Infection and Propagation of Astrovirus VA1 in Cell Culture

Abstract

Astrovirus VA1/HMO‐C (VA1) is the representative genotype of mamastrovirus 9, a species of the single‐stranded, positive‐sense RNA viral family, Astroviridae. Astroviruses have been traditionally considered pathogens of the gastrointestinal tract but they have been recently associated with neurological diseases in humans, cattle, mink, sheep, and pigs. VA1 is the astrovirus genotype most commonly identified from human cases of meningoencephalitis and has been recently propagated in cell culture. VA1 can now be used as a model system to study pathogenesis of the neurological diseases associated with astrovirus infection. In this article, we describe two fundamental assays to quantify replication and propagation of VA1, a quantitative reverse transcription‐PCR (qRT‐PCR) to measure viral RNA and a 50% tissue culture infectious dose (TCID50) assay to measure infectious viral particles. © 2018 by John Wiley & Sons, Inc.

Differential In Vitro Infection of Neural Cells by Astroviruses

“…VA1 infections were performed as previously described (31, 55), and the same conditions were used for VEEV TC83 infections. For infections performed with HAstV4, an aliquot of the virus was activated with 100 μg/ml of trypsin for 30 min (56).…”

“…Using a previously published VA1 stock (C-P5) (31), Caco-2 cells grown in T175 flasks were infected with C-P5 at an MOI of 0.006 and incubated for 7 days to generate a new stock (C-P6) (55). The flasks were freeze-thawed three times to generate whole-cell/supernatant lysate.…”

“…Next, C-P6 was inoculated into T175 flasks containing confluent Caco-2 cells at an MOI of 0.01 and incubated for 5 days. Flasks were freeze-thawed three times, and the lysate (C-P7) was quantified by a TCID 50 assay (3.16 × 10 7 TCID 50 units/ml) and used in all subsequent experiments (55).…”

“…RNA from the cellular or supernatant fractions was extracted by the use of Direct-zol 96-well plates (Zymo Research). VA1 gRNA was quantified using a previously published TaqMan-based qRT-PCR assay (31, 55). To quantify HAstV4 RNA, we adapted a previously published qRT-PCR assay (16, 57).…”

“…We used a previously published qRT-PCR-based TCID 50 assay to quantify VA1 or HAstV4 infectious particles (31, 55). All viral infections were performed in triplicate with completion of two independent experiments.…”