Department of Microbiology and Plant Pathology, University of California, Riverside, CA, 92521, USA.
United States Department of Agriculture, Agricultural Research Service, Parlier, CA, 93648, USA.
Virol J. 2018 Sep 15;15(1):141. doi: 10.1186/s12985-018-1041-4.
The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses.
The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis.
Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes.
The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.
RNA 病毒基因组两端的非翻译区具有多种功能，并可能表现出不同程度的核苷酸（nt）异质性。然而，柑橘碎叶病毒（CTV）基因组两端极端 nt 的异质性程度尚未得到全面描述。本研究旨在对来自加利福尼亚州的两种广泛流行的 CTV 基因型 T36-CA 和 T30-CA 进行特征描述，这两种基因型尚未进行测序或深入分析。获得的信息将用于我们正在进行的构建这些病毒的感染性互补（c）DNA 克隆的工作。
通过对使用 5'和 3'快速 cDNA 末端扩增生成的病毒双链 (ds) RNA 的正链和/或负链 cDNA 克隆进行测序，确定病毒基因组的末端 nt。使用反转录-聚合酶链反应生成对应于两种病毒完整基因组序列的克隆 cDNA，对其进行测序，并进行系统发育分析。
在所鉴定的主要末端 nt 中，有些与 GenBank 中的共识序列相同，而有些则不同或独特。值得注意的是，T36-CA 的主要 5'nt 变体之一包含了高度保守的胞苷酸，在所有其他全长 T36 序列中都存在，但在其中缺失。正如预期的那样，但之前从未系统地验证过，在 T36-CA 和 T30-CA 的 5'末端共识 nt 上游也发现了带有额外 nt（s）的独特变体。与极端 5'末端 nt 相比，T36-CA 和 T30-CA 的极端 3'末端 nt 与参考序列相比更为保守，尽管也发现了 nt 变体。值得注意的是，在许多 T36-CA 序列中发现了极端 3'末端的额外胸腺嘧啶核苷酸。最后，基于成对比较和与多个参考序列的系统发育分析，发现这两种病毒的完整序列与各自基因型的序列高度保守。
鉴定了 T36-CA 和 T30-CA 基因组的极端末端 nt，揭示了这些 CTV 基因组区域异质性的新见解。T36-CA 和 T30-CA 分别是源自加利福尼亚州的 CTV 的第一个和第二个基因型，是第一个和第二个完全测序和分析的 CTV 基因型。
