J Vet Sci.  2018 Jan;19(1):35-43. 10.4142/jvs.2018.19.1.35.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene

Affiliations
  • 1Indian Council of Agricultural Research-Indian Veterinary Research Institute, Uttar Pradesh 243122, India. malikyps@ivri.res.in
  • 2Department of Biomedical Sciences, One Health Center for Zoonoses and Tropical Veterinary Medicine, Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, Federation of Saint Kitts and Nevis.
  • 3Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
  • 4Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest 1143, Hungary.

Abstract

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.

Keyword

enterotoxins; nsp4 gene evolution; porcine; rotavirus; viral nonstructural proteins

MeSH Terms

Animals
Classification
Clone Cells
Enterotoxins*
Epitopes
Genetic Variation
Genotype
Humans
Protein Folding
Protein Processing, Post-Translational
Rotavirus*
Sequence Analysis
Viral Nonstructural Proteins
Virulence
Enterotoxins
Epitopes
Viral Nonstructural Proteins

Figure

  • Fig. 1 Homology of the interspecies variable domains (ISVD) in NSP4 enterotoxins from porcine and human isolates. The red area indicates the ISVD where no apparent changes in the amino acid residues were observed. The present study included porcine NSP4s isolated from diarrheic (PoRVA-C3, 30, 32, 34, and 36) and non-diarrheic viral specimens (PoRVA-172, 173, 174, and 175). Porcine reference strain OSU-C5111 (E1 genotype, KJ450851) was included for better representation of homology.

  • Fig. 2 Phylogenetic analysis of nucleotide (A) and amino acid sequences (B) of porcine rotavirus group A (RVA) enterotoxins. RVA isolates with E2 and E9 genotypes were represented as empty (Δ) and solid (▲) triangles, respectively. Isolates with the E2 genotype were robustly separated from those with the E1 and E9 genotypes. Porcine RVA isolates with the E9 genotype were relatively close to those with the E1 genotype and constituted a separate sub-clade suggesting multiphyletic origin of members with E1/E9 and E2 genotypes. All PoRVA isolates identified in the study were clustered together except PoRVA-C3. Round dots (●) represent the diarrheic isolates, while square dots (■) show non-diarrheic PoRVA isolates. Reference prototype porcine strain of OSU-C5111 (E1 genotype, KJ450851), human reference strains Wa (E1 genotype, AF200225) and KUN (HuRVA reference strain, E2 genotype, D88829) are represented by asterisks (*).

  • Fig. 3 Haplotype networks of nsp4 genes with different genotypes of human and porcine origin. (A) Minimum spanning network showing closeness of E1 and E9 (▲) genotypes in reference to the E2 (Δ) genotype. (B) Parsimony splits network exhibiting diversity among members of the E1 and E9 genotypes. Grouping of human and porcine isolates of Indian and Chinese origin suggest possible re-assortment events among them. Black asterisks represent reference RVA strains. (C) The genealogy of nsp4 genes from porcine and human RVA isolates of E1 and E9 genotypes. The TCS network showing 27 haplotypes connected parsimoniously. Individual discs indicate haplotypes with the size of the disc proportional to the number of accessions in the haplotype. RVA strains with E1 and E9 genotypes exhibited distance from each other. The PoRVA-30 strain (encircled in eclipse) isolated in the study was identified as the basal (ancestral) haplotype. Hatch marks represent nucleotide changes during evolution.


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