If an application has 45 subcontract sites, should each PI from each site submit a Letter of Support? Are there any limits to the number of letters that should be included?

Yes, the PI from each site should write a Letter of Support. There is no limit of the number of Letters of Support that can be submitted with an application. 

For more information, please be sure to read the Submission Instructions (formerly known as application guidelines) for your specific PFA and cycle.