Janowski Andrew B, Bauer Irma K, Holtz Lori R, Wang David
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.
Department of Molecular Microbiology and Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA
J Virol. 2017 Sep 12;91(19). doi: 10.1128/JVI.00740-17. Print 2017 Oct 1.
Astrovirus VA1/HMO-C (VA1; mamastrovirus 9) is a recently discovered astrovirus genotype that is divergent from the classic human astroviruses (mamastrovirus 1). The gastrointestinal tract is presumed to be the primary site of infection and pathogenicity for astroviruses. However, VA1 has been independently detected in brain tissue of five cases of human encephalitis. Studies of the pathogenicity of VA1 are currently impossible because there are no reported cell culture systems or models that support VA1 infection. Here, we describe successful propagation of VA1 in multiple human cell lines. The initial inoculum, a filtered clinical stool sample from the index gastroenteritis case cluster that led to the discovery of VA1, was first passaged in Vero cells. Serial blind passage in Caco-2 cells yielded increasing copies of VA1 RNA, and multistep growth curves demonstrated a >100-fold increase in VA1 RNA 72 h after inoculation. The full-length genomic and subgenomic RNA strands were detected by Northern blotting, and crystalline lattices of viral particles of ∼26-nm diameter were observed by electron microscopy in infected Caco-2 cells. Unlike other human astrovirus cell culture systems, which require addition of exogenous trypsin for continued propagation, VA1 could be propagated equally well with or without the addition of trypsin. Furthermore, VA1 was sensitive to the type I interferon (IFN-I) response, as VA1 RNA levels were reduced by pretreatment of Caco-2 cells with IFN-β1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1. Astroviruses are an emerging cause of central nervous system infections in mammals, and astrovirus VA1/HMO-C is the most prevalent astrovirus in cases of human encephalitis. This virus has not been previously propagated, preventing elucidation of the biology of this virus. We describe the first cell culture system for VA1, a key step necessary for the study of its ability to cause disease.
星状病毒VA1/HMO-C（VA1；乳多空病毒9型）是一种最近发现的星状病毒基因型，与经典人类星状病毒（乳多空病毒1型）不同。胃肠道被认为是星状病毒感染和致病的主要部位。然而，在5例人类脑炎患者的脑组织中独立检测到了VA1。目前无法对VA1的致病性进行研究，因为尚无支持VA1感染的细胞培养系统或模型报道。在此，我们描述了VA1在多种人类细胞系中的成功增殖。最初的接种物是来自导致VA1发现的首例胃肠炎病例群的经滤过的临床粪便样本，首先在Vero细胞中传代。在Caco-2细胞中连续盲传产生了越来越多的VA1 RNA拷贝，多步生长曲线显示接种后72小时VA1 RNA增加了100倍以上。通过Northern印迹法检测到全长基因组和亚基因组RNA链，在感染的Caco-2细胞中通过电子显微镜观察到直径约26nm的病毒颗粒晶格。与其他需要添加外源性胰蛋白酶才能持续增殖的人类星状病毒细胞培养系统不同，无论是否添加胰蛋白酶，VA1都能同样良好地增殖。此外，VA1对I型干扰素（IFN-I）反应敏感，因为用IFN-β1a预处理Caco-2细胞可降低VA1 RNA水平。在细胞培养中增殖VA1的能力将有助于对VA1的嗜神经性和神经发病机制的研究。星状病毒是哺乳动物中枢神经系统感染的一个新兴病因，而星状病毒VA1/HMO-C是人类脑炎病例中最常见的星状病毒。该病毒此前尚未增殖，阻碍了对其生物学特性的阐明。我们描述了首个VA1细胞培养系统，这是研究其致病能力的关键必要步骤。
