Mubemba B, Thompson P N, Odendaal L, Coetzee P, Venter E H
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.
Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, South Africa.
J Virol Methods. 2017 May;243:10-14. doi: 10.1016/j.jviromet.2017.01.014. Epub 2017 Jan 22.
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 G 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.
裂谷热（RVF）由布尼亚病毒科中一种节肢动物传播的白蛉病毒引起，是一种影响反刍动物和人类的出血性疾病。由于该病毒的人畜共患性质，病毒分离需要在生物安全3级实验室进行。新鲜和冷冻样本是分离病毒及获取序列数据的首选样本类型。然而，这些样本不仅稀缺，还会对实验室人员构成健康风险。存档的福尔马林固定石蜡包埋（FFPE）组织样本安全且易于获取，然而，FFPE衍生的RNA在大多数情况下会降解，并在肽键处发生交联，而且尚不清楚这种样本类型是否适合作为回顾性系统发育研究的参考材料。将针对裂谷热病毒（RVFV）M片段结构G糖蛋白编码基因490 nt部分的逆转录聚合酶链反应（RT-PCR）检测应用于从存档的RVFV阳性FFPE样本中提取的总RNA。多次尝试获取目标扩增子均未成功。然后使用新一代测序（NGS），即TruSeq（Illumina）对FFPE样本进行分析，并在Miseq基因组分析仪（Illumina）上进行测序。通过参考映射，将不同程度浅深度的缺口病毒序列数据与参考序列进行比对。然而，NGS未能产生足够长的重叠群，无法在所有样本中始终覆盖相同的基因组区域以进行系统发育分析。
