Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. Rowland / Severe acute respiratory syndrome (SARS) is caused by an enveloped, positive-stranded RNA virus, the SARS coronavirus (SARS-CoV). Coronaviruses along with the arteriviruses are placed in the order, Nidovirales. Even though nidovirus replication is restricted to the cytoplasm, the nucleocapsid protein (N) of several coronaviruses and arteriviruses, localize to the nucleolus during infection. Confocal microscopy of N protein localization in Vero cells infected with the SARS-CoV or transfected with the SARS-CoV N gene failed to show presence of N in the nucleoplasm or nucleolus. Recombinant N remained cytoplasmic after the addition of leptomycin B (LMB), a drug that inhibits nuclear export. SARS-CoV N possesses a unique lysine-rich domain, located between amino acids 369-389, which possesses several nuclear localization signal (NLS) and nucleolar localization signal (NoLS) motifs. A chimeric protein composed of the 369-389 peptide substituted for the NLS of equine infectious anemia virus (EIAV) Rev protein (ERev) showed no nuclear localization activity. Three negatively charged amino acids, located at positions 372, 377 and 379 in SARS-CoV N were hypothesized to play a role in the loss of nuclear targeting. Substitution of aspartic acid-372 with alanine restored nuclear localization to the chimeric protein. A full-length recombinant SARS-N protein with the alanine-372 substitution localized to the nucleus. Therefore, the presence of an aspartic acid at position 372 is sufficient to retain N in the cytoplasm The mechanistic basis for how aspartic acid-372 interrupts nuclear transport is unknown, but may lie in the electrostatic repulsion with negatively charged amino acids located within the NLS binding pocket of importin-alpha.

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.

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.

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

Coronaviruses are a diverse group of large, single-stranded RNA virus that cause respiratory and enteric diseases in mammalian and avian species. Phylogenetic analysis shows that SARS-CoV is an unique branch of coronavirus showing no close relationship to other groups of coronaviruses. The genome size of SARS-CoV is about 30 kilobase and the genome, like other coronaviruses, is composed of replicase (rep), spike (S), envelope (E), membrane (M) and nucleocapsid (N) and 8 additional unknown open reading frames (ORFs) (ORF 3a, ORF 3b, ORF 6, ORF 7a, ORF 7b, ORF8a, ORF 8b and ORF 9b). The 3a gene, the largest unknown ORF, encodes a viral protein which is predicted to be a transmembrane protein. In this study, we showed that the 3a protein was expressed in SARS patients' lung and intestinal tissues, and it is localized to the endoplasmic reticulum (ER) in 3a-transfected monkey kidney Vero E6 cells. Results from experiments including chromatin condensation, DNA fragmentation and antibody microarray suggest that the 3a protein may trigger apoptosis through a caspase-8-dependent pathway and possibly a PKR-mediated FADD-caspase-8 pathway. Our data show that over-expression of the SARS-CoV protein can induce apoptosis in vitro. / Severe acute respiratory syndrome (SARS), an atypical form of pneumonia, is first recognized in Guangdong Province, China in November 2002 and later spread to Hong Kong in mid February 2003. It is believed that the etiological agent of SARS is a previously unknown coronavirus - SARS-CoV. Over 8,400 cases and 789 deaths were reported to World Health Organization (WHO) from over 28 countries around the world including Hong Kong. Up to now, there are still no efficient antiviral drugs to treat the disease, and the detailed pathology of SARS-CoV infection and the host response to the viral infection are still unknown. During the epidemic, we have done complete genome sequencing for five SARS-CoV isolates and we postulate that at least two SARS-CoV strains with distinct etiological origins exist in the environment during the epidemic. / Law Tit-wan Patrick. / "Aug 2005." / Adviser: Stephen K. W. Tsui. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 156-172). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Thesis (M.A. (Health Social Science))--Mahidol University.

Em março de 2009, foram identificados os primeiros casos de vírus influenza A, não subtipado anteriormente e que levou a comunidade internacional a enfrentar a primeira pandemia do século XXI, na vigência do novo Regulamento Sanitário Internacional de 2005. No intervalo das semanas epidemiológicas 16 e 33, foram notificados 34.506 casos de síndrome respiratória aguda grave no Brasil. No Brasil, a maior incidência ocorreu nas faixas etárias de crianças ≤ 5 anos (3.8/100.000) e com idades entre 20-29 anos (4.6/100.000). Neste período os casos ficaram concentrados nas regiões sul e sudeste, com 94% dos casos notificados. A taxa de mortalidade na população durante este período foi 0.39/100.000 habitantes. Pessoas que apresentaram comorbidades relacionadas apresentaram o dobro de risco de evolução para o óbito, quando comparado às pessoas sem comorbidade. (RR = 1,89 IC 95% 1,64-2,18). Apesar do clima tropical, o Brasil foi um dos países mais afetados pela pandemia. No entanto, este evento está possibilitando o fortalecimento das ações de vigilância e assistência que serão úteis em todas as situações de emergências de saúde pública de importância nacional e internacional. / In March 2009, identified the first cases of a new influenza A virus, not subtyped previously. Without immunity, the international community suffered the first pandemic of the century, the term of the International Health Regulations 2005. Between epidemiological weeks 16 and 33, were reported 34,506 cases of severe acute respiratory syndrome in Brazil. In Brazil, the highest incidence occurred in younger children ≤ 5 year (3.8/100,000) and one at ages 20-29 years (4.6/100,000). Ninety-four percent of cases concentrated in two of Brazil’s five geographic regions – the south and southeast. . The mortality rate in the population during this period was 0.39/100,000 inhabitants. Cases with a reported comorbidity had approximately twice the risk of those without (RR=1.89; 95%CI 1.64 – 2.18). Despite the tropical climate, Brazil was one of the countries most affected by the pandemic. However, this event is allowing the strengthening of surveillance and assistance that will be useful in all situations of public health emergencies of national and international concern.

Em março de 2009, foram identificados os primeiros casos de vírus influenza A, não subtipado anteriormente e que levou a comunidade internacional a enfrentar a primeira pandemia do século XXI, na vigência do novo Regulamento Sanitário Internacional de 2005. No intervalo das semanas epidemiológicas 16 e 33, foram notificados 34.506 casos de síndrome respiratória aguda grave no Brasil. No Brasil, a maior incidência ocorreu nas faixas etárias de crianças ≤ 5 anos (3.8/100.000) e com idades entre 20-29 anos (4.6/100.000). Neste período os casos ficaram concentrados nas regiões sul e sudeste, com 94% dos casos notificados. A taxa de mortalidade na população durante este período foi 0.39/100.000 habitantes. Pessoas que apresentaram comorbidades relacionadas apresentaram o dobro de risco de evolução para o óbito, quando comparado às pessoas sem comorbidade. (RR = 1,89 IC 95% 1,64-2,18). Apesar do clima tropical, o Brasil foi um dos países mais afetados pela pandemia. No entanto, este evento está possibilitando o fortalecimento das ações de vigilância e assistência que serão úteis em todas as situações de emergências de saúde pública de importância nacional e internacional. / In March 2009, identified the first cases of a new influenza A virus, not subtyped previously. Without immunity, the international community suffered the first pandemic of the century, the term of the International Health Regulations 2005. Between epidemiological weeks 16 and 33, were reported 34,506 cases of severe acute respiratory syndrome in Brazil. In Brazil, the highest incidence occurred in younger children ≤ 5 year (3.8/100,000) and one at ages 20-29 years (4.6/100,000). Ninety-four percent of cases concentrated in two of Brazil’s five geographic regions – the south and southeast. . The mortality rate in the population during this period was 0.39/100,000 inhabitants. Cases with a reported comorbidity had approximately twice the risk of those without (RR=1.89; 95%CI 1.64 – 2.18). Despite the tropical climate, Brazil was one of the countries most affected by the pandemic. However, this event is allowing the strengthening of surveillance and assistance that will be useful in all situations of public health emergencies of national and international concern.

Em março de 2009, foram identificados os primeiros casos de vírus influenza A, não subtipado anteriormente e que levou a comunidade internacional a enfrentar a primeira pandemia do século XXI, na vigência do novo Regulamento Sanitário Internacional de 2005. No intervalo das semanas epidemiológicas 16 e 33, foram notificados 34.506 casos de síndrome respiratória aguda grave no Brasil. No Brasil, a maior incidência ocorreu nas faixas etárias de crianças ≤ 5 anos (3.8/100.000) e com idades entre 20-29 anos (4.6/100.000). Neste período os casos ficaram concentrados nas regiões sul e sudeste, com 94% dos casos notificados. A taxa de mortalidade na população durante este período foi 0.39/100.000 habitantes. Pessoas que apresentaram comorbidades relacionadas apresentaram o dobro de risco de evolução para o óbito, quando comparado às pessoas sem comorbidade. (RR = 1,89 IC 95% 1,64-2,18). Apesar do clima tropical, o Brasil foi um dos países mais afetados pela pandemia. No entanto, este evento está possibilitando o fortalecimento das ações de vigilância e assistência que serão úteis em todas as situações de emergências de saúde pública de importância nacional e internacional. / In March 2009, identified the first cases of a new influenza A virus, not subtyped previously. Without immunity, the international community suffered the first pandemic of the century, the term of the International Health Regulations 2005. Between epidemiological weeks 16 and 33, were reported 34,506 cases of severe acute respiratory syndrome in Brazil. In Brazil, the highest incidence occurred in younger children ≤ 5 year (3.8/100,000) and one at ages 20-29 years (4.6/100,000). Ninety-four percent of cases concentrated in two of Brazil’s five geographic regions – the south and southeast. . The mortality rate in the population during this period was 0.39/100,000 inhabitants. Cases with a reported comorbidity had approximately twice the risk of those without (RR=1.89; 95%CI 1.64 – 2.18). Despite the tropical climate, Brazil was one of the countries most affected by the pandemic. However, this event is allowing the strengthening of surveillance and assistance that will be useful in all situations of public health emergencies of national and international concern.