Propagation of Astrovirus VA1, a Neurotropic Human Astrovirus, in Cell Culture

Abstract

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 in vivo 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 Ͼ100-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 ϳ26-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-␤1a. The ability to propagate VA1 in cell culture will facilitate studies of the neurotropism and neuropathogenesis of VA1.IMPORTANCE 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.KEYWORDS astrovirus, astrovirus VA1, cell culture, electron microscopy, encephalitis, pathogenesis, subgenomic RNA, viral propagation I nitially identified in 1975, members of the single-stranded, positive-sense RNA viral family Astroviridae have been frequently detected in vertebrate stool samples (1, 2). In humans, the first identified astrovirus species was mamastrovirus 1 (classic human astrovirus), and eight serotypes of this virus have been described (2-4). Most humans are exposed to the classic human astroviruses with seroprevalence as high as 94% to specific serotypes (5-9). The classic human astroviruses predominantly cause a selflimited gastrointestinal illness and have been characterized as the third-to fifth-mostcommon viral etiology of diarrhea and gastroenteritis in humans (10)(11)(12)(13)(14).Astroviruses contain three open reading frames (ORFs) and based on conserved genomic elements are hypothesized to share common mechanisms of replication (2). Astroviruses encode a slippery sequence and stem-...

Infection and Propagation of Astrovirus VA1 in Cell Culture

“…Monitoring the increase in viral RNA is also an important readout when quantifying the effect of antiviral interventions. We have previously demonstrated a twenty-fold decrease in viral RNA 96-hr post-inoculation when pretreating Caco-2 cells with interferon-β (Janowski et al, 2017).…”

“…We have described some of the biology of VA1 in cell culture and VA1 appears to have divergent in vitro biology when compared to mamastrovirus 1 (Janowski et al, 2017). The addition of trypsin to cell culture medium is necessary for propagation of mamastrovirus 1 (Lee & Kurtz, 1981).…”

5 challenges in understanding the role of the virome in health and disease

“…The lack of culture systems for viruses identified in virome studies, for both eukaryotic viruses and phages, is pronounced. As an example, although dozens of novel eukaryotic viruses have been identified in the mammalian enteric tract by metagenomic sequencing, culture systems for only a very limited number have been described to date [14,15]. Likewise, for phages, genomic sequences of thousands of novel phages can be identified in a single study [16,17], but very few have been isolated [18].…”

A decade of RNA virus metagenomics is (not) enough

“…A number of options for functional characterization of these viruses are available but they unfortunately require work. Additional culture models are needed to handle all the new discoveries and genetic engineering methods for more easily establishing cell lines from exotic species are worth pursuing (Ettayebi et al, 2016;Finkbeiner et al, 2012;Stenglein et al, 2012;Bell-Sakyi and Attoui, 2016;Janowski et al, 2017). Focusing discovery on genetically tractable organisms has allowed for relatively rapid functional studies of the new discovered C. elegans Orsay virus Jiang et al, 2017).…”