Evidence for genetic linkage between the gene segments encoding NSP4 and VP6 proteins in common and reassortant human rotavirus strains

Abstract

NSP4-encoding genes of 78 human rotavirus strains of common or reassortant genotypes were characterized by reverse transcription-PCR followed by sequencing and phylogenetic analysis. It was found that all the human strains characterized clustered into only two of the five known NSP4 genotypes. Linkage between NSP4 genotypes and VP6 subgroups was 100%, NSP4 genotype A being linked to VP6 of subgroup I (SGI) and NSP4 of genotype B being linked to VP6 of SGII. The diversity among the NSP4- and VP6-encoding genes was significantly less than that among the VP7 and VP4 genes in cocirculating human rotavirus strains. Whereas G and P types appear to be shared among different animal species and humans, the NSP4- and VP6-encoding genes appear to segregate according to their host of origin, suggesting that these two proteins may be host restriction determinants. The NSP4-VP6 association may be structurally determined during rotavirus replication (morphogenesis).

FIG. 1.

Unrooted dendrogram constructed by using rotavirus NSP4 nucleotide sequences (coding region) and the maximum parsimony method. The NSP4 cDNA sequences of the 78 human rotavirus strains characterized in this study are highlighted. Sequences representative of the different existing NSP4 genotypes were included for comparison (accession numbers: EC, U96337; A131, AF144798; ALA, AF144793; CP-1, AF448854; CH1, AB065287; AU1, D89873; FR3-348, AB 048203). The calibration bar indicates the number of base conversions.

FIG. 2.

Phylogenetic tree of rotavirus NSP4 sequences constructed by the maximum parsimony method. The genotype-defining clusters were confirmed by bootstrap values of >90% (not shown). Sequences available from GenBank/EMBL from human and animal rotavirus strains (accession numbers and strain identifiers are in the first column) were included alongside representative NSP4 nucleotide sequences of human strains from this study for comparison. G and P type (where available) and host of origin are indicated after each strain identifier or accession number.

FIG. 3.

Phylogenetic tree of rotavirus NSP4 sequences (maximum parsimony method) derived from human strains of SGI or SGII; G and P type specificities, where available, are indicated. Sequences available at GenBank/EMBL were included for comparison. Lineages identified within NSP4 genotypes A or B, confirmed by bootstrap values of >90% (not shown), are indicated with horizontal lines. The NSP4 gene sequence of the human strain AU1 (accession number D89873) was included as an outlier.

FIG. 4.

Alignment of the deduced amino acid sequences of the genes encoding NSP4 genogroups A and B of human rotavirus strains. The DLP binding domain is boxed.