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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

M.P.H., Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: Herpes Simplex Virus type 2 (HSV-2) is a common cause of genital ulcers worldwide and has emerged as a co-factor in human immunodeficiency virus (HIV) acquisition and transmission. A study was conducted to determine the prevalence of HSV-2, its correlates, the accuracy of reported history of genital ulcer disease (GUD) to predict HSV-2 infection and the extent of symptom recognition in a clinic population in Johannesburg. Methods: 210 women aged 18 years or older were interviewed and socio-demographic, sexual behaviour and clinical information collected. Serological testing for HSV-2 and HIV infections was performed, but only where sera were available for the latter. Factors associations with HSV-2 infection were assessed using logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI). The sensitivity, specificity, predictive values and likelihood ratios of a history of GUD were calculated. Results: The estimated sero prevalence of HSV-2 was 73% (95% CI 67% - 79%). Few participants, 13/206 (6%) participants had knowledge of genital herpes. Only 9/203 (4%) participants recognised lesions of genital herpes following education and counselling about HSV-2 infection. HSV-2 infection was associated with older age(>25 years of age) OR 2.6 (95% CI 1.4-5.0), spending more than 2 nights away from home, OR 6.0 (95% CI 1.0-62.7), having more than 2 sexual lifetime partners, OR 2.2 (95% CI 1.1-3.9), a history of an STI in the past 3 months ,OR 3.6 (95% CI 1.2-9.5) and HIV infection, OR3.3( 95%CI 1.4-7.9). A history of genital ulceration performed poorly as a predictor of HSV-2 seropositivity; the sensitivity was 7% and specificity was 96%. Conclusion: HSV-2 prevalence was high and few participants were aware of their infection. HVS-2 infection was associated with risky sexual behaviour .A history of genital ulcer disease was not sufficient as a diagnostic tool for HSV-2 infection. Public health interventions should focus on behavioural modification and increasing awareness of genital herpes. HSV-2 management should be incorporated into HIV care and STI protocols.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-06-10T14:20:01Z No. of bitstreams: 1 Dissertação - Tatiana Amaral Pires de Almeida.pdf: 3233406 bytes, checksum: e04eb0576f711f1ed5bbe73ae78b4932 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-11T18:30:48Z (GMT) No. of bitstreams: 1 Dissertação - Tatiana Amaral Pires de Almeida.pdf: 3233406 bytes, checksum: e04eb0576f711f1ed5bbe73ae78b4932 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-11T19:08:12Z (GMT) No. of bitstreams: 1 Dissertação - Tatiana Amaral Pires de Almeida.pdf: 3233406 bytes, checksum: e04eb0576f711f1ed5bbe73ae78b4932 (MD5) / Made available in DSpace on 2015-06-11T19:08:12Z (GMT). No. of bitstreams: 1 Dissertação - Tatiana Amaral Pires de Almeida.pdf: 3233406 bytes, checksum: e04eb0576f711f1ed5bbe73ae78b4932 (MD5) Previous issue date: 2010-08-20 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / The Herpes Simplex Virus type 2 (HSV-2) is a widespread, incurable agent that establishes latency in nervous cells and is responsible for the majority of genital herpes cases. The UL23 is a polymorphic gene, which encodes the viral thymidine kinase (TK) of the Herpes virus. TK has six conserved regions, including two active sites, which plays an important role in acyclovir (ACV) metabolism. Therefore, some mutations in UL23 are implicated with ACV resistance phenoty pe. In this work, we analyzed 67 samples collected between Ap ril 2008 and September 2009 at the STI/STD clinic of “Alfredo da Matta” Foundation, in Manaus, Amazonas, Brazil obtained from HSV-2 genital ulcer patients. In order to characterize polymorphisms, all sp ecimens were processed for total nucleic acid isolation, submitted to PCR amplification targeting the complete ORF of UL23 gene followed by automated nucleotide sequencing with dideoxy chain terminators. Sequences analysis shown that 45 sequences were 100% similar to the reference strain “HG52”. The remaining 22 sequences (32.8%) showed at least one nucleotide substitution; none insertions or deletions were found. Amino acids substitutions were detected in 19 out of 22. The mutation Gly39Glu was the most frequently detected (16/19), followed by Asn78Asp (5/19); Gly39Glu and Asn78Asp were simultaneously found in four samples. Deduced amino acids sequences were aligned with 66 available on GenBank from African and American continents. A total of 23 variable sites were identified and, once more, Gly39Glu mutation was the most frequent found (74/133). Substitutions previously associated with ACV resistance were not detected, however, Gly39Glu was recently described as a probable resistant mutation. Another four mutations observed: His4Tyr, Met70Arg, Asn245Ser and Ala366Val had not been previously described. The theoretical models of TK’s structure built for seven samples showed that amino acids substitutions were found away from the conserved and/or catalytic sites of TK, with the exception of Arg338Gln which was nearly from ATP-binding site. Mutations associated with ACV resistance were not detected. However one of the mutations found, Gly39Glu, was p reviously associated with this resistance phenoty pe. Another four mutations found in his work: His4Tyr, Met70Arg, Asn245Ser and Ala366Val had not been previously described elsewere. To our knowledge, this is the first study on UL23 polymorphisms on Brazilian HSV-2 samples, a research field considered as a priority by WHO HIV/STI program. / latência em células nervosas sendo responsável pela maioria dos casos de herpes genital. O UL23 é um gene polimórfico que codifica a timidinaquinase viral (TK) do Herpesvírus. A TK possui seis regiões conservadas, incluindo dois sítios ativos; esta enzima desempenha um papel importante no metabolismo do aciclovir (ACV). Consequentemente, algumas mutações no gene UL23 estão relacionadas a fenótipos de resistência ao ACV. Nesta dissertação, analisamos 67 amostras coletadas entre Abril de 2008 e Setembro de 2009 na clínica de DST/IST da Fundação “Alfredo da Matta”, em Manaus, Amazonas, Brasil, obtidas de pacientes com úlceras genitais por HSV-2. Com o objetivo de se caracterizar polimorfismos, todos os espécimes foram processados para extração total de ácido nucleico e submetidos à amplificação da CDS do gene UL23 pela técnica de PCR, seguida de sequenciamento nucleotídico automatizado utilizando-se terminadores dideóxi. As análises de sequências mostraram que 45 delas foram 100% similares à cepa-referência “HG52”. As 22 sequências restantes (32,8%) mostraram pelo menos uma substituição de nucleotídeo; nenhuma inserção ou deleção foi encontrada. Substituições de aminoácidos foram detectadas em 19 dessas 22 sequências. A mutação Gly39Glu foi a mais frequente (16/19), seguida pela Asn78Asp (5/19); Gly39Glu e Asn78Asp foram encontradas simultaneamente em quatro amostras. Sequências deduzidas de aminoácidos foram alinhadas com 66 sequências Africanas e Americanas disponíveis no GenBank. Um total de 23 sítios variáveis foram identificados e, mais uma vez, a mutação Gly39Glu foi a mais frequente (74/133). Substituições previamente associadas à resistência ao ACV não foram detectadas, entretanto, a Gly39Glu foi recentemente descrita como uma provável mutação de resistência. Outras quatro mutações observadas, His4Tyr, Met70Arg, Asn245Ser e Ala366Val, foram descritas pela primeira vez por este trabalho. Nos modelos teóricos da estrutura da TK, construídos para sete amostras, foi possível visualizar que as substituições de aminoácidos estavam situadas longe dos sítios ativos/conservados desta enzima, com exceção da mutação Arg338Gln que apresentou maior proximidade com o sítio de ligação de ATP. Não foram detectadas mutações sugestivas de resistência ao ACV. Ao nosso conhecimento, este é o primeiro estudo de polimorfismos do gene UL23 do HSV-2 em amostras brasileiras, um campo considerado prioridade pelo Programa de HIV/IST da OM S.

DNA damage response (DDR) pathways are essential in maintaining genomic integrity in cells, but many DDR proteins have other important functions such as in the innate immune sensing of cytoplasmic DNA. Some DDR proteins are known to be beneficial or restrictive to viral infection, but most remain uncharacterised in this respect. Non-homologous end joining (NHEJ) is a mechanism of double stranded DNA (dsDNA) repair that functions to rapidly mend broken DNA ends. The NHEJ machinery is well characterised in the context of DDR but recent studies have linked the same proteins to innate immune DNA sensing and, hence, anti-viral responses. The aim of this thesis is to further investigate the interplay between herpes simplex virus 1 (HSV-1), a dsDNA virus, and two NHEJ proteins, DNA protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX). PAXX was first described in the literature as a NHEJ protein in 2015, but whether it has any role in the regulation of virus infection has not been established. Here we show that PAXX acts as a restriction factor for HSV-1 because PAXX-/- (KO) cells produce a consistently higher titre of HSV-1 than the respective wild type (WT) cells. We hypothesised that this could be due to a role of PAXX binding viral DNA and directly inhibiting HSV-1 replication or activating an anti-viral innate immune response. We have been able to, at least partially, rule out both of these initial hypotheses by showing that there was a reduced number of viral genomes present in KO cells during active lytic infection, and that an identical level of type I interferons are produced from WT and KO cells during HSV-1 infection. Although further characterisation of HSV-1 infection in WT and KO cells has not defined the molecular mechanism of restriction of HSV-1 by PAXX, we have uncovered a potential role for PAXX in mitogen-activated protein kinase (MAPK) signalling. In addition, and consistent with its function in restriction of HSV-1 infection, we show that infection with this virus in WT cells induces a loss of nuclear PAXX protein. Preliminary data suggest that these changes in localisation may occur as a result of stimulation of the cells with DNA, but not the RNA analogue poly(I:C). The role of PAXX in the regulation of HSV-1 infection in vivo was investigated by studying KO mice. Despite previous observations that mice lacking NHEJ proteins have brain defects related to autoinflammatory pathology, there were no obvious defects in the development of Paxx-/- mice, and they had brains of normal weight. No significant difference in viral spread or viral protein expression was observed between WT and KO HSV-1 infected mice, and KO mice did not exhibit abnormal pathology. There were, however, small but significant differences in the cellular immune response to infection which might be explained by reduced MAPK signalling in KO cells. DNA-PKcs is another component of the NHEJ machinery that acts to assist in dsDNA break repair in the nucleus and as an innate sensor of cytoplasmic viral DNA, but the effect of DNA-PKcs on HSV-1 infection has not been fully explored. Murine skin fibroblasts (MSFs) derived from wild type and PRKDC-/- (DNA-PKcs deficient) mice were cultured ex vivo and used for innate immune studies. Although HSV-1 was able to infect and stimulate these cells, no differences in the stimulation of innate immune gene expression between the two genotypes was observed, suggesting that DNA-PKcs does not contribute to HSV-1 sensing in MSFs. It has previously been reported that the HSV-1 protein ICP0 targets DNA-PKcs for degradation, although the reason for this is unknown. We confirmed these data, although found it to be cell-type specific and explored this interaction further using PRKDC-/- RPE-1 cells created using CRISPR/Cas9. HSV-1 infection in these cells followed unusual dynamics, and the development of cytopathic effect was accelerated as compared to WT cells. Together these observations confirm that DNA-PKcs regulates HSV-1 infection, but more work is required to fully understand the mechanisms involved.

The egress pathway of herpes simplex virus-1 (HSV-1) is a complicated process mediated by co-ordinated activity of several virus glycoproteins. The virions are first assembled and enveloped at trans-Golgi-network (TGN) or endosome membranes and then travel through a guided pathway that is directed towards the cell adherent points for secretion. Once secreted the vast majority of virions remain associated with the extracellular membrane of cells and very few free virions are released into the culture medium (< 1%). The mechanisms that mediate both the targeted secretion of newly assembled virions at cell contact points and post-secretion attachment of virions with the extracellular surface of cells are poorly understood, and were the topics of this research. In this thesis, an HSV-1 passage mutant of increased virion secretion phenotype had been studied. Genome sequencing of the mutant virus identified mutations in three viral envelope proteins. Study of recombinant viruses that were constructed based on those three mutations revealed that a single amino acid change in glycoprotein I (gI) of glycine to arginine at residue 39 is responsible for the increased release of virus. The result suggests the principal effect of this mutation is to modify the secretory pathway used by virions during their release from infected cells. Data also suggests a role of gC in the attachment of virions to the extracellular surface of cells after egress. In the context of HSV-1 envelopment and egress glycoprotein E (gE), which forms a heterodimeric complex with gI (gE/gI), is known to be important. The gE/gI complex has been shown to interact with many tegument proteins and have a redundant role in secondary envelopment. The gE/gI complex has been also proposed to colocalise with various cellular components and sort the nascent virions to cell contact points. However, there is little understanding of the cellular proteins that gE/gI interact with, or the mechanisms that mediate targeted secretion of virions. This research has identified a novel interactome of gE/gI by mass-spectrometric analysis utilising stable isotope labelling with amino acids in cell culture (SILAC) medium. Among the cellular interactome obtained, Nipsnap1 was validated by co-precipitation assays from both infected and transfected cells, and furthermore using cell free systems, suggesting gE and Nipsnap1 directly interact. Nipsnap1 and its homologue Nipsnap2 have been proposed to contribute in vesicle transport and membrane fusion in cells. Using CRISPR-Cas9 technology these proteins were knocked out in a keratinocyte cell line (HaCaT) to investigate their role in HSV-1 egress. However, little or no effect on HSV-1 egress could be observed upon loss of either or both of these proteins suggesting the biological significance of gE-Nipsnap1 interaction may not be directly linked to any egress function of gE/gI. Two further interesting 'hits' from the gE/gI interactome were interferon-induced transmembrane protein type-2 (IFITM2), a virus restriction factor, and Myoferlin that has a putative role in endocytic vesicle recycling. This study could validate gE-Myoferlin interaction and co-localisation in infected or transfected cells however, functional significance of this interaction remains to be determined. Overall, the research of this thesis has provided a better understanding of the role of the gE/gI complex in HSV-1 egress and investigated the role of some interesting cellular proteins in the context of virion egress.

The herpesvirus life cycle involves a step where newly formed capsids leave the nucleus by translocating across the intact nuclear envelope (NE). Little is known about the role of cellular factors during nuclear egress. We sought to identify novel cellular proteins that interact with the conserved herpes simplex virus-1 (HSV-1) pUL34 by performing a yeast two-hybrid screen. pUL34 was chosen due to its crucial and multifunctional role during nuclear egress. From 42 cellular factors that interacted with pUL34 in yeast, twelve were further evaluated in mammalian cells by co-localization studies using immunofluorescence. No specific co-location between the tested cellular factors and pUL34 was observed in infected cells, thus the screen failed to convincingly identify novel pUL34 interactors. In the second part of the thesis we addressed the functional significance of the cellular protein torsinA (TA) in the HSV-1 life cycle. We became interested in TA due to its role in maintaining normal NE morphology. We showed that perturbing the normal function of TA through overexpression impaired HSV-1 replication and caused a defect in capsid nuclear egress. In mouse embryonic fibroblasts that failed to express TA (TA-/-MEFs), HSV-1 replication was also inhibited, but a defect in capsid nuclear egress was not apparent. Strikingly, infection in TA-null MEFs induced a NE breakdown, the extent of which was dependent on viral products involved in nuclear egress. The viral growth defect and NE envelope breakdown, however, seem to be TA-null cell line specific rather than a functional consequence of TA loss as indicated by TA-/-MEFs reconstituted with TA and 293T with reduced TA levels. In conclusion, overexpression and loss of TA have different effects on the HSV-1 life cycle.