A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, in partial fulfilment of the requirements for the degree Master of Medicine (Virology) / This study was undertaken to determine the role of human parvovirus B19 (B19) in chronic anaemia of HIV infected TB patients. Patlents were selected from an existing databank of 307 patients included ln a MRC HIV/TB study. Twenty-nine patients, 15 colnfected with HIV/TB and 14 Infected with TB only, were identified for further evaluation. These patients' sera were subjected to serological and DNA detection studies using IgG and IgM ELISA methods and a nested polymerase chain reaction (PCR) assay. The selection of the nested PCR was based on comparative evaluation of a new rapid 99 cycle PCR method recommended for hepatitis B DNA detection and the nested PCR method established for B19. The nested assay was shown to be the more sensitive system in the context of B19 DNA detection. Serological evaluation of these 29 patients suggested that a greater proportion of HIV/TB patients with chronic anaemia had evidence of recent or past exposure to B19 than those not experiencing anaemia. The nested PCR demonstrated the presence of circulating B19 DNA in 2 coinfected individuals with haematological pictures compatible with persistent B19 infection. B19 DNA was also demonstrated in a TB only patient without anaemia; further haematological and serological evidence in this patient suggested recent exposure to B19. The serological and DNA amplification assay results of these 29 patients would suggest a possible role - either causal or co-factorial - for persistent B19 infection in the establishment of chronic anaemia in HIV/TB patients. / Andrew Chakane 2019

Bovine parvovirus (BPV) is a helper-independent parvovirus. It has a small icosahedral capsid with a single stranded DNA genome. It is a highly stable virus with a narrow host range. It causes acute gastroenteritis in calves. It is considered to be a cytolytic virus because it kills the host cells. However, the mechanism by which the virus causes cell death is not known. The work described in this thesis assessed different parameters of cell death in BPV infected embryonic bovine tracheal (EBTr) cells. There are several ways for viruses to induce cell death. Viruses can induce apoptosis in the infected cell. They can also kill the host cell by necrosis. Several approaches were used in this work to look for evidence of apoptosis and necrosis. Cells undergoing apoptosis exhibit cardinal signs that distinguish them from other dying cells. Among these signs are the exposure of phosphatidylserine to the outer surface of the plasma membrane, DNA fragmentation into non-random DNA sections that are multimers of 180bp, nuclear morphology changes and caspase activation. These signs were studied in this research and data collected from these experiments did not show any positive sign of apoptosis in infected cells due to virus infection. Cells undergoing a necrotic cell death have a different pattern. The cells swell then burst releasing their cytoplasmic contents. The DNA is fragmented in a random fashion. Cellular morphology was studied in this research and the data suggested that BPV infected cells swell, then shrink and detach from the surface of the culture vessel. Moreover, formation of apoptotic bodies was not detected in dying infected cells. Release of cytoplasmic contents was also assessed by looking at concentrations of LDH enzyme, viral haemagglutinin, and the number of infectious viral particles in the media of infected cells. Data from the different approaches employed in this study do not support the hypothesis that BPV kills the infected EBTr cell by apoptosis, rather, infected cells in culture become necrotic, swell, release their cytoplasmic contents, and detach.

Memoria para optar al Título Profesional de Médico Veterinario. / La infección por parvovirus canino tipo 2 (CPV-2) es una de las principales causas de enteritis hemorrágica en perros de todo el mundo y contar con una técnica de diagnóstico que sea altamente sensible es fundamental para Médicos Veterinarios, dueños y criadores de perros. En este trabajo se implementó un protocolo que utiliza la Reacción en Cadena de la Polimerasa (PCR) convencional para detectar un fragmento del ADN de CPV-2 a partir de heces de perros con signología clínica correspondiente a parvovirosis canina. En total se recolectaron y analizaron 12 muestras de heces que resultaron positivas con la PCR convencional, lo que fue confirmado mediante la secuenciación de los fragmentos obtenidos y contrastados con las secuencias de las distintas variantes de CPV-2 descritas en la base de datos GenBank. Las mismas muestras fueron analizadas con una prueba rápida, que corresponde a una técnica de inmunocromatografía (IC) de uso rutinario en la consulta veterinaria. En este caso de las 12 muestras analizadas, sólo un 41,7% resultaron positivas, evidenciando una menor sensibilidad que la técnica molecular para el diagnóstico de parvovirus canino. Adicionalmente, se hizo un análisis de las secuencias nucleotídicas obtenidas arrojando una variabilidad promedio de 0,7%. Los resultados de este trabajo permiten establecer que la PCR convencional es una técnica de diagnóstico recomendable para la detección de parvovirus canino, no así las pruebas rápidas utilizadas en la consulta veterinaria que en este y otros estudios han mostrado constantemente su baja sensibilidad. Es importante destacar que el presente trabajo es la primera aproximación molecular al parvovirus canino tipo 2 en Chile / Infection with canine parvovirus type 2 (CPV-2) is one of the main causes of hemorrhagic enteritis in dogs worldwide and having a diagnostic technique that is highly sensitive is essential for veterinarians, dog owners and breeders. In this work, a protocol was implemented that uses the conventional Polymerase Chain Reaction (PCR) to detect a CPV-2 DNA fragment from feces of dogs with clinical signology corresponding to canine parvovirus. In total, 12 stool samples that were positive with conventional PCR were collected and analyzed, which was confirmed by sequencing the fragments obtained and contrasted with the sequences of the different variants of CPV-2 described in the GenBank database. The same samples were analyzed with a rapid test, which corresponds to a routine immunochromatography (IC) technique in the veterinary practice. In this case of the 12 samples analyzed, only 41.7% were positive, showing a lower sensitivity than the molecular technique for the diagnosis of canine parvovirus. Additionally, an analysis of the nucleotide sequences obtained was made, yielding an average variability of 0.7%. The results of this work allow to establish that conventional PCR is a recommended diagnostic technique for the detection of canine parvovirus, but not the rapid tests used in the veterinary practice that in this and other studies have consistently shown low sensitivity. It is important to note that the present work is the first molecular approach to canine parvovirus type 2 in Chile

Parvoviruses cause dreadful enteritis in waterfowls and lead to tremendous financial losses. This study aims at developing effective way to prevent waterfowl parvoviral infection. Duck parvoviruses (DPVs) and goose parvoviruses (GPVs) were isolated from organs of infected waterfowls. The presence of virus in the specimens was identified using polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis. To reveal the genetic variation of viral capsid proteins (VPs), full length VPs gene were amplified and sequenced. The sequence data indicated the sequences diverge 4.1 to 4.4% among viral strains isolated during 1990 to 1999. The variant amino acids cluster in the common regions of VP3 at residues 203-266 and 482-534, which overlaps with the regions proposed to expose on the outer surfaces of parvoviral particles. These data implying that selective pressure from host immune system might play a part. The nucleotide sequences of VPs also reveal that DPV and GPV share 77 % similarity at the DNA, and 84.6% at the protein level. The most variable regions reside in the N-terminal of VP2 before the initiation codon of VP3 with 35% (19/54) amino acids divergence. This study also reveals the presence of conserved strain-specific residues in VPs and these residues seldom vary among different isolates of the same virus, suggesting that they might be important in maintaining viral structure or host specificity which worth further investigation. To investigate the antigenicity of VPs, the GPV genomic DNA encoding common region of VPs was fused in frame with glutathione S-transferase (GST) gene for the expression of GST-GPV (248-516) fusion protein in bacterial cells. Purified fusion protein was used as immunogen for the generation of rabbit anti-GPV (248-516) antiserum. The potential diagnostic usage was confirmed by the fact that this antiserum was able to differentiate between viral infected and uninfected primary embryonic fibroblast cells by immunocytochemical analysis. In addition, VPs in purified DPV and GPV virions were analyzed by Western blotting. This antiserum detected two prominent proteins bands with the molecule weight of 80 and 70 kilodaltons, which correspond to the sizes of VP1 and VP2 reported in the literature. The fact that VP1 of DPV reacts weakly with this antiserum suggests the existence of antigenic discrepancy between DPV and GPV. For the purpose of developing subunit vaccine for the control of Derzy's disease, recombinant full length VPs were expressed using both prokaryotic, GST and histidine-tagged fusion proteins, and eukaryotic, baculovirus and mammalian vero cell, expression systems. After large- scale production and purification, same amount of 4 recombinant VPs were individually used to immunize 1-week-old geese. The antibodies induced after immunization were then evaluated by enzyme-linked immunosorbent assay (ELISA). All four recombinant proteins stimulate approximately 7 to 8 folds increases of ELISA antibodies titers, and together with preliminary data of safety tests suggest a potential usage as subunit vaccine for the control of parvoviral infection.

Reproduction de : Thèse d'exercice : Médecine : Nancy 1 : 2004. / Titre provenant de l'écran-titre.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Orientadores: Sandra Regina Muchinechi Fernandes, Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T16:42:34Z (GMT). No. of bitstreams: 1 Sachetto_Zoraida_M.pdf: 4729475 bytes, checksum: 274c907aab485d19facfe60e402f092a (MD5) Previous issue date: 2005 / Resumo: Poliarterite nodosa (PAN) é uma vasculite sistêmica necrosante de pequenas e médias artérias e a poliangiíte microscópica de pequenos vasos (arteríolas, vênulas e capilares) caracterizada por glomerulonefrite necrosante pauci-imune rapidamente progressiva e envolvimento pulmonar que estão ausente na PAN. Uma possível relação etiológica entre as vasculites sistêmicas e infecção viral tem sido considerada. O vírus da hepatite B está comprovadamente associado à etiopatogênese em alguns casos de PANe outros agentes virais são também apontados como prováveis desencadeadores de PAN, tais como os vírus HIV, HTLV1, citomegalovírus e parvovirus B19 (B19). Na PAN existem relatos de casos comprovando a presença concomitante de atividade da doença e testes sorológicos IgM positivos para o B19 e pela presença do DNA do B19 no soro, medula óssea e músculo em um paciente, pesquisado por reação em cadeia de polimerase (PCR). Não há investigação referente à pesquisa de DNA do parvovírus B19 em amostras teciduais de PANe poliangiíte microscópica. A pesquisa desta associação se toma importante na abordagem terapêutica destas doenças pela necessidade de corticosteróides e imunossupressores podendo agravar as manifestações de uma parvovirose associada. Nosso objetivo foi pesquisar a presença do DNA viral do B19 em tecidos fixados em parafina e com evidência histopatológica de vasculite em pacientes com PANe poliangiíte microscópica e se há relação com as manifestações clínicas, índices de atividade (BVAS) e gravidade (FFS) da doença. Foram investigados todos os pacientes com diagnóstico histopatológico de PAN ou poliangiíte microscópica, com blocos de parafina disponíveis no Departamento de Anatomia Patológica e acompanhados no Ambulatório de Vasculites do Hospital das Clínicas/Disciplina de Reumatologia da FCM!Unicamp no período de 1985 a 2003. A investigação foi feita pela pesquisa da presença de seqüência de ácidos nucléicos do B19 por PCR. Foram encontrados 21 pacientes com amostras teciduais e blocos de parafina disponíveis para estudo. Destas 21 amostras, 17 apresentaram DNA de boa qualidade para realização do PCR para o B19. Dez com diagnóstico de PAN (média da idade 29,3:!::15,1; M:F=I,5:1) e sete de poliangiíte microscópica (média da idade 41,9 :!::18,5; M:F=1:1,3). A média do BVAS, no início da doença, foi de 15,7 :!:: 7,7 com mínimo de 5 e máximo de 26 para PANe de 20,7 :f: 7,1 com mínimo de 13 e máximo de 31 para poliangiíte microscópica. Na PAN, FFS=1 em 60% e FFS=O em 40% dos casos, com média de 0,6 :f: 0,5. Na poliangiíte microscópica FFS=2 em 14%, FFS=1 em 43% e FFS=O também em 43% dos casos, com média de 0,7 :f:0,8. . I Nenhum dos tecidos avaliados apresentou positividade para o DNA do B19. Este resultado sugere que o B19, provavelmente, não está associado a etiopatogenia da PAN idiopática e da poliangiíte microscópica / Abstract: Polyarteritis nodosa (PAN) is a well-known form of sistemic necrotizing vasculitis. lndividualized ftom PAN, microscopic polyangiitis (MPA) is a systemic vasculitis of small-sized vessels (arterioles, venules or capillaries) characterized by the presence of rapidly progressive glomerulonephritis and pulmonary involvement, absent in PAN. Nowadays, a role of viral infections in primary systemic vasculitis has been considered. The hepatitis B virus is associated with the ethiopathogenesis ofsome cases ofPAN. HIV, HTLVl, CMV and parvovirus B19 (BI9) may also be found, eventually, in some patients with PAN. Evidence of the B19 involvement in primary systemic vasculitis is based on some anedoctal reports of cases of Wegener' s granulomatosis and PAN and studies with giant-cell arteritis. Regarding in PAN, there are few reports showing an active disease and positive serological tests for the B19. B19DNA is the serum, marrow bone and muscle was found in one patient with PAN. To our knowledgment, there is no research related to B19DNA in tissue samples of patients with PAN and MPA Our objective was to identify the presence ofB19 DNA in fixed, paraffin-embedded tissue samples obtained ftom patients with PAN and MPA We investigated the patients with histological diagnosis of PAN or MPA who were attended at the university hospital ftom 1985 to 2003. AlI fixed, paraffin-embedded tissue samples available, with evidence of vasculitis, were screened for B19 DNA using the polymerase chain reaction (PCR) method. ,Among all patients, we found 17 tissue samples available for the study. Ten had PAN (mea nage 29.3 :t: 15.1yrs.; M:F sex ratio 1.5:1)and seven had microscopicpolyangiitis (mean age 41.9 :t: 18.5 yrs.; M:F sex ratio 1:1,3). AlI of the patients investigatedwere negative for parvovirus B19 DNA .J These results suggest that the parvovirus B19 is not involved in the pathogenesis of idiopathic PAN and MPA / Mestrado / Clinica Medica / Mestre em Clinica Medica

Das porzine Parvovirus (PPV), 2013 vom International Committee on taxonomy of Viruses (ICTV) in ungulate Protoparvovirus 1 umbenannt, ist ein unbehülltes, einzelsträngiges DNA Virus und gehört innerhalb der Familie Parvoviridae zur Subfamilie Parvovirinae. Es ist weltweit in allen Bereichen der Schweinehaltung endemisch und verursacht große wirtschaftliche Verluste in den Betrieben (TRUYEN und STRECK 2012). Anders als die verwandten caninen und felinen Parvoviren (seit 2013 arnivore Protoparvovirus 1) ist es nicht durch zum Teil tödlich verlaufende Durchfallerkrankungen, sondern durch Fruchtbarkeitsstörungen wie Abort, Mumifikation und Unfruchtbarkeit, auch bekannt als SMEDI – Syndrom (Stillbirth = Totgeburt, Mummification =Mumifikation, Embryonic Death = embryonaler Tod und Infertility = Unfruchtbarkeit), gekennzeichnet. Die Schwere des Verlaufs hängt dabei wesentlich vom Zeitpunkt sowie von dem, für die Infektion verantwortlichen Isolats ab. Als besonders gefährdet gelten ungeimpfte Jungsauen, die innerhalb der ersten 70 Tage der Trächtigkeit in Kontakt mit dem Virus treten. Das Virus verfügt über eine ausgesprochen hohe Tenazität gegenüber äußeren Einflüssen. Es ist hitzestabil, unempfindlich gegenüber pH-Werten zwischen 3-9 sowie äther- und chloroformresistent (CARTWRIGHT und HUCK 1967, MAYR et al. 1968, JOHNSON und COLLINGS 1969, BACHMANN 1970, MORIMOTO 1972). Einmal im Bestand bleibt es somit über Monate infektiös. Es stehen für die Bekämpfung nur wenige Mittel zur Verfügung. Eine entscheidende Möglichkeit ist die Einhaltung eines strikten Impfregimes, wobei Impfstoffe zum Einsatz kommen, die seit etwa 3 Jahrzehnten auf den gleichen inaktivierten Virus-Isolaten beruhen. In den letzten zehn Jahren wurden zunehmend neue Isolate entdeckt, die sich, wie das hochvirulente Isolat Kresse und das wenig virulente Isolat NADL2, nur in wenigen Aminosäuren unterscheiden. Zum Teil weisen sie aber gravierende Unterschiede in ihrer Pathogenität auf. Daraus ergeben sich neben dem dringenden Rat zur Beobachtung der aktuellen Entwicklung mehrere Fragen hinsichtlich der zukünftigen Handhabung des Virus (SOARES et al. 2003, ZIMMERMANN et al. 2006). So sollte geklärt werden: • wie verbreitet sind diese neuen Isolate • was könnte ihre Entwicklung begünstigt haben • wie effizient ist der Schutz, den herkömmliche Impfstoffe gegen die neuen Isolate bieten • kann eines der Isolate eine Grundlage für einen neuen, effizienteren Impfstoff liefernDiese Dissertation umfasst insgesamt drei Veröffentlichungen, welche versuchen, die gestellten Fragen zu beantworten. Im ersten Artikel wird die Wirksamkeit eines neuen Impfstoffes auf Grundlage des hochvirulenten, vorherrschenden Isolat 27a untersucht. Im zweiten Manuskript wird mit Hilfe von in vitro- und in silico- Modellen die Populationsdynamik demonstriert. Die dritte Veröffentlichung widmet sich der Beschreibung der neuen Parvotypen (PPV2, PPV3 und PPV4), welche aus Herzen und Tonsillen von deutschen, klinisch gesunden Schlachtschweinen isoliert werden konnten.

A parvovirose canina é uma doença causada pelo parvovírus canino, um vírus pertencente à família Parvoviridae. A doença causa quadros agudos de gastroenterite hemorrágica altamente contagiosa, e é responsável por altas taxas de morbidade e mortalidade, principalmente, em cães jovens. O principal agente etiológico desencadeador da doença é o parvovírus canino tipo 2 (CPV 2). As vacinas parenterais comercializadas contra esse vírus não são adequadas para filhotes com menos de 45 dias de idade. Além disso, essa doença não apresenta um tratamento especifico, sendo a profilaxia de grande importância. O objetivo desse trabalho foi desenvolver um antígeno de entrega vacinal de modo a proteger o animal antes do seu desenvolvimento imunológico. Através do aumento da produção de IgA total a partir da primeira imunização e da resposta sistêmica de IgG especifica a partir da segunda imunização em camundongos, foi possível verificar que as superfícies de mucosa são ativas imunologicamente, desde o nascimento do animal como também capazes de estimular tanto a resposta local quanto a sistêmica em camundongos imaturos e adultos. No intuito de proteger também esses jovens animais, a imunização por via sublingual mostrou-se uma técnica promissora. Em comparação com os grupos de camundongos imunizados com a amostra vacinal e o hidrogel líquido, o grupo que recebeu o hidrogel liofilizado teve uma melhor resposta imunológica, uma vez que a técnica de liofilização aumentou a característica de mucoadesividade da quitosana e consequentemente aumentou o tempo de permanência do hidrogel na mucosa sublingual. / Canine parvovirus is a virus that belongs to the Parvoviridae family. The disease causes acute hemorrhagic gastroenteritis, which is highly contagious and responsible for high rates of morbidity and mortality, especially in puppies. The main etiologic agent of this disease is canine parvovirus type 2 (CPV 2). Nowadays, there is a commercial parenteral vaccine against this virus, but it is not suitable for puppies under 45 days old. This disease has no specific treatment and the prophylaxis has a great importance. The aim of this study was to develop a sublingual delivery vaccine that protect the animals before their 45 days. By increasing the total IgA production from the first immunization and systemic specific IgG response after the second immunization in mice, it demonstrated that mucosal surfaces are immunologically active since birth and capable of stimulating both systemic and local response in puppies and adults. In order to protect these puppies, mucosal immunization is a promising technique. The group that received lyophilized hydrogel had a better immune response, since the lyophilization technique increased the mucoadhesive of chitosan and consequently the residence time of the lyophilized hydrogel at sublingual mucosa.

A parvovirose canina é uma doença causada pelo parvovírus canino, um vírus pertencente à família Parvoviridae. A doença causa quadros agudos de gastroenterite hemorrágica altamente contagiosa, e é responsável por altas taxas de morbidade e mortalidade, principalmente, em cães jovens. O principal agente etiológico desencadeador da doença é o parvovírus canino tipo 2 (CPV 2). As vacinas parenterais comercializadas contra esse vírus não são adequadas para filhotes com menos de 45 dias de idade. Além disso, essa doença não apresenta um tratamento especifico, sendo a profilaxia de grande importância. O objetivo desse trabalho foi desenvolver um antígeno de entrega vacinal de modo a proteger o animal antes do seu desenvolvimento imunológico. Através do aumento da produção de IgA total a partir da primeira imunização e da resposta sistêmica de IgG especifica a partir da segunda imunização em camundongos, foi possível verificar que as superfícies de mucosa são ativas imunologicamente, desde o nascimento do animal como também capazes de estimular tanto a resposta local quanto a sistêmica em camundongos imaturos e adultos. No intuito de proteger também esses jovens animais, a imunização por via sublingual mostrou-se uma técnica promissora. Em comparação com os grupos de camundongos imunizados com a amostra vacinal e o hidrogel líquido, o grupo que recebeu o hidrogel liofilizado teve uma melhor resposta imunológica, uma vez que a técnica de liofilização aumentou a característica de mucoadesividade da quitosana e consequentemente aumentou o tempo de permanência do hidrogel na mucosa sublingual. / Canine parvovirus is a virus that belongs to the Parvoviridae family. The disease causes acute hemorrhagic gastroenteritis, which is highly contagious and responsible for high rates of morbidity and mortality, especially in puppies. The main etiologic agent of this disease is canine parvovirus type 2 (CPV 2). Nowadays, there is a commercial parenteral vaccine against this virus, but it is not suitable for puppies under 45 days old. This disease has no specific treatment and the prophylaxis has a great importance. The aim of this study was to develop a sublingual delivery vaccine that protect the animals before their 45 days. By increasing the total IgA production from the first immunization and systemic specific IgG response after the second immunization in mice, it demonstrated that mucosal surfaces are immunologically active since birth and capable of stimulating both systemic and local response in puppies and adults. In order to protect these puppies, mucosal immunization is a promising technique. The group that received lyophilized hydrogel had a better immune response, since the lyophilization technique increased the mucoadhesive of chitosan and consequently the residence time of the lyophilized hydrogel at sublingual mucosa.