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Fiorentina players Patrick Cutrone and German Pezzella are among three new confirmed cases of coronavirus at the club.
Physio Stefano Dainelli has also tested positive for COVID-19, the Serie A side announced on Saturday.
All three "are in good health at their homes in Florence", Fiorentina said via their official Twitter account.
On Friday, striker Dusan Vlahovic became the Viola's first confirmed case, although the club said he was at home and did not have any symptoms.
"The club is in the process of undertaking all the necessary isolation procedures in accordance with the regulations, starting with identifying all the people who have had contact with the player," Fiorentina said.
Juventus defender Daniele Rugani was the first Serie A player to test positive for the virus, which has spread alarmingly quickly in the country.
Sampdoria striker Manolo Gabbiadini was the second confirmed case in Serie A and the club announced a further five positive tests on Friday.
First-team players Omar Colley, Albin Ekdal, Antonino La Gumina and Morten Thorsby, as well as club doctor Amedeo Baldari, have all contracted the virus.
As of March 14, Italy has seen 17,660 confirmed cases of coronavirus and 1,266 deaths.
The Italian government has suspended all sport until April 3 due to the outbreak and placed the country in lockdown, with all shops except food stores and pharmacies closing.
Fiorentina currently lie 13th is Serie A on 30 points, five clear of the relegation zone. They were undefeated in three games before the suspension of activities.
Football is not the only sport to have been locked down by the pandemic, with the Six Nations rugby, which was due to be concluded this weekend, and the Masters golf, which was slated for later in the month, among the major events to have been postponed or cancelled.