Goebel Scott, Snyder Beth, Sellati Timothy, Saeed Mohammad, Ptak Roger, Murray Michael, Bostwick Robert, Rayner Jonathan, Koide Fusataka, Kalkeri Raj
Department of Infectious Diseases Research, Drug Development, Southern Research, Frederick, MD, United states.
Department of Infectious Diseases, Drug Discovery Division, Southern Research, Birmingham, AL, United states.
J Virol Methods. 2016 Dec;238:13-20. doi: 10.1016/j.jviromet.2016.09.015. Epub 2016 Oct 3.
Despite the rapid spread of Zika virus (ZIKV) infection and associated neurological complications in the America's, prophylactic or therapeutic countermeasures are not currently available. This is mostly due to the fact that until recently there was no presumed need for medical intervention since there was no association between ZIKV infection and significant human morbidity. Consequently, there are currently no tools due mostly to the lack of sensitive cell based assays amenable for identification of ZIKV inhibitors. To address this unmet need we have developed a cell based virus yield assay suitable for testing antivirals against Zika virus. Using bioinformatics, several isolates of ZIKV from the Americas, Africa, and Asia were analyzed for sequence similarity. The alignment data were then used to design primers targeting a ZIKV genomic region that was highly conserved among all the ZIKV isolates. Subsequently, primers were used in a sensitive, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to detect ZIKV RNA. The qRT-PCR assay was found to be highly sensitive (lower limit of detection between-10-100 copies) and reproducible. Evaluation of the primers and probes used for ZIKV against another flavivirus (Dengue virus) demonstrated specificity of detection. To evaluate potential of qRT-PCR assay as an antiviral screening tool against ZIKV, Vero cells pretreated with Type I Interferons (IFN α) were infected with virus, followed by measurement of ZIKV RNA found in the cell culture supernatants using qRT-PCR assay. Dose-dependent antiviral activity of Type I Interferons and mycophenolic acid (MPA) against Zika virus in this cell culture system was confirmed using qRT-PCR. Due to reproducible assay performance, qPCR associated higher sensitivity and short duration of the assay time, this novel cell based assay will be very useful for confirming the activity of antivirals against ZIKV.
尽管寨卡病毒（ZIKV）感染及其相关神经并发症在美洲迅速传播，但目前尚无预防性或治疗性对策。这主要是因为直到最近，由于寨卡病毒感染与严重人类发病之间没有关联，所以认为没有医疗干预的必要。因此，目前由于缺乏适用于鉴定寨卡病毒抑制剂的基于细胞的敏感检测方法，尚无可用工具。为满足这一未被满足的需求，我们开发了一种基于细胞的病毒产量检测方法，适用于测试针对寨卡病毒的抗病毒药物。利用生物信息学，对来自美洲、非洲和亚洲的几种寨卡病毒分离株进行了序列相似性分析。然后，比对数据被用于设计靶向寨卡病毒基因组一个在所有寨卡病毒分离株中高度保守区域的引物。随后，这些引物被用于一种灵敏的定量逆转录聚合酶链反应（qRT-PCR）检测方法中以检测寨卡病毒RNA。发现该qRT-PCR检测方法高度灵敏（检测下限在10至100拷贝之间）且可重复。用于寨卡病毒的引物和探针针对另一种黄病毒（登革病毒）的检测评估显示了检测的特异性。为评估qRT-PCR检测方法作为针对寨卡病毒的抗病毒筛选工具的潜力，用I型干扰素（IFNα）预处理的非洲绿猴肾细胞（Vero细胞）被病毒感染，随后使用qRT-PCR检测方法测量细胞培养上清液中发现的寨卡病毒RNA。在该细胞培养系统中，使用qRT-PCR证实了I型干扰素和霉酚酸（MPA）对寨卡病毒具有剂量依赖性抗病毒活性。由于检测性能可重复、qPCR具有更高的灵敏度且检测时间短，这种新型的基于细胞的检测方法对于确认抗病毒药物对寨卡病毒的活性将非常有用。
