Evaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis

•The aim of the study was to evaluate the potential of RVFV positive FFPE samples as a source of sequence data for retrospective analysis.•A RT-PCR targeting a 490 nt portion of the GN glycoprotein gene of RVFV was applied to total RNA extracted from RVFV positive FFPE samples.•Attempts to obtain ta...

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Bibliographic Details
Published in:Journal of virological methods 2017-05, Vol.243, p.10-14
Main Authors: Mubemba, B., Thompson, P.N., Odendaal, L., Coetzee, P., Venter, E.H.
Format: Article
Language:English
Subjects:
Disinfectants
Fixatives
Formaldehyde
Formalin-fixed paraffin-embedded
Humans
Next generation sequencing
Paraffin
Pathology, Molecular - methods
Phylogeny
Retrospective Studies
Reverse Transcriptase Polymerase Chain Reaction - methods
Rift Valley Fever - virology
Rift Valley fever virus
Rift Valley fever virus - classification
Rift Valley fever virus - genetics
Rift Valley fever virus - isolation & purification
Sequence Analysis, DNA
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Summary:•The aim of the study was to evaluate the potential of RVFV positive FFPE samples as a source of sequence data for retrospective analysis.•A RT-PCR targeting a 490 nt portion of the GN glycoprotein gene of RVFV was applied to total RNA extracted from RVFV positive FFPE samples.•Attempts to obtain target amplicons were unsuccessful. FFPE samples were then analysed using next generation sequencing.•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. 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 GN glycoprotein 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2017.01.014