Human Astrovirus (HAstV) Replication Systems
Dr. Christiane Wobus
Human astroviruses (HAstV) are understudied positive-sense RNA viruses that cause viral gastroenteritis in immunocompromised people. HAstV are classified into three clades: VA/HMO, classical human AstV (HAstV) and MLB. In the Wobus laboratory, we recently established human intestinal enteroids (HIE) as an in vitro replication system for all the three astrovirus clades. Here, we used HIE to study the effects of triptolide (RNA polymerase inhibitor), ruxolitinib (JAK inhibitor) and 2’-C-methylcytidinine (2’CMC - nucleoside analogue) on HAstV replication. HIE monolayers were pretreated with these drugs overnight and infected with VA1 for 3 days. Virus titer was then determined by reverse transcription quantitative polymerase chain reaction (RTqPCR). Furthermore, we started the establishment of the reverse genetics system for MLB1 and HAstV1 in our lab. This is a process of obtaining virus stock by transfecting permissive cells with RNA encoding the viral genome. To do this, plasmids encoding MLB1 and HAstV1 viral genome were amplified in bacteria, purified with QIAGEN kit and transcribed into RNA in vitro. The RNA was later used to transfect Caco-2 and HEK293T cells followed by a 48 hour incubation. Virus was harvested from cell lysates after 3 freeze-thaw cycles. Finally, to establish a small animal model for HAstV, we infected polymeric immunoglobulin receptor knockout mice with VA1 and HAstV5 per orally and intraperitoneally. Virus titer was determined at 24 and 48 hours post infection from homogenized mice tissues. Results are currently being analyzed. Our data will provide information on HAstV replication and enhance the study of AstV biology.
Townsend, Sydney, "Human Astrovirus (HAstV) Replication Systems" (2019). Undergraduate Research and Creative Inquiry Symposia. 86.