Orientadores: Ricardo Pasquini, Marilda Mendonça Siqueira / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Medicina Interna / Resumo: Cistite hemorrágica (CH) é uma das mais freqüentes complicações em pacientes submetidos a transplante de medula óssea (TMO). Normalmente é atribuída ao emprego da droga ciclofosfamida durante o regime de condicionamento. Contudo, infecções virais também podem causar CH e, pacientes transplantados são de alto risco para estas infecções devido ao comprometimento de seu sistema imunológico. CH associada ao adenovirus tem se tornado uma seqüela reconhecida da imunossupressão. As manifestações clínicas mais freqüentes são febre, disúria, dor suprapúbica e hematúria macroscópica. Apesar de ser uma doença habitualmente autolimitada, ela freqüentemente está associada a um período de internamento prolongado, alto custo, significante desconforto para o paciente e necessidade de múltiplas transfusões sangüíneas. Neste trabalho realizou-se um estudo prospectivo para detecção de adenovirus em pacientes com CH nos primeiros 100 dias pós-transplante, compararam-se diferentes métodos laboratoriais e procurou-se uma correlação definitiva entre a excreção urinária de adenovirus e a CH. Estudaram-se 75 pacientes que receberam TMO no Hospital de Clínicas da UFPR, no período de julho de 1996 e junho de 1997. Realizou-se análise das amostras de urina de todos os pacientes na fase pré-transplante e apenas daqueles com hematúria micro ou macroscópica no pós-transplante. As urinas foram processadas para a detecção de adenovirus pelos métodos de cultura celular, ensaio imunoenzimático (EIE) e reação de polimerização em cadeia (PCR). Foram processadas 143 amostras de urina, 75 pré-transplante como ou sem hematúria e 68 pós-transplante apenas nas urinas com hematúria. Após o TMO a hematúria ocorreu em 38,9% dos pacientes, sendo com maior freqüência em paciente com transplante alogênico não consangüíneo e no primeiro mês póstransplante. No período pré-transplante, adenovirus foi isolado em um paciente que estava assintomático e, em três pacientes no pós-transplante. Esses apresentavam CH grau 3 ou 4 (grave), e estavam entre o segundo e terceiro mês pós-transplante. A sensibilidade do EIE para a detecção de adenovirus em urina foi de 30,0% e especificidade de 98,5%. A sensibilidade do PCR foi de 88,9% e especificidade de 59,0%. A imunohistoquímica (IH) dos pacientes com CH e adenovirus na urina que evoluíram para óbito demonstrou grande quantidade de antígenos desse vírus em túbulos renais confirmando a doença renal. / Abstract: Hemorrhagic cystitis is a major cause of morbidity during bone marrow transplantation (BMT) and is usually attributed to the use of high-dose cyclophosphamide in the preparation for BMT. However, viral infection can also cause hemorrhagic cystitis and, transplant patients are at high risk for infection because of the compromised immune system. Adenovirus-associated hemorrhagic cystitis has become a recognized sequels of immunossupression. Acute hemorrhagic cystitis due adenovirus has most frequently been found clinically manifested by fever, disuria, suprapubic pain and gross hematuria. Although adenovirus hemorrhagic cystitis is usually a self-limited disease, it's often associated with a prolonged and expensive hospital stay, significant patient discomfort and multiple blood product transfusion. The diagnosis of viral infection is usually determined by viral cultures. In recipients of BMT viremia and viruria are often observed in recipients without any symptoms. We designed a prospective study of adenovirus detection attempted 100 days after BMT from urine, compare different laboratory techniques and found the definite correlation between urinary adenovirus excretion and HC. The study included 75 patients who received BMT at the Clinical Hospital of Federal University of Parana from July 1996 to June 1997. The analysis of samples of urine was made of all patients before transplantation and every 10 days after BMT, if it was presented microscopic or macroscopic hematuria, until 100 days. The urine specimens were processed for adenovirus detection by culture in HEp-2 cells, PCR and enzyme-linked assay. It was processed 143 samples of urine, 75 pre-transplant with and without hematuria and 68 post-transplant, only of the urine with hematuria. After of BMT, hematuria occurred in 38,9% of patients, was more frequent in unrelated donor transplant and during first month period (60,7%). Adenovirus was isolated at one pre-transplant patient that was without symptoms and, in three post-transplant patients. All of them had hemorrhagic cystitis grade 3 or 4 (severe), and were at month 2 or 3 post-transplant. The sensitivity of EIA was 30,0% and specificity was 98,5% to adenovirus detection in urine samples. The sensitivity of PCR tests was 88,9% and specificity was 59,0%. The immunohistochemistry of kidneys of patients that died demonstrated adenovirus antigens in renal tubules thus confirming the renal disease.

Abstract Adenoviral gene transfer is a valuable tool in molecular biology research. In order to be an efficient and safe vector, adenovirus structure and infection mechanism as well as molecular biology of the used transgene need to be well studied. The aim of this study was to evaluate the role of adenovirus as a gene transfer vector from several perspectives. Adenovirus uses receptor-mediated endocytosis in order to enter the target cell. The effect of Rab5 GTPase on adenovirus entry and gene transfer efficiency was examined first. Next, adenovirus was used as an investigatory tool in the cardiovascular research, focused on clarifying the role of adrenomedullin (AM) in heart and vascular remodeling. Finally, a model of adenoviral gene transfer into skin fibroblasts was used. The role of Rab5 GTPase in the adenovirus endocytosis was examined in HeLa cells using Cy3-labeled adenovirus, and gene transfer efficiency using β-galactosidase encoding adenovirus. Rab5 increased both adenovirus uptake and gene transfer, whereas dominant negative Rab5S34N decreased both endocytosis and gene transfer. The data indicate that Rab5 is needed in mediating the adenovirus uptake into the target cell. In the rat heart, adenovirus-mediated AM gene transfer transiently improved systolic function both in vivo and in vitro. AM caused activation of translocation of protein kinases C ε and δ, whereas phosphorylation of p38 mitogen activated protein kinase was decreased in the left ventricle. AM significantly attenuated the development of angiotensin II-induced cardiac hypertrophy. In rats with myocardial infarction, AM enhanced dilatation of left ventricle and thinning of anterior wall. The role of AM in neointima formation was evaluated in rat artery after endothelial injury. Intravascular AM gene transfer decreased neointimal growth and increased neointimal myofibroblasts apoptosis. These results show that AM regulates left ventricular systolic function and remodeling in the heart, and plays a role in pathological vascular remodeling. Adenovirus-mediated lysyl hydroxylase (LH) gene transfer into skin fibroblasts of type VI Ehlers-Danlos syndrome patient and rat skin increased functional LH production, elevated LH activity, and human LH mRNA production both in vitro and in vivo. LH gene replacement therapy may thus lead to possibilities to improve skin wound healing in Ehlers-Danlos syndrome patients.

Abstract The progress in recombinant DNA technology has made possible the introduction of exogenous genetic material into cells. Gene therapy aims to correct a defective gene, introduce a therapeutic exogenous gene or a counteracting gene into somatic cells without modification of the germ-cell line. The most important technical interests in the field of gene therapy research have pertained to the development of safe and effective vectors and suitable methods for the delivery of the exogenous gene carrying vectors into the target cells. The aim of this study was to evaluate surgical methods used for gene delivery and to develop an effective gene transfer method for organ-specific gene transfer, primarily into the renal glomeruli. There are genetic and acquired diseases that are candidates for gene therapy. Alport syndrome is an X-chromosome-linked disease caused by a mutation in the type IV collagen α5 chain gene, which causes a defect of the glomerular basement membrane in the kidney, leading to progressive renal failure in males. This manifestation could theoretically be prevented by the transfer of a normal α5 chain gene into the renal glomerular cells. Cystic fibrosis and α1-antitrypsin deficiency are examples of pulmonary diseases and genetic lysosomal storage diseases that are candidates for splenic gene transfer. The gene transfer strategies used so far have proved relatively ineffective. Recombinant adenovirus, retrovirus, adeno-associated virus and liposomes have been previously used as vectors. Direct injection, intra-arterial, intravenous and intratracheal delivery of vectors have been the most extensively studied methods. This preclinical experimental work for marker gene transfer into the kidney, spleen, lung and mammary gland was done by using rabbits, pigs and goats as test animals. The adenoviral vector carrying a β-galactosidase reporter gene was first infused in the renal artery of rabbits and pigs in vivo with or without pharmacological agents. This did not result in any remarkable gene transfer into the kidney. Next, the incubation time between the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys for 12 hours. Perfusion at room temperature did not improve gene transfer. When the perfusion temperature was raised to 37°C, improved and mostly glomerular gene transfer was observed, with up to 80% of the glomeruli showing β-galactosidase expression in four ex vivo experiments. A closed-circuit organ perfusion method for in vivo gene transfer was developed in this study. The surgical perfusion experiment was tested successfully in ten in vivo perfusions of the kidney, eight of the spleen and eight of the lung in a porcine model. This method led to effective, up to 75% gene transfer into the renal glomeruli as assessed after four days. In the spleen, the perfusion method resulted in relatively effective gene transfer into perifollicular splenic cells, mostly macrophages and endothelial cells. Lung perfusion yielded transgene expression in alveolar epithelial cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar endothelial cells and alveolar macrophages. Perfusion of the goats mammary gland using a retroviral vector in three experiments resulted in growth hormone secretion into the milk. The gene transfer operation was well tolerated by the animals, and no clinical signs of inflammation were observed. No remarkable humoral immunological response against adenovirus or β-galactosidase was elicited in the kidney experiments, but histological signs of inflammation as mononuclear cell clusters in the kidney and lung were seen four and seven days after the experiments. The spleen showed no macroscopic or microscopic pathologic alterations after the perfusion.

Spinal muscular atrophy (SMA) is the most commonly inherited neurodegenerative disease that leads to infant mortality worldwide. There are no known cures for SMA, but small increase in protein levels of SMN can be beneficial. We have developed adenoviral (Ad) vectors that express a human transgene of SMN and have tested their safety in vitro. We have demonstrated that these viruses can effectively express the transgene following cell entry and that the levels are relative to the virus dose. The viruses do not appear to impact the health and function of the cells, and are capable of increasing the number of Gems. We also attempted to change the tropism of the viruses through fiber protein modifications in order to target muscles and motor neurons. Our results suggest that a therapy based on an Ad-SMN fiber-modified vector may ultimately be successful in treating patients of SMA.

The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

Hajj is the annual gathering of Islam practitioners in Mecca, Saudi Arabia. During the event, gastrointestinal infections are usually experienced and outbreaks have always been a concern; nevertheless, a deep and integrative study of the etiological agents has never been carried out. Here, I describe for the first time the epidemiology of pathogenic enteric viruses during Hajj 2011, 2012 and 2013. The focus of this study was the common enteric viruses Astrovirus, Norovirus, Rotavirus and Adenovirus. An enzyme Immunoassay established their presence in 14.9%, 15.0% and 6.6% of the reported cases of acute diarrhea for 2011, 2012 and 2013, respectively. For the three years of study, Astrovirus accounted for the majority of the viral infections. To our knowledge, this is the first time an epidemiological study depicts Astrovirus as the main viral agent of gastroenteritis in a mass gathering event. Hajj is rich in strains of Astrovirus, Norovirus and Rotavirus. A first screening by RT-PCR resulted in ten different genotypes. Strains HAstV 2, HAstV 1 and HAstV 5 were identified for Astrovirus. GI.6, GII.3, GII.4 and GII.1 were described for Norovirus and G1P[8], G4P[8] and G3P[8] were found for Rotavirus. The majority of the Astrovirus isolates could not be genotyped suggesting the presence of a new variant(s). Cases like this encourage the use of metagenomics (and nextgeneration sequencing) as a state-of-the-art technology in clinical diagnosis. A sample containing Adenovirus particles is being used to standardize a process for detection directly from stool samples and results will be obtained in the near future. The overall findings of the present study support the concept of Hajj as a unique mass gathering event that potentiates the transmission of infectious diseases. The finding of Norovirus GII.4 Sydney, a variant originated from Australia, suggests that Hajj is a receptor of infectious diseases worldwide. This work is part of the Hajj project, a collaborative effort with the Ministry of Health of the Kingdom of Saudi Arabia in order to describe entirely the epidemiology of gastrointestinal diseases in Hajj. It is expected that the results of this study will serve in the refinement of public health policies.

No description available.

Adenoviruses are non-enveloped, dsDNA tumor viruses responsible for a breadth of pathogenesis including acute respiratory disease and viral myocarditis. Gap junctions, which are formed by connexin proteins, directly couple the cytoplasms of apposed cells enabling immunological, metabolic, and electrical intercellular communication. The gap junction protein connexin43 (Cx43; gene name – GJA1) is the most widely expressed human connexin protein and is the predominant connexin in the working myocardium. Given the immunological role for Cx43 gap junctions, we hypothesized that gap junctions would be targeted during adenoviral infection. We find reduced Cx43 protein due to suppression of GJA1 transcription dependent upon β-catenin during adenoviral infection, with viral protein E4orf1 sufficient to induce β-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation at residues previously demonstrated to alter gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation, with complementary studies in human induced pluripotent stem cell derived-cardiomyocytes revealing Cx43 gap junction remodeling concomitant with reduced ZO-1 complexing. These findings demonstrate specific targeting of gap junction function by Ad5 leading to disruptions in intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts. Intercellular junction proteins belonging to classically defined unique junctions exhibit extensive cross-talk and interdependency for expression and localization. We find reduced connexin43 (Cx43) phosphorylation at a known internalization motif, leading us to hypothesize that gap junctions are maintained during adenoviral infection in order to stabilize intercellular junctions and adenoviral receptors therein. Utilizing immunofluorescence confocal microscopy, we demonstrate that Cx43 reductions are primarily cytosolic with Cx43 preservation at the plasma membrane. Click-IT chemistry, a non-radioactive pulse-chase technique, reveals that Cx43 ½ life is extended during adenoviral infection. In order to test if remaining Cx43 exists in de facto gap junctions (i.e. not undocked or cytosolic connexons) we utilized 1 % Triton X-100 solubility fractionation and find Cx43 is indeed primarily junctional during adenoviral infection. Having demonstrated increases in junctional Cx43, we next asked how tightly coupled cells were during adenoviral infection and by ECIS measurements of electrical resistance we demonstrate a transient increase in mechanical coupling during infection. Our future aims are to uncover changes in Coxsackievirus and adenovirus receptor (CAR) protein localization to determine if adenoviral-induced changes to subcellular architecture predisposes neighboring cells to infection and enhances viral spread. These findings will add to the existing model of adenoviral infection and more broadly, contribute to the therapeutic design of adenoviral vectors for cancer and gene therapy. / Doctor of Philosophy / The human heart will beat more than 3 billion times during the average lifetime. This is accomplished by billions of individual heart muscle cells, called cardiomyocytes, contracting in synchrony. Cardiomyocytes require direct cell to cell communication in order to receive the proper cues and work in concert. Outside of the heart, including the lining of the lungs which acts as a first line of defense against invading pathogens, direct cell to cell communication is important for mounting proper immune responses. A primary means by which cells communicate directly with neighboring cells is through gap junctions which are formed of proteins called connexins. Six connexin proteins form a channel in the cell surface that binds to a similar channel on an apposing cell to create a continuous gap junction channel, coupling the cell interiors directly. The most widely expressed human connexin, and the most abundant connexin in the heart, is connexin43 (Cx43; gene name – GJA1). Adenoviruses are pathogens commonly associated with respiratory illnesses in addition to more serious diseases including viral myocarditis, or infection of the heart. Given that Cx43 gap junctions enable direct intercellular communication important in initiating immune responses, we hypothesized that adenovirus would target Cx43 and gap junctions during infection. We find reduced Cx43 protein in cells infected with human adenovirus, and revel that the expression of the GJA1 gene is suppressed. We next focused on potential signaling pathways that are changed during adenoviral infection. β-catenin is a factor with several cellular roles including regulating expression of specific genes including GJA1 (Cx43). We demonstrate β-catenin is activated during adenoviral infection and that this is necessary for reducing Cx43 transcripts. A pathway that activates β-catenin in this manner is the PI3K/Akt signaling axis, which has previously been shown to be turned on during adenovirus infection by a viral protein called E4orf1. We find the adenoviral protein E4orf1 is sufficient to induce β-catenin activation revealing a potential therapeutic target for future studies. We next determined that direct cell to cell communication through gap junctions is reduced before loss of the gap junction protein Cx43 during infection. Gap junctions are modified by the cell to change their ability to couple cells independently of protein levels alone and we find gap junction modifications consistent with altered communication ability. Furthermore, the gap junction protein Cx43 interacts with the cellular skeleton protein Zonula Occludens-1 (ZO-1) during movement into and out of gap junction clusters. We determined alterations in Cx43/ZO-1 interactions consistent with gap junction remodeling. In complimentary studies we find the same gap junction remodeling in cardiomyocytes revealing arrhythmogenic potential during acute adenoviral infection in human heart cells. Localized with gap junctions are several other junction proteins including the Coxsackievirus and adenovirus receptor (CAR) which is critical in cardiac development and also the primary receptor for species C adenoviruses (used in our studies). CAR expression has been demonstrated to alter Cx43 levels and indeed, many junctional proteins influence the expression and/or localization of other junctional proteins. Interestingly, despite reduced Cx43 levels and reduced gap junction function (cell to cell communication), we detected decreases in a gap junction modification that is associated with gap junction degradation, suggesting that new gap junction protein Cx43 is not being made but already synthesized Cx43 is degraded more slowly. We hypothesized Cx43 is maintained during adenoviral infection in order to recruit other junctional components, principally CAR, on uninfected neighbor cells to predispose them to infection. We observed using microscopy that Cx43 reductions are primarily inside the cell but Cx43 is preserved on the cell surface and at junctions between cells. We next asked if the protein is being degraded more slowly and find Cx43 exists for longer in infected cells signifying that it is being degraded more slowly. Utilizing a fractionation technique to separate gap junction connexin from connexon that is non-junctional or inside the cell, we detect an increase in junctional Cx43, revealing maintenance of Cx43 gap junction structures. Having now identified adenoviral-mediated maintenance of Cx43 gap junction structures, we next wanted to test for changing in mechanical coupling (i.e. how tightly are the cells connected to one another) where we demonstrate an increase in mechanical coupling during adenoviral infection. Our future directions are to determine if this increase in Cx43 gap junction maintenance and mechanical coupling is concomitant with changes in CAR expression/localization on uninfected neighboring cells and if altered, does this predispose uninfected neighbors of infected cells to infection.

Cardiovascular disease is the leading cause of death world-wide, with 42% of sudden cardiac death in young adults caused by myocarditis. Viruses represent the main cause of myocarditis, with adenovirus being a leading pathogen. However, it is not understood how adenoviruses cause sudden cardiac arrest. Myocarditis is defined by two phases, acute and chronic. The acute phase involves viral-mediated remodeling of subcellular structures in the myocardium, which is thought to contribute to arrhythmogenesis. The chronic phase is immune response-mediated, where the host immune system causes damage that induces gross remodeling of the heart, which can result in cardiac arrest or heart failure. Electrical impulses of the heart are propagated by cardiomyocytes, via gap junctions, ion channels, and intracellular junctions, creating the healthy heartbeat. Cx43, the primary gap junction protein in the myocardium, not only propagates electrical signals, but also anti-viral molecules. Viral targeting of gap junction function leads to reduced anti-viral responses in neighboring cells. However, reduced cellular communication would dangerously alter cardiac conduction. Using a cardiotropic adenovirus, MAdV-3, we find that viral genomes are significantly enriched in the heart, with a decrease of gap junction and ion channel mRNA in infected hearts, however, their protein levels were unchanged. Phosphorylation of Cx43 at serine 368, known to reduce gap junction open probability, was increased in infected hearts. Ex vivo optical mapping illustrated decreased conduction velocity in the infected heart and patch clamping of isolated cardiomyocytes revealed prolonged action potential duration, along with decreased potassium current density during infection. Pairing mouse work with human induced pluripotent stem cell-derived cardiomyocytes, we found that human adenovirus type-5 infection increased pCx43-Ser368 and perturbation of intercellular coupling, as we observed with in vivo MAdV-3 infection. Allowing adenovirus infection to progress in vivo, we find myocardium remodeling and immune cell infiltration. Together, these data demonstrate the complexity of cardiac infection from viral-infection induced subcellular alterations in electrophysiology to immune-mediated cardiomyopathy of cardiac adenoviral infection. Our data describe virally induced mechanisms of arrhythmogenesis, which could lead to the development of new diagnostic tools and therapies, to help protect patients from arrhythmia following infection. / Doctor of Philosophy / Viral infection has long thought to be a cause of unexplained sudden cardiac death, especially in young adults. Viruses have been identified to cause many cases of deleterious remodeling of the heart, which can result in heart failure. The heart relies on electrical signaling that moves in a coordinated fashion to contract and pump blood throughout the body. The cells within the heart that do this are called cardiomyocytes, and they join end-to-end to communicate with each other via gap junctions. Gap junctions are tunnels that allow for ions that create electrical impulses to pass, and molecules, such as ones that are important in immune responses to infection. In addition to gap junctions in the heart, ion channels, which are highly selective to allow only one ion flow, unlike gap junctions, create the healthy heartbeat. The most common gap junction in the heart comprises Cx43 proteins. If a virus were to alter how Cx43 connects to a neighboring cell, this would cause a better environment for the virus, as this would keep anti-viral surveillance low, however, this would change how the electrical signal moves throughout the heart, creating arrhythmias. Adenoviruses are a common cold virus, but have been found in the hearts of many cardiac arrest patients. However, little is known on how adenoviruses may cause cardiac arrest, because human adenoviruses are only successful in humans, and mouse adenoviruses are only successful in mice. This creates a challenge when studying the dynamic heart, which does not translate well to cells in a dish. A mouse adenovirus, called Mouse Adenovirus Type-3 (MAdV-3) was reported to favor infecting the heart in mice, but no research has been published on if this virus can answer how adenoviruses change the heart. Because of this virus, and our prior research that adenoviruses can decrease Cx43 within skin cells in a dish, we used MAdV-3 to understand if, how adenoviruses could cause sudden cardiac arrest, and if longer infection could change the overall structure of the heart. We find that MAdV-3 infection prefers the heart to other organs, and that early stages, reduce both the speed of the electrical signal moves through heart and, looking within a cardiomyocyte, how it creates that electrical signal. These changes are arrhythmogenic and accompany modification of Cx43 that would close the gap junction between two cells, changing how ions and molecules move between cells. Using a human adenovirus infection in human cardiomyocytes created from stem cells, this result is also observed. If infection is allowed to continue in the mouse to cause chronic infection, the heart itself changes shape and is diseased. Together, this work shows that adenoviruses create a diseased heart, first the virus changes how the electrical signal moves and then later, causes thinning of the heart muscle. These data illustrate the role viruses play in causing cardiac arrest and could lead to diagnostic or drug targets.

This study was undertaken to examine the specificity of the cell-mediated immune response to vesicular stomatitis virus in mice, using the recombinant adenovirus vector AdG12. AdG12 contains the coding region for VSV glycoprotein (G) within the genome of adenovirus type 5. Ultimately, these studies attempted to provide a model for the use of adenovirus vectors to elicit specific CTL responses in mice against an inserted foreign protein. Cell-mediated immunity was examined using a standard ⁵¹Cr release assay. Splenocyte effectors from VSV or AdG12 primed mice were tested for their ability to lyse labelled infected target cells. A number of target cell lines were analyzed for productive infection by AdG12 and expression of VSV-G. Of the lines tested, B10.D2 (H-2ᵈ) and PAK (H-2ᵇ) lines were shown to be infectible with AdG12 and expressed VSV-G 36 hours post infection. Cell lines P815 (H-2ᵈ) and EL-4 (H-2ᵇ) did not appear to be AdG12 infectible. Responses were measured in mice intravenously infected with AdG12. Results demonstrated that peak cytotoxic activity from AdG12 primed mice occurred six days post-infection against syngeneic target cells infected with AdG12, Ad5 wt or VSV. However, these effectors also significantly lysed allotargets infected with VSV, implying that VSV infected targets were lysed in a non-MHC restricted manner. In subsequent experiments, it was discovered that VSV infected B10.D2 and PAK targets were markedly lysed by effectors from immunized and non-immunized Balbic, C57B1/6 and CBA/J mice. Thus, it appeared that these mouse strains contained an inherent or natural cytotoxic activity against VSV infected targets that was unlike classical CTL killing. Depletion experiments showed that this activity was not due to adherent or Thy1 bearing cells within spleen cell populations. To further characterize this activity, splenocyte effectors were tested for their ability to lyse NK-sensitive YAC-1 targets, but no significant lysis was demonstrated. However, despite these results, it appeared that this activity was that of an NK-like effector. The presence of NK-like cytotoxicity against VSV infected targets precluded efforts to define specific anti-VSV responses in these mice. / Thesis / Master of Science (MS)