L'établissement de latence du virus de l'Herpès simplex 1 (HSV1) est contrôlé par les corps nucléaires PML (PML-NBs) mais leur implication exacte reste encore confuse. Une des caractéristiques majeures de la latence du virus est l'interaction entre le génome viral et les PML-NBs formant des structures nommées viral DNA-containing PML-NBs (vDCP-NBs). L'utilisation d'un modèle d'infection de fibroblastes primaires humains, qui reproduit la formation des vDCP-NBs, combinée à une approche par immuno-FISH, a permis de montrer que les vDCP-NBs contiennent l'histone H3.3 et ses chaperons, les complexes DAXX-ATRX et HIRA. La protéine HIRA a été également observé au sein des vDCP-NBs dans les neurones des ganglions trijumeaux de souris infectées par HSV1. Des expériences de ChIP-qPCR dans des cellules exprimant H3.3 ou H3.1 tagguées, nous a permis de déterminer que le génome viral est spécifiquement chromatinisé avec l'histone H3.3. La déplétion d'une seule protéine des complexes chaperons de H3.3 affecte légèrement l'incorporation de H3.3 dans les génomes viraux latents. Au contraire, l'absence de PML diminue significativement la chromatinisation H3.3 du génome HSV-1 latent sans remplacement par H3.1. Cette étude démontre une régulation épigénétique du génomes HSV1 latent par une chromatinisation dépendante de H3.3 impliquant les complexes chaperons DAXX-ATRX et HIRA. De plus, cette étude révèle un rôle majeur des PML-NBs dans la chromatinisation H3.3 des génomes HSV1 latent / Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by PML nuclear bodies (PML-NBs) although their exact implication is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML-NBs leading to the formation of viral DNA-containing PML-NBs (vDCP-NBs). Using a replication defective HSV-1 infected human primary fibroblast model reproducing the formation of vDCP-NBs, combined with an IF-FISH approach developed to detect latent HSV-1, we show that vDCP-NBs contain both histone H3.3 and its chaperone complexes, i.e. the DAXX/ATRX and the HIRA complex. HIRA was also detected co-localizing with vDCP-NBs present in trigeminal ganglia neurons from HSV-1 infected WT mice. ChIP-qPCR performed on fibroblasts stably expressing tagged H3.3 or H3.1 show that latent HSV1 genomes are chromatinized almost exclusively with H3.3. Depletion of single proteins from the H3.3 chaperone complexes only mildly affects H3.3 deposition on the latent HSV1 genome. In contrast, absence of PML significantly impacts on the chromatinization of the latent genomes with H3.3 without replacement with H3.1. Consequently, the study demonstrates a specific epigenetic regulation of latent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving both H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML-NBs are major actors of the latent HSV-1 H3.3 chromatinization through a PML-NBs/histone H3.3/H3.3 chaperones axis

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Department of Microbiology / PhD (Microbiology) / Human herpes virus type 8 (HHV-8), also known as Kaposi’s sarcoma associated herpes virus (KSHV), is the etiologic agent of Kaposi’s sarcoma (KS), and AIDS related Kaposi’s sarcoma (AIDS-KS). HHV-8 which is a member of the Herpesviridae family, exhibits extensive genetic diversity globally. In endemic regions, infection with HHV-8 occurs very early on in life, which is an indication of both environmental and vertical routes of transmission. The advent of HIV leads to the classification of an AIDS-KS defining condition in HIV infections. This suggests that in regions where HIV and HHV-8 are endemic, KS may become common in a mature HIV epidemic. Just like the prevalence of HIV in Northern South Africa is generally high as in most regions of the country, as the HIV epidemic matures in South Africa, it is important to understand the burden and distribution of HHV-8 infection, and the likely genotypes infecting the population. The main objective of the thesis was to establish the epidemiology and infecting genotypes of HHV-8 in Northern South Africa (Limpopo Province), where no data exists. First, a systematic review of the literature was carried out for the entire African continent to determine the seroprevalence and genotype distribution of HHV-8 in all African countries (n=53). In this review, Sudan and South Sudan were considered as one country. Articles were searched using the PRISMA guideline and exported using an article grid. More than two-thirds (64%) of the studies reported on seroprevalence, 29.3% on genotypes; and 9.5% were on both seroprevalence and genotypes. About 45% (24/53) of the African countries had data on HHV-8 seroprevalence exclusively, and more than half (53%) had data on either seroprevalence or genotypes. Almost half (47%) of the countries had no data on HHV-8 infection. There was high heterogeneity in the types of tests and interpretation algorithms used in determining HHV-8 seropositivity across the different studies. Generally, seroprevalence ranged from 2.0% in a group of young children in Eritrea to 100% in a small group of individuals with KS in the Central Africa Republic and a larger group of KS in individuals in Morocco. Approximately, 16% of all the studies reported on children. The difference in seroprevalence across the African region was not significant (95% CI, X2 =0.86; p =0.35), although specifically, a relatively significant ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|VIII level of infection was observed in HIV-infected children. About 38% of the countries had data on K1 genotypes A, A5, B, C, F and Z which occurred at frequencies of 5.3%, 26.3%, 42.1%, 18.4%, 5.3% and 2.6% respectively. Twenty-three percent of the countries had data for K15 genotypes, whereas genotypes P, M and N occurred at frequencies of 52.2%, 39.1% and 8.7% respectively. Data on HHV-8 inter-genotype recombinant is scanty. Our finding suggests that HHV-8 is endemic on the entire African continent, and in HIV endemic regions, but there is need for a harmonized testing protocol for better understanding of HHV-8 seropositivity. HHV-8 genotype A5 and B for K1 gene and genotype P and M for K15 gene are the most predominant genotypes in Africa. The review, for the first time, has provided information on HHV-8 burden on the entire African continent, and suggests that vaccine development efforts for Africa should focus on genotypes B and P. The second component of the investigation focused on the burden of HHV-8 in an HIV population in Northern South Africa (Limpopo Province). Plasma from 3501 HIV infected individuals from 5 districts in Limpopo Province were assessed for antibodies to both the lytic antigen (ORFK8.1) and the latent antigen (ORF73). The distribution of infection was analyzed based on demographic, socioeconomic, and immunological parameters. Statistical inferences for significant differences were determined by Chisquare at a confidence interval of 95%. P-values less than 0.05 were considered significant. About 19.0% of the study population was positive for antibodies to either the lytic or latent antigens or both. Prevalence of antibodies to the lytic antigen was significantly higher than prevalence of antibodies to the latent antigen (17.3% vs 4.1%; p=0.0001). Significant differences were observed for age groups, racial population groups, districts and year of sample collection (p=<0.0001, p=<0.0001, p=<0.0001 and p=0.0385) respectively. Associations were found between both antigens in comparison to the different variables such as age group, racial population groups and districts (R2 value ranging between 0.886 and 1.0). The burden of HHV-8 has now been established for the first time in Northern South Africa. The third aspect of the investigation was a meta-analysis of HHV-8 seroprevalence in Southern Africa in order to understand the impact of geographical location (urban vs rural) on infection. The analysis revealed a significant association between urban settings and HHV-8 infection (p=0.0001). ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|IX The fourth component of the thesis examined the detection of HHV-8 antigen through polymerase chain reaction (PCR) in 534 participants in HIV infected and HIV noninfected populations. A selection of mouthwash DNA samples were subjected to Next Generation Sequencing (NGS) for subsequent genotype inference. Mouth wash samples were obtained from each consenting individual before eating or smoking, and their DNA was purified. A 233bp fragment of the ORF26 gene of HHV-8 was amplified by PCR. HHV-8 was detected in 150 of the 534 participants (28.1%). A significant difference in detection was observed for gender, HIV status, district and the level of education (p=0,0003; p=0.0094; p=0.0002 and p=0.0095) respectively. Consensus sequences were derived from NGS reads for 13 samples. The genotyping results revealed that genotype Q, B, E and N are the genotypes predominant in the study population. As such no mixed infections were detected. Therefore, from the investigations foregoing have demonstrated for the first time the following: (1) HHV-8 is endemic in the entire African continent, which suggest a coendemicity in regions already endemic for HIV; (2) HHV-8 is endemic in Northern South Africa; (3) Urban settings in Southern Africa are associated with high HHV-8 infection; (4) HHV-8 genotypes Q, B, E and N may be predominant in Northern South Africa, with B and P common on the entire African continent. Hence, studies should focus on the generation of full length HHV-8 genomes of the common genotypes to support the selection of genes for vaccine design and development. / NRF

Les infections virales en particulier celles dues aux virus de la famille des Herpesviridae pendant la période d’aplasie et de lymphopénie à la suite d’une greffe de cellules souches hématopoïétiques (GCSH) peuvent occasionner des complications très graves, souvent associées à une morbidité et mortalité élevées. Les recommandations cliniques actuelles préconisent l’utilisation des antiviraux pour la prévention de certaines de ces infections. L’efficacité du famciclovir et de l’acyclovir contre les virus de l’herpès simplex (HSV), le virus varicella-zoster (VZV) et l’herpésvirus humain de type 6 (HHV-6) est bien reconnue, cependant il nous manque des données quant à leur effet contre le virus Epstein-Barr (EBV) et le cytomégalovirus (CMV) dans la population pédiatrique. L’objectif principal de ce projet de maitrise a été de mesurer l’incidence de l’infection aux virus HSV, VZV, EBV, CMV et HHV-6 et de mesurer l’association entre l’utilisation de la prophylaxie antivirale (acyclovir et famciclovir) et l’infection (virémie asymptomatique et maladie) avec le CMV et l’EBV dans une cohorte pédiatrique de GCSH allogéniques. Les données d'une cohorte de sujets ayant subis pour la première fois une GCSH enrôlés dans quatre centres de greffes pédiatriques au Canada entre juillet 2013 et mars 2017 (Étude TREASuRE) ont été utilisées. Le recrutement a été effectué au : CHU Sainte-Justine (Montréal) (n=86), British Columbia Children’s Hospital (Vancouver) (n=31), Winnipeg Children's Hospital and CancerCare Manitoba (n=28) et Alberta Children’s Hospital (n=11). Le suivi des patients avait débuté 1 mois avant la greffe et avait duré 13 mois. L’âge médian des patients au recrutement était de 6,3 ans. Les courbes de Kaplan-Meier ont permis d’estimer l'incidence cumulée des infections CMV et EBV avec intervalle de confiance (IC) à 95% à 100 jours post-greffe en fonction de la prophylaxie antivirale (acyclovir ou famciclovir). Les modèles multivariés de régression de Cox à risques proportionnels ont permis de mesurer l'association entre la prise d’antiviraux (acyclovir ou famciclovir) et le développement de ces infections. L’étude a inclus 156 sujets âgés de 0 à 21 ans. Les incidences cumulées de la virémie des virus de HSV, VZV, EBV, CMV et HHV-6 à 100 jours de suivi ont été respectivement de 2.5% (IC 95% : 0.8–7.6), 0.8% (IC 95% : 0.1–6.1), 34.5% (IC 95% : 27.6–42.6), 19.9% (IC 95% : 14.5-27.1) et 3.4% (IC 95% : 1.2–9.1). Les incidences cumulées pour CMV et EBV n’ont pas montré de différence statistiquement significative entre les groupes ayant reçu la prophylaxie antivirale (acyclovir ou famciclovir) et ceux qui ne l’ont pas reçu. Les analyses de Cox n’ont montré aucun effet significatif des antiviraux sur le CMV avec un HR ajusté de 0.55 (IC 95% : 0.24–1.26) pour l’acyclovir et de 0.82 (IC 95% : 0.30–2.29) pour le famciclovir. Il en était de même pour l’EBV avec un HR ajusté de 1.41 (IC 95% : 0.63–3.14) pour l’acyclovir et de 0.79 (IC 95% : 0.36–1.72) pour le famciclovir. Notre étude n’a montré aucune preuve d’effet de la prophylaxie antivirale avec le famciclovir et l’acyclovir contre l’EBV et le CMV. Très peu de cas de HSV et de VZV ont été diagnostiqués dans cette cohorte ce qui est conforme avec l’idée selon laquelle l’acyclovir et le famciclovir sont efficaces pour ces virus. / Viral infections, especially those involving members of the Herpesviridae during the period of aplasia and lymphopenia following allogeneic hematopoietic stem cell transplantation (HSCT), cause very serious complications, often associated with high morbidity and mortality. Current clinical guidelines recommend prophylactic use of antivirals, which has proven to be effective against certain viruses. The efficacy of famciclovir and acyclovir against herpes simplex viruses (HSV), varicella zoster virus (VZV) and human herpesvirus type 6 (HHV-6) is well-recognized, however, we lack data on their effects against Epstein-Barr virus (EBV) and cytomegalovirus (CMV) in the pediatric population. The main objective of this master's project was to measure the incidence of herpes virus infection, specifically by HSV, VZV, EBV, CMV and HHV-6, and to measure the association between the use of antiviral prophylaxis (acyclovir and famciclovir) and infection (including both asymptomatic viremia and disease) by CMV and EBV in a pediatric cohort of allogeneic HSCT. We used data from the TREASuRE cohort, which includes patients enrolled for a first allogeneic HSCT in four pediatric centers in Canada between July 2013 and March 2017. Recruitment was carried out at: CHU Sainte-Justine (Montreal) (n = 86), British Columbia Children's Hospital (Vancouver) (n = 31), Winnipeg Children's Hospital and CancerCare Manitoba (n = 28) and Alberta Children's Hospital (n = 11). Patient follow-up began 1 month before transplant and lasted 13 months. Median patient age at recruitment was 6.3 years. Kaplan-Meier curves were used to estimate the cumulative incidence of CMV and EBV infections with 95% confidence interval (CI) at 100 days post-transplant according to antiviral prophylaxis (acyclovir or famciclovir). Multivariate proportional hazards Cox regression models were used to measure the association between antiviral use (acyclovir or famciclovir) and the detection of these infections. The study included 156 subjects aged 0 to 21 years. The cumulative incidences of viremia due to HSV, VZV, EBV, CMV and HHV-6 at day 100 of follow-up were respectively 2.5% (CI 95%: 0.8–7.6), 0.8% (CI 95%: 0.1-6.1), 34.5% (CI 95%: 27.6-42.6), 19.9% (CI 95%: 14.5-27.1) and 3.4% (95% CI: 1.2-9.1). The cumulative incidences for CMV and EBV did not show a statistically significant difference between the groups who received antiviral prophylaxis (acyclovir or famciclovir) and those who did not. Cox analyses showed no significant effect of antivirals on CMV with an adjusted HR of 0.55 (95% CI: 0.24–1.26) for acyclovir and 0.82 (95% CI: 0.30–2.29) for famciclovir. The same was true for EBV with an adjusted HR of 1.41 (95% CI: 0.63–3.14) for acyclovir and 0.79 (95% CI: 0.36–1.72) for famciclovir. Our study showed no evidence of an effect with use of famciclovir or acyclovir prophylaxis on EBV and CMV infections. Very few cases of HSV and VZV infections were diagnosed in this cohort, which is consistent with the idea that acyclovir and famciclovir are effective against the latter viruses.