Mubemba, B., Thompson, P.N., Odendaal, L., Coetzee, P., and Venter, E.H. (2017) Evaluation of formalin-fixed paraffin-embedded tissue samples as a source of sequence data for phylogenetic analysis of Rift Valley fever virus. Journal of Virological Methods, 243. pp. 10-14.

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Abstract

Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haem-orrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafetylevel 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sam-ple type for isolation and acquisition of sequence data. However, these samples are scarce in additionto posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tis-sue samples are safe and readily available, however FFPE derived RNA is in most cases degraded andcross-linked in peptide bonds and it is unknown whether the sample type would be suitable as referencematerial for retrospective phylogenetic studies. A RT-PCR assay targeting a 490 nt portion of the struc-tural GNglycoprotein encoding gene of the RVFV M-segment was applied to total RNA extracted fromarchived RVFV positive FFPE samples. Several attempts to obtain target amplicons were unsuccessful.FFPE samples were then analysed using next generation sequencing (NGS), i.e. Truseq®(Illumina) andsequenced on the Miseq®genome analyser (Illumina). Using reference mapping, gapped virus sequencedata of varying degrees of shallow depth was aligned to a reference sequence. However, the NGS did notyield long enough contigs that consistently covered the same genome regions in all samples to allowphylogenetic analysis.

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