1. Have any drivers been updated since it was working properly? Possibly automated driver downloads running in the background if you were connected to the internet recently?
2. I assume there's no virus protection running on this PC, correct?
3. When you say you've checked all the hardware and everything's running fine - does that mean you ran diagnostics and the result was ok, or have you also done performance monitoring and all are humming along with very low utilization? (or both?) If you haven't run diagnostics on your disks and RAM(especially your RAM), I'd highly recommend that.
4. Have you replaced your USB cable to your audio interface? It would be odd if a cable were suddenly bad, but I've seen stranger things.
If all that fails, maybe your audio driver has become corrupt? Have you tried reinstalling / updating your audio driver?
Hope that helps - if none of that works, let us know - maybe we can come up with other ideas.
Russ