Lijuan Yuan, Viginia Tech

Charlotte Nyblade1, Alice Leruth1, Facundo Gabriel Cuba2, Peng Zhou1, Lauren Lavoie1, Judy Qiu3,4, Xiao-Li Pang4, Lijuan Yuan1* 1.Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA, USA 2.National Center for Genomics and Bioinformatics, Argentina 3.Public Health Laboratory, Alberta Precision Laboratories, Edmonton, Alberta, Canada 4.Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada

Vaccines

A human rotavirus (HRV) strain was isolated from a human stool sample (AB-NOV-GII-4) using gnotobiotic pigs. When six-day-old gnotobiotic pigs were inoculated with AB-NOV-GII-4, the inoculum induced norovirus shedding (peak titer: 1.64 × 10⁴ genomic RNA copies/g of GII.4/P[16]) and diarrhea lasting 6.5 days. However, when 27-day-old gnotobiotic pigs were inoculated, norovirus shedding was absent, yet severe diarrhea persisted for 6–8 days. Viral RNA and DNA extracted from pig fecal samples underwent deep sequencing (RNAseq and DNAseq via NovaSeq SP v1.5, 300 Cycle), revealing that only HRV was present in the older pigs. Genomic analysis confirmed the strain as Rotavirus A with the genome constellation R1-C1-M1-P[8]-A1-I1-T1-N1-G1-E1-H1, closely related to the virulent HRV Wa G1P[8] strain. This novel strain was designated VTR-1 (GenBank accession numbers PQ039531–PQ039541). Following serial passaging in MA104 and Vero cell cultures, viral titers measured by RT-qPCR increased from ~10³/mL (passages 2–8) to >10⁶-7/mL (passages 14–16), demonstrating successful adaptation to cell culture. The virulent and cell culture-adapted variants of VTR-1 HRV offer a valuable model for rotavirus challenge studies, facilitating the evaluation of next-generation rotavirus vaccines and the production of large HRV stock pools. This study also underscores the utility of gnotobiotic pigs for isolating and purifying mixed viruses from human stool samples.