Aaron Ramsey has added to Ryan Giggs' selection headache by being ruled out of the Nations League trip to Bulgaria.
Wales boss Giggs defended the decision not to strengthen his squad at a lunchtime press conference after losing four players for the Group B4 game in Sofia.
But little more than two hours later Juventus playmaker Ramsey – who captained Wales in the 0-0 draw against the Republic of Ireland on Sunday – was also out of the game.
"Aaron Ramsey has withdrawn from the squad due to injury and will not travel to Bulgaria," the Football Association of Wales posted on its Twitter account.
Tottenham defender Ben Davies captained Wales in the friendly defeat to England at Wembley last Thursday and is set to wear the armband again.
Wales have now lost five players from an original 27-man squad and are short of forward options, with David Brooks, Kieffer Moore and Hal Robson-Kanu dropping out alongside suspended midfielder Joe Morrell.
Giggs says being in a Covid-19 bubble has made the situation "more problematic", although other countries have called up players after being hit by withdrawals.
But he insisted he has enough players at his disposal without attempting to draft others in to bolster his squad.
"We all got tested (for coronavirus) after the Ireland game on Sunday," Giggs said.
"After that it would have been very difficult (to bring players in). Players would have had fly to Dublin and then Bulgaria.
"We just have to deal with the players we've got, which is fine."
Asked if he should have called up a replacement when Robson-Kanu was ruled out with a broken arm two days before the England game, Giggs said: "We had 26 players so that was fine.
"I felt with Dan James, Harry Wilson and Brooksy not involved in the England game that they would be three new players coming into two games.
"So we felt we were fine in that respect.
"It has been more problematic because of the Covid situation. But that wasn't solely the reason, we felt we had enough players."
Wales head to Eastern Europe top of Group B4 with seven points from three Nations League games and yet to concede in the competition.
Giggs has reined back on his initial attacking approach – Wales scored 10 goals in his first four games – to tighten up a defence which has seen goalkeeper Wayne Hennessey keep five clean sheets in six games.
"I always feel that we need to get the defensive aspect as we do have players who can hurt teams," Giggs said.
"It is always about getting the balance. You don't want to give chances away but we need to be better going forward.
"That is about creating relationships between players, whether full-back, winger, number 10, centre-forward, midfielders, and that is difficult with the turnover of players we get. But it will come."
Bournemouth defender Chris Mepham has boosted Wales by being declared fit after missing Sunday's goalless draw with the Republic of Ireland in Dublin.
Mepham picked up a knee injury in the England defeat at Wembley.
"It's not easy with this being the third game," Giggs said. "But every country's in the same boat so you have to deal with it.
"There is a lot of juggling with players and making sure they've had enough minutes, but also that they're still sharp.
"We have to be ready and we have been in the two (Nations League) games so far. We've done well, a draw and a win, and we've been solid."