Oluwasemowo Olukunle O, Graham Monica E, Murugesh Deepa K, Borucki Monica K
Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
Microbiol Spectr. 2025 Jan 7;13(1):e0162424. doi: 10.1128/spectrum.01624-24. Epub 2024 Nov 29.
Plaque assay is the gold standard for the quantification of viable cytopathic viruses like Zika virus (ZIKV). Some strains of ZIKV produce plaques that are very difficult to accurately visualize and count on the commonly used Vero cell line. From data generated in our lab, we became curious if Vero/TMPRSS2 cells may be a better alternative; therefore, we compared the plaque forming units of two strains of ZIKV on Vero/TMPRSS2 cells to those produced by Vero cells. We also compared the virus stock titer generated on Vero/TMPRSS2 cells to that generated by the Vero cell line. Although the Vero cells generated higher quantity of ZIKV stocks, the Vero/TMPRSS2 cells produced plaques with significantly improved morphology and visibility and may, therefore, be a better alternative to use for performing plaque assays for strains of ZIKV that are more difficult to titer on regular Vero cells.
While there are several methods of viral quantification, the plaque assay remains the gold standard for accurate quantification of replication-competent, cytopathic viruses including Zika virus (ZIKV). Vero cells are commonly used to titer ZIKV stock via the plaque assay. Prior to the initiation of this study, we observed that ZIKV-PRV strain plaque assays using Vero cells often yielded plaques that were very small, overly diffuse, and hard to count. We also observed that the ZIKV-CAM strain often did not produce plaques at all on Vero cells. This study shows that Vero cells expressing TMPRSS2 improve the morphology and visibility of plaques produced by ZIKV-PRV and ZIKV-CAM compared to regular Vero cells and may, therefore, be a better alternative to use for performing plaque assays for these strains and perhaps for other strains of ZIKV that are difficult to titer. However, Vero cells proved to be superior for generating high titer stock.
蚀斑测定法是对寨卡病毒（ZIKV）等有活力的致细胞病变病毒进行定量的金标准。一些寨卡病毒株产生的蚀斑在常用的非洲绿猴肾（Vero）细胞系上很难准确观察和计数。根据我们实验室生成的数据，我们好奇非洲绿猴肾/TMPRSS2细胞是否可能是更好的选择；因此，我们比较了两种寨卡病毒株在非洲绿猴肾/TMPRSS2细胞上形成的蚀斑单位与在非洲绿猴肾细胞上产生的蚀斑单位。我们还比较了在非洲绿猴肾/TMPRSS2细胞上产生的病毒原液滴度与在非洲绿猴肾细胞系上产生的病毒原液滴度。尽管非洲绿猴肾细胞产生的寨卡病毒原液数量更多，但非洲绿猴肾/TMPRSS2细胞产生的蚀斑形态和可见性显著改善，因此对于在常规非洲绿猴肾细胞上更难滴定的寨卡病毒株，可能是进行蚀斑测定的更好选择。
虽然有几种病毒定量方法，但蚀斑测定法仍然是准确量化包括寨卡病毒（ZIKV）在内具有复制能力的致细胞病变病毒的金标准。非洲绿猴肾细胞常用于通过蚀斑测定法滴定寨卡病毒原液。在本研究开始之前，我们观察到使用非洲绿猴肾细胞进行寨卡病毒 - PRV株蚀斑测定时，通常产生的蚀斑非常小、过度弥散且难以计数。我们还观察到寨卡病毒 - CAM株在非洲绿猴肾细胞上通常根本不产生蚀斑。这项研究表明，与常规非洲绿猴肾细胞相比，表达TMPRSS2的非洲绿猴肾细胞改善了寨卡病毒 - PRV和寨卡病毒 - CAM产生的蚀斑的形态和可见性，因此对于这些毒株以及可能对于其他难以滴定的寨卡病毒株，可能是进行蚀斑测定的更好选择。然而，事实证明非洲绿猴肾细胞在产生高滴度原液方面更具优势。
