Rotavirus Strain Isolation and Propagation

The human rotavirus WI61 strain was first isolated from an 18-month-old child with gastroenteritis in Children’s Hospital of Philadelphia in 1983.^{68 (link)} Rotarix (RV1, GSK) and RotaTeq (RV5, Merck) are kindly shared by Dr. Kristen Ogden at Vanderbilt University. Murine rotavirus rD6/2-2 g strain was generated using an optimized, plasmid-based rotavirus reverse genetics system,^{69 (link)} and tested for its genetic stability, intestinal replication, and virulence. African Green Monkey kidney epithelial cell line MA104 (CRL-2378.1) was obtained from American Type Culture Collection (ATCC) and cultured in Medium 199 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (VWR), 100 units/ml penicillin and 100 μg/ml streptomycin. Rotavirus strains were propagated as described previously^{27 (link)} and the titers of virus concentrate were determined in MA104 cells using a standard plaque assay.^{70 (link)} Rotavirus inoculum was prepared by activating the stock with 5 μg/ml trypsin for 20 min at 37°C and diluting it into 50% CM with 10 μM Y-27632 up to the total volume of 100 μl, with 50 μg/ml soybean trypsin inhibitor.

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Greigert V., Saraav I., Son J., Zhu Y., Dayao D., Antia A., Tzipori S., Witola W.H., Stappenbeck T.S., Ding S, & Sibley L.D. (2024). Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus. Gut Microbes, 16(1), 2297897.

Publication 2024

RotaTeq MA104 Medium 199

Corresponding Organization : Washington University in St. Louis

Other organizations : Tufts University, University of Illinois Urbana-Champaign, Cleveland Clinic Lerner College of Medicine

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Variable analysis

independent variables

Rotavirus strains: human rotavirus WI61 strain, Rotarix (RV1, GSK), RotaTeq (RV5, Merck), and murine rotavirus rD6/2-2 g strain

dependent variables

Genetic stability of murine rotavirus rD6/2-2 g strain
Intestinal replication of murine rotavirus rD6/2-2 g strain
Virulence of murine rotavirus rD6/2-2 g strain

control variables

African Green Monkey kidney epithelial cell line MA104 (CRL-2378.1) cultured in Medium 199 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (VWR), 100 units/ml penicillin and 100 μg/ml streptomycin
Rotavirus strains propagated as described previously
Rotavirus inoculum prepared by activating the stock with 5 μg/ml trypsin for 20 min at 37°C and diluting it into 50% CM with 10 μM Y-27632 up to the total volume of 100 μl, with 50 μg/ml soybean trypsin inhibitor

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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