Porcine Reproductive and Respiratory Syndrome (PRRS) is a global disease which takes a significant toll on the pork industry and the welfare of pigs. The causative agent – PRRS virus (PRRSV) – is a single-stranded, positive-sense RNA virus of the Nidovirales order. In the process of replication, PRRSV induces the rearrangement of cellular membranes to form double-membrane vesicles (DMVs). These structures are thought to have a role in a) concentrating viral structures to increase their chances of interacting with one another, and b) preventing elements of the cellular immune response from detecting viral structures. Previous work has suggested that the DMV originates from the autophagy pathway – a highly-conserved mechanism for cells to recycle extraneous organelles and proteins during times of stress. Other work suggests that the DMV may be a co-opted EDEMosome – a recently-discovered vesicle which is involved in regulating the level of endoplasmic reticulum-associated degradation (ERAD). This thesis explores these possibilities – using immunofluorescent imaging as well as examining the proteomic and ribonucleic acid composition of the DMV as isolated by flow cytometry or separated from other organelles by density gradient – calling both candidate pathways into question and suggesting other candidate structures such as exosomal vesicles.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Science in Epidemiology (Epidemiology and Biostatistics). 2016 / Background Reducing severe acute respiratory illness (SARI)-associated mortality in African children remains a public health priority and an immense challenge. The pneumococcal conjugate vaccine (PCV) was introduced into the South African routine immunization programme in 2009. The objectives of this study were: I. To describe the demographic characteristics, clinical presentation, respiratory pathogens of children aged <5 years hospitalized with SARI in an urban (Chris Hani-Baragwanath Hospital, Soweto) and a rural (Matikwana and Mapuleng Hospitals, Mpumalanga) setting in South Africa from 2009-2013 and II. To compare the factors associated with mortality among children aged <5 years hospitalized with SARI in these two sites separately. Methods Hospitalized children with SARI were enrolled into an active, prospective sentinel surveillance program. Clinical and epidemiologic data were collected until discharge. Nasopharyngeal aspirates were tested for influenza (A and B) and eight other respiratory viruses. In-hospital case-fatality proportion (CFP) and risk factors for mortality were determined for each hospital site separately using unconditional logistic regression. Results The in-hospital CFP was significantly higher in the rural (6.9%, 103/1486) than the urban (1.3%, 51/3811) site (p<0.001). This was observed among both HIV-infected (urban: 6.6%, 17/257) vs. (rural: 12.9%, 30/233) (p=0.019) and HIV-uninfected children (urban: 0.6%, 13/2236) vs. (rural: 4.2% 36/857) (p<0.001). In the urban site the only factor that is independently associated with death on multivariate analysis was HIV infection (odds ratio (OR) 12.1, 95% confidence interval (CI) 5.8-25.2). In the rural site HIV infection (OR 3.5, 95% CI 1.7-6.9), age <1 year (OR 3.5, 95% CI 2.0-6.1) vs. 1-4 years, any respiratory virus detected (OR 0.4, 95% CI 0.2-0.6), pneumococcal infection(OR 4.5, 95% CI 1.8-10.8) and malnutrition (OR 12.8, 95%CI 1.2-134.6) were independently associated with mortality. Conclusion SARI mortality was higher in the rural setting. Even in the era of PCV availability pneumococcus is still associated with mortality in rural areas. Efforts to prevent and treat HIV infections in children and reduce malnutrition may reduce SARI deaths. / MT2017

Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first manifested in humans in China in November 2002 and has subsequently spread worldwide. According to the World Health Organization, 8098 cases occurred during the outbreak, and healthcare workers accounted for 1707 (21%) of the cases. To determine the prevalence of SARS infection of healthcare workers in Taiwan, we performed a serosurvey by the recombinant protein-based enzyme-linked immunosorbent assay (ELISA) to test for immunoglobulin (Ig) G antibodies to the SARS coronavirus (SARS-CoV) among 1525 healthcare workers in 26 hospitals that admitted SARS patients in mid-May, 2003. Then, a case-control study was carried out to evaluate the risk factors of SARS infection among the healthcare workers. A total of 52 infected staffs and 78 hospital and age matched non-infected controls were recruited. The seroprevalence rate was 3.68% (58/1525) for healthcare workers. Univariate analysis showed that with the habit of drinking coffee or tea, taking care of fever patients more than 8 days, ever practice of CPR, suction of sputum, taking patient¡¦s temperature, use of P100 mask, use of N95 mask, use of face cover, use of goggles, use of gown, removing gloves after work, working in isolation area or fever screen station were significantly protective factors. In addition, eating jujube was a risk factor for SARS infection. Then, the multivariate analysis showed that use of P100 ¡]OR: 0.056, 95%CI: 0.019-0.162, p value: <0.001¡^and working in isolation area ¡]OR: 0.153, 95%CI: 0.029-0.810, p value: 0.027¡^or fever screen station¡]OR: 0.103, 95%CI: 0.011-0.963, p value: 0.046¡^were the most important protective factors for SARS infection. These findings suggest that nosocomial infection of SARS can be prevented effectively by use of P100 and the triage screening in emergency departments.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

In part ONE of this thesis, including the most up to date information on SARS virology, disease transmission, pathogenesis and laboratory diagnosis will be summarized and presented, including the results of many studies in which I have participated (these references will be underlined as they appear in text). This of course summarizes knowledge that is now known in 2006 but was largely unknown during the initial outbreak. In part TWO, six original clinical studies performed at PWH will be presented: study (1) describes the clinical manifestations and severity of SARS, and its potential to cause major hospital outbreaks; (2) demonstrates the importance of epidemiological linkage in diagnosing SARS; (3) reports the clinical outcomes of a stepwise treatment protocol, which includes the use of corticosteroid therapy as an immunomodulant; (4) demonstrates that corticosteroid therapy can retard viral clearance, and should be used judiciously; (5) demonstrates that a more robust humoral response is associated with severe SARS, thus indicating that passive immunity treatment strategies seem only suitable either during early illness or as prophylaxis; and (6) shows that SARS has few early discriminating laboratory features compared to other causes of community-acquired pneumonia, thus a high index of suspicion is needed to recognize this infection in the absence of worldwide transmission. A thorough review of the relevant published material will be included in the discussion section of each study. / Severe Acute Respiratory Syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. It caused a global outbreak in 2003, resulting in more than 8000 infections, 700 deaths, and major social and economic disruption. In the initial phase of the SARS outbreak, the medical profession had no knowledge regarding the responsible pathogen, nor the clinical manifestations of SARS and the course of illness. There was no reliable diagnostic tool and no known effective therapy. But for the first time in medical history, we witnessed the rapid accumulation of knowledge on a disease as it evolved, which in turn assisted its management and control. / Since conducting randomized-controlled trials during the 2003 crisis was almost impossible, most of the presented studies are either descriptive or case-controlled in design. However, these studies have laid foundations for recent and future research into the clinical diagnosis and management of SARS. Moreover, the construction of the SARS clinical database has contributed to the work of other investigators, which has resulted in over thirty-six publications. It is my hope that these research endeavors can contribute to the understanding of this emerging, deadly disease. / Lee Lai Shun, Nelson. / "April 2006." / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0205. / Thesis (M.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 264-292). / 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. / Abstracts in English and Chinese. / School code: 1307.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a controlled disease in South Africa. This disease is caused by an Arterivirus and occurs commonly in Europe (European serotype) and in the United States of America (American serotype); therefore PRRS is not a trade sensitive disease. However, the disease has severe economic implications for the producer and the local pork industry and the decision was made by the Department of Agriculture, Fisheries and Forestry in association with the South African Pork Producers’ Organization (SAPPO) to eradicate the disease when the first outbreak occurred in 2004 in the Western Cape. Severe disease leading to acute mortalities and almost 100% mortality and morbidity rate in a few pig units in the Jacobsdal area (Kuilsrivier district) in the Cape Town peninsula, alarmed local veterinary consultants during the autumn of 2004. A first diagnosis of Salmonella choleraesuis was confirmed at the Provincial Veterinary Laboratory in Stellenbosch. Antibiotic treatment did not resolve the clinical picture. Sows still aborted and died and young pigs still died from acute respiratory distress. The syndrome was similar to “blue ear disease” because of severe cyanosis visible on the extremities of affected pigs. The first suspected diagnosis of Porcine Reproductive and Respiratory Syndrome virus (PRRSV) was made on post mortem examination on 10 June 2004 and was confirmed on 17 June 2004 with positive ELISA (Idexx Herdchek PRRSV Ab test kit 2XR) results. A stamping out procedure immediately followed through slaughtering of all affected pigs showing clinical signs of PRRSV infection. Pigs in close proximity with possible contact and infection risk were also slaughtered. The movement of pigs was only allowed under Red Cross permit and all pig auctions were stopped. A local and countrywide serological survey was implemented immediately. The results of this survey luckily showed that the outbreak was limited to a few districts in the Western Cape. The probable source of infection is suspected to be uncooked swill originating from the Cape Town Harbour or the Cape Town International Airport, which was fed to pigs. The PRRSV responsible for the outbreak was confirmed on 5 July 2004 as the American serotype by RT-PCR test done at Lelystad, Netherlands. The National Department of Agriculture (NDA)* agreed to compensate pig owners for slaughtered pigs. This decision was further made possible by funding from SAPPO to protect the rest of the commercial pig herd in South Africa to ensure food safety and security. A total of 32 pig units were affected by PRRS of which only one was a commercial unit. All affected pigs were slaughtered by the end of August 2004. Units were cleaned and disinfected by the staffs of the Boland and Swartland State veterinary departments with approved disinfectants which is effective against PRRSV. Cleaned units had to stay empty of pigs for at least 8 weeks after disinfection was completed. Restocking was only allowed from known PRRS-free pig suppliers and regular monitoring was implemented of all previously infected sites and units in high risk areas. On-going serological monitoring revealed no more positive cases since May 2005. It seems that the stamping out procedure and a temporary ban on movement and auctions of live pigs played a primary role in eradication of the PRRSV outbreak in South Africa in 2004. * The name of the NDA has been changed to the National Department of Agriculture, Forestry and Fisheries (DAFF) in 2009, but because the NDA was the applicable name when the PRRS outbreak occurred, NDA will be used in this document. / Dissertation (MMedVet)--University of Pretoria, 2010. / Production Animal Studies / unrestricted

Coronaviruses have caused high pathogenic epidemics within the human population on two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-MERS). Four other species of coronavirus circulate endemically in the human population (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics can be used to study an organism’s phenotype by logical mutation of its genotype. Construction of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This system should aid in answering remaining questions of coronavirus genetics and evolution as well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS. / October 2015

Porcine Reproductive and Respiratory Syndrome (PRRS) is, globally, one of the costliest of diseases to the pig industry. Despite enormous efforts, methods such as vaccination strategies and herd management have failed to fully control the disease. Exploiting the genetic variation in host response could be included as part of a multifaceted approach to mitigate the devastating impact of this disease. Establishing the presence of genetic variation and its underlying genetic architecture are key to implementing genomic selection, which is considered a viable and safe long-term disease control strategy. This thesis explores the effect of natural PRRSV outbreaks on the reproductive performance of sows, and the underlying genetic influences on it. Litter records were available from two farms, where Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) outbreaks had been confirmed using ELISA. One farm had full pedigree information, but for both farms 60K SNP genotypes were available. In both farms, performance records could be partitioned into an epidemic and non-epidemic phase using a previously established threshold method. The partitioning also identified a period of high reproductive failure not coinciding with a diagnosed PRRSV outbreak on one farm. This period was isolated and analysed separately. Linear mixed models were used to explore both genetic and non-genetic factors contributing to differences in reproductive performance associated with the two phases. This analysis identified five disease indicator traits identified showing significant differences (>95% CI) in least squares means between the epidemic and non-epidemic phase. These were the number of mummified, stillborn, dead and alive piglets per litter and the fraction of the total born dead. Alternative statistical models that accounted for differences in the severity of the individual PRRSV outbreaks were also considered throughout. Despite differences in the estimates associated with different models and farms, in general very low heritability estimates were obtained for these disease indicator traits during the non-epidemic phase, whereas the traits were found moderately heritable during the epidemic phase. Two genome wide association analyses methods were used to explore the distribution of the genetic effects throughout the genome: Family-based Score Test for Association (FASTA) and Genome-wide Rapid Analysis using Mixed Model and Regression (GRAMMAR). In addition, regional associations were studied using Regional Heritability Mapping (RHM). Associations were then further characterised using Measured Genotype (MG) analyses. Genome-wide significant associations were identified for five SNPs and one region. The regional association spans the region previously identified in an experimental challenge experiment of growing pigs, in association with viral load and weight gain. Different patterns of linkage disequilibrium (LD) are observed which may explain why this study and others failed to find single SNP effects at this location. One genome wide significant SNP on SSC15 was found between two previously identified SNPs associated with PRRSV mortality. Five further putative SNP associations are indicated by RHM and subsequent measured genotype analysis, two of which flank previously reported associations and indicate an epistatic effect, observed in several traits. In summary, this study showed that reproductive performance of sow is considerably reduced during PRRSV outbreaks and the genetics of the sow significantly affects variance in survival and mortality. Several novel genomic regions associated with the reproductive performance of sows in the absence and during PRRSV outbreaks have been identified in this study. In addition to these, the results suggest the region on SSC4 previously associated with PRRSV viral load and weight gain may also affect foetal mortality. These results demonstrate the potential for genomic selection to be used to mitigate PRRSV related reproductive losses, the greatest financial exposure faced by the pig industry. In addition, RHM is directly shown to capture genetic variance, where single SNP methods fail to identify an effect, highlighting the usefulness of this tool as a method to identify genomic regions with significant effect on production traits.

The thesis examines a mediated crisis and how The Straits Times and The Australian approach the reporting of Severe Acute Respiratory Syndrome (SARS). It looks at how this mediated crisis exemplifies the culture of the national newspaper and in turn how the national newspaper has an historical influence on the national psyche. A total of 649 reports and headlines and 141 letters about SARS in The Straits Times (including The Straits Time Interactive) were examined from April 2003 to November 2003 as were 125 headlines from The Australian. The early sections of the thesis discuss how a crisis makes news; examine how the media report a crisis and what emphasis is given to aspects such as: actors, primary definers, vocabulary, lexical choices, subjects, themes, issues and value dimension or stance. The first chapter defines crisis, journalism and crisis journalism and discusses where the latter sits within the continuing expansion and development of major theoretical frameworks, including living in a risk society. The implication here is that crisis and risk have a symbiotic relationship. Historical perspectives of news are discussed in Chapter 2, and the newspaper is placed within the context of contemporary media. The chapter discusses how newspapers are aligned with the concept of the national mind and demonstrates the roles and formations of the two newspapers in relation to the SARS crisis. Chapter 3 codes the headlines, article titles and subtitles of The Straits Times and The Australian and using content analysis of the headlines, analyses the reporting of a serious health crisis SARS that lasted from March to November, 2003. The quantification within content analysis enables a researcher to read and interpret questions that relate to the intensity of meaning in texts, their social impact, the relationships between media texts and the realities and representations they reflect (Hansen et al, 1998). The theory and method of content analysis is used in this chapter to consider differences between The Straits Times and The Australian and to exemplify the media’s representation of the narratives of SARS as it happened in the countries of Singapore and Australia. Aspects of crisis and risk, the newspaper and the national mind, narratives, presentations, and post SARS events are discussed in the last chapter. It is concluded from these discussions there is a world narrative that tells the story of how the human condition likes to live and rely on a safe social environment always being available. The relationship between a mediated crisis and risk are also discussed. In addition, it is maintained that reporting in 2003 was not just about SARS but a way of reporting that allowed one to view journalism as an aid to good governance, particularly with regard to living in a risk and crisis-ridden society.

Recently, progress was made in collecting, classifying, and characterizing the genetic diversity of type 2 porcine reproductive and respiratory syndrome virus (PRRSV) using all known and publically available sequencing information. Despite this voluminous attempt, these analyses were largely na?ve of the Canadian contribution to circulating viruses. This represented a vital omission in the study of molecular epidemiology due to the fact that Canada had recorded the earliest evidence of the existence of type 2 PRRSV. To this end, the genetic diversity and evolutionary aspects of PRRSVs distributed in the Province of Ontario in Canada were characterized to abridge this existing knowledge gap on type 2 PRRSV. Genotyping of type 2 strains is primarily based on either a phylogenetic or restriction fragment length polymorphism (RFLP) approach. Classification of Ontario PRRSV field isolates (n = 505) from 1999 to 2010, based on a global type 2 PRRSV ORF5 phylogenetic framework, revealed genetic diversity comparable to PRRSV in the USA, with sequences assigned to five of nine lineages (1, 2, 5, 8 and 9). A majority (~85%) of these isolates were typed to the first two lineages (1 and 2). Despite a relatively smaller sample size to the USA, the topology of the phylogenetic tree indicated Canadian origins of these two lineages. Mapping RFLP patterns of Ontario isolates onto the phylogenetic tree revealed numerous examples of different patterns located within the same phylogenetic cluster. Examples of the non-specificity of RFLP patterns to any particular lineage or sub-lineage were abound. Statistical analysis showed occurrences where similar RFLP patterns masked diverse genetic distances and instances of close genetic proximity with divergent RFLP patterns. An examination of the most abundant 15 RFLP patterns revealed that the discrepancy between RFLP typing and genetic distances was not attributable to a single or few patterns but was rather a permeating feature. Importantly, the tree topology also indicated a Canadian ancestry for the highly virulent MN184-related strains that first emerged in 2001 in the USA. Selective pressure analyses highlighted a handful of positively selected sites most of which were located in the ORF5 ectodomains of outbreak strains, implicating the host immune system as the possible selective agent. This was in contrast to the closely-related Ontario strains which were subject to strong purifying selection. A broader survey of transmission dynamics in North America unveiled a higher virus flow from Canada to the USA with the primary targets being the Lake States and Corn Belt. In turn, these regions served to disseminate viruses to other swine production regions in the USA. Virus flow from the USA to Canada occurred on a much smaller scale. Collectively, extensive genetic diversity prevails in type 2 PRRSV in one region of the North American swine industry and it is not described adequately by RFLP typing which might have some value in differentiating strains at the local farm level, instead. For diagnostic and research purposes, phylogenetic typing should be the preferred method. Finally, stronger surveillance needs to be adopted to minimize cross-border virus transmission. / published_or_final_version / Biological Sciences / Master / Master of Philosophy