Dissertations / Theses on the topic 'Viruses transmission'

Insects are host to a diverse range of vertically transmitted micro-organisms, but while their bacterial symbionts are well-studied, little is known about their vertically transmitted viruses. The sigma virus (DMelSV) is currently the only natural hostspecific pathogen to be described in Drosophila melanogaster. In this thesis I have examined; the diversity and evolution of sigma viruses in Drosophila, their transmission and population dynamics, and their ability to host shift. I have described six new rhabdoviruses in five Drosophila species — D. affinis, D. obscura, D. tristis, D. immigrans and D. ananassae — and one in a member of the Muscidae, Muscina stabulans (Chapters two and four). These viruses have been tentatively named as DAffSV, DObsSV, DTriSV, DImmSV, DAnaSV and MStaSV respectively. I sequenced the complete genomes of DObsSV and DMelSV, the L gene from DAffSV and partial L gene sequences from the other viruses. Using this new sequence data I created a phylogeny of the rhabdoviruses (Chapter two). The sigma viruses form a distinct clade which is closely related to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they may deserve to be recognised as a new genus. Furthermore, this analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing me to reconstruct the major transitions that have occurred during the evolution of the family. This data suggests that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects. In Chapter three I examined whether the new sigma viruses in Drosophila affinis and Drosophila obscura are both vertically transmitted. As is the case for DMelSV, both males and females can transmit these viruses to their offspring. Males transmit lower viral titres through sperm than females transmit through eggs, and a lower proportion of their offspring become infected. I then examined natural populations of D. obscura in the UK; 39% of flies were infected and the viral population shows clear evidence of a recent expansion, with extremely low genetic diversity and a large excess of rare polymorphisms. Using sequence data I estimate that the virus has swept across the UK within the last ~11 years, during which time the viral population size doubled approximately every 9 months. Using simulations based on lab estimates of transmission rates, I show that the biparental mode of transmission allows the virus to invade and rapidly spread through populations, at rates consistent with those measured in the field. Therefore, as predicted by the simulations, the virus has undergone an extremely rapid and recent increase in population size. In Chapter four I investigated for the first time whether vertically transmitted viruses undergo host shifts or cospeciate with their hosts. Using a phylogenetic approach I show that sigma viruses have switched between hosts during their evolutionary history. These results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts. In Chapter five I examined the ability of three Drosophila sigma viruses to persist and replicate in 51 hosts sampled across the Drosophilidae phylogeny. I used a phylogenetic mixed model to account for the non-independence of host taxa due to common ancestry, which additionally allows integration over the uncertainty in the host phylogeny. In two out of the three viruses there was a negative correlation between viral titre and genetic distance from the natural host. Additionally the host phylogeny explains an extremely high proportion of the variation (after considering genetic distance from the natural host) in the ability of these viruses to replicate in novel hosts (>0.8 for all viruses). There were strong phylogenetic correlations between all the viruses (>0.65 for all pairs), suggesting a given species’ level of resistance to one virus is strongly correlated with its resistance to other viruses. This suggests the host phylogeny, and genetic distance from the natural host, may be important in determining viruses ability to host switch. This work has aimed to address fundamental questions relating to host-parasite coevolution and pathogen emergence. The data presented suggests that sigma viruses are likely to be widespread vertically transmitted insect viruses, which have dynamic interactions with their hosts. These viruses appear to have switched between hosts during their evolutionary history and it is likely the host phylogeny is a determinant of such host shifts.

Pandemic influenza viruses are responsible for many deaths and much suffering as well as large costs accumulated through healthcare provision and lost productivity. The swine-origin H1N1 influenza virus that was at the root of the pandemic in 2009, although considered mild, in the UK alone caused the deaths of 361 people, subjected 1,700 to critical care and led to hospital admission of 7,879, before summer 2010. This virus is now embedded in the human population and will remain there, inducing further mortality, morbidity, and cost until it is replaced by a new pandemic influenza virus and the cycle repeats. However, if we improve our understanding of the characteristics of virus/host interactions that support transmission, we may be able to identify potentially problematic strains, improve our preparedness and ultimately prevent future pandemics. The ability of an influenza virus to transmit from host to host is multifactorial. One role proven to be important because its inhibition by drugs prevents replication is the ability of neuraminidase (NA) to cleave sialic acid (SA). NAs of recently circulating influenza viruses were analysed with the aim of identifying characteristics that might support new or previous pandemic influenza virus strains. NA stalk truncation, induced by influenza virus transmission through gallinaceous poultry, was found to hinder transmission through the air of an otherwise transmissible virus. While this genetic ablation did not directly affect sialidase activity, when measured using a small soluble substrate, it did reduce the ability of virions to release complex tethered substrates, separate from one another and penetrate through mucus. This work suggests that the next pandemic virus is unlikely to have a short stalk NA. Previously, loss of the ability of NA to haemabsorb (Hb) chicken red blood cells (CRBSs) was associated with the adaptation of an influenza virus from avian to human hosts and a single amino acid mutation was shown to reinstate the haemabsorption ability of an NA from a prototypic pandemic H2N2 virus of avian-origin. This thesis shows that the human-adapted 2009 pandemic N1 NA, which arose from the introduction of an avian influenza virus into swine, did not Hb CRBCs, as expected. However, unexpectedly, many back mutations to make the 2009 N1 resemble an NA that did Hb also did not alter this characteristic. Further investigations showed that some NAs from both avian and swine influenza viruses also did not Hb, despite the fact that NA haemabsorption increased viral growth in airway culture cells. Thus we conclude that loss of Hb occurs within birds, before a virus can cross the species barrier into mammals and this may be due to a reduction, rather than a gain, in activity.

Viruses cause 60% of human infections and are probably the most common cause of infectious disease acquired indoors. Rapid spread of viral illness in indoor establishments facilitates disease morbidity and mortality. The goal of this dissertation is to clarify the role of fomites in the viral infection cycle. Research methods include investigation of published literature, and the use of polymerase chain reaction (PCR) for viral detection. The Appendix A study reviewed published literature to assess the significance of fomites in the transmission of ten common respiratory and enteric viruses (rhinovirus, respiratory syncytial virus (RSV), influenza A, parainfluenza 1 (HPIV1), coronavirus, rotavirus, calicivirus, hepatitis A virus (HAV), astrovirus and adenovirus). Results suggest that fomites play an important role in the transmission of common viral pathogens, and the use of disinfectants may limit the spread of viral disease. The Appendix B study examined PCR primer detection limits by determining the time length viruses can be isolated on fomites. Results indicated that poliovirus 1 and hepatitis A virus could be detected for up to 60 days. Parainfluenza 1 virus isolation yielded detection at 30 days and 50 days. Norovirus isolation yielded detection at 20 days and 30 days. Influenza virus isolation results were inconsistent, yielding no initial detection and detection up to 20 days. Appendix C assessed the occurrence of human parainfluenza 1 virus (HPIV1) on surfaces in office settings. HPIV1 was detected on 37% of fomites. HPIV1 was detected most on desktops (47%), and least on light switches (19%). Study results indicated a statistically significant difference between positive fomites in different buildings (Chi-square p < 0.011), and between building cubicles and conference room fomites (Chi-square p < 0.011). Appendix D evaluated the prevalence of influenza A virus on surfaces in day care and home settings. Influenza A was isolated on 23% of fall day care fomites and 53% of spring day care fomites. Influenza was isolated on 59% of home fomites sampled during March, and no influenza was detected on home fomites sampled during the summer. Overall, Influenza A virus was isolated on over 50% of fomites in homes and day care centers.

Introduction: Respiratory Syncytial Virus is the major viral cause of lower respiratory tract disease in young children worldwide, with the greatest burden of disease in infants aged 1-3 months. Consequently, vaccine development has centered on a vaccine to directly protect the infants in this age group. The fundamental problem is that these young infants are poor responders to candidate RSV vaccines. This thesis focuses on the use of mathematical models to explore the merits of vaccination. Methods: Following development and analysis of a simple non-age-structured ODE model, we elaborate this to a Realistic Age Structured model (RAS) capturing the key epidemiological characteristics of RSV and incorporating age-specific vaccination options. The compartmental ODE model was calibrated using agespecific and time series hospitalization data from a rural coastal Kenyan population. The determination of Who Acquires Infection From Whom (WAIFW) matrix was done using social contact data from 1) a synthetic mixing matrix generated from primarily household occupancy data and 2) a diary study that we conducted in the Kilifi Health and Demographic Surveillance System (KHDSS). The vaccine was assumed to elicit partial immunity equivalent to wild type infection and its impact was measured by the ratio of hospitalized RSV cases after to before introduction. of vaccination. Uncertainty and sensitivity analysis were undertaken using Latin Hypercube Sampling (LHS) and partial rank correlation respectively. Given the importance of households in the transmission of respiratory infections, an exploratory household model was developed to capture the transmission dynamics of RSV A and B in a population of households. Results: From the analytical work of the simple ODE model, we have demonstrated that the model has the potential to exhibit a backward bifurcation curve within realistic parameter ranges. Both the diary and the synthetic mixing matrices had similar characteristics i.e. strong assortative mixing in individuals less than 30 years old and strong mixing between children less than 5 years and adults between 20 and 50 years old. When the two matrices were jointly linearly regressed, their elements were well correlated with an R2 ~ 0.6. The RAS model was capable of capturing the age-specific disease and the temporal epidemic nature of RSV in the specified location. Introduction of routine universal vaccination at ages varying from the first month of life to the 10th year of life resulted in optimal long-term benefit at 7 months (for the diary contact model) and 5 months (for the synthetic contact model). The greatest benefit arose under the assumption of age-related mixing with the contact diary data with no great deal of effectiveness lost when the vaccine is delayed between 5 and 12 months of age from birth. Vaccination was also shown to change the temporal dynamics of RSV hospitalizations and also to increase the average age at primary infection. From the sensitivity analysis, we identified the duration of RSV specific maternal antibodies, duration of primary and tertiary infections as the most important parameters in explaining the imprecision observed in predicting both the age specific hospitalizations and the optimal month at vaccination. Results from the household model have demonstrated that the household epidemic profile may be different from the general population with strong interaction of the viruses in the household that do not necessarily reflect at the population level. Conclusion: The synthetic matrix method would be a preferable alternative route in estimating mixing patterns in populations with the required socio-demographic data. Retrospectively, the synthetic mixing data can be used to reconstruct contact patterns in the past and therefore beneficial in assessing the effect of demographic transition in disease transmission. Universal infant vaccination has the potential to significantly reduce the burden of RSV associated disease, even with delayed vaccination between 5 and 12 months. This age class represents the group that is being targeted by vaccines that are currently under development. More accurate data measuring the duration of RSV specific maternal antibodies and the duration of infections are required to reduce the uncertainty in the model predictions.

Kyoto University (京都大学)
0048
新制・課程博士
博士(人間・環境学)
甲第7910号
人博第53号
10||135(吉田南総合図書館)
新制||人||14(附属図書館)
UT51-99-G504
京都大学大学院人間・環境学研究科人間・環境学専攻
(主査)教授 速水 正憲, 教授 中村 榮太郎, 教授 丸山 圭蔵
学位規則第4条第1項該当

Influenza is a widespread disease occurring in seasonal epidemics, and each year is responsible for up to 500,000 deaths worldwide. Influenza can develop into strains which cause severe symptoms and high mortality rates, and could potentially reach pandemic status if the virus’ properties allow easy transmission. Influenza is transmissible via contact with the virus, either directly (infected people) or indirectly (contaminated objects); via reception of large droplets over short distances (one metre or less); or through inhalation of aerosols containing the virus expelled by infected individuals during respiratory activities, that can remain suspended in the air and travel distances of more than one metre (the aerosol route). Aerosol transmission of viruses involves three stages: production of the droplets containing viruses; transport of the droplets and ability of a virus to remain intact and infectious; and reception of the droplets (via inhalation). Our understanding of the transmission of influenza viruses via the aerosol route is poor, and thus our ability to prevent a widespread outbreak is limited. This study explored the fate of viruses in droplets by investigating the effects of some physical factors on the recovery of both a bacteriophage model and influenza virus. Experiments simulating respiratory droplets were carried out using different types of droplets, generated from a commonly used water-like matrix, and also from an ‘artificial mucous’ matrix which was used to more closely resemble respiratory fluids. To detect viruses in droplets, we used the traditional plaque assay techniques, and also a sensitive, quantitative PCR assay specifically developed for this study. Our results showed that the artificial mucous suspension enhanced the recovery of infectious bacteriophage. We were able to report detection limits of infectious bacteriophage (no bacteriophage was detected by the plaque assay when aerosolised from a suspension of 103 PFU/mL, for three of the four droplet types tested), and that bacteriophage could remain infectious in suspended droplets for up to 20 minutes. We also showed that the nested real-time PCR assay was able to detect the presence of bacteriophage RNA where the plaque assay could not detect any intact particles. Finally, when applying knowledge from the bacteriophage experiments, we reported the quantitative recoveries of influenza viruses in droplets, which were more consistent and stable than we had anticipated. Influenza viruses can be detected up to 20 minutes (after aerosolisation) in suspended aerosols and possibly beyond. It also was detectable from nebulising suspensions with relatively low concentrations of viruses.

Thesis (Ph. D.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Virginia-Maryland Regional College of Veterinary Medicine. Maryland Campus. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Insect and rodent samples were collected from suspected VSV-NJ enzootic areas over 2 consecutive field seasons (1996-1997) and from southern Arizona only during 1998. Insect samples were screened for arboviruses, and rodent sera were tested for the presence of VSV-NJ and VSV-IN neutralizing antibodies. Vesicular stomatitis virus New Jersey serotype was isolated from a pool of Culicoides sp. collected in 1997 near Belen, New Mexico. All rodent sera were negative for specific VSV-NJ and VSV-IN antibodies. Genetic analysis of the hypervariable region of the phosphoprotein gene demonstrated that the 1997 Belen VSV-NJ isolate was more closely related to viruses isolated from livestock during the 1982-83 western U.S. epizootic than to other VSV-NJ isolates. This suggests that VSV-NJ may be enzootic in the western U.S. Simulium vittatum was shown to be a competent vector of VSV-NJ. Virus-infected females were allowed to feed on laboratory mice and on deer mice. All laboratory mice seroconverted by day 21 post-exposure. Neutralizing antibody titers increased from an average of 1:4 at baseline to >1:1,024 on day 21. An age-related effect on viral pathogenesis was evident in Peromyscus maniculatus following VSV-NJ exposure by black fly bite. Lethal encephalomyelitis was evident in all 6-week-old mice, but in only one 6-month-old mouse. Peromyscus maniculatus did not meet the standard definition of a reservoir host for VSV-NJ because a viremia, was not detected. Nonetheless, P. maniculatus may play a role in virus maintenance since non-infected black flies became infected while co-feeding with infected black flies on the same non-viremic host. These results represent the first example of a western U.S. insect species becoming infected with VSV-NJ by feeding on a host. Simulium vinatum and S. notatum were shown to be competent laboratory vectors of VSV-IN. Saliva from experimentally infected Simulium vittatum and S. notatum was collected and tested for the presence of infectious virus. Virus was detected in the saliva of both species following oral infection. Independent experiments were conducted to determine if transovarial transmission of VSV-NJ and VSV-IN occurs in black flies. Transovarial transmission was not detected. Transstadial transmission of both virus serotypes was detected.

Powassan (POW) virus, the cause of human encephalitis in the northeastern U.S. and Canada, is transmitted by tick bite. Since the geographic and host distribution patterns of Ixodes dammini Spielman, et al, 1979 and POW virus overlap, the potential of this tick species to transmit POW virus was explored. Transmission experiments were conducted with hamsters and rabbits which fed immature and adult ticks, respectively, from a POW-free colony. Oral infection rates in larvae and nymphs fed on POW-infected hamsters were 10% and 40%, respectively; in females fed on POW-infected rabbits, 57%. Transstadial transmission rates for nymphs exposed to POW virus as larvae, adults exposed as larvae, and adults exposed as nymphs, were 9.5%, 10% and 54%, respectively. Evidence of transovarial transmission was acquired when 2 clean hamsters feeding F$ sb2$ larvae and nymphs originally exposed to virus in the F$ sb1$ nymphal stage seroconverted to POW virus with hemagglutination inhibition titers of 80 and 5120, respectively, on week 4 post-tick-drop-off. The transovarial transmission rate was 16.6%. All developmental stages were able to transmit POW virus orally to clean hosts regardless of when the ticks were originally exposed to virus.
These results indicate that I. dammini is a competent vector of POW virus under experimental conditions. Field studies are necessary to determine if the same holds true under natural conditions.

Norovirus is the commonest cause of viral gastroenteritis, affecting all age groups worldwide. Outbreaks frequently occur in semi-closed communities such as schools, cruise ships, prisons and hospitals. Within the healthcare environment, the economic and logistical burdens and the inconvenience caused by norovirus is significant, since ward closure remains central to infection control. The aim of this study was to investigate norovirus transmission dynamics during hospital outbreaks. The ultimate goal was to provide information that could, in future, lead to the development of novel, less disruptive approaches to curtailing the spread of infection. The study explored the application of 'next generation' high throughput DNA sequencing technologies to the determination of large numbers of norovirus genomes. Whole genome sequences provide the highest possible level of discrimination among viruses, information which is essential to the identification of linked and independent cases of infection. The approach exploits the high norovirus mutation rate, which is typical of RNA viruses. Consequently, viruses within a single ward which differ by more than a few SNVs can be considered to represent independent introductions, rather than a single outbreak. Whole genome sequence data (determined for noroviruses collected between 2009 and 2013) were combined with epidemiological data, providing further insights into transmission dynamics. These data identified multiple independent virus introductions during single ward outbreaks. The possible origin of such outbreaks in Oxfordshire hospitals were investigated using viruses originating in the local community, and in other healthcare environments distributed throughout the UK. Whole genome sequences of noroviruses from consecutive years were genetically divergent, confirming the rapid evolution of the virus over time and excluding the possibility of prolonged environmental contamination as a reservoir of infection. Such detailed information on norovirus transmission within the healthcare environment could inform alternative future approaches to optimising infection control within the healthcare setting.

The cacao swollen shoot disease of Theobroma cacao L. (cacao) is caused by Cacao swollen shoot virus (CSSV; genus, Badnavirus, family, Caulimoviridae). The virus is endemic to West Africa, where it poses a serious threat to cocoa production. Despite efforts to control CSSV spread by replacement of infected trees with tolerant cultivars and mealybug vector management, the disease is widespread in West Africa. In Trinidad, leaf mosaic and vein-banding symptoms have been observed in cacao plants in the field since the 1940s, and recently at the International Cocoa Genebank (ICGT), a custodian of cacao germplasm resources. The strains A and B of the suspect Cacao Trinidad virus (CTV) caused the symptoms, and were thought to be related to CSSV, however, viral causality was not demonstrated, until now. To develop molecular detection methods for CSSV in infected plants, polymerase chain reaction (PCR) amplification of eight regions of the CSSV genome was implemented. The PCR results showed variable amplification frequencies of 19 - 42% at each region, for 124 isolates collected in Cote d'Ivoire and Ghana. Pairwise nucleotide (nt) analyses of the eight regions showed 66-99% shared identities, indicating that CSSV isolates exhibit extensive variability with respect to primer design. The results provided preliminary evidence for the existence of a CSSV complex consisting of four divergent species. The full length genome of 14 CSSV isolates from cacao determined using the Illumina HiSeq platform showed 70-99% shared nt identities. The pairwise nt identities placed CSSV sequences into a group of four distinct species, one of which represented a previously undescribed species. Moreover, the full-length genomes grouped phylogenetically with other badnaviruses and revealed two CSSV subclades with three types of genome arrangements; four, five or six open reading frames (ORFs). Predicted functional protein domains were conserved on each ORF. Two distinct, full-length genome sequences were determined using the Illumina HiSeq platform, from DNA isolated from cacao leaves exhibiting distinct symptoms in Trinidad. The sequences were validated by PCR-amplification and sequencing of overlapping viral genome fragments. Pairwise nt analysis indicated that each genome shared 52-62% nt identities with CSSV and other badnaviruses, suggesting that the two are distinct species. Phylogenetic analysis indicated that the two sequences are not strains of the same virus, as supposed, but they represent two previously undescribed species in the genus, Badnavirus, and they have been named Cacao mild mosaic virus (CaMMV) and Cacao yellow-vein-banding virus (CYVBV). Despite sharing the same host and causing similar symptoms in cacao, CSSV, CaMMV, and CYVBV are phylogenetically-distinct species. The discovery of a CSSV species complex and the identification of three new cacao-infecting badnavirus species will support the development of molecular detection tools using the partial and complete genome sequences determined in this study. The ability to develop validated molecular tools for the detection of CSSV and related viruses, CaMMV and CYVBV, in cacao will aid quarantine efforts and safe movement of germplasm from the ICGT in Trinidad to cacao-growing countries, worldwide. Also, molecular diagnostics tools are expected to be useful in efforts underway to develop CSSV-resistant planting material for countries in West Africa, which are currently experiencing continued or new disease outbreaks.

At present the origins of major human pathogens associated with hepatic disease are poorly understood. The absence of such information pertaining to the evolutionary history of hepatitis B virus (HBV) and hepatitis C virus (HCV) and its genetically related viruses impacts upon the development of vaccines and effective eradication strategies. Studies are currently limited by the absence of historical samples from which to date the emergence of human infections and therefore the evolution of human hepatic viruses relies on epidemiological studies and genetic analysis of contemporary virus populations worldwide. Approximately one third of the world’s population is infected with HBV, and despite the availability of a vaccine, the virus is attributed with over 1 million deaths per year through liver disease. HBV variants infecting humans show genetic and antigenic heterogeneity and are currently classified into 8 genotypes A-H with nucleotide divergence of between 9-13%. In addition to these variants, recombination has been detected between genotypes A and D, and B and C, which can generate novel variants. The past 10 years has seen the detection of HBV in chimpanzees, gorillas and other non-human primates (NHPs) at frequencies comparable to those observed in regions of endemic human HBV infection. Despite the genetic divergence between human and NHP HBV variants the detection of recombination between human genotype C and chimpanzee and gibbon variants suggests that HBV can share hosts in nature. The evolutionary process that may have given rise to the distinct species-specific variants of NHP HBV within overlapping geographical regions has not been reconciled, with evidence supporting both allopatric speciation and co-speciation. HCV a member of the Flaviviridae family currently infects approximately 3% of the world’s population and is one of the major causes of chronic liver disease, hepatocellular carcinoma and liver cirrhosis. Human pegivirus (HPgV) a member of the Pegivirus genus of the Flaviviridae family infects approximately 5% of the world’s population, although it is of unknown disease association. Very recently, several studies of wild rodent and bat populations have revealed much greater viral diversity of members of both Hepacivirus and Pegivirus genera. Homologues of HCV have been detected in a range of species including domestic dogs (canine hepacivirus [CHV]) and horses (non-primate hepaciviruses [NPHV]). Similarly, several new pegiviruses have been described in horses (equine pegivirus, [EPgV] and Theiler’s Disease Associated Virus [TDAV]), several species of rodents (rodent pegivirus [RPgV]), and further species of bats (bat pegivirus, [BPgV]). Despite the differences in pathogenicity between HCV and HPgV infections, they share similar genomic organisation and are capable of establishing persistent infections in humans. Studies into bat, horse and rodent homologs of HCV and HPgV have yet to determine disease associations, transmission routes and seroprevalence. Studies presented within this thesis broaden our understanding of the clinical presentations and host range of NPHV and EPgV. Screening to determine the level of active and past infection to both viruses provides novel insight into infection frequencies, host range, disease progression and examines the correlation between infections and the presence or absence of hepatic disease. Research examining HBV variants circulating in NHPs in Cameroon provides novel evidence for the occurrence of recombination and cross species transmission between NHP variants of HBV and examines the role these findings play in expanding our understanding of the evolution of HBV.

The European honeybee (Apis mellifera) is a managed insect pollinator of global economic importance. Over the last few decades honeybees have been undergoing a major health crisis, with one of the biggest causes the parasitic mite, Varroa destructor and its role in changing the viral landscape of deformed wing virus (DWV), which consists of two major variants: DWV-A and DWV-B. Prior to the start of this project there was limited information known about the mechanisms behind the relationships between Varroa, DWV and honeybees. The overarching aim of this project was to further enhance our understanding of these complex relationships, focusing on the impact of Varroa DWV transmission and differences between the main DWV variants. One of the initial obstacles to understanding these complex interactions was the inability to accurately quantify DWV variants. Prior to the start of this project, there was a need for an accurate assay for the quantification of DWV-A, DWV-B and total DWV, allowing the role of both variants in viral transmission and establishment to be investigated. While primers did exist for DWV quantification, the majority did not distinguish between variants, or provide accurate levels of DWV. Given these challenges in variant detection, a new assay for the quantification of DWV-A, DWV-B and total DWV was designed and validated. The assay consists of an external plasmid standard with distinct sections, for the detection of variants and total DWV. This DWV variant plasmid assay was essential for further transmission studies in this project. DWV variant transmission was explored using a variety of different methods. A new in vitro feeding system was used, to allow investigations into Varroa DWV variant transmission in isolation. The feeding system utilises locust haemolymph, allowing changes in DWV transmission to be detected. In multiple feeding experiments significant changes in DWV transmission were detected. Significant changes in DWV composition within feeding Varroa were detected with decreased levels of DWV, and changing variant levels. Switches in variant composition within mites and transmission rates occurred during Varroa in vitro feeding. These variant switches occurred in both directions from DWV-A to DWV-B, and DWV-B to DWV-A dominance. These changes in mite variant composition corresponded to changes in levels of replicating strands. iv These changes in DWV transmission, composition and replicating strand detection were only seen due to the use of this in vitro feeding system. The in vitro work provided valuable information about Varroa variant transmission and composition changes during feeding but this is not a natural system. Honeybee pupae from a Varroa-free area with extremely low DWV titres provided the opportunity to investigate Varroa variant transmission and pupal DWV establishment. Over 96 hours total DWV levels underwent a 1339408X fold increase, within pupae following Varroa feeding, with a sharp increase after 12 hours, followed by a plateau after 60 hours. Within this time period, DWV-A underwent a similar increase, while DWV-B increased at a much slower rate (33X fold change). In contrast to the in vitro work, mite DWV levels did not decrease during feeding. The impact of natural Varroa cell infestation on L5 larvae was investigated, showing no significant effects between pupal total DWV levels and mite density, and DWV levels between infested and none-infested larvae. However, this lack of significance could be attributed to the use of L5 larvae, which had only undergone a maximum of 24 hours Varroa feeding within the cell. Additionally, the use of two drug treatments (ribavirin and hydroxyurea) to reduce DWV levels was explored. Both drug treatments were tested against Varroa and honeybees, using a variety of methods: immersion (Varroa), injections (honeybees) and feeding (both). While neither drug treatment resulted in consistent DWV decreases, some reduction in DWV levels were seen following Varroa soaking in drug solutions. A significant decrease in DWV was seen in honeybees following bolus and ad libitum feeding of drug treatments. Overall, information and insights have been gained regarding the complex relationship between Varroa, honeybees and DWV. A new DWV variant qPCR assay was developed and utilised in subsequent studies. DWV variant switches in both transmission rates and mite composition were found to occur in in vitro studies. Differences in DWV variant establishment within honeybees were detected following Varroa in vivo feeding, in low DWV pupae. Though the tested drug treatments did not affect DWV levels, this highlights the difficultly facing the establishment of any DWV treatment.

Within the paradigm of HIV-1 infection, macrophages play a crucial role as long-lived viral reservoirs. However, cell-free virus infection is inefficient and is unlikely to explain the levels of infection observed in vivo. To investigate the hypothesis that macrophages might be infected via direct contact with HIV-1-infected T cells, macrophage and HIV-1-infected T cell cocultures were imaged in real time. I observed that macrophages preferentially phagocytosed HIV-1-infected T cells and, using long-term culture assays, I established that following coculture the macrophage became productively infected. Phagocytosis of HIV-1-infected cells occurred independently of viral tropism; however, productive infection following T cell phagocytosis was restricted by viral tropism. Imaging flow cytometry showed that macrophages primarily phagocytose dying HIV-1-infected T cells. However, a significant population of HIV-1-infected 'healthy' cells were also taken up. Furthermore, ICAM-1 was identified as mediating the uptake of HIV-1-infected T cells. These results indicate that apoptosis plays a significant, but not sufficient, role in the mechanism for recognition and uptake of HIV-1-infected T cells. The response of macrophages to HIV-1 infection remains controversial. Using both primary macrophages and a monocyte/macrophage NFκB reporter line assay, I demonstrated that macrophages are activated in response to HIV-1-infected T cells. In addition, during coculture with HIV-1-infected T cells, macrophages upregulated secretion of Th1 cytokines, with associated dysregulation of regulatory cytokines. Finally, data presented suggest that polarisation of macrophages towards M1 and M2 phenotypes alters the susceptibility to HIV-1 infection in the cell-to-cell route.

La découverte des virus géants il y a une dizaine d’années a véritablement bouleversé notre perception du monde viral. Cette découverte a ouvert un débat sur l’origine et l’histoire évolutive de ces virus, et ravivé celui portant sur la nature des virus : peuvent-ils être considérés comme vivants ?J'ai caractérisé un nouveau Marseilleviridae, isolé à partir d’un échantillon prélevé en Nouvelle-Calédonie, appelé Noumeavirus. Les Marseilleviridae sont des grands virus à ADN, qui possèdent des particules à symétrie icosaédrique d’environ 200 nm de diamètre, et des génomes à ADN double-brin de plus de 300 kb. Différentes approches de génomique, protéomique, ainsi que l’étude du cycle infectieux ont montré que le cycle infectieux de ces virus n’était pas indépendant du noyau cellulaire mais recrutait transitoirement les fonctions nucléaires à l’usine virale.J'ai également caractérisé un nouveau Pandoravirus, isolé à partir d’un échantillon prélevé en Nouvelle-Calédonie, appelé Pandoravirus neocaledonia. Les Pandoravirus possèdent une morphologie unique au sein des virus, ainsi qu’un génome colossal à ADN double-brin de 2.5 Mb. Des études comparatives avec d’autres Pandoravirus ont été réalisées en combinant plusieurs approches, afin de mieux comprendre les caractéristiques de ces virus inédits. La morphologie étonnante de ces virus nous a poussés à étudier la nature de leur enveloppe, constituée d’un réseau de fibres formant des structures lamellaires. Se pourrait-il que les Pandoravirus, contrairement aux autres virus, détournent la machinerie cellulaire pour construire leurs particules ? Dans ce cas, quelle serait leur place dans l’histoire évolutive des virus ?
The discovery of giant viruses about a decade ago has truly shaken our perception of the viral world. This discovery has initiated a debate on the origin and evolutionary history of these viruses, and it revived the debate on their nature: are viruses alive?I characterized a new Marseilleviridae, isolated from a sample collected in New-Caledonia, named Noumeavirus. The Marseilleviridae are large DNA viruses that have icosahedral particles of about 200 nm in diameter, and double-stranded DNA genomes of more than 300 kb. Various approaches, such as genomics, proteomics and the study of the infectious cycle, allowed us to reveal that the infectious cycle of these viruses was not independent from the cell nucleus as we thought, but was transiently recruiting nuclear functions to the viral factory.I also characterized a new Pandoravirus, isolated from a sample collected in New-Caledonia, named Pandoravirus neocaledonia. Pandoraviruses have a unique morphology and a gigantic double-stranded DNA genome of about 2.5 Mb. Comparative studies with other Pandoraviruses were performed using several approaches to better understand the characteristics of these original viruses. The astonishing morphology of these viruses led us to investigate the nature of their envelope, which is made of a mesh of fibers forming lamellar structures. Is it possible that Pandoraviruses, unlike other viruses, hijack the cellular machinery to build their particles? In this case, what would be their place in the evolutionary history of viruses?

Les virus entérotropes sont des virus ubiquitaires infectant une large catégorie de vertébrés dont l’homme et les primates non-humains (PNHs). Ils se transmettent principalement par voie féco-orale directe ou indirecte à la suite de laquelle ils atteignent les entérocytes et s’y multiplient. Bien que parfois asymptomatiques, les infections causées par les virus entérotropes peuvent se manifester par des gastroentérites très fréquentes chez les enfants de moins de 5 ans. Ces mêmes virus peuvent être responsables de pathologies sévères telles que les maladies respiratoires, encéphalitiques, cardiaques, neurologiques. À partir des années 1950, de nombreux virus entérotropes ont été isolés de tissus de PNHs couramment utilisés en cultures cellulaires et en recherche biomédicale. Dès lors, de nombreuses études ont été conduites sur la caractérisation des virus entérotropes principalement chez les PNHs captifs ou en contact avec l’homme. En milieu naturel, en dehors des entérovirus et des adénovirus, leur circulation, leur épidémiologie et leur diversité restent encore peu connues. L’objectif de cette thèse est donc de rechercher et caractériser les virus entérotropes chez les PNHs d’Afrique Centrale. Ainsi à partir de 600 échantillons de fèces de PNHs collectés dans des forêts et réserves naturelles au Gabon, nous avons pu mettre en évidence la circulation de différentes espèces d’entérovirus (EVs) chez les mandrills et les chimpanzés. Cette caractérisation a également permis de mettre en évidence des EVs proches d’EVs infectant l’homme ainsi que deux nouveaux sérotypes chez un chimpanzé et chez un mandrill. Nous avons également mis en évidence un astrovirus (AstV) totalement divergent d’AstVs référencés chez un gorille. En dehors de leur circulation en milieu naturel, les virus entérotropes sont également présents chez les PNHs en contact fréquents avec l’homme. De ce fait à partir d’échantillons fécaux d’un groupe de 12 chimpanzés du Sanctuaire de Tchimpounga, nous avons caractérisé l’EV-C99 responsable de cas de paralysie chez l’homme et probablement responsable de celle observée chez un chimpanzé. De plus, deux sapovirus (SaVs) très proches d’un SaV identifié chez l’homme ont également été caractérisés. L’Afrique Centrale est donc caractérisée par une diversité de virus entérotropes qui circulent chez les PNHs. L’identification chez les PNHs de virus entérotropes proches en milieu naturel de ceux infectant l’homme soulève l’existence d’une probabilité de transmission inter-espèce entre les PNHs et l’homme dont le sens reste encore à déterminer. Par contre chez les PNHs du sanctuaire, la susceptibilité à ces virus humains peut être responsable de pathologies graves comme la paralysie observée chez les chimpanzés
The enteric viruses are ubiquitous virus infecting a broad range of vertebrates, including humans and non-human primates (NHPs). They are spread by direct or indirect fecal-oral route following which they reach the enterocytes and multiply. Even though infections caused by these viruses are asymptomatic, enteric viruses could be responsible for frequent gastroenteritis in children under 5 years of age. These viruses may be responsible for severe pathologies such as respiratory, encephalitic, cardiac and neurological diseases. In the 1950s, many viruses have been isolated from NHPs species commonly used in cell culture and biomedical research. Since, many studies have been conducted to characterize, then enteric viruses have been mainly identified in captive NHPs or those living in close contact with humans. Little is known concerning the circulation, epidemiology and diversity of enteric viruses in the wild, except for enteroviruses and adenoviruses. The objective of this thesis is to investigate and characterize the enteric virus in NHPs of Central Africa. Thus from 600 samples of feces of NHPs collected in natural forests and reserves in Gabon, we highlighted the circulation of different species enteroviruses (EVs) in mandrills and chimps. We also identified EVs close to those infecting humans as well as two new serotypes in a chimpanzee and in a mandrill. We have highlighted an astrovirus (AstV) completely divergent from those referenced in a gorilla. Apart from their outstanding natural environment, enteric viruses are also present in NHPs in frequent contact with humans. Therefore fecal samples from a group of 12 chimpanzees from the Tchimpounga Sanctuary, we characterized the EV-C99 responsible for cases of paralysis in humans and probably responsible for that observed in a chimpanzee. In addition, two sapovirus (SaVs) very close to a SaV identified in humans have also been characterized. Central Africa is therefore characterized by a diversity of enteric virus circulating in NHPs. The identification in the wild of enteric virus in NHPs close to those infecting humans raises probability of cross-species transmissions between NHPs and humans whose sense remains to be determined. However in NHPs in the sanctuary, susceptibility to these human viruses can be responsible for severe diseases such as paralysis observed in chimpanzee

Diarrhea caused by rotavirus and Giardia is a major health problem among children attending day-care centers because of inadequate personnel hygiene. Epidemiological evidence suggesting person-to-person transmission of enteric pathogens has long been recognized. This study was initiated to investigate the effectiveness of handwashing for the removal of rotavirus and Giardia from contaminated hands. The palms of participant hands were innoculated with approximately 103 Giardia cysts or 105 plaque forming units of rotavirus and the effect of washing using tap water alone, a liquid soap or a bar soap on their removal was assessed. Handwashing with liquid soap was found to be very effective in the removal of rotavirus and Giardia cysts as compared to washing with bar soap or tap water alone. The overall recovery of viruses in both bar soap and liquid soap was low (0.03-22.5%), probably due to virus inactivation by the detergent.

The goal of my dissertation is to characterize the contribution of industrial food animal production to between-farm transmission of zoonotic influenza A viruses and transmission of these viruses from industrial food animals to humans. The intention of this research is to improve the capacity of public health policies in the United States to prevent the emergence of pandemic influenza viruses.

Preventing and controlling outbreaks within animal populations and avoiding human infection with zoonotic influenza A viruses can reduce the risk of emergence of pandemic influenza viruses in human populations. Industrial food animal production, which dominates the market in the United States and much of the developed world – and increasingly, the developing world as well – has long been considered biosecure. However, emerging research indicates that these industrial systems are vulnerable to disease incursions and suggests that they may play a central role in driving the emergence of zoonotic diseases. The implications of these industrial systems for human influenza risk, particularly the emergence of novel zoonotic influenza A viruses, remains largely unaddressed in the current literature and in health policy strategies in the United States.

Chapter 1 of this dissertation outlines my research goals and provides background on my central research themes and topics. Chapter 2 documents the limits of biosecurity within industrial systems, highlighting risks to food animal workers. Chapter 3 details a cross-sectional serology study of a cohort of industrial poultry workers and community members (n=99) in the Delmarva Peninsula, a tri-state area of intense poultry production in the Mid-Atlantic region of the United States. No evidence of infection with avian influenza viruses is observed in this population.

Chapter 4 contains a quantitative modeling study to estimates risk of between-farm transmission of avian influenza viruses among industrial poultry farms. This study concluded that company affiliation was a significant source of exposure risk from vehicular transmission. Chapter 5 is a policy analysis of the limitations of current pandemic preparedness policy in the United States to adequately incorporate primary prevention. The central results of this dissertation, their significance to public health and opportunities for further research are highlighted in Chapter 6.

Orientador: Helaine Maria Besteli Pires Milanez
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Objetivo: avaliar a transmissão vertical (TV) do HIV e fatores associados em gestantes soropositivas acompanhadas em um serviço universitário brasileiro (CAISM/UNICAMP) entre 2000 e 2009. Sujeitos e Métodos: coorte histórica de 452 gestações e seus recém-nascidos. Os dados foram coletados dos prontuários e registrados em fichas específicas. Crianças sem seguimento foram convocadas para definição diagnóstica. Análise dos dados: análise descritiva através de distribuição percentual e de médias; teste de X², exato de Fisher, t de Student, Mann-Whitney e ANOVA, razão de risco e intervalo de confiança. Resultados: A TV foi de 3,6%. A idade média das gestantes foi 27 anos; principal categoria de exposição foi a sexual (86,5%); 55% já apresentava o diagnóstico prévio à gravidez. Sessenta e dois por cento não estavam em uso de TARV ao engravidar. CD4 médio inicial foi de 474 células/ml e 70.3% apresentaram carga viral indetectável no terceiro trimestre. Como TARV, 55% usaram esquemas com IP e 35% com nevirapina. Monoterapia com AZT foi utilizada em 5,5%. Idade gestacional média no parto foi de 37,2 semanas e em 92% a via foi cesárea; 97,2% receberam AZT endovenoso. Os fatores associados à TV foram: baixa contagem de CD4, elevada carga viral, tempo reduzido de TARV, presença de alterações gestacionais (anemia, RCF, oligoâmnio), coinfecções durante o pré-natal (CMV e toxoplasmose) e presença de trabalho de parto. Uso de TARV potente, parto por cesárea e uso do AZT pelo RN foram fatores protetores. Má adesão ao tratamento esteve presente em 13 dos 15 casos infectados; em sete houve presença de coinfecção neonatal (CMV e toxoplasmose). Conclusão: Fatores de risco para TV foram comprometimento do estado imunológico da gestante, menor tempo de terapia, coinfecções (CMV e toxoplasmose) e presença de trabalho de parto. O uso de TARV potente e a realização de cesárea foram fatores protetores para a TV do HIV.
Abstract: Objectives: to evaluate mother-to-child transmission (MTCT) rates and related factors in HIV-infected pregnant women from CAISM/UNICAMP between 2000 and 2009. Subjects and methods: cohort of 452 HIV-infected pregnant women and their newborns. Data was collected from recorded files and undiagnosed children were enrolled for investigation. Statistical analysis: qui-square test, Fisher exact test, Student t test, Mann-Whitney test, ANOVA, risk ratio and confidence intervals. Results: MTCT occurred in 3.6%. The study population displayed a mean age of 27 years; 86.5% were found to have acquired HIV through sexual contact; 55% were aware of the diagnosis prior to the pregnancy; 62% were not using HAART. Mean CD4 cell-count was 474 cells/ml and 70.3% had undetectable viral loads in the third trimester. HAART included nevirapine in 35% of cases and protease inhibitors in 55%; Zidovudine monotherapy was used in 5.5%. Mean gestational age at delivery was 37.2 weeks and in 92% by caesarian section; 97.2% received intravenous zidovudine. Implicated factors related to MTCT were: low CD4 cell counts, elevated viral loads, maternal aids, shorter periods receiving HAART, maternal concurring illnesses (anemia, IUGR, oligodydramnium), coinfections (CMV and toxoplasmosis) and the occurrence of labor. Use of HAART for longer periods, caesarian delivery and oral zidovudine for the newborns were associated with a decreased risk. Poor adhesion to treatment was present in 13 of the 15 cases of transmission; in 7, co-infections were diagnosed (CMV and toxoplasmosis). Conclusion: Use of HAART and caesarian delivery are protective factors in mother-to-child transmission of HIV. Maternal coinfecctions and maternal concurring illnesses were risk factors for MTCT.
Universidade Estadual de Campi
Ciencias Biomedicas
Mestre em Tocoginecologia

Les polérovirus infectent une large gamme de plantes d’intérêt économique. Ils sont transmis par un insecte vecteur, le puceron, selon le mode circulant non-multipliant. Le virus, acquis par le puceron lors de l’ingestion de sève sur une plante infectée, traverse l’épithélium des cellules intestinales puis celui des glandes salivaires par un mécanisme de transcytose impliquant des récepteurs encore inconnus. Le récepteur de l’éphrine (Eph) est une protéine membranaire dont un domaine est capable de se lier dans la levure aux protéines structurales des polérovirus. En développant des techniques basées sur l’ARN interférence, nous avons montré que l’acquisition orale d’ARN double brin ciblant Eph chez le puceron Myzus persicae permet de réduire de manière reproductible l’internalisation des polérovirus dans le corps du puceron. Les pucerons ainsi traités transmettent le virus avec une efficacité réduite. Eph pourrait donc assurer la fonction de récepteur des polérovirus chez M. persicae
Poleroviruses infect a wide range of economically important plants. They are transmitted in a circulative and non-propagative mode by an insect vector, the aphid. The virus particles are acquired by aphids when ingesting the sap from an infected plant and cross successively the epithelia of the midgut and the salivary gland cells by a transcytosis mechanism that relies on the presence of unknown receptors.The ephrin receptor (Eph) is a membrane protein which contains a domain able to bind in yeast to the structural proteins of poleroviruses. By developing methods based on RNA interference, we have shown that oral acquisition of double-stranded RNA targeting Eph in the aphid Myzus persicae can reproducibly reduce polerovirus internalization into the aphid's body. Such treated aphids transmit the virus to plants with a lower efficiency. Eph could therefore function as a receptor for poleroviruses in M. persicae

East Africa has been experiencing an increase in the occurrence of emerging infectious diseases such as yellow fever (YF) and dengue (DEN). Increasing frequency of YF activity in East Africa constitutes a re-emergence that was not detected for over 40 years. Additionally, DEN outbreaks have also increased in frequency and continue to be detected in Kenya and in neighboring countries like Tanzania, Somalia, Djibouti, Eritrea and South Sudan. The renewed vigor of YF and dengue fever (DF) re-emergence in East Africa presents a new challenge to public health in spite of the availability of a safe and effective vaccine for YF. However, there is need to understand the potential for YF and DEN transmission along the border areas of Kenya, because Kenya is classified among countries with medium to high risk for YF transmission. This classification was mainly based on historical data, proximity to countries reporting recent YF outbreaks, the presence of non-human primates known reservoirs for these viruses, unrestricted human movement and presence of potential vector mosquito species. Both YF and DEN share a similar niche in the ecosystem and are associated with Aedes mosquito species of the subgenus Stegomyia. While the factors leading to the re-emergence of these diseases are poorly understood, a better epidemiologic understanding relating to disease ecology including presence of potential vectors, their host blood feeding preferences, the vector competence in transmission of these viruses and evidence of virus circulation in human population, will guide assessment of disease risk in the target areas and help to prevent or mitigate severe outbreaks in this region.
Thesis (PhD)--University of Pretoria,2020
National Institute of Health Sciences L'oreal- UNESCO for women in science
Medical Virology
PhD in Medical Virology
Restricted

Hepatitis E virus (HEV) is an important human pathogen, with pigs and likely other animal species serving as natural reservoirs. There are currently four recognized HEV genotypes that infect humans within the genus Hepevirus of the family Hepeviridae. Genotypes 1 and 2 are human viruses that are associated with waterborne and fecal-oral transmission in developing countries, while genotypes 3 and 4 have been identified in humans and other animal species and are zoonotic and endemic in both industrialized and developing countries. In my dissertation research, we identified the first strain of HEV from rabbits in the United States. We subsequently determined the complete genome sequence of the virus. Phylogenetic analyses of the full-length sequence indicated that U.S. rabbit HEV is a distant member of the zoonotic genotype 3, thus raising a potential concern for zoonotic infection. In order to investigate the cross-species potential of rabbit HEV, we then determined its antigenic cross-reactivity with other animal strains of HEV. Additionally, we demonstrated that the novel rabbit HEV could cross species barriers and infect pigs under experimental conditions. Finally, we attempted to determine the risk factors and sources of foodborne HEV infection in the United States. We detected HEV for the first time from non-liver pork commercial products in the United States and demonstrated consumption of undercooked meat a risk factor for HEV infection. HEV sequences of genotype 3 origin were detected from pork products purchased from grocery stores in Southwest Virginia. Approximately 6.3% (21/335) of university students tested seropositive for HEV antibodies and, importantly, those with a history of consuming undercooked meats were 13 times more likely to be seropositive. These results further underscore the importance of cooking pork thoroughly and using proper hygiene when preparing meals.
Ph. D.

This thesis presents and tests a novel hypothesis that attempts to predict the relative humidity (RH) dependent survival of airborne respiratory viruses in protein-enriched saline aerosols. The hypothesis proposed that virus-laden respiratory aerosols exist in either an effloresced (solid) or deliquesced (liquid) state, depending on the ambient air RH and that the survival of viruses embedded in such aerosols changes with that state. Experiments confirmed as predicted, that rhinovirus and influenza virus exhibited a V-shaped surviving fraction dependence on RH. Implications concerning the survival of these viruses under seasonal conditions are discussed along with strategies to control indoor airborne infection.

El productes de plasma dessecat per esprai (SDP) tenen un alt contingut proteic i són components útils per a moltes aplicacions, principalment com a productes valuosos per la nutrició animal. El plasma s'obté a partir de sang de porcs sans aptes pel consum humà. Durant l’obtenció, s’afegeixen anticoagulants i la sang s’emmagatzema en tancs refrigerats. Un cop a la planta de processament, la sang es centrifuga, per separar el plasma de la fracció cel∙lular. El plasma es deshidrata en un procés d'assecat per polvorització per produir un producte final en pols. El dispositiu assecador crea microgotes per aspersió del plasma a alta pressió, l’aigua s'evapora per l'entrada d'aire a 170‐250°C i una temperatura de sortida a 80°C. L'assecat per esprai o polvorització produeix diferents efectes simultanis de deshidratació, canvis de temperatura i altres efectes com canvis osmòtics, dany oxidatiu i desnaturalització proteica que podrien contribuir a explicar un procés encara poc entès d'inactivació microbiana. L'objectiu d'aquesta tesi va ser avaluar si el plasma porcí assecat per aerosol (SDPP), en forma de producte comercial i obtingut a partir de lots de milers de porcs y sotmès a tractament, podria transmetre o no alguns dels virus més resistents a altes temperatures y que per altra banda, de forma molt freqüent, afecten a la producció porcina. Atès que la majoria d'elles poden produir infeccions inaparents, fins i tot a l’edat de final d’engreix, el risc que es puguin trobar a a la sang de recollida d’escorxador no és menyspreable. En el primer estudi es va fer el seguiment de la transmissió del Parvovirus porcí (PPV) durant el subministra d’SDPP comercial, com a model de detecció de virus d'alta resistència tèrmica, en porcs susceptibles. Trenta‐sis porcs Landrace x Duroc deslletats (28 d d'edat) van ser alimentats amb dietes que contenien 0 o 8% SDPP. El lot d’SDPP utilitzat contenia anticossos (ABs) per PPV (títol 1: 400). Es varen prendre mostres de sang dels porcs els dies 0 i 63 per a determinar si l'alimentació SDPP havia causat el desenvolupament d'ABs contra PPV, Virus de la síndrome reproductiva i respiratòria (PRRSV) o el Virus de la malaltia d'Aujeszky (ADV). La inclusió de SDPP a la dieta va millorar el creixement dels porcs sense seroconversió contra els virus estudiats. L'objectiu del segon estudi fou avaluar si l’SDPP comercial que conté genoma del Circovirus porcí tipus 2 (PCV2) podia ser un vehicle de transmissió d'aquest virus. Es varen utilitzar garrins Landrace recent deslletats de truges no virèmiques i seleccionats per baixos títols d'ABs. En aquest estudi, l'absència d'ABs enfront PCV2 en porcs experimentals no era necessari ja que els anticossos materns són comuns a les granges comercials. L’SDPP es va incloure en les dietes d'assaig a 0 o 8%. El lot SDPP utilitzat a l'estudi contenia 2,47 x 105 còpies d'ADN de PCV2 / ml mesurats per PCR quantitativa en temps real (qrt‐PCR). Els porcs van ser mostrejats a dies 0, 10, 35 i 45 . No es va observar virèmia o seroconversió enfront PCV2 en porcs alimentats amb SDPP i tampoc no es va observar seroconversió a cap altre dels virus analitzats (PPV, el virus de la malaltia vesicular porcina i ADV). En el tercer estudi, l’SDPP que contenia ADN de PCV2 es va utilitzar per provar la potencial transmissió del PCV2 de porcs inoculats amb el PRRSV. El PRRSV és un virus amb efectes immunomoduladors que normalment facilita les infeccions concurrents. Vint‐i‐tres porcs Landrace de 3,5 setmanes d'edat distribuïts en un arranjament factorial 2 x 2 i assignats a corralines de nivell de bioseguretat 3 (BSL3) per evitar la contaminació creuada per PRRSV (ja que és un virus molt estès en la producció comercial). Les dietes contenien 0 o 8% SDPP. El lot comercial específic de SDPP utilitzat en aquest estudi contenia 7,56 x 105 còpies del genoma de PCV2 per gram. Els porcs varen ser mostrejats a 0, 14 i 28 dies després de l'exposició PRRSV. Tant els grups desafiats amb PRRSV com amb SDPP no varen donar lloc a la transmissió de PCV2. El quart estudi es va dirigir a avaluar si l’SDPP comercial podia estar involucrat en la transmissió del Virus de l'hepatitis E (VHE), una infecció viral freqüent entre la població de porcs i que ha estat reconegut com a zoonotic potencial. Es van trobar ABs enfront HEV en el 100% de les 84 mostres analitzades de diferents lots comercials d’SDPP d'origen espanyol, mentre que només el 22,4% de les mateixes mostres varen ser positives a l'ARN del VHE. En conseqüència, era un motiu de preocupació saber si SDPP pot contribuir a la transmissió del VHE per SDPP. Es varen analitzar mostres de sèrum d'estudis previs en els quals s'havien alimentat els porcs amb dietes comercials SDPP al 0 o 8% per detectar HEV. L’edat dels porcs va variar de 3 a 15 setmanes d’edat i la durada d'alimentació va ser entre 4 a 9 setmanes, depenent de l'experiment. En una de les mostres d’SDPP es va confirmar que contenia VHE ARN. No es va detectar seroconversió en cap dels animals pertanyents als diferents estudis, el que va portar a la conclusió que l’SDPP no representa un risc per a la transmissió del VHE. Es pot concloure que els resultats dels estudis abans esmentats van contribuir a aclarir que SDPP no sembla ser un vector de patògens i, per tant, és un ingredient d’alt contingut de proteïna natural segur i d'alta qualitat per al seu ús en l'alimentació animal.
El plasma desecado por aerosol (SDP) tiene un alto contenido proteico, que lo convierte en un componente útil para muchas aplicaciones, y de alto valor para la nutrición animal. El plasma se obtiene a partir de sangre de cerdos sanos, aptos para el consumo humano. Durante su obtención, se añaden anticoagulantes y la sangre se almacena en tanques refrigerados. Una vez en la planta de procesado, la sangre se centrifuga, para separar el plasma de la fracción celular. El plasma se deshidrata mediante un proceso de secado por pulverización en aerosol para producir un producto final en polvo. El dispositivo crea un espray de microgotas por aspersión del plasma a alta presión, el agua se evapora por la entrada de aire a 170‐250°C y una temperatura de salida de 80°C. El secado por pulverización produce distintos efectos simultáneos, incluyendo: deshidratación, cambios bruscos de temperatura y otros efectos tales como cambios osmóticos, estrés oxidativo y desnaturalitzación proteica que podrían contribuir a explicar un proceso aún poco entendido de inactivación microbiana. El objetivo de esta tesis fue el de evaluar si el plasma porcino desecado por aerosol (SDPP) en forma de producto comercial y obtenido a partir de lotes de miles de cerdos podría transmitir o no algunos de los virus más resistentes a altas temperaturas y que frecuentemente afectan a la producción porcina. Dado que la mayoría de ellos pueden producir infecciones inaparentes, incluso a la edad de final de engorde, el riesgo que puedan encontrarse en la sangre recogida en matadero no es despreciable. En el primer estudio se realizó un seguimiento de transmisión del Parvovirus porcino (PPV) durante el aporte de SDPP comercial en la dieta, como un modelo de detección de virus de alta resistencia térmica, en cerdos susceptibles. Treinta y seis cerdos Landrace x Duroc recién destetados (28 de edad) se alimentaron con dietas que contenían 0 o 8% SDPP. El lote de SDPP utilizado contenía anticuerpos (ABs) para PPV (título 1: 400). Se tomaron muestras de sangre de cerdos en el día 0 y 63 para determinar si la inclusión de SDPP en el alimento había causado el desarrollo de ABs frente PPV, el Virus del síndrome reproductivo y respiratorio porcino (PRRSV) o el Virus de la enfermedad de Aujeszky (ADV). La inclusión de SDPP en la dieta mejoró el crecimiento de los cerdos sin seroconversión frente a los virus estudiados. El objetivo del segundo estudio consistió en evaluar si el SDPP comercial que contenía genoma de Circovirus porcino tipo 2 (PCV2) podía ser un vehículo de transmisión de este virus. Se utilizaron lechones Landrace recién destetados de cerdas no virémicas y seleccionados por presentar bajos títulos de ABs. En este estudio, la ausencia de ABs frente PCV2 en cerdos experimentales no era una condición excluyente, ya que los anticuerpos maternos son un factor común en los cerdos de granjas comerciales. El SDPP se incluyó en las dietas de ensayo a 0 o 8%. El lote de SDPP utilizado en el estudio contenía 2,47 x 105 copias de ADN de PCV2 / ml medidos por PCR cuantitativa de tiempo real PCR (qrtPCR). Los cerdos fueron muestreados a 0, 10, 35 y 45 días. No se observó viremia o seroconversión frente PCV2 en los cerdos alimentados con SDPP y tampoco se observó seroconversión frente a ninguno de los otros virus analizados (PPV, el virus de la enfermedad vesicular porcina, y ADV). En el tercer estudio, el SDPP que contenía ADN de PCV2 se utilizó para probar la potencial transmisión del PCV2 en cerdos inoculados con el PRRSV. El PRRSV es un virus con efectos inmunomoduladores que normalmente facilita las infecciones concurrentes. Veintitrés cerdos Landrace de 3,5 semanas de edad fueron distribuidos en un diseño factorial 2 x 2 y asignados a corralinas BSL3 para evitar la contaminación cruzada por PRRSV (un virus ampliamente extendido en condiciones de producción comercial). Las dietas contenían 0 o 8% SDPP. El lote comercial específico de SDPP utilizado en este estudio contenía 7,56 x 105 copias del genoma de PCV2 por gramo. Los cerdos se muestrearon a 0, 14 i 28 d después de la exposición al PRRSV. Los grupos desafiados con PRRSV y alimentados con SDPP no dieron lugar a transmisión del PCV2. El cuarto estudio se dirigió a evaluar si el SDPP comercial estaba involucrado en la transmisión del Virus de la Hepatitis E (VHE), una infección viral frecuente entre la población de cerdos que ha sido reconocido como un virus con potencial zoonótico. Se encontraron ABs frente a HEV en el 100% de las 84 muestras analizadas de distintos lotes comerciales de SDPP de origen español, mientras que solo el 22,4% de las mismas muestras fueron positivas por ARN del VHE. En consecuencia, era un motivo de preocupación conocer si el SDPP puede contribuir a la transmisión del el VHE. Se analizaron muestras de estudios previos en los que se habían alimentado los cerdos con dietas comerciales con SDPP al 0 o 8% para detectar HEV. La edad de los cerdos varió entre 3 a 15 semanas de edad y la duración de la alimentación fue entre 4 a 9 semanas, dependiendo del experimento. Una de las muestras de SDPP se confirmó que contenía ARN del VHE. No se detectó la seroconversión en ninguno de los animales pertenecientes a los distintos estudios, lo que condujo a la conclusión que el SDPP no representa un riesgo para la transmisión del VHE. Se concluyó que los resultados de los estudios mencionados contribuyeron a aclarar que el SDPP no parece ser un vector de los agentes patógenos estudiados y, por lo tanto, según los estudios realizados se trata de un ingrediente natural seguro de alta calidad con un alto contenido de proteínas para su uso en nutrición animal.
Spray dried plasma (SDP) products have high protein contents and, therefore, are useful components for many applications, mainly as valuable products for animal nutrition. Plasma is obtained from blood of healthy pigs fit for slaughter for human consumption. Blood is pooled from many animals, collected in tanks with anticoagulant and chilled. In the processing plant, blood is centrifuged to separate the plasma from cellular fraction and dehydrated by a spray‐drying process to produce a powered ingredient. The spray dryer device creates micro‐drops and evaporates water by inlet air at 170‐250°C and outlet temperature at 80°C. Spray‐drying produces concurrent effects of dehydration, changes of temperature and others effects such as osmotic changes, oxidative damage, and protein denaturizing stress that could contribute to explain a poorly understood process of microbial inactivation. The objective of this thesis was to evaluate if commercial spray dried porcine plasma (SDPP) obtained from batches of thousands of pigs could transmit or not some of most common high heat resistant viruses that affect swine production. Since most of them may produce unapparent infections at slaughter age, the risk to be found in collected blood at slaughterhouse is not negligible. In the first study, Porcine parvovirus (PPV) transmission throughout commercial SDPP, as model of high thermally resistant virus, was explored in susceptible naïve pigs. Thirty‐six Landrace × Duroc weanling pigs (28 d of age) were fed with diets containing either 0 or 8% SDPP. The SDPP lot used contained antibodies (ABs) to PPV (titer 1:400). Blood samples were collected from pigs on d0 and 63 to determine whether feeding SDPP caused development of ABs against PPV, Porcine reproductive and respiratory syndrome virus (PRRSV) or Aujezsky disease virus (ADV). Inclusion of SDPP in the diet improved growth of pigs without seroconversion against studied viruses. The objective of the second study was to assess if commercial SDPP containing Porcine circovirus type 2 (PCV2) genome may be a vehicle of transmission for this virus. Weaned Landrace piglets from non viremic sows and selected for low ABs titres were used. In this study, absence of ABs against PCV2 in experimental pigs was not required because maternal antibodies are widespread in commercial farms. SDPP were included in the test diets at 0 or 8%. The SDPP lot used in the study contained 2.47 x 105 PCV2 DNA copies/ml measured by quantitative real time PCR (qPCR). Pigs were sampled at 0, 10, 35 and 45 d. No viremia or seroconversion against PCV2 was observed in pigs fed with SDPP and also no seroconversion to other virus analyzed (PPV, ADV and Swine vesicular disease virus, SVDV) was observed. In the third study, SDPP containing PCV2 DNA was used to test the potential transmission of PCV2 to pigs challenged with PRRSV. PRRSV is a virus with immunomodulatory effects that usually facilitates concurrent infections. Twenty‐three Landrace pigs of 3.5 weeks (w) of age were distributed in a 2 x 2 factorial arrangement and allocated in BSL3 boxes to avoid PRRSV cross‐contamination (since it is a widely spread virus under commercial production conditions). The diets contained 0 or 8% SDPP. The specific commercial lot of SDPP used in this study contained 7.56 x 105 PCV2 genome copies per gram. Pigs were sampled at 0, 14 and 28 d post PRRSV challenge. PRRSV challenged groups and SDPP groups, did not result in PCV2 transmission. The fourth study was addressed to assess if commercial SDPP was involved in the transmission of Hepatitis E virus (HEV), a prevalent viral infection within the pig population that has been recognized with zoonotic potential. HEV ABs were found in 100% of 84 samples of different commercial SDPP lots from Spanish origin, while only 22.4% of the same samples were positive for HEV RNA. Accordingly, it was of concern to know if SDPP may contribute to HEV transmission by SDPP. Serum samples from previous studies in which naïve pigs were fed with commercial SDPP diets at 0 or 8% were analysed to detect HEV. Age of pigs ranged from 3 to 15w of age and the feeding duration was between 4 to 9w, depending on the experiment. One of the lots of SDPP was confirmed to contain HEV RNA. HEV seroconversion was not detected in any of the animals belonging to the different studies, leading to the conclusion that SDPP does not represent a risk for HEV transmission. It can be concluded that the results of the above mentioned studies contributed to clarify that SDPP seems not to be a vector for pathogens and, therefore, it is a natural, safe, high‐quality ingredient with high protein contents for use in animal nutrition.

The aim of this quasi-experimental study was to examine the impact of a health education intervention on knowledge about HIV/Hepatitis B transmission, attitudes, beliefs and intentions towards condom use. Research has shown tht small group discussion, single sex groups, age proximity of health educators, and HIV prevention integrated in the broader sexual health context, increased the effectiveness of health education with regard to safer sexual practices.

Between 1994 to 1998 the ANRS 049a DITRAME trial was conducted during which a short regimen of ZDV demonstrated for the first time acceptability, tolerance and efficacy on reduction of mother-to-child transmission (MTCT). Our major aim was to analyze certain virological characteristics of infected women in this cohort and their association to HIV-1 transmission. On the one hand, we analyzed the HIV-1 replication capacity in different physiological compartments :blood, vaginal fluids (VF) and breast milk (BM) related to MTCT were investigated by nested case control studies in the DITRAME cohort. We demonstrated the relationship between plasma viral load (VL), at 34 weeks of amenorrheae and at Day 8 post partum, and MTCT in Africa where the probability to be exclusively breastfed for an one year infant is 46.6%. We also analyzed relationship between plasma VL and ZDV treatment. Additionally, we demonstrated that MTCT is essentially the consequence of a high proviral load in VF in our context. Moreover, reduced levels of HIV-1 RNA in milk at Day 8 were observed in mothers receiving ZDV therapy rather than in mothers under placebo. For the first time, the association between BMVL and postnatal transmission has been studied. We observed a highly significative difference between BMVL of women who transmitted the virus and those who did not. Moreover, univariate and multivariate analyzes clearly indicated that early breastfeeding log10 HIV-RNA at Day8 is an independent factor significatively associated to MTCT. Decreased median BMVL from 1608 copies/mL (c/ml) at Day8 to 346 c/ml at Day45 were found for mothers who transmitted the virus during the postpartum and who received placebo. Nevertheless, for those who received ZDV, median BMVL increased from 56 c/ml at Day8 to 470.5 c/ml at Day45. This marked trend to a rebound effect of BMVL could be the consequence of the treatment withdrawal as observed for adults at HAART withdrawal.

On the other hand, we studied the variability of HIV and its association with MTCT. First, we analyzed HIV-1 diversity in African women in France and Burkina Faso. In a second step, we demonstrated that HMA was an adapted tool for co and super-infections studies for adults. By this way, we identified two superinfections among 147 women within our commercial sex workers cohort. Additionally, we used this tool to analyze children of the DITRAME cohort who were infected in utero and who could be superinfected during the delivery or later by breastfeeding. We identified seven children, among 18 who were infected in utero, displaying HMA profiles suspicions for co-infections, and who had a more important mortality rate than normally. Their proviral env sequences are currently analyzed.

Moreover, we confirmed the fact that the rate of vitamin A has no influence on MTCT.

De 1994 à 1998, s’est déroulé l’essai clinique DITRAME ANRS 049a qui a démontré, pour la première fois, l’acceptabilité, la tolérance et l’efficacité d’un traitement court de zidovudine (ZDV) sur la diminution de la TME. Notre travail s’est inscrit dans le cadre de cet essai et a eu pour but d’en analyser certains des aspects virologiques et leur rapport avec la transmission de la mère à l’enfant du VIH (TME). D’une part, nous avons analysé les niveaux de réplication virale dans différents compartiments physiologiques :le sang, les sécrétions cervico-vaginales (SCV) et le lait maternel (LM) et leur rapport avec la transmission, par des études cas-témoins nichées dans la cohorte DITRAME. Nous avons démontré le rapport entre la charge virale libre (CV) dans le plasma à 34 semaines d’aménorrhée et à J8 postpartum et la TME dans le contexte africain où la probabilité d’avoir un allaitement exclusif à un an est de 46,6%, et analysé leur rapport avec le traitement ZDV. Nous avons également démontré que la TME est essentiellement due à une charge provirale plus élevée dans les SCV dans notre contexte. De plus, grâce à la mise au point d’une technique, nous avons démontré que la ZDV avait un effet global marqué sur la diminution de la CV libre dans le LM. Il s’agit de la première étude mettant en relation la CV dans le lait avec la transmission postnatale. De même, nous avons observé une différence très hautement significative entre les charges virales libres des femmes ayant transmis le VIH et les non transmettrices. De plus, nos analyses univariée et multivariée démontrent que la CVlm mesurée en log10 de la lactation précoce (J8) est un facteur indépendant très significativement associé à la TME. Chez les femmes ayant transmis le virus durant le post-partum et non traitées à la ZDV, la CVlm médiane a décru de 1608 copies/mL (c/ml) à J8 à 346 c/ml à J45. Par contre, chez les femmes ayant transmis le virus mais ayant reçu un traitement ZDV, la CVlm médiane évolue de 56 c/ml à J8 à 470,5 c/ml à J45. Cette tendance marquée à un effet rebond de la CVlm à J45 laisse penser que la TME qui a lieu chez les femmes traitées à la ZDV pourrait être une conséquence de l’arrêt de ce traitement, comme observé chez les adultes après arrêt du traitement HAART.

D’autre part, nous avons étudié la variabilité du VIH en fonction de la TME. Dans un premier temps, nous avons analysé la diversité du VIH-1 chez des mères africaines vivant en France, et par après au Burkina Faso. Ensuite, grâce à l’élaboration d’une nouvelle technique, nous avons démontré que le HMA pouvait être un outil adapté à l’étude des co- et sur-infection chez l’adulte. Nous avons identifié de cette manière deux surinfections parmi 147 femmes analysées au sein d’une cohorte de femmes à haut risque de surinfection. Nous avons ensuite utilisé ce moyen pour étudier des enfants de la cohorte DITRAME infectés in utero qui auraient pu se surinfecter durant le peripartum ou ensuite par l’allaitement. Sept enfants parmi 18 analysés, présentant des profils HMA à suspicion de coinfection et qui présentaient un taux de mortalité plus élevé que la normale, ont été identifiés. Leurs séquences provirales env sont en cours d’analyse actuellement.

Par ailleurs, nous avons confirmé le fait que le taux de vitamine A n’a pas d’influence sur la TME.

Doctorat en Sciences agronomiques et ingénierie biologique
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Ce travail est réparti en deux parties différentes issues de deux études différentes.

La première partie décrit l’étude AMATA conçue en 2005 au Rwanda, étude prospective basée sur le suivi d’une cohorte répartie en deux groupes d’intervention postnatale. Cette étude avait pour objectif de tester l’hypothèse que l’allaitement maternel (AM) sous trithérapie antirétrovirale maternelle (HAART) pouvait être une prévention aussi efficace que le lait artificiel (LA) afin de réduire drastiquement la transmission du virus VIH de la mère à l’enfant avec une moindre mortalité infantile. Cette intervention permettait de préserver les avantages de l’AM, connue pour offrir une prévention naturelle minimisant les infections graves, en particulier les gastro-entérites et diminuant le taux de malnutrition protéino-énergétique (MPE). Dans la cohorte « AMATA », un groupe d’enfants était allaité exclusivement durant six mois, les mères étant sous trithérapie antirétrovirale systématique et un autre groupe d’enfants était nourri au LA durant les six premiers mois de vie. L’intervention débutait durant la grossesse à partir de la 28ème semaine d’âge gestationnel, une trithérapie antirétrovirale étaient donnée à toutes ces femmes enceintes infectées par le VIH participant à l’étude, quel que soit leur stade immunitaire ou clinique. Cette trithérapie était poursuivie à vie pour les femmes nécessitant cette combinaison de traitements antirétroviraux pour des raisons cliniques et/ou immunitaires et non poursuivie pour les autres femmes, avec un schéma d’interruption minimisant les résistances aux ARVs.

Les critères d’évaluation de comparaison des deux interventions postnatales étaient la survie à 9 mois des enfants non infectés, le taux d’infection par le VIH et la mortalité des enfants dans chaque groupe. La présence de facteurs confondants a été recherchée en effectuant une analyse de variance car la randomisation était impossible pour des raisons éthiques.

Dans l’étude AMATA, parmi les 532 enfants inclus, 227 (43%) étaient allaités et 305 (57%) recevaient du LA, 7 enfants furent infectés par le VIH (1,3%) dont 6 in utero (3 enfants par groupe). Un enfant fut infecté par l’AM correspondant à un risque cumulatif postnatal de 0,5% [IC95% 0,1–3,4%; P 0,24]. Ce taux de transmission reste parmi les plus bas dans un pays à ressources limitées même en comparant avec d’autres études où la trithérapie fut aussi utilisée durant l’AM. Ces études furent publiées après le début de l’enrôlement des patientes dans l’étude rwandaise AMATA en 2005.

La différence de mortalité à 9 mois n’était pas statistiquement différente dans les 2 groupes avec 3,3% (95% IC 1,6–6,9%) pour les enfants allaités et 5,7% (95% IC 3,6–9,2%) pour les enfants recevant du LA (P= 0,20).

Cette étude renforce la notion que l’AM sous trithérapie antirétrovirale (HAART) reste une approche à recommander dans les contextes où la mortalité infantile est élevée. Cette prévention postnatale permet non seulement de réduire très efficacement la transmission du VIH de la mère à l’enfant en préservant les avantages de l’AM et en évitant les risques du LA distribué dans des contextes d’hygiène précaire où un accès à l’eau potable est difficile.

Dans cette étude, l’efficacité de ces 2 interventions postnatales était comparable avec des taux de transmission et de mortalité semblables statistiquement.

La deuxième partie de ce travail, basée sur les résultats d’une cohorte d’enfants âgés de moins de 18 mois nés de mères infectées par le VIH permettait d’évaluer les signes cliniques présomptifs proposés par l’OMS en 2005. Ces signes

étaient créés afin de pouvoir effectuer le diagnostic clinique d’infection par le VIH chez les enfants exposés au virus VIH

dans les pays où les techniques moléculaires de PCR n’étaient pas accessibles. Les enfants nés de mères infectées par le

VIH gardent parfois des anticorps anti-VIH maternels jusqu’à l’âge de 18 mois sans être pourtant contaminés par le VIH/SIDA. Avant cet âge, la confirmation de l’infection par le VIH repose sur la démonstration de la présence d’ADN proviral ou ARN par la technique PCR. La mortalité précoce des nourrissons infectés par le VIH est élevée, il est important de pouvoir bénéficier d’ARVs dès le diagnostic précoce de l’infection.

Les signes cliniques de présomption d’infection par le VIH chez l’enfant exposé (sérologie VIH +) de moins de 18 mois ont été proposés en 2005 par l’OMS et modifiés en 2006 mais ne furent jamais évalués.

Cette étude transversale comprenant 236 enfants de moins de 18 mois ayant une sérologie VIH positive consistait à évaluer la sensibilité (76,6%) et la spécificité (52,7%) de ces signes cliniques en confirmant leur statut infectieux réel par le test PCR pour le VIH, test de référence.

Cette spécificité basse inquiétante était liée aux enfants présentant des signes cliniques similaires bien que non infectés par le VIH mais souvent carencés par manque d’apport calorique et/ou souffrant d’une forme avancée de tuberculose extra pulmonaire ou d’autres affections chroniques. Ces enfants cachectiques pouvaient présenter les mêmes signes cliniques que les enfants infectés par le VIH car ils avaient une baisse de leur immunité cellulaire due à la MPE.

Dans la première partie de ce travail, l’étude AMATA a montré 2 façons efficaces de diminuer la transmission du VIH de la mère à l’enfant.

Dans la deuxième partie, on a évalué une méthode de diagnostic clinique précoce proposé par l’OMS afin de détecter les enfants infectés par le VIH en l’absence de test virologique PCR mais la basse spécificité indique la nécessité d’améliorer cette méthode diagnostique.

Doctorat en Sciences médicales
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Thesis (PhD)-- Stellenbosch University, 2013.
ENGLISH ABSTRACT: The emergence and re-emergence of viral human pathogens from wildlife sources in the recent past has led to increased studies and surveillance of wildlife for potentially zoonotic agents in order to gain a better understanding of the pathogens, their sources as well as events that may lead to viral emergence. Of the >1407 known human pathogens, 13% are classified as emerging or re-emerging, and 58% as zoonotic; 37% of the (re-)emerging and 19% of the zoonotic pathogens are RNA viruses, accounting for the majority of recently emerged infectious diseases with a zoonotic origin, such as HIV, Ebola, Hendra, Nipah, Influenza and SARS. This study focusses on potentially zoonotic viruses hosted by rodents (Muridae family), shrews (order previously known as Insectivora/Soricomorpha, now reclassified as Eulipotyphla) and bats (order Chiroptera). Rodents and bats represent the largest (~40%) and second largest (~25%) mammalian orders and both occur on every continent except Antarctica. Together, the three mammalian orders investigated represent the most relevant potential sources of new zoonoses. In this study I investigated the occurrence of astroviruses, arenaviruses, coronaviruses and hantaviruses in South African small mammal species belonging to the orders mentioned above. These viruses have either been implicated in recent emerging zoonotic events or are considered to have the potential to cause cross-species transmissions resulting in a zoonotic event. In the first part of the study specimens collected from various bat, rodent and shrew species were screened for viral sequences by broadly reactive PCRs; positive samples were characterised by sequencing and sequence analysis. A separate part of the study focussed on hantavirus disease in humans: a seroprevalance survey was conducted to determine the presence of hantavirus antibodies in the local population. Additionally, acutely ill patients with potential hantavirus disease were tested in an attempt to identify possible acute infections and define clinical hantavirus disease in South Africa. Screening of rodent and shrew specimens resulted in the identification of eight novel arenavirus sequences. Seven of the sequences are related to Merino Walk virus, a recently identified South African arenavirus, and the eighth sequence represents a novel lineage of Old World arenaviruses. Screening of bat specimens resulted in the identification of highly diverse novel astrovirus and coronavirus sequences in various South African bat species, including the identification of a viral sequence closely related to the recently emerged Middle East Respiratory Syndrome coronavirus. While the study did not identify hantavirus infections in any of the acutely ill patients, it found seroprevalences similar to those observed in Europe and West Africa. The results obtained highlight the importance of small mammals in the emergence of potential zoonoses and further reinforce the importance of viral surveillance of relevant wildlife species. Further in-depth studies of naturally infected reservoir host populations are required in order to gain a better understanding of virus-host dynamics and the events that lead to virus emergence.
German Research Foundation (DFG) (project number: KR1293/9-1/13-1)
The Polio Research Foundation and the NHLS Research
Harry Crossley Foundation, the Polio Research Foundation and Stellenbosch University for granting scholarships and bursaries for PhD.

The convergence of compelling evidence that transmission of HIV from a pregnant woman living with HIV to her foetus can be significantly interrupted due to advances in antiretroviral and obstetrical interventions, and worrisome epidemiologic data documenting a rise in HIV infection among Canadian women, spurred the development in Canada and world wide of policies and programmes aimed at increasing the number of pregnant women who are tested for HIV. Responding to innovative therapy reducing perinatal HIV transmission risk by increasing the number of pregnant women who agree to test for HIV is clearly an important prevention objective. However, the process must be accomplished in a way that is of most benefit to the pregnant woman herself and in a way that does not compromise a pregnant woman's rights to the established Canadian principles of HIV counselling and testing.
Working with pregnant women in Ontario, the province with the highest level of HIV infection among Canadian women, this thesis articulates and interprets their experiences of prenatal HIV counselling and testing and details their perspectives on best practices. The pregnant women's evidence-based recommendations for the re-design of prenatal HIV testing programmes are provided. These unique data have important utility for federal and provincial policy makers as HIV counselling and testing policies and programmes that encompass and are grounded in pregnant womens' experiences and perspectives are likely to be maximally acceptable and thereby increase the number of pregnant women who can be apprised of prophylactic treatment to take care of their own health needs as well as those of their unborn children.
In order for pregnant women to increase control over their own health and that of their unborn children, there is clear value in all pregnant women being afforded the opportunity to know their HIV status. However, the voices of the women in this study suggest that the autonomy rights of pregnant women may well be at risk in a programme in which the current emphasis is on potential HIV infection of the foetus rather than on potential or actual infection of the pregnant woman.

Orientadores: Egberto Ribeiro Turato, Helaine Maria Besteti Pires Mayer Milanez
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O objetivo geral deste estudo foi interpretar as significações psicossociais relatadas pelas mulheres no momento da descoberta da infecção pelo HIV na gestação, durante consulta de pré-natal especializado no Centro de Atenção Integral à Saúde da Mulher da Universidade Estadual de Campinas (Caism/UNICAMP). Os objetivos específicos foram conhecer as angústias e conflitos gerados após a informação sobre a presença da infecção pelo HIV, e discutir os significados da maternidade e a impossibilidade do aleitamento materno no contexto da infecção pelo HIV. Sujeitos e Métodos: Estudo exploratório, na particular abordagem Clínico-Qualitativa. A amostra de sujeitos foi construída pela técnica de saturação de dados, através de entrevista semidirigida de questões abertas. O grupo de sujeitos foi composto por gestantes com 22 a 31 anos de idade, entre 15 e 38 semanas de gestação, e que receberam o diagnóstico de HIV no início da gestação, sendo a maioria multípara, com situação conjugal estável e ocupação do lar. Resultados e Discussão: Nas falas destas mulheres ficou evidente que o momento da revelação do diagnóstico de HIV durante a gestação é carregado de intensas emoções, seguidas de fantasias e sentimentos conflitantes, com resistência da aceitação dos mesmos e medo intenso da transmissão vertical, mesmo sabendo que, utilizando a terapia anti-retroviral, a chance de infectar o bebê seria mínima. Emergiram também sentimentos de angústia e estigma relacionados ao impedimento da amamentação. Considerações Finais: Acreditamos que estar grávida na presença da infecção pelo HIV exige um duplo trabalho de redefinição subjetiva, na medida em que a mulher precisa se reconhecer como mãe e como portadora do HIV. Os resultados apontam que essas mulheres se deparam com circunstâncias adversas que envolvem o desejo de amamentar e, ao mesmo tempo, preservar a saúde de seus filhos
Abstract: Objectives - The general purpose of this study was to interpret psycho-social conceptions reported by women as they discover an HIV-infection during prenatal care at a specialized facility in a Woman's Health Center (Centro de Atenção Integral à Saúde da Mulher) of the University of Campinas (Caism/UNICAMP). Specific objectives were to recognize conflicts and afflictions generated after receiving an HIV diagnosis and discuss meanings of motherhood and proscription of breastfeeding in the setting of HIV infection. Methods: Exploratory study conducted in a clinical-qualitative way; the sample size was defined by the saturation of collected information. The sample was composed of recently diagnosed HIVinfected pregnant women (ages from 22 to 31 years, gestational ages from 15 to 38 weeks, mostly multiparous, housewives, and reporting stable relationships. Results and Discussion: From the analyzed data, it became clear that the experience of a newly found HIV infection diagnosis during pregnancy gives rise to intense emotions, fantasies and conflicted feelings, along with resistance in accepting emotions and great fear of mother-to-child transmission of HIV, even in face of minimal risks achievable by antiretroviral prophylaxis. Also emerged feelings of affliction and stigmatization related to suppression of breastfeeding. Final Considerations: We believe that pregnancy in the setting of HIV infection demands double amounts of effort in redefining one's self, in the sense that the woman must acknowledge herself as both a mother and a carrier of HIV. Results show that these women are faced with adverse circumstances involving the desire to breastfeed and, concomitantly, to preserve the health of their children
Mestrado
Ciencias Medicas
Mestre em Tocoginecologia

Magister Public Health - MPH
Human Immune-deficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) is a major public health problem. Globally, the number of new HIV infections is decreasing but the total number of people living with the disease is increasing. An estimated 5.7 million South Africans are currently living with the disease. The life expectancy of people living with HIV (PLHIV) in South Africa has slowly increased due to the availability of Anti-Retroviral Therapy (ART). The progressive "chronicity" of HIV may be associated with a variety of impairments and disabilities for people living with HIV. This emphasising the increasingly important role that physiotherapists play to minimize the disabling impact of the disease and improve quality of life for PLHIV. The aim of study was to determine the HIV/AIDS knowledge, attitudes and the occupational risk perception of physiotherapists practicing in the Eastern Cape Province, South Africa. This study utilized a cross sectional descriptive quantitative survey to collect data. The data was collected via a structured self-administered postal questionnaire. The questionnaires were captured in Microsoft Excel and analysed statistically using CDC Epi-Info version 3.5.1. Data was analysed descriptively and the chi-square test, T-tests and ANOVA was used to identify any statistically significant relationship between variables. The results of the study identified that the physiotherapists in the study have "high" general HIV related knowledge, although major gaps regarding HIV prevention and transmission still exists. The physiotherapists expressed a positive attitude towards PLHIV, while they perceive themselves to be at low risk of HIV transmission risk when managing PLHIV. The physiotherapists with more than 10 years' experience had significantly better HIV related knowledge compared to those with less than 10 years' experience while the attitudes of married physiotherapists towards PLHIV were significantly less favourable than those who were not married. There is a need for intervention strategies to address the HIV knowledge gaps of physiotherapists. Intervention strategies need to address physiotherapists HIV prevention and transmission knowledge.

Les virus de l'enroulement de la vigne (Grapevine leafroll-associated virus, GLRaV) sont répandus mondialement et transmis à la vigne uniquement par cochenilles (Coccoidea). En France, l'enroulement viral affecte particulièrement les vignobles des régions septentrionales.L'approche biologique de la vection a montré la capacité de Phenacoccus aceris à transmettre à la vigne les GLRaV-1, -3, -4, -5, -6, -9 et ceux du bois strié Grapevine virus A et B. Cette étude est la première démonstration de la transmission du GLRaV-6 et confirme l'absence de spécificité des cochenilles dans la transmission des Ampelovirus. Les larves néonates de P. aceris et de Neopulvinaria innumerabilis représentent un stade de développement efficace pour la transmission de ces virus. En conséquence, leurs capacités vectrices, associées à leur fort potentiel de dissémination anémophile, impliquent un risque important de dispersion naturelle de ces virus dans un vignoble infesté. Les relevés sur quatre parcelles distinctes montrent que Parthenolecanium corni, Pulvinaria vitis, Heliococcus bohemicus et P. aceris sont communes, chaque vignoble différant par la diversité spécifique, le taux de ceps infestés et l'abondance des cochenilles. L'étude épidémiologique prouve le rôle des cochenilles dans la dispersion de l'enroulement viral dans les vignobles septentrionaux. A Bonzon, la responsabilité de P. aceris dans la diffusion rapide du GLRaV-1 est mise en évidence. Cette découverte représente la première preuve en Europe d'une dispersion naturelle du GLRaV-1. A Marsannayla-Côte, l'incidence du GLRaV-1 reste faible, la colonie de P. aceris ne semblant avoir qu'un rôle très limité dans la diffusion de la maladie. L'épidémiologie moléculaire à Bonzon révèle une diversité génétique importante du GLRaV-1 à l'échelle parcellaire et fournit pour la première fois des données sur le polymorphisme génétique d'une population de GLRaV-1 ayant été dispersée par des cochenilles.

Thesis (MBA (Business Management))--Stellenbosch University, 2008.
The government has identified the need to transform the South African economy from one that is primarily resource based to one that is knowledge-based and has formulated a 10 year plan in order to accomplish this objective. The plan involves the creation and funding of five theme-specific consortium-based centres of competence that focus on the five top national health priorities, linked to the growth of the local pharmaceutical industry. This research study proposed that if collaboration and communication between academic researchers and the biotechnology industry in South Africa was improved it would lead to an increase in the development of innovative products for HIV/AIDS prevention and treatment. The objective of the study was the development of a strategy for a centre of competence for HIV research and development that brings together academic researchers and industry in a public private partnership and that will enable the proposal to be tested. Centre of competence programmes in both developed and developing countries, including Sweden, Austria and Estonia, were reviewed. The success factors for the various programmes were discussed. The strategic planning analysis began by considering the mandate of the CoC for HIV R&D. The requirements and expectations of the DST in establishment of the centres of competence were examined. An analysis of the external environment relevant to the South African biotechnology industry was then performed. This involved a detailed macro-environmental analysis in which political, economic, social, technological and environmental factors were considered. It was followed by an analysis of the current biotechnology industry in South Africa. The industry’s dominant economic features were identified as were its future driving forces. In a competitive environment analysis the South African biotechnology industry was found to be extremely competitive. Two industry issues, price controls and access to capital, were identified and discussed. The industry key success factors identified included access to large and sustained capital, attracting and retaining talented employees, an efficient and high quality regulatory authority, continued government support, productive and appropriate partnerships and skilled intellectual property management. An internal environment analysis was performed which identified competencies and resource strengths of the CoC for HIV R&D, including the high level of academic research in the HIV/AIDS field and expertise in clinical trials of HIV/AIDS products. Competitive deficiencies and resource weaknesses identified included shortages of skills and talent and the lack of co-ordination for funding of HIV/AIDS research. The analysis of the internal environment continued with the examination of the internal value chain of the CoC for HIV R&D. This consisted of discovery, pre-clinical development and clinical development stages. Gaps in the value chain were identified, including the lack of facilities for high-throughput screening of compounds for anti-HIV activity, lack of pre-clinical testing facilities and lack of manufacturing plants capable of producing products for use in clinical trials. The results of the external and internal environment analysis were used in a SWOC analysis and a number of strategies were identified to capitalise on opportunities and to address challenges. A subsequent competitive strength assessment identified a competitive advantage in the formation of the CoC for HIV R&D. In addition a number of strategic issues facing the centre were identified and ways to address or manage the issues were proposed. The strategic planning process was completed by the selection of a strategic approach for the CoC for HIV R&D. The study concluded that a PPP of public and private organisations operating under a corporate strategy of related diversification developed and implemented by the CoC for HIV R&D, would be suitable for testing the Proposal. The study’s conclusion also highlighted the need to ensure that the CoC for HIV R&D receives a long term commitment of funding from public sources, and that is managed by an experienced team with strong leadership skills. Important strategies emerging from the study and specifically from the SWOC analysis were development of a national HIV research plan and funding of the highest priority projects; focusing research funding on research with greatest potential for generation of HIV/AIDS products; and establishment of new technology platforms to fill gaps in the value chain. Finally, a number of recommendations were made for implementation of the results of this study or as the basis for further study.