruses are enveloped, single stranded, positive sense RNA viruses with genomes ranging between 27-32kb in size. The 5’ two-thirds of the genome encodes for the 1a/b polyprotein, while the 3’ one-third of the genome encodes for the structural proteins that mediate viral entry into the host cell. These structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The nucleocapsid protein is expressed at high levels within an infected cell. Studies have shown that this protein plays a key regulatory role in different cellular pathways, including the inhibition of interferon production and the up-regulation of the AP1 signal transduction pathway, amongst others. Also, the N protein is vital in the formation of the ribonucleocapsid core by binding to the viral RNA during virion assembly. The focus of this study is the immune response in whole blood cultures to the presence of human coronavirus (HCoV) NL63 N protein. To characterise the stimulation of the immune activity against HCoV-NL63 N in blood cultures, the HCoV-NL63 N gene was expressed in a bacterial system. In this pilot study, GSTtagged N constructs were then purified and used to treat whole blood cultures from three volunteers. ELISAs were used to measure the cytokine response in these treated whole blood cultures. Results showed that the nucleocapsid protein has an inflammatory response on whole blood cultures. These results have generated vital information in the potential function of the HCoV-NL63 N protein on the immune system. It is suffice to say that the HCoV-NL63 N protein is able to elicit an effective inflammatory response within the host cell. Future studies into the cellular pathways affected by the HCoV-NL63 N protein will clarify its exact role in stimulating the host immune system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the coronavirus disease of 2019 (COVID-19) pandemic, which has infected millions of people worldwide. To date, several vaccines and antivirals have been developed against SARS-CoV-2; however, its tendency to mutate rapidly poses a continued threat to human health. As such, the development of better pan-coronavirus therapeutics is still required. Recently, the SARS-CoV-2 non-structural protein 13 (Nsp13) helicase has been shown to be an attractive therapeutic target given its high conservation rate among coronaviruses and indispensable role in viral replication. Based on this, we sought to further study the biochemical mechanisms behind Nsp13's binding and unwinding activities, along with its interactions with host cells, to provide further insight for future therapeutic development. To study the binding and unwinding activity of Nsp13, we site-specifically incorporated the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into Nsp13 to act as a bioorthogonal handle for fluorescent labelling. We identified five potential sites for AzF incorporation in Nsp13 and assessed their reactivities towards a conjugated Cy5 fluorophore through strain-promoted alkyne-azide cycloaddition (SPAAC). Further experiments were also performed to ensure that the unwinding activity was not adversely affected before these Nsp13-AzF constructs were utilized in fluorescence resonance energy transfer (FRET)-based binding assays. Ultimately, the F81AzF construct was identified to be the most suitable for monitoring the binding of Nsp13 to a series of nucleic acid substrates in a distance-dependent manner by FRET. The next steps of this project would be to implement F81AzF in single-molecule FRET (smFRET) experiments to directly monitor the positioning and dynamics of this helicase on its substrate. In addition, interactions between Nsp13 and host cellular and biological processes were investigated to provide further insight into potential mechanisms that can be exploited for novel therapeutic development. From transcriptomic profiling analyses of A549 cells, we uncovered that Nsp13 influences microRNA (miRNA) expression and signalling pathways; in particular, miR-146a, a potent mediator of inflammation and immune responses, was found to be induced upon Nsp13-overexpression. Further experiments revealed that this may lead to the inhibition of NF-κB signalling, through the repression of the upstream targets TRAF6 and IRAK1, to suppress the production of proinflammatory cytokines and facilitate viral infection. Collectively, from this work, we propose that further exploration of these miR-146a-mediated signalling pathways may present alternative strategies for antiviral investigations.

Heat stress and immune challenge are costly issues to the swine industry causing significant loss in production and health including reduced efficiency in muscle accretion and energy utilization. Alterations to metabolism and immune response may participate in these shortcomings. The study objectives were to examine the metabolic profiles and immune status of swine subjected to a dual challenge of thermal stress and porcine reproductive and respiratory syndrome virus (PRRSV). To determine this, pigs were subjected to four treatments: thermo-neutral (22° C; TN), thermo-neutral PRRSV infected (TP), heat stress (HS), and heat stress PRRSV infected (HP), during two experimental phases. The first phase consisted of infecting half the experimental group with PRRSV while the rest remained infection free in thermo-neutral conditions. A second phase further divided infected and non-infected into heated conditions for three days of constant heat (35° C) or TN conditions. Venous blood was collected prior to each phase and before sacrifice to analyze for metabolites. At sacrifice liver and longissimus dorsi skeletal muscle samples were collected for gene expression analysis. Pigs in challenged conditions had increased body temperatures, reduced feed intake, and lighter body weights compared to controls, with greatest detriment to dual challenged pigs. In addition, challenged pigs had increased markers of muscle degradation. In challenged pigs, differences (p<0.05) were observed in the metabolic and cytokine gene expression profiles suggesting heat stress blunts the immune response of viral infection in muscle and liver. In conclusion, heat stress and immune challenge directly and indirectly affect metabolism and cytokine expression and both variables may contribute to decreased growth parameters. / Master of Science

Porcine reproductive and respiratory syndrome (PRRS) is the most significant infectious disease currently affecting the swine industry worldwide. Several inactivated and modified live vaccines (MLV) have been developed to curb PRRSV infections. The unsatisfactory efficacy and safety of these vaccines, drives for the development of new generation PRRS universal vaccines. Virus like particles (VLPs) based vaccines are gaining increasing acceptance compared to subunit vaccines, as they present the antigens in more veritable conformation and are even readily recognized by the immune system. Hepatitis B virus (HBV) core antigen (HBcAg) is very well studied and has been successfully used as a carrier for more than 100 other viral sequences. In this study, hybrid HBcAg VLPs are generated by fusion of the conserved protective epitopes of PRRSV and expressed in E. coli. An optimized purification protocol that overcomes issues from ultracentrifugation is developed to obtain hybrid HBcAg VLP protein from the inclusion bodies. This hybrid HBcAg VLP protein self assembled to 23nm VLPs that were shown to block virus infection of susceptible cells when tested on MARC 145 cells. Therefore, the safety of non-infectious and non-replicable VLPs and production through low-cost E. coli fermentation may make this vaccine competitive against current vaccines on both efficacy and cost. / Master of Science

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine. To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV. We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5. / Ph. D.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include a diverse group of pathogens that cause a wide array of diseases that can range from sub-clinical to lethal. These viruses infect humans and mammals as well as a variety of other hosts. For their successful replication, viruses interact closely with host cells from the binding to the receptor to the exit as complete viral progenies. During the events, viruses are dependent on host factors for receptor bindings, genome synthesis, and trafficking of viral genome and proteins. Thus there have been major efforts on the studies of understanding the virus-host interactions in the field of virology. In my PhD program, I have studied the host factors that regulate the replication of viruses using porcine reproductive and respiratory syndrome virus (PRRSV) and hepatitis C virus (HCV). I found that modulation of either the viral receptor or cellular signaling pathways had pronounced effects in the replication of PRRSV or HCV respectively. Using PRRSV, I found that the modulation of the level of the putative receptor CD163 on cells with cytokines significantly influence virus replication, suggesting the importance of cytokine presence in environments to determine the replication and pathogenicity of PRRSV via receptor expression in vivo. With HCV, I found that the enhancement of the virus replication occurs through the activation of the epidermal growth factor receptor/extracellular signal-regulated kinase pathway by bile acids which are abundant in the liver where the virus targets in vivo. Furthermore, I found that the bile acid-mediated signaling pathway significantly inhibited the antiviral activities against HCV. These results indicate the importance of environmental factors such as bile acids and signaling pathways in the replication and pathogenicity of HCV in vivo.

A síndrome do desconforto respiratório agudo (SDRA) e a malária placentária (MP) são complicações da malária, cujos mecanismos imunopatogênicos pouco compreendidos. Neste trabalho, mostramos que camundongos MyD88-/- e Casp1/11-/- não desenvolveu SDRA, morrendo devido ao quadro de anemia severa associada à hiperparasitemia. Posteriormente, demonstrou-se que, embora a patogênese da doença dependa do inflamassoma AIM2, não depende dos inflamassomas NLRP3 e NLRC4 e do eixo IL1. Em uma segunda etapa do projeto foi mostrado que a progressão da PM são decorrentes da ativação das vias de sinalização TLR4/9/MyD88, mas não do TLR2. Ademais, evidenciou-se a participação dos inflamassomas NLRP3 e AIM2, porém não do NLRC4, nesse processo. Por fim, os dados obtidos sugerem que a ativação dessas vias culmina com a liberação de IL-1&#946; que, ao agir em seu receptor, inibe a expressão de transportadores de aminoácidos e glicose, com consequente disfunção do desenvolvimento do feto em camundongos grávidas com MP. Em conclusão, este trabalho apresenta, pela primeira vez, uma associação entre a ativação da via MyD88 e dos inflamassomas pelo plasmódio e a progressão da SDRA e MP. / Acute respiratory distress syndrome (ARDS) and placental malaria (PM) are complications of the malaria, whose the immunopathogenic mechanisms are poorly understood. In that study we showed that MyD88-/- and Casp 1/11-/- mice did not develop ARDS, dying due to severe anemia associated to hyperparasitemia. Subsequently, it was been shown that although such mechanism depends on the AIM2 inflammasome, it does not depend on the NLRP3 and NLRC4 inflammasomes and the IL-1 axis. In a second stage of the project, it should be noted that those complications are due to the activation of the TLR4/9/MyD88 signaling pathways, but not the TLR2. In addition, the participation of the NLRP3 and AIM2 inflammosomes, but not the NLRC4 was shown in that process. Finally, the data suggest that the activation of these pathways culminates with the release of the IL-1&#946; which acts on its receptor inhibiting the amino acid expression and glucose transporters with a consequent dysfunction in the fetal development of pregnant mice with MP. In conclusion, this work makes for the first time an association between the MyD88 pathway activation and inflammasomes by plasmodium and the progression of the ARDS and MP.