Department of Biomedical Engineering, University of California, Davisgrid.27860.3b, California, USA.
Department of Pathology, University of California, San Diego, California, USA.
Microbiol Spectr. 2022 Aug 31;10(4):e0121122. doi: 10.1128/spectrum.01211-22. Epub 2022 Jul 25.
Feline calicivirus (FCV) is a major cause of upper respiratory disease in cats and is often used as a model for human norovirus, making it of great veterinary and human medical importance. However, questions remain regarding the route of entry of FCV . Increasing work has shown that extracellular vesicles (EVs) can be active in viral infectivity, yet there is no work examining the role of EVs in FCV infection. Here, we begin to address this knowledge gap by characterizing EVs produced by a feline mammary epithelial cell line (FMEC). We have confirmed that EVs are produced by infected and mock-infected FMECs and that both virions and EVs are coisolated with standard methods of virus purification. We also show that they can be enriched differentially by continuous iodixanol density gradient. EVs were enriched at a density of 1.10 g/mL confirmed by tetraspanin expression, size profile, and transmission electron microscopy (TEM). Maximum enrichment of FCV at a density of 1.18 g/mL was confirmed by titration, quantitative reverse transcriptase PCR (q-RT PCR), and TEM. However, infectious virus was recovered from nearly all samples. When used to infect epithelium, both EV-rich and virus-rich fractions had the same levels of infectiousness as determined by percentage of wells infected or titer achieved postinfection. These findings highlight the importance of coisolates during viral purification, showing that EVs may represent a parallel route of entry that has previously been overlooked. Additional experiments are necessary to explore the role of EVs in FCV infection. Feline calicivirus (FCV) is a common cause of upper respiratory infection in cats. Both healthy and infected cells produce small particles called extracellular vesicles (EVs), which are nanoparticles that act as messengers between cells and can be hijacked during viral infection. Historically, the role of EVs in viral infection has been overlooked, and subsequently no group has studied the role of EVs in FCV infection. We hypothesized that EVs may play a role in FCV infection. Here, we show that EVs are copurified with FCV when collecting virus. To study their individual effects, we successfully enrich for viral particles and EVs separately by taking advantage of their different densities. Our initial studies show that EV-enriched versus virus-enriched fractions are equally able to infect cells in culture. These findings highlight the need to both consider the purity of virus after purification and to further study EVs' role in natural FCV infection.
猫杯状病毒 (FCV) 是猫上呼吸道疾病的主要病因，常被用作人类诺如病毒的模型，因此对兽医和人类医学具有重要意义。然而，FCV 的进入途径仍存在一些问题。越来越多的研究表明，细胞外囊泡 (EVs) 在病毒感染中具有活性，但目前尚无研究探讨 EVs 在 FCV 感染中的作用。在这里，我们通过研究猫乳腺上皮细胞系 (FMEC) 产生的 EVs 开始解决这一知识空白。我们已经证实，感染和模拟感染的 FMEC 都会产生 EVs，并且可以通过标准的病毒纯化方法共分离出病毒粒子和 EVs。我们还表明，它们可以通过连续碘克沙醇密度梯度进行差异富集。EVs 在密度为 1.10 g/mL 处被富集，通过四跨膜蛋白表达、大小分布和透射电子显微镜 (TEM) 确认。通过滴定、定量逆转录 PCR (q-RT-PCR) 和 TEM 确认 FCV 在密度为 1.18 g/mL 处的最大富集。然而，几乎所有样本中都能回收感染性病毒。当用于感染上皮细胞时，EV 丰富和病毒丰富的部分具有相同的感染水平，通过感染后感染孔的百分比或达到的滴度来确定。这些发现强调了在病毒纯化过程中共分离物的重要性，表明 EVs 可能代表了以前被忽视的平行进入途径。需要进行额外的实验来探索 EVs 在 FCV 感染中的作用。猫杯状病毒 (FCV) 是猫上呼吸道感染的常见原因。健康细胞和感染细胞都会产生称为细胞外囊泡 (EVs) 的小颗粒，这些纳米颗粒充当细胞之间的信使，并且可以在病毒感染期间被劫持。历史上，EVs 在病毒感染中的作用一直被忽视，因此没有研究小组研究过 EVs 在 FCV 感染中的作用。我们假设 EVs 可能在 FCV 感染中发挥作用。在这里，我们表明在收集病毒时，EVs 与 FCV 一起被共纯化。为了研究它们各自的作用，我们通过利用它们的不同密度成功地分别富集病毒粒子和 EVs。我们的初步研究表明，EV 富集与病毒富集的部分在培养细胞中同样能够感染细胞。这些发现强调了在病毒纯化后不仅要考虑病毒的纯度，还要进一步研究 EVs 在自然 FCV 感染中的作用。
