Abstract
John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.
Highlights
John Cunningham virus (JCV) is the etiological agent of Progressive Multifocal Leukoencephalopathy (PML), a demyelinating disease of the brain [1]
Intracellular viral DNA was extracted from 1 × 106 COS-7 cells after infection at the selected sampling time and used in quantitative PCR (Q-PCR) for JCV TAg in order to evaluate the efficiency of JCV replication
In order to test whether an infectious progeny has been produced during infection in COS-7 cells, total protein extracts from COS-7 cells were used to assess capsid protein VP1 production using Western blot analysis
Summary
John Cunningham virus (JCV) is the etiological agent of Progressive Multifocal Leukoencephalopathy (PML), a demyelinating disease of the brain [1]. The JCV genome has two conserved protein-coding regions (early and late) transcribed in opposite directions starting from a common regulatory region known as the Non-coding Control Region (NCCR), the most variable portion of the viral genome. It is responsible of tissue tropism, contains the origin of viral DNA replication and promoter/ enhancer elements [2]. It controls expression of early and late proteins including large T-antigen (TAg). JCV and PML investigation have been severely hampered by the lack of an animal model and the host cell range of archetype JCV is strictly restricted in cultured cells
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