Scotland also lost a player for the tournament when a groin problem ended Grant Gilchrist's involvement as the brutal nature of the first two weeks of matches begins to take its toll.
Hours after Wales were officially listed as second in the world rankings on the back of the 28-25 Twickenham victory -- their highest-ever position -- their worst fears were realised when centre Williams (knee) and winger Amos (dislocated shoulder) were ruled out.
The backs duo will be replaced in the squad by James Hook and Gareth Anscombe.
Fullback Liam Williams, meanwhile, will not be considered for Thursday's Cardiff clash with Fiji after being concussed at Twickenham.
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England flanker Tom Wood escaped with a warning for the accidental kick to the head that caused the injury and is free to play in the now pivotal Pool A match against Australia on Saturday, as is veteran number eight Nick Easter.
The 37-year-old Easter, who has not started for England since the 2011 World Cup, was called up to the squad to replace Vunipola, who damaged knee ligaments against Wales.
Easter is likely to be on the bench after Ben Morgan was given a clean bill of health after missing the Wales game with a knee injury.
While both England and Wales camps were doing their best to focus on their next games, the sports sections of Monday's newspapers were still dominated by last Saturday's match and England's decision not to kick for goal from a late penalty that could have levelled the score.
Special celebratory pictorial front pages were the order of the day in the regional papers of Wales, but The Times described England captain Chris Robshaw's option to go for a lineout as a "moment of madness" while former England flyhalf-turned pundit Stuart Barnes said the decision was the "worst piece of captaincy I can remember in my playing or media days".
One team getting some much-needed good news on the injury front were Italy, after it was announced that their inspirational captain Sergio Parisse will join the squad on Tuesday after being given the all-clear on the calf injury that ruled him out of the first two games of the tournament.
The number eight had surgery to drain a haematoma in his calf three weeks ago but is fit to play against Ireland on Sunday in a game Italy must win to have any realistic chance of reaching the quarter-finals for the first time.
Ireland's victory over Romania at Wembley on Sunday was watched by a World Cup record crowd of 89,297, but the tournament's smallest venue takes centre stage for the next match, when Exeter's 12,000-capacity Sandy Park hosts the Pool C game between Tonga and Namibia on Tuesday.
Until five years ago and Exeter's promotion to the Premiership, Sandy Park was the home to second-tier rugby, but it has a special place in the sport's history as the venue for New Zealand's first game in Britain, when the "Originals" beat Devon 55-4 in 1905.
(Editing by David Goodman)
