Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.4322/2179-443X.0671 http://hdl.handle.net/11449/206085 |
Resumo: | Hepatitis B virus (HBV) research has been hampered by the lack of suitable and reproducible cell culture systems that reliably mimic the viral life cycle. Several infection stages and metabolic aspects related to HBV cycle still need to be elucidated. The aim of this research was the study of techniques aiming at developing a sustainable in vitro platform of hepatitis B virus infection, in a simplified and low maintenance approach, evaluating the continuity of infection in cell culture throughout many passages, using a positive serum pool to the virus in human hepatocellular carcinoma. The viral load was quantified by real-time polymerase chain reaction. The cells underwent a freeze and thaw cycle, followed by seeding, and the new culture was analyzed to quantify the viral load. An aliquot was used to detect the surface antigen (HBsAg), by chemiluminescence. The detection of the core antigen (HBcAg) was performed by flow cytometry and by immunofluorescence microscopy. Viral load remained detectable throughout the studied period, 50 days after initial infection. The process of freezing and seeding produced detectable viral load for a 7-day period. HBsAg was reagent in the infected cells, confirming the maintenance of infection. The flow cytometry result indicated 11.85% of HBcAg positive cells, which demonstrates that new viral particles were at the assembly stage. Indirect immunofluorescence using epiluminescence microscopy allowed the detection of viral HBcAg in the interior of infected cells, confirming the results obtained by flow cytometry. The platform for infection in cell culture was successfully obtained during the studied period, which represents the possibility to apply this model in a continuous practice, to support several biotechnological purposes. |
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Repositório Institucional da UNESP |
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Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesisCell CultureHepatitis BHepatocellular Carcinoma CellsIn vitro InfectionHepatitis B virus (HBV) research has been hampered by the lack of suitable and reproducible cell culture systems that reliably mimic the viral life cycle. Several infection stages and metabolic aspects related to HBV cycle still need to be elucidated. The aim of this research was the study of techniques aiming at developing a sustainable in vitro platform of hepatitis B virus infection, in a simplified and low maintenance approach, evaluating the continuity of infection in cell culture throughout many passages, using a positive serum pool to the virus in human hepatocellular carcinoma. The viral load was quantified by real-time polymerase chain reaction. The cells underwent a freeze and thaw cycle, followed by seeding, and the new culture was analyzed to quantify the viral load. An aliquot was used to detect the surface antigen (HBsAg), by chemiluminescence. The detection of the core antigen (HBcAg) was performed by flow cytometry and by immunofluorescence microscopy. Viral load remained detectable throughout the studied period, 50 days after initial infection. The process of freezing and seeding produced detectable viral load for a 7-day period. HBsAg was reagent in the infected cells, confirming the maintenance of infection. The flow cytometry result indicated 11.85% of HBcAg positive cells, which demonstrates that new viral particles were at the assembly stage. Indirect immunofluorescence using epiluminescence microscopy allowed the detection of viral HBcAg in the interior of infected cells, confirming the results obtained by flow cytometry. The platform for infection in cell culture was successfully obtained during the studied period, which represents the possibility to apply this model in a continuous practice, to support several biotechnological purposes.Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista (UNESP)Instituto de Química Universidade Estadual Paulista (UNESP)Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista (UNESP)Instituto de Química Universidade Estadual Paulista (UNESP)Universidade Estadual Paulista (Unesp)da Cruz, Carla Rios [UNESP]Lopes, Rute [UNESP]Santana, Moema de Souza [UNESP]da Costa, Paulo Inácio [UNESP]2021-06-25T10:26:16Z2021-06-25T10:26:16Z2020-01-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.4322/2179-443X.0671Revista de Ciencias Farmaceuticas Basica e Aplicada, v. 41.2179-443X1808-4532http://hdl.handle.net/11449/20608510.4322/2179-443X.06712-s2.0-85102939408Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengRevista de Ciencias Farmaceuticas Basica e Aplicadainfo:eu-repo/semantics/openAccess2021-10-22T20:56:11Zoai:repositorio.unesp.br:11449/206085Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T18:50:10.165399Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
title |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
spellingShingle |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis da Cruz, Carla Rios [UNESP] Cell Culture Hepatitis B Hepatocellular Carcinoma Cells In vitro Infection |
title_short |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
title_full |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
title_fullStr |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
title_full_unstemmed |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
title_sort |
Hepatitis B virus: A new platform for in vitro studies of infection, replication cycle and pathogenesis |
author |
da Cruz, Carla Rios [UNESP] |
author_facet |
da Cruz, Carla Rios [UNESP] Lopes, Rute [UNESP] Santana, Moema de Souza [UNESP] da Costa, Paulo Inácio [UNESP] |
author_role |
author |
author2 |
Lopes, Rute [UNESP] Santana, Moema de Souza [UNESP] da Costa, Paulo Inácio [UNESP] |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
da Cruz, Carla Rios [UNESP] Lopes, Rute [UNESP] Santana, Moema de Souza [UNESP] da Costa, Paulo Inácio [UNESP] |
dc.subject.por.fl_str_mv |
Cell Culture Hepatitis B Hepatocellular Carcinoma Cells In vitro Infection |
topic |
Cell Culture Hepatitis B Hepatocellular Carcinoma Cells In vitro Infection |
description |
Hepatitis B virus (HBV) research has been hampered by the lack of suitable and reproducible cell culture systems that reliably mimic the viral life cycle. Several infection stages and metabolic aspects related to HBV cycle still need to be elucidated. The aim of this research was the study of techniques aiming at developing a sustainable in vitro platform of hepatitis B virus infection, in a simplified and low maintenance approach, evaluating the continuity of infection in cell culture throughout many passages, using a positive serum pool to the virus in human hepatocellular carcinoma. The viral load was quantified by real-time polymerase chain reaction. The cells underwent a freeze and thaw cycle, followed by seeding, and the new culture was analyzed to quantify the viral load. An aliquot was used to detect the surface antigen (HBsAg), by chemiluminescence. The detection of the core antigen (HBcAg) was performed by flow cytometry and by immunofluorescence microscopy. Viral load remained detectable throughout the studied period, 50 days after initial infection. The process of freezing and seeding produced detectable viral load for a 7-day period. HBsAg was reagent in the infected cells, confirming the maintenance of infection. The flow cytometry result indicated 11.85% of HBcAg positive cells, which demonstrates that new viral particles were at the assembly stage. Indirect immunofluorescence using epiluminescence microscopy allowed the detection of viral HBcAg in the interior of infected cells, confirming the results obtained by flow cytometry. The platform for infection in cell culture was successfully obtained during the studied period, which represents the possibility to apply this model in a continuous practice, to support several biotechnological purposes. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-01-01 2021-06-25T10:26:16Z 2021-06-25T10:26:16Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.4322/2179-443X.0671 Revista de Ciencias Farmaceuticas Basica e Aplicada, v. 41. 2179-443X 1808-4532 http://hdl.handle.net/11449/206085 10.4322/2179-443X.0671 2-s2.0-85102939408 |
url |
http://dx.doi.org/10.4322/2179-443X.0671 http://hdl.handle.net/11449/206085 |
identifier_str_mv |
Revista de Ciencias Farmaceuticas Basica e Aplicada, v. 41. 2179-443X 1808-4532 10.4322/2179-443X.0671 2-s2.0-85102939408 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Revista de Ciencias Farmaceuticas Basica e Aplicada |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808128988341600256 |