Mason Mount and Ben Chilwell are out of England's European Championship clash with the Czech Republic and must remain in isolation until the start of next week.
The Three Lions' preparations for Tuesday's final Group D match were thrown into chaos when it emerged that the Chelsea pair had been in contact with Scotland's Billy Gilmour, who tested positive for coronavirus.
Mount and Chilwell will now miss the pool decider at Wembley and could also miss England's last-16 match, with qualification assured but their place in the group up in the air.
The Football Association said in a statement: "We can confirm that Ben Chilwell and Mason Mount must isolate up to and including next Monday (June 28). This decision has been taken in consultation with Public Health England.
"The pair were confirmed overnight as close contacts of Scotland's Billy Gilmour after his positive test following last Friday's match."
The statement continued: "Chilwell and Mount will isolate and train individually in private areas at England's training base St. George's Park, with the rest of the squad returning there after tonight's fixture against Czech Republic at Wembley.
"We will continue to follow all Covid-19 protocols and the UEFA testing regime, while remaining in close contact with PHE.
"The entire England squad and staff had lateral flow tests on Monday and all were again negative, as was the case with Sunday's UEFA pre-match PCR tests. Further tests will be carried out as and when appropriate."
