Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces

Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces

Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/μL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/μL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.

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Main Authors: Quintero-Gil, Carolina, Mirazo, Santiago, Parra-Suescún, Jaime, López-Herrera, Albeiro, Mainardi, Victoria, Arbiza, Juan, Orduz, Sergio
Format: Digital revista
Language:eng
Published: Universidad Nacional de Colombia - Sede Bogotá - Facultad de Ciencias - Departamento de Biología 2019
Online Access:https://revistas.unal.edu.co/index.php/actabiol/article/view/79351
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record_format ojs
institution UNAL
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access En linea
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language eng
format Digital
author Quintero-Gil, Carolina
Mirazo, Santiago
Parra-Suescún, Jaime
López-Herrera, Albeiro
Mainardi, Victoria
Arbiza, Juan
Orduz, Sergio
spellingShingle Quintero-Gil, Carolina
Mirazo, Santiago
Parra-Suescún, Jaime
López-Herrera, Albeiro
Mainardi, Victoria
Arbiza, Juan
Orduz, Sergio
Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces
author_facet Quintero-Gil, Carolina
Mirazo, Santiago
Parra-Suescún, Jaime
López-Herrera, Albeiro
Mainardi, Victoria
Arbiza, Juan
Orduz, Sergio
author_sort Quintero-Gil, Carolina
title Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces
title_short Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces
title_full Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces
title_fullStr Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces
title_full_unstemmed Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces
title_sort cell culture isolation of hepatitis e virus genotype 3 strain obtained from human feces
description Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/μL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/μL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection.
publisher Universidad Nacional de Colombia - Sede Bogotá - Facultad de Ciencias - Departamento de Biología
publishDate 2019
url https://revistas.unal.edu.co/index.php/actabiol/article/view/79351
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spelling oai:www.revistas.unal.edu.co:article-793512020-04-17T16:26:30Z Cell culture isolation of Hepatitis E Virus Genotype 3 Strain obtained from human feces Aislamiento de una cepa genotipo 3 del virus de la hepatitis E obtenida de heces humanas Quintero-Gil, Carolina Mirazo, Santiago Parra-Suescún, Jaime López-Herrera, Albeiro Mainardi, Victoria Arbiza, Juan Orduz, Sergio A549 cells hepevirus isolation RNA virus Aislamiento células A549 hepevirus virus ARN Hepatitis E virus (HEV) is considered one of the leading causes of acute viral hepatitis worldwide, and about 20 million infections and approximately 57 000 deaths occurred every year. However, little is known about the replicative virus cycle due to the absence of a consensus cell culture model. A549 cell line is considered susceptible to HEV genotype 3, however, both viral strain and cell culture conditions could affect the viral isolation in vitro. The objective of this work was to isolate in vitro an HEV-3 strain obtained from human feces. To this, a genotype 3 HEV strain previously identified by genetic characterization was inoculated in A549 monolayers, and incubated for two hours at 37 °C. Five days post-infection, cells were passaged (subcultured) for the first time, and serial passages were done on average every four days during 41 days. HEV replication was evaluated through RT-qPCR in each passage, and reinfection of the cell line with the viral progeny derived from A549 infected monolayers was assessed through immunofluorescence and RT-qPCR. Viral RNA was detected in each passage from infected monolayers, and the highest amount was found after 26 days (2 x 106 copies/μL). In reinfection assay, capsid antigen was detected perinuclearly and forming foci, and 1x104 copies/μL of viral RNA was detected after 96 hours post infection. This shows that HEV recovered from the cell lysate monolayers was infectious. This viral isolate offers a critical tool to study the unknown aspect of HEV infection. El virus de la hepatitis E (HEV) se considera como una de las principales causas de hepatitis viral aguda en el mundo; cada año ocurren aproximadamente 20 millones de infecciones y 57 000 muertes. Debido a la ausencia de un modelo de cultivo celular consenso, se sabe poco sobre el ciclo replicativo del virus. La línea celular A549 se considera susceptible al genotipo 3 de HEV, pero tanto la cepa viral como las condiciones del cultivo celular podrían afectar el aislamiento viral in vitro. Por tanto nos propusimos aislar in vitro una cepa genotipo 3 del HEV. Para ello, se inocularon células A549 con una cepa HEV-3 identificada previamente por caracterización genética, y se incubó durante dos horas a 37 °C. Cinco días después de la infección, las células se pasaron (subcultivaron) por primera vez, y se realizaron pases seriados cada cuatro días en promedio, durante 41 días. En cada pase se evalúo la replicación del HEV mediante RT-qPCR. La reinfección de la línea celular con progenie viral derivada de monocapas de A549 infectadas se evaluó mediante inmunofluorescencia y RT-qPCR. Se detectó ARN viral en cada pase a partir de monocapas, y el pico máximo se alcanzó a los 26 días post infección (2 x 106 copias/μL). En el ensayo de reinfección, se detectó antígeno de cápside perinuclearmente y formando focos, y se detectaron 1 x 104 copias/μL de RNA viral a las 96 horas post infección. El HEV recuperado de lisado de monocapas fue infeccioso. Este aislado viral ofrece una herramienta importante para estudiar aspectos desconocidos de la infección por HEV. Universidad Nacional de Colombia - Sede Bogotá - Facultad de Ciencias - Departamento de Biología 2019-09-01 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion application/pdf application/xml https://revistas.unal.edu.co/index.php/actabiol/article/view/79351 10.15446/abc.v24n3.79351 Acta Biológica Colombiana; Vol. 24 No. 3 (2019); 503-508 Acta Biológica Colombiana; Vol. 24 Núm. 3 (2019); 503-508 1900-1649 0120-548X eng https://revistas.unal.edu.co/index.php/actabiol/article/view/79351/pdf https://revistas.unal.edu.co/index.php/actabiol/article/view/79351/xml Derechos de autor 2019 Acta Biológica Colombiana https://creativecommons.org/licenses/by-nc-sa/4.0

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