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®

Abstract

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 storage of samples above 0°C for an extended time, yet preserves the viral RNA in a state which allows its detection by reverse transcription-polymerase chain reaction (RT-PCR), and even rescue of infectious virus after transfection of the extracted RNA into susceptible cells. Supernatants from infected cell cultures as well as organs from diseased animals were stored in Trizol® for 1–4 weeks at −20°C, 4°C, room temperature, or 37°C. RNA was then extracted and used subsequently for RT-PCR, as well as transfection into susceptible cells to initiate the replication of progeny virus. Formaldehyde-fixed samples were also included in this study. Storage up to 4 weeks at 37°C in Trizol® still yielded positive RT-PCR results and rescue of infectious virus upon RNA transfection. In contrast, formaldehyde fixation reduced drastically the detectability of viral RNA. This method represents a safe and inexpensive alternative to −70°C (dry ice) storage or transport of samples, and abolishes the biosafety risks involved in shipping deep-frozen infectious materials.