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Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin- and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells. Nota: la publicación fue aceptada el día 16 de Diciembre del 2014. Incluyo aquí la carta por parte de la editora de la revista en la que nos comunica que solo eran necesarios unos cambios mínimos de redacción. From:jacomine.krijnse@bioquant.uni-heidelberg.de To:ldelgui@fcm.uncu.edu.ar CC: Subject:Cellular Microbiology - CMI-14-0223.R1 Body:Dear Dr. Delgui, Your manuscript, "Infectious Bursal Disease Virus uptake Involves Macropinocytosis and Trafficking to Early Endosomes in a Rab5-dependent Manner", has been reviewed by the same two referees. Their reports are included below. As you will see both reviewers are very satisfied with the changes made. However, the second reviewer recommends a minor text change related to the requirement of cholesterol. Please resubmit your revised manuscript to me at ManuscriptCentral, https://mc.manuscriptcentral.com/cell-micro. Please include a letter stating how you have addressed this minor comment upon which I will accept the manuscript in due course. To help the editor and reviewers, please upload two versions of your manuscript text in Word format, one ´clean´ version and one with the changes made highlighted in yellow. To maintain current scientific reviews, revisions of manuscripts should be resubmitted within 3 months of the editor´s decision. Additional time for resubmission required to conduct specific experimental protocols may be requested in advance. I look forward to receiving your revised manuscript. Best regards, Jacomine Krijnse Locker Referees comments: Referee: 1 Comments to the Author (There are no comments.) Referee: 3 Comments to the Author The addition of controls for the various internalization assays, and the inclusion of GTPase experiments, additional compounds and dextran uptake assays have served to greatly improve the manuscript. The role of macropinocytosis in entry of IBD is convincingly demonstrated. Minor comment: The authors imply that due to the effect seen with cholesterol depleting drugs that perhaps Caveolea is involved in entry. I do not understand this link as dynamin depletion (which is required for caveolea endocytosis) has no impact on infection and cholesterol is required for macropinocytosis (Grimmer, S., van Deurs, B. & Sandvig, K. Membrane ruffling and macropinocytosis in A431 cells require cholesterol. J. Cell Sci. 115, 2953?2962 (2002). Why do the authors not think the cholesterol requirement is more in support of their data on macropinocytosis? Date Sent:16-Dec-2014