Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity. The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as "fitness") in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background. Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs. In peripheral blood mononuclear cells, replication rates (means +/- standard deviations) of RT recombinants were 72.5% +/- 27.3% and replication rates of PR recombinants were 60.5% +/- 33.6% of the rates of NL4-3. PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% +/- 23.5% of NL4-3 replication rates. Cloning of the cognate RT improved fitness of some PR mutant clones. For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% +/- 10.7%, and integrase content diminished by 23.3% +/- 18.4%), a novel mechanism for a decreased-fitness phenotype. Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT. Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure. These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Wolbachia density and virulence attenuation after transfer into a novel host

The factors that control replication rate of the intracellular bacterium Wolbachia pipientis in its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain of Wolbachia that is known to over-replicate and shorten the lifespan of its Drosophila melanogaster host, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachia association. After transinfection (the transfer of a Wolbachia strain into a different species) of the over-replicating Wolbachia popcorn strain from D. metanogaster to Drosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect in D. simulans attenuated only slightly and was comparable to that induced in D. metanogaster. This study reveals a strong host involvement in Wolbachia replication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.

Dissecting the molecular interaction between hepatocytes and Plasmodium liver parasites

Dissertation presented to obtain the Ph.D degree in Biology

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Discordant increases in CD4+ T cells in human immunodeficiency virus-infected patients experiencing virologic treatment failure: role of changes in thymic output and T cell death.

Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ~319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.

Organ tropism during the acute and chronic phases of Trypanosoma cruzi infection in BALB/c mice

The aim of the present study was to investigate the presence of Trypanosoma cruzi in the heart, liver, lung, and kidneys, using hemoculture and PCR analysis, of mice infected with different parasite strains during the acute and chronic phases of infection. Parasitemia curves revealed strain-specific biological behaviors. For the Y and JLP strains, the acute phase of infection started at days six and ten post-infection, parasitemia peaked at days seven and 15 post-infection, the chronic phase started at days nine and 28 post-infection, and animals started dying at days 19 and 120 post-infection, respectively. When the two strains were compared, the JLP strain exhibited reduced and slower replication rates associated with a delayed peak of parasitism and reduced parasite burdens. However, parasites were detected in all studied organs using PCR analysis. The capacity of both strains to infect different organs likely influences disease pathogenesis.

Análises moleculares da região controle do DNA mitocondrial de astrocitomas na população paraense

O câncer do sistema nervoso central representa 2% de todas as neoplasias malignas na população mundial e 23% dos casos de câncer infantil. No Brasil, estimam-se 4.820 casos deste câncer em homens e 4.450 em mulheres para o ano de 2012. Os gliomas são tumores do sistema nervoso central formados a partir de células da glia e somam mais de 70% do tumores cerebrais. A propriedade mais importante dos gliomas é sua capacidade de evasão imunológica. Idade, etnia, gênero e ocupação podem ser considerados fatores de risco para o surgimento de gliomas, e são duas vezes mais frequentes em afro-americanos. O astrocitoma é o tumor glial mais frequente, constituindo cerca de 75% dos casos de gliomas. Estes tumores são classificados em quatro graus, de acordo com a Organização Mundial de Saúde. O DNA mitocondrial está relacionado com o desenvolvimento e a progressão de vários tipos de tumores. A mitocôndria é responsável pelo balanço energético celular e está envolvida no disparo da apoptose em resposta ao estresse oxidativo. Mutações na D-LOOP podem alterar a taxa de replicação do DNA e aumentar o risco do desenvolvimento do câncer. Neste estudo foram analisadas 29 amostras de astrocitoma classificados de acordo com a OMS. Nossos dados sugerem que os astrocitomas de baixo grau podem estar relacionados à herança genética, tornando portadores de alguns polimorfismos ou mutações específicas, mais suscetíveis ao risco de desenvolver a doença, e os de alto grau podem estar relacionados à exposição prolongada aos agentes carginógenos. Foram identificados polimorfismos e mutações onde alguns apresentaram relação com o risco do desenvolvimento de astrocitomas e com a progressão da doença. A inserção de dois ou mais nucleotídeos nas regiões de microssatélites pode causar sua instabilidade e contribuir com o surgimento do câncer. A deleção no sítio 16132 pode ser um marcador para astrocitoma de alto grau, assim como a inserção de duas ou mais citosinas no sítio 16190 pode ser um marcador específico para astrocitomas. As mutações heteroplásmicas podem ser determinantes para o surgimento e/ou progressão de astrocitomas de alto grau.

Precision of Usual Food Intake Estimates According to the Percentage of Individuals with a Second Dietary Measurement

Background Statistical methods for estimating usual intake require at least two short-term dietary measurements in a subsample of the target population. However, the percentage of individuals with a second dietary measurement (replication rate) may influence the precision of estimates, such as percentiles and proportions of individuals below cut-offs of intake. Objective To investigate the precision of the usual food intake estimates using different replication rates and different sample sizes. Participants/setting Adolescents participating in the continuous National Health and Nutrition Examination Survey 2007-2008 (n=1,304) who completed two 24-hour recalls. Statistical analyses performed The National Cancer Institute method was used to estimate the usual intake of dark green vegetables in the original sample comprising 1,304 adolescents with a replication rate of 100%. A bootstrap with 100 replications was performed to estimate CIs for percentiles and proportions of individuals below cut-offs of intake. Using the same bootstrap replications, four sets of data sets were sampled with different replication rates (80%, 60%, 40%, and 20%). For each data set created, the National Cancer Institute method was performed and percentiles, Cl, and proportions of individuals below cut-offs were calculated. Precision estimates were checked by comparing each Cl obtained from data sets with different replication rates with the Cl obtained from original data set. Further, we sampled 1,000, 750, 500, and 250 individuals from the original data set, and performed the same analytical procedures. Results Percentiles of intake and percentage of individuals below the cut-off points were similar throughout the replication rates and sample sizes, but the Cl increased as the replication rate decreased. Wider CIs were observed at 40% and 20% of replication rate. Conclusions The precision of the usual intake estimates decreased when low replication rates were used. However, even with different sample sizes, replication rates >40% may not lead to an important loss of precision. J Acad Nutr Diet. 2012;112:1015-1020.

HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus)

Cotton rats (Sigmodon hispidus and S. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat and S. fulviventer is more susceptible than S. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorly in vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 × 105 cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. Neonate S. fulviventer were not more susceptible to infection than adults. In vitro culture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase–PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improved in vivo.

In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

l-Arginine-dependent suppression of apoptosis in Trypanosoma cruzi: Contribution of the nitric oxide and polyamine pathways

Until recently, a capacity for apoptosis and synthesis of nitric oxide (⋅NO) were viewed as exclusive to multicellular organisms. The existence of these processes in unicellular parasites was recently described, with their biological significance remaining to be elucidated. We have evaluated l-arginine metabolism in Trypanosoma cruzi in the context of human serum-induced apoptotic death. Apoptosis was evidenced by the induction of DNA fragmentation and the inhibition of [3H]thymidine incorporation, which were inhibited by the caspase inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD-CHO). In T. cruzi exposed to death stimuli, supplementation with l-arginine inhibited DNA fragmentation, restored [3H]thymidine incorporation, and augmented parasite ⋅NO production. These effects were inhibited by the ⋅NO synthase inhibitor Nω-nitroarginine methyl ester (l-NAME). Exogenous ⋅NO limited DNA fragmentation but did not restore proliferation rates. Because l-arginine is also a substrate for arginine decarboxylase (ADC), and its product agmatine is a precursor for polyamine synthesis, we evaluated the contribution of polyamines to limiting apoptosis. Addition of agmatine, putrescine, and the polyamines spermine and spermidine to T. cruzi sustained parasite proliferation and inhibited DNA fragmentation. Also, the ADC inhibitor difluoromethylarginine inhibited l-arginine-dependent restoration of parasite replication rates, while the protection from DNA fragmentation persisted. In aggregate, these results indicate that T. cruzi epimastigotes can undergo programmed cell death that can be inhibited by l-arginine by means of (i) a ⋅NO synthase-dependent ⋅NO production that suppresses apoptosis and (ii) an ADC-dependent production of polyamines that support parasite proliferation.

Phosphatidylserine-containing liposomes:modulators of rhinovirus induced inflammatory responses

Background: Human rhinoviral infections are major contributors to the healthcare burden associated with acute exacerbations of asthma. We, and others have recently demonstrated that rhinovirus (RV)-induced inflammatory responses are mediated by multiple signalling mechanisms, such as IL-1/MyD88 (1) and TLR3/RIGI (2). We have also previously published work showing that TLR signalling is effectively inhibited by phosphatidylserine-containing liposomes (SAPS), through the disruption of membrane microdomains (3). Evidence has also suggested that membrane microdomains may influence infections with RV. In this study, we explored the ability of SAPS to modulate responses to the natural viral pathogens, RV-1B and RV-16. Method: The immortalized bronchial epithelial cell line, BEAS-2B or primary bronchial epithelial cells were infected with RV-1B or RV-16 at a TCID50/ml of 19107 for 1 h. Immediately following infection, various concentrations of SAPS were added and changes in cytokine release were measured at 24 h. SAPS remained present throughout. Type I and III interferon (IFN) expression and rates of viral replication were measured by quantitative PCR. Virus quantification was also performed using a viral CPE assay, and IFN signalling was measured by western blot. Liposome stability was characterised and intracellular trafficking of fluorescently labelled SAPS in BEAS-2B cells was investigated using confocal microscopy. For in vivo studies, female wt Balb/c mice were pre-treated with SAPS for 2 h prior to infection with RV as previously described and changes in BAL cell number, BAL cytokine production and viral replication were quantified (4). Results: Characterisation of SAPS liposomes by mass spectrometry showed no obvious signs of oxidation over the time period tested, and liposome size remained constant. Preliminary confocal studies revealed that SAPS was rapidly internalised within the cell and was found to associate with intracellular compartments such as the early endosome and golgi. Viral infected BEAS-2B cells co-incubated with SAPS, showed notably impaired responses to RV as assessed by release of CXCL8 and CCL5. SAPS also reduced RV-induced IFNb production and STAT-1 phosphorylation, without significantly influencing viral replication rates. Modest increases in viral particle production were only observed at 48 and 72 h time points. Suppression of viral-induced cytokine production was also observed in primary bronchial epithelial cells and pilot in vivo studies showed that SAPS results in reduced KC production at 24 h post viral infection, and this was associated with reduced neutrophil numbers within the BAL fluid. Conclusion: Our data demonstrates a potential means of modulating inflammatory responses induced by human rhinovirus.

RNA Virus Evolution: a Cross-scale, Disease Dynamic Perspective

RNA viruses are an important cause of global morbidity and mortality. The rapid evolutionary rates of RNA virus pathogens, caused by high replication rates and error-prone polymerases, can make the pathogens difficult to control. RNA viruses can undergo immune escape within their hosts and develop resistance to the treatment and vaccines we design to fight them. Understanding the spread and evolution of RNA pathogens is essential for reducing human suffering. In this dissertation, I make use of the rapid evolutionary rate of viral pathogens to answer several questions about how RNA viruses spread and evolve. To address each of the questions, I link mathematical techniques for modeling viral population dynamics with phylogenetic and coalescent techniques for analyzing and modeling viral genetic sequences and evolution. The first project uses multi-scale mechanistic modeling to show that decreases in viral substitution rates over the course of an acute infection, combined with the timing of infectious hosts transmitting new infections to susceptible individuals, can account for discrepancies in viral substitution rates in different host populations. The second project combines coalescent models with within-host mathematical models to identify driving evolutionary forces in chronic hepatitis C virus infection. The third project compares the effects of intrinsic and extrinsic viral transmission rate variation on viral phylogenies.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ~319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.