Association of NSP4 mutation and VP4 rotavirus genotype in children with severe-moderate diarrhea

Context: Rotavirus is the main cause of acute diarrhea among children under five years old. Meanwhile, NSP4 has a role in the development of secretory diarrhea. The NSP4 genotype is divided into 21 genogroups (E1-E21). Information related to rotavirus infection and the role of NSP4 pathogenesis in humans has not yet been described clearly, along with the limited information on the association between NSP4 and VP4. Aims: To determine the genotype and mutation of NSP4 and analyze the mutation of NSP4 with VP4 genotype and diarrhea severity. Methods: A total of 51 rotavirus-positive samples were collected from children with acute diarrhea between August-December 2016 and June-August 2018 from two hospitals and two primary healthcare centers in Bandar Lampung, Indonesia. The severity of diarrhea data was collected by observation, interviews with the parents or guardians, and medical records based on Ruuska and Vesikari's scores. Identification of the VP4 gene was completed using primers from the WHO Manual of RV Detection and Characterization Methods protocols. P types of these samples were then determined by sequencing methods. DNA amplification for NSP4 gene was carried out using the primers described previously. Lastly, the amplified viral genes were subjected to sequence analysis. Results: In this study, amino acids substitution was found in H1, H2, H3 domain, enterotoxin region, VP4-binding site, and VP6-binding site from E1 and E2 genotype. Unusual combination occurred between P[8] genotype and E2 with amino acid substitution at position 72 in children with severe diarrhea. The enterotoxin region mutation at position 129 and hydrophobic domain H1 at position 14 mostly existed in the NSP4 sequence associated with P[4] and in children with severe diarrhea. Conclusion: This finding indicated that amino acid variation in the NSP4 sequence related to VP4 genotype but had no association with diarrhea severity.