A newly discovered enteric virus has recently been associated with large outbreaks of waterborne gastroenteritis. Most commonly referred to as the Norwalk agent, this virus appears to be morphologically and biophysically similar ·to the parvoviruses. Presently there is very little known about the fate of parvoviruses in environmental systems. In this study the parvovirus H-1, a putative human virus containing single-stranded DNA (ssDNA), was used as a model virus for chlorine inactivation experiments. The purpose of this research was two-fold: first, to investigate the kinetics of inactivation of parvovirus H-1 by low levels of free chlorine (0.05 - 0.20 mg L⁻¹) at pH 7 and at 5, 10, 20, and 30°C; and secondly, to determine the mechanism by which chlorine inactivates this virus. Inactivation occurred in the usual dose-response relationship, that is, increasing the chlorine dose caused an increase in the rate of inactivation. The results indicated that perhaps more than one reaction mechanism was responsible for inactivation, and the reaction mechanism was a function of temperature. The energy required for the inactivation reaction using 0.05 mg L⁻¹ free chlorine from 5 to 30°C was graphically determined to be 2.4 Kcal mole⁻¹. The change in entropy was calculated to be -52.34 entropy units. From the mechanism study it was concluded that the initial action of chlorine on parvovirus H-1 was on the capsid. Alterations in the two major capsid proteins caused the virion to rupture, and, as evidenced by electron microscopy the ssDNA was exposed. Also, the adsorption ability of the chlorine-treated virions to host cells was significantly inhibited. This was presumably due to the effect on the spatial arrangement of the capsid proteins in their entirety rather than a loss of, or change, in only one polypeptide. The sedimentation rate of the chlorine-treated whole virus decreased from 116S to 43S. The chlorine caused certain sites on the capsid proteins to become highly reactive which facilitated the formation of higher molecular weight aggregates as detected by fluorographs of electrophoretic protein patterns in polyacrylamide gels. Most significant was the discovery that the ssDNA remained undamaged and was still capable of in vitro replication even after 60 minutes of exposure to 5 mg L⁻¹ of sodium hypochlorite at pH 7. / Ph. D.

The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus. / Ph. D. / incomplete_metadata

Porcine parvovirus (PPV) is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are some of the clinical signs commonly associated with PPV infection in a herd. The virus genome is considered to be conservative, with substitution rates near to that of their host. However, it has been shown that some parvoviruses exhibit a substitution rate close to that commonly determined for RNA viruses. In this scenario, new PPV phenotypes may reduce the effectiveness of the currently used vaccines, recommending the continuous monitoring of the currently prevalent PPV strains. In addition, a number of novel porcine parvoviruses have been described during the last decade, but the importance and characteristics of these viruses remain unknown. In the present dissertation, three studies were performed to address the PPV genetic variability, to monitor the emergence of new PPV strains and the prevalence of novel parvoviruses. In the first study, recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analyzed. These samples, together with sequences retrieved from GenBank, were included in three datasets (viral protein complete gene, viral protein partial gene and non-structural protein complete gene). For each dataset, the nucleotide substitution rate was determined and a molecular clock estimated. The analysis revealed that for the new strains, the amino acids substitutions were located mainly in the viral capsid loops. Only the capsid protein datasets present the higher suitability for phylogenetic analysis. In them, a higher divergence was found, with three well defined clusters. By inferring the evolutionary dynamics of the PPV sequences, a nucleotide substitution rate of approximately 10 -4 substitutions per site per year was found for these datasets. An association of the phylogenetic tree with the molecular clock revealed that the main divergence of the PPV strains for the viral protein ocurred in the last 30 years. In the second study, the population dynamic of PPV isolates from swine herds was analyzed using PPV complete protein gene and partial sequences deposited in GenBank. The population dynamic of the virus was calculated using a Bayesian approach with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed by twenty-one consecutives passages of the Challenge strain (a virulent field strain) and NADL2 strain (a vaccine strain) in PK15 cell-line supplemented with polyclonal antibodies raised against the vaccine strain (negative control was not supplemented). The Bayesian analysis indicated a decrease in the population diversity over the years and the predominance of some PPV strains. In agreement, the in vitro study revealed that a lower number of mutations appeared for both viruses in the presence of anti-PPV antibodies in comparison with the control passages without antibodies. In the third study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for PPV, porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3) and porcine parvovirus 4 (PPV4). Positive samples of PPV, PPV2 and PPV3 were sequenced. PPV was observed in 60/100 hearts and 61/100 tonsils and PPV2 in 55/100 hearts and 78/100 tonsils. PPV3 and PPV4 could not be detected in the heart samples but 20/100 and 7/100, respectively, of the tonsils were tested positive. The phylogenetic analysis of the PPV, PPV2 and PPV3 sequences revealed that the German samples could be divided in at least two clusters or clades for each virus. Altogether, it can be concluded that PPV is continuously evolving. Apparently, PPV vaccines largely used in the last 30 years probably have reduced the genetic diversity of the virus and induced the predominance of strains with distinct capsid profile from the original vaccine-based strain. Moreover, the high prevalence of the PPV, PPV2 and PPV3 and their genetic diversity highlight the importance of the continuous monitoring of these viruses.

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-25Bitstream added on 2014-06-13T20:35:26Z : No. of bitstreams: 1 ribeiro_mcm_me_botfmvz.pdf: 287673 bytes, checksum: dec8c154bcb68842908b9737b4e6eeb8 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cinomose e a parvovirose canina são duas enfermidades infecto-contagiosas de grande importância para a clínica de pequenos animais onde a PCR vem sendo aplicada com ótimos resultados no diagnóstico. No entanto, para o sucesso da técnica, é necessário que o ácido nucléico esteja o mais puro possível e livre de inibidores das polimerases (Transcriptase reversa e/ou Taq DNA polimerase), desejando-se um método de extração simples e rápido. O teste de coaglutinação utilizando o Staphylococcus aureus (COA) é baseado na propriedade da proteína A de se ligar especificamente à porção Fc da imunoglobulina G de alguns mamíferos e algumas subclasses de IgG de camundongos. Assim, neste trabalho utilizou-se a coglutinação para obtenção de DNA ou RNA livres de inibidores, com capacidade de concentração de partículas virais dispersas nas amostras biológicas e de forma simples, rápida e de baixo custo. Para tanto, 10 amostras de fezes positivas para o vírus da parvovirose canina e 17 amostras de urina positivas para o vírus da cinomose foram submetidas à extração de ácidos nucléicos utilizando o COA e kits comerciais para posteriormente serem analisadas pela PCR em tempo real e PCR convencional respectivamente. As amostras de fezes foram diluídas de 1: 10 a 1: 100 000 e as amostras de urina foram utilizadas puras. A metodologia desenvolvida foi eficiente na extração dos dois tipos de amostra. O método proposto demonstrou ser confiável e de baixo custo para a preparação de DNA e RNA viral para o diagnóstico molecular. / PCR presents excellent results for the diagnosis of canine distemper and canine parvoviruses, two important infectious and contagious diseases for small animal internal medicine. However, success of technique depends on nucleic acid samples free of polymerase inhibitors (Reverse Transcriptase and / or Taq DNA polymerase). The coagglutination test using Staphylococcus aureus (COA) is based on the property of specific binding of protein A to the Fc portion of immunoglobulin G of some mammals and some of IgG subclasses of mice. This work was carried out the coagglutination procedure to obtain nucleic acid inhibitors free, with capacity for viral particle concentration dispersed in biological samples, simply, quickly and low cost. For this purpose, 10 canine parvovirus positive stool samples and 17 canine distemper virus positive urine samples were submitted to the preparation of nucleic acids using the COA and commercial kits in order to be analyzed by real-time PCR or conventional PCR respectively. Fecal specimens were diluted from 1: 10 to 1: 100 000 and urine samples were used pure. The developed methodology was efficient in extracting the two types of sample. The method proposed demonstrated to be reliable and cheap to prepare viral DNA or RNA for molecular diagnosis.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves : 144-159). Also available on the Internet.

Porcine parvovirus (PPV) is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are some of the clinical signs commonly associated with PPV infection in a herd. The virus genome is considered to be conservative, with substitution rates near to that of their host. However, it has been shown that some parvoviruses exhibit a substitution rate close to that commonly determined for RNA viruses. In this scenario, new PPV phenotypes may reduce the effectiveness of the currently used vaccines, recommending the continuous monitoring of the currently prevalent PPV strains. In addition, a number of novel porcine parvoviruses have been described during the last decade, but the importance and characteristics of these viruses remain unknown. In the present dissertation, three studies were performed to address the PPV genetic variability, to monitor the emergence of new PPV strains and the prevalence of novel parvoviruses. In the first study, recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analyzed. These samples, together with sequences retrieved from GenBank, were included in three datasets (viral protein complete gene, viral protein partial gene and non-structural protein complete gene). For each dataset, the nucleotide substitution rate was determined and a molecular clock estimated. The analysis revealed that for the new strains, the amino acids substitutions were located mainly in the viral capsid loops. Only the capsid protein datasets present the higher suitability for phylogenetic analysis. In them, a higher divergence was found, with three well defined clusters. By inferring the evolutionary dynamics of the PPV sequences, a nucleotide substitution rate of approximately 10 -4 substitutions per site per year was found for these datasets. An association of the phylogenetic tree with the molecular clock revealed that the main divergence of the PPV strains for the viral protein ocurred in the last 30 years. In the second study, the population dynamic of PPV isolates from swine herds was analyzed using PPV complete protein gene and partial sequences deposited in GenBank. The population dynamic of the virus was calculated using a Bayesian approach with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed by twenty-one consecutives passages of the Challenge strain (a virulent field strain) and NADL2 strain (a vaccine strain) in PK15 cell-line supplemented with polyclonal antibodies raised against the vaccine strain (negative control was not supplemented). The Bayesian analysis indicated a decrease in the population diversity over the years and the predominance of some PPV strains. In agreement, the in vitro study revealed that a lower number of mutations appeared for both viruses in the presence of anti-PPV antibodies in comparison with the control passages without antibodies. In the third study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for PPV, porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3) and porcine parvovirus 4 (PPV4). Positive samples of PPV, PPV2 and PPV3 were sequenced. PPV was observed in 60/100 hearts and 61/100 tonsils and PPV2 in 55/100 hearts and 78/100 tonsils. PPV3 and PPV4 could not be detected in the heart samples but 20/100 and 7/100, respectively, of the tonsils were tested positive. The phylogenetic analysis of the PPV, PPV2 and PPV3 sequences revealed that the German samples could be divided in at least two clusters or clades for each virus. Altogether, it can be concluded that PPV is continuously evolving. Apparently, PPV vaccines largely used in the last 30 years probably have reduced the genetic diversity of the virus and induced the predominance of strains with distinct capsid profile from the original vaccine-based strain. Moreover, the high prevalence of the PPV, PPV2 and PPV3 and their genetic diversity highlight the importance of the continuous monitoring of these viruses.

Wild-type viruses with intrinsic oncolytic capacity in human includes DNA viruses like some autonomous parvoviruses and many RNA viruses. Recent advances in molecular biology have allowed the design of several genetically modified viruses, such as adenovirus and herpes simplex virus that specifically replicate in, and kill tumor cells. However, still several hurdles regarding clinical limitations and safety issues should be overcome before this mode of therapy can become of clinical relevance. It includes limited virus spread in tumor masses, stability of virus in the blood, trapping within the liver sinusoids, transendothelial transfer, and/or vector diffusion of viral particles to tumor cells, limited tumor transduction, immune-mediated inactivation or destruction of the virus. For replication-competent vectors without approved antiviral agents, suicide genes might be used as fail-safe mechanism. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. Therefore, viruses that target the defective self-renewal pathways in cancer cells might lead to improved outcomes.<p>In this thesis, data we generated in the field of oncolytic autonomous parvoviruses are presented.<p>We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population, (CAT ELISA) or at the single cell level, (FACS analysis of Green Fluorescent Protein). Cat expression was substantial (up to 10000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells, (with the exception of an expression slightly above background in fibroblasts.). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP positive normal cells could be detected with the exception of very few (less than 0.1%) human fibroblast cells expressing high level of GFP. We also replace capsid genes by genes coding for the costimulatory molecules B7-1 and B7-2 and show that, upon infection with B7 recombinant virions, only tumor cells display the costimulatory molecules and their immunogenicity was increased without any effect on normal cells. Using a recombinant MVM containig the Herpes Simplex thymidine kinase gene, we could get efficient killing of most tumor cell types in the presence of ganciclovir, whithout affecting normal proliferating cells. We also produced tetracycline inducible packaging cell lines in order to improve recombinant vectors yields. The prospects and limitations of these different strategies will be discussed.<p>An overview is given of the general mechanisms and genetic modifications by which oncolytic viruses achieve tumor cell-specific replication and antitumor efficacy. However, as their therapeutic efficacy in clinical trials is still not optimal, strategies are evaluated that could further enhance the oncolytic potential of conditionally replicating viruses in conjunction with other standard therapies. <p>Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells to deliver oncolytic viruses to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will explore some ways deserving further study in order to be able to utilize various oncolytic viruses for effective cancer treatment. <p><p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

L’immunothérapie anticancéreuse est basée sur la capacité du système immunitaire à reconnaître les cellules tumorales comme étrangères et à les éliminer. Les stratégies immunothérapeutiques abordées dans ce travail, incluent l’activation du système immunitaire par l’expression de facteurs immunomodulateurs (l’interleukine-2) via l’utilisation d’un vecteur dérivé du parvovirus MVM, ou par présentation des antigènes tumoraux par la machinerie des cellules dendritiques (DC), via la génération d’hybrides entre DC et cellules tumorales (TC).<p>L’intérêt majeur du parvovirus autonome MVM en tant que vecteur pour la thérapie génique du cancer vient de son expression préférentielle dans les cellules transformées (oncotropisme) et de son aptitude à lyser celles-ci (oncolyse). Les vecteurs générés au laboratoire conservent l’unité de transcription NS et expriment l’IL2 humaine sous contrôle du promoteur P38, à la place des protéines de capside. Malgré les améliorations apportées à la production de vecteurs recombinants, la faible concentration des stocks reste un problème. Il a été montré que, de nombreux virus sont mieux produits en conditions de faible tension en oxygène (hypoxie). Nous avons tenté d’améliorer les titres des vecteurs en les produisant sous faible tension d’oxygène mais sans y parvenir (annexe 1). Dans un modèle in vivo utilisant la lignée de mélanome K-1735 dans des souris immunocompétentes, des cellules tumorales infectées in vitro avant leur implantation en sous-cutané ont montré un effet anti-tumoral du vecteur MVM/IL2 (annexe 2). Afin de mettre en évidence l’apport de l’oncolyse parvovirale dans l’activité anti-tumorale, nous avons mis au point des expériences, dans le même modèle de tumeur, visant à comparer l’efficacité du vecteur MVM/IL2 à celle d’autres vecteurs, Ad/IL2 et Rétrovirus/IL2, ne possédant pas d’activité oncolytique. Dans le but de mettre en évidence une éventuelle réponse immune in vivo, nous avons utilisé le modèle de tumeur TC-1 mais ce modèle s’est montré moins sensible à l’effet du vecteur MVM/IL2 et nous n’avons pas pu démontrer d’activation de cellules cytotoxiques spécifiques de la tumeur.<p>Il a été proposé d’utiliser des hybrides entre DC/TC pour la vaccination anti-tumorale pour optimaliser la présentation des antigènes tumoraux. Une lignée cellulaire exprimant la protéine fusogène du virus de la leucémie du Gibbon (GaLV-FMG, Gibbon ape leukemia virus) a été dérivée de la lignée cellulaire CHO (cellules ovariennes de hamster chinois) au laboratoire. Cette lignée CHO-FMG, utilisée comme partenaire intermédiaire, a permis la fusion entre cellules tumorales et dendritiques (annexe 3). Nous avons montré que l’expression transitoire après infection par un vecteur AAV-FMG ou après transfection transitoire ne génère pas un pourcentage significatif d’hybrides. En effet, le niveau d’expression ainsi que le pourcentage de cellules transduites exprimant FMG s’est révélé trop faible. Ceci a mis en valeur l’efficacité de la lignée stable CHO-FMG comme intermédiaire de la fusion. De plus, nous avons intégré dans la lignée fusogène, le gène de l’interleukine-2, qui devrait permettre d’augmenter l’efficacité de l’induction de la réponse immune. <p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le parvovirus adéno-associé (AAV) possède un génome à ADN linéaire simple brin de 4,7kb encadré par deux séquences palindromiques inversées et identiques de 145 nucléotides appelées ITRs, requises en cis pour la réplication et l’encapsidation de l’ADN viral. Dans un AAV recombinant (rAAV), la totalité de la partie codante du génome viral est remplacée par une cassette d’expression et seuls les ITRs sont conservés.<p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished