Extracellular Vesicles (EVs) Are Copurified with Feline Calicivirus, yet EV-Enriched Fractions Remain Infectious.
Extracellular Vesicles (EVs) Are Copurified with Feline Calicivirus, yet EV-Enriched Fractions Remain Infectious.
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DOI：
10.1128/spectrum.01211-22
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发表时间：
2022-08-31
影响因子：
3.7
通讯作者：
Carney, Randy P.
中科院分区：
文献类型：
--
作者：
Mizenko, Rachel R.;Brostoff, Terza;Jackson, Kenneth;Pesavento, Patricia A.;Carney, Randy P.
猫杯状病毒（FCV）是猫上呼吸道疾病的主要病因，经常被用作人类诺如病毒的模型，使其具有重要的兽医和人类医学意义。然而，关于FCV在体内的进入途径仍然存在疑问。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，但没有研究EVs在FCV感染中的作用。在这里，我们开始通过表征由猫乳腺上皮细胞系（FMEC）产生的ev来解决这一知识差距。我们已经证实，EVs是由感染和模拟感染的fmec产生的，并且病毒粒子和EVs都是用标准的病毒纯化方法共同分离的。我们还发现，它们可以通过连续的碘二醇密度梯度进行差异富集。通过tetraspanin表达、大小谱和透射电镜（TEM）证实，EVs在1.10 g/mL的密度下富集。通过滴定、定量逆转录酶PCR （q-RT PCR）和透射电镜（TEM）验证了FCV在1.18 g/mL密度下的最大富集。然而，从几乎所有样本中都发现了传染性病毒。当用于感染体外上皮时，通过感染孔的百分比或感染后达到的滴度来确定富ev和富病毒组分具有相同的传染性水平。这些发现强调了共分离物在病毒纯化过程中的重要性，表明ev可能代表了以前被忽视的平行进入途径。需要进一步的实验来探索ev在FCV感染中的作用。重要性：猫杯状病毒（FCV）是猫上呼吸道感染的常见病因。健康细胞和受感染细胞都会产生被称为细胞外囊泡（EVs）的小颗粒，这是一种纳米颗粒，充当细胞之间的信使，在病毒感染期间可以被劫持。从历史上看，ev在病毒感染中的作用一直被忽视，随后也没有小组研究ev在FCV感染中的作用。我们假设ev可能在FCV感染中起作用。在这里，我们发现在收集病毒时，电动汽车与FCV进行了聚合。为了研究它们的个体效应，我们成功地利用病毒颗粒和电动汽车的不同密度分别富集。我们的初步研究表明，富集ev和富集病毒的组分在培养中感染细胞的能力是一样的。这些发现表明，需要考虑纯化后病毒的纯度，并进一步研究ev在自然FCV感染中的作用。
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 in vivo. 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 in vitro 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. IMPORTANCE 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.
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影响因子：
64.8
作者：
Feng Z;Hensley L;McKnight KL;Hu F;Madden V;Ping L;Jeong SH;Walker C;Lanford RE;Lemon SM
通讯作者：
Lemon SM
DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
4
作者：
Nava, P;López, S;González-Mariscal, L
通讯作者：
González-Mariscal, L
影响因子：
4.6
作者：
Brennan, K.;Martin, K.;Mc Gee, M. M.
通讯作者：
Mc Gee, M. M.
影响因子：
6.4
作者：
Morris-Love, Jenna;Gee, Gretchen V.;Atwood, Walter J.
通讯作者：
Atwood, Walter J.