Severe acute respiratory syndrome (SARS) is a severe pulmonary infection that has been identified in multiple outbreaks around the world after emerging from mainland China in early 2003, The syndrome is caused by SARS-associated coronavirus, a novel human infection. SARS-associated coronavirus is spread by multiple mechanisms, including direct contact and large-droplet aerosolization, and may be spread by droplet nuclei as well. Clinical disease is characterized by fever, dry cough, interstitial infiltrates, and variable progression to respiratory failure, No treatment has clearly been shown to be effective. Aggressive infection control measures to prevent viral spread are key to outbreak management.

Atrial fibrillation, long QT syndrome, and Middle East Respiratory Syndrome are three deadly human diseases for which genetic animal models are needed. From elucidating disease pathogenesis to facilitating the development of treatments, animal models are crucial for studying human disease. One of the most effective ways to generate specific animal models is through genetic modification. Historically, mice have been most widely used as genetically modified models, despite a number of limitations. New gene editing technologies such as CRISPR/Cas9 have made developing alternative genetic models that better recapitulate some human diseases better and more feasible. In this thesis, I describe my efforts to develop genetically modified goat and hamster models for atrial fibrillation and long QT syndrome, and genetically modified hamster models for Middle East Respiratory Syndrome. For long QT syndrome model development, I knocked out the KCNQ1 gene in goat fetal fibroblast cells and baby hamster kidney cells using the CRIPSR/Cas9 system. The knockout results in loss-of-function mutations, a known cause of human long QT syndrome. The edited goat fibroblast cells will be nuclear donors for future cloning experiments to produce live goats possessing the KCNQ1 knockout. The CRISPR gene targeting sgRNA/Cas9 vector, specific for the hamster KCNQ1, has been used for pronuclear injections to produce KCNQ1 knockout hamsters. For atrial fibrillation model development, I designed a single-stranded donor oligonucleotide that generates a KCNQ1 gainof-function mutation resulting in the disease. This oligonucleotide was injected into hamster embryos along with the KCNQ1 sgRNA/Cas9-expressing vector to generate hamsters containing the gain-of-function mutation. Finally, for Middle East Respiratory Syndrome model development, I established a breeding colony of human DPP4 transgenic hamsters in the STAT2 knockout background. Human DPP4 transgenic hamsters are susceptible to MERS-CoV infection, showing mild clinical signs and allowing viral replication in lung tissue. Giving these hamsters a STAT2 knockout background should promote a more severe disease progression. For all three diseases, the foundations for the development of genetic animal models have been laid.

Bakgrund   Svår akut respiratorisk sjukdom [SARS] var en ny typ av atypisk lunginflammation som orsakades av ett coronavirus som tidigare enbart var känt att smittade mellan djur. År 2002 muterade detta virus och började infektera människor. 2000-talets första epidemi drog igång och över 8000 personer konstaterades smittade. Många personer som smittades blev svårt sjuka och behövde söka vård på grund av andningssvårigheter och hypoxi.  Syfte  Syftet var att belysa sjuksköterskans upplevelser av att vårda personer med svår akut respiratorisk sjukdom på sjukhus. Metod Den valda metoden för detta arbete var en icke-systematisk litteraturöversikt där 15 vetenskapliga artiklar inkluderades. Artiklarna som användes var både kvalitativa och kvantitativa och har kvalitetsgranskats med hjälp av Sophiahemmets Högskolas bedömningsunderlag. Insamling av artiklar har gjorts från databaserna Cinahl, PubMed och PsycINFO och analyserades med integrerad dataanalys.  Resultat Resultatet visade att sjuksköterskorna upplevde stor stress, rädsla och maktlöshet. Adekvat skyddsutrustning och kunskap om smittan saknades. Sjuksköterskorna hamnade i en situation där de var tvungna att välja att antingen stanna kvar i sin profession eller leta efter arbete med mindre yrkesrisker. Många valde att stanna, detta för att de insåg att det inte fanns någon annan som kunde ta hand om dessa personer. Slutsats Trots de psykiska påfrestningarna som drabbade sjuksköterskorna kunde de med stöd av varandra ta sig igenom epidemin. Många kände att de växte i sin roll som sjuksköterska och kunde ta med sig nya lärdomar inför framtiden. Dock kunde stora brister uppdagas i sjukvården och dessa kunskapsluckor behöver fyllas med lärdomar från tidigare epidemier och pandemier inför framtida utbrott. / Background Severe acute respiratory syndrome was a new type of atypical pneumonia caused by a coronavirus that previously was known only to spread amongst animals. During late 2002 the virus mutated and started to infect people. The 21st century’s first epidemic began and over 8000 people were confirmed infected with the new disease. A lot of people became severely ill and had to seek medical care due to breathing difficulties and hypoxia. Aim The purpose was to shed light on the nurse's experiences of caring for persons with severe acute respiratory syndrome in hospitals. Method The chosen method for this paper was a non-systematic literature review in which 15 scientific articles were included. The articles included were both qualitative and quantitative and were quality reviewed with the help of Sophiahemmets University’s assessment tool. The articles were collected using the databases CINAHL, PubMed and PsycINFO and analyzed with an integrated data analysis. Results The results show that nurses experienced a high level of stress, fear and powerlessness. There was a lack of adequate protective equipment and knowledge about the disease. The nurses were forced to choose, either to stay in their profession or search for a different occupation with less risk of getting infected. A lot of nurses chose to stay, they realized that there were no one else who could take care of the patients.  Conclusions Despite the psychological symptoms that affected the nurses, they realized that they could get through the epidemic with the support of each other. Many nurses felt a growth in their profession and could bring a lot of new knowledge with them for the future. A lack of knowledge was discovered in the healthcare system and there are a lot off lessons to be learned for future epidemic and pandemic outbreaks.

For over thirty years, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has been a major contributor to morbidity and mortality in the commercial swine industry across the globe. This highly mutagenic RNA virus causes significant economic losses wherever it is prevalent, leading to $664 million in annual losses in the United States. Unfortunately, the current prevention and diagnostic techniques available have proven to be insufficient in controlling the spread of this disease. We describe an alternative diagnostic method exploiting the rapid turnaround time and long-read capacity of Oxford Nanopore Technology's MinION next-generation sequencer. We have developed a novel primer set designed to span Open Reading Frames 3 through 7 of the PRRSV genome, which has allowed for multiplexing of samples, thus reducing individual cost of testing, while yielding significantly more information than previously available. This novel primer pair and sequencing technique have distinguished mixed infections within individual animals and may be used to determine vaccination status. This new approach will help producers and veterinarians make better-informed decisions about co-mingling of animals and vaccination strategies, thus reducing the emergence of new, pathogenic strains of PRRSV. / Master of Science / Porcine reproductive and respiratory syndrome virus (PRRSV) is a common, economically important pathogen in commercial swine production. The virus was first identified in the late 1980's during outbreaks in the United States and Europe. In female pigs, the disease is characterized by abortion storms, and the delivery of mummified fetuses or very weak, ill piglets. Neonates often display signs of pneumonia, respiratory distress, and many die from hypoxia. Surviving piglets are highly susceptible to other diseases and are poor growers compared to other, unaffected piglets. Boars may show signs of respiratory disease and can also have decreased libido and reproductive success for months at a time. The virus is prone to mutating once a pig is infected, preventing herds from mounting sufficient immunity to protect against new, mutant strains. Identifying infected pigs early and accurately is crucial to managing PRRSV outbreaks. Currently available diagnostic tests for PRRSV have many limitations, thus we have developed a new diagnostic test using next-generation sequencing technology. Oxford Nanopore Technology provides a commercially available nanopore sequencer, the MinION, that can read long DNA strands in real-time. With this technology we have expanded the area of the PRRSV genome that can be sequenced, which allows us to better identify and distinguish strains of PRRSV in infected, and vaccinated pigs. This new testing method will allow veterinarians and practitioners across the country to better identify and predict outbreaks in their herds, helping them develop better management strategies against PRRSV.

No description available.

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases facing the global swine industry. Porcine circovirus type 2 (PCV2) is the primary and essential causative agent of PCVAD, but development of clinical disease typically requires co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). The specific mechanisms of co-infection that lead to clinical disease are not fully understood, but immune modulation by the co-infecting viruses is thought to play a critical role. The ability of dendritic cells (DC) infected with PRRSV, PCV2 or both to induce regulatory T cells (Tregs) was evaluated in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p<0.05). This Treg induction was found to be dependent on TGF-β and not IL-10. Further investigation of the in vivo swine immune response to acute co-infection with PCV2 and PRRSV failed to detect activation of Tregs in peripheral blood mononuclear cells (PBMCs) or bronchoalveolar lavage samples. The Treg response to in vitro and in vivo PRRSV challenge in pigs persistently infected with PCV2 or vaccinated against PCV2 was evaluated. There was no significant difference in Tregs in PBMCs among chronically PCV2-infected, vaccinated PCV2 challenged or negative control pigs. However, following in vitro infection of monocyte-derived dendritic cells with PCV2, PRRSV, or both viruses, co-cultured lymphocytes from chronically infected and PCV2 vaccinated pigs had significantly (p<0.05) decreased Treg expression in the virus infected groups compared to the negative controls. In separate experiments, pigs vaccinated against PCV2 and subsequently challenged with an attenuated PRRSV strain and its pathogenic parental strain developed increased CD4+CD25+FoxP3+ Tregs (p<0.05) in PBMC samples compared to uninfected controls, and this correlated with increased suppressor activity and IL-10 expression. The findings from these studies indicate that the interaction of PCV2 and PRRSV in swine modulates the host immune response mediated in part through the activity of Tregs. However, the extent to which Tregs orchestrate a dysregulated immune response in the pathogenesis of PCVAD in vivo remains to be determined. / Ph. D.

Since emerging in Europe and the US, PRRS has spread globally and become the most significant infectious disease currently devastating the swine industry. In the US alone, the economic losses caused by this disease amount to more than 560 million US dollars every year. Modified-live PRRSV vaccines (MLV) are the most effective option currently available for the control of the disease. MLVs can confer solid protection against homologous re-infection and have significant effects in reducing viral shedding. But the vaccine efficacy varies upon heterologous challenge. None of the current vaccines are able to completely prevent respiratory infection, transplacental transmission, as well as pig-to-pig transmission of the virus. More importantly, the intrinsic risk of MLV vaccine to revert to virulent virus under farm conditions poses a great safety concern. The unsatisfactory efficacy and safety of current PRRSV vaccines drives the continuous efforts of developing a new generation of vaccines. The strategy we focus on for novel PRRSV vaccine development is subunit vaccine. The reasons for choosing this strategy are: 1) subunit vaccines only contain the immunogenic fragments of a pathogen. Administration of such pathogen fragments eliminates the risk of pathogens reverting back to their virulent form as in the case of modified live vaccines. 2) Subunit vaccines have advantages in terms of vaccine production since a well-defined pathogen fragment can more easily be produced consistently. To achieve of our goal of developing safe and efficacious subunit vaccines against PRRSV, three projects were completed. First, a scalable process for purification of PRRSV particles from cell culture was developed. This process produced purified viral particles for ELISA and cell-based assays used in vaccine development. Second, a plant-made oral subunit vaccine against PRRSV was developed. Administration of the plant-made vaccine, the vaccinated animals produced virus-specific serum and intestine mucosal antibodies with neutralization activity, as well as cellular immune responses with a preference of virus-specific IFN-γ production. Since neutralization antibodies and virus-specific IFN-γ response are the crucial factors contributing to protection against PRRSV infection, the plant-made oral subunit vaccine strategy is an attractive strategy for developing a new generation of the vaccine to control PRRS disease. Third, a chimeric protein consisting of the ectodomains of viral M and GP5 proteins was expressed and purified. The protein product showed a single band on a silver-stained gel and contained an endotoxin level of less than 10 EU/mg protein. In addition, the purified protein showed expected bioactivities. It was antigenic, could bind to a cellular receptor for the virus (heparan sulfate), and could block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV. / Ph. D.

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

This thesis is about the politics and the possibilities of aetiology. Firstly, the possibilities. Does an infectious disease have one, single pathogenic cause or many, interacting causes? In the medical microbiological sciences, there is no definitive answer, one way or another, to this question: there, the conditions of aetiological possibility exist in a curious tension. Ever since the birth of the 'germ theory of disease' and the concomitant birth of the singular aetiological object, these conditions have allowed for the co-existence of a very different, and far less well understood kind of object: the multifactorial object. That SARS was caused by one, singular viral agent, a coronavirus (CoV), is now entrenched as microbiological fact. And yet, the curious thing about SARS is that the history of the 2003 outbreak is littered with moments at which the possibility of the multifactorial object presented itself to, and was actively considered by, medical microbiologists. So how did we get here - to SARS-CoV, an infectious disease that could be understood and storied in this, the most singular of ways? And what happened along the way to deny the multifactorial aetiological object any kind of existence at all? In an attempt to grapple with these questions, the thesis seeks to recover the possibility of the multifactorial object through a deep, ethnomethodological reading of the moments at which it flared up precise/y as a possibility for medical microbiologists investigating the outbreak. What emerges from that recovery operation is a sense that the multifactorial object was never actually ruled out or disproved in any way, but rather, was vanished. Put another way, the suggestion is that various medical microbiological practices and interventions, whilst establishing singularity, were serving, at the same time, to create an illusion of multifactorality's non-existence; an illusion behind which the issue of multifactorality, its possibility, could be discarded without ever having to be resolved, one way or the other. In the closing sections of this thesis a move is made towards suggesting that SARS-Co V, the singular disease, was the product of a choice-, a choice that was made to explore one aetiological possibility at the expense of another. And that is where the politics comes in. For if politics, the realm of the political, can be taken to arise in situations where various possibilities exist but not all possibilities can be chosen, then it follows that what this thesis provides is an opportunity to foreground the politics bound up with the practical doing of aetiology. As a result, and based on the experience of attempting to recover the vanished multifactorial object from the 2003 SARS outbreak, the thesis concludes with an attempt to inhabit the present in such a way as to make it possible to think, in a little more detail, about where aetiology, as understood by medical microbiologists, might be heading in the future: might recent shifts in practical, everyday, seemingly innocuous microbiological technique, have begun to make it easier to coax the multifactorial object out into a space of visibility? Might those shifts actually herald the crossing of an epistemological threshold in the medical sciences? And might the conditions of aetiological possibility be changing, and changing in ways that would drastically alter what it meant to speak of a 'disease', an 'infection' and a 'pathogen'?

Choy Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / Table of contents --- p.xi / List of figures --- p.xv / List of tables --- p.xviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe acute respiratory syndrome (SARS) - An overview --- p.1 / Chapter 1.1.1 --- Epidemiology of SARS --- p.1 / Chapter 1.1.2 --- Clinical presentation of SARS --- p.2 / Chapter 1.1.3 --- Diagnostic tests of SARS --- p.5 / Chapter 1.1.4 --- Treatment of SARS --- p.7 / Chapter 1.2 --- Severe acute respiratory syndrome coronavirus (SARS- CoV) --- p.8 / Chapter 1.2.1 --- The etiological agent of SARS --- p.8 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- Genome of SARS-CoV --- p.11 / Chapter 1.3 --- Spike (S) glycoprotein of SARS-CoV --- p.14 / Chapter 1.3.1 --- Functions of SARS-CoV S glycoprotein --- p.15 / Chapter 1.3.2 --- Receptors for S glycoprotein of SARS-CoV --- p.17 / Chapter 1.4 --- 3C-like protease (3CLPro) of SARS-CoV --- p.20 / Chapter 1.4.1 --- Extensive proteolytic processing of SARS-CoV replicase polyproteins --- p.20 / Chapter 1.4.2 --- SARS-CoV 3CLPro --- p.21 / Chapter 1.4.3 --- Substrate specificity of SARS-CoV 3CLPro --- p.22 / Chapter 1.5 --- Combating SARS - Vaccine and drug development --- p.24 / Chapter 1.5.1 --- Vaccine development against SARS --- p.24 / Chapter 1.5.2 --- Drug development against SARS --- p.25 / Chapter 1.6 --- Project objectives of this thesis --- p.27 / Chapter 1.6.1 --- Synthetic Peptide Studies on SARS-CoV S glycoprotein --- p.27 / Chapter 1.6.2 --- Synthetic Peptide Studies on SARS-CoV 3CLPro --- p.28 / Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.30 / Chapter 2.1.1 --- Bioinformatics analyses of SARS-CoV S gly- coprotein --- p.30 / Chapter 2.1.2 --- Peptide design and molecular modeling --- p.32 / Chapter 2.1.3 --- Solid phase peptide synthesis (SPPS) --- p.33 / Chapter 2.1.4 --- Peptide conjugation --- p.35 / Chapter 2.1.5 --- Immunization in rabbits and monkeys --- p.36 / Chapter 2.1.6 --- ELISA analysis --- p.37 / Chapter 2.1.7 --- Immunofluorescent confocal microscopy --- p.39 / Chapter 2.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.40 / Chapter 2.2.1 --- Protein expression and purification --- p.40 / Chapter 2.2.2 --- Solid phase peptide synthesis (SPPS) --- p.41 / Chapter 2.2.3 --- Peptide cleavage assay --- p.44 / Chapter 2.2.4 --- Molecular docking --- p.46 / Chapter 3 --- Results --- p.48 / Chapter 3.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.48 / Chapter 3.1.1 --- General features and structural analyses of the S glycoprotein --- p.48 / Chapter 3.1.2 --- Peptides design and synthesis --- p.53 / Chapter 3.1.3 --- ELISA analysis and immunofluorescent con- focal microscopy --- p.55 / Chapter 3.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.62 / Chapter 3.2.1 --- Substrate specificity of SARS-CoV 3CLPro . . --- p.62 / Chapter 3.2.2 --- Molecular docking of SARS-CoV 3CLPro and peptide substrates --- p.74 / Chapter 4 --- Discussion --- p.78 / Chapter 4.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.78 / Chapter 4.1.1 --- Synthetic peptides elicited SARS-CoV specific antibodies --- p.78 / Chapter 4.1.2 --- Factors affecting the specificity and antigenic- ity of synthetic peptides --- p.80 / Chapter 4.1.3 --- Next step towards vaccine development --- p.83 / Chapter 4.1.4 --- A synthetic peptide-based approach --- p.84 / Chapter 4.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.86 / Chapter 4.2.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.87 / Chapter 4.2.2 --- Sequence comparison between SARS-CoV 3CLpro cleavage sites --- p.90 / Chapter 4.2.3 --- A rapid and high throughput approach to screen protease substrate specificity --- p.94 / Bibliography --- p.98