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Reverse transcription-real-time polymerase chain reaction (RT-qPCR) for the VP6 gene was used to study group A rotavirus shedding in children with symptomatic and asymptomatic rotavirus infection. Sequential stool samples (n = 345) from 10 children with rotavirus associated diarrhea and from five children (n = 161) with asymptomatic rotavirus infection were collected over a period of 2 months. A RT-qPCR assay on the samples using a rotavirus VP6 plasmid standard demonstrated high reproducibility, with an inter-assay coefficient of variation (CV) of 1.40–2.97% and an intra-assay CV of 0.03–3.03%. The median duration of shedding was longer in children with diarrhea compared to asymptomatic children (24 days vs. 18 days; P = 0.066). The median quantitation cycle (Cq) at presentation in symptomatic children was 17.21 compared to 30.98 in asymptomatic children (P = 0.086). The temporal pattern in symptomatic children consisted of a high initial viral shedding coinciding with the duration of diarrhea, followed by a rapid fall, and then a small increase in secondary shedding 21 days later. Compared to children with rotavirus diarrhea, those with asymptomatic infection shed lower quantities of virus throughout the observation period. No difference was noted between the G and P genotypes of samples collected at onset of infection and during the shedding period. Shedding was intermittent in a subset of children in both groups. RT-qPCR is a useful method to characterize shedding patterns. J. Med. Virol. 85:1661–1668, 2013. © 2013 Wiley Periodicals, Inc.

Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. Two unusual rotavirus strains not previously reported in India, G11P[25] (CRI 10795) and G3P[3] (CRI 33594) were isolated from faecal samples of asymptomatic children in India. The strains were characterized by sequence analysis of the genes encoding the VP7, VP4, VP6, and NSP4. The G11P[25] strain was closely related to the human G11P[25] strains from Bangladesh (with 98% identity at the nucleotide [nt] level and the amino acid [aa] level for the VP7 gene and 96% identity at the nt and 98% at the aa level for the VP4 gene). The G3P[3] strain was found to be related to a G3P[3] strain isolated in Thailand (CMH222; 88% identity at the nt level and 97% at aa level for the VP7 gene and 84% identity at the nt level and 90% at the aa level for the VP4 gene). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G11P[25] strain was of VP6 subgroup II and NSP4 genotype B. The G3P[3] strain was identified as NSP4 genotype C and the VP6 gene showed 97% identity at the deduced amino acid level with strain CMH222 (Thailand) strain but did not cluster with sequences of SGI, SGII, SGI+II or SG-nonI/nonII. Both strains had gene segments of animal rotavirus origin suggesting inter-species transmission of rotavirus, and in the case of G11P[25] possibly underwent reassortment subsequently with human strains resulting in an animal-human hybrid strain.

Reverse transcription-real-time polymerase chain reaction (RT-qPCR) for the VP6 gene was used to study group A rotavirus shedding in children with symptomatic and asymptomatic rotavirus infection. Sequential stool samples (n = 345) from 10 children with rotavirus associated diarrhea and from five children (n = 161) with asymptomatic rotavirus infection were collected over a period of 2 months. A RT-qPCR assay on the samples using a rotavirus VP6 plasmid standard demonstrated high reproducibility, with an inter-assay coefficient of variation (CV) of 1.40-2.97% and an intra-assay CV of 0.03-3.03%. The median duration of shedding was longer in children with diarrhea compared to asymptomatic children (24 days vs. 18 days; P = 0.066). The median quantitation cycle (C(q)) at presentation in symptomatic children was 17.21 compared to 30.98 in asymptomatic children (P = 0.086). The temporal pattern in symptomatic children consisted of a high initial viral shedding coinciding with the duration of diarrhea, followed by a rapid fall, and then a small increase in secondary shedding 21 days later. Compared to children with rotavirus diarrhea, those with asymptomatic infection shed lower quantities of virus throughout the observation period. No difference was noted between the G and P genotypes of samples collected at onset of infection and during the shedding period. Shedding was intermittent in a subset of children in both groups. RT-qPCR is a useful method to characterize shedding patterns.