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16-bit MS-DOS Subsystem error while installing or running a DOS application

Symptoms

If you try to start or install an MS-DOS-based or a 16-bit Windows-based program on your Windows XP-based computer, you may receive an error message that is similar to one of the following:

16-bit MS-DOS Subsystem path to the program that you are trying to start or install C:\Windows\System32\config.nt The system file is not suitable for running MS-DOS and Microsoft Windows applications. Choose 'Close' to terminate the application.

You may encounter to other similar error messages, but with different file names config.nt and Autoexec.nt. This issue may occur if one or more of the following files are missing or damaged:

Config.nt
Autoexec.nt
Command.com

Restoring the missing files

To restore these files, download the above three files from here. Unzip and copy the missing file(s) to Windows\System32 folder. For more information, see article Error message when you install or start an MS-DOS or 16-bit Windows-based program

This issue which is showing-up lately, seems to be caused by a Trojan (may be TrojanDownloader.Win32.Dia.a) which seems to any of the three files indicated above. Reference: See this thread for more information.

Recommended action

Clean-up the Windows startup applications. Reboot and then run a full system virus scan with updated definitions. If you're unable to run anti-virus program locally, use the Online virus scan services listed in this page. Re-create the three files mentioned above.

Run a scan from Safe Mode with fully updated anti-virus reference files. Additionally, scan with Ad-aware, SpyBot and CWShredder. Update Ad-aware, SpyBot while in Normal mode . Restart Windows in Safe Mode to successfully scan and remove ad-ware, trojan etc.

The Parasite Fight - Quick Fix Protocol

Get a copy of Autoruns tool (latest version allows managing the Startup locations for other user profiles).
How to manage Windows Startup?