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Amplification and titration of stocks of clinical strains of BK virus
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Contextualização: O poliomavírus BK (BKPyV) infeta cerca de 80% da população mundial,
estabelecendo uma infeção persistente sobretudo nas células epiteliais do trato renal e nas
células epiteliais de transição do trato urinário. O BKPyV replica-se de forma discreta, não
apresentando quaisquer manifestações clínicas em indivíduos imunocompetentes. No entanto,
em situações de transplante renal, a replicação do BKPyV pode conduzir a casos de nefropatia.
Até à data, não existe no mercado nenhum fármaco eficaz contra o BKPyV, pelo que a estratégia
clínica utilizada para impedir o desenvolvimento da nefropatia consiste na redução da terapia
imunossupressora. Neste enquadramento, a pesquisa de um antiviral eficaz é fundamental. De
forma a assegurar a eficácia global do fármaco é necessário que sejam realizados estudos pré clínicos utilizando as mais variantes estirpes do BKPyV.
Objetivo: Desenvolvimento de um modelo in vitro mais eficaz para a replicação e amplificação
de estirpes isoladas do BKPyV, em comparação com os modelos já desenvolvidos.
Métodos: As células Vero e as células MRC-5 foram cultivadas e incubadas com a estirpe
216x2X durante um mês. De seguida, as partículas virais foram extraídas e purificadas. Estes
modelos foram avaliados quanto à sua eficácia através do cálculo da titulação dos stocks
produzidos e quanto à sua infectividade através da infeção de células epiteliais tubulares
proximais renais humanas (RPTEC).
Resultados: Os resultados demonstram que ambas as linhas celulares são permissíveis à estirpe
216x2X. No entanto, quando utilizando as células Vero, estas não foram capazes de produzir
novos viriões, ao contrário do que aconteceu quando se usou as células MRC-5. Assim,
podemos aferir que a estirpe 216x2X pode ser adaptada a um modelo in vitro utilizando as
células MRC-5.
Conclusão: A falta de antivirais no combate à infeção pelo BKPyV apresenta-se como um
desafio na gestão de doentes em condições pós-transplante. A procura deste fármaco ideal
continua a ser condicionada pela falta de informação consequente da inexistência de bons
modelos in vitro e in vivo. Além disso, a utilização da estirpe Dunlop apresenta como problema
adjacente a raridade da mesma na população humana. A investigação deve continuar a rumar
no sentindo de conseguir-se não só isolar-se estirpes presentes em doentes como também no
desenvolvimento de um fármaco eficaz para a população em geral.
Background: The BK polyomavirus (BKPyV) infects approximately 80% of the world’s population, establishing a persistent infection mostly in renal tubular epithelial cells and the urinary tract transitional epithelial cells. BKPyV replicates discreetly, causing no clinical manifestations in immunocompetent individuals. However, BKPyV replication can cause nephropathy in kidney transplant recipients. To date, there is no effective drug on the market to combat BKPyV, so the clinical strategy for preventing nephropathy is to reduce immunosuppressive therapy. In this context, the search for an effective antiviral is critical. Pre clinical investigations using the most diverse strains of BKPyV are required to ensure the overall efficiency of the antiviral. Objective: Development of a more effective in vitro model for the replication and amplification of isolated BKPyV strains, compared to models already developed. Methods: For one month, Vero and MRC-5 cells were cultivated and incubated with the 216x2X strain. After that, the virus particles were isolated and purified. These models were evaluated for their efficacy by calculating the titration of the stocks produced and for their infectivity by infecting human renal proximal tubular epithelial cells (RPTEC). Results: The results show that both cell lines are permissible to the 216x2X strain. However, when Vero cells were used, they were unable to produce new virions, unlike MRC-5 cells. We can therefore see that the 216x2X strain can be adapted to an in vitro model using MRC-5 cells. Conclusion: The lack of antivirals to combat BKPyV infection is a challenge in the management of post-transplant patients. The search for this ideal antiviral continues to be conditioned by the lack of information resulting from the absence of good in vitro and in vivo models. Furthermore, the use of the Dunlop strain presents the adjacent problem of its rarity in the human population. Continued research is required not just to isolate strains present in patients, but also to develop an effective drug for the general population.
Background: The BK polyomavirus (BKPyV) infects approximately 80% of the world’s population, establishing a persistent infection mostly in renal tubular epithelial cells and the urinary tract transitional epithelial cells. BKPyV replicates discreetly, causing no clinical manifestations in immunocompetent individuals. However, BKPyV replication can cause nephropathy in kidney transplant recipients. To date, there is no effective drug on the market to combat BKPyV, so the clinical strategy for preventing nephropathy is to reduce immunosuppressive therapy. In this context, the search for an effective antiviral is critical. Pre clinical investigations using the most diverse strains of BKPyV are required to ensure the overall efficiency of the antiviral. Objective: Development of a more effective in vitro model for the replication and amplification of isolated BKPyV strains, compared to models already developed. Methods: For one month, Vero and MRC-5 cells were cultivated and incubated with the 216x2X strain. After that, the virus particles were isolated and purified. These models were evaluated for their efficacy by calculating the titration of the stocks produced and for their infectivity by infecting human renal proximal tubular epithelial cells (RPTEC). Results: The results show that both cell lines are permissible to the 216x2X strain. However, when Vero cells were used, they were unable to produce new virions, unlike MRC-5 cells. We can therefore see that the 216x2X strain can be adapted to an in vitro model using MRC-5 cells. Conclusion: The lack of antivirals to combat BKPyV infection is a challenge in the management of post-transplant patients. The search for this ideal antiviral continues to be conditioned by the lack of information resulting from the absence of good in vitro and in vivo models. Furthermore, the use of the Dunlop strain presents the adjacent problem of its rarity in the human population. Continued research is required not just to isolate strains present in patients, but also to develop an effective drug for the general population.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
BK polyomavirus In vitro models Amplification Strains Kidney transplant Mestrado Integrado - 2023