Conor Coady and Marcus Rashford will miss England’s Nations League double-header against Belgium and Iceland.
Wolves defender Coady has been forced to withdraw and isolate after being identified as a close contact to someone who has tested positive for coronavirus via track and trace.
Although the 27-year-old has tested negative twice with the contact coming prior to meeting up with the England squad, he still has to isolate in line with Government guidelines.
Meanwhile, Manchester United's Marcus Rashford misses out with a shoulder injury sustained during the win over Everton on Saturday.
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In a statement, the Football Association said: "Coady has been forced to self-isolate having discovered he had inadvertently come into contact with somebody prior to camp who has tested positive for Covid-19 via an NHS Test and Trace alert.
"The Wolverhampton Wanderers defender had returned two negative tests as part of The FA and UEFA's protocol but, in line with UK Government guidelines, has to adhere to a period of self-isolation.
"Meanwhile, Rashford's arrival into camp had initially been delayed to allow for further assessment on an injury sustained last weekend but the Manchester United forward will now not meet up."
On his withdrawal, Wolves skipper Coady said he is "absolutely gutted" to have to pull out of the squad.
"I contracted and recovered from the virus myself back in March, and have tested negative twice in camp, but of course it is important that I abide by the rules set by the government and observe a period of self-isolation," the defender said.
"It's so frustrating to have to miss games because of this, but these are the rules that are set out and the most important thing is to make sure my team-mates are not put at risk.
"My next priority will be to keep my fitness and hope I can be back and available for selection for the next Wolves fixture.
"For now, I will be supporting the lads from afar and I want to wish them all the very best of luck for the next two games."
Southgate added in his post-match press conference following the 3-0 win over Republic of Ireland: "To lose both is disappointing. We had some doubts about Marcus following the weekend, we hoped he might progress but that hasn't happened.
"For Conor, it's one of those weird ones where he has to isolate when he's more than likely not at risk. I think we're OK on numbers. We think we've got the cover, but let's hope that is the case."
England face Belgium in Leuven on Sunday before hosting Iceland at Wembley on Wednesday in the final two games of their Nations League group.