Human Cytomegalovirus (HCMV), the prototypic β-herpesvirus, is the most common cause of congenital infections as well as morbidity and mortality in immunocompromised patients. The anti-HCMV drugs currently available have a number of drawbacks (i.e. detrimental side-effects and/or the appearance of drug resistant strains), which limit their clinical usefulness. Therefore, a better understanding of host-virus interactions is important to develop new, safe and effective ways to treat HCMV. HCMV has evolved various strategies to make the host cell more conducive for the replication process, many of these involve modulation of host signalling pathways through proteins or non-coding RNAs. The focus of this thesis is on the regulation of one class of non-coding RNA, microRNAs (miRNA) by HCMV as well as murine CMV (MCMV). miRNAs are short ~22 nucleotide RNA sequences, which negatively regulate the stability and translational efficiency of specific target messenger RNAs (mRNAs). It has been previously shown that three host-encoded miRNAs, miR-199-3p, miR-199-5p and miR-214, are down-regulated in both MCMV and HCMV infected cells. Despite the biological and genomic differences between the two viruses, this down-regulation occurs in both infections, suggesting a possible conserved antiviral role of the miRNAs in mouse and human cells. Consistent with this, miR-199-3p and miR-214 manifest antiviral properties against MCMV and HCMV when over-expressed in vitro. This thesis investigates two hypotheses: 1) CMV down-regulates the expression of these host miRNAs through a mechanism involving viral factors, 2) The down-regulation of miR-199-3p leads to the up-regulation of its targets and this influences the cell in a way that favours some aspect of the viral life cycle. The first part of this project examined the regulation of miR-199-3p, miR-199-5p, and miR-214, which derive from a single primary transcript (pri-miRNA). The down-regulation of all three miRNAs was found to occur at the transcriptional level by 4 hours post infection. The promoter of the miR-199a/214 cluster was therefore cloned into a reporter vector in order to interrogate the factors regulating transcription of pri-miRNA in infection; this was carried out in the murine model based on availability of reagents. The reduction in the pri-miRNA was found to correlate with a decrease in the transcriptional activity of miR-199a/214 promoter in infected cells. Further analysis revealed the presence of a sequence between -421 to -273 relative to the transcription start site (TSS) that was critical for promoter activity. This sequence contains a putative serum response element (SRE), which includes two binding sites for the SRF dimer (serum response factor) and a binding site for a molecule of TCF (ternary complex factor), ELK-1. Initial knock-down studies suggest that these transcription factors are required for basal activity but it remains unknown whether they are involved in the differential expression of miR-199a/214 observed during infection. Another binding site for the transcription factor TWIST-1 was found outside this region, which is known to regulate the miR-199a/214 cluster in other cell types. Western blot analysis showed reduced expression of TWIST-1 in cells infected with HCMV and MCMV infections, by 24 and 48 hours, respectively, suggesting a role of TWIST-1 in regulating miR-199a/214 cluster during these infections. This regulation seems to be dependent on viral gene expression, as a replication deficient viral mutant fails to repress the promoter function and subsequent pri-miRNA production. Taken together, these results suggest an active viral mechanism for transcriptional repression of the miR-199a/214 promoter. To understand the antiviral function of miR-199-3p, the second part of this thesis examined whether miR-199-3p regulates host signalling pathways important for CMV replication and/or the life cycle. A microarray analysis was carried out with samples from cells transfected with miR- 199-3p mimic versus inhibitor. This revealed 198 genes significantly down-regulated by the miRNA. From the 198 genes, Ingenuity pathway analysis (IPA) software identified several host pathways with a potential role in HCMV infection including: PI3K/AKT signalling, the ERK-MAPK cascade, and prostaglandin production. This thesis examined the role of miR-199-3p in regulating the PI3K/AKT pathway in HCMV infection. It was found that miR-199-3p modulates the phosphorylation of the central regulator of PI3K/AKT signalling, AKT. Transfection of miR-199-3p before the infection impedes the complete phosphorylation of AKT, which is known to be required for the immediate early viral gene expression and replication. This provides an explanation for the antiviral function of miR-199-3p, through its ability to modulate AKT phosphorylation. An open question, however, is how the natural down-regulation of miR-199-3p from 24 to 72 hours post infection naturally affects AKT phosphorylation. Several predicted targets of miR-199-3p, such as PIK3CB, ITGA3, and ITGA6 were shown to be up-regulated at these late time points, correlating with the miR-199-3p down-regulation. The interaction of miR-199-3p with target sites in the 3′UTRs of PIK3CB and ITGA3 was validated by luciferase reporter assays and western blotting and qRT-PCR results indicated that protein and mRNA levels of ITGA6 were regulated by miR-199-3p mimic transfection. However, the knock-down of these three targets did not result in a significant decrease of the viral growth, and thus cannot alone explain the antiviral function of miR-199-3p. Overall, this study suggests that the transcriptional repression of miR- 199a/214 is likely a strategy employed by CMV to support its own growth through attenuating the biological effect of miR-199-3p within the host cell.

Memoria para optar al Titulo Profesional de Médico Veterinario / En consideración a que: a) se desconoce si las alpacas en Chile se han infectado con el virus herpes equino tipo 1 (VHE-1) o un virus antigénicamente relacionado; b) en Chile se ha aislado el VHE-1 desde equinos naturalmente infectados; c) el VHE-1 produce una enfermedad de alto significado económico en el ganado equino; d) se ha descrito una enfermedad de importancia en asociación con una infección por virus herpes similar al VHE-1 en alpacas de Nueva York, procedentes de Chile, en este estudio se planteó detectar en forma indirecta la existencia de infección por VHE-1, o un virus antigénicamente relacionado, en camélidos sudamericanos (CS) a través de la pesquisa de animales que han respondido inmunológicamente al agente viral. El total de muestras correspondió a 204 muestras de sueros sanguíneos, de los cuales 98 pertenecen a alpacas (Lama pacos); 44 a llamas (Lama glama); 37 a guanacos (Lama guanicoe); y 25 a vicuñas (Vicugna vicugna), procedentes de 14 rebaños de las Regiones I, IV, V, XII y Metropolitana. La presencia y cuantificación de anticuerpos se realizó mediante la prueba de seroneutralización dilución punto final (SNDPF), enfrentando diluciones en base dos del suero del animal, a 100 dosis infecciosas cultivo de tejido 50% (DICT50) del VHE-1. Del total de muestras analizadas, 52 (25,5 %) presentaron anticuerpos seroneutralizantes para el VHE-1. La positividad por especie animal fue: 19/98 alpacas (19,4 %); 10/44 llamas (22,7 %); 9/37 guanacos (24,3 %); y 14/25 vicuñas (56 %). Los títulos de anticuerpos oscilaron entre 2,8 y 89 con una media de 10. La media fue para: las alpacas 6,1; las llamas 12; los guanacos 12; y para las vicuñas 17. Se concluye que en el país existen CS infectados con VHE-1 o en su defecto con un virus herpes que comparte antígenos comunes con el VHE-1

Memoria para optar al Título Profesional de Médico Veterinario / Considerando el impacto productivo y reproductivo que causa la enfermedad producida por el virus herpes equino 1 (VHE-1) dentro de la población equina, el conocimiento de la susceptibilidad de los camélidos sudamericanos (CS) a la infección y a la enfermedad producida por un virus similar al VHE-1, el antecedente de la presencia de anticuerpos neutralizantes para el VHE-1 en CS de diferentes regiones del país, el desconocimiento de la situación de infección por el VHE-1 en alpacas y llamas de la Región de Tarapacá y la importancia de estas especies para la población aymara; el objetivo de este estudio es detectar, indirectamente, la existencia de infección por VHE-1, o por un virus antigénicamente relacionado, en camélidos sudamericanos (CS) de la Región de Tarapacá, a través de la pesquisa de animales que han respondido inmunológicamente al agente viral. Asumiendo que el 1% de los CS poseen anticuerpos que reaccionan con el VHE-1, se analizó un total de 192 muestras (75 de vicuñas, 75 de llamas y 42 de alpacas) las cuales se obtuvieron, entre los meses de febrero y abril del año 2006, de diferentes localidades de la Provincia de Parinacota (Caquena, Cruzani/Parinacota, Surire, Guallancallan/Gral Lagos, Guallatire, Visviri, Chuslluta/Gral Lagos, Humaquenque, Chislluma/Ankara, Caquena Culicculine, Putre, Visluvia, Colchane) en la Región de Tarapacá, excepto 18 muestras de alpacas que fueron obtenidas el año 1987 y no se registró la localidad de origen. La presencia de anticuerpos se determinó mediante la prueba de seroneutralización dilución punto final (SNDPF), enfrentando diluciones en base dos del suero del animal, a 100 dosis infectantes cultivo de tejido 50% (DICT50) del VHE-1. Del total de muestras analizadas, ninguna presentó anticuerpos neutralizantes para el VHE-1 o para algún otro virus antigénicamente relacionado. Se concluye que menos del 1% de los CS ubicados en la localidades de Caquena, Cruzani/Parinacota, Surire, Guallancallan/Gral Lagos, Guallatire, Visviri, Chuslluta/Gral Lagos, Humaquenque, Chislluma/Ankara, Caquena Culicculine, Putre, Visluvia, Colchane, de la Provincia de Parinacota, en la Región de Tarapacá podrían estar infectados con VHE- 1 o en su defecto con un virus herpes que comparte antígenos con el VHE-1

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Anatomical Pathology Johannesburg, 2016 / Introduction: Plasmablastic lymphoma (PBL) is an uncommon variant of aggressive B-cell non-Hodgkin lymphoma that occurs in immune-compromised individuals, most commonly secondary to HIV infection. This tumour classically occurs in the oral cavity but has also been described in a variety of extra-oral locations. This is a clinicopathological study of 45 cases of extra-oral PBL (EPBL). Aim: To define the clinical parameters, histology and immunophenotypic features of extra-oral plasmablastic lymphoma (EPBL) and assess the extent to which Epstein–Barr virus (EBV) is associated with this tumour. Materials and Methods: This retrospective study on archival cases of EPBL included the patients‟ age, gender, race, HIV status where available and the site of tumour presentation. Of 49 archival cases retrieved, 4 were discounted owing to reclassification as diffuse large B-cell lymphoma (1 case), multiple myeloma or extramedullary plasma cell tumour (3 cases). The remaining 45 cases were reviewed histologically and classified according to whether they displayed a pure plasmablastic (PBm) morphology or a plasmablastic morphology with plasmacytic differentiation (PBm+PCd), and assessed immunohistochemically with CD45 (LCA), CD20, CD79a, PAX5, CD138, MUM1, BLIMP1, VS38c, Ki-67, BCL6, CD10, HHV8 and CyclinD1 using standard automated procedures. The presence of EBV was assessed by chromogenic in-situ hybridisation. Ethical clearance was obtained (M10750 and M120993). Results: 27of the 45 cases had a pure plasmablastic morphology. The remaining 18 cases showed plasmacytic differentiation. There was no site predilection according to histological pattern. 60% of the tumours were reported in males and 40% in females and all were black African patients. The anus was the favoured extra-oral site of presentation (13 of 45 cases, 28%), followed by soft tissue (11 of 45 cases, 24%). There was no significant difference in the age of presentation between males (38.5 years) and females (35.4 years). Of the 18 patients of known HIV status, 17 were HIV positive (94%). The immunohistochemical profile of EPBL recapitulated that found for both oral and extra-oral PBL in the literature, except for CD45 (leucocyte common antigen) which signalled positively in a higher percentage of cases. 36 of 42 cases (85.7%) were positive for CD45. The positive membrane signal for CD45 was of variable intensity, between 5 and 100% of tumour cells. EBV was positive by in situ hybridisation in 37 of 40 cases tested (92.5%). Conclusion: EPBL is identical to its oral counterpart in gender and age distribution, HIV status, morphological appearances, immunophenotypic profile and association with EBV. The high association with EBV as assessed by in-situ hybridisation studies mirrors that of oral-based PBL reported in the literature. EPBL should be regarded as the same tumour as that arising within the oral cavity. A peculiarity observed within this case cohort is the high level of expression of CD45 (leucocyte common antigen). This has been reported to be of low or near absent expression in most cases of PBL, as defined by the 2008 WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues. / MT2016

According to the UNAIDS global report 2013, an estimated 6.1 million people are living with human immunodeficiency virus (HIV) in South Africa. The incidence of Kaposi sarcoma (KS) has increased dramatically since the Acquired Immunodeficiency Syndrome (AIDS) epidemic. Of the estimated 66 200 cases of KS worldwide, 58 800 are thought to have occurred in SSA (Parkin 2002). However, there remains a paucity of published data about KS from South Africa. This retrospective study was conducted to describe the epidemiology of KS at Chris Hani Baragwanath Academic Hospital (CHBAH) and to determine possible links among the CD4 counts, intensity and distribution of human herpes virus 8 latency- associated nuclear antigen 1 (HHV8 LNA-1) immunohistochemical staining and the stage of KS. Nine hundred and thirty eight histopathology reports of KS diagnosed in 901 patients at CHBAH between 2005 and 2009 were reviewed and demographic data (age, gender, topographic site, CD4 count, HIV status, KS stage, HHV8 LNA-1 staining, concomitant pathology) were recorded. The H&E stained sections and HHV8 LNA-1 immunostains of a cohort of 127 cases were subsequently reviewed and categorised with regard to intensity and distribution of staining. The male:female ratio was 1,2:1. The mean age was 36,8 years (standard deviation {SD} 10,2 years) and the median CD4 count 127,5 cells/mm3 (quartile range {QR} 184,5 cells/mm3). Lower limb skin biopsies accounted for 49,6% of cases. Concomitant pathology was seen in 4,6% of cases. Infections and inflammatory dermatoses were the most frequently diagnosed concomitant pathology in cutaneous biopsies. Paediatric, visceral and endemic KS accounted for only limited proportions of cases (1,44% of patients; 1,4% and 1,3% respectively). There was a significant difference in the distribution of HHV8 LNA-1 staining in patch versus nodular KS (p = 0,011). The CD4 counts were not predictive of KS Page | v stage (p = 0,701) or the intensity (p = 0,877) and distribution (p = 0,846) of HHV8 LNA-1 immunohistochemical staining. This study highlights the epidemiology of KS and the variability in HHV8 LNA-1 immunohistochemical staining across CD4 counts and stages of KS.

In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult. The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

Koi Herpesvirus (KHV), a species-specific DNA virus of the family Herpesviridae, is responsible for mass mortalities of common carp (Cyprinus carpio carpio L.) throughout the world. KHV’s broad geographical distribution and relatively high mortality rate among infected fish, creates significant disposal issues when die-offs occur, especially taking into account the body burden of contaminants in the fish. In locales where adequate disposal facilities are unavailable, or are unable to accommodate additional loadings of contaminated fish carcasses, concerns regarding human and environmental health are raised. During the summer of 2008, residents of the Lake Simcoe Region of southern Ontario, Canada, were faced with a massive die-off of carp, infected with KHV. Carp within the Great Lakes and much of the world are known to bioaccumulate (and biomagnify) contaminants, such as, polychlorinated biphenyls (PCBs), pesticides (e.g., dichlorodiphenyltrichloroethane, DDT, and toxic metals (e.g., mercury). These contaminants have been associated with numerous adverse effects on both human and environmental health, and are thus of important considerations when planning for large-scale carcass disposal, following fish die-offs. Although suites of microbiological tests and water quality assessments are frequently conducted to identify causative factors during extensive fish-kills - assessments of relative contaminant burdens in the carcasses, which should dictate the most appropriate method of carcass disposal - are rarely performed. A case study on Snake Island, Lake Simcoe, Ontario was conducted to further examine the implications of this policy. Soil samples from two known disposal sites and three presumed control locations were sampled on Snake Island and sent to the Analytical Services Unit of Queen’s University for chemical analysis. Although none of the soil samples exceeded any legal guidelines in the present study, there is still concern as future die-offs of other fish species or piscivorous birds and the disposal of large numbers of carcasses may be an issue.

Bovine herpesvirus 1 (BHV-1) is a member of the Alphaherpesvirinae subfamily and is the prototype ruminant herpesvirus. BHV-1 causes a number of complications in cattle including upper respiratory tract disorders, conjunctivitis, genital disorders, abortions, and immune suppression. Like all herpesviruses, reactivation from latency can occur throughout the animal’s life. Of particular economic importance is the bovine respiratory disease complex (BRDC) or ‘shipping fever’, in which BHV-1 plays a major role. BRDC is an enormous economic concern as it costs the US cattle industry approximately one billion dollars annually. In order to generate improved gene-deleted vaccines against BHV-1, there is a need to understand the contributions of viral gene products during infection. US3 is a serine/threonine kinase present in BHV-1 and is thought to play major roles during viral infection. As in other herpesviruses, US3 in BHV-1 is expected to phosphorylate several cellular and/or viral proteins. We recently presented evidence that BHV-1 US3 phosphorylates both VP8 and VP22; however, further functional characteristics of BHV-1 US3 during viral infection have not been elucidated. The hypothesis of this project is that the deletion of the US3 gene leads to reduced BHV-1 fitness. To explore this hypothesis, we generated a US3-deleted (ΔUS3) and subsequent US3-rescued (US3R) BHV-1 virus. Using these viral mutants, we characterized the growth properties of the viruses, evaluated the effect of the US3 deletion on major structural BHV-1 proteins, characterized the protein composition of the mature virions, and, identified viral processes that were impaired in the deletion mutant. Initially, the ∆US3 virus was generated through a 3-step PCR strategy which replaced the gene of interest with an antibiotic resistance cassette. Following this, the US3 gene was rescued via a two-step en passant mutagenesis strategy which has been previously used to generate insertions, deletions, and substitutions in herpesvirus-containing bacterial artificial chromosome (BAC) DNA. In vitro characterization of ∆US3 BHV-1 has demonstrated that US3 deletion affects BHV-1 growth characteristics, expression kinetics of major structural proteins, mature virion composition, cell to cell spread, and the subcellular localization of key viral proteins during infection. Growth kinetics of ∆US3 BHV-1 were impaired compared to wild-type (WT) BHV-1, especially at late times post-infection. Plaque sizes formed by ∆US3 BHV-1 were significantly smaller than those formed by either WT or US3R BHV-1, demonstrating that US3 is important for cell to cell spread. The expression kinetics of major structural and regulatory BHV-1 proteins were different between cells infected with ∆US3 or WT BHV-1, and incorporation of these proteins into the mature viruses differed, demonstrating that US3 is instrumental in ensuring proper protein expression and mature virus composition in vitro. Of particular importance, glycoprotein B (gB), was shown to be expressed in higher quantities earlier during infection in the absence of US3, and that this protein was incorporated in significantly higher amounts in mature virions which lacked US3. Qualitative analysis of ∆US3 BHV-1 infected monolayers suggested the abolishment of cell to cell projections characteristic of WT BHV-1 infection. Finally, the disruption of gB in ∆US3 BHV-1 infected cells was confirmed by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Through confocal microscopy, evidence was provided that infection with ∆US3 BHV-1 possibly results in earlier expression of gB on the surface of cells and less intracellular accumulation of this protein during late stages of infection. The observed effect on the localization of intracellular gB in ∆US3 BHV-1 infected cells was quantified by flow cytometry. ∆US3 BHV-1 infected cells had approximately 25% higher gB expression on the surface of cells and a corresponding 25% decrease in intracellular gB. Although these differences have not yet been demonstrated to be statistically significant and not confirmed through infection with US3R BHV-1, this suggests that US3 may influence the synthesis and cellular trafficking of gB in vitro.

Con el objetivo de tener una información general sobre la presencia de agentes virales neumotrópicos, se muestreó 345 alpacas adultas procedentes de 31 hatos, pertenecientes a medianos y pequeños criadores de la provincia de Canchis - Cusco. Las muestras de sueros sanguíneos fueron procesadas para la detección de anticuerpos neutralizantes para los virus: Parainfluenza tipo 3 (PI3), Respiratorio Sincitial Bovino (RSB) y el Herpes virus bovino tipo 1 (VHB1). Todas las muestras fueron trabajadas mediante la prueba de neutralización viral utilizando cultivos celulares secundarios de origen bovino (cornetes nasales) y cepas virales citopáticas mantenidas en el laboratorio. Las pruebas de laboratorio evidencian que los 31 hatos muestreados tienen animales seropositivos a los virus RSB y PI3; y solamente 1/30 se encontró animales reactores al VHB1. El estudio, además determino una seroprevalencia de 80.16% ± 6.96% (101/126) y 67.54% ± 4.94% (233/345) del VRSB y el PI3 respectivamente. Estos resultados sugieren que los animales muestreados han estado expuestos a varios agentes potencialmente neumopatogénicos, y evidencian una activa replicación viral al momento del muestreo que coincidió con una severa ola de baja temperatura en la zona y mortalidad de animales por neumonías en el campo, quedando por demostrarse la presencia directa de estos agentes en muestras patológicas y verificar el posible rol causal en procesos neumónicos. / The objective of the present study was determine the presence of respiratory virus. Three hundred forty five serum samples from thirty one farms were tested by virus neutralization test to detect neutralizing antibodies against PI-3, BHV1 and BRSV. The farms were small farms from the province of Canchis - Cusco. All the samples were worked by means of the test of viral neutralization using secondary cellular cultures of bovine origin and cytopathic virus from the laboratory. Thirty one farms had seroreactive animals to BRSV and PI-3; and one farm had seroreactive animals to BHV1. The 80,16% ± 6,96% (101/126) and 67,54% ± 4,94% (233/345) of the samples had antibodies against BRSV and PI-3 respectively. These results suggest that sample animals have been exposed potentially to respiratory virus, and demonstrate an active viral replication at the time of the sampling that agreed with a severe low temperature in the zone and mortality of animals by pneumonia in the field.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 146-195).