Made available in DSpace on 2014-06-11T19:34:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-18Bitstream added on 2014-06-13T20:27:47Z : No. of bitstreams: 1 moura_mf_dr_botfca.pdf: 469933 bytes, checksum: eccaa27134acf7a66cc0606cbff1deaa (MD5) / Os potyvírus constituem cerca de 90% das espécies conhecidas da família Potyviridae. No Brasil ocasionam sérios entraves em alface (Lactuca sativa L.) e em pimentão (Capsicum annuum L.), onde se pode citar o Lettuce mosaic virus – LMV e o Pepper yellow mosaic virus (PepYMV), respectivamente. Com o intuito de melhor compreender o reservatório natural de potyvírus em plantas invasoras, amostras foram coletadas em áreas produtoras de pimentão e analisadas utilizando-se antissoro anti-potyvirus (Agdia). Entre estas plantas positivas, destacou-se Solanum americanum Mill, onde foi verificada infecção mista do Cucumber mosaic virus e do Potato virus Y, e Commelina benghalensis L. em que foi encontrado um possível novo potyvírus com a maior identidade de nucleotídeos da proteína capsidial (62%) com a espécie Hardenbergia mosaic virus. Este potyvírus não foi transmitido por extrato vegetal, bem como por afídeos para plantas de pimentão e Nicotiana tabaccum TNN. Na região codificadora para a proteína capsidial do potyvirus não foi encontrado o domínio DAG, relacionado a transmissão por afídeos. Visando encontrar possíveis fontes de resistência ao Lettuce mosaic virus - LMV, genótipos foram inoculados com o isolado LMV-AF-199 (LMV-Most) e o fator de iniciação de tradução eucariótico eIF4E destes genótipos analisado. Em Calona e Salinas-88, conhecidas previamente como portadoras dos genes recessivos mol1 e mol2 foram observados sintomas em todas as plantas inoculadas e verificado o padrão típico do eIF4E1 e eIF4E2, respectivamente... / The Potyvirus genus corresponds to 90% of known species of the Potyviridae family. In Brazil potyviruses causes serious problems in lettuce (Lactuca sativa L) and in pepper crops (Capsicum annuum L.), which we can highlight Lettuce mosaic virus – LMV and Pepper yellow mosaic virus (PepYMV), respectively. To increase knowledge about the natural reservoir of potyviruses in weeds, samples were collected from a pepper producer area and analyzed for potyvirus using antiserum anti-potyvirus (Agdia). Solanum americanum Mill was identified as a host for Cucumber mosaic virus and Potato virus Y. In Commelina benghalensis L. a possible new species of potyvirus was found with higher nucleotide identity of the coat protein (62%) with Hardenbergia mosaic virus. This potyvirus could not be transmitted by aphids to sweetpepper and... (Complete abstract click electronic access below)

It is shown here that the ability of B lymphocytes to act as supportive host cells for virus infections requires they be activated from the resting Gostage of the cell cycle. I have used a series of activation regimens, which allow B cells to progress to different stages in their activation/differentiation pathway toward antibody secretion, in order to evaluate the extent of activation required to support vesicular stomatitis or Newcastle disease virus infections. At least three distinct phases during B cell activation which affected VSV infection were defined. Freshly isolated resting murine splenic B cells in the Go phase of the cell cycle do not support VSV, assessed by protein synthesis, infectious center formation, and PFU production. Small B cells cultured for 48 hours without stimulation still do not support VSV. B cells stimulated with the lymphokines found in Con A activated supernatants from splenic T cells or cloned T cell lines transited into the G1 phase of the cell cycle but remain refractory to VSV. These VSV non-supportive B cell populations do take up virus particles and transcribe viral mRNAs which can be translated in vitro, suggesting a translational block to VSV. B cells stimulated into the S phase of the cell cycle with anti-immunoglobulin synthesize VSV proteins and increased numbers of infectious centers, but only low level PFU synthesis (center) is observed. Co-stimulation with anti-Ig and lymphokines, which supports differentiation to antibody secretion, enhanced PFU synthesis without further increasing the number of infected B cells. LPS, which activates B cells directly to antibody secretion by a pathway different from anti-Ig, induced infectious centers, and PFUs at levels comparable to those seen when stably transformed permissive cell lines are infected. Co-stimulation of LPS activated B cells with the same lymphokine populations that enhance PFU production when anti-Ig is used as a stimulator suppresses PFU production completely, suggesting that anti-Ig and LPS activated B cells are differentially responsive to lymphokines. NDV infection of murine B cells differed markedly from VSV infection, as all B cell populations examined gave a similar response pattern. NDV viral proteins were synthesized by B cells in each of the activation states previously described, even freshly isolated B cells. Infectious center formation increased up to 5-fold over the levels observed with unstimulated B cells after anti-Ig or LPS activation. However, PFU synthesis was low (center) for all B cell populations. These results suggest that these two similar viruses may be dependent on different host cell factors and that these factors are induced for VSV but not NDV by the B cell activators employed here or that the process of infection of B cell by these two viruses induces different cellular responses.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology, 2012 / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi. Routine water quality analysis however, does not include monitoring for viral contaminants, as this process is hampered by the lack of simple, reliable, time- and cost-effective testing methods to concentrate and detect viral pathogens. The primary aim of this study was thus to establish and optimise routine monitoring techniques for the detection of rota-, adeno- and enteroviruses in the Berg- and Plankenburg Rivers, Western Cape. Initially, various concentration and extraction methods were compared for the optimum recovery of viruses from spiked water samples. One hundred milliliter water samples were spiked with one milliliter rotavirus and two milliliters adenovirus control virions (Coris Bioconcept, Gembloux, Belgium). Optimisation testing of enterovirus was however, not completed due to the unavailability of a positive control. Four viral concentration techniques, namely the Silicon dioxide (SiO2) method, positively charged, negatively charged and the mixed-ester filters, were compared. Various nucleic acid extraction methods were also employed to establish which method would provide optimum yields for both DNA and RNA nucleic acids. The extraction techniques included the TRIzol method (Invitrogen, California, USA) for RNA extraction, the Roche High Pure PCR Template Preparation kit (Roche, Mannheim, Germany) for DNA extraction, and the QIAamp Ultrasens Virus kit (Qiagen GmbH, Hilden, Germany) for simultaneous RNA and DNA extraction. The use of virus specific primers within the PCR technique was also optimised. In addition, gene specific primers and oligo(dT)15 primers were tested and compared to establish which primers would yield the best results since gene specific primers are said to be more sensitive than oligo(dT)15 primers (van Pelt-Verkuil et al., 2008) when synthesising cDNA (rotavirus). The SiO2 concentration method yielded variable results when it was used with the various nucleic acid extraction techniques in this study, since positive PCR results were obtained when used in combination III with some techniques, while negative results were obtained with others. Similarly, variable results were also obtained when negatively charged filters were used to concentrate virus particles, and when this method was used in conjunction with various virus nucleic acid extraction techniques to identify different viruses by RT-PCR and PCR. Results for the non-charged mixed-ester filter were comparable to the positively charged filters when used in conjunction with the various nucleic acid extraction techniques in this study. Both these techniques yielded the highest viral particle concentration from the spiked water samples. Pilot study results indicated the presence of rotavirus and adenovirus detected by RT-PCR and PCR respectively, when filtering through the positively charged filter. The positively charged filter/QIAamp UltraSens virus kit combination was found to be the optimum combination when analysing the spiked water results and was then employed for the concentration of virus particles in the river water samples collected from the Plankenburg- and Berg River systems throughout the study period. The expected PCR product of 346 bp for rotavirus was absent in all 72 river water samples analysed for both river systems. In contrast to the PCR results obtained for rotavirus, the expected product of 261 bp for adenovirus was detected in 22 (30.5%) samples collected throughout the study period. Fifteen of the 22 adenovirus positive samples were found in the Plankenburg River (distributed over all sites), while seven of the 22 adenovirus positive samples were found in the Berg River (all sites). A nested PCR was used to detect enterovirus in the river water samples collected from both river systems throughout the study period. In the first round of the enterovirus PCR 15 river water samples (at various sites for both river systems) yielded a faint 513 bp product. Further amplification by nested PCR then yielded 13 (18.1%) positive nested PCR products of 297 bp. The incidence of adenovirus and enterovirus in river waters reported in the current study and the Van Heerden et al. (2003) investigation motivates for similar studies to be conducted in drinking water, dam water used for recreational purposes as well as rainwater, which is gaining popularity as a sustainable water source.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2012. / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi.

L'infection par le cytomégalovirus humain (HCMV) est une cause majeure de mortalité chez les patients immunodéprimés et représente la première cause d'infection congénitale. HCMV est un virus complexe qui s'est adapté au système immunitaire humain en développant de multiples mécanismes d'évasion. L'infection primaire à HCMV est associée à une réplication virale prolongée avant l'établissement de la latence. Il a été montré que cette intense réplication lors de la phase initiale de l'infection était associée à une épuisement fonctionnel des lymphocytes T CD4 spécifiques du virus. Alors que les anticorps jouent un rôle dans la limitation de la dissémination virale et la prévention de l'infection à HCMV, les réponses des lymphocytes B sont peu caractérisées. Dans le présent travail, nous avons étudié l'impact de l'infection à HCMV sur le phénotype et la fonctionnalité des sous-populations de LB du sang circulant chez une cohorte de femme enceintes avec une primo-infection par HCMV en utilisant comme contrôles des sujets sains séropositifs et séronégatifs pour HCMV ainsi que des femmes enceintes séronégatives. Nous montrons que l'infection primaire par HCMV induit une expansion significative et prolongée de deux sous-populations de LB :les LB mémoires activés (CD27+CD21low) et mémoires atypiques (CD27-CD21low), précédemment décrites lors d'infection chroniques. Les LB mémoires atypiques démontrent des signes d'épuisement fonctionnel comme en témoigne une expression élevée de récepteurs inhibant le BCR et une moindre réponse à la stimulation in vitro mesurée par la production de TNF-α. Les expansions de ces deux sous-populations sont corrélées entre elles et liées à la virémie. Ces résultats contribuent à la compréhension de la régulation des réponses des LB lors d'infections virales, en montrant que l'épuisement fonctionnel de LB, précédemment décrit lors d'infections chroniques, peut également survenir lors d'infections primaires.<p>Dans un deuxième temps, nous avons étudié l'acquisition des réponses B mémoires spécifiques de HCMV dirigées contre la principale glycoprotéine de surface, la glycoprotéine B (gB), et deux polypeptides du tégument. Lors de l'infection primaire par HCMV, la production d'anticorps neutralisant le virus, dirigés contre les glycoprotéines d'enveloppe, est retardée par rapport aux anticorps dirigés contre le tégument qui sont non neutralisant. Nous montrons que le phénotype des LB mémoires spécifiques de gB est différent de celui des LB mémoires spécifiques du tégument. La majorité des LB mémoires spécifiques de gB exprime un phénotype CD27+CD21+ alors que la majorité de ceux du tégument exprime le phénotype CD27+CD21low. Nous montrons par la suite chez des sujets sains que ces deux sous-populations de LB mémoires présentent des différences phénotypiques, au niveau de l'expression de récepteurs liés au "trafficking" cellulaire ainsi qu'au niveau de la fonctionnalité. Les LB mémoires CD21low, contrairement au LB mémoires CD21high, expriment des taux bas des récepteurs CXCR5 et CCR7, qui permettent la migration vers les centres germinatifs, mais des taux élevés de CD11c promouvant la migration vers les tissus périphériques. Après stimulation in vitro, les LB mémoires CD21low vont avoir une capacité de production d'immunoglobulines immédiate mais une réponse proliférative plus faible comparée aux LB mémoires CD21+. Nous démontrons la relevance de cette division des LB mémoires sur base de l'expression du CD21 dans un modèle de vaccination de rappel contre la toxoïde tétanique (TT). Après rappel, nous observons une expansion significative de LB mémoires spécifiques de la TT exprimant un phénotype CD27+CD21lowCXCR5lowCD11chigh. Nous proposons ainsi un nouveau mécanisme de manipulation des réponses humorales par des pathogènes qui se traduit par une limitation de l'induction de réponses B effectrices. Nos travaux permettraient également une meilleure approche des réponses B mémoires physiologiques chez l'homme en proposant une classification des LB mémoires basées sur leur fonctionnalité et leur phénotype.<p><p>Human cytomegalovirus (HCMV) infection is a major cause of mortality in immunocompromised patients and is the first cause of congenital infection worldwide. HCMV is a complex virus that has developed multiples immune evasions mechanisms during its co-evolution with mankind. Although often asymptomatic, primary HCMV infection is associated with an intense and prolonged viral replication. It has been previously shown that this intense viral replication is associated with functional exhaustion of virus-specific CD4+ T cells. Although neutralizing antibodies limits viral dissemination and play a role in the prevention of HCMV infection, B cell responses during HCMV infection have been poorly studied so far.<p>In this work, we have studied the impact of HCMV infection on the phenotype and functionality of peripheral-blood B cell subsets in a cohort of pregnant women with a primary HCMV infection. Controls were healthy seronegative and seropositive HCMV donors and HCMV seronegative pregnant women. We show that primary HCMV infection induces a significant and prolonged expansion of two B-cell subsets, previously described in chronic infections :activated memory B cells (MBC) (CD27+CD21low) and atypical MBC (CD27-CD21low). Atypical MBC display signs of functional exhaustion with increased expression of inhibitory receptors and a lower response to in vitro stimulation as assessed by TNF-α production. Expansion of these two subsets are correlated and higher in subjects with detectable viremia. These results contribute to the understanding of the regulation of B cell responses during viral infections and indicate that B cell exhaustion, previously described during chronic infections, can be observed in primary infection.<p>Next, we have characterized the acquisition of HCMV-specific B cell responses directed against envelope glycoprotein B (gB) and two tegument polypeptides (pp150 and pp52). During primary HCMV infection, the production of neutralizing antibodies targeting envelope glycoproteins is delayed when compared to non-neutralizing anti-tegument antibodies. We show that gB and tegument-specific MBC have distinct phenotype during primary HCMV infection. The majority of gB-specific MBC have a CD27+CD21+ phenotype while the majority of tegument-specific MBC have a CD27+CD21low phenotype. We show that CD27+CD21+ and CD27+CD21low MBC express different pattern of chemokine receptors pattern but also have distinct functionality. CD27+CD21low MBC, on the contrary to CD27+CD21+ MBC, express low levels of CXCR5 and CCR7 that favor migration to lymph nodes and germinal centers but express high levels of CD11c that promotes migration to inflammatory tissues.<p>In vitro stimulation of sorted subsets of healthy individuals indicates that CD27+CD21low MBC have higher capacity of immediate immunoglobulin production but a lower proliferative potential as compared to CD27+CD21+ MBC. We further show the relevance of a division of MBC subsets based on CD21 expression in a model of TT booster immunization. Following booster immunization, a significant expansion of TT-specific MBC expressing the phenotype CD27+CD21lowCXCR5lowCD11chigh is observed. <p>We propose that HCMV manipulates the host humoral response by limiting the induction of gB-specific CD27+CD21low "effector" MBC. Our work also indicates that human MBC physiological responses should be studied according to their respective phenotype and functions.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Abstract Viral infections are common in childhood and a usual cause for hospitalization. Viruses are easily transmitted among children both in pediatric wards and in other child care facilities, like child day-care centers. Hand hygiene is an important part of prevention of the transmission of viruses. Since hospitalization times are getting shorter, hospital-associated infections often manifest after discharge. The aim of the study was to evaluate the magnitude of hospital-associated infections during and after hospitalization in pediatric wards with a focus on the effect of the ward structure. Data were collected during two periods of two years in the pediatric infectious diseases ward in Oulu University Hospital; data collection in the latter period was done using electronic follow-up methods. A two-year study period was also carried out in University Children’s hospital in Basel and in North Karelia Central Hospital in Joensuu. Paper questionnaires and electronic questionnaires were compared as methods of doing continuous surveillance of hospital-associated infections during and after hospitalization. The safety of alcohol-based hand gels in children’s use was studied using alcometer measurements after hand rub. Experiences on the use of alcohol-based hand gels in child day-care centers were collected by interviewing the personnel with questionnaires. Altogether 5.8 to 17.5% of hospitalized children (N=7046) got a hospital-associated infection; 65 to 93% of the infections became evident after discharge. The number of hospital-associated infections was lowest in wards where single rooms and cohorting based on infection etiology were used. Increased risk for hospital-associated infection was associated with young age, longer hospitalization time and a shared room. A higher response rate was achieved with electronic follow-up compared with questionnaires on paper, 84 vs. 61%. The costs of follow-up were €13.61 and €15.07 per patient in electronic and conventional follow-up, respectively. Electronic follow-up decreased annual expenses by 17.1%. Alcohol-based hand gels were found to be safe in children’s use, as no absorption was detected despite several contacts between hands and mucous membranes. Personnel in child day-care centers were active in using hand rubs and found them useful and easy to use. Earlier, there had been one incident with fire when using matches while hands were still wet with alcohol. The majority of hospital-associated infections in children become evident after discharge, and electronic follow-up is useful in evaluating their magnitude. The number of hospital-associated infections can be decreased with single room bedding and careful infection control. Alcohol-based hand gels are safe in children’s hand hygiene. / Tiivistelmä Lapset sairastavat usein virusinfektioita, jotka ovat yleinen sairaalahoidon syy. Virukset leviävät herkästi lasten keskuudessa, lastentautien osastoilla ja lapsiryhmissä, kuten päiväkodeissa. Virusten leviämistä voidaan estää hyvällä käsihygienialla. Lyhyiden hoitoaikojen vuoksi osa virusten aiheuttamista sairaalainfektioista ilmenee vasta kotona. Tutkimuksen tarkoituksena oli selvittää sairaalainfektioiden määrä hoidon aikana ja kotiutuksen jälkeen sekä osastorakenteen vaikutus sairaalainfektioihin lastentautien osastoilla. Sairaalainfektioaineisto kerättiin Oulun yliopistollisen sairaalan lasten infektio-osastolla kahtena kahden vuoden jaksona, joista jälkimmäisessä käytettiin sähköistä seurantajärjestelmää. Lisäksi kahden vuoden aineistot kerättiin Pohjois-Karjalan keskussairaalan lastentautien osastolla ja Baselin yliopistollisen sairaalan lastenosastoilla. Paperikyselylomakkeilla ja sähköisesti tehdyn sairaalainfektioseurannan toteutusta verrattiin. Lisäksi tutkittiin alkoholikäsihuuhteiden käytön turvallisuutta lapsilla päiväkotiolosuhteissa. Alkoholin imeytymistä tutkittiin poliisin tarkkuusalkometrillä käsihuuhteen käytön jälkeen. Oulun kaupungin päiväkodeista kysyttiin käsihuuhteiden käyttökokemuksista kyselylomakkeilla. Sairaalainfektion sai 5,8-17,1&#160;% sairaalassa hoidetuista lapsista (N=7046). Infektioista 65-93&#160;% tuli oireisiksi kotiutuksen jälkeen. Sairaalainfektioiden määrä oli pienin osastoilla, jossa käytettiin yhden hengen huoneita ja potilaiden kohortointia taudinaiheuttajan mukaan. Sairaalainfektion riskiä lisäsivät lapsen nuori ikä, pitkä sairaalahoitoaika ja jaettu potilashuone. Sähköisessä sairaalainfektioseurannassa oli parempi kotiutuksen jälkeinen vastausprosentti kuin paperilomakkeilla, 84&#160;% vrt. 61&#160;%. Potilasta kohden kuluja tuli sähköisessä seurannassa 13,61 euroa ja paperilomakkeilla tehdyssä seurannassa 15,07 euroa. Sähköisen seurannan käyttö laski vuosikuluja 17,1&#160;%. Alkoholikäsihuuhteiden käyttö todettiin turvalliseksi lapsilla. Useista limakalvokontakteista huolimatta käsihuuhteen käytön jälkeen alkoholia ei imeytynyt verenkiertoon. Käsihuuhteiden käyttö päiväkodeissa on aktiivista, ja henkilökunta koki sen helpoksi ja hyödylliseksi. Aiemmin oli tapahtunut yksi vaaratilanne tulen kanssa tulitikkua sytytettäessä käsien ollessa vielä käsihuuhteesta märät. Lasten sairaalainfektioista suuri osa ilmenee kotiutuksen jälkeen, ja näiden infektioiden määrää voidaan arvioida sähköisellä seurantajärjestelmällä. Sairaalainfektioiden määrää voidaan vähentää käyttämällä yhden hengen huoneita ja huolehtimalla hyvästä hygieniasta. Alkoholihuuhteiden käyttö lasten käsihygieniassa on turvallista.

South Africa‘s high prevalence of HIV/AIDS coupled with a high crime rate and incidence of sexual violence necessitated the enquiry and study into the role of criminal law to address the wilful transmission of HIV.1 This study shows that criminal law can be used to punish offenders for wrongdoing and therefore finds application in the wilful transmission of HIV.2 The study distinguishes the dividing line between the justifiable use of criminal law and where use of the criminal law becomes discriminatory in nature and counterproductive to public health measures. The United Nations (hereinafter referred to as the UN) laid down guiding principles for countries to adopt when using the criminal law and stated that countries should use existing criminal law offences to prosecute intentional HIV infections.3 The South African Law Commission (hereinafter referred to as the SALC) endorses this approach. South Africa‘s use of the criminal law, in response to harmful HIV behaviour is in line with the UN recommendations as it uses the existing common law offences to prosecute the wilful transmission of HIV, namely murder, attempted murder and assault. Drawing from the writer‘s comparative study in Chapter Six below, South Africa, members of the Zimbabwean parliament, Canada, as well as the American Bar Association have all concluded that the use of specific HIV-related legislation creates some a form of stigmatization towards people living with HIV and is therefore not warranted. This study shows that criminal law has a role to play in the wilful transmission of HIV; however the creation of HIV specific legislation is not recommended and existing criminal law offences should be used to address harmful HIV related behaviour. Such an approach is in line with the guiding principles laid down by the UN and SALC.

Health is often defined in terms of absence of disease or pathological processes, but this is a definition of exclusion and incomplete. For example, SARS-CoV-2 viral load does not reliably predict disease severity, and so individuals must vary in their ability to control inflammation and maintain normal tissue homeostasis. This host defense strategy is called disease tolerance, and better understanding of disease tolerance mechanisms could change the way that we treat disease and work to maintain health. The first project presented in this dissertation found that after accounting for effects of age and sex, innate lymphoid cells (ILCs), but not T cells, were lower in adults and children sick with COVID-19 or MIS-C, independent of lymphopenia. Furthermore, abundance of ILCs, but not of T cells, correlated inversely with disease severity. These blood ILCs were shown to produce amphiregulin, a protein implicated in disease tolerance and tissue homeostasis, and the percentage of amphiregulin-producing ILCs was lower in males. These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance accounts for increased COVID-19 severity with age and in males. The second project describes a novel mouse model of epigenetic inheritance wherein paternal influenza A virus (IAV) infection results in less severe influenza disease in IAV infected offspring. This offspring phenotype was not attributable to differences in viral load, indicating a possible difference in disease tolerance. Paternal caloric deprivation decreased, and influenza B virus infection increased, offspring influenza disease severity, and in vitro fertilization demonstrated sperm are sufficient to transfer IAV-associated epigenetic inheritance phenotypes. These findings represent a foundation for further work that, by continuing to elucidate the mechanisms of disease tolerance and epigenetic inheritance, could provide novel therapeutic interventions to help promote and maintain health.

Since the first cases were reported over thirty years ago, great strides have been made to control disease progression in people living with HIV/AIDS. However, current estimates report that there are about 34 million individuals infected with HIV worldwide. Critical in the ongoing fight against this pandemic is the continuing development of highly active anti-retroviral therapies, ideally those with novel mechanisms of action. Currently, there are no medications approved for use that exploit the HIV-1 MA protein, despite its central role in multiple stages of the virus life cycle. This thesis sought to examine whether a highly conserved glutamate residue at position 99 in the understudied C-terminal helix of MA is required for HIV-1 replication. I characterized a panel of mutant viruses that contain different amino acid substitutions at this position using viral infectivity studies, virus-cell fusion assays, and immunoblotting. In doing so, I found that substitution of this glutamate with either a valine (E99V) or lysine (E99K) residue disrupted Env incorporation into nascent HIV particles, and abrogated their ability to fuse with target-cell membranes. In determining that the strain of HIV could affect the magnitude of E99V-associated defects, I identified a compensatory substitution at MA residue 84 that rescued both E99V- and E99K-associated impairments. I further characterized the MA E99V and E99K mutations by truncating HIV Env and pseudotyping with heterologous envelope proteins in an attempt to overcome the Env incorporation defect. Unexpectedly, I found that facilitating fusion at the plasma membrane was not sufficient to reverse the severe impairments in virus infectivity. Using quantitative PCR, I determined that an early post-entry step is disrupted in these particles that contain the E99V or E99K MA substitutions. However, allowing entry of mutant virus particles into cells through an endosomal route conferred a partial rescue in infectivity. As the characterization of this post-entry defect was limited by established virological methods, I designed a novel technique to analyze post-fusion events in retroviral infection. Thus, I present preliminary data regarding the development of a novel PCR-based assay that monitors trafficking of the viral reverse transcription complex (RTC) in an infected cell. The data presented in this thesis indicate that a single residue in MA, E99, has a previously unsuspected and key role in multiple facets of HIV-1 MA function. The pleiotropic defects that arise from specific substitutions of this amino acid implicate a hydrophobic pocket in MA in Env incorporation and an early post-entry function of the protein. These findings suggest that this understudied region of MA could be an important target in the development of a novel antiretroviral therapy.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.