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 parvovlrus B19 (B19) in chronic anaemia of HIV infected TB patients. Patlents were selected from an existing databank of 307 patients included in 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 patient's era 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

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

Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies. Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

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

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

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

A síndrome da má absorção (SMA), caracterizada pelo mau desenvolvimento e desuniformidade do lote de aves, causa importantes prejuízos econômicos à avicultura comercial. No entanto, por se tratar de uma doença multifatorial e possivelmente polimicrobiana, o envolvimento do parvovírus de galinha (ChPV) na ocorrência da SMA ainda é pouco conhecido. Com o propósito de elucidar a possível associação entre a presença do ChPV e a ocorrência da SMA, foram desenvolvidas e aplicadas ferramentas moleculares para a detecção e quantificação do ChPV em amostras de frangos comerciais no Estado do Rio Grande do Sul. Uma PCR quantitativa foi desenvolvida para detectar e quantificar cópias do genoma (CG) do ChPV em amostras de suabes de cloaca de 59 frangos saudáveis e 68 frangos com sinais clínicos sugestivos de SMA. Os resultados revelaram que todas as amostras dos dois grupos investigados, continham o genoma do ChPV. No entanto, a carga viral em frangos com SMA foi significativamente (p≤0,0001) maior (1x105 CG/100 ng DNA) do que em frangos saudáveis (1,3x103 CG/100 ng DNA). Adicionalmente às amostras de cloaca, o ChPV também foi investigado em amostras de tecidos (fígado, timo, baço, bursa de Fabricius - BF e intestino) e soros provenientes de nove frangos saudáveis e 50 frangos com sinais indicativos da SMA. O ChPV foi encontrado tanto em aves saudáveis como nas aves com SMA, no entanto, observou-se uma diferença na distribuição deste agente nos tecidos analisados. O genoma do vírus foi mais frequentemente detectado na BF, baço e intestino das aves com SMA, sendo que o intestino foi o tecido que apresentou maior carga viral. Os resultados encontrados nestes estudos demonstraram que o genoma viral estava altamente disseminado nas aves investigadas. Além disso, observou-se uma maior carga viral em frangos de corte com SMA quando comparado com aves sadias. Com base nos resultados encontrados, sugere-se que a maior carga viral de ChPV existente em aves com SMA, em relação a aves saudáveis, seja um dos fatores que favoreça a ocorrência da síndrome. / The malabsorption syndrome (MAS), characterized by the poor development and lack of uniformity of chicken flocks, causes significant economic losses to commercial poultry. However, because it is a multifactorial and possibly polymicrobial disease, the involvement of Chicken parvovirus (ChPV) in the MAS occurrence is still not clear. In order to elucidate the possible association between the presence of ChPV and the occurrence of MAS, molecular tools were developed and applied for the detection and quantification of ChPV DNA in commercial poultry samples in the state of Rio Grande do Sul. A quantitative PCR was developed to detect and quantify the ChPV genome copies (GC) in cloacal swab samples of 59 healthy broilers and 68 broilers with clinical signs suggestive of MAS. The results showed that all investigated samples of the two groups contained the genome ChPV. However, viral loads in MAS-affected animals were significantly (p≤0.0001) higher (1x105 GC/100 ng DNA) than in healthy broilers (1.3x103 GC/100 ng DNA). In addition to the cloacal samples, the presence of ChPV DNA was also investigated in tissue samples (liver, thymus, spleen, bursa of Fabricius - BF and intestine) and sera from nine healthy broilers and 50 broilers with signals indicative of MAS. The ChPV was found in healthy avian as well MAS-affected, however, there was a difference in the distribution of this agent in those tissues. The virus genome was more frequently detected in the BF, spleen and intestines of the MAS-affected broilers, and the intestines contained the highest viral loads, in comparison with other tissues. Our results demonstrated that the viral genome can be found in both healthy and MAS-affected broilers. In addition, higher viral loads were detected in broilers with signs suggestive of MAS compared to healthy birds. Based on these results, it is suggested that the greatest viral load of ChPV existing in MAS-affected broilers when compared to healthy birds, is one of the factors that favor the occurrence of the syndrome.