Instituto Superior Politécnico do Kuanza Sul (ISPKS), Rua 12 de Novembro, Sumbe, Angola.
Centro Nacional de Investigação Científica (CNIC), 201 Ho Chi Min Avenue, CP 34, Luanda, Angola.
Virol J. 2021 Mar 23;18(1):62. doi: 10.1186/s12985-021-01535-x.
Citrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Controls should be applied to nurseries, which could act as diversity hotspots for CTV. Early detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is particularly relevant in those growing regions where no dedicated certification programs are currently in use.
Double-stranded RNA extracted from Citrus spp. samples, collected in two locations in Angola, were pooled and submitted to a random-primed RNA-seq. This technique was performed to acquire a higher amount of data in the survey, before the amplification and sequencing of genes from single plants. To confirm the CTV infection in individual plants, as suggested by RNA-seq information from the pooled samples, the analysis was integrated with multiple molecular marker amplification (MMM) for the main known CTV strains (T30, T36, VT and T3).
From the analysis of HTS data, several assembled contigs were identified as CTV and classified according to their similarity to the established strains. By the MMM amplification, only five individual accessions out of the eleven pooled samples, resulted to be infected by CTV. Amplified coat protein genes from the five positive sources were cloned and sequenced and submitted to phylogenetic analysis, while a near-complete CTV genome was also reconstructed by the fusion of three overlapping contigs.
Phylogenetic analysis of the ORF1b and CP genes, retrieved by de novo assembly and RT-PCR, respectively, revealed the presence of a wide array of CTV strains in the surveyed citrus-growing spots in Angola. Importantly, molecular variants among those identified from HTS showed high similarity with known severe strains as well as to recently described and emerging strains in other citrus-growing regions, such as S1 (California) or New Clade (Uruguay).
柑橘产业在世界范围内受到柑橘衰退病毒（CTV）爆发的严重影响。应在苗圃中采取防治措施，因为苗圃可能成为 CTV 的多样性热点。早期检测和鉴定这种病毒的危险或新兴株系，对预防疾病爆发有很大帮助。在那些目前没有专门认证计划的种植地区，这一点尤为重要。
从安哥拉两个地点收集的柑橘属样本中提取双链 RNA，进行随机引物 RNA-seq。这项技术是在对单个植物的基因进行扩增和测序之前进行的，旨在调查中获得更多的数据。为了确认混合样本的 RNA-seq 信息所提示的个别植物感染 CTV，分析整合了主要已知 CTV 株系（T30、T36、VT 和 T3）的多种分子标记扩增（MMM）。
从 HTS 数据分析中，鉴定出几个组装的连续序列为 CTV，并根据与已建立的株系的相似性进行分类。通过 MMM 扩增，在 11 个混合样本中，只有 5 个个体样本被 CTV 感染。从五个阳性来源中扩增出的外壳蛋白基因被克隆和测序，并提交进行系统发育分析，同时通过融合三个重叠连续序列，也重建了近完整的 CTV 基因组。
通过从头组装和 RT-PCR 分别获得的 ORF1b 和 CP 基因的系统发育分析表明，在所调查的安哥拉柑橘种植区存在广泛的 CTV 株系。重要的是，从 HTS 鉴定出的分子变异与已知的严重株系以及其他柑橘种植地区（如加利福尼亚的 S1 或乌拉圭的新株系）最近描述和新兴株系具有高度相似性。
