Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol((R))

A method for storing samples containing classical swine fever virus (CSFV) or foot-and-mouth disease virus (FMDV), respectively, was developed, which abolishes the infectivity of both plus strand RNA viruses, and allows stora ge of samples above 0 degrees C for an extended time, yet preserves the vir al RNA in a state which allows its detection by reverse transcription-polym erase chain reaction (RT-PCR), and even rescue of infectious virus after tr ansfection of the extracted RNA into susceptible cells. Supernatants from i nfected cell cultures as well as organs from diseased animals were stored i n Trizol((R)) for 1-4 weeks at - 20 degrees C, 4 degrees C, room temperatur e, or 37 degrees C. RNA was then extracted and used subsequently for RT-PCR , as well as transfection into susceptible cells to initiate the replicatio n of progeny virus. Formaldehyde-fixed samples were also included in this s tudy. Storage up to 4 weeks at 37 degrees C in Trizol((R)) still yielded po sitive RT-PCR results and rescue of infectious virus upon RNA transfection. In contrast, formaldehyde fixation reduced drastically the detectability o f viral RNA. This method represents a safe and inexpensive alternative to - 70 degrees C (dry ice) storage or transport of samples, and abolishes the biosafety risks involved in shipping deep-frozen infectious materials. (C) 2000 Elsevier Science B.V. All rights reserved.