Alice awakens in a deserted mansion with amnesia. She dresses and checks the mansion and she is subdued by an unknown person. A group of commandos break into the mansion and arrest the person, who introduces himself as Matt Addison, who has just transferred as a cop in Raccoon P.D. The commandos explain that everyone in the group, except Matt, is an employee of the Umbrella Corporation, and that Alice and her partner Spence are guards for a Hive entrance under the disguise of a couple living in the mansion. Five hours prior, The Red Queen shut down the entire facility and released amnesia inducing gas. The commando team does not know why the Red Queen sealed the facility. The group travels to the underground train under the mansion that leads to the Hive, where they find Spence. They start the train and travel into the facility.
They reach the Queen's chamber, but it is protected by a laser defense system that kills four of the commandos. Kaplan disables the Red Queen and the power fails, opening all of the doors in the Hive. This releases the zombified staff and containment units containing Lickers, mutated animals. During a battle with the zombies, Rain is bitten and J.D is killed. Alice begins regaining her memories, but Matt and Alice are separated from the group. Matt looks for information about his sister Lisa, while Alice encounters several infected dogs and surprises herself when she reflexively defends herself with martial arts.
Matt finds his sister zombified. Alice saves him and Matt explains that he and Lisa were environmental activists. Lisa infiltrated Umbrella to smuggle out evidence of illegal experiments. Alice remembers she was Lisa's contact in the Hive and was intending to bring down Umbrella, but does not tell Matt. The survivors reunite at the Queen's chamber. The commandos explain that they have one hour before the Hive will seal automatically, trapping them inside. Alice and Kaplan activate the Red Queen in order to find an exit and rig the Red Queen's circuit breaker so they can shut her down remotely to force her cooperation. As they escape through maintenance tunnels, they are ambushed by zombies. Kaplan is bitten and separated from the group.
Alice remembers that an anti-virus is in the lab, but when they arrive it is missing. Spence regains his memory, realizing he stole and released the virus. He hid the T-virus and anti-virus on the train. Spence is bitten, traps the survivors in the lab, and heads to the train. He retrieves the anti-virus, but is ambushed and killed by a Licker as the survivors watch on a security monitor. The Red Queen offers to spare Alice and Matt if they kill Rain, whose health is fading and who has been infected too long for the anti-virus to work reliably. As the Licker attempts to reach them, Rain tells Alice to kill her. Alice refuses, then a power outage occurs. The lab door opens to reveal Kaplan, who disabled the Red Queen to open the door. They reach the train, where Alice dispatches a zombified Spence and takes the anti-virus.
On the train, they inject Rain and Kaplan with the anti-virus. However, the Licker is hiding on the train, and attacks them, clawing Matt. The licker kills Kaplan and hurls him from the train. Alice and Matt battle the Licker. Rain turns into a zombie and attacks Matt, but he kills her. They open a trapdoor, dropping the Licker under the train, killing it. Matt and Alice escape the Hive as the containment doors close.
At the mansion, Matt's wound begins mutating. Before Alice can give him the anti-virus the mansion doors burst open and a group of Umbrella scientists and commandos seize them. They subdue Alice and take Matt away, revealing he is to be put into the Nemesis Program and they intend to re-open the Hive. Alice attempts to fight them off, but is knocked unconscious.
Some time later, Alice awakens at the Raccoon City Hospital strapped to an examination table, with no memory of what happened since her capture. After escaping, she goes outside to find Raccoon City abandoned. A newspaper clipping shows that the T-virus spread to the surface after Umbrella reopened the Hive, creating an army of undead that devastated the city. Alice arms herself with a shotgun from an abandoned police car.
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