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The Denver Broncos lost 31-3 to the New Orleans Saints as practice squad receiver Kendall Hinton was called up to fill in at quarterback.
All four of the Broncos' quarterbacks were absent after Jeff Driskel tested positive for Covid-19 on Thursday.
Team-mates Drew Lock, Brett Rypien and Blake Bortles are now in isolation after all being deemed "high-risk" close contacts.
Hinton, 23, previously played quarterback in college.
An undrafted player who has been on the team's practice squad since last month, he made his NFL debut on less than a day's notice and made 1-for-9 in passing for 13 yards as well as two interceptions.
"I can easily say this has been the most eventful 24 hours of my life," said Hinton. "But when I got the call it was pure excitement, of course there was nerves and disbelief."
He added that he had only had time to learn 20-30 plays before the game.
"Coming in I knew that I had no idea what the state of the game was like. I am familiar with our concepts but it's a lot different from quarterback," he said.
"We knew it would be a tough situation but it was a challenge I was willing to take."
The San Francisco 49ers will play their next two home games at the Arizona Cardinals' State Farm Stadium in Glendale because of local Covid-19 restrictions.
Santa Clara County, where the 49ers' stadium and training facilities are based, has issued a declaration to help curb the spread of coronavirus, which includes a three-week ban on "all recreational activities that involve physical contact".
The 49ers will face the Buffalo Bills in Arizona on Monday followed by the Washington Football Team on 13 December.