title: Evaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis.
abstract: Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies. A RT-PCR assay targeting a 490 nt portion of the structural GNglycoprotein encoding gene of the RVFV M-segment was applied to total RNA extracted from archived 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) and sequenced on the Miseq®genome analyser (Illumina). Using reference mapping, gapped virus sequence data of varying degrees of shallow depth was aligned to a reference sequence. However, the NGS did not yield long enough contigs that consistently covered the same genome regions in all samples to allow phylogenetic analysis.
authors.affiliation: Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa; Department of Zoology and Aquatic Sciences, School of Natural Resources, Copperbelt University, Zambia. Electronic address: mubembab85@yahoo.co.uk.
authors.forename: B
authors.initials: B
authors.lastname: Mubemba
authors.affiliation: Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, South Africa.
authors.forename: P N
authors.initials: PN
authors.lastname: Thompson
authors.affiliation: Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, South Africa.
authors.forename: L
authors.initials: L
authors.lastname: Odendaal
authors.affiliation: Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa.
authors.forename: P
authors.initials: P
authors.lastname: Coetzee
authors.affiliation: Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa.
authors.forename: E H
authors.initials: EH
authors.lastname: Venter
mesh_terms: D004202:Disinfectants; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D010232:Paraffin; D057089:Pathology, Molecular / Q000379:methods*; D010802:Phylogeny; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012295:Rift Valley Fever / Q000821:virology*; D012296:Rift Valley fever virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D004202:Disinfectants; D005404:Fixatives; D005557:Formaldehyde; D010232:Paraffin
keywords: Formalin-fixed paraffin-embedded; Next generation sequencing; Rift Valley fever virus