Murine Norovirus: Additional Protocols for Basic and Antiviral Studies

Abstract

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity. Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR. Support Protocol 1: Preparation of standard. Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging. Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER). Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus. Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells. Support Protocol 3: Purification of the norovirus NS1-2 protein. Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus. Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus. Support Protocol 5: Preparation of conditioned medium for enteroids culture. Support Protocol 6: Isolation of crypts for enteroids generation. Support Protocol 7: Enteroid culture passaging and maintenance. Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections. Alternate Protocol 1: Intragastric inoculation of neonatal mice. Alternate Protocol 2: Scoring colon contents.