[Jaws]

Three other things to look for:

1. Make sure that Calibre is not starting up at boot time. This isn't Calibre, but the we're-going-to-pretend-that-all-Windows-machines-are-shared relics of Windows 95 still buried in the OS's model. The short version is that Python and boot-time startup sometimes don't play nice together, leading to later file-access-priority issues. (Some of this is Python's responsibility, too; but I'm one of those old-school "nothing runs until the OS is fully loaded and integrity-checked" guys who is obviously not kewl enuf to deal with Modern Programming Practices. Unfortunately, the Python developers are kewl, just ask them.)

2. Make sure that no other instances of Calibre are running, even from a prior session. I like ProcessExplorer for this, it's clearer than Task Manager (especially when trying to close down multiple instances), but squashing everything saying Calibre and then immediately rebooting should clear any lingering "but it's already open in another instance of Calibre!" flags hiding in there.

3. Clear out all of your caches and prefetches, reboot, and see if the problem persists. If so, there's a decent chance that something else in your startup sequence is locking the file; one frequent culprit is the search indexer, but it's not predictably so, and really no more predictable than any other program that might be putting its dirty digital fingers on the file (perhaps some other CBR/CBZ reader? perhaps the vendor's interface file from where you obtained the ebook?).