Generation and Quantitation of Infectious Hepatitis C Virus Derived from Cell Culture (HCVcc)

Paulson, Matthew

doi:10.1002/0471141755.ph13b04s51

Cited by 2 publications

(1 citation statement)

References 29 publications

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“…Cell culture-derived, infectious type 2a hepatitis C viruses (HCV) (HCVcc) were generated by transfection of Huh-7.5.1 cells with JFH-1 viral RNA following the protocols described by Paulson with modifications. 43 Briefly, HCV viral RNA was transcribed in vitro from the plasmid pJFH-1 using MEGAscript T7 Transcription kit (AM1333M, Life Technologies) and was then used to transfect Huh-7.5.1 cells using the TransIT-mRNA Transfection kit (Mirus Bio LLC, Madison WI, USA). Cells were split every 3 days.…”

Section: Methodsmentioning

confidence: 99%

Design and evaluation of novel interferon lambda analogs with enhanced antiviral activity and improved drug attributes

Zhao

Dong

et al. 2016

DDDT

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Type III interferons (IFNs) (also called IFN-λ: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4) are critical players in the defense against viral infection of mucosal epithelial cells, where the activity of type I IFNs is weak, and unlike type I IFNs that are associated with severe and diverse side effects, type III IFNs cause minimal side effects due to the highly restricted expression of their receptors, and thus appear to be promising agents for the treatment and prevention of respiratory and gastrointestinal viral infection. However, the antiviral potency of natural type III IFNs is weak compared to type I and, although IFN-λ3 possesses the highest bioactivity among the type III IFNs, IFN-λ1, instead of IFN-λ3, is being developed as a therapeutic drug due to the difficulty to express IFN-λ3 in the prokaryotic expression system. Here, to develop optimal IFN-λ molecules with improved drug attributes, we designed a series of IFN-λ analogs by replacing critical amino acids of IFN-λ1 with the IFN-λ3 counterparts, and vice versa. Four of the designed analogs were successfully expressed in Escherichia coli with high yield and were easily purified from inclusion bodies. Interestingly, all four analogs showed potent activity in inducing the expression of the antiviral genes MxA and OAS and two of them, analog-6 and -7, displayed an unexpected high potency that is higher than that of type I IFN (IFN-α2a) in activating the IFN-stimulated response element (ISRE)-luciferase reporter. Importantly, both analog-6 and -7 effectively inhibited replication of hepatitis C virus in Huh-7.5.1 cells, with an IC50 that is comparable to that of IFN-α2a; and consistent with the roles of IFN-λ in mucosal epithelia, both analogs potently inhibited replication of H3N2 influenza A virus in A549 cells. Together, these studies identified two IFN-λ analogs as candidates to be developed as novel antiviral biologics.

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Section: Methodsmentioning

confidence: 99%