USB and VM latencies are both questionable here, I would think.

ISA motherboards still work and have been mass produced in the hundreds of millions. (I have about a dozen in my retro closet.)

The esoteric ISA expansion cards would be the bottleneck, not the motherboards.

All those layers add some latency, but 8 mhz is really slow. USB 4 can do extremely low latency, otherwise PCIe over USB wouldn't work.