Abstract
Worldwide cooperation between laboratories working with classical swine fever virus (CSFV) requires exchange of virus isolates. For this purpose, shipment of CSFV RNA is a safe alternative to the exchange of infectious material. New techniques using desiccation have been developed to store RNA at room temperature and are reported as effective means of preserving RNA integrity. In this study, we evaluated the stability and integrity of dried CSFV RNA stored at room temperature. First, we determined the stability of CSFV RNA covering CSFV genome regions used typically for the detection of viral RNA in diagnostic samples by reverse transcription-polymerase chain reaction (RT-PCR). To this end, different concentrations of in vitro-transcribed RNAs of the 5’-untranslated region and of the NS5B gene were stored as dried RNA at 4, 20, and 37oC for two months. Aliquots were analyzed every week by CSFV-specific quantitative real-time RT-PCR. Neither the RNA concentration nor the storage temperature did affect CSFV RNA yields at any of the time evaluated until the end of the experiment. Furthermore, it was possible to recover infectious CSFV after transfection of SK-6 cells with dried viral RNA stored at room temperature for one week. The full-length E2 of CSFV was amplified from all the recovered viruses, and nucleotide sequence analysis revealed 100% identity with the corresponding sequence obtained from RNA of the original material. These results show that CSFV RNA stored as dried RNA at room temperature is stable, maintaining its integrity for downstream analyses and applications.
Highlights
Classical swine fever (CSF) is a contagious viral disease of swine affecting both domestic and wild Suidae of all breeds and ages
CSF is caused by classical swine fever virus (CSFV), an enveloped single-stranded, positivesense RNA virus belonging to the Pestivirus genus of the Flaviviridae family (Thiel et al, 2005)
The 5 ́UTR and NS5B fragments of the ‘Pinar del Río’ (CSF1058) strain were amplified by reverse transcription-polymerase chain reaction (RTPCR) using the primer pairs reported by Hoffmann et al (2005) and Díaz de Arce et al (1998), respectively
Summary
Classical swine fever (CSF) is a contagious viral disease of swine affecting both domestic and wild Suidae of all breeds and ages. It is a notifiable disease to the World Organization for Animal Health (OIE) due to the highly contagious nature of the virus, the high mortality associated with the acute forms of the disease, and the huge economic losses incurred by the affected countries, along with the impact on the international trade (Haines et al, 2013). CSF is caused by classical swine fever virus (CSFV), an enveloped single-stranded, positivesense RNA virus belonging to the Pestivirus genus of the Flaviviridae family (Thiel et al, 2005).
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