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Hepatitis C virus infection in phenotypically distinct Huh7 cell lines.

ABSTRACT: In 2005, the first robust hepatitis C virus (HCV) infectious cell culture system was developed based on the HCV genotype 2a JFH-1 molecular clone and the human-derived hepatoma cell line Huh7. Although much effort has been made to dissect and expand the repertoire of JFH-1-derived clones, less attention has been given to the host cell despite the intriguing facts that thus far only Huh7 cells have been found to be highly permissive for HCV infection and furthermore only a limited number of Huh7 cell lines/stocks appear to be fully permissive. As such, we compiled a panel of Huh7 lines from disparate sources and evaluated their permissiveness for HCV infection. We found that although Huh7 lines from different laboratories do vary in morphology and cell growth, the majority (8 out of 9) were highly permissive for infection, as demonstrated by robust HCV RNA and de novo infectious virion production following infection. While HCV RNA levels achieved in the 8 permissive cell lines were relatively equivalent, three Huh7 lines demonstrated higher infectious virion production suggesting these cell lines more efficiently support post-replication event(s) in the viral life cycle. Consistent with previous studies, the single Huh7 line found to be relatively resistant to infection demonstrated a block in HCV entry. These studies not only suggest that the majority of Huh7 cell lines in different laboratories are in fact highly permissive for HCV infection, but also identify phenotypically distinct Huh7 lines, which may facilitate studies investigating the cellular determinants of HCV infection.

SUBMITTER: Sainz B

PROVIDER: S-EPMC2720605 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAirborne transmitted pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), need to interact with host cells of the respiratory tract in order to be able to enter and disseminate in the host organism. Pulmonary alveolar macrophages (PAM) and MA104 derived monkey kidney MARC-145 cells are known to be permissive to PRRSV infection and replication and are the most studied cells in the literature. More recently, new cell lines developed to study PRRSV have been genetically modified to make them permissive to the virus. The SJPL cell line origin was initially reported to be epithelial cells of the respiratory tract of swine. Thus, the goal of this study was to determine if SJPL cells could support PRRSV infection and replication in vitro.ResultsThe SJPL cell growth was significantly slower than MARC-145 cell growth. The SJPL cells were found to express the CD151 protein but not the CD163 and neither the sialoadhesin PRRSV receptors. During the course of the present study, the SJPL cells have been reported to be of monkey origin. Nevertheless, SJPL cells were found to be permissive to PRRSV infection and replication even if the development of the cytopathic effect was delayed compared to PRRSV-infected MARC-145 cells. Following PRRSV replication, the amount of infectious viral particles produced in SJPL and MARC-145 infected cells was similar. The SJPL cells allowed the replication of several PRRSV North American strains and were almost efficient as MARC-145 cells for virus isolation. Interestingly, PRRSV is 8 to 16 times more sensitive to IFN? antiviral effect in SJPL cell in comparison to that in MARC-145 cells. PRRSV induced an increase in IFN? mRNA and no up regulation of IFN? mRNA in both infected cell types. In addition, PRRSV induced an up regulation of IFN? and TNF-? mRNAs only in infected MARC-145 cells.ConclusionsIn conclusion, the SJPL cells are permissive to PRRSV. In addition, they are phenotypically different from MARC-145 cells and are an additional tool that could be used to study PRRSV pathogenesis mechanisms in vitro.

Project description:Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-?-, IFN-?-, and NF-?B-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells.IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.

Project description:Hepatitis C Virus is a leading cause of chronic liver disease. The identification and characterisation of key host cellular factors that play a role in the HCV replication cycle is important for the understanding of disease pathogenesis and the identification of novel anti-viral therapeutic targets. Gene expression profiling of HCV infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defence mechanisms (apoptosis, proliferation and anti-oxidant responses), cellular metabolism (lipid and protein metabolism) and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV associated pathogenesis. These include an increase in pro-inflammatory and pro-apoptotic signalling and a decrease in the anti-oxidant response pathways of the infected cell. 5x105 Huh7 cells were seeded in 25cm2 culture flasks and infected in triplicate either with the genotype 2a HCV clone, JFH-1 at a multiplicity of infection (MOI) of 3 or mock infected with an equal volume of concentrated conditioned growth medium. At 6, 12, 18, 24 and 48 hours post-infection, cellular RNA was extracted using TRIzol reagent (Invitrogen). Trizol lysates were shipped to Expression Analysis (NC, USA) where RNA was purified, quality tested using the Agilent Bioanalyser and hybridised onto Human U133 Plus 2.0 Affymetrix microarray chips for fluorescence data acquisition. In summary, a total of 30 RNA samples were analysed including 3x mock infected samples taken at 6, 12, 18, 24 and 48 hours post-treatment and 3x JFH-1 infected samples taken at 6, 12, 18, 24 and 48 hours post-infection. Two samples (Mock_6hrs_1 and JFH-1_6hrs_1) did not pass our data quality control measures and were therefore excluded from the statistical analysis.

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Hepatitis C virus infection in phenotypically distinct Huh7 cell lines.

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