Early events associated with infection of Epstein-Barr virus infection of primary B-cells
Authors: Pankaj Kumar
Subhash C Verma
Erle S Robertson
Masanao Murakami
Fuming Yi
Sabyasachi Halder
Journal: PLoS ONE (2009)
Discipline: Multidisciplinary
Keywords: Epstein Barr virus; EBV; BAC; GFP; HEK 293T
Affiliations: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

Abstract:  Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection. 

Retracted: No

PubPeer comments:
 Figure 9B. * Red boxes: The 24h and 72h Flourescence (sic) panels appear to look the same. They both show a little dot in the right half of the photo that is not visible in any of the other fluorescence images. Could the authors please check? ![file] , 
 Figure 10A. * Orange boxes: In the 96hrs row, the ACV0 and ACV25 DAPI panels appear to look the same, while the MERGE panels look different. Could the authors please check? ![file] , 
 2021 Expression of Concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0256674 Following the publication of this article [1], concerns were raised regarding results presented in Figs 3,u00a09, andu00a010. Specifically,In Fig 3A, irregularities have been detected in the background signal surrounding bands in the following lanes:u00a0u00a0u00a0u00a0u25cb BamHI T panel: lanes 1u20137u00a0u00a0u00a0u00a0u25cb BamHI K panel: lanes 2u20137u00a0u00a0u00a0u00a0u25cb BamHI E panel: lane 7u00a0u00a0u00a0u00a0u25cb BamHI H panel: lanes 3u20137u00a0u00a0u00a0u00a0u25cb BamHI C panel: lanes 1, 3, 5, 7u00a0u00a0u00a0u00a0u25cb Puro panel: lanes 3, 5, 6u00a0u00a0u00a0u00a0u25cb EGFP panel: lanes 1, 5, 7Inu00a0Fig 9B, the ACV25 24hrs Fluorescence panel appears similar to the ACV25 72 hrs Fluorescence panel.Inu00a0Fig 10A, the ACV 0 96 hrs DAPI panel appears similar to the ACV 25 96 hrs DAPI panel.The authors clarified that theu00a0Fig 9Bu00a0ACV25 24hour panel and theu00a0Fig 10Au00a0ACV 0 (96h) DAPI panel were inadvertently duplicated during figure preparation and provided the updated Figsu00a09u00a0andu00a010u00a0below to correct the duplicated panels. Furthermore, the authors indicated that the irregularities in the Fig 3A results are due to the resolution of the camera, dust or particulate matter on the gel, and the way the images were captured in the gel documentation system. They explained that raw data underlying the published PCR results are no longer available due to the Gel Doc system having been replaced in the time since the experiments were conducted. In the absence of the original data underlying the Fig 3A results, the concerns cannot be resolved.The original data underlying the results presented in Figs 3A andu00a09Au00a0are no longer available. The individual level data underlyingu00a0Fig 9C and 9Du00a0are provided in theu00a0S1u00a0andu00a0S2u00a0Files below. The data underlying the remaining results presented in this article are available from the corresponding author upon request.Theu00a0PLOS ONEu00a0Editors issue this Expression of Concern to notify readers of the above concerns and relay the supporting data and updated figures provided by the corresponding author.Reference1.Halder S, Murakami M, Verma SC, Kumar P, Yi F, Robertson ES (2009) Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells. PLoS ONE 4(9): e7214.u00a0https: doi.org 10.1371 journal.pone.0007214u00a0pmid:19784370 ,