Li Min, Green Patrick L
Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
J Virol Methods. 2007 Jun;142(1-2):159-68. doi: 10.1016/j.jviromet.2007.01.023. Epub 2007 Mar 6.
HTLV-1 and HTLV-2 are highly related delta-retroviruses that infect and transform T-lymphocytes, but have distinct pathogenic properties. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the comparative levels of all known HTLV-1 and HTLV-2 viral mRNAs in different transformed cell lines and at different stages of virus infection have not been assessed. In this study, we compiled a series of oligonucleotide primer pairs and probes to quantify both HTLV-1 and HTLV-2 mRNA species using real-time RT-PCR. The optimized reaction for detection of each mRNA had amplification efficiency greater than 90% with a linear range spanning 25-2.5 x 10(7) copies. The R(2)'s of all standard curves were greater than 0.97. Quantitation of HTLV mRNAs between different cell lines showed variability (gag/pol>or=tax/rex>env>or=accessory proteins), but the overall levels of each mRNA relative to each other within a cell line were similar. These results provide a method to quantify all specific mRNAs from both HTLV-1 and HTLV-2, which can be used to evaluate further viral gene expression and correlate transcript levels to key stages of the virus life cycle and ultimately, pathogenesis.
人类嗜T淋巴细胞病毒1型（HTLV-1）和人类嗜T淋巴细胞病毒2型（HTLV-2）是高度相关的δ逆转录病毒，它们感染并转化T淋巴细胞，但具有不同的致病特性。HTLV的复制和存活需要从未剪接的以及一系列高度相关的可变剪接mRNA种类中表达多种基因产物。迄今为止，尚未评估在不同转化细胞系以及病毒感染不同阶段中所有已知的HTLV-1和HTLV-2病毒mRNA的相对水平。在本研究中，我们设计了一系列寡核苷酸引物对和探针，用于通过实时逆转录聚合酶链反应（RT-PCR）对HTLV-1和HTLV-2的mRNA种类进行定量分析。检测每种mRNA的优化反应的扩增效率大于90%，线性范围为25至2.5×10⁷个拷贝。所有标准曲线的R²均大于0.97。不同细胞系之间HTLV mRNA的定量分析显示存在差异（gag/pol≥tax/rex＞env≥辅助蛋白），但同一细胞系内每种mRNA相对于彼此的总体水平相似。这些结果提供了一种对HTLV-1和HTLV-2的所有特定mRNA进行定量分析的方法，可用于进一步评估病毒基因表达，并将转录水平与病毒生命周期的关键阶段以及最终的发病机制相关联。
