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Production and Purification of Cell Culture-generated Hepatitis B Virus by Transient Transfection and Density Gradient.

ABSTRACT: An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line-derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture-generated HBV (HBVcc) by the transient transfection of the HBV molecular clone, a large amount of purified viruses is needed, because such viruses exhibit limited infection efficiencies in cell culture. Here, we describe how to generate and purify HBVcc by the transient transfection of HBV molecular clones. This system provides a powerful tool for studying the infection and propagation of HBV and for developing anti-viral agents against HBV.

SUBMITTER: Murayama A

PROVIDER: S-EPMC10366991 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Production and Purification of Cell Culture-generated Hepatitis B Virus by Transient Transfection and Density Gradient.

Murayama Asako A Akari Hirofumi H Kato Takanobu T

Bio-protocol 20230720 14

An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line-derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture-generated HBV (HBVcc) by the transient transf ...[more]

PMID: 37497458

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Project description:BackgroundPlasmodium falciparum zygotes develop in the mosquito midgut after an infectious blood meal containing mature male and female gametocytes. Studies of mosquito-produced P. falciparum zygotes to elucidate their biology and development have been hampered by high levels of contaminating mosquito proteins and macromolecules present in zygote preparations. Thus, no zygote-specific surface markers have been identified to date. Here, a methodology is developed to obtain large quantities of highly purified zygotes using in vitro culture, including purification methods that include magnetic column cell separation (MACS) followed by Percoll density gradient centrifugation. This straightforward and effective approach provides ample material for studies to enhance understanding of zygote biology and identify novel zygote surface marker candidates that can be tested as transmission blocking vaccine (TBV) candidates.MethodsPlasmodium falciparum gametocyte cultures were established and maintained from asexual cultures. Gametocytes were matured for 14 days, then transferred into zygote media for 6 h at 27 ± 2 °C to promote gamete formation and fertilization. Zygotes were then purified using a combination of MACS column separation and Percoll density gradient centrifugation. Purity of the zygotes was determined through morphological studies: the parasite body and nuclear diameter were measured, and zygotes were further transformed into ookinetes. Immunofluorescence assays (IFA) were also performed using the ookinete surface marker, Pfs28.ResultsAfter stimulation, the culture consisted of transformed zygotes and a large number of uninfected red blood cells (RBCs), as well as infected RBCs with parasites at earlier developmental stages, including gametes, gametocytes, and asexual stages. The use of two MACS columns removed the vast majority of the RBCs and gametocytes. Subsequent use of two Percoll density gradients enabled isolation of a pure population of zygotes. These zygotes transformed into viable ookinetes that expressed Pfs28.ConclusionThe combined approach of using two MACS columns and two Percoll density gradients yielded zygotes with very high purity (45-fold enrichment and a pure population of zygotes [approximately 100%]) that was devoid of contamination by other parasite stages and uninfected RBCs. These enriched zygotes, free from earlier parasites stages and mosquito-derived macromolecules, can be used to further elucidate the biology and developmental processes of Plasmodium.

Project description:The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

Dataset Information

Production and Purification of Cell Culture-generated Hepatitis B Virus by Transient Transfection and Density Gradient.

Publications

Production and Purification of Cell Culture-generated Hepatitis B Virus by Transient Transfection and Density Gradient.

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