No abstract available. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / Unrestricted

O parvovírus B19 é o agente causador de eritemas infecciosos em crianças, hidropsia fetal em mulheres gestantes, esse vírus pode causar anemia crônica e crise aplástica transitória respectivamente. A levedura P. pastoris é um sistema de expressão usado na produção de várias proteínas heterólogas. O objetivo deste trabalho foi expressar as proteínas VP1 e VP2 do parvovírus humano B19 em levedura P. pastoris. As seqüências gênicas VP1 e VP2 foram amplificadas por PCR, usando DNA do vírus B19, os produtos obtidos foram inicialmente subclonados no vetor pGEM-TEasy. Os fragmentos de DNA foram digeridos com enzima de restrição EcoRI e NotI , purificados e inseridos no vetor de expressão e excreção pPIC9K de P. pastoris, entre os sítios EcoRI e NotI. Para expressão das proteínas recombinantes VP1 e VP2 de parvovírus humano B19, os transformantes foram crescidos em glicerol e induzidos pela adição de metanol. As expressões dos antígenos recombinantes foram analisadas por SDS-PAGE e atividade biológica foram confirmadas pelos ensaios imunológicos ELISA, Dot-Blot e Western Blot. / Human Parvovirus B19 is the causative agent of erythema infectiosum in children, hydrops fetalis in pregnant women, B19 may cause chronic anemia and aplastic crisis, respectively. The yeast P. pastoris expression system is being used for the production of various recombinant heterologous proteins. The objective of this work was to express the VP1 and VP2 proteins of the human parvovirus B19 in the yeast Pichia pastoris. The coding sequence of VP1 and VP2 were amplified by PCR, using DNA virus of B19. PCR-products were initially subcloned in the vector pGEM-TEasy. The DNA fragment EcoRI and NotI was excised, purified, and inserted between the sites EcoRI and NotI of P. pastoris expression-secretion vector pPIC9K.For heterologous expression of the proteins VP1 and VP2 Human parvovirus B19, the transformants were growth on glycerol and induced by the addition of methanol. The expressed recombinant antigens VP1 and VP2 were analyzed by SDS-PAGE and its biological activity were confirmed through Enzyme immunoassay EIA, Dot-Blot e Western Blot.

Mouse polyomavirus (MPyV) is a small non-enveloped virus. Its capsid consists of 72 pentamers of the major capsid protein VP1. The central cavity of each VP1 pentamer contains one minor capsid protein, either VP2, or VP3. The minor capsid proteins are dispensable for capsid formation, but their presence is required for infection of the host cell, presumably because of their anticipated functions during virus entry. After internalization, MPyV virions traffic to endoplasmic reticulum (ER). VP2 and VP3 have been proposed to function as factors responsible for penetration of ER membranes, which is required for subsequent delivery of the viral DNA into the nucleus, a key step of the early phase of MPyV infection. Three hydrophobic domains were predicted in the sequence of VP2 and VP3. First in the unique Nterminal part of VP2, second and third in the common part of VP2 and VP3. The third domain corresponds to C-terminal VP1binding alpha-helix. It has been previously found in our laboratory, that VP2 and VP3 fused to N-terminus of EGFP, when expressed in mammalian cells, display properties similiar to the wild-type VP2 and VP3, namely affinity to intracellular membranes and high cytotoxicity. Expression plasmids carrying mutated VP2 and VP3 fused to Nterminus of EGFP were prepared to determine the hydrophobic...

Die Welpensterblichkeit wird auf 10-20 % der lebend geborenen Welpen geschätzt. Zwei Virusinfektionen scheinen bei Welpensterblichkeit und Fruchtbarkeitsstörungen des Hundes eine wesentliche Rolle zu spielen: das canine Herpesvirus Typ 1 (CHV-1) und das Canine Minute Virus (CnMV, synonym Minute Virus of Canines). Ziel der Arbeit war es, die Verbreitung dieser beiden Viren unter Zuchthunden in Deutschland zu untersuchen. Das klinische Erkrankungsbild ist bei beiden Viren ähnlich. Bei erwachsenen Tieren verläuft die Infektion meist unauffällig. Infektionen während der Trächtigkeit können zu Fruchtresorption, Aborten, Missbildungen und Welpensterblichkeit führen. Infizierte Welpen erkranken meist in den ersten drei Lebenswochen und sind apathisch, zeigen Saugunlust, haben Atemwegsprobleme, Erbrechen und leiden oft auch unter Durchfall. Die Mortalität ist hoch. Zwischen Juli 2004 und Oktober 2005 wurden 429 Serumproben gesammelt. Die Bestimmung der Seroprävalenz von CHV-1 erfolgt im Serumneutralisationstest (SNT) mit Madin Darby Canine Kidney (MDCK)-Zellen ohne den Zusatz von Komplement. CnMV-spezifische Antikörper wurden mittels indirekter Immunofluoreszenz (IFT) auf zuvor mit CnMV infizierten Walter Reed Canine Zellen (WRCC) nachgewiesen. Des Weiteren wurden 37 Abstriche, 34 Spermaproben, 16 Kotproben und 37 verstorbene Welpen aus 14 Würfen mittels PCR und Virusisolierung auf CHV-1 und CnMV direkt untersucht. 27,7 % (119/429) aller Serumproben hatten neutralisierende Antikörper gegen CHV-1. Die Seroprävalenz für CnMV beträgt 5,6 % (24/429). Die statistische Auswertung ergab nur für das Geschlecht einen signifikanten Einfluss auf den Serostatus von CHV-1. Alter, Zwingergröße, Anzahl Ausstellungsbesuche und Rasse spielten keine Rolle für die Seroprävalenz von CHV-1. Bei den klinischen Vorberichten Welpensterblichkeit (n = 24) und Fruchtresorptionen (n = 5) ist die Seroprävalenz von CHV-1 mit 37,5 % und 40 % bedeutend höher als die ermittelte Gesamtprävalenz von 27,7 %. Die Untersuchung der Abstriche, Sperma-, Kot- und Organproben von verstorbenen Welpen mittels PCR und zum Teil Virusisolierung auf CnMV und CHV-1 verlief in allen Fällen mit negativem Ergebnis. Der Nachweis von CnMV ist bis heute nur in wenigen Speziallabors möglich. Um die Nachweismöglichkeiten von CnMV zu verbessern und zu vereinfachen, wurde das Virusprotein-2-(VP2)-Gen von CnMV in den Vektor pET22b(+) kloniert. Das am C-terminalen Ende mit einem 6x His-Tag markierte Protein wurde im E.coli-Stamm BL21CodonPlus(DE3)RIL exprimiert, aufgereinigt und als Antigen im ELISA getestet. Die prokaryotische Genexpression des VP2 von CnMV ist für die Etablierung eines diagnostischen ELISA nicht geeignet. Während der Expression aggregierte das Protein zu unlöslichen Inclusion Bodies. Nach Aufreinigung ist das Protein immer noch mit E.coli-Proteinen kontaminiert. Im ELISA zeigte sich das rekombinante Antigen wenig spezifisch. In Fällen von Welpensterblichkeit (n = 14), waren diese in weit weniger Fällen als vermutet CHV-1 oder CnMV bedingt. Insgesamt scheint CnMV in Deutschland weit weniger verbreitet zu sein als in anderen Ländern der Welt.

The spread of bluetongue virus (BTV) to previously disease-free regions which prohibit the use of the current BTV live-attenuated vaccine has highlighted the need for a new generation of vaccines (Ferrari, De Liberato et al. 2005; Veronesi, Hamblin et al. 2005). Subunit vaccines are one of the attractive alternative strategies. Subunit vaccines against BTV would target the outercapsid protein VP2, the main neutralization-specific antigen (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). A subunit vaccine based on the use of BTV-VP2 may be achieved by either using VP2 by itself or by means of virus-like particles (VLPs) on which VP2 proteins are exposed. In VLPs, the VP2 is co-expressed with other capsid and core proteins to form a particle that resembles the intact BTV. The BTV-VLP vaccine strategy is advantageous since it presents the neutralizing epitopes of more than one viral protein in a more authentic manner as found on the virus itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However there are difficulties associated with large scale production and a decrease in the stability of the particles over time (Berg, Difatta et al. 2005; Wang, Zhao et al. 2006). Studies have already demonstrated the vaccine potential of BTVVP2 by itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However if BTV-VP2 is to be used by itself as a single subunit vaccine, it is important that the protein is expressed under conditions where it is correctly folded and soluble. Solubility refers to the capacity of the expressed antigen to fold into an ordered tertiary structure that authentically exposes the neutralizing epitopes to the immune system (Dinner, Sali et al. 2000; Dobson 2003). However non-native interactions within and between in vitro synthesized viral proteins such as BTV-VP2 often leads to protein aggregation or insolubility. The immune response against aggregated or insoluble proteins is generally very poor. This problem of aggregation and insolubility may be alleviated to an extent by generating truncated versions of the protein from which hydrophobic regions that promote aggregation have been deleted leaving only the major neutralizing epitopes of the antigen (Fukumoto, Xuan et al. 2003; Bonafe, Rininger et al. 2009; Liu, Zeng et al. 2009; Seo, Pyo et al. 2009). The focus of the research presented in this dissertation was to evaluate the solubility of full-length BTV(10)-VP2 and truncated versions thereof after expression in a prokaryotic and baculovirus-Sf9 expression system. The full-length BTV(10)-VP2 (956 amino acids) gene and genes encoding truncated versions of BTV(10)-VP2 i.e. BTV(10)-VP2(aa450) (amino acid 1 to 450) and BTV(10)- VP2(aa650) (amino acid 1 to 650) were cloned into the bacterial expression vector pET160-DEST and the baculovirus expression vector pDEST™8. The C-terminal hydrophobic regions which might contribute to aggregation or insolubility of the protein when expressed in vitro were deleted from these truncated BTV(10)-VP2 proteins. The truncated proteins however still contained BTV neutralizing epitopes that were predicted from literature. The prokaryotic expression of the full-length BTV(10)-VP2 and the other truncated recombinant BTV(10)-VP2 proteins was carried out in E. coli BL21 Star DE3 expression strain. The initial pilot expression study confirmed high level expression of the recombinant proteins. The study also revealed that these proteins were insoluble. The optimization of the prokaryotic expression in order to increase the yield of soluble proteins by means of differential inducer concentrations, fermentation temperature and harvesting times did not produce soluble BTV(10)-VP2 and truncated BTV(10)-VP2 proteins. Previous studies have demonstrated the role of L-arginine in the recovery of soluble proteins from aggregation by reversing aggregation (Tsumoto, Umetsu et al. 2003). However in the current study, arginine treatment of the inclusion body and bacterial lysate containing the BTV(10)- VP2 and truncated recombinant proteins did not release soluble proteins. No soluble recombinant BTV(10)-VP2 proteins were detected when the recombinant proteins were expressed in BL21 host cells over-expressing heat-shock proteins (hsps) and chemical chaperones. However when the different recombinant proteins were co-expressed with the molecular chaperones dnaK-dnaJ-GrpE, it resulted in a fraction of soluble recombinant BTV(10)-VP2 proteins. In particular, approximately 50% of the total expressed BTV(10)-VP2(aa450) protein was soluble while approximately 20% of the total expressed BTV(10)-VP2(aa650) and full-length BTV(10)-VP2 were found soluble when coexpressed with dnaK-dnaJ-GrpE chaperones. These recombinant proteins could be eluted from a nickel affinity column further confirming that these proteins are in fact soluble. Interestingly the coexpression of the BTV(10)-VP2(aa450) protein with the above chaperones in combination with chaperones groEL-groES or only groEL-groES did not produce any soluble proteins. Baculovirus-insect expression of the aforementioned BTV(10)-VP2 recombinant proteins was carried out in Spodoptera frugiperda 9 (Sf9) cells. High level expression of the recombinant proteins was confirmed by an initial pilot expression study conducted at 42 hours post infection (p.i.). The pilot study also revealed that the recombinant proteins were insoluble. Arginine treatment of the lysate released a small fraction of soluble BTV(10)-VP2(aa450) and BTV(10)-VP2(ORF) proteins only detectable with immunoblot analysis using the anti-BTV(10) IgY antibodies. The amount of solubilized proteins was however too small to justify the cost associated with this expression system. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted

Les entérovirus (EV) humains, membres de la famille des Picornaviridae, comprennent plus de 100 génotypes appartenant à 4 espèces : Enterovirus A, B, C et D. Ces virus sont à l’origine de pathologies très variées et occupent une place importante en santé publique. La méthode conventionnelle de typage des EV consiste en une réaction de séroneutralisation avec des antisérums spécifiques à partir de souches isolées en culture cellulaire ; cette technique est longue, coûteuse et limitée par sa capacité à identifier correctement les variants antigéniques et les nouveaux génotypes. De plus, elle est limitée aux génotypes cultivables. De nouvelles méthodologies de typage moléculaire par séquençage partiel du génome ont été récemment développées ; elles consistent à analyser une partie variable de la région codant une des protéines de capside (VP1 ou alternativement VP2 ou VP4). Cependant ces techniques sont le plus souvent réalisées à partir de souches isolées en culture cellulaire. Le but de ce travail a été de développer une technique de typage des EV directement sur des prélèvements cliniques en se basant sur le séquençage partiel de la région VP2 dont le laboratoire avait montré précédemment l’intérêt (Nasri et al., 2007). Pour le dessin des amorces, nous avons utilisé la stratégie CODEHOP (COnsensus DEgenerate Hybrid Oligonucleotide Primer) de manière à améliorer à la fois la spécificité et la sensibilité de la méthode d’amplification. Nous présentons ici un premier article décrivant la nouvelle technique de typage VP2 et rapportons son application au typage d’échantillons cliniques trouvés positifs par une PCR ciblant la région 5’ non codante du génome des EV sur une période de trois ans. Le deuxième article présente pour la première fois l’application d’une technique de typage direct à des échantillons environnementaux d’eaux usées. Le troisième article montre l’intérêt de coupler deux techniques de typage ciblant des régions différentes (VP1 et VP2) pour l’identification de souches d’EV isolées par culture cellulaire en Centre-Afrique. Malgré des problèmes de sensibilité, cette nouvelle technique de typage directement à partir d’échantillons peut rendre de grands services tant en clinique humaine que pour la surveillance environnementale / Human enteroviruses (EV), members of the Picornaviridae family, comprise more than 100 genotypes belonging to four species: Enterovirus A, B, C and D. These viruses are responsible for a wide range of pathologies and play an important role in Public Health. The classic method for typing EVs consists in a seroneutralisation assay with specific antisera using strains isolated by cell culture; this technique is cumbersome, expensive and unable to type currently antigenic variants and new serotypes. In addition, it is limitated to culturable serotypes. New methods of molecular typing by partial sequencing of the genome have been recently developed; they consist in analysing a variable part of the region coding for capsid protein (VP1 or alternatively VP2 or VP4). However, these techniques are usually performed on strains isolated by cell culture. The aim of this work was to develop a typing method able to work from clinical specimens by partial sequencing of the VP2 region, which had been shown to exhibit a good typing performance (Nasri et al., 2007). For the design of primers, we used the CODEHOP (COnsensus DEgenerate Hybrid Oligonucleotide Primer) strategy on order to improve the sensitivity and the specificity of the amplification assay. We present herein a first article that describes in details the new VP2 typing method and requests its use for typing clinical specimens found positive by a PCR assay targeting the 5’ non coding region of EVs over a period of three years. The second paper describes for the first time the direct use of a typing method on environtmental wastewater samples. The third article shows the interest of coupling 2 typing techniques targeting different regions (VP1 and VP2) of the EV genome for the identification of strains isolated by cell culture in Republic of Central Africa. Despite a loss of sensitivity, the new VP2 typing method used directly on specimens was found to be of great help both for human diagnosis and environmental surveillance

Mouse polyomavirus is a member and model virus of Polyomaviridae family. In order to infect cells and produce viral progeny, the viral chromosome must be transported to the nucleus. Several studies suggest that virions are transporeted to the endoplasmic reticulum, from which they are transferred to the cytosol with assistace of host proteins. Two of these proteins are the chaperon, BiP (binding immunoglobulin protein) and the cochaperone, DNAJ B14. Polyomaviruses probably enter the nucleus through nuclear pores with the assistence of importins. These processes were mainly studied with SV40. In this work, we show that MPyV infection induces a change in distribution of the DNAJ B14 protein, which became clustered into foci, where it co-localizes with the viral capsid protein, VP1. The occurrence of foci varies during infection. With use of proximity ligation assay, we have shown that during an early fase of MPyV infection, DNAJ B14 and BiP get in the close proximity with VP1. It is suggested that negatively charged amino acids at the N-terminus of the minor capsid protein, VP2, are required for targeting virions to translocon and proteins associated with ERAD. We created MPyV with VP2 mutated in these amino acids. The negatively charged amino acid at position 17 is not necessary for successful...

Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....

Das canine Parvovirus (CPV-2)wurde erstmals bei Hunden im Jahr 1978 beschrieben. Dieses Virus verbreitete sich innerhalb weniger Monate weltweit in einer schweren Pandemie. Retrospektiv wird angegnommen, dass es sich aud dem Felinen Panleukopenie-Virus oder einem nah verwandten Virus entwickelt hat. Ziel dieser Arbeit war es, durch Untersuchungen von archivierten paraffineingebetteten Gewebeproben von Hunden und Katzen aus den frühen 1970iger Jahren einen möglichen Anzestor des caninen Parvovirus zu identifizieren und die Hypothese zu überprüfen, dass CPV-2 schon vor 1978 in den Hundepopulationen zirkulierte.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-19T11:33:37Z No. of bitstreams: 1 resumo.pdf: 14552 bytes, checksum: c7feb5483902e97430fd0ecfd37fc98b (MD5) / Made available in DSpace on 2017-06-19T11:33:37Z (GMT). No. of bitstreams: 1 resumo.pdf: 14552 bytes, checksum: c7feb5483902e97430fd0ecfd37fc98b (MD5) Previous issue date: 2005-03-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O vírus da doença infecciosa bursal é um importante agente patogênico de aves e desde 1962 tem estado relacionado com grandes prejuízos econômicos. Ainda hoje não foi estabelecido um protocolo de vacinação realmente eficiente e falhas na imunização das aves são geralmente descritas. Os motivos dessas falhas vem sendo pesquisados e as causas alegadas são desde o surgimento de cepas patogênicas por mutações na região do gene que codifica para a VP2, uso mal assessorado de vacinas pouco atenuadas e a reversão vacinal. Este trabalho teve como objetivo pesquisar e comparar a patogenicidade de isolados de campo com a de cepas vacinais. Foram testadas 21 amostras de bursa que foram submetidas à Unidade de Sanidade Avícola da Universidade Federal de Viçosa com suspeita de doença infecciosa bursal e três amostras vacinais. O isolamento viral foi realizado em células VERO, sendo o vírus isolado de 14,28% (3/21) das amostras de campo e de todas as amostras vacinais. As amostras foram padronizadas pela habilidade de provocar efeito patogênico em cultivo celular e foram inoculadas em ovos embrionados de 10 dias de idade. Amostras de fígado, rins, bursa, baço e intestino de cada um dos embriões foram coletadas durante 8 dias e as alterações macroscópicas avaliadas. Metade de cada amostra coletada foi mantida à 4°C e metade foi fixada com formol 10%. As amostras fixadas foram processadas pelo método de inclusão em parafina, coradas com hematoxilina e eosina e as alterações microscópicas avaliadas. Das amostras mantidas a 4°C foi extraído o RNA total pelo método do TRIzol e testadas pela técnica de RT- PCR. Nas observações anatomopatológicas os embriões inoculados apresentavam tamanho reduzido quando comparado aos embriões controle e órgãos com lesões sugestivas de danos vasculares. Nas análises histopatológicas, os órgãos linfóides apresentaram pronunciada depleção linfóide, os hepátocitos vacuolização citoplasmática e os rins estruturas basófilas dentro de túbulos. No teste de RT-PCR foi possível detectar a presença do ácido nucléico viral em todos os tecidos, sendo que a distribuição tecidual dos isolados de campo foi maior quando comparada aos isolados vacinais. Esses resultados revelaram não haver grande diferença quando comparadas as lesões causadas pelos isolados de campo com as lesões causadas por algumas amostras vacinais. Porém há distintos padrões de distribuição em tecidos que poderia indicar uma variação na atenuação de algumas amostras vacinais. Esses resultados sugerem que as amostras vacinais podem causar lesões características da doença e que o uso de determinada cepa, mais ou menos invasiva, deve ser avaliada com cuidado pelo profissional no campo. / The infectious bursal disease virus is an important pathological agent of poultry and since 1962 it has been related to great economical losses. Until now, an efficient vaccination protocol has not been established and faults in poultry immunization are generally described. These faults motifs are being searched and various causes have been quoted, from the emergence of pathogenic strains by mutations in the gene responsible for coding VP2, to bad usage of vaccines attenuated for low passage and vaccinal reversion. The aim of this work was to examine and compare field isolates pathogenicity with vaccinal strains. Twenty-one bursa samples suspicious of infectious bursal disease submitted to Unidade de Sanidade Avícola da Universidade Federal de Viçosa and three vaccinal samples were tested. The viral isolation was accomplished in VERO cells, the virus was isolated in 14,28% (3/21) of field samples and in all vaccinal samples. The samples were standardized by its ability to provoke pathogenic effect in cellular culture and were inoculated in 10 days embryonic eggs. Liver, kidney, bursa, spleen and gut samples of each embryo were collected during 8 days and the macroscopic alterations evaluated. Half of each collected sample was stored at 4°C and the other half was fixed with 10% formol. The fixed samples were processed by paraffin xinclusion method, dyed with hematoxilin and eosin and the microscopic alterations evaluated. Total RNA extraction was performed on those samples stored at 4°C by TRIzol method and tested by RT-PCR technique. In anatomopathological observations inoculated embryos presented reduced size when compared to control embryos and organs with characteristics lesions of vascular injury. In histopathological analysis lymphoid organs presented an accentuated lymphoid depletion, hepatocytes presented citoplasmatic vacuolization and kidneys basophiles structures inside tubules. In RT-PCR test was possible to detect the presence of viral nucleic acid in each and every tissues, and the distribution of field isolates was larger when compared to vaccinal isolates. The results revealed that there is not much difference between lesions caused by field isolates with lesions caused by some of vaccinal samples. However, there are distinct distribution patterns in tissues that could indicate a variation on attenuation in some vaccinal samples. These results suggest that vaccinal samples could cause characteristics disease lesions and that the use of a determinate strain more or less invasive should be evaluated with care by the field professional.